Synthesis of Silica-Based Solid-Acid Catalyst Material as a Potential Osteochondral Repair Model In Vitro

For osteochondral damage, the pH value change of the damaged site will influence the repair efficacy of the patient. For better understanding the mechanism of the acid-base effect, the construction of in vitro model is undoubtedly a simple and interesting work to evaluate the influence. Here, a novel porous silica-based solid-acid catalyst material was prepared by additive manufacturing technology, exhibiting improved eliminating effects of the residue. SEM, FTIR, and TGA were used to characterize the morphology, structure, and thermal stability of the synthesized 3D material. The reaction between 4-methoxybenzyl alcohol and 3, 4-dihydro-2H-pyran was used as a template reaction to evaluate the eliminating performance of the 3D porous material. Solvents were optimized, and three reaction groups in the presence of 3D SiO2, 3D SiO2-SO3H, and 3D SiO2-NH-SO3H, as well as one without catalyst, were compared. In addition, in consideration of the complicated situation of the physiological environment in vivo, universality of the synthesized 3D SiO2-NH-SO3H catalyst material was studied with different alcohols. The results showed that the sulfonic acid-grafted 3D material had excellent catalytic performance, achieving a yield over 95% in only 20 min. Besides, the catalyst material can be recycled at least 10 times, with yields still higher than 90%. Such a solid catalyst material is expected to have great potential in additive manufacturing because the catalyst material is easy-recyclable, renewable and biocompatible. The 3D material with connective channels may also be utilized as an in vitro model for environment evaluation of osteochondral repair in the future.


INTRODUCTION
For decades, many people suffer from various bone and cartilage damages such as arthritis. To treat these diseases, tissue engineering provides a promising and alternative method in orthopedic surgery and biomedical engineering (Wei et al., 2021a). For example, Wei et al. fabricated a 3D printed polycaprolactone scaffold modified by insulin-releasing PLGA nanoparticles for osteochondral repair, and the results indicate that both of the in vitro and in vivo studies show that the insulin-releasing scaffolds significantly improve the osteogenic differentiation of rabbit bone mesenchymal stem cells and the proliferation of chondrocytes, and further improve the repair of cartilage and subchondral bone after 8-and 12-weeks implantation in rabbit osteochondral defects (Wei et al., 2021b). However, several factors have great influence on tissue regeneration, for example, such as the degradation of the biomaterials will result in the acid-base concentration change at the treatment site Xue et al., 2021), and the enrichment and diffusion of calcium ions could promote neovascularization and repair of the critical defect (Xing et al., 2021). Thus, to understand the influence of these factors is an important issue for bone and cartilage repair.
In vitro model provides an indirect and convenient platform to assess the repair effect and scrutinize different molecular and possible therapeutic targets for better understanding the mechanisms and osteoarthritis therapeutics (Hopkins et al., 2021;Singh et al., 2021). Consequently, The aim of this work is to establish an in vitro model to evaluate the effect of acidbase by preparing a novel porous silica-based solid-acid catalyst material using additive manufacturing technology which may exhibit improved eliminating effects. To prepare the model, in the synthesis of some complex compounds, molecule intermediates often carry multiple functional groups, and sometimes, these functional groups react with other reactants before the intended product(s) can be formed. Therefore, protecting some functional groups of the molecule from reaction is necessary, while leaving other functional groups free to participate in the reaction (Rajkumari et al., 2017). One important example is the protection of hydroxyl groups. Until now, many ways have been discovered to achieve alcohol protection. Among these methods, ether-forming protection of hydroxyl groups (reaction of an alcohol with 3,4-dihydro-2H-pyran) is considered the most popular and convenient solution (Kumar et al., 2014;Khder et al., 2018). This is because, first, the protective agent is easier to prepare than other protective agents. Further, the corresponding ether pair reaction conditions and some reagents used, such as the format reagent, hydride, strong alkali, and other organometallic reagents, have good stability (Azzena et al., 2018). Lastly, many kinds of catalysts have been reported for tetrahydropyranylation of alcohols, including ionic liquids (Rafael et al., 2001), silica chloride (Ravindranath et al., 2001), Al(OTf) 3 (Williams et al., 2010), silica sulfonic acid (Shimizu et al., 2004), and modified zeolites (Narender et al., 2010;Shin et al., 2017). However, many of them have disadvantages, such as low efficiency, instability, harsh reaction conditions, and an inability to be recycle. Preparing catalysts free from these disadvantages is an important research direction (Nakhaei et al., 2019;Sajjadifar et al., 2019). According to previous work, advanced continuous porous structures help control and increase the distribution of catalytically active components, enhancing catalytic performance (Stuecker et al., 2004;Ludwig et al., 2018). However, traditional porous materials are obtained by a template method and vapor deposition, as well as others, and suffer from long preparation cycles and complicated steps (Xintong and Chang-jun, 2017). Additive manufacturing technology, also called 3D printing, is a powerful tool for designing advanced structures (Wang et al., 2020). Silica is preferred as a carrier for its stability in most solvents, and Niknam and coworkers (Niknam et al., 2010) successfully prepared silica-bonded S-sulfonic acid (SBSSA) and employed it as a recyclable catalyst for the condensation reaction of aromatic aldehydes with 3-methyl-l-phenyl-5-pyrazolone. With continued catalyst development, Pourghasemi-Lati and colleagues (Pourghasemi-Lati et al., 2018) immobilized butane-1-sulfonic acid on magnetic Fe 3 O 4 @SiO 2 nanoparticles for the synthesis of 5-arylidine barbituric acids and pyrano [2,3-d] pyrimidine derivatives. In addition, silica-based biomaterials have been widely studied in bone and cartilage tissue engineering due to high mechanical stiffness, high biocompatibility, and special bioactivities including stimulating osteogenesis and angiogenesis by enhancing corresponding gene expressions (Li et al., 2000;Du et al., 2015;Ishikawa et al., 2018). For example, silica based inorganic/organic hybrids could promote collagen Type II, Sox9 and Aggrecan production from chondrocytes (Nelson et al., 2021) or have mechanically strong and elastic characteristics (Yu et al., 2021).
Here, a new silica carrier is described, fabricated by 3D printing and combined with advanced structural perspectives. Further, chemical modifications were carried out, introducing many sulfonic acid groups, and the catalyst material was applied to hydroxyl protection reactions as model test. In addition, in consideration of the complicated situation of the physiological environment in vivo, universality of the synthesized material was studied. The new material is anticipated to be utilized as an in vitro model for environment evaluation of osteochondral repair in the future.

