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<front>
<journal-meta>
<journal-id journal-id-type="publisher-id">Front. Bioeng. Biotechnol.</journal-id>
<journal-title>Frontiers in Bioengineering and Biotechnology</journal-title>
<abbrev-journal-title abbrev-type="pubmed">Front. Bioeng. Biotechnol.</abbrev-journal-title>
<issn pub-type="epub">2296-4185</issn>
<publisher>
<publisher-name>Frontiers Media S.A.</publisher-name>
</publisher>
</journal-meta>
<article-meta>
<article-id pub-id-type="publisher-id">1179332</article-id>
<article-id pub-id-type="doi">10.3389/fbioe.2023.1179332</article-id>
<article-categories>
<subj-group subj-group-type="heading">
<subject>Bioengineering and Biotechnology</subject>
<subj-group>
<subject>Original Research</subject>
</subj-group>
</subj-group>
</article-categories>
<title-group>
<article-title>Micronutrient optimization for tissue engineered articular cartilage production of type II collagen</article-title>
<alt-title alt-title-type="left-running-head">Cruz et al.</alt-title>
<alt-title alt-title-type="right-running-head">
<ext-link ext-link-type="uri" xlink:href="https://doi.org/10.3389/fbioe.2023.1179332">10.3389/fbioe.2023.1179332</ext-link>
</alt-title>
</title-group>
<contrib-group>
<contrib contrib-type="author">
<name>
<surname>Cruz</surname>
<given-names>Maria A.</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
<uri xlink:href="https://loop.frontiersin.org/people/2235099/overview"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Gonzalez</surname>
<given-names>Yamilet</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
<uri xlink:href="https://loop.frontiersin.org/people/2251069/overview"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname>V&#xe9;lez Toro</surname>
<given-names>Javier A.</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Karimzadeh</surname>
<given-names>Makan</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Rubbo</surname>
<given-names>Anthony</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
<uri xlink:href="https://loop.frontiersin.org/people/2238057/overview"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Morris</surname>
<given-names>Lauren</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Medam</surname>
<given-names>Ramapaada</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Splawn</surname>
<given-names>Taylor</given-names>
</name>
<xref ref-type="aff" rid="aff2">
<sup>2</sup>
</xref>
<uri xlink:href="https://loop.frontiersin.org/people/840205/overview"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Archer</surname>
<given-names>Marilyn</given-names>
</name>
<xref ref-type="aff" rid="aff3">
<sup>3</sup>
</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Fernandes</surname>
<given-names>Russell J.</given-names>
</name>
<xref ref-type="aff" rid="aff3">
<sup>3</sup>
</xref>
<uri xlink:href="https://loop.frontiersin.org/people/1115103/overview"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Dennis</surname>
<given-names>James E.</given-names>
</name>
<xref ref-type="aff" rid="aff2">
<sup>2</sup>
</xref>
</contrib>
<contrib contrib-type="author" corresp="yes">
<name>
<surname>Kean</surname>
<given-names>Thomas J.</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
<xref ref-type="aff" rid="aff2">
<sup>2</sup>
</xref>
<xref ref-type="corresp" rid="c001">&#x2a;</xref>
<uri xlink:href="https://loop.frontiersin.org/people/839235/overview"/>
</contrib>
</contrib-group>
<aff id="aff1">
<sup>1</sup>
<institution>Biionix Cluster</institution>, <institution>Internal Medicine</institution>, <institution>University of Central Florida College of Medicine</institution>, <addr-line>Orlando</addr-line>, <addr-line>FL</addr-line>, <country>United States</country>
</aff>
<aff id="aff2">
<sup>2</sup>
<institution>Baylor College of Medicine</institution>, <addr-line>Houston</addr-line>, <addr-line>TX</addr-line>, <country>United States</country>
</aff>
<aff id="aff3">
<sup>3</sup>
<institution>Department of Orthopaedics and Sports Medicine, University of Washington</institution>, <addr-line>Seattle</addr-line>, <addr-line>WA</addr-line>, <country>United States</country>
</aff>
<author-notes>
<fn fn-type="edited-by">
<p>
<bold>Edited by:</bold> <ext-link ext-link-type="uri" xlink:href="https://loop.frontiersin.org/people/60075/overview">Oommen Varghese</ext-link>, Uppsala University, Sweden</p>
</fn>
<fn fn-type="edited-by">
<p>
<bold>Reviewed by:</bold> <ext-link ext-link-type="uri" xlink:href="https://loop.frontiersin.org/people/892370/overview">Ron June</ext-link>, Montana State University, United States</p>
<p>
<ext-link ext-link-type="uri" xlink:href="https://loop.frontiersin.org/people/622324/overview">Solvig Diederichs</ext-link>, Heidelberg University Hospital, Germany</p>
</fn>
<corresp id="c001">&#x2a;Correspondence: Thomas J. Kean, <email>thomas.kean@ucf.edu</email>
</corresp>
</author-notes>
<pub-date pub-type="epub">
<day>06</day>
<month>06</month>
<year>2023</year>
</pub-date>
<pub-date pub-type="collection">
<year>2023</year>
</pub-date>
<volume>11</volume>
<elocation-id>1179332</elocation-id>
<history>
<date date-type="received">
<day>06</day>
<month>03</month>
<year>2023</year>
</date>
<date date-type="accepted">
<day>23</day>
<month>05</month>
<year>2023</year>
</date>
</history>
<permissions>
<copyright-statement>Copyright &#xa9; 2023 Cruz, Gonzalez, V&#xe9;lez Toro, Karimzadeh, Rubbo, Morris, Medam, Splawn, Archer, Fernandes, Dennis and Kean.</copyright-statement>
<copyright-year>2023</copyright-year>
<copyright-holder>Cruz, Gonzalez, V&#xe9;lez Toro, Karimzadeh, Rubbo, Morris, Medam, Splawn, Archer, Fernandes, Dennis and Kean</copyright-holder>
<license xlink:href="http://creativecommons.org/licenses/by/4.0/">
<p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</p>
</license>
</permissions>
<abstract>
<p>Tissue Engineering of cartilage has been hampered by the inability of engineered tissue to express native levels of type II collagen <italic>in vitro</italic>. Inadequate levels of type II collagen are, in part, due to a failure to recapitulate the physiological environment in culture. In this study, we engineered primary rabbit chondrocytes to express a secreted reporter, <italic>Gaussia</italic> Luciferase, driven by the type II collagen promoter, and applied a Design of Experiments approach to assess chondrogenic differentiation in micronutrient-supplemented medium. Using a Response Surface Model, 240 combinations of micronutrients absent in standard chondrogenic differentiation medium, were screened and assessed for type II collagen promoter-driven <italic>Gaussia</italic> luciferase expression. While the target of this study was to establish a combination of all micronutrients, alpha-linolenic acid, copper, cobalt, chromium, manganese, molybdenum, vitamins A, E, D and B7 were all found to have a significant effect on type II collagen promoter activity. Five conditions containing all micronutrients predicted to produce the greatest luciferase expression were selected for further study. Validation of these conditions in 3D aggregates identified an optimal condition for type II collagen promoter activity. Engineered cartilage grown in this condition, showed a 170% increase in type II collagen expression (Day 22 Luminescence) and in Young&#x2019;s tensile modulus compared to engineered cartilage in basal media alone.Collagen cross-linking analysis confirmed formation of type II-type II collagen and type II-type IX collagen cross-linked heteropolymeric fibrils, characteristic of mature native cartilage. Combining a Design of Experiments approach and secreted reporter cells in 3D aggregate culture enabled a high-throughput platform that can be used to identify more optimal physiological culture parameters for chondrogenesis.</p>
</abstract>
<kwd-group>
<kwd>primary chondrocyte</kwd>
<kwd>media optimization</kwd>
<kwd>tissue engineered cartilage</kwd>
<kwd>design of experiments (DoE)</kwd>
<kwd>scaffold-free tissue engineered cartilage</kwd>
<kwd>vitamins and minerals</kwd>
<kwd>extracellular matrix reporter</kwd>
<kwd>type II collagen heteropolymer</kwd>
</kwd-group>
<contract-sponsor id="cn001">National Institute of Arthritis and Musculoskeletal and Skin Diseases<named-content content-type="fundref-id">10.13039/100000069</named-content>
</contract-sponsor>
<contract-sponsor id="cn002">University of Central Florida<named-content content-type="fundref-id">10.13039/100007900</named-content>
</contract-sponsor>
<contract-sponsor id="cn003">College of Medicine, University of Central Florida<named-content content-type="fundref-id">10.13039/100012874</named-content>
</contract-sponsor>
<custom-meta-wrap>
<custom-meta>
<meta-name>section-at-acceptance</meta-name>
<meta-value>Biomaterials</meta-value>
</custom-meta>
</custom-meta-wrap>
</article-meta>
</front>
<body>
<sec id="s1">
<title>Introduction</title>
<p>Osteoarthritis (OA) is the most common degenerative musculoskeletal disease and is projected to increase in prevalence (<xref ref-type="bibr" rid="B55">Mueller and Tuan, 2011</xref>; <xref ref-type="bibr" rid="B65">Sacitharan, 2019</xref>). OA is characterized by progressive degeneration of articular cartilage in the joints of the hands, knees, and hip due to an imbalance of cartilage matrix anabolism and catabolism (<xref ref-type="bibr" rid="B55">Mueller and Tuan, 2011</xref>; <xref ref-type="bibr" rid="B65">Sacitharan, 2019</xref>). Articular cartilage is a form of specialized connective tissue, primarily composed of type II collagen, water, and proteoglycans with sparsely distributed chondrocytes (<xref ref-type="bibr" rid="B73">Sophia Fox et al., 2009</xref>). Cartilage has limited healing and regenerative abilities given that its avascular nature limits access to circulating progenitor cells following physical insult (<xref ref-type="bibr" rid="B73">Sophia Fox et al., 2009</xref>; <xref ref-type="bibr" rid="B34">Kean and Dennis, 2015</xref>). Currently, there are no disease-modifying treatments for OA (<xref ref-type="bibr" rid="B34">Kean and Dennis, 2015</xref>; <xref ref-type="bibr" rid="B65">Sacitharan, 2019</xref>). Available therapeutics offer short-lived relief of acute symptoms and do not prevent endpoint joint damage, therefore there is a strong need for the development of new disease-modifying therapeutics (<xref ref-type="bibr" rid="B34">Kean and Dennis, 2015</xref>; <xref ref-type="bibr" rid="B65">Sacitharan, 2019</xref>; <xref ref-type="bibr" rid="B13">Dennis et al., 2020a</xref>).</p>
<p>Tissue engineering of cartilage has the potential to revolutionize the field by providing improved <italic>in vitro</italic> models for drug discovery and/or a biological replacement (<xref ref-type="bibr" rid="B13">Dennis et al., 2020a</xref>). Tissue engineering incorporates the use of components such as cells, scaffolds, growth factors, and physical stimulation to generate biomimetic tissue (<xref ref-type="bibr" rid="B22">Francis et al., 2018</xref>). However, tissue engineering of cartilage has been hampered by an inability to recapitulate the properties of native cartilage tissue, which we hypothesize is primarily due to insufficient type II collagen production. Whereas 90%&#x2013;95% of collagen in native tissue is type II collagen, several studies have reported much lower type II collagen levels in engineered tissue with values hovering around 20% of dry weight despite modifications to increase collagen deposition (<xref ref-type="bibr" rid="B85">Vunjak-Novakovic et al., 1999</xref>; <xref ref-type="bibr" rid="B25">Gemmiti and Guldberg, 2006</xref>; <xref ref-type="bibr" rid="B5">Bian et al., 2011</xref>; <xref ref-type="bibr" rid="B69">Sato et al., 2017</xref>; <xref ref-type="bibr" rid="B86">Whitney et al., 2018</xref>; <xref ref-type="bibr" rid="B13">Dennis et al., 2020a</xref>). We postulate that part of this deficiency in type II collagen is due to sub-optimal formulation of the culture medium used for cartilage engineering <italic>in vitro</italic>.</p>
<p>Differentiation media traditionally used for chondrocyte cell culture was noted to lack several micronutrients which are known to be physiologically essential for a host of biological processes (<xref ref-type="bibr" rid="B14">Dennis et al., 2020b</xref>). Although the specific roles that some of these micronutrients have in chondrogenesis remain undefined, there are several findings that point towards these biomolecules having significant effects on cartilage generation and maintenance (<xref ref-type="bibr" rid="B38">Koyano et al., 1996</xref>; <xref ref-type="bibr" rid="B7">Booth and Mayer, 1997</xref>; <xref ref-type="bibr" rid="B42">Litchfield et al., 1998</xref>; <xref ref-type="bibr" rid="B10">Connor, 1999</xref>; <xref ref-type="bibr" rid="B21">Finch, 2015</xref>; <xref ref-type="bibr" rid="B9">Chin and Ima-Nirwana, 2018</xref>; <xref ref-type="bibr" rid="B14">Dennis et al., 2020b</xref>). In our ATDC5 study, we found the combination of copper and vitamin K to have the most beneficial effect on type II collagen production (<xref ref-type="bibr" rid="B13">Dennis et al., 2020a</xref>). We hypothesize the addition of these vitamins and minerals to basal differentiation medium will promote type II collagen production <italic>in vitro</italic> and better mimic the physiological environment.</p>
<p>A Design of Experiments (DoE) approach was implemented in this study to screen different combinations of vitamins and minerals. DoE is a statistical technique that facilitates systematic optimization by producing experimental design models to study interactions of multiple factors on a desired outcome or response. DoE allows for a multi-factor, rather than a one-factor approach, that evaluates synergistic effects, and can predict optimal conditions while reducing the burden of conducting repetitive experiments. DoE has provided significant benefits to other fields of engineering and biotechnology but has rarely been used in cartilage tissue engineering and regenerative medicine (<xref ref-type="bibr" rid="B15">Enochson et al., 2012</xref>; <xref ref-type="bibr" rid="B39">Kuterbekov et al., 2019</xref>). A defined chondrogenic media containing all the missing vitamins and minerals, essential for human health, has not been established prior to this study.</p>
<p>In this study, we have identified, for the first time, an optimal supplementation of physiologically necessary micronutrients to chondrogenic media, using a streamlined platform that includes a type II collagen promoter-driven <italic>Gaussia</italic> luciferase construct in primary rabbit articular chondrocytes combined with a DoE approach. This optimized chondrogenic media significantly enhances type II collagen expression in primary rabbit chondrocytes cultured in 3D cell aggregates and engineered cartilage sheets.</p>
</sec>
<sec sec-type="results" id="s2">
<title>Results</title>
<sec id="s2-1">
<title>Stimulation of type II collagen promoter activity by TGF-&#x3b2;1 in primary rabbit chondrocytes</title>
<p>To characterize the TGF-&#x3b2;1 response of engineered type II collagen promoter-driven <italic>Gaussia</italic> luciferase reporter (COL2A1-Gluc) in primary rabbit chondrocytes, cells were cultured in 3D aggregates in defined chondrogenic media supplemented with 0&#x2013;10&#xa0;ng/mL of TGF-&#x3b2;1, a known stimulator of type II collagen (<xref ref-type="bibr" rid="B26">Han et al., 2005</xref>; <xref ref-type="bibr" rid="B31">Jennifer et al., 2010</xref>; <xref ref-type="bibr" rid="B13">Dennis et al., 2020a</xref>). Conditioned media, containing the secreted <italic>Gaussia</italic> luciferase, was assayed for luminescence over 3&#xa0;weeks. Dose response curves were generated from luminescence data at Day 7 (<xref ref-type="fig" rid="F1">Figure 1A</xref>) and Day 21 (<xref ref-type="fig" rid="F1">Figure 1B</xref>). As seen in <xref ref-type="fig" rid="F1">Figure 1</xref>, there was a dose dependent increase in luminescence with a calculated 50% effective concentration (EC50) of 0.17&#xa0;ng/ml and 0.10&#xa0;ng/ml for Day 7 and Day 21 respectively. Curve fit was tested with an extra sum-of-squares F test (GraphPad Prism) and <italic>p</italic> &#x3c; 0.0001 that the data could not be fit to a single curve.</p>
<fig id="F1" position="float">
<label>FIGURE 1</label>
<caption>
<p>Dose effect of TGF-&#x3b2;1 on COL2A1-GLuc reporter rabbit chondrocytes. <bold>(A</bold>, <bold>B)</bold> Primary COL2A1-GLuc rabbit chondrocytes were grown in aggregate culture in the presence of different concentrations of TGF-&#x3b2;1 (0&#x2013;10&#xa0;ng/mL). Dose response curves were generated from transformed luminescence data at day 7 <bold>(A)</bold> and day 21 <bold>(B)</bold> after seeding. EC50s for each day are shown within each curve. Values are the mean &#xb1; S.D. n &#x3d; 4.</p>
</caption>
<graphic xlink:href="fbioe-11-1179332-g001.tif"/>
</fig>
</sec>
<sec id="s2-2">
<title>Response Surface Model and subsequent ANOVA analysis identified interactions between micronutrients that increased type II collagen promoter-driven expression of <italic>Gaussia</italic> luciferase</title>
<p>To identify potential interactions between factors and their effect on type II collagen expression, COL2-GLuc rabbit chondrocytes were seeded in 3D aggregate culture with DoE generated combinations of vitamins and minerals. Media was sampled and replaced every 2&#x2013;3&#xa0;days over 3&#xa0;weeks. Combinations and concentrations are defined by the parameters set in the response surface model (<xref ref-type="table" rid="T1">Table 1</xref>) and are listed in <xref ref-type="sec" rid="s12">Supplementary Table S2</xref> in <xref ref-type="sec" rid="s12">Supplementary Data Sheet S2</xref>.</p>
<table-wrap id="T1" position="float">
<label>TABLE 1</label>
<caption>
<p>Concentrations of micronutrients absent in chondrogenic media with input parameters for DoE screen.</p>
</caption>
<table>
<thead valign="top">
<tr>
<th rowspan="2" align="center">Term</th>
<th rowspan="2" align="center">Name</th>
<th rowspan="2" align="center">Abbreviation</th>
<th rowspan="2" align="center">Manufacturer</th>
<th rowspan="2" align="center">Maximal serum concentration<xref ref-type="table-fn" rid="Tfn1">
<sup>1</sup>
</xref>
</th>
<th colspan="2" align="center">DoE screen (pg/mI)</th>
</tr>
<tr>
<th align="center">Minimum</th>
<th align="center">Maximum</th>
</tr>
</thead>
<tbody valign="top">
<tr>
<td align="center">A</td>
<td align="center">Linolenic Acid</td>
<td align="center">ALA</td>
<td align="center">Sigma-Aldrich</td>
<td align="center">54&#xa0;mg/L</td>
<td align="center">1.0E-15</td>
<td align="center">270.43</td>
</tr>
<tr>
<td align="center">B</td>
<td align="center">Chromium</td>
<td align="center">Cr</td>
<td align="center">Sigma-Aldrich</td>
<td align="center">28&#xa0;&#x03BC;g/L</td>
<td align="center">1.0E-15</td>
<td align="center">0.14</td>
</tr>
<tr>
<td align="center">C</td>
<td align="center">Cobalt</td>
<td align="center">Co</td>
<td align="center">Sigma-Aldrich</td>
<td align="center">0.9 &#x03BC;g/L</td>
<td align="center">1.0E-15</td>
<td align="center">4.52E-03</td>
</tr>
<tr>
<td align="center">D</td>
<td align="center">Copper</td>
<td align="center">Cu</td>
<td align="center">Sigma-Aldrich</td>
<td align="center">670 &#x03BC;g/L</td>
<td align="center">1.0E-15</td>
<td align="center">3.35</td>
</tr>
<tr>
<td align="center">E</td>
<td align="center">Iodine</td>
<td align="center">I</td>
<td align="center">Alfa-Aesar</td>
<td align="center">92&#xa0;&#x03BC;g/L</td>
<td align="center">1.0E-15</td>
<td align="center">0.46</td>
</tr>
<tr>
<td align="center">F</td>
<td align="center">Manganese</td>
<td align="center">Mn</td>
<td align="center">Sigma-Aldrich</td>
<td align="center">12&#xa0;&#x03BC;g/L</td>
<td align="center">1.0E-15</td>
<td align="center">0.06</td>
</tr>
<tr>
<td align="center">G</td>
<td align="center">Molybdenum</td>
<td align="center">Mo</td>
<td align="center">Sigma-Aldrich</td>
<td align="center">2.0 &#x03BC;g/L</td>
<td align="center">1.0E-15</td>
<td align="center">0.01</td>
</tr>
<tr>
<td align="center">H</td>
