These authors have contributed equally to this work
This article was submitted to Biomaterials, a section of the journal Frontiers in Bioengineering and Biotechnology
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Nowadays, 3D printing technology has been applied in dentistry to fabricate customized implants. However, the biological performance is unsatisfactory. Polydopamine (PDA) has been used to immobilize bioactive agents on implant surfaces to endow them with multiple properties, such as anti-infection and pro-osteogenesis, benefiting rapid osseointegration. Herein, we fabricated a PDA coating on a 3D-printed implant surface (3D-PDA)
Simplification of the manufacturing process and clinical procedures, and decreases in treatment time are the trends of implant dentistry. When an immediate implant technique is implemented, the implant needs to be inserted into the tooth socket immediately after tooth extraction, which can effectively preserve the height and width of the alveolar and minimize the margin bone adsorption during the healing stage of extraction socket (
Our former study found that a 3D-printed Ti6Al4V implant could obviously improve osseointegration in a rat condyle implant model compared to the implant prepared by traditional machining technology after implantation for 3 weeks. However, there was no significant difference between them in the longer term. Moreover, compared to a sand-blasting with large grit and acid-etching titanium implant (SLA), which is prevalently used in clinic, the osseointegration performance of a 3D-printed Ti6Al4V implant was inferior and not ideal (
Lots of research has confirmed that the surface characteristics play essential roles in dominating implant fate (
In effect, the above studies have mainly focused on providing the osteogenic property to implants for the sake of enhancing the osseointegration performance. Actually, many other factors participate in the implant osseointegration process (
With the emergence and development of biochemical surface modification, bioactive agents, such as protein, peptide, growth factor, and drugs have been tentatively applied to implant surfaces. Nowadays, many techniques can immobilize the above bioactive agents, mainly classified into three kinds, protein adsorption, covalent immobilization by physical methods, or by chemical methods (
PDA is the self-polymerized product of dopamine in an alkaline aqueous solution in the dark. Previous research confirmed that polydopamine can powerfully adhere to a large variety of material surfaces and apparently enhance hydrophilicity and biocompatibility, which is a universal method to functionalize material surfaces (
Overall, as mentioned before, our previous study has successfully designed and fabricated a customized 3D-printed implant. In the current research, we primarily focused on studying the biocompatibility and
The 3D-printed Ti6Al4V implants (Ø 2 mm × 4 mm) and plates (side length: 10 mm, height: 2 mm) were fabricated by an EOS laser printing system (EOS GmbH Munchen, Germany) as previously demonstrated (
The surface topography of the 3D-PDA and 3D groups were characterized
Sprague Dawley (SD) rats approximately 2 weeks old were purchased from the Shanghai SLAC Laboratory Animal Co. Ltd (Shanghai, China) and used in the current study. All the animal experiments and procedures were performed according to the approval and guidance of the Institutional Animal Care and Use Committee of Tongji University (Shanghai, China). The process of isolating BMSCs coincided with former research (
To evaluate the form of BMSCs on the two group surfaces, 1 × 104 cells were cultivated on each specimen. After culturing for 24 h, the cells were rinsed three times with phosphate buffer saline (PBS) and immobilized with 4% paraformaldehyde (PFA) overnight at 4°C. Then the samples were dehydrated with hierarchical ethanol series sequentially, freezing dried, and sprayed with gold before scanning
For the cell proliferation assay, 2 × 104 cells were cultured on the 3D-PDA and 3D groups. At days 1, 4 and 7, samples were rinsed with PBS three times and cultured in medium added with 10% CCK-8 (CCK-8, Beyotime, China) working solution for 3 h at 37°C in the dark. Afterward, the solution absorbance was detected at a 405 nm wavelength by utilizing a microplate reader (Biotek, United States).
BMSCs were cultivated (2 × 104/well) on the two kinds of sample surfaces to measure the ALP activity performance at days 4 and 7. For quantitative analysis of ALP activity, samples were flushed with PBS and soaked in 1% TritonX-100 (Beyotime, China). After centrifuging at 4°C (12,000 rpm × 10 min), supernatants were obtained. The ALP kit (JianCheng Bioengineering Institute, Nanjing, China) and the BCA protein trial kit (Beyotime, China) were used to test the ALP activity and total protein concentration, respectively. Ultimately, the results were computed and normalized to the total protein level.
BMSCs were cultivated on the different groups for 4 and 7 days to detect the osteogenesis-related gene expression level. Subsequently, the cells from each group were dissolved in Trizol reagent (Invitrogen, United States) and the total RNA was refined. Afterward, 1 μg of RNA was transcribed in reverse into cDNA by using a Prime-ScriptTM RT reagent kit (Takara Bio, Japan). Primers utilized in this research were synthesized by Sangon Biotech (Shanghai) Co., Ltd and are listed in
Primer sequences utilized for RT-PCR.