Preparation of Porous SiO 2
SiO 2 powder was first dispersed in ethanol and, then, loaded in a syringe and installed on the 3D printer. Next, the porous silica structure was printed under a pressure of 0.5 MPa and speed of Frontiers in Bioengineering and Biotechnology | www.frontiersin.org December 2021 | Volume 9 | Article 790139 6 mm/s. Porous SiO 2 was obtained via a sintering process at 1,500°C. In the following research, two kinds of porous silicabased solid-acid catalysts were prepared: 3D SiO 2 -SO 3 H (catalyst 1) and 3D SiO 2 -NH-SO 3 H (catalyst 2).

Preparation of Porous Silica-Based Solid-Acid Catalyst 1
Three-dimensional SiO 2 was first activated by 15 ml of 30% H 2 O 2 under 150°C to increase the number of hydroxyl groups. After drying, three pieces of 3D SiO 2 were mixed with chlorosulfonic acid (0.5 ml) and reacted in CHCl 3 (8 ml) at 0°C for 4 h. After drying in the vacuum drying oven, catalyst 1 (3D SiO 2 -SO 3 H, Scheme 1) was obtained.

Preparation of Porous Silica-Based Solid-Acid Catalyst 2
A schematic diagram of the preparation of 3D SiO 2 -NH-SO 3 H is presented in Figure 1. Briefly, the carrier was first activated by 15 ml of 30% H 2 O 2 at 150°C to increase the number of hydroxyl groups (Díaz-Marta et al., 2018). Then, (3chloropropyl)triethoxysilane (0.5 ml) was used to modify the surface of porous SiO 2 , yielding the chlorine-modified porous carrier (3D SiO 2 -Cl, Scheme 2 in Figure 1). To obtain more sulfonic acid groups, 3D SiO 2 -Cl (three pieces) were put in a round-bottomed flask with diethylenetriamine (5 mmol), triethylamine (5.5 mmol), and DMF (10 ml) and reacted at 100°C for 12 h. After drying, the intermediate product, with double terminal amino groups, was obtained (3D SiO 2 -NH 2 , Scheme 3 in Figure 1). Finally, three pieces of 3D SiO 2 -NH 2 were mixed with chlorosulfonic acid (0.5 ml) in CHCl 3 (8 ml) for 4 h at 0°C, and then, catalyst 2 (3D SiO 2 -NH-SO 3 H, Scheme 4 in Figure 1) was obtained via drying in the vacuum drying oven.