<td align="center">Thyroxine</td>
<td align="center">T4</td>
<td align="center">Sigma-Aldrich</td>
<td align="center">17&#xa0;ng/L</td>
<td align="center">1.0E-15</td>
<td align="center">0.12</td>
</tr>
<tr>
<td align="center">J</td>
<td align="center">Vitamin A</td>
<td align="center">Vit A</td>
<td align="center">Alfa-Aesar</td>
<td align="center">780 &#x03BC;g/L</td>
<td align="center">1.0E-15</td>
<td align="center">0.08</td>
</tr>
<tr>
<td align="center">K</td>
<td align="center">Vitamin B12</td>
<td align="center">Vit B12</td>
<td align="center">Sigma-Aldrich</td>
<td align="center">914&#xa0;ng/L</td>
<td align="center">1.0E-15</td>
<td align="center">4.56E-03</td>
</tr>
<tr>
<td align="center">L</td>
<td align="center">Biotin</td>
<td align="center">Vit B7</td>
<td align="center">Alfa-Aesar</td>
<td align="center">3,004&#xa0;ng/L</td>
<td align="center">1.0E-15</td>
<td align="center">0.01</td>
</tr>
<tr>
<td align="center">M</td>
<td align="center">Vitamin D</td>
<td align="center">Vit D</td>
<td align="center">Selleck Chemicals</td>
<td align="center">86&#xa0;ng/L</td>
<td align="center">1.0E-15</td>
<td align="center">4.31E-04</td>
</tr>
<tr>
<td align="center">N</td>
<td align="center">Vitamin E</td>
<td align="center">Vit E</td>
<td align="center">Sigma-Aldrich</td>
<td align="center">18.4&#xa0;mg/L</td>
<td align="center">1.0E-15</td>
<td align="center">91.84</td>
</tr>
<tr>
<td align="center">0</td>
<td align="center">Vitamin K</td>
<td align="center">Vit K</td>
<td align="center">Sigma-Aldrich</td>
<td align="center">2.2&#xa0;&#x03BC;g/L</td>
<td align="center">1.0E-15</td>
<td align="center">0.01</td>
</tr>
<tr>
<td align="center">P</td>
<td align="center">Zinc</td>
<td align="center">Zn</td>
<td align="center">Alfa-Aesar</td>
<td align="center">1.2&#xa0;mg/L</td>
<td align="center">1.0E-15</td>
<td align="center">5.99</td>
</tr>
</tbody>
</table>
<table-wrap-foot>
<fn id="Tfn1">
<label>
<sup>1</sup>
</label>
<p>Note: References for serum concentrations: <xref ref-type="bibr" rid="B13">Dennis et al. (2020a)</xref>, <xref ref-type="bibr" rid="B22">Francis et al. (2018)</xref>, <xref ref-type="bibr" rid="B25">Gemmiti and Guldberg (2006)</xref>, <xref ref-type="bibr" rid="B7">Booth and Mayer (1997)</xref>, <xref ref-type="bibr" rid="B9">Chin and Ima-Nirwana (2018)</xref>, <xref ref-type="bibr" rid="B21">Finch (2015)</xref>, <xref ref-type="bibr" rid="B38">Koyano et al. (1996)</xref>, <xref ref-type="bibr" rid="B2">Badr et al. (2007)</xref>, <xref ref-type="bibr" rid="B57">Pacifici et al. (1980)</xref>, <xref ref-type="bibr" rid="B76">Sumitani et al. (2020)</xref>, <xref ref-type="bibr" rid="B16">Farndale et al. (1982)</xref>, <xref ref-type="bibr" rid="B46">Makris et al. (2013)</xref>, <xref ref-type="bibr" rid="B68">Sardesai (1993)</xref>, <xref ref-type="bibr" rid="B53">Mello and Tuan (2006)</xref>, <xref ref-type="bibr" rid="B41">Lind et al. (2013)</xref>, <xref ref-type="bibr" rid="B49">Masuda et al. (2015)</xref>, <xref ref-type="bibr" rid="B74">Stabler (2013)</xref>, <xref ref-type="bibr" rid="B83">Tsonis (1991)</xref>, <xref ref-type="bibr" rid="B88">Wluka et al. (2002)</xref>, <xref ref-type="bibr" rid="B51">MayoClinic (2022)</xref>.</p>
</fn>
</table-wrap-foot>
</table-wrap>
<p>In the surface response model, each vitamin or mineral is introduced as an independent variable and is defined in Design-Expert (V.12, StatEase) as a model term. Luminescence signal over time, cumulative luminescence and resazurin data are defined as responses. The response surface study was designed as a quadratic model. <xref ref-type="fig" rid="F2">Figures 2A, B</xref> shows the normal probability plot after the data was transformed to fit the quadratic model for week 2 and week 3. The residuals are the deviation of each sample compared to its predicted value. For the residuals to be normally distributed they must show a linear trend, indicated by the red line, with little variation outside of it. As seen in <xref ref-type="fig" rid="F2">Figures 2A, B</xref> the residuals are normally distributed for all timepoints.</p>
<fig id="F2" position="float">
<label>FIGURE 2</label>
<caption>
<p>Dose effect of basal chondrogenic media supplemented with DoE micronutrient combinations on COL2A1-GLuc reporter Rabbit chondrocytes. Normal probability plots of the residuals for <italic>Gaussia</italic> Luciferase signal at weeks 2 <bold>(A)</bold> and 3 <bold>(B)</bold> after seeding. 3D surface response plots for interactive effects between vitamin A and linolenic acid at indicated weeks after seeding. End of week 2 <bold>(C)</bold>, and week 3 <bold>(D)</bold>.</p>
</caption>
<graphic xlink:href="fbioe-11-1179332-g002.tif"/>
</fig>
<p>ANOVA analysis of luminescence expression for week 2 and week 3 of chondrogenesis identified significant model terms, i.e., factors that have significant effects on the type II collagen responses (<xref ref-type="table" rid="T2">Table 2</xref>).</p>
<table-wrap id="T2" position="float">
<label>TABLE 2</label>
<caption>
<p>ANOVA results of surface response model for micronutrients significantly regulating COL2A1-GLuc reporter activity in rabbit chondrocytes.</p>
</caption>
<table>
<thead valign="top">
<tr>
<td rowspan="2" align="left">Source</td>
<th colspan="2" align="center">Week 1</th>
<th colspan="2" align="center">Week 2</th>
<th colspan="2" align="center">Week 3</th>
</tr>
<tr>
<td align="left">F- Value</td>
<td align="left">
<italic>p</italic>-value</td>
<td align="left">F- Value</td>
<td align="left">
<italic>p</italic>-value</td>
<td align="left">F- Value</td>
<td align="left">
<italic>p</italic>-value</td>
</tr>
</thead>
<tbody valign="top">
<tr>
<td align="left" style="background-color:#FFFF00">Model</td>
<td align="center" style="background-color:#FFFF00">2.480</td>
<td align="center" style="background-color:#FFFF00">&#x3c;0.0001</td>
<td align="center" style="background-color:#FFFF00">2.730</td>
<td align="center" style="background-color:#FFFF00">&#x3c;0.0001</td>
<td align="center" style="background-color:#FFFF00">2.491</td>
<td align="center" style="background-color:#FFFF00">&#x3c;0.0001</td>
</tr>
<tr>
<td align="left" style="background-color:#90EE90">ALA</td>
<td align="center" style="background-color:#90EE90">9.980</td>
<td align="center" style="background-color:#90EE90">&#x3c;0.0001</td>
<td align="center" style="background-color:#90EE90">30.420</td>
<td align="center" style="background-color:#90EE90">&#x3c;0.0001</td>
<td align="center" style="background-color:#90EE90">33.971</td>
<td align="center" style="background-color:#90EE90">0.001</td>
</tr>
<tr>
<td align="left" style="background-color:#90EE90">Cu</td>
<td align="center" style="background-color:#90EE90">11.020</td>
<td align="center" style="background-color:#90EE90">0.001</td>
<td align="center" style="background-color:#90EE90">13.760</td>
<td align="center" style="background-color:#90EE90">0.000</td>
<td align="center" style="background-color:#90EE90">10.709</td>
<td align="center" style="background-color:#90EE90">0.001</td>
</tr>
<tr>
<td align="left" style="background-color:#90EE90">Vit A</td>
<td align="center" style="background-color:#90EE90">42.650</td>
<td align="center" style="background-color:#90EE90">&#x3c;0.0001</td>
<td align="center" style="background-color:#90EE90">46.190</td>
<td align="center" style="background-color:#90EE90">&#x3c;0.0001</td>
<td align="center" style="background-color:#90EE90">30.202</td>
<td align="center" style="background-color:#90EE90">&#x3c;0.0001</td>
</tr>
<tr>
<td align="left" style="background-color:#FFA07A">ALA<sup>2</sup>
</td>
<td align="center" style="background-color:#FFA07A">3.390</td>
<td align="center" style="background-color:#FFA07A">0.067</td>
<td align="center" style="background-color:#FFA07A">10.370</td>
<td align="center" style="background-color:#FFA07A">0.002</td>
<td align="center" style="background-color:#FFA07A">11.496</td>
<td align="center" style="background-color:#FFA07A">0.001</td>
</tr>
<tr>
<td align="left" style="background-color:#FFA07A">Cu<sup>2</sup>
</td>
<td align="center" style="background-color:#FFA07A">3.540</td>
<td align="center" style="background-color:#FFA07A">0.061</td>
<td align="center" style="background-color:#FFA07A">4.840</td>
<td align="center" style="background-color:#FFA07A">0.029</td>
<td align="center" style="background-color:#FFA07A">4.534</td>
<td align="center" style="background-color:#FFA07A">0.034</td>
</tr>
<tr>
<td align="left" style="background-color:#90EE90">Vit A<sup>2</sup>
</td>
<td align="center" style="background-color:#90EE90">32.520</td>
<td align="center" style="background-color:#90EE90">&#x3c;0.0001</td>
<td align="center" style="background-color:#90EE90">36.430</td>
<td align="center" style="background-color:#90EE90">&#x3c;0.0001</td>
<td align="center" style="background-color:#90EE90">23.391</td>
<td align="center" style="background-color:#90EE90">&#x3c;0.0001</td>
</tr>
<tr>
<td align="left" style="background-color:#FFA07A">ALA and Vit E</td>
<td align="center" style="background-color:#FFA07A">3.770</td>
<td align="center" style="background-color:#FFA07A">0.054</td>
<td align="center" style="background-color:#FFA07A">5.420</td>
<td align="center" style="background-color:#FFA07A">0.021</td>
<td align="center" style="background-color:#FFA07A">5.185</td>
<td align="center" style="background-color:#FFA07A">0.024</td>
</tr>
<tr>
<td align="left" style="background-color:#FFA07A">Cr and Co</td>
<td align="center" style="background-color:#FFA07A">3.510</td>
<td align="center" style="background-color:#FFA07A">0.062</td>
<td align="center" style="background-color:#FFA07A">5.600</td>
<td align="center" style="background-color:#FFA07A">0.019</td>
<td align="center" style="background-color:#FFA07A">5.174</td>
<td align="center" style="background-color:#FFA07A">0.024</td>
</tr>
<tr>
<td align="left" style="background-color:#90EE90">Mn and Mo</td>
<td align="center" style="background-color:#90EE90">9.980</td>
<td align="center" style="background-color:#90EE90">0.002</td>
<td align="center" style="background-color:#90EE90">10.790</td>
<td align="center" style="background-color:#90EE90">0.001</td>
<td align="center" style="background-color:#90EE90">11.034</td>
<td align="center" style="background-color:#90EE90">0.001</td>
</tr>
<tr>
<td align="left" style="background-color:#90EE90">Co and Vit D</td>
<td align="center" style="background-color:#90EE90">7.610</td>
<td align="center" style="background-color:#90EE90">0.006</td>
<td align="center" style="background-color:#90EE90">5.720</td>
<td align="center" style="background-color:#90EE90">0.018</td>
<td align="center" style="background-color:#90EE90">8.165</td>
<td align="center" style="background-color:#90EE90">0.005</td>
</tr>
<tr>
<td align="left" style="background-color:#FFA07A">T4 and Vit D</td>
<td align="center" style="background-color:#FFA07A">3.310</td>
<td align="center" style="background-color:#FFA07A">0.070</td>
<td align="center" style="background-color:#FFA07A">6.620</td>
<td align="center" style="background-color:#FFA07A">0.011</td>
<td align="center" style="background-color:#FFA07A">8.878</td>
<td align="center" style="background-color:#FFA07A">0.003</td>
</tr>
<tr>
<td align="left" style="background-color:#FFA07A">Vit B7 and Zn</td>
<td align="center" style="background-color:#FFA07A">3.860</td>
<td align="center" style="background-color:#FFA07A">0.051</td>
<td align="center" style="background-color:#FFA07A">3.070</td>
<td align="center" style="background-color:#FFA07A">0.081</td>
<td align="center" style="background-color:#FFA07A">4.086</td>
<td align="center" style="background-color:#FFA07A">0.045</td>
</tr>
<tr>
<td align="left">Lack of Fit</td>
<td align="center">0.592</td>
<td align="center">0.897</td>
<td align="center">0.416</td>
<td align="center">0.983</td>
<td align="center">0.515</td>
<td align="center">0.945</td>
</tr>
</tbody>
</table>
<table-wrap-foot>
<fn>
<p>Yellow indicates the model was significant at all timepoints, green - that component was significant at all timepoints and orange - that component was significant at one or two time points.</p>
</fn>
</table-wrap-foot>
</table-wrap>
<p>Results of this analysis include, F-values, <italic>p</italic>-values, and lack of fit test, which indicate how well the responses fit the model. As shown in <xref ref-type="table" rid="T2">Table 2</xref> the F- and P- values of the model, as well as the lack of fit test, over the 3&#xa0;weeks support that the model is significant and thus the analysis for the associated terms is valid. <xref ref-type="table" rid="T2">Table 2</xref> displays model terms that were significant for at least one of the timepoints indicated shown by a <italic>p</italic>-value &#x3c;0.05. Significant single terms for all timepoints were linolenic acid, copper, and vitamin A. Several interactions were defined as significant for at least one of the timepoints shown (orange highlighted), while others including: chromium and cobalt, manganese and molybdenum, and cobalt and vitamin D were significant for all timepoints.</p>
<p>To contrast these results, testing micronutrients in a one factor at a time approach previously identified basal media supplemented with cobalt, chromium, thyroxine, or Vitamin B7, as having higher luminescence at multiple concentrations as compared to basal media alone (<xref ref-type="sec" rid="s12">Supplementary Figure S1A</xref>). Combinations of these factors, with factors previously shown to have an effect (<xref ref-type="bibr" rid="B13">Dennis et al., 2020a</xref>), are shown in <xref ref-type="sec" rid="s12">Supplementary Figure S1B</xref>. Copper and Vitamin B7 alone or in combination with each other seemingly had no effect on luminescence, &#x223c;40,000 RLU; however, in combination with thyroxine they significantly increased luminescence &#x223c;70,000 RLU as compared to basal media alone, &#x223c;40,000 RLU. This supports that there a synergistic effect between copper, biotin, and thyroxine. Furthermore, this supports the use of a Design of Experiments approach over a one factor a time approach as the DoE screen also identified factors that were significant in interactions but were not identified as individually significant.</p>
<p>Using a response surface model allows us to determine significant interactions between terms as well as determine and predict optimal concentrations of the terms within the parameters input into the initial model. 3D surface plots in <xref ref-type="fig" rid="F2">Figures 2C, D</xref> show the dose effect of two terms (linolenic acid and vitamin A) in relation to each other and to the response (luminescence) at week 2 (<xref ref-type="fig" rid="F2">Figure 2C</xref>) and week 3 (<xref ref-type="fig" rid="F2">Figure 2D</xref>) of chondrogenesis. During week 2 (<xref ref-type="fig" rid="F2">Figure 2C</xref>) there is a predicted optimal concentration for linolenic acid and vitamin A (approximately 3 &#xd7; 10<sup>&#x2212;6</sup>&#xa0;fg/mL and 5 &#xd7; 10<sup>&#x2212;1</sup>&#xa0;fg/mL respectively) that results in a maximal response that increases in week 3 (<xref ref-type="fig" rid="F2">Figure 2D</xref>), from 10<sup>1.8</sup> RLU to 10<sup>2.4</sup> RLU, although the optimal concentrations of the terms remain the same. Interestingly, the DoE model predicts that there is an optimal concentration for these in the femtomolar range (<xref ref-type="fig" rid="F2">Figures 2C, D</xref>) while each of the two factors, when analyzed as sole additives, were detrimental to chondrogenesis at higher concentrations (<xref ref-type="sec" rid="s12">Supplementary Figure S1A</xref>).</p>
<p>
<xref ref-type="fig" rid="F2">Figures 2C, D</xref> is a representation of only two of the terms and a predicted optimal for each individual timepoint. Through Design-Expert, multiple terms and responses can be analyzed together to derive predicted optimal concentrations for all terms. These predicted optima account for individual responses as well interactions between terms. Out of the generated predicted optimal conditions, 5 were selected for validation, designated as conditions 12, 25, 52, 72, and 89 (<xref ref-type="sec" rid="s12">Supplementary Table S2</xref> in <xref ref-type="sec" rid="s12">Supplementary Data Sheet S2</xref>).</p>
</sec>
<sec id="s2-3">
<title>DoE predicted conditions improved <italic>Gaussia</italic> luciferase expression over basal media</title>
<p>To validate the Design-Expert generated predicted conditions, COL2A1-GLuc cells in aggregate culture were maintained in media supplemented with the predicted conditions (<xref ref-type="sec" rid="s12">Supplementary Table S2</xref> in <xref ref-type="sec" rid="s12">Supplementary Data Sheet S2</xref>) for 3&#xa0;weeks. As seen in <xref ref-type="fig" rid="F3">Figure 3A</xref>, all conditions tested had increased luminescence over basal media control for all timepoints after day 8, which suggests that predicted conditions have an anabolic effect early in chondrogenesis. Cumulative luminescence seen in <xref ref-type="fig" rid="F3">Figure 3B</xref> is the sum of luminescence signal over all days in culture and confirms that increased luminescence at each timepoint results in an overall significant increase in type II collagen promoter-driven activity for all predicted conditions, with condition 25 having a higher cumulative signal of &#x223c;1 &#xd7; 10<sup>6</sup> RLU as compared to basal media, &#x223c;6 &#xd7; 10<sup>5</sup> RLU. Single day luminescence shown for day 10 (<xref ref-type="fig" rid="F3">Figure 3C</xref>) and day 22 (<xref ref-type="fig" rid="F3">Figure 3D</xref>) supports an increase in luminescence that is statistically significant for all conditions tested as compared to basal media with condition 25 having an average luminescence signal that is twice of that in basal media, &#x223c;2 &#xd7; 10<sup>5</sup> RLU vs. 1 &#xd7; 10<sup>5</sup> RLU respectively, for both timepoints.</p>
<fig id="F3" position="float">
<label>FIGURE 3</label>
<caption>
<p>Validation of DoE predicted optimal conditions. <bold>(A&#x2013;D)</bold> Conditions predicted by DoE analysis were tested in aggregate culture of COL2A1-GLuc reporter rabbit chondrocytes over 22&#xa0;days. Results are shown as luminescence over 22&#xa0;days sampled <bold>(A)</bold>, as well as cumulative luminescence signal <bold>(B)</bold>. To explore temporal effects, data was also analyzed at single day luminescence shown here for Day 10 <bold>(C)</bold> and Day 22 <bold>(D)</bold>. At day 22, aggregate cultures were assessed for total DNA <bold>(E)</bold>, glycosaminoglycan <bold>(F)</bold> and collagen <bold>(G)</bold> content. Results are also shown as total glycosaminoglycan and collagen normalized to DNA content <bold>(H, I)</bold> and to each other <bold>(J)</bold>. Alternatively, aggregates were fixed, embedded in paraffin and sectioned. <bold>(K)</bold> Sections were analyzed for type II collagen. Scale Bars, 200&#xa0;um. <bold>(A&#x2013;D)</bold> N &#x3d; 6. <bold>(E&#x2013;J)</bold> N &#x3d; 5. Replicates or means &#xb1; S.D. and &#x2a; p &#x3c;0.05, &#x2a;&#x2a; p &#x3c;0.01 and &#x2a;&#x2a;&#x2a; p &#x3c;0.001 vs. Basal Media control.</p>
</caption>
<graphic xlink:href="fbioe-11-1179332-g003.tif"/>
</fig>