Gene | Primer sequences (F = forward; R = reverse) |
---|---|
BSP | F: 5′-AGAAAGAGCAGCACGGTTGAGT-3′ |
R: 5′-GACCCTCGTAGCCTTCATAGCC-3′ | |
OCN | F: 5′-GCCCTGACTGCATTCTGCCTCT-3′ |
R: 5′-TCACCACCTTACTGCCCTCCTG-3′ | |
COL-1 | F: 5′-GCCTCCCAGAACATCACCTA-3′ |
R: 5′-GCAGGGACTTCTTGAGGTTG-3′ | |
OPN | F: 5′-CCAAGCGTGGAAACACACAGCC-3′ |
R: 5′-GGCTTTGGAACTCGCCTGACTG-3′ | |
β-actin | F: 5′-GTAAAGACCTCTATGCCAACA-3′ |
R: 5′-GGACTCATCGTACTCCTGCT-3′ |
In order to evaluate the osseointegration performance of the two kinds of implants, 10 SD rats weighing approximately 280 g were used in the trial. All the animal assays were authorized and performed according to the instructions of the Institutional Animal Care and Use Committee of Tongji University (Shanghai, China). The rat femoral condyle model was used in the current study and the surgical procedures were demonstrated previously (
Hard-tissue slicing was utilized for histological and histomorphometric observation and analysis. Briefly, the harvested samples were fixed in 4% PFA for 24 h. Then they were washed in distilled deionized water and then dehydrated in grade ethanol series from 70 to 100%. Subsequently, the samples were embedded in a light-curing one-component resin (Technovit 7200VLC, kulzers, Friedrichsdorf, Germany). After 15 h, the polymerized samples were cut longitudinally into 200-μm-thick sections with a diamond circular saw system (Exakt 300 CL, Exakt Apparatebau, Germany), and then ground and polished to around 30 μm thickness using a grinding system (Exakt 400 CS, Exakt Apparatbau, Germany).
After being stained by Van Gieson’s picrofuchsine, the sections were observed and photographed by a microscope (Olympus, Japan). And Image-Pro Plus 6.0 software was used to perform histomorphometric analysis and compute the bone-implant contact (BIC) percentage in the cancellous bone. BIC was calculated as the percentage of the length of direct contact to the total length of the implant surface.
Using SPSS 22.0 statistical software, all the data were demonstrated as means ± standard deviation (SD). Independent
SEM images of 3D
Surface free energy (SFE) dominates the interplay between the surface, aqueous biological environment, and proteins. What is more, SFE can be reflected by water contact angle (WCA) measurements. In general, based on a WCA >90° or <90°, a surface could be classified as hydrophobic (low SFE) or hydrophilic (high SFE). And at WCA >150° or <5°, the surface could be further defined as super-hydrophilic and super-hydrophobic (
The SEM results show the effect of the PDA-coated surface on the adhesion of BMSCs after seeding for 24 h (
SEM images of adherent cells on the 3D
The proliferation of BMSCs cultured on 3D and 3D-PDA samples were detected by the CCK-8 test (
Propagation of BMSCs on the 3D and 3D-PDA samples by CCK-8 test after seeding for 1, 4, and 7 days
After being cultivated for 4 and 7 days, the ALP activity of BMSCs cultured on the two kinds of specimens was evaluated (
The
Histological images of hard-tissue slicing of 3D
Taking the
It is essential to improve surface characteristics, promoting the biocompatibility and osseointegration performance of personalized 3D-printed dental implants. In the present research, the 3D-printed Ti6Al4V implant surface was successfully modified with a PDA coating, which apparently increased the hydrophilicity. Furthermore, the BMSCs adhesion, propagation, and osteogenic differentiation
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of Tongji University (Shanghai, China).
HW performed the experiment and wrote the original draft. CY helped to prepare the manuscript and gave some conceptualization on this manuscript. KL, RZ and SZ led the conceptualization and project administration, and supervised the writing and editing of the manuscript. All authors contributed to the article and approved the submitted version.
The 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.
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.
The authors acknowledge the support provided by the National Natural Science Foundation of China (82072396, 81871490), the Science and Technology Commission of Shanghai Municipality (21490711700), the Interdisciplinary Program of Shanghai Jiao Tong University (YG2021ZD12), the Program of Shanghai Academic/Technology Research Leader (19XD1434500), the Open Project of State Key Laboratory of Oral Diseases (SKLOD2021OF01), and Two-Hundred Talent (20191819).