Catalytic Reaction of the 3D Porous Catalyst
Catalytic activity was investigated via the reaction between 4methoxybenzyl alcohol (5 mmol) and 3, 4-dihydro-2H-pyran (5.5 mmol). To begin, different solvents (10 ml) were tested, to determine the most efficient one. Further, the material, obtained via direct sulfonation of activated SiO 2 (3D SiO 2 -SO 3 H), was compared with 3D SiO 2 -NH-SO 3 H. Under optimized solvent conditions in the presence of 3D SiO 2 -NH-SO 3 H, different alcohols were reacted with 3, 4-dihydro-2H-pyran, and product yields and corresponding reaction times were recorded.

Physiochemical Characterization
Scanning electron microscopy (SEM; Phenom Pro, Phenom-World Co., Ltd. Eindhoven, Netherlands) was used to observe the material micro-surface. Fourier transform infrared spectroscopy (FTIR; NICOLET IS 50, Thermo Fisher Scientific Co., Ltd. Massachusetts, United States) was utilized to determine whether the sulfonic acid group successfully grafted onto SiO 2 . Thermogravimetric analysis (TGA; TGA-50, Shimadzu Co., Ltd. Kyoto, Japan) was also used to confirm the molecules were grafted on 3D SiO 2 . Catalytic reaction products were characterized by FTIR and 1 H NMR spectroscopy (NMR; Advance III HD 500MHz, Bruker Technology Co., Ltd. New York, United States).

Cytotoxicity Test
C518 cells were seeded in a 96-well plate at a density of 5 × 10 3 cells per well and cultured for 24 h at 37°C. Then, the medium was removed, and cells were incubated with different concentrations of sample leaching solution (12.5, 25, 50, 100, and 200 μg/ml). Cell