<p>To corroborate results seen by luminescence output, endpoint biochemical assays were performed at day 22 of the experiment to quantitate DNA, glycosaminoglycan (GAG) and total collagen content. <xref ref-type="fig" rid="F3">Figure 3E</xref> shows an average of &#x223c;0.4&#xa0;&#x3bc;g of DNA per sample with no significant difference between conditions tested, which suggests that predicted conditions have no effect on cell proliferation or viability over 22&#xa0;days. Total glycosaminoglycan content is shown in <xref ref-type="fig" rid="F3">Figure 3F</xref> and as amount per microgram of DNA in <xref ref-type="fig" rid="F3">Figure 3H</xref>. As expected, DoE predicted conditions did not significantly affect glycosaminoglycan production over 22&#xa0;days, although condition 89 shows large variability between samples as compared to other conditions. Quantification of total collagen, as seen in <xref ref-type="fig" rid="F3">Figure 3G</xref> shows an increase in aggregates cultured in predicted conditions to &#x223c;10&#xa0;&#xb5;g over basal media (&#x223c;6&#xa0;&#xb5;g). However, when normalized to micrograms of DNA (<xref ref-type="fig" rid="F3">Figure 3I</xref>) only conditions 12, 25 and 89, show increased collagen as compared to aggregates cultured in basal media with significant variability within each group leading to lack of statistical significance. Glycosaminoglycan to collagen ratio (<xref ref-type="fig" rid="F3">Figure 3J</xref>) further support an increase in collagen with no change in GAG content (see <xref ref-type="sec" rid="s12">Supplementary Data Sheet S2</xref> for GAG histology). Immunohistochemistry for type II collagen (<xref ref-type="fig" rid="F3">Figure 3K</xref>) confirms the presence of type II collagen for cell aggregates in all conditions at day 22 with similar staining pattern across all conditions.</p>
</sec>
<sec id="s2-4">
<title>No single factor from optimized condition 25 significantly impacts type II collagen promoter activity in primary rabbit chondrocytes</title>
<p>When using a one factor at a time approach, thyroxine (T4) was required in the tested combinations for type II collagen stimulation over basal media (<xref ref-type="sec" rid="s12">Supplementary Figure S1B</xref>). To test if one factor was solely responsible for the enhancement in luminescence seen over basal media, aggregates were cultured in the predicted DoE condition 25, or combinations where one factor was removed from condition 25 for 22&#xa0;days <xref ref-type="fig" rid="F4">Figure 4A</xref> represents luminescence data for all days tested with basal media shown by a thicker black line and complete condition 25 shown by the thicker red line. All conditions had increased luminescence as compared to basal media after day 8. For statistical analysis, day 10 luminescence (<xref ref-type="fig" rid="F4">Figure 4B</xref>) is shown for all conditions tested.</p>
<fig id="F4" position="float">
<label>FIGURE 4</label>
<caption>
<p>Effect of a single micronutrient removal from DoE predicted condition 25 on type II collagen driven expression of <italic>Gaussia</italic> Luciferase. Aggregates of COL2A1-GLuc primary rabbit chondrocytes were cultured with condition 25 or condition 25 with a single micronutrient removed as indicated. Results are shown as luminescence over 22&#xa0;days <bold>(A)</bold>. Luminescence is shown for a single timepoint, Day 10 <bold>(B)</bold>. <italic>&#x3c6;</italic> indicates <italic>p</italic> &#x3c; 0.05 vs Basal Media control. N &#x3d; 7. Mean &#xb1; S.D.</p>
</caption>
<graphic xlink:href="fbioe-11-1179332-g004.tif"/>
</fig>
<p>Aggregates cultured in all conditions had increased luminescence. However, effects by condition 25 with Linolenic acid, vitamin A or with vitamin D removed, were not statistically significant as compared to basal media, suggestive of a major role for these biomolecules. Formulations where any of the other factors were removed from Condition 25 had significant increases in luminescence as compared to basal media alone. There was no statistical significance between any of the conditions with a single factor removed as compared to complete condition 25. These results are evidence that no single factor within DoE predicted condition 25 is solely responsible for the higher stimulation of type II collagen promoter activity observed.</p>
</sec>
<sec id="s2-5">
<title>Relative concentrations of vitamins and minerals, regardless of absolute concentrations, in DoE predicted conditions play a significant role in type II collagen stimulation in primary rabbit chondrocytes</title>
<p>To determine if ratios, regardless of the absolute concentration, within the DoE predicted conditions were sufficient for type II collagen stimulation, aggregate cultures were treated with the DoE predicted conditions at the concentrations given or at 1/15th of the predicted concentrations. Two-way ANOVA analysis of luminescence at Day 10 (<xref ref-type="fig" rid="F5">Figure 5A</xref>) and Day 17 (<xref ref-type="fig" rid="F5">Figure 5B</xref>) determined that media conditions at 1x or 1/15x of their DoE predicted concentrations had an overall <italic>p</italic> &#x3d; 0.0149 for 6a and <italic>p</italic> &#x3d; 0.0004 for 6b but that none of the individual comparisons had <italic>p</italic> &#x3c; 0.05.</p>
<fig id="F5" position="float">
<label>FIGURE 5</label>
<caption>
<p>Effect of absolute versus relative concentration of micronutrients on Type II collagen driven expression of <italic>Gaussia</italic> Luciferase. Aggregates of COL2A1-GLuc primary rabbit chondrocytes were cultured with predicted DoE conditions (1x) or the same ratios of these conditions at 1/15th the optimal predicted concentration (1/15x) over 22&#xa0;days. Luminescence results are shown for day 10 <bold>(A)</bold> and day 17 <bold>(B)</bold>. <italic>&#x3c6;</italic> indicates <italic>p</italic> &#x3c; 0.05 vs Basal Media control. N &#x3d; 5 for DoE at 1/15x and N &#x3d; 6 for DoE at 1x. Individual replicates and the mean &#xb1; S.D. are shown.</p>
</caption>
<graphic xlink:href="fbioe-11-1179332-g005.tif"/>
</fig>
<p>Interestingly, while aggregates cultured at the 1x DoE predicted conditions had significantly higher luminescence as compared to those in basal media, aggregates cultured in most conditions at 1/15th did not. Only condition 25&#xa0;at 1/15th the concentration showed a significantly higher level of luminescence at both days vs Basal Media, &#x223c;1.5 to &#x223c;1.7 &#xd7; 10<sup>5</sup> RLU vs. &#x223c;1 &#xd7; 10<sup>5</sup> RLU. This suggests that the combinatorial effect of the vitamins and minerals plays a significant role in type II collagen promoter activity in primary rabbit chondrocytes, but concentrations as predicted by the DoE are optimal for type II collagen stimulation.</p>
</sec>
<sec id="s2-6">
<title>DoE predicted condition 25 stimulates type II collagen in tissue engineered rabbit cartilage</title>
<p>To determine if condition 25, the best performing DoE predicted condition, could have an effect in cartilage tissue engineering, we cultured COL2A1-GLuc or Non-transduced (NonTr) primary rabbit chondrocytes in custom, 3D printed bioreactors adapted from Whitney GA, et al. shown in <xref ref-type="fig" rid="F6">Figures 6A&#x2013;C</xref> over 22&#xa0;days (<xref ref-type="bibr" rid="B87">Whitney et al., 2012</xref>; <xref ref-type="bibr" rid="B14">Dennis et al., 2020b</xref>).</p>
<fig id="F6" position="float">
<label>FIGURE 6</label>
<caption>
<p>Biochamber for the generation of tissue engineered cartilage sheets Custom 3D printed ABS biochambers were designed to generate tissue engineered articular cartilage sheets. <bold>(A)</bold> Model of the biochamber assembly. <bold>(B)</bold> Photo of printed biochamber and Nalgene container fitted with sterile filter top. <bold>(C)</bold> Diagram of cell seeding and media top up media levels.</p>
</caption>
<graphic xlink:href="fbioe-11-1179332-g006.tif"/>
</fig>
<p>At the end of this culture period, 1.2&#xa0;cm<sup>2</sup> cartilage sheets were collected as shown in <xref ref-type="fig" rid="F7">Figure 7A</xref>. Over the 22&#xa0;days bioreactors housing COL2A1-Gluc cells were regularly sampled for luminescence. <xref ref-type="fig" rid="F7">Figure 7B</xref> represents luminescence data for all days tested.</p>
<fig id="F7" position="float">
<label>FIGURE 7</label>
<caption>
<p>Tissue engineered cartilage sheet response to supplementation with condition 25. COL2A1-GLuc primary rabbit chondrocytes were cultured in bioreactors with condition 25 (1x) or condition 25 at 1/15th the concentration (1/15x) over 22&#xa0;days <bold>(A)</bold> On day 22, engineered sheets were collected. <bold>(B)</bold> Luminescence signal over 22&#xa0;days in culture. Max tensile force <bold>(C)</bold> and tensile modulus <bold>(D)</bold> are shown. DNA <bold>(E)</bold>, glycosaminoglycan <bold>(F)</bold> and collagen content <bold>(G)</bold> of the tissue were assessed. Data is also calculated as GAG/DNA <bold>(H)</bold>, collagen/DNA <bold>(I)</bold> and GAG/collagen <bold>(J)</bold>.</p>
</caption>
<graphic xlink:href="fbioe-11-1179332-g007.tif"/>
</fig>
<p>Similar to results seen when conditions were tested in aggregates (<xref ref-type="fig" rid="F3">Figure 3</xref>), both condition 25 at 1x and at 1/15th concentration showed increased luminescence as compared to basal media after day 8. A different curve in luminescence is observed in <xref ref-type="fig" rid="F7">Figure 7B</xref> as compared to <xref ref-type="fig" rid="F3">Figure 3A</xref> because samples were collected from inside of the biochamber for day 1 and day 3. After day 3, medium was added to increase exchange between the inside and outside of the chamber, thus samples collected are from a larger volume resulting in a decrease in luminescence from day 3 to day 8. After sheets were collected, several biopsy punches of the sheets were obtained for endpoint analysis. To determine whether increased COL2A1 reporter activity translates to improved mechanical properties of engineered cartilage, we assessed tissue elasticity via tensile testing. As expected, we observed a marked increase in the Tensile modulus (<xref ref-type="fig" rid="F7">Figure 7C</xref>) as well as maximal tensile force (<xref ref-type="fig" rid="F7">Figure 7D</xref>), in sheets generated in condition 25 media at 1x and 1/15th of the concentration as compared to basal media, from &#x223c;6&#xa0;MPa in basal media to &#x223c;8&#xa0;MPa in condition 25 and &#x223c;10&#xa0;MPa in condition 25 at 1/15th concentration. In contrast, compression testing resulted in a marked decrease in stiffness for sheets generated in conditions 25 regardless of concentration (<xref ref-type="sec" rid="s12">Supplementary Figure S2</xref>).</p>
<p>To evaluate whether the luminescence results reflected matrix accumulation, biochemical assays were performed at day 22 of the experiment to quantitate DNA, glycosaminoglycan (GAG) and total collagen content from bioreactors seeded with COL2A1-Gluc and non-transduced cells. Both COL2A1-gLuc and non-transduced cell generated sheets had similar trends in DNA (<xref ref-type="fig" rid="F7">Figure 7E</xref>), GAG (<xref ref-type="fig" rid="F7">Figure 7F</xref>), and total collagen (<xref ref-type="fig" rid="F7">Figure 7G</xref>) except for the sheet generated with COL2A1-Gluc cells in condition 25 at (1x), which shows lower collagen as compared to its non-transduced counterpart (<xref ref-type="fig" rid="F7">Figure 7G</xref>). Sheets in supplemented groups had overall higher collagen content per milligram of tissue, as well as per mi-crogram of DNA (<xref ref-type="fig" rid="F7">Figures 7G,I</xref>). Total glycosaminoglycan content is relatively constant for all groups at ~40 &#x03bc;g per mg of wet tissue weight (<xref ref-type="fig" rid="F7">Figure 7F</xref>), was potentially stimulated in condition 25 when normalized to DNA (<xref ref-type="fig" rid="F7">Figure 7H</xref>). When normalized to collagen content (<xref ref-type="fig" rid="F7">Figure 7J</xref>) there is a noticeable decrease in both reporter cell and non-transduced sheets generated in condition 25 (both 1x and 1/15x) suggesting that condition 25 specifically affects collagen content and not glycosaminoglycan content.</p>
<p>Immunofluorescence of sections of the engineered cartilage confirmed the presence of type II collagen (<xref ref-type="fig" rid="F8">Figure 8A</xref>). Interestingly, condition 25 (1/15th) had a more even distribution of type II collagen signal as compared to basal media alone which showed an uneven pattern of staining. Collagen heteropolymer analysis was also carried out on samples of the engineered cartilage sheets and compared to native rabbit articular cartilage to further analyze collagen content. <xref ref-type="fig" rid="F8">Figure 8B</xref> shows that the major pepsin-resistant Coomassie blue-stained band in both transfected and non-transfected articular chondrocyte cultures migrated identically to the &#x3b1;1(II) chain of type II collagen in the adult rabbit articular cartilage. &#x3b2;1(II) chains (dimers of &#x3b1;1(II) chains) were also observed in all lanes (<xref ref-type="fig" rid="F8">Figure 8B</xref>). Two faintly stained bands migrating slightly slower than that of the &#x3b1;1(II) chains, are the &#x3b1;1 (XI) and &#x3b1;2 (XI) chains of type XI collagen as previously identified by mass spectrometry and described by McAlinden et al. (<xref ref-type="bibr" rid="B19">Fernandes et al., 2007</xref>; <xref ref-type="bibr" rid="B52">McAlinden et al., 2014</xref>). The &#x3b1;1(I) and &#x3b1;2(I) chains of type I collagen were neither detected in engineered cartilages nor in the control articular cartilage (if present the &#x3b1;2(I) chain migrates slightly faster than the &#x3b1;1(II) chain), indicating that type II collagen and type XI collagen were the major collagens synthesized by the cultured chondrocytes and the cartilage collagen phenotype was maintained (<xref ref-type="fig" rid="F8">Figure 8B</xref>). The Western blot shown in <xref ref-type="fig" rid="F8">Figure 8C</xref> confirms that the Coomassie blue stained bands were indeed &#x3b1;1(II) chains of type II collagen.</p>
<fig id="F8" position="float">
<label>FIGURE 8</label>
<caption>
<p>Analysis of Type II collagen and heteropolymer formation with Type IX collagen in tissue engineered cartilage. <bold>(A)</bold> Engineered cartilage was analyzed for type II collagen. Scale Bars, 300&#xa0;um. <bold>(B)</bold> Coomassie blue-stained SDS-PAGE gel of pepsin solubilized collagen showing &#x3b2;1(II), &#x3b1;1 (XI), &#x3b1;2 (XI) and &#x3b1;1(II) chains. Equivalent dry weight (25&#xa0;&#xb5;g) was loaded. <bold>(C)</bold> Western blot of samples equivalent to those in <bold>(B)</bold> and probed with anti-type II collagen antibody (1C10). <bold>(D)</bold> Western blot of samples equivalent to those electrophoresed in <bold>(B)</bold> (above) and probed with mAb 10F2. This antibody specifically recognizes the C- telopeptide domain of type II collagen when it is cross-linked to another &#x3b1;1(II) collagen chain. <bold>(E)</bold> Western blot of samples identical to those in <bold>(B)</bold> probed with antibody 5890. This antibody specifically recognizes N-telopeptide domain of &#x3b1;1 (XI) collagen when cross-linked to chains of &#x3b1;1(II) and &#x3b2;1(II). X denotes crosslinks.</p>
</caption>
<graphic xlink:href="fbioe-11-1179332-g008.tif"/>
</fig>
<p>Using mAb 1C10, which specifically recognizes type II collagen chains (<xref ref-type="bibr" rid="B17">Fernandes et al., 2001</xref>), intense staining of both the &#x3b1;1(II) and &#x3b2;1(II) chains were observed in all the cultures and the adult rabbit cartilage. Since equivalent engineered cartilage dry weights loads were electrophoresed in all the lanes, densitometry of the Western blot revealed increased levels of type II collagen (&#x3b1;1(II) &#x2b; &#x3b2;1(II)) reactivity under condition 25 in both transfected and non-transfected cultures compared to basal conditions. Condition 25 (1/15x) however showed highest levels of reactivity indicating highest type II collagen retained in the Condition 25 (1/15x) sheet. This is consistent with the analytical results in <xref ref-type="fig" rid="F7">Figure 7G</xref> showing highest collagen content under this condition on a per mass basis. Using a refined Western blot method (<xref ref-type="bibr" rid="B14">Dennis et al., 2020b</xref>) we were able to identify precise domains of collagen chains that were cross-linked in these cultures. As seen in <xref ref-type="fig" rid="F8">Figure 8D</xref>, Western blotting using mAb 10F2 (<xref ref-type="bibr" rid="B18">Fernandes et al., 2003</xref>; <xref ref-type="bibr" rid="B52">McAlinden et al., 2014</xref>) recognized the &#x3b1;1(II) and &#x3b2;1(II) chains in all the cultures and the adult rabbit cartilage. This is evidence that the C-telopeptide of the &#x3b1;1(II) chain specifically recognized by this antibody was cross-linked to the helical lysine (K87) residues in a fraction of &#x3b1;1(II) collagen chains and, thus, type II to type II collagen cross-links had formed in these cultures (<xref ref-type="bibr" rid="B14">Dennis et al., 2020b</xref>). It must be reiterated that pepsin-extracted &#x3b1;1(II) collagen chains are devoid of telopeptides unless they are cross-linked to the lysine residues in the helical regions of &#x3b1;1(II) chains (<xref ref-type="bibr" rid="B52">McAlinden et al., 2014</xref>; <xref ref-type="bibr" rid="B14">Dennis et al., 2020b</xref>).</p>
<p>To examine if type II and type XI collagen molecules in these cultures were stabilized by these cross-links, we used the pAb 5890 (<xref ref-type="bibr" rid="B19">Fernandes et al., 2007</xref>; <xref ref-type="bibr" rid="B52">McAlinden et al., 2014</xref>). As seen in <xref ref-type="fig" rid="F8">Figure 8E</xref>, this antibody also recognized the &#x3b1;1(II) chains and the &#x3b2;1(II) chains of type II collagen in the tissue engineered sheets and adult rabbit cartilage. As shown before (<xref ref-type="bibr" rid="B52">McAlinden et al., 2014</xref>), this means that the N-telopeptide of the &#x3b1;1 (XI) chain to which this antibody was raised was cross-linked to the helical lysine (K930) residue in a fraction of &#x3b1;1(II) chains of type II collagen molecules and thus a hetero-polymer of type II and type XI collagens had formed in all these cultures. A faint reactivity of the &#x3b1;1 (XI) chain was observed in some of the engineered cartilage cultures that probably indicates that N-telopeptides of &#x3b1;1 (XI) chain are cross-linked to helical lysine of another &#x3b1;1 (XI) chain and a homo-polymer in a fraction of type XI collagen had also formed in these cultures. We have published on the locations of the antibody epitopes in the &#x3b1;1(II) chain or in telopeptide stubs crosslinked to the collagen chains in type II-IX-XI heteropolymer (<xref ref-type="bibr" rid="B14">Dennis et al., 2020b</xref>).</p>
<p>The data confirms that a polymer of type II collagen had formed in tissue engineered cartilage sheets and a mature collagenous heteropolymer of cross-linked type II-XI collagen fibrils had formed as seen for control type II collagen extracted from rabbit articular cartilage (<xref ref-type="fig" rid="F8">Figures 8C&#x2013;E</xref>, last lane).</p>
</sec>
</sec>
<sec sec-type="discussion" id="s3">
<title>Discussion</title>
<p>Our previous efforts to optimize defined chondrogenic media, tested the effect of 15 different micronutrients and thyroxine on murine chondrocytes using proposed concentrations based on physiologic levels in a one-factor at a time approach (<xref ref-type="bibr" rid="B13">Dennis et al., 2020a</xref>). In that work, we identified copper, vitamin A and linolenic acid as having a positive effect on chondrogenesis. Overall, we found that combinations of these micronutrients were able to increase the type II collagen promoter activity when tested temporally and in a dose-dependent manner (<xref ref-type="bibr" rid="B13">Dennis et al., 2020a</xref>). While our previous work showed that vitamins and minerals affect type II collagen production in murine chondrocytes <italic>in vitro</italic>, we noted several limitations of using a traditional one factor at a time approach. This approach consists of experimental runs that are executed to hold every factor constant except for the variable of interest. This approach poorly reflects the complexity of <italic>in vivo</italic> conditions by failing to account for important interactions and largely relies on iterative experiments and trial and error for optimization. In the current study, we combined a non-destructive reporter, primary rabbit chondrocytes, 3D culture in 96-well plates, automated pipetting, and Design of Experiments approach as an efficient high throughput platform. We were able to not only identify interactions of micronutrients that had an effect on type II collagen promoter activity, but to also derive an optimal combination containing all missing factors as a supplement to traditional basal media, that simultaneously increased type II collagen expression.</p>
<p>There are several advantages to the platform we implemented in this study. 1) we made use of primary rabbit articular chondrocytes as a model for healthy cartilage. Primary cells have the advantage of being more relevant in orthopedic research than cell lines and thus more likely to mimic responses <italic>in vivo</italic> (<xref ref-type="bibr" rid="B30">Horton et al., 1988</xref>; <xref ref-type="bibr" rid="B82">Thenet et al., 1992</xref>; <xref ref-type="bibr" rid="B75">Steimberg et al., 1999</xref>; <xref ref-type="bibr" rid="B67">Santoro et al., 2015</xref>). 2) using 3D cell aggregates adapted to 96-well plates cultured in physioxic conditions, which we adapted for use in an automated system, allows chondrocytes to maintain their phenotype as compared to 2D culture (<xref ref-type="bibr" rid="B4">Benya and Shaffer, 1982</xref>; <xref ref-type="bibr" rid="B6">Bonaventure et al., 1994</xref>; <xref ref-type="bibr" rid="B91">Yoo et al., 1998</xref>; <xref ref-type="bibr" rid="B61">Penick et al., 2005</xref>; <xref ref-type="bibr" rid="B79">Takahashi et al., 2007</xref>). 3) We made use of a secreted <italic>Gaussia</italic> Luciferase reporter (<xref ref-type="bibr" rid="B81">Tannous et al., 2005</xref>; <xref ref-type="bibr" rid="B2">Badr et al., 2007</xref>; <xref ref-type="bibr" rid="B64">Ruecker et al., 2008</xref>; <xref ref-type="bibr" rid="B89">Wurdinger et al., 2008</xref>; <xref ref-type="bibr" rid="B80">Tannous, 2009</xref>). Traditional biochemical assays to evaluate chondrogenesis typically rely on destructive endpoint analysis, and due to the length of culture of the samples, low cell number in aggregates, and long and laborious processes, can result in high variability as seen in <xref ref-type="fig" rid="F3">Figures 3E&#x2013;J</xref>. Using a secreted reporter allows us to sample the media without lysis of the aggregate and thus provides the ability to examine the temporal effects of the treatment conditions on chondrogenesis. Because media is replaced every 2&#x2013;3&#xa0;days <italic>Gaussia</italic> luciferase readings provide a readout of the activity of the type II collagen promoter at early, mid and late timepoints in chondrogenesis. This was seen and confirmed by the TGF-&#x3b2;1 dose response curve where we have shown, for the first time, that the effective dose of TGF-&#x3b2;1 on type II collagen stimulation differs throughout the process of chondrogenesis. Furthermore, type II collagen expression levels are 66% higher at early timepoints with a decrease in activity at later timepoints of chondrogenesis (<xref ref-type="fig" rid="F1">Figures 1A, B</xref>). As maximal type II collagen production was achieved with 1&#xa0;ng/ml TGF&#x3b2;1 (<xref ref-type="fig" rid="F1">Figure 1</xref>), this concentration was used to screen for micronutrient effects. The <italic>Gaussia</italic> luciferase assay is simple, sensitive and fast to perform and thus reduces variability between samples.</p>
<p>Using this platform, we successfully screened 240 combinations of vitamins and minerals for their ability to promote type II collagen. Previous studies have shown that several of the micronutrients we tested can play a role in chondrogenesis (<xref ref-type="bibr" rid="B57">Pacifici et al., 1980</xref>; <xref ref-type="bibr" rid="B3">Barone et al., 1991</xref>; <xref ref-type="bibr" rid="B62">Ren et al., 2007</xref>; <xref ref-type="bibr" rid="B9">Chin and Ima-Nirwana, 2018</xref>; <xref ref-type="bibr" rid="B86">Whitney et al., 2018</xref>; <xref ref-type="bibr" rid="B13">Dennis et al., 2020a</xref>; <xref ref-type="bibr" rid="B76">Sumitani et al., 2020</xref>). Previously, vitamins D and K were shown to play a role in the development and regulation of chondrogenesis, while vitamin A exhibited inhibitory action on <italic>in vitro</italic> chondrogenic differentiation (<xref ref-type="bibr" rid="B3">Barone et al., 1991</xref>; <xref ref-type="bibr" rid="B10">Connor, 1999</xref>). Additionally, vitamin E has exhibited oxidative stress inhibition during <italic>in vivo</italic> and clinical studies (<xref ref-type="bibr" rid="B9">Chin and Ima-Nirwana, 2018</xref>). Other trace minerals such as copper and zinc promote extracellular matrix formation and deficiencies in selenium and iodine have been shown to impair bone and growth formation (<xref ref-type="bibr" rid="B62">Ren et al., 2007</xref>). Molecules like linoleic acid are known to enhance the metabolic activity of differentiating cells, while thyroxine was shown to increase type II collagen expression and glycosaminoglycan (GAG) deposition in scaffold-free engineered cartilage tissue (<xref ref-type="bibr" rid="B10">Connor, 1999</xref>; <xref ref-type="bibr" rid="B86">Whitney et al., 2018</xref>). In this study we identified linolenic acid, copper, and vitamin A, as well as interactions between various vitamins and minerals (<xref ref-type="table" rid="T2">Table 2</xref>) as having significant effects on type II collagen stimulation promoter activity. With both vitamin A and linolenic acid there was an optimal concentration above which deleterious effects on type II collagen production were evident (<xref ref-type="fig" rid="F2">Figure 2</xref>).</p>
<p>The role of each micronutrient in chondrogenesis, particularly in the production of type II collagen, is not clear but effects of the highlighted micronutrients on cell function are briefly described here. Linolenic acid, an essential omega-3 fatty acid, can be synthesized into eicosanoids potentially leading to an anti-inflammatory effect or, through cell membrane integration, alter lipid raft composition, membrane transport and signaling (<xref ref-type="bibr" rid="B20">Finch and Finch, 2007</xref>; <xref ref-type="bibr" rid="B71">Shaikh, 2012</xref>). Chromium affects metabolism through insulin activity and glucose oxidation, both components of defined chondrogenic media, and could therefore promote cartilage anabolism (<xref ref-type="bibr" rid="B44">Lukaski, 2000</xref>; <xref ref-type="bibr" rid="B59">Pechova and Pavlata, 2007</xref>). Copper is a co-factor in many enzymes, perhaps the most relevant in cartilage tissue engineering is lysyl oxidase, where supplementation has enhanced collagen crosslinking (<xref ref-type="bibr" rid="B47">Makris et al., 2013a</xref>; <xref ref-type="bibr" rid="B13">Dennis et al., 2020a</xref>). Interestingly, copper also regulates HIF-1&#x3b1; binding to hypoxic response elements, significant due to the chondrogenic activity of HIF-1&#x3b1; (<xref ref-type="bibr" rid="B43">Liu et al., 2018</xref>). Cobalt, while mainly known for its vitamin B12 functions, has several noncorrin-cobalt containing enzymes including prolidase which could increase collagen synthesis; cobalt is also able to stabilize HIF-1&#x3b1; (<xref ref-type="bibr" rid="B36">Kobayashi and Shimizu, 1999</xref>; <xref ref-type="bibr" rid="B77">Surazynski et al., 2008</xref>). Manganese is a cofactor for many enzymes including superoxidase dismutase, potentially protecting the cartilage against reactive oxygen (<xref ref-type="bibr" rid="B11">Crowley et al., 2000</xref>). Additionally, the level of manganese was higher in hip articular cartilage than in the bone (<xref ref-type="bibr" rid="B8">Brodziak-Dopierala et al., 2013</xref>). Molybdenum is a cofactor of more than 50 enzymes including sulfite oxidases which play a role in sulfated glycosaminoglycans, an essential component of cartilage (<xref ref-type="bibr" rid="B60">Pecora et al., 2006</xref>; <xref ref-type="bibr" rid="B29">Hille et al., 2014</xref>). Vitamin A, known for its anti-chondrogenic properties (<xref ref-type="bibr" rid="B57">Pacifici et al., 1980</xref>), was stimulatory to type II collagen production in the femtomolar range potentially through an FGF/Dlx mechanism (<xref ref-type="bibr" rid="B23">Frenz et al., 2010</xref>). Vitamin E has a complex and poorly understood role overall, with little data in cartilage where it seems to have an anti-inflammatory/anti-oxidant effect (<xref ref-type="bibr" rid="B24">Galli et al., 2017</xref>; <xref ref-type="bibr" rid="B9">Chin and Ima-Nirwana, 2018</xref>; <xref ref-type="bibr" rid="B28">Hassan et al., 2019</xref>). Vitamin D, well known for its promotion of bone homeostasis, also has a role in chondrogenesis&#x2014;increasing cartilage thickness, proteoglycan content, type II collagen and lubricin expression (<xref ref-type="bibr" rid="B40">Li et al., 2016</xref>; <xref ref-type="bibr" rid="B78">Szychlinska et al., 2019</xref>). Vitamin B7&#x2019;s role in chondrogenesis is unclear; however, its role in gluconeogenesis, amino acid metabolism and fatty acid synthesis would undoubtedly impact chondrogenesis (<xref ref-type="bibr" rid="B66">Saleem and Soos, 2023</xref>).</p>
<p>These findings relied on the use of Response Surface Methodology based on Design of Experiments which significantly reduces the number of trials, accounts for errors in the model, and for interactions between factors (<xref ref-type="bibr" rid="B27">Hanrahan and Lu, 2006</xref>; <xref ref-type="bibr" rid="B56">Myers et al., 2016</xref>; <xref ref-type="bibr" rid="B90">Yolmeh and Jafari, 2017</xref>; <xref ref-type="bibr" rid="B1">Aydar, 2018</xref>). Statistical analysis with this approach allowed us to predict an optimal combination of vitamins and minerals, condition 25, that when tested <italic>in vitro</italic> showed significant increases in type II collagen as compared to the basal media control. Furthermore, we removed one factor at a time from condition 25 and confirmed the importance of linolenic acid, vitamin A and vitamin D and their interactions in type II collagen stimulation, further confirming the validity of the DoE results.</p>
<p>While Response Surface Methodology has significant advantages, its effectiveness does rely on the data fitting a second order polynomial model, thus fit statistics are crucial to ensure that the data fits the model (<xref ref-type="bibr" rid="B37">Ko&#xe7; and Kaymak-Ertekin, 2017</xref>; <xref ref-type="bibr" rid="B1">Aydar, 2018</xref>). In addition, the validation of any findings is essential. Other aspects to consider include parameter selection for optimization of factors and response, as well as examining predicted values before validation. In our study, this is seen by our predicted condition 25, while having a predicted desirability of 0.595, it was selected for validation due to the high predicted individual responses during analysis. When tested <italic>in vitro</italic>, it showed similar if not better responses than other selected conditions. There are few studies that have used Design of Experiments to look at biological processes, typically investigating fewer factors (<xref ref-type="bibr" rid="B15">Enochson et al., 2012</xref>; <xref ref-type="bibr" rid="B63">Renner and Liu, 2013</xref>; <xref ref-type="bibr" rid="B39">Kuterbekov et al., 2019</xref>). To date, this study is the first to apply a response surface model to primary chondrocytes.</p>
<p>After validation of condition 25 in aggregates we explored this supplementation in tissue engineered cartilage. Effects generated in one tissue engineered system are often found to be context dependent. To broaden the impact of this study and provide proof of concept data for the screening strategy, we used custom 3D printed bioreactors adapted from Whitney, GA et al. to generate cartilage sheets <italic>in vitro</italic> (<xref ref-type="fig" rid="F6">Figures 6</xref>, <xref ref-type="fig" rid="F7">7A</xref>) (<xref ref-type="bibr" rid="B87">Whitney et al., 2012</xref>; <xref ref-type="bibr" rid="B33">Kean, 2019</xref>; <xref ref-type="bibr" rid="B14">Dennis et al., 2020b</xref>). Similar to our findings in cell aggregates, type II collagen promoter-driven expression of <italic>Gaussia</italic> Luciferase was significantly increased as compared to cells in basal media in engineered cartilage. Biochemical studies supported an increase in total collagen content. Western blots of pepsin extracted samples confirm the increase is type II collagen, specifically, in sheets supplemented with condition 25 (<xref ref-type="fig" rid="F8">Figure 8C</xref>). Collagen x-link analysis supports the formation of type II collagen to type II collagen and type II collagen to type IX collagen heteropolymers, as in native rabbit cartilage (<xref ref-type="fig" rid="F8">Figures 8D, E</xref>). These crosslinks are characteristic of mature cartilage. This is significant as cell processes, particularly in tissue engineering, are often context dependent (<xref ref-type="bibr" rid="B12">Day et al., 2016</xref>; <xref ref-type="bibr" rid="B84">Vermeulen et al., 2020</xref>; <xref ref-type="bibr" rid="B45">Luo and Li, 2022</xref>). It is interesting to note that condition 25 at 1/15th was optimal for type II collagen expression as compared to condition 25 at 1x, as seen by luminescence, immunofluorescence and Western blot. It is possible that higher concentrations are not needed by chondrocytes and could even be detrimental for chondrogenesis resulting in greater type II collagen expression when the concentrations are decreased. Multiple cell types, like osteoblasts, endothelial cells and vascular smooth muscle cells, have specialized mechanisms to recycle and fully utilize vitamins and minerals, as these cannot be synthesized by humans (<xref ref-type="bibr" rid="B32">Kagan and Tyurina, 1998</xref>; <xref ref-type="bibr" rid="B50">May et al., 2003</xref>; <xref ref-type="bibr" rid="B70">Schurgers et al., 2013</xref>; <xref ref-type="bibr" rid="B72">Shearer and Newman, 2014</xref>). Investigation of micronutrient recycling in chondrocytes has not been well studied and was beyond the scope of this work.</p>
<p>Supplementation with condition 25 also altered the mechanical properties of the engineered cartilage. While it increased the tensile modulus of engineered cartilage, unexpectedly, we observed a decrease in Young&#x2019;s modulus in compressive tests as compared to basal media (<xref ref-type="sec" rid="s12">Supplementary Figure S2</xref>), suggestive of decreased stiffness. While it is thought that type II collagen generally increases the tensile properties of cartilage, there is no clear correlation between type II collagen and stiffness (<xref ref-type="bibr" rid="B48">Mansour, 2009</xref>). Furthermore, mechanical testing of live biological tissue is also confounded by the method of testing. Patel JM et al. (<xref ref-type="bibr" rid="B58">Patel et al., 2019</xref>), has explored the inconsistencies present with various modes of mechanical testing which make any comparison of our findings to previous literature extremely difficult. Despite a decrease in the compressive modulus the engineered cartilage generated with condition 25 shows mechanical and biochemical properties closer to that of native cartilage than engineered cartilage generated in basal media alone.</p>
</sec>
<sec sec-type="conclusion" id="s4">
<title>Conclusion</title>
<p>This study demonstrates that the physiologic environment of micronutrients to culture chondrocytes has a far greater impact on chondrogenesis than previously appreciated. Supplementation of culture medium with 15 micronutrients, that are physiologically present in the articular joint, can be tailored to improve <italic>in vitro</italic> chondrogenesis, and the biochemical and mechanical properties of tissue engineered cartilage. Our results show that the presence and concentrations of seemingly minor components of culture medium can have a major impact on chondrogenesis. Furthermore, we established a streamlined process using Design of Experiments and primary reporter chondrocytes as a way to identify optimal chondrogenic conditions <italic>in vitro</italic>.</p>
</sec>
<sec sec-type="materials|methods" id="s5">
<title>Experimental procedures</title>
<sec id="s5-1">
<title>Rabbit primary chondrocyte isolation</title>
<p>Rabbits were euthanized under American Veterinary Medical Association guidelines and knees were isolated within 2&#xa0;h of euthanasia. This study uses rabbits obtained from University of Texas Health Sciences (Dr. Steven Mills). Baylor College of Medicine did not require the study to be reviewed or approved by an ethics committee because we received dead animals. The articular knee joints were dissected under sterile conditions, and articular cartilage was isolated from both the femoral condyle and the tibial plateau. Isolated cartilage was diced into &#x3c;1&#xa0;mm (<xref ref-type="bibr" rid="B55">Mueller and Tuan, 2011</xref>) pieces before sequential digest, first in hyaluronidase for 30&#xa0;min (660 Units/ml Sigma, H3506; in DMEM/F12 with pen/strep/amphotericin B, 30&#xa0;mL), followed by collagenase type II for &#x223c;16&#xa0;h at 37&#xb0;C (583 Units/ml Worthington Biochemical Corp.; in DMEM/F12 with 10% FBS, 1% pen/strep/fungizone, 30&#xa0;mL). The digest was then filtered through a 70&#xa0;&#x3bc;m cell strainer, washed with DMEM/F12, and resuspended in growth media (DMEM/F12 supplemented with 10% FBS, 1% pen/strep). Cells were subsequently infected as described below or cryopreserved (95% FBS, 5% DMSO).</p>
</sec>
<sec id="s5-2">
<title>Lentiviral construct</title>
<p>Lentivirus was generated as previously described (<xref ref-type="bibr" rid="B13">Dennis et al., 2020a</xref>). Briefly, an HIV based lentiviral third generation system from GeneCopoeia was used to generate pseudolentiviral particles. Custom ordered COL2A1-<italic>Gaussia</italic> Luciferase plasmid (1.4&#xa0;kb promoter, HPRM22364-LvPG02, GeneCopoeia, Inc., <xref ref-type="sec" rid="s12">Supplementary Data Sheet S2</xref>), envelope (pMD2. G) and packaging (psPAX2) plasmids were amplified in <italic>Escherichia coli</italic> (GCI-L3, GeneCopoeia) and silica column purified (Qiagen Maxiprep) before being co-transfected into HEK293Ta (GeneCopoeia) cells via calcium chloride precipitation. Pseudolentiviral particles were harvested from conditioned media after 48&#xa0;h and concentrated via ultracentrifugation (10,000 RCF, 4&#xb0;C, overnight). Titers for COL2A1-Gluc lentivirus were estimated via real-time PCR and aliquots stored at &#x2212;80&#xb0;C.</p>
</sec>
<sec id="s5-3">
<title>Lentivirus infection of primary rabbit chondrocytes</title>
<p>Isolated rabbit chondrocytes were seeded at 6,200 cells/cm<sup>2</sup> in growth media and allowed to adhere overnight (&#x223c;20% confluency). Cells were infected with lentivirus (COL2A1-GLuc; MOI 25 in growth media) in the presence of 4&#xa0;&#x3bc;g/ml polybrene (Opti-mem, Gibco) for 12&#xa0;h. Lentiviral medium was replaced with growth medium and cells expanded to &#x223c;90% confluency. Cells were subsequently plated on flasks coated in porcine synoviocyte matrix (<xref ref-type="bibr" rid="B34">Kean and Dennis, 2015</xref>; <xref ref-type="bibr" rid="B35">Kean et al., 2019</xref>) and selected with puromycin (2&#xa0;&#x3bc;g/mL) when 70% confluent for 48&#xa0;h. Culturing of rabbit chondrocytes during infection was done in physioxic (37&#xb0;C, 5% O<sub>2</sub>, 5% CO<sub>2</sub>) conditions. Newly generated COL2A1-GLuc cells were cryopreserved at the end of this first passage (95% FBS, 5% DMSO). These cells were used for all subsequent studies.</p>
</sec>
<sec id="s5-4">
<title>Chondrogenic culture</title>
<p>Rabbit COL2A1-GLuc were thawed and seeded in growth media at 6000 cells/cm<sup>2</sup> and expanded to 90%&#x2013;100% confluence in physioxia. Cells were trypsinized (0.25% Trypsin/EDTA; Corning), resuspended in basal chondrogenic media (93.24% High-Glucose DMEM (Gibco), 1% dexamethasone 10<sup>&#x2212;5</sup>&#xa0;M (Sigma), 1% ITS &#x2b; premix (Becton-Dickinson), 1% Glutamax (Hyclone), 1% 100&#xa0;mM Sodium Pyruvate (Hyclone), 1% MEM Non-Essential Amino Acids (Hyclone), 0.26% L-Ascorbic Acid Phosphate 50&#xa0;mM (Wako), 0.5% Fungizone (Life Technologies) with TGF-&#x3b2;1 (Peprotech) and seeded as described below.</p>