RESULTS AND DISCUSSION
Three-dimensional SiO 2 was observed, together with fresh 3D SiO 2 -NH-SO 3 H, and 3D SiO 2 -NH-SO 3 H after one catalytic cycle in Figure 2. Figures 2A,D,G show optical images of asprepared 3D SiO 2 , fresh 3D SiO 2 -NH-SO 3 H, and 3D SiO 2 -NH-SO 3 H which catalysed the reaction of 1-octanol with 3, 4dihydro-2H-pyran. Compared with the white colour of the treated, blank 3D SiO 2 , the surface of 3D SiO 2 -NH-SO 3 H was light yellow with some black dots, which were used for catalysing the reaction. The scanning electron microscope (SEM) images of as-prepared 3D SiO 2 ( Figures 2B,C), fresh 3D SiO 2 -NH-SO 3 H ( Figures 2E,F), and 3D SiO 2 -NH-SO 3 H after one catalytic cycle ( Figures 2H,I) showed obvious differences. The micro-surface of the carrier and catalyst showed consequent holes, beneficial for introducing more sulfonic acid groups. Furthermore, grey parts existed on the smooth carrier surface, which could be a series of grafted molecules and sulfonic acid groups. The presence of sulfonic acid groups on the surface of 3D porous silicon was confirmed by FTIR (Figure 3). The broad peak near 3,490 cm −1 was assigned to N-H stretching vibrations, while the peaks at 2,936 and 2,873 cm −1 were attributed to C-H stretching vibrations. The peak at 1,084 cm −1 arises from the stretching vibration of Si-O-Si, but due to the overlap of S O and Si-O-Si characteristic peaks around 1,100 cm −1 , the half-peak at 1,118 cm −1 was considered to be the stretching vibration peak of S O (Pourghasemi-Lati et al., 2018). In the low wavenumber region, some characteristic sulfonic acid peaks also overlapped with SiO 2 , and the peaks at 617 and 616 cm −1 could be stretching vibrations of S-O. TGA was also completed to determine the stability of the solid-acid catalyst material (Figure 4). The curve indicated that weight loss from room temperature to 100°C was a rapid, decreasing process, which may be mainly water evaporation. As the temperature continued to rise, no plateau was seen, which may indicate the material began to decompose. At about 600°C, the grafted molecules were completely decomposed.
To optimize reaction conditions for the synthesized material, we used the reaction of 4-methoxybenzyl alcohol and 3, 4dihydro-2H-pyran as a template and explored the effect of different solvents on the catalytic yield in the presence of catalyst material (Table 1). In all solvents examined, methylene chloride showed the highest yield of 96% after 20 min. Furthermore, most of the non-polar solvents were promoting and exhibited better catalytic effects.
Frontiers in Bioengineering and Biotechnology | www.frontiersin.org December 2021 | Volume 9 | Article 790139 6 experiments. All reaction products were detected by thin-layer chromatography (TLC) per 5 min. Table 2 shows that the reaction either did not take place or was minimal in the absence of catalyst material (Entry 3) or in the presence of 3D SiO 2 (Entry 4), respectively. However, high reaction yields were obtained in 20 min with both porous catalysts (Entries 1 and 2). Of the two materials, 3D SiO 2 -NH-SO 3 H exhibited a better catalytic effect, with a 96% yield, which may be due to the presence of more sulfonic acid groups compared to 3D SiO 2 -SO 3 H ( Table 2).
To gain a deeper understanding of the materials' effect in protecting a series of alcohols, various alcohols were reacted with 3, 4-dihydro-2H-pyran in the presence of 3D SiO 2 -NH-SO 3 H. Catalytic reaction products were characterized by FTIR and 1H NMR spectroscopy (see Supplementary  Material). The experiment showed that the porous silica-based solid-acid material had broad catalytic effects; many alcohol reactions were catalysed with high yields of 89% or more, with 1-hexadecanol (Entry 5) achieving a 98% yield (Table 3).
Lastly, the recyclability of 3D SiO 2 -NH-SO 3 H was investigated by the model reaction of 4-methoxybenzyl alcohol with 3, 4dihydro-2H-pyran ( Figure 5), demonstrating that the material could be used at least 10 times, while still achieving high catalytic yields. Other research also focuses on the reusable behaviour of SiO 2 catalyst (Kamble et al., 2021).
Meantime, in consideration of the new material is anticipated to be utilized as an in vitro model for osteochondral repair in the future work, the cytobiocompatibility is important because for the future evaluation, the cells must be seeded into the 3D scaffold. Thus the cytotoxicity test was performed using C518 cells as osteochondral model cell. As can be seen in Figure 6, both 3D SiO 2 and 3D catalyst exhibit excellent cell viability at different concentrations at 24 h, while at 48 h, the material shows limited cytotoxicity (<80%) only at high concentration (200 μg/ml), which indicates that the material is suitable for model construction in vitro to evaluate the potential influence of different factors.

CONCLUSION
Some achievements have been obtained in bone and cartilage repair including clinical trial and applications when using 3D printing as an efficient tool to control the accurate microstructure of the scaffold in tissue engineering. However, the cure rate still remains a problem due to the complicated situation of the in vivo physiological environment, especially the microenvironment surrounding the damaged site. To study the influence of the microenvironment, a catalytic strategy was proposed to eliminate the residue of the scaffold degradation segment. In addition, an in vitro model will provide a simple and  practical platform to evaluate these changes. Therefore, a porous silica-based solid-acid catalyst material was successfully prepared by additive manufacturing technology and the results showed excellent catalytic performance. The catalytic strategy by constructing an in vitro model supplies an alternative way in environment evaluation of osteochondral repair and it also shows potential for the enzymatic catalytic research during the body's metabolism process in the future studies.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

AUTHOR CONTRIBUTIONS
All authors contributed significantly to this work. YL, RL and SW conceptualized the system and designed the studies. YJ performed the experiments with assistance from JG, GR, XG, XX, YW, RY and ZL. RL, JG and GR performed the experiments for the revision. YJ wrote the draft and finished the manuscript with assistance from YL for revision. RL wrote the revision.

FUNDING
Financial support from the National Natural Science Foundation of China (32171337) and National Marine Economic Innovation and Development Project (16PYY007SF17) is gratefully acknowledged. This research is also supported by the Program for Innovative Research Team in Science and Technology at Fujian Province University. We also thank Instrumental Analysis Center of Huaqiao University for the support of the work.