</sec>
<sec id="s5-5">
<title>Generation and maintenance of 3D aggregates</title>
<p>To generate 3D aggregates, cells were seeded at 50,000 cells per well (in 96-well cell repellent u-bottom plates, GreinerBio) and then centrifuged at 500 RCF, 5&#xa0;min. For the TGF-&#x3b2;1 dose response studies, that serve as positive controls for the reporter cells, aggregates were cultured in basal chondrogenic media and different concentrations of TGF-&#x3b2;1 ranging from 0&#x2013;10&#xa0;ng/mL. For the DoE studies, aggregates were cultured in basal chondrogenic media (1&#xa0;ng/ml TGF-&#x3b2;1) as a control or basal media supplemented with vitamins and minerals (<xref ref-type="table" rid="T1">Table 1</xref>; <xref ref-type="sec" rid="s12">Supplementary Tables S1, S2</xref>). Plates were cultured for 3&#xa0;weeks in physioxia, media was sampled and replaced three times a week with respective medium. An OT-2 (Opentrons) python coded robotic pipette, programmed at an aspiration height of 2&#xa0;mm from the bottom of the wells and aspiration rate of 40&#xa0;&#x3bc;L/s was utilized for media preparation, cell feeding, and media sampling for luciferase assay (<xref ref-type="sec" rid="s12">Supplementary File S1</xref>). After 3&#xa0;weeks, cell aggregates were either fixed in neutral buffered formalin for histology or medium removed and aggregates frozen dry (&#x2212;20&#xb0;C) for biochemical assays.</p>
</sec>
<sec id="s5-6">
<title>Tissue engineered cartilage sheets</title>
<sec id="s5-6-1">
<title>Biochamber sterilization and assembly</title>
<p>Custom 3D printed biochambers (<xref ref-type="bibr" rid="B33">Kean, 2019</xref>) that produce 1.2&#xa0;cm<sup>2</sup> cartilage sheets are shown in <xref ref-type="fig" rid="F6">Figure 6</xref>. The chambers are made of an acrylonitrile butadiene styrene (ABS) seeding chamber and a 10&#xa0;&#xb5;m pore polyester membrane (Sterlitech). Screws, a silicone washer and ABS frits hold everything securely and prevent any leaking in between the different pieces. Furthermore, they keep the chamber elevated to allow medium to reach the membrane from the top and bottom for efficient media exchange. The chambers are contained within Nalgene containers modified to have a 0.2&#xa0;&#xb5;m sterile filter on the top to allow gas exchange.</p>
<p>The Nalgene containers along with screws, silicone washer, polyester membrane and nuts were autoclaved and sterile filters fitted to the containers in a biosafety cabinet. ABS pieces were placed in a sealable container for sterilization by immersion in a 10% bleach solution, water rinse, followed by a 10% sodium thiosulfate treatment to neutralize any remaining chlorine, sterile water and isopropanol wash before drying in the biosafety cabinet. Biochambers were assembled as shown in <xref ref-type="fig" rid="F6">Figure 6A</xref> inside a biosafety hood using sterile surgical gloves and autoclaved surgical tools to handle biochamber parts. Once assembled, the polyester membrane was coated with fibronectin (50&#xa0;&#x3bc;g/cm<sup>2</sup>, Corning, in PBS) and allowed to dry in a biosafety cabinet for 1h.</p>
</sec>
<sec id="s5-6-2">
<title>Generation and culture of tissue engineered cartilage sheets</title>
<p>COL2A1-Gluc cells or uninfected primary rabbit chondrocytes were seeded at 5 &#xd7; 10<sup>6</sup> cells/cm<sup>2</sup> in ABS biochambers with a 1.2&#xa0;cm<sup>2</sup> seeding area in basal chondrogenic media alone or in basal media with condition 25 at 1x and 1/15x (<xref ref-type="sec" rid="s12">Supplementary Table S2</xref>) (<xref ref-type="bibr" rid="B33">Kean, 2019</xref>). Media were added in the Nalgene container outside of the biochamber making sure it did not reach the top of the biochamber and combine with the cell suspension inside the biochamber in order to allow cells to adhere to the membrane. After 1&#xa0;day, medium was added to the top of the biochamber so that media exchange occurs with the inside of the whole biochamber (<xref ref-type="fig" rid="F6">Figure 6C</xref>). These were cultured in physioxia on a shaker (10 RPM) for 3-week with media changes three times a week. During media replacement, samples from COL2A1-Gluc biochambers were assessed for luciferase. After 3&#xa0;weeks, cartilage sheets were collected, four (4&#xa0;mm) biopsy punches were taken for mechanical assessment, and collagen cross-linking analysis, remaining pieces of the sheets were frozen (&#x2212;80&#xb0;C) for biochemical assessment or stored in formalin for histology.</p>
</sec>
</sec>
<sec id="s5-7">
<title>Luciferase assay</title>
<p>Cell culture medium sampled from the seeded 96-wells (20&#xa0;&#xb5;L/well) was assessed using a stabilized <italic>Gaussia</italic> Luciferase buffer at a final concentration of 0.09&#xa0;M MES, 0.15M Ascorbic Acid, and 4.2&#xa0;&#xb5;M Coelenterazine in white 96-well plates. Luminescence was measured in a plate reader (25&#xb0;C, relative light units, EnVision plate reader). An OT-2 (Opentrons) python coded robotic pipette was utilized for luciferase buffer addition to white plates (GreinerBio).</p>
</sec>
<sec id="s5-8">
<title>Immunohistochemistry/immunofluorescence</title>
<p>At the end of 3-week culture, cell aggregates were fixed in 10% Neutral Buffered Formalin overnight, embedded in paraffin wax and sectioned (5&#xa0;&#xb5;m sections). Sections were deparaffinized and hydrated, followed by treatment with pronase (1&#xa0;mg/ml, Sigma P5147, in PBS with 5&#xa0;mM CaCl<sub>2</sub>) for epitope retrieval and incubation with primary anti-Collagen Type II (DSHB II-II6B3) followed by a biotinylated secondary and Streptavidin-HRP (BD Biosciences). II-II6B3 was deposited to the DSHB by Linsenmayer, T.F. (DSHB Hybridoma Product II-II6B3). Sections were stained with a chromogen-based substrate kit (Vector labs, VIP substrate vector kit). Engineered cartilage sheet sections were also treated with pronase and primary anti-Collagen Type II (DSHB II-II6B3) followed by VectaFluor R.T.U Antibody Kit DyLight<sup>&#xae;</sup> 488 (Vector Labs DI-2788) following manufacturer&#x2019;s protocol. All sections were imaged at &#xd7;10 magnification.</p>
</sec>
<sec id="s5-9">
<title>Biochemical assays</title>
<p>Frozen cell aggregates, or pieces of engineered cartilage were thawed in PBS, and enzymatically digested with Papain (25&#xa0;&#x3bc;g/mL, Sigma, P4762, in 2&#xa0;mM cysteine; 50&#xa0;mM sodium phosphate; 2&#xa0;mM EDTA at a pH 6.5, 100&#xa0;&#xb5;L) at 65&#xb0;C overnight. During digestion, plates were covered with a qPCR adhesive sealing film (USA Scientific), a silicone sheet, and steel plates clamped to the plate to prevent evaporation. After digestion half of the digest was transferred to another plate and frozen for hydroxyproline assessment. For the remaining half of the digest, papain was inactivated with 0.1M NaOH (50&#xa0;&#xb5;L) followed by neutralization (100&#xa0;mM Na2HPO4, 0.1 N HCL, pH 1.82, 50&#xa0;&#xb5;L). To assess DNA, samples of the digests (20&#xa0;&#xb5;L) were combined with buffered Hoechst dye (&#x23;33258, 667&#xa0;ng/ml, phosphate buffer pH 8, 100&#xa0;&#xb5;L) and fluorescence measured at an excitation of 365&#xa0;nm and emission of 460&#xa0;nm. For GAG assessment, samples of aggregate digest (5&#xa0;&#xb5;L) were combined with a 1,9-Dimethyl-methylene blue solution (195&#xa0;&#xb5;L) and absorbance was measured at 595&#xa0;nm and 525&#xa0;nm (<xref ref-type="bibr" rid="B16">Farndale et al., 1982</xref>). Absorbance readings were corrected by subtracting 595&#xa0;nm reading from 525&#xa0;nm. Total micrograms of DNA and GAG were calculated using a Calf thymus DNA standard (Sigma) and Chondroitin Sulfate standard (Seikagaku Corp.), respectively.</p>
<p>For hydroxyproline (HP), the frozen digest was thawed and incubated overnight at 105&#xb0;C with 6M HCL (200&#xa0;&#xb5;L). Plates were covered as described above to prevent evaporation. Samples were subsequently dried at 70&#xb0;C overnight with a hydroxyproline standard (Sigma). Copper sulfate (0.15M, 10&#xa0;&#xb5;L) and sodium hydroxide (2.5&#xa0;M, 10&#xa0;&#xb5;L) were added to each well and incubated at 50&#xb0;C for 5&#xa0;min, followed by hydrogen peroxide (6%, 10&#xa0;&#xb5;L) for 10&#xa0;min. Sulfuric acid (1.5 M, 40&#xa0;&#x3bc;L) and Ehrlich&#x2019;s reagent (<xref ref-type="bibr" rid="B14">Dennis et al., 2020b</xref>) (20&#xa0;&#xb5;L) were added and samples further incubated at 70&#xb0;C for 15&#xa0;min before reading absorbance at 505&#xa0;nm. Total micrograms of hydroxyproline were calculated from the standard curve. Total collagen was calculated by the following formula (<xref ref-type="bibr" rid="B14">Dennis et al., 2020b</xref>) (&#xb5;g of HP X 7.6 &#x3d; &#xb5;g Total Collagen). (<xref ref-type="bibr" rid="B14">Dennis et al., 2020b</xref>).</p>
</sec>
<sec id="s5-10">
<title>Collagen cross-link analysis</title>
<p>After harvest, samples were frozen at &#x2212;80&#xb0;C until use. Samples were lyophilized, and dry weights obtained. Proteoglycans were extracted using 4M guanidine hydrochloride (GuHCl) in 50&#xa0;mM Tris buffer pH 7.4. The residue was exhaustively rinsed using MilliQ water to remove residual GuHCl, lyophilized and weighed. The cross-linked collagen network was depolymerized using equal volumes of pepsin (0.5&#xa0;mg/mL in 0.5M acetic acid) (<xref ref-type="bibr" rid="B18">Fernandes et al., 2003</xref>). Equivalent aliquots of dry weights were loaded on 6% polyacrylamide gels. Pepsin-extracted type II collagen from adult rabbit articular cartilage was used as a control. After electrophoresis, collagen chains were stained using Coomassie Blue. For Western blots, following SDS-PAGE the separated collagen chains were transferred, by electrophoresis, onto a polyvinyl difluoride (PVDF) membrane and probed with monoclonal antibody (mAb) 10F2 raised against a synthetic peptide EKGPDPLQ (<xref ref-type="bibr" rid="B18">Fernandes et al., 2003</xref>) to identify the C-telopeptide of &#x3b1;1(II) collagen chains cross-linked to &#x3b1;1(II) chains. Another blot was probed with polyclonal antibody (pAb) 5890 to identify the N-telopeptide of &#x3b1;1 (XI) chains cross-linked to &#x3b1;1(II) chains (<xref ref-type="bibr" rid="B14">Dennis et al., 2020b</xref>). This blot was then stripped and probed with mAb 1C10 to identify &#x3b1;1(II) chains (<xref ref-type="bibr" rid="B14">Dennis et al., 2020b</xref>). As we have described before, this determines if a heteropolymer of type II and type XI collagen had formed (<xref ref-type="bibr" rid="B18">Fernandes et al., 2003</xref>). All three antibodies (10F2, 5890 and 1C10) were a gift from Dr. David R. Eyre, University of Washington, Seattle. Type II collagen from adult rabbit articular cartilage was extracted using pepsin as above and used as a control.</p>
</sec>
<sec id="s5-11">
<title>Mechanical testing</title>
<sec id="s5-11-1">
<title>Compression testing</title>
<p>Biopsy punches (4&#xa0;mm) were thawed in Tyrode&#x2019;s solution (Sigma) with protease inhibitors (Sigma, P8465) and equilibrated to room temperature. Using a TA. XT<italic>Plus connect</italic> Texture analyzer a trigger force of 0.1&#xa0;g determined the height of the tissue, then 5%&#x2013;20% strain was applied in 5% increments at 0.1&#xa0;mm/s with a 20-min hold to reach equilibrium. From these results, the equilibrium force was calculated, and a stress vs. strain curve generated (<xref ref-type="bibr" rid="B14">Dennis et al., 2020b</xref>). Young&#x2019;s modulus in compression was determined from the slope of these curves.</p>
</sec>
<sec id="s5-11-2">
<title>Tensile testing</title>
<p>Biopsy punches (4&#xa0;mm) were thawed in Tyrode&#x2019;s solution (Sigma) with protease inhibitors and equilibrated to room temperature. As previously described, a custom dog-bone punch was made from biopsy punches and punches taken from the 4&#xa0;mm punch (<xref ref-type="bibr" rid="B14">Dennis et al., 2020b</xref>). Custom holders were made from laminate projector sheets and dog-bone punches attached using cyanoacrylate glue (Ultra Gel Control, Loctite), tissue was continuously bathed in PBS during this process (<xref ref-type="bibr" rid="B14">Dennis et al., 2020b</xref>). Using a TA. XTPlus connect Texture analyzer with a trigger force of 0.1 G, tissues were stretched to failure at 3&#xa0;mm/s, the equilibrium tensile force was calculated and a stress vs. strain curve generated (<xref ref-type="bibr" rid="B14">Dennis et al., 2020b</xref>). Young&#x2019;s modulus in tension was determined from the slope of these curves.</p>
</sec>
</sec>
<sec id="s5-12">
<title>Design of experiment response surface design</title>
<p>Design-Expert 12 (StatEase) was used to generate a surface response model to assess the effect of 15 factors: linoleic acid, cobalt, copper, chromium, iodine, manganese, molybdenum, thyroxine, vitamin A, vitamin B12, vitamin B7, vitamin D, vitamin E, vitamin K, and zinc. <xref ref-type="table" rid="T1">Table 1</xref> shows minimum and maximum concentrations input into Design-Expert. The response surface I-optimal blocked design generated 240 total conditions to assess the response (<xref ref-type="sec" rid="s12">Supplemental Table S1</xref>).</p>
</sec>
<sec id="s5-13">
<title>Design of experiments analysis</title>
<p>At the end of this experiment, responses (luminescence, metabolic activity and aggregate area) from the screen of 240 conditions as well as results from the one factor at a time approach, were analyzed (Design-Expert, StatEase). Analysis of the results suggested a quadratic model as the best fit. After transformation of the data to fit a quadratic model, Analysis of Variance (ANOVA) was used to identify the positive and negative effects on chondrogenesis as well as fit statistics for the model. The optimization module of the software was used to generate five predicted optimal combinations of factors (<xref ref-type="sec" rid="s12">Supplementary Table S2</xref>, <xref ref-type="sec" rid="s12">Supplementary Data Sheet S2</xref>). Two sets of parameters were used to generate predicted conditions. For condition 25 from <xref ref-type="sec" rid="s12">Supplementary Table S2</xref>, all vitamins and minerals were targeted at 75% serum max except for linolenic acid, vitamin A, copper and vitamin D which are set at their predicted optima. For the other predicted conditions vitamins and minerals were set between 0.01% of serum max and serum max except for vitamin A, E and linolenic acid which were at their approximate optima. All conditions were selected to maximize luminescence for week two and week three as well as aggregate area for week three. Condition 25 also had a target of 0.2, for Resazurin (metabolic activity), the average measurement for chondrocyte aggregates, at week three.</p>
</sec>
<sec id="s5-14">
<title>Ethics</title>
<p>Rabbits were euthanized under American Veterinary Medical Association guidelines for another study (Dr. Steven Mills (University of Texas Health Sciences)).</p>
</sec>
<sec id="s5-15">
<title>Statistical analysis</title>
<p>Statistical analysis for all experiments except for the Design of Experiments screen (analysis described above) were performed using GraphPad Prism 9 and One-way or two-way ANOVA. All data passed tests for normality. In all figures &#x2a; indicates <italic>p</italic>-value &#x3c;0.05, &#x2a;&#x2a; indicates <italic>p</italic>-value &#x3c;0.01, and &#x2a;&#x2a;&#x2a; indicates <italic>p</italic>-value &#x3c;0.001.</p>
</sec>
</sec>
</body>
<back>
<sec sec-type="data-availability" id="s6">
<title>Data availability statement</title>
<p>The original contributions presented in the study are included in the article/<xref ref-type="sec" rid="s12">Supplementary Material</xref>, further inquiries can be directed to the corresponding author.</p>
</sec>
<sec id="s7">
<title>Ethics statement</title>
<p>Rabbits were euthanized under American Veterinary Medical Association guidelines and knees were isolated within 2&#xa0;hours of euthanasia. This study uses rabbits obtained from University of Texas Health Sciences (Dr. Steven Mills). Baylor College of Medicine did not require the study to be reviewed or approved by an ethics committee because we received dead animals.</p>
</sec>
<sec id="s8">
<title>Author contributions</title>
<p>Research design: MC, RF, JD, and TK. Conducted experiments: MC, YG, JV, MK, AR, LM, RM, TS, MA, and TK. Analyzed and interpreted data: MC, AR, RF, and TK. Prepared the manuscript: MC, YG, and TK. Contributed to the discussion, language and proofreading: MC, RF, JD, and TK. All authors contributed to the article and approved the submitted version.</p>
</sec>
<sec id="s9">
<title>Funding</title>
<p>Supplied by the University of Central Florida (TK), University of Central Florida College of Medicine (TK), the Rolanette and Berdon Lawrence Bone Disease Program (TK), NIH grant AR057025 (RF) and the Ernest M. Burgess Endowed Chair research program at the University of Washington (RF).</p>
</sec>
<ack>
<p>We would like to thank Dr. Steven Mills (University of Texas Health Sciences) for the donation of rabbit tissue. The plasmids, pMD2. G, and psPAX2, were a gift from Didier Trono (Addgene plasmid &#x23; 12259; <ext-link ext-link-type="uri" xlink:href="http://n2t.net/addgene:12259">http://n2t.net/addgene:12259</ext-link>; <ext-link ext-link-type="uri" xlink:href="https://scicrunch.org/resolver/RRID:Addgene_12259">RRID:Addgene_12259</ext-link>) and (Addgene plasmid &#x23; 12260; <ext-link ext-link-type="uri" xlink:href="http://n2t.net/addgene:12260">http://n2t.net/addgene:12260</ext-link>; <ext-link ext-link-type="uri" xlink:href="https://scicrunch.org/resolver/RRID:Addgene_12260">RRID:Addgene_12260</ext-link>) respectively. This article was published as a pre-print on BioRxiv (<xref ref-type="bibr" rid="B92">Cruz, 2022</xref>). Article processing charges were provided in part by the UCF College of Graduate Studies Open Access Publishing Fund.</p>
</ack>
<sec sec-type="COI-statement" id="s10">
<title>Conflict of interest</title>
<p>TK and MC are inventors named on a patent application on &#x201c;Anabolic Drugs Stimulating Type II Collagen Production from Chondrocytes or their Progenitors&#x201d; is in process and is tangentially related.</p>
<p>The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p>
</sec>
<sec sec-type="disclaimer" id="s11">
<title>Publisher&#x2019;s note</title>
<p>All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.</p>
</sec>
<sec id="s12">
<title>Supplementary material</title>
<p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type="uri" xlink:href="https://www.frontiersin.org/articles/10.3389/fbioe.2023.1179332/full#supplementary-material">https://www.frontiersin.org/articles/10.3389/fbioe.2023.1179332/full&#x23;supplementary-material</ext-link>
</p>
<supplementary-material xlink:href="DataSheet2.PDF" id="SM1" mimetype="application/PDF" xmlns:xlink="http://www.w3.org/1999/xlink"/>
<supplementary-material xlink:href="DataSheet1.PDF" id="SM2" mimetype="application/PDF" xmlns:xlink="http://www.w3.org/1999/xlink"/>
<supplementary-material xlink:href="Table1.XLSX" id="SM3" mimetype="application/XLSX" xmlns:xlink="http://www.w3.org/1999/xlink"/>
</sec>
<ref-list>
<title>References</title>
<ref id="B1">
<citation citation-type="book">
<person-group person-group-type="author">
<name>
<surname>Aydar</surname>
<given-names>A. Y.</given-names>
</name>
</person-group> (<year>2018</year>). <source>Utilization of response surface methodology in optimization of extraction of plant materials</source>. <publisher-name>IntechOpen</publisher-name>.</citation>
</ref>
<ref id="B2">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Badr</surname>
<given-names>C. E.</given-names>
</name>
<name>
<surname>Hewett</surname>
<given-names>J. W.</given-names>
</name>
<name>
<surname>Breakefield</surname>
<given-names>X. O.</given-names>
</name>
<name>
<surname>Tannous</surname>
<given-names>B. A.</given-names>
</name>
</person-group> (<year>2007</year>). <article-title>A highly sensitive assay for monitoring the secretory pathway and ER stress</article-title>. <source>PLoS ONE</source> <volume>2</volume>, <fpage>e571</fpage>. <pub-id pub-id-type="doi">10.1371/journal.pone.0000571</pub-id>
</citation>
</ref>
<ref id="B3">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Barone</surname>
<given-names>L. M.</given-names>
</name>
<name>
<surname>Owen</surname>
<given-names>T. A.</given-names>
</name>
<name>
<surname>Tassinari</surname>
<given-names>M. S.</given-names>
</name>
<name>
<surname>Bortell</surname>
<given-names>R.</given-names>
</name>
<name>
<surname>Stein</surname>
<given-names>G. S.</given-names>
</name>
<name>
<surname>Lian</surname>
<given-names>J. B.</given-names>
</name>
</person-group> (<year>1991</year>). <article-title>Developmental expression and hormonal regulation of the rat matrix Gla protein (MGP) gene in chondrogenesis and osteogenesis</article-title>. <source>J. Cell Biochem.</source> <volume>46</volume>, <fpage>351</fpage>&#x2013;<lpage>365</lpage>. <pub-id pub-id-type="doi">10.1002/jcb.240460410</pub-id>
</citation>
</ref>
<ref id="B4">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Benya</surname>
<given-names>P. D.</given-names>
</name>
<name>
<surname>Shaffer</surname>
<given-names>J. D.</given-names>
</name>
</person-group> (<year>1982</year>). <article-title>Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gels</article-title>. <source>Cell</source> <volume>30</volume>, <fpage>215</fpage>&#x2013;<lpage>224</lpage>. <pub-id pub-id-type="doi">10.1016/0092-8674(82)90027-7</pub-id>
</citation>
</ref>
<ref id="B5">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Bian</surname>
<given-names>L.</given-names>
</name>
<name>
<surname>Zhai</surname>
<given-names>D. Y.</given-names>
</name>
<name>
<surname>Mauck</surname>
<given-names>R. L.</given-names>
</name>
<name>
<surname>Burdick</surname>
<given-names>J. A.</given-names>
</name>
</person-group> (<year>2011</year>). <article-title>Coculture of human mesenchymal stem cells and articular chondrocytes reduces hypertrophy and enhances functional properties of engineered cartilage</article-title>. <source>Tissue Eng. Part A</source> <volume>17</volume>, <fpage>1137</fpage>&#x2013;<lpage>1145</lpage>. <pub-id pub-id-type="doi">10.1089/ten.tea.2010.0531</pub-id>
</citation>
</ref>
<ref id="B6">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Bonaventure</surname>
<given-names>J.</given-names>
</name>
<name>
<surname>Kadhom</surname>
<given-names>N.</given-names>
</name>
<name>
<surname>Cohen-Solal</surname>
<given-names>L.</given-names>
</name>
<name>
<surname>Ng</surname>
<given-names>K.</given-names>
</name>
<name>
<surname>Bourguignon</surname>
<given-names>J.</given-names>
</name>
<name>
<surname>Lasselin</surname>
<given-names>C.</given-names>
</name>
<etal/>
</person-group> (<year>1994</year>). <article-title>Reexpression of cartilage-specific genes by dedifferentiated human articular chondrocytes cultured in alginate beads</article-title>. <source>Exp. Cell Res.</source> <volume>212</volume>, <fpage>97</fpage>&#x2013;<lpage>104</lpage>. <pub-id pub-id-type="doi">10.1006/excr.1994.1123</pub-id>
</citation>
</ref>
<ref id="B7">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Booth</surname>
<given-names>S. L.</given-names>
</name>
<name>
<surname>Mayer</surname>
</name>
</person-group> (<year>1997</year>). <article-title>Skeletal functions of vitamin K-dependent proteins: Not just for clotting anymore</article-title>. <source>Nutr. Rev.</source> <volume>55</volume>, <fpage>282</fpage>&#x2013;<lpage>284</lpage>. <pub-id pub-id-type="doi">10.1111/j.1753-4887.1997.tb01619.x</pub-id>
</citation>
</ref>
<ref id="B8">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Brodziak-Dopierala</surname>
<given-names>B.</given-names>
</name>
<name>
<surname>Kwapulinski</surname>
<given-names>J.</given-names>
</name>
<name>
<surname>Sobczyk</surname>
<given-names>K.</given-names>
</name>
<name>
<surname>Wiechula</surname>
<given-names>D.</given-names>
</name>
</person-group> (<year>2013</year>). <article-title>The content of manganese and iron in hip joint tissue</article-title>. <source>J. Trace Elem. Med. Biol.</source> <volume>27</volume>, <fpage>208</fpage>&#x2013;<lpage>212</lpage>. <pub-id pub-id-type="doi">10.1016/j.jtemb.2012.12.005</pub-id>
</citation>
</ref>
<ref id="B9">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Chin</surname>
<given-names>K. Y.</given-names>
</name>
<name>
<surname>Ima-Nirwana</surname>
<given-names>S.</given-names>
</name>
</person-group> (<year>2018</year>). <article-title>The role of vitamin E in preventing and treating osteoarthritis - a review of the current evidence</article-title>. <source>Front. Pharmacol.</source> <volume>9</volume>, <fpage>946</fpage>. <pub-id pub-id-type="doi">10.3389/fphar.2018.00946</pub-id>
</citation>
</ref>
<ref id="B10">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Connor</surname>
<given-names>W. E.</given-names>
</name>
</person-group> (<year>1999</year>). <article-title>&#x3b1;-Linolenic acid in health and disease</article-title>. <source>Am. J. Clin. Nutr.</source> <volume>69</volume>, <fpage>827</fpage>&#x2013;<lpage>828</lpage>. <pub-id pub-id-type="doi">10.1093/ajcn/69.5.827</pub-id>
</citation>
</ref>
<ref id="B11">
<citation citation-type="book">
<person-group person-group-type="author">
<name>
<surname>Crowley</surname>
<given-names>J.</given-names>
</name>
<name>
<surname>Traynor</surname>
<given-names>D.</given-names>
</name>
<name>
<surname>Weatherburn</surname>
<given-names>D.</given-names>
</name>
</person-group> (<year>2000</year>). <source>Metal ions in biological systems</source>, <volume>37</volume>. <publisher-loc>New York</publisher-loc>: <publisher-name>Marcel Dekker</publisher-name>.</citation>
</ref>
<ref id="B92">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Cruz</surname>
<given-names>M. A.</given-names>
</name>
<name>
<surname>Gonzalez</surname>
<given-names>Y.</given-names>
</name>
<name>
<surname>V&#x00e9;lez Toro</surname>
<given-names>J. A.</given-names>
</name>
<name>
<surname>Karimzadeh</surname>
<given-names>M.</given-names>
</name>
<name>
<surname>Rubbo</surname>
<given-names>A.</given-names>
</name>
<name>
<surname>Morris</surname>
<given-names>L.</given-names>
</name>
<etal/>
</person-group> (<year>2022</year>). <article-title>Micronutrient optimization using design of experiments approach in tissue engineered articular cartilage for production of type II collagen</article-title>. <source>BioRxiv</source> <volume>2022</volume>, <fpage>519522</fpage>. <pub-id pub-id-type="doi">10.1101/2022.12.07.519522</pub-id>
</citation>
</ref>
<ref id="B12">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Day</surname>
<given-names>E. K.</given-names>
</name>
<name>
<surname>Sosale</surname>
<given-names>N. G.</given-names>
</name>
<name>
<surname>Lazzara</surname>
<given-names>M. J.</given-names>
</name>
</person-group> (<year>2016</year>). <article-title>Cell signaling regulation by protein phosphorylation: A multivariate, heterogeneous, and context-dependent process</article-title>. <source>Curr. Opin. Biotechnol.</source> <volume>40</volume>, <fpage>185</fpage>&#x2013;<lpage>192</lpage>. <pub-id pub-id-type="doi">10.1016/j.copbio.2016.06.005</pub-id>
</citation>
</ref>
<ref id="B13">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Dennis</surname>
<given-names>J. E.</given-names>
</name>
<name>
<surname>Splawn</surname>
<given-names>T.</given-names>
</name>
<name>
<surname>Kean</surname>
<given-names>T. J.</given-names>
</name>
</person-group> (<year>2020</year>). <article-title>High-throughput, temporal and dose dependent, effect of vitamins and minerals on chondrogenesis</article-title>. <source>Front. Cell Dev. Biol.</source> <volume>8</volume>, <fpage>92</fpage>. <pub-id pub-id-type="doi">10.3389/fcell.2020.00092</pub-id>
</citation>
</ref>
<ref id="B14">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Dennis</surname>
<given-names>J. E.</given-names>
</name>
<name>
<surname>Whitney</surname>
<given-names>G. A.</given-names>
</name>
<name>
<surname>Rai</surname>
<given-names>J.</given-names>
</name>
<name>
<surname>Fernandes</surname>
<given-names>R. J.</given-names>
</name>
<name>
<surname>Kean</surname>
<given-names>T. J.</given-names>
</name>
</person-group> (<year>2020</year>). <article-title>Physioxia stimulates extracellular matrix deposition and increases mechanical properties of human chondrocyte-derived tissue-engineered cartilage</article-title>. <source>Front. Bioeng. Biotechnol.</source> <volume>8</volume>, <fpage>590743</fpage>. <pub-id pub-id-type="doi">10.3389/fbioe.2020.590743</pub-id>
</citation>
</ref>
<ref id="B15">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Enochson</surname>
<given-names>L.</given-names>
</name>
<name>
<surname>Brittberg</surname>
<given-names>M.</given-names>
</name>
<name>
<surname>Lindahl</surname>
<given-names>A.</given-names>
</name>
</person-group> (<year>2012</year>). <article-title>Optimization of a chondrogenic medium through the use of factorial design of experiments</article-title>. <source>Biores Open Access</source> <volume>1</volume>, <fpage>306</fpage>&#x2013;<lpage>313</lpage>. <pub-id pub-id-type="doi">10.1089/biores.2012.0277</pub-id>
</citation>
</ref>
<ref id="B16">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Farndale</surname>
<given-names>R. W.</given-names>
</name>
<name>
<surname>Sayers</surname>
<given-names>C. A.</given-names>
</name>
<name>
<surname>Barrett</surname>
<given-names>A. J.</given-names>
</name>
</person-group> (<year>1982</year>). <article-title>A direct spectrophotometric microassay for sulfated glycosaminoglycans in cartilage cultures</article-title>. <source>Connect. Tissue Res.</source> <volume>9</volume>, <fpage>247</fpage>&#x2013;<lpage>248</lpage>. <pub-id pub-id-type="doi">10.3109/03008208209160269</pub-id>
</citation>
</ref>
<ref id="B17">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Fernandes</surname>
<given-names>R. J.</given-names>
</name>
<name>
<surname>Hirohata</surname>
<given-names>S.</given-names>
</name>
<name>
<surname>Engle</surname>
<given-names>J. M.</given-names>
</name>
<name>
<surname>Colige</surname>
<given-names>A.</given-names>
</name>
<name>
<surname>Cohn</surname>
<given-names>D. H.</given-names>
</name>
<name>
<surname>Eyre</surname>
<given-names>D. R.</given-names>
</name>
<etal/>
</person-group> (<year>2001</year>). <article-title>Procollagen II amino propeptide processing by ADAMTS-3</article-title>. <source>J. Biol. Chem.</source> <volume>276</volume>, <fpage>31502</fpage>&#x2013;<lpage>31509</lpage>. <pub-id pub-id-type="doi">10.1074/jbc.m103466200</pub-id>
</citation>
</ref>
<ref id="B18">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Fernandes</surname>
<given-names>R. J.</given-names>
</name>
<name>
<surname>Schmid</surname>
<given-names>T. M.</given-names>
</name>
<name>
<surname>Eyre</surname>
<given-names>D. R.</given-names>
</name>
</person-group> (<year>2003</year>). <article-title>Assembly of collagen types II, IX and XI into nascent hetero-fibrils by a rat chondrocyte cell line</article-title>. <source>Eur. J. Biochem.</source> <volume>270</volume>, <fpage>3243</fpage>&#x2013;<lpage>3250</lpage>. <pub-id pub-id-type="doi">10.1046/j.1432-1033.2003.03711.x</pub-id>
</citation>
</ref>
<ref id="B19">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Fernandes</surname>
<given-names>R. J.</given-names>
</name>
<name>
<surname>Weis</surname>
<given-names>M.</given-names>
</name>
<name>
<surname>Scott</surname>
<given-names>M. A.</given-names>
</name>
<name>
<surname>Seegmiller</surname>
<given-names>R. E.</given-names>
</name>
<name>
<surname>Eyre</surname>
<given-names>D. R.</given-names>
</name>
</person-group> (<year>2007</year>). <article-title>Collagen XI chain misassembly in cartilage of the chondrodysplasia (cho) mouse</article-title>. <source>Matrix Biol.</source> <volume>26</volume>, <fpage>597</fpage>&#x2013;<lpage>603</lpage>. <pub-id pub-id-type="doi">10.1016/j.matbio.2007.06.007</pub-id>
</citation>
</ref>
<ref id="B20">
<citation citation-type="book">
<person-group person-group-type="author">
<name>
<surname>Finch</surname>
<given-names>C. E.</given-names>
</name>
</person-group> (<year>2007</year>). &#x201c;<article-title>Chapter 2 - infections, inflammogens, and drugs</article-title>,&#x201d; in <source>The biology of human longevity</source>. Editor <person-group person-group-type="editor">
<name>
<surname>Finch</surname>
<given-names>C. E.</given-names>
</name>
</person-group> (<publisher-loc>Burlington</publisher-loc>: <publisher-name>Academic Press</publisher-name>), <fpage>113</fpage>&#x2013;<lpage>173</lpage>.</citation>
</ref>
<ref id="B21">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Finch</surname>
<given-names>C. W.</given-names>
</name>
</person-group> (<year>2015</year>). <article-title>Review of trace mineral requirements for preterm infants: What are the current recommendations for clinical practice?</article-title> <source>Nutr. Clin. Pract.</source> <volume>30</volume>, <fpage>44</fpage>&#x2013;<lpage>58</lpage>. <pub-id pub-id-type="doi">10.1177/0884533614563353</pub-id>
</citation>
</ref>
<ref id="B22">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Francis</surname>
<given-names>S. L.</given-names>
</name>
<name>
<surname>Di Bella</surname>
<given-names>C.</given-names>
</name>
<name>
<surname>Wallace</surname>
<given-names>G. G.</given-names>
</name>
<name>
<surname>Choong</surname>
<given-names>P. F. M.</given-names>
</name>
</person-group> (<year>2018</year>). <article-title>Cartilage tissue engineering using stem cells and bioprinting technology&#x2014;barriers to clinical translation</article-title>. <source>Front. Surg.</source> <volume>5</volume>, <fpage>70</fpage>. <pub-id pub-id-type="doi">10.3389/fsurg.2018.00070</pub-id>
</citation>
</ref>
<ref id="B23">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Frenz</surname>
<given-names>D. A.</given-names>
</name>
<name>
<surname>Liu</surname>
<given-names>W.</given-names>
</name>
<name>
<surname>Cvekl</surname>
<given-names>A.</given-names>
</name>
<name>
<surname>Xie</surname>
<given-names>Q.</given-names>
</name>
<name>
<surname>Wassef</surname>
<given-names>L.</given-names>
</name>
<name>
<surname>Quadro</surname>
<given-names>L.</given-names>
</name>
<etal/>
</person-group> (<year>2010</year>). <article-title>Retinoid signaling in inner ear development: A "goldilocks" phenomenon</article-title>. <source>Am. J. Med. Genet. A</source> <volume>152A</volume>, <fpage>2947</fpage>&#x2013;<lpage>2961</lpage>. <pub-id pub-id-type="doi">10.1002/ajmg.a.33670</pub-id>
</citation>
</ref>
<ref id="B24">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Galli</surname>
<given-names>F.</given-names>
</name>
<name>
<surname>Azzi</surname>
<given-names>A.</given-names>
</name>
<name>
<surname>Birringer</surname>
<given-names>M.</given-names>
</name>
<name>
<surname>Cook-Mills</surname>
<given-names>J. M.</given-names>
</name>
<name>
<surname>Eggersdorfer</surname>
<given-names>M.</given-names>
</name>
<name>
<surname>Frank</surname>
<given-names>J.</given-names>
</name>
<etal/>
</person-group> (<year>2017</year>). <article-title>Vitamin E: Emerging aspects and new directions</article-title>. <source>Free Radic. Biol. Med.</source> <volume>102</volume>, <fpage>16</fpage>&#x2013;<lpage>36</lpage>. <pub-id pub-id-type="doi">10.1016/j.freeradbiomed.2016.09.017</pub-id>
</citation>
</ref>
<ref id="B25">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Gemmiti</surname>
<given-names>C. V.</given-names>
</name>
<name>
<surname>Guldberg</surname>
<given-names>R. E.</given-names>
</name>
</person-group> (<year>2006</year>). <article-title>Fluid flow increases type II collagen deposition and tensile mechanical properties in bioreactor-grown tissue-engineered cartilage</article-title>. <source>Tissue Eng.</source> <volume>12</volume>, <fpage>060320122646001</fpage>&#x2013;<lpage>060320122646479</lpage>. <pub-id pub-id-type="doi">10.1089/ten.2006.12.ft-53</pub-id>
</citation>
</ref>
<ref id="B26">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Han</surname>
<given-names>F.</given-names>
</name>
<name>
<surname>Adams</surname>
<given-names>C. S.</given-names>
</name>
<name>
<surname>Tao</surname>
<given-names>Z.</given-names>
</name>
<name>
<surname>Williams</surname>
<given-names>C. J.</given-names>
</name>
<name>
<surname>Zaka</surname>
<given-names>R.</given-names>
</name>
<name>
<surname>Tuan</surname>
<given-names>R. S.</given-names>
</name>
<etal/>
</person-group> (<year>2005</year>). <article-title>Transforming growth factor-&#x3b2;1 (TGF-&#x3b2;1) regulates ATDC5 chondrogenic differentiation and fibronectin isoform expression</article-title>. <source>J. Cell. Biochem.</source> <volume>95</volume>, <fpage>750</fpage>&#x2013;<lpage>762</lpage>. <pub-id pub-id-type="doi">10.1002/jcb.20427</pub-id>
</citation>
</ref>
<ref id="B27">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Hanrahan</surname>
<given-names>G.</given-names>
</name>
<name>
<surname>Lu</surname>
<given-names>K.</given-names>
</name>
</person-group> (<year>2006</year>). <article-title>Application of factorial and response surface methodology in modern experimental design and optimization</article-title>. <source>Crit. Rev. Anal. Chem.</source> <volume>36</volume>, <fpage>141</fpage>&#x2013;<lpage>151</lpage>. <pub-id pub-id-type="doi">10.1080/10408340600969478</pub-id>
</citation>
</ref>
<ref id="B28">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Hassan</surname>
<given-names>W. N.</given-names>
</name>
<name>
<surname>Bin-Jaliah</surname>
<given-names>I.</given-names>
</name>
<name>
<surname>Haidara</surname>
<given-names>M. A.</given-names>
</name>
<name>
<surname>Eid</surname>
<given-names>R. A.</given-names>
</name>
<name>
<surname>Heidar</surname>
<given-names>E. H. A.</given-names>
</name>
<name>
<surname>Dallak</surname>
<given-names>M.</given-names>
</name>
<etal/>
</person-group> (<year>2019</year>). <article-title>Vitamin E ameliorates alterations to the articular cartilage of knee joints induced by monoiodoacetate and diabetes mellitus in rats</article-title>. <source>Ultrastruct. Pathol.</source> <volume>43</volume>, <fpage>126</fpage>&#x2013;<lpage>134</lpage>. <pub-id pub-id-type="doi">10.1080/01913123.2019.1627446</pub-id>
</citation>
</ref>
<ref id="B29">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Hille</surname>
<given-names>R.</given-names>
</name>
<name>
<surname>Hall</surname>
<given-names>J.</given-names>
</name>
<name>
<surname>Basu</surname>
<given-names>P.</given-names>
</name>
</person-group> (<year>2014</year>). <article-title>The mononuclear molybdenum enzymes</article-title>. <source>Chem. Rev.</source> <volume>114</volume>, <fpage>3963</fpage>&#x2013;<lpage>4038</lpage>. <pub-id pub-id-type="doi">10.1021/cr400443z</pub-id>
</citation>
</ref>
<ref id="B30">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Horton</surname>
<given-names>W. E.</given-names>
</name>
<name>
<surname>Cleveland</surname>
<given-names>J.</given-names>
</name>
<name>
<surname>Rapp</surname>
<given-names>U.</given-names>
</name>
<name>
<surname>Nemuth</surname>
<given-names>G.</given-names>
</name>
<name>
<surname>Bolander</surname>
<given-names>M.</given-names>
</name>
<name>
<surname>Doege</surname>
<given-names>K.</given-names>
</name>
<etal/>
</person-group> (<year>1988</year>). <article-title>An established rat cell line expressing chondrocyte properties</article-title>. <source>Exp. Cell Res.</source> <volume>178</volume>, <fpage>457</fpage>&#x2013;<lpage>468</lpage>. <pub-id pub-id-type="doi">10.1016/0014-4827(88)90414-4</pub-id>
</citation>
</ref>
<ref id="B31">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Jennifer</surname>
<given-names>L.</given-names>
</name>
<name>
<surname>Puetzer</surname>
<given-names>J. N. P.</given-names>
</name>
<name>
<surname>Loboa</surname>
<given-names>E. G.</given-names>
</name>
</person-group> (<year>2010</year>). <article-title>Comparative review of growth factors for induction of three-dimensional <italic>in vitro</italic> chondrogenesis in human mesenchymal stem cells isolated from bone marrow and adipose tissue</article-title>. <source>Tissue Eng. Part B Rev.</source> <volume>16</volume>, <fpage>435</fpage>&#x2013;<lpage>444</lpage>. <pub-id pub-id-type="doi">10.1089/ten.teb.2009.0705</pub-id>
</citation>
</ref>
<ref id="B32">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Kagan</surname>
<given-names>V. E.</given-names>
</name>
<name>
<surname>Tyurina</surname>
<given-names>Y. Y.</given-names>
</name>
</person-group> (<year>1998</year>). <article-title>Recycling and redox cycling of phenolic antioxidants</article-title>. <source>Ann. N. Y. Acad. Sci.</source> <volume>854</volume>, <fpage>425</fpage>&#x2013;<lpage>434</lpage>. <pub-id pub-id-type="doi">10.1111/j.1749-6632.1998.tb09921.x</pub-id>
</citation>
</ref>
<ref id="B33">
<citation citation-type="book">
<person-group person-group-type="author">
<name>
<surname>Kean</surname>
<given-names>T. J.</given-names>
</name>
</person-group> (<year>2019</year>). <source>1 cm^2 biochamber</source>. <comment>Available at: <ext-link ext-link-type="uri" xlink:href="https://3dprint.nih.gov/discover/3dpx-010438%202022">https://3dprint.nih.gov/discover/3dpx-010438.2022</ext-link>
</comment>.</citation>
</ref>
<ref id="B34">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Kean</surname>
<given-names>T. J.</given-names>
</name>
<name>
<surname>Dennis</surname>
<given-names>J. E.</given-names>
</name>
</person-group> (<year>2015</year>). <article-title>Synoviocyte derived-extracellular matrix enhances human articular chondrocyte proliferation and maintains Re-differentiation capacity at both low and atmospheric oxygen tensions</article-title>. <source>PLoS One</source> <volume>10</volume>, <fpage>e0129961</fpage>. <pub-id pub-id-type="doi">10.1371/journal.pone.0129961</pub-id>
</citation>
</ref>
<ref id="B35">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Kean</surname>
<given-names>T. J.</given-names>
</name>
<name>
<surname>Ge</surname>
<given-names>Z.</given-names>
</name>
<name>
<surname>Li</surname>
<given-names>Y.</given-names>
</name>
<name>
<surname>Chen</surname>
<given-names>R.</given-names>
</name>
<name>
<surname>Dennis</surname>
<given-names>J. E.</given-names>
</name>
</person-group> (<year>2019</year>). <article-title>Transcriptome-wide analysis of human chondrocyte expansion on synoviocyte matrix</article-title>. <source>Cells</source> <volume>8</volume>, <fpage>85</fpage>. <pub-id pub-id-type="doi">10.3390/cells8020085</pub-id>
</citation>
</ref>
<ref id="B36">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Kobayashi</surname>
<given-names>M.</given-names>
</name>
<name>
<surname>Shimizu</surname>
<given-names>S.</given-names>
</name>
</person-group> (<year>1999</year>). <article-title>Cobalt proteins</article-title>. <source>Eur. J. Biochem.</source> <volume>261</volume>, <fpage>1</fpage>&#x2013;<lpage>9</lpage>. <pub-id pub-id-type="doi">10.1046/j.1432-1327.1999.00186.x</pub-id>
</citation>
</ref>
<ref id="B37">
<citation citation-type="book">
<person-group person-group-type="author">
<name>
<surname>Ko&#xe7;</surname>
<given-names>B.</given-names>
</name>
<name>
<surname>Kaymak-Ertekin</surname>
<given-names>F.</given-names>
</name>
</person-group> (<year>2017</year>). <source>Response surface methodology and food processing applications</source>. <publisher-loc>G&#x131;da</publisher-loc>. <comment>(Turkish with English Abstract)</comment>.</citation>
</ref>
<ref id="B38">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Koyano</surname>
<given-names>Y.</given-names>
</name>
<name>
<surname>Hejna</surname>
<given-names>M.</given-names>
</name>
<name>
<surname>Flechtenmacher</surname>
<given-names>J.</given-names>
</name>
<name>
<surname>Schmid</surname>
<given-names>T. M.</given-names>
</name>
<name>
<surname>Thonar</surname>
<given-names>E. J. M. A.</given-names>
</name>
<name>
<surname>Mollenhauer</surname>
<given-names>J.</given-names>
</name>
</person-group> (<year>1996</year>). <article-title>Collagen and proteoglycan production by bovine fetal and adult chondrocytes under low levels of calcium and zinc ions</article-title>. <source>Connect. tissue Res.</source> <volume>34</volume>, <fpage>213</fpage>&#x2013;<lpage>225</lpage>. <pub-id pub-id-type="doi">10.3109/03008209609000700</pub-id>
</citation>
</ref>
<ref id="B39">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Kuterbekov</surname>
<given-names>M.</given-names>
</name>
<name>
<surname>Machillot</surname>
<given-names>P.</given-names>
</name>
<name>
<surname>Baillet</surname>
<given-names>F.</given-names>
</name>
<name>
<surname>Jonas</surname>
<given-names>A. M.</given-names>
</name>
<name>
<surname>Glinel</surname>
<given-names>K.</given-names>
</name>
<name>
<surname>Picart</surname>
<given-names>C.</given-names>
</name>
</person-group> (<year>2019</year>). <article-title>Design of experiments to assess the effect of culture parameters on the osteogenic differentiation of human adipose stromal cells</article-title>. <source>Stem Cell Res. Ther.</source> <volume>10</volume>, <fpage>256</fpage>. <pub-id pub-id-type="doi">10.1186/s13287-019-1333-7</pub-id>
</citation>
</ref>
<ref id="B40">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Li</surname>
<given-names>S.</given-names>
</name>
<name>
<surname>Niu</surname>
<given-names>G.</given-names>
</name>
<name>
<surname>Wu</surname>
<given-names>Y.</given-names>
</name>
<name>
<surname>Du</surname>
<given-names>G.</given-names>
</name>
<name>
<surname>Huang</surname>
<given-names>C.</given-names>
</name>
<name>
<surname>Yin</surname>
<given-names>X.</given-names>
</name>
<etal/>
</person-group> (<year>2016</year>). <article-title>Vitamin D prevents articular cartilage erosion by regulating collagen II turnover through TGF-&#x3b2;1 in ovariectomized rats</article-title>. <source>Osteoarthr. Cartil.</source> <volume>24</volume>, <fpage>345</fpage>&#x2013;<lpage>353</lpage>. <pub-id pub-id-type="doi">10.1016/j.joca.2015.08.013</pub-id>
</citation>
</ref>
<ref id="B41">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Lind</surname>
<given-names>T.</given-names>
</name>
<name>
<surname>Sundqvist</surname>
<given-names>A.</given-names>
</name>
<name>
<surname>Hu</surname>
<given-names>L.</given-names>
</name>
<name>
<surname>Pejler</surname>
<given-names>G.</given-names>
</name>
<name>
<surname>Andersson</surname>
<given-names>G.</given-names>
</name>
<name>
<surname>Jacobson</surname>
<given-names>A.</given-names>
</name>
<etal/>
</person-group> (<year>2013</year>). <article-title>Vitamin a is a negative regulator of osteoblast mineralization</article-title>. <source>PloS one</source> <volume>8</volume>, <fpage>e82388</fpage>. <pub-id pub-id-type="doi">10.1371/journal.pone.0082388</pub-id>
</citation>
</ref>
<ref id="B42">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Litchfield</surname>
<given-names>T. M.</given-names>
</name>
<name>
<surname>Ishikawa</surname>
<given-names>Y.</given-names>
</name>
<name>
<surname>Wu</surname>
<given-names>L. N.</given-names>
</name>
<name>
<surname>Wuthier</surname>
<given-names>R. E.</given-names>
</name>
<name>
<surname>Sauer</surname>
<given-names>G. R.</given-names>
</name>
</person-group> (<year>1998</year>). <article-title>Effect of metal ions on calcifying growth plate cartilage chondrocytes</article-title>. <source>Calcif. Tissue Int.</source> <volume>62</volume>, <fpage>341</fpage>&#x2013;<lpage>349</lpage>. <pub-id pub-id-type="doi">10.1007/s002239900442</pub-id>
</citation>
</ref>
<ref id="B43">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Liu</surname>
<given-names>X.</given-names>
</name>
<name>
<surname>Zhang</surname>
<given-names>W.</given-names>
</name>
<name>
<surname>Wu</surname>
<given-names>Z.</given-names>
</name>
<name>
<surname>Yang</surname>
<given-names>Y.</given-names>
</name>
<name>
<surname>Kang</surname>
<given-names>Y. J.</given-names>
</name>
</person-group> (<year>2018</year>). <article-title>Copper levels affect targeting of hypoxia-inducible factor 1&#x3b1; to the promoters of hypoxia-regulated genes</article-title>. <source>J. Biol. Chem.</source> <volume>293</volume>, <fpage>14669</fpage>&#x2013;<lpage>14677</lpage>. <pub-id pub-id-type="doi">10.1074/jbc.ra118.001764</pub-id>
</citation>
</ref>
<ref id="B44">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Lukaski</surname>
<given-names>H. C.</given-names>
</name>
</person-group> (<year>2000</year>). <article-title>Magnesium, zinc, and chromium nutriture and physical activity</article-title>. <source>Am. J. Clin. Nutr.</source> <volume>72</volume>, <fpage>585S</fpage>&#x2013;<lpage>593S</lpage>. <pub-id pub-id-type="doi">10.1093/ajcn/72.2.585s</pub-id>
</citation>
</ref>
<ref id="B45">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Luo</surname>
<given-names>J.</given-names>
</name>
<name>
<surname>Li</surname>
<given-names>P.</given-names>
</name>
</person-group> (<year>2022</year>). <article-title>Context-dependent transcriptional regulations of YAP/TAZ in stem cell and differentiation</article-title>. <source>Stem Cell Res. Ther.</source> <volume>13</volume>, <fpage>10</fpage>. <pub-id pub-id-type="doi">10.1186/s13287-021-02686-y</pub-id>
</citation>
</ref>
<ref id="B46">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Makris</surname>
<given-names>E. A.</given-names>
</name>
<name>
<surname>Hu</surname>
<given-names>J. C.</given-names>
</name>
<name>
<surname>Athanasiou</surname>
<given-names>K. A.</given-names>
</name>
</person-group> (<year>2013</year>). <article-title>Hypoxia-induced collagen crosslinking as a mechanism for enhancing mechanical properties of engineered articular cartilage</article-title>. <source>Osteoarthr. Cartil.</source> <volume>21</volume>, <fpage>634</fpage>&#x2013;<lpage>641</lpage>. <pub-id pub-id-type="doi">10.1016/j.joca.2013.01.007</pub-id>
</citation>
</ref>
<ref id="B47">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Makris</surname>
<given-names>E. A.</given-names>
</name>
<name>
<surname>MacBarb</surname>
<given-names>R. F.</given-names>
</name>
<name>
<surname>Responte</surname>
<given-names>D. J.</given-names>
</name>
<name>
<surname>Hu</surname>
<given-names>J. C.</given-names>
</name>
<name>
<surname>Athanasiou</surname>
<given-names>K. A.</given-names>
</name>
</person-group> (<year>2013</year>). <article-title>A copper sulfate and hydroxylysine treatment regimen for enhancing collagen cross-linking and biomechanical properties in engineered neocartilage</article-title>. <source>FASEB J.</source> <volume>27</volume>, <fpage>2421</fpage>&#x2013;<lpage>2430</lpage>. <pub-id pub-id-type="doi">10.1096/fj.12-224030</pub-id>
</citation>
</ref>
<ref id="B48">
<citation citation-type="book">
<person-group person-group-type="author">
<name>
<surname>Mansour</surname>
<given-names>J. M.</given-names>
</name>
</person-group> (<year>2009</year>). &#x201c;<article-title>Biomechanics of cartilage</article-title>,&#x201d; in <source>Kinesiology: The mechanics and pathomechanics of human movement</source> (<publisher-name>Lippincott Williams and Wilkins</publisher-name>).</citation>
</ref>
<ref id="B49">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Masuda</surname>
<given-names>E.</given-names>
</name>
<name>
<surname>Shirai</surname>
<given-names>K.</given-names>
</name>
<name>
<surname>Maekubo</surname>
<given-names>K.</given-names>
</name>
<name>
<surname>Hirai</surname>
<given-names>Y.</given-names>
</name>
</person-group> (<year>2015</year>). <article-title>A newly established culture method highlights regulatory roles of retinoic acid on morphogenesis and calcification of mammalian limb cartilage</article-title>. <source>BioTechniques</source> <volume>58</volume>, <fpage>318</fpage>&#x2013;<lpage>324</lpage>. <pub-id pub-id-type="doi">10.2144/000114300</pub-id>
</citation>
</ref>
<ref id="B50">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>May</surname>
<given-names>J. M.</given-names>
</name>
<name>
<surname>Qu</surname>
<given-names>Z.-c.</given-names>
</name>
<name>
<surname>Neel</surname>
<given-names>D. R.</given-names>
</name>
<name>
<surname>Li</surname>
<given-names>X.</given-names>
</name>
</person-group> (<year>2003</year>). <article-title>Recycling of vitamin C from its oxidized forms by human endothelial cells</article-title>. <source>Biochimica Biophysica Acta (BBA) - Mol. Cell Res.</source> <volume>1640</volume>, <fpage>153</fpage>&#x2013;<lpage>161</lpage>. <pub-id pub-id-type="doi">10.1016/s0167-4889(03)00043-0</pub-id>
</citation>
</ref>
<ref id="B51">
<citation citation-type="book">
<collab>MayoClinic</collab> (<year>2022</year>). <source>Mayo medical laboratories rochester test catalog</source>. <comment>Available at: <ext-link ext-link-type="uri" xlink:href="https://www.mayocliniclabs.com/test-catalog/print-catalog.html">https://www.mayocliniclabs.com/test-catalog/print-catalog.html</ext-link>
</comment>.</citation>
</ref>
<ref id="B52">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>McAlinden</surname>
<given-names>A.</given-names>
</name>
<name>
<surname>Traeger</surname>
<given-names>G.</given-names>
</name>
<name>
<surname>Hansen</surname>
<given-names>U.</given-names>
</name>
<name>
<surname>Weis</surname>
<given-names>M. A.</given-names>
</name>
<name>
<surname>Ravindran</surname>
<given-names>S.</given-names>
</name>
<name>
<surname>Wirthlin</surname>
<given-names>L.</given-names>
</name>
<etal/>
</person-group> (<year>2014</year>). <article-title>Molecular properties and fibril ultrastructure of types II and XI collagens in cartilage of mice expressing exclusively the &#x3b1;1(IIA) collagen isoform</article-title>. <source>Matrix Biol.</source> <volume>34</volume>, <fpage>105</fpage>&#x2013;<lpage>113</lpage>. <pub-id pub-id-type="doi">10.1016/j.matbio.2013.09.006</pub-id>
</citation>
</ref>
<ref id="B53">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Mello</surname>
<given-names>M. A.</given-names>
</name>
<name>
<surname>Tuan</surname>
<given-names>R. S.</given-names>
</name>
</person-group> (<year>2006</year>). <article-title>Effects of TGF-&#x3b2;1 and triiodothyronine on cartilage maturation: <italic>In vitro</italic> analysis using long-term high-density micromass cultures of chick embryonic limb mesenchymal cells</article-title>. <source>J. Orthop. Res.</source> <volume>24</volume>, <fpage>2095</fpage>&#x2013;<lpage>2105</lpage>. <pub-id pub-id-type="doi">10.1002/jor.20233</pub-id>
</citation>
</ref>
<ref id="B54">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Mobasheri</surname>
<given-names>A. L. I.</given-names>
</name>
<name>
<surname>Platt</surname>
<given-names>N.</given-names>
</name>
<name>
<surname>Thorpe</surname>
<given-names>C.</given-names>
</name>
<name>
<surname>Shakibaei</surname>
<given-names>M.</given-names>
</name>
</person-group> (<year>2006</year>). <article-title>Regulation of 2-Deoxy-d-Glucose transport, lactate metabolism, and MMP-2 secretion by the hypoxia mimetic cobalt chloride in articular chondrocytes</article-title>. <source>Ann. N. Y. Acad. Sci.</source> <volume>1091</volume>, <fpage>83</fpage>&#x2013;<lpage>93</lpage>. <pub-id pub-id-type="doi">10.1196/annals.1378.057</pub-id>
</citation>
</ref>
<ref id="B55">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Mueller</surname>
<given-names>M. B. M. D.</given-names>
</name>
<name>
<surname>Tuan</surname>
<given-names>R. S. P.</given-names>
</name>
</person-group> (<year>2011</year>). <article-title>Anabolic/catabolic balance in pathogenesis of osteoarthritis: Identifying molecular targets</article-title>. <source>PM R</source> <volume>3</volume>, <fpage>S3</fpage>&#x2013;<lpage>S11</lpage>. <pub-id pub-id-type="doi">10.1016/j.pmrj.2011.05.009</pub-id>
</citation>
</ref>
<ref id="B56">
<citation citation-type="book">
<person-group person-group-type="author">
<name>
<surname>Myers</surname>
<given-names>R. H.</given-names>
</name>
<name>
<surname>Montgomery</surname>
<given-names>D. C.</given-names>
</name>
<name>
<surname>Anderson-Cook</surname>
<given-names>C. M.</given-names>
</name>
</person-group> (<year>2016</year>). <source>Response surface methodology: Process and product optimization using designed experiments</source>. <publisher-loc>Incorporated, New York</publisher-loc>: <publisher-name>John Wiley and Sons</publisher-name>.</citation>
</ref>
<ref id="B57">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Pacifici</surname>
<given-names>M.</given-names>
</name>
<name>
<surname>Cossu</surname>
<given-names>G.</given-names>
</name>
<name>
<surname>Molinaro</surname>
<given-names>M.</given-names>
</name>
<name>
<surname>Tato</surname>
<given-names>F.</given-names>
</name>
</person-group> (<year>1980</year>). <article-title>Vitamin A inhibits chondrogenesis but not myogenesis</article-title>. <source>Exp. Cell Res.</source> <volume>129</volume>, <fpage>469</fpage>&#x2013;<lpage>474</lpage>. <pub-id pub-id-type="doi">10.1016/0014-4827(80)90517-0</pub-id>
</citation>
</ref>
<ref id="B58">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Patel</surname>
<given-names>J. M.</given-names>
</name>
<name>
<surname>Wise</surname>
<given-names>B. C.</given-names>
</name>
<name>
<surname>Bonnevie</surname>
<given-names>E. D.</given-names>
</name>
<name>
<surname>Mauck</surname>
<given-names>R. L.</given-names>
</name>
</person-group> (<year>2019</year>). <article-title>A systematic review and guide to mechanical testing for articular cartilage tissue engineering</article-title>. <source>Tissue Eng. Part C Methods</source> <volume>25</volume>, <fpage>593</fpage>&#x2013;<lpage>608</lpage>. <pub-id pub-id-type="doi">10.1089/ten.tec.2019.0116</pub-id>
</citation>
</ref>
<ref id="B59">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Pechova</surname>
<given-names>A.</given-names>
</name>
<name>
<surname>Pavlata</surname>
<given-names>L.</given-names>
</name>
</person-group> (<year>2007</year>). <article-title>Chromium as an essential nutrient: A review</article-title>. <source>Veterin&#xe1;rn&#xed; medic&#xed;na</source> <volume>52</volume>, <fpage>1</fpage>&#x2013;<lpage>18</lpage>. <pub-id pub-id-type="doi">10.17221/2010-vetmed</pub-id>
</citation>
</ref>
<ref id="B60">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Pecora</surname>
<given-names>F.</given-names>
</name>
<name>
<surname>Gualeni</surname>
<given-names>B.</given-names>
</name>
<name>
<surname>Forlino</surname>
<given-names>A.</given-names>
</name>
<name>
<surname>Superti-Furga</surname>
<given-names>A.</given-names>
</name>
<name>
<surname>Tenni</surname>
<given-names>R.</given-names>
</name>
<name>
<surname>Cetta</surname>
<given-names>G.</given-names>
</name>
<etal/>
</person-group> (<year>2006</year>). <article-title>
<italic>In vivo</italic> contribution of amino acid sulfur to cartilage proteoglycan sulfation</article-title>. <source>Biochem. J.</source> <volume>398</volume>, <fpage>509</fpage>&#x2013;<lpage>514</lpage>. <pub-id pub-id-type="doi">10.1042/bj20060566</pub-id>
</citation>
</ref>
<ref id="B61">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Penick</surname>
<given-names>K. J.</given-names>
</name>
<name>
<surname>Solchaga</surname>
<given-names>L. A.</given-names>
</name>
<name>
<surname>Welter</surname>
<given-names>J. F.</given-names>
</name>
</person-group> (<year>2005</year>). <article-title>High-throughput aggregate culture system to assess the chondrogenic potential of mesenchymal stem cells</article-title>. <source>Biotechniques</source> <volume>39</volume>, <fpage>687</fpage>&#x2013;<lpage>691</lpage>. <pub-id pub-id-type="doi">10.2144/000112009</pub-id>
</citation>
</ref>
<ref id="B62">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Ren</surname>
<given-names>F. L.</given-names>
</name>
<name>
<surname>Guo</surname>
<given-names>X.</given-names>
</name>
<name>
<surname>Zhang</surname>
<given-names>R.</given-names>
</name>
<name>
<surname>Wang</surname>
<given-names>S.</given-names>
</name>
<name>
<surname>Zuo</surname>
<given-names>H.</given-names>
</name>
<name>
<surname>Zhang</surname>
<given-names>Z.</given-names>
</name>
<etal/>
</person-group> (<year>2007</year>). <article-title>Effects of selenium and iodine deficiency on bone, cartilage growth plate and chondrocyte differentiation in two generations of rats</article-title>. <source>Osteoarthr. Cartil.</source> <volume>15</volume>, <fpage>1171</fpage>&#x2013;<lpage>1177</lpage>. <pub-id pub-id-type="doi">10.1016/j.joca.2007.03.013</pub-id>
</citation>
</ref>
<ref id="B63">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Renner</surname>
<given-names>J. N.</given-names>
</name>
<name>
<surname>Liu</surname>
<given-names>J. C.</given-names>
</name>
</person-group> (<year>2013</year>). <article-title>Investigating the effect of peptide agonists on the chondrogenic differentiation of human mesenchymal stem cells using design of experiments</article-title>. <source>Biotechnol. Prog.</source> <volume>29</volume>, <fpage>1550</fpage>&#x2013;<lpage>1557</lpage>. <pub-id pub-id-type="doi">10.1002/btpr.1808</pub-id>
</citation>
</ref>
<ref id="B64">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Ruecker</surname>
<given-names>O.</given-names>
</name>
<name>
<surname>Zillner</surname>
<given-names>K.</given-names>
</name>
<name>
<surname>Groebner-Ferreira</surname>
<given-names>R.</given-names>
</name>
<name>
<surname>Heitzer</surname>
<given-names>M.</given-names>
</name>
</person-group> (<year>2008</year>). <article-title>Gaussia-luciferase as a sensitive reporter gene for monitoring promoter activity in the nucleus of the green alga Chlamydomonas reinhardtii</article-title>. <source>Mol. Genet. genomics MGG</source> <volume>280</volume>, <fpage>153</fpage>&#x2013;<lpage>162</lpage>. <pub-id pub-id-type="doi">10.1007/s00438-008-0352-3</pub-id>
</citation>
</ref>
<ref id="B65">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Sacitharan</surname>
<given-names>P. K.</given-names>
</name>
</person-group> (<year>2019</year>). <article-title>Ageing and osteoarthritis</article-title>. <source>Subcell. Biochem.</source> <volume>91</volume>, <fpage>123</fpage>&#x2013;<lpage>159</lpage>. <pub-id pub-id-type="doi">10.1007/978-981-13-3681-2_6</pub-id>
</citation>
</ref>
<ref id="B66">
<citation citation-type="book">
<person-group person-group-type="author">
<name>
<surname>Saleem</surname>
<given-names>F.</given-names>
</name>
<name>
<surname>Soos</surname>
<given-names>M. P.</given-names>
</name>
</person-group> (<year>2023</year>). &#x201c;<article-title>Biotin deficiency</article-title>,&#x201d; in <source>StatPearls (treasure island (FL)</source>.</citation>
</ref>
<ref id="B67">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Santoro</surname>
<given-names>A.</given-names>
</name>
<name>
<surname>Conde</surname>
<given-names>J.</given-names>
</name>
<name>
<surname>Scotece</surname>
<given-names>M.</given-names>
</name>
<name>
<surname>Abella</surname>
<given-names>V.</given-names>
</name>
<name>
<surname>L&#xf3;pez</surname>
<given-names>V.</given-names>
</name>
<name>
<surname>Pino</surname>
<given-names>J.</given-names>
</name>
<etal/>
</person-group> (<year>2015</year>). <article-title>Choosing the right chondrocyte cell line: Focus on nitric oxide: Choosing the right chondrocyte cell line: Focus</article-title>. <source>J. Orthop. Res.</source> <volume>33</volume>, <fpage>1784</fpage>&#x2013;<lpage>1788</lpage>. <pub-id pub-id-type="doi">10.1002/jor.22954</pub-id>
</citation>
</ref>
<ref id="B68">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Sardesai</surname>
<given-names>V. M.</given-names>
</name>
</person-group> (<year>1993</year>). <article-title>Molybdenum: An essential trace element</article-title>. <source>Nutr. Clin. Pract.</source> <volume>8</volume>, <fpage>277</fpage>&#x2013;<lpage>281</lpage>. <pub-id pub-id-type="doi">10.1177/0115426593008006277</pub-id>
</citation>
</ref>
<ref id="B69">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Sato</surname>
<given-names>K.</given-names>
</name>
<name>
<surname>Mera</surname>
<given-names>H.</given-names>
</name>
<name>
<surname>Wakitani</surname>
<given-names>S.</given-names>
</name>
<name>
<surname>Takagi</surname>
<given-names>M.</given-names>
</name>
</person-group> (<year>2017</year>). <article-title>Effect of epigallocatechin-3-gallate on the increase in type II collagen accumulation in cartilage-like MSC sheets</article-title>. <source>Biosci. Biotechnol. Biochem.</source> <volume>81</volume>, <fpage>1241</fpage>&#x2013;<lpage>1245</lpage>. <pub-id pub-id-type="doi">10.1080/09168451.2017.1282809</pub-id>
</citation>
</ref>
<ref id="B70">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Schurgers</surname>
<given-names>L. J.</given-names>
</name>
<name>
<surname>Uitto</surname>
<given-names>J.</given-names>
</name>
<name>
<surname>Reutelingsperger</surname>
<given-names>C. P.</given-names>
</name>
</person-group> (<year>2013</year>). <article-title>Vitamin K-dependent carboxylation of matrix gla-protein: A crucial switch to control ectopic mineralization</article-title>. <source>Trends Mol. Med.</source> <volume>19</volume>, <fpage>217</fpage>&#x2013;<lpage>226</lpage>. <pub-id pub-id-type="doi">10.1016/j.molmed.2012.12.008</pub-id>
</citation>
</ref>
<ref id="B71">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Shaikh</surname>
<given-names>S. R.</given-names>
</name>
</person-group> (<year>2012</year>). <article-title>Biophysical and biochemical mechanisms by which dietary N-3 polyunsaturated fatty acids from fish oil disrupt membrane lipid rafts</article-title>. <source>J. Nutr. Biochem.</source> <volume>23</volume>, <fpage>101</fpage>&#x2013;<lpage>105</lpage>. <pub-id pub-id-type="doi">10.1016/j.jnutbio.2011.07.001</pub-id>
</citation>
</ref>
<ref id="B72">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Shearer</surname>
<given-names>M. J.</given-names>
</name>
<name>
<surname>Newman</surname>
<given-names>P.</given-names>
</name>
</person-group> (<year>2014</year>). <article-title>Recent trends in the metabolism and cell biology of vitamin K with special reference to vitamin K cycling and MK-4 biosynthesis</article-title>. <source>J. Lipid Res.</source> <volume>55</volume>, <fpage>345</fpage>&#x2013;<lpage>362</lpage>. <pub-id pub-id-type="doi">10.1194/jlr.r045559</pub-id>
</citation>
</ref>
<ref id="B73">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Sophia Fox</surname>
<given-names>A. J.</given-names>
</name>
<name>
<surname>Bedi</surname>
<given-names>A.</given-names>
</name>
<name>
<surname>Rodeo</surname>
<given-names>S. A.</given-names>
</name>
</person-group> (<year>2009</year>). <article-title>The basic science of articular cartilage: Structure, composition, and function</article-title>. <source>Sports Health</source> <volume>1</volume>, <fpage>461</fpage>&#x2013;<lpage>468</lpage>. <pub-id pub-id-type="doi">10.1177/1941738109350438</pub-id>
</citation>
</ref>
<ref id="B74">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Stabler</surname>
<given-names>S. P.</given-names>
</name>
</person-group> (<year>2013</year>). <article-title>Vitamin B12 deficiency</article-title>. <source>N. Engl. J. Med.</source> <volume>368</volume>, <fpage>149</fpage>&#x2013;<lpage>160</lpage>. <pub-id pub-id-type="doi">10.1056/nejmcp1113996</pub-id>
</citation>
</ref>
<ref id="B75">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Steimberg</surname>
<given-names>N.</given-names>
</name>
<name>
<surname>Viengchareun</surname>
<given-names>S.</given-names>
</name>
<name>
<surname>Biehlmann</surname>
<given-names>F.</given-names>
</name>
<name>
<surname>Gu&#xe9;nal</surname>
<given-names>I.</given-names>
</name>
<name>
<surname>Mignotte</surname>
<given-names>B.</given-names>
</name>
<name>
<surname>Adolphe</surname>
<given-names>M.</given-names>
</name>
<etal/>
</person-group> (<year>1999</year>). <article-title>SV40 large T antigen expression driven by col2a1 regulatory sequences immortalizes articular chondrocytes but does not allow stabilization of type II collagen expression</article-title>. <source>Exp. Cell Res.</source> <volume>249</volume>, <fpage>248</fpage>&#x2013;<lpage>259</lpage>. <pub-id pub-id-type="doi">10.1006/excr.1999.4478</pub-id>
</citation>
</ref>
<ref id="B76">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Sumitani</surname>
<given-names>Y.</given-names>
</name>
<name>
<surname>Uchibe</surname>
<given-names>K.</given-names>
</name>
<name>
<surname>Yoshida</surname>
<given-names>K.</given-names>
</name>
<name>
<surname>Weng</surname>
<given-names>Y.</given-names>
</name>
<name>
<surname>Guo</surname>
<given-names>J.</given-names>
</name>
<name>
<surname>Yuan</surname>
<given-names>H.</given-names>
</name>
<etal/>
</person-group> (<year>2020</year>). <article-title>Inhibitory effect of retinoic acid receptor agonists on <italic>in vitro</italic> chondrogenic differentiation</article-title>. <source>Anat. Sci. Int.</source> <volume>95</volume>, <fpage>202</fpage>&#x2013;<lpage>208</lpage>. <pub-id pub-id-type="doi">10.1007/s12565-019-00512-3</pub-id>
</citation>
</ref>
<ref id="B77">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Surazynski</surname>
<given-names>A.</given-names>
</name>
<name>
<surname>Miltyk</surname>
<given-names>W.</given-names>
</name>
<name>
<surname>Palka</surname>
<given-names>J.</given-names>
</name>
<name>
<surname>Phang</surname>
<given-names>J. M.</given-names>
</name>
</person-group> (<year>2008</year>). <article-title>Prolidase-dependent regulation of collagen biosynthesis</article-title>. <source>Amino Acids</source> <volume>35</volume>, <fpage>731</fpage>&#x2013;<lpage>738</lpage>. <pub-id pub-id-type="doi">10.1007/s00726-008-0051-8</pub-id>
</citation>
</ref>
<ref id="B78">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Szychlinska</surname>
<given-names>M. A.</given-names>
</name>
<name>
<surname>Imbesi</surname>
<given-names>R.</given-names>
</name>
<name>
<surname>Castrogiovanni</surname>
<given-names>P.</given-names>
</name>
<name>
<surname>Guglielmino</surname>
<given-names>C.</given-names>
</name>
<name>
<surname>Ravalli</surname>
<given-names>S.</given-names>
</name>
<name>
<surname>Di Rosa</surname>
<given-names>M.</given-names>
</name>
<etal/>
</person-group> (<year>2019</year>). <article-title>Assessment of vitamin D supplementation on articular cartilage morphology in a Young healthy sedentary rat model</article-title>. <source>Nutrients</source> <volume>11</volume>, <fpage>1260</fpage>. <pub-id pub-id-type="doi">10.3390/nu11061260</pub-id>
</citation>
</ref>
<ref id="B79">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Takahashi</surname>
<given-names>T.</given-names>
</name>
<name>
<surname>Ogasawara</surname>
<given-names>T.</given-names>
</name>
<name>
<surname>Asawa</surname>
<given-names>Y.</given-names>
</name>
<name>
<surname>Mori</surname>
<given-names>Y.</given-names>
</name>
<name>
<surname>Uchinuma</surname>
<given-names>E.</given-names>
</name>
<name>
<surname>Takato</surname>
<given-names>T.</given-names>
</name>
<etal/>
</person-group> (<year>2007</year>). <article-title>Three-dimensional microenvironments retain chondrocyte phenotypes during proliferation culture</article-title>. <source>Tissue Eng.</source> <volume>13</volume>, <fpage>1583</fpage>&#x2013;<lpage>1592</lpage>. <pub-id pub-id-type="doi">10.1089/ten.2006.0322</pub-id>
</citation>
</ref>
<ref id="B80">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Tannous</surname>
<given-names>B. A.</given-names>
</name>
</person-group> (<year>2009</year>). <article-title>Gaussia luciferase reporter assay for monitoring biological processes in culture and <italic>in vivo</italic>
</article-title>. <source>Nat. Protoc.</source> <volume>4</volume>, <fpage>582</fpage>&#x2013;<lpage>591</lpage>. <pub-id pub-id-type="doi">10.1038/nprot.2009.28</pub-id>
</citation>
</ref>
<ref id="B81">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Tannous</surname>
<given-names>B. A.</given-names>
</name>
<name>
<surname>Kim</surname>
<given-names>D. E.</given-names>
</name>
<name>
<surname>Fernandez</surname>
<given-names>J. L.</given-names>
</name>
<name>
<surname>Weissleder</surname>
<given-names>R.</given-names>
</name>
<name>
<surname>Breakefield</surname>
<given-names>X. O.</given-names>
</name>
</person-group> (<year>2005</year>). <article-title>Codon-optimized Gaussia luciferase cDNA for mammalian gene expression in culture and <italic>in vivo</italic>
</article-title>. <source>Mol. Ther.</source> <volume>11</volume>, <fpage>435</fpage>&#x2013;<lpage>443</lpage>. <pub-id pub-id-type="doi">10.1016/j.ymthe.2004.10.016</pub-id>
</citation>
</ref>
<ref id="B82">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Thenet</surname>
<given-names>S.</given-names>
</name>
<name>
<surname>Benya</surname>
<given-names>P. D.</given-names>
</name>
<name>
<surname>Demignot</surname>
<given-names>S.</given-names>
</name>
<name>
<surname>Feunteun</surname>
<given-names>J.</given-names>
</name>
<name>
<surname>Adolphe</surname>
<given-names>M.</given-names>
</name>
</person-group> (<year>1992</year>). <article-title>SV40-immortalization of rabbit articular chondrocytes: Alteration of differentiated functions</article-title>. <source>J. Cell. physiology</source> <volume>150</volume>, <fpage>158</fpage>&#x2013;<lpage>167</lpage>. <pub-id pub-id-type="doi">10.1002/jcp.1041500121</pub-id>
</citation>
</ref>
<ref id="B83">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Tsonis</surname>
<given-names>P. A.</given-names>
</name>
</person-group> (<year>1991</year>). <article-title>1,25-dihydroxyvitamin D3 stimulates chondrogenesis of the chick limb bud mesenchymal cells</article-title>. <source>Dev. Biol.</source> <volume>143</volume>, <fpage>130</fpage>&#x2013;<lpage>134</lpage>. <pub-id pub-id-type="doi">10.1016/0012-1606(91)90060-g</pub-id>
</citation>
</ref>
<ref id="B84">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Vermeulen</surname>
<given-names>S.</given-names>
</name>
<name>
<surname>Roumans</surname>
<given-names>N.</given-names>
</name>
<name>
<surname>Honig</surname>
<given-names>F.</given-names>
</name>
<name>
<surname>Carlier</surname>
<given-names>A.</given-names>
</name>
<name>
<surname>Hebels</surname>
<given-names>D. G.</given-names>
</name>
<name>
<surname>Eren</surname>
<given-names>A. D.</given-names>
</name>
<etal/>
</person-group> (<year>2020</year>). <article-title>Mechanotransduction is a context-dependent activator of TGF-beta signaling in mesenchymal stem cells</article-title>. <source>Biomaterials</source> <volume>259</volume>, <fpage>120331</fpage>. <pub-id pub-id-type="doi">10.1016/j.biomaterials.2020.120331</pub-id>
</citation>
</ref>
<ref id="B85">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Vunjak-Novakovic</surname>
<given-names>G.</given-names>
</name>
<name>
<surname>Martin</surname>
<given-names>I.</given-names>
</name>
<name>
<surname>Obradovic</surname>
<given-names>B.</given-names>
</name>
<name>
<surname>Treppo</surname>
<given-names>S.</given-names>
</name>
<name>
<surname>Grodzinsky</surname>
<given-names>A. J.</given-names>
</name>
<name>
<surname>Langer</surname>
<given-names>R.</given-names>
</name>
<etal/>
</person-group> (<year>1999</year>). <article-title>Bioreactor cultivation conditions modulate the composition and mechanical properties of tissue-engineered cartilage</article-title>. <source>J. Orthop. Res.</source> <volume>17</volume>, <fpage>130</fpage>&#x2013;<lpage>138</lpage>. <pub-id pub-id-type="doi">10.1002/jor.1100170119</pub-id>
</citation>
</ref>
<ref id="B86">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Whitney</surname>
<given-names>G. A.</given-names>
</name>
<name>
<surname>Kean</surname>
<given-names>T. J.</given-names>
</name>
<name>
<surname>Fernandes</surname>
<given-names>R. J.</given-names>
</name>
<name>
<surname>Waldman</surname>
<given-names>S.</given-names>
</name>
<name>
<surname>Tse</surname>
<given-names>M. Y.</given-names>
</name>
<name>
<surname>Pang</surname>
<given-names>S. C.</given-names>
</name>
<etal/>
</person-group> (<year>2018</year>). <article-title>Thyroxine increases collagen type II expression and accumulation in scaffold-free tissue-engineered articular cartilage</article-title>. <source>Tissue Eng. Part A</source> <volume>24</volume>, <fpage>369</fpage>&#x2013;<lpage>381</lpage>. <pub-id pub-id-type="doi">10.1089/ten.tea.2016.0533</pub-id>
</citation>
</ref>
<ref id="B87">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Whitney</surname>
<given-names>G. A.</given-names>
</name>
<name>
<surname>Mera</surname>
<given-names>H.</given-names>
</name>
<name>
<surname>Weidenbecher</surname>
<given-names>M.</given-names>
</name>
<name>
<surname>Awadallah</surname>
<given-names>A.</given-names>
</name>
<name>
<surname>Mansour</surname>
<given-names>J. M.</given-names>
</name>
<name>
<surname>Dennis</surname>
<given-names>J. E.</given-names>
</name>
</person-group> (<year>2012</year>). <article-title>Methods for producing scaffold-free engineered cartilage sheets from auricular and articular chondrocyte cell sources and attachment to porous tantalum</article-title>. <source>Biores Open Access</source> <volume>1</volume>, <fpage>157</fpage>&#x2013;<lpage>165</lpage>. <pub-id pub-id-type="doi">10.1089/biores.2012.0231</pub-id>
</citation>
</ref>
<ref id="B88">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Wluka</surname>
<given-names>A. E.</given-names>
</name>
<name>
<surname>Stuckey</surname>
<given-names>S.</given-names>
</name>
<name>
<surname>Brand</surname>
<given-names>C.</given-names>
</name>
<name>
<surname>Cicuttini</surname>
<given-names>F. M.</given-names>
</name>
</person-group> (<year>2002</year>). <article-title>Supplementary vitamin E does not affect the loss of cartilage volume in knee osteoarthritis: A 2 year double blind randomized placebo controlled study</article-title>. <source>J. rheumatology</source> <volume>29</volume>, <fpage>2585</fpage>&#x2013;<lpage>2591</lpage>.</citation>
</ref>
<ref id="B89">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Wurdinger</surname>
<given-names>T.</given-names>
</name>
<name>
<surname>Badr</surname>
<given-names>C.</given-names>
</name>
<name>
<surname>Pike</surname>
<given-names>L.</given-names>
</name>
<name>
<surname>de Kleine</surname>
<given-names>R.</given-names>
</name>
<name>
<surname>Weissleder</surname>
<given-names>R.</given-names>
</name>
<name>
<surname>Breakefield</surname>
<given-names>X. O.</given-names>
</name>
<etal/>
</person-group> (<year>2008</year>). <article-title>A secreted luciferase for <italic>ex vivo</italic> monitoring of <italic>in vivo</italic> processes</article-title>. <source>Nat. methods</source> <volume>5</volume>, <fpage>171</fpage>&#x2013;<lpage>173</lpage>. <pub-id pub-id-type="doi">10.1038/nmeth.1177</pub-id>
</citation>
</ref>
<ref id="B90">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Yolmeh</surname>
<given-names>M.</given-names>
</name>
<name>
<surname>Jafari</surname>
<given-names>S. M.</given-names>
</name>
</person-group> (<year>2017</year>). <article-title>Applications of response surface methodology in the food industry processes</article-title>. <source>Food bioprocess Technol.</source> <volume>10</volume>, <fpage>413</fpage>&#x2013;<lpage>433</lpage>. <pub-id pub-id-type="doi">10.1007/s11947-016-1855-2</pub-id>
</citation>
</ref>
<ref id="B91">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Yoo</surname>
<given-names>J. U.</given-names>
</name>
<name>
<surname>Barthel</surname>
<given-names>T. S.</given-names>
</name>
<name>
<surname>Nishimura</surname>
<given-names>K.</given-names>
</name>
<name>
<surname>Solchaga</surname>
<given-names>L.</given-names>
</name>
<name>
<surname>Caplan</surname>
<given-names>A. I.</given-names>
</name>
<name>
<surname>Goldberg</surname>
<given-names>V. M.</given-names>
</name>
<etal/>
</person-group> (<year>1998</year>). <article-title>The chondrogenic potential of human bone-marrow-derived mesenchymal progenitor cells</article-title>. <source>J. Bone Jt. Surg. Am.</source> <volume>80</volume>, <fpage>1745</fpage>&#x2013;<lpage>1757</lpage>. <pub-id pub-id-type="doi">10.2106/00004623-199812000-00004</pub-id>
</citation>
</ref>
</ref-list>
</back>
</article>