I. INTRODUCTION

Section:

ChooseTop of pageABSTRACTI. INTRODUCTION <<II. RESULTSIII. DISCUSSION AND CONCL...IV. METHODSSUPPLEMENTARY MATERIALBone marrow mesenchymal stem cells (BMSCs) are among the most promising and commonly used elements in tissue engineering and regenerative medicine due to their ability to differentiate into a variety of cell types, including osteoblasts, chondrocytes, and adipocytes.1–51. M. F. Pittenger, A. M. Mackay, S. C. Beck, R. K. Jaiswal, R. Douglas, J. D. Mosca et al., “ Multilineage potential of adult human mesenchymal stem cells,” Science 284(5411), 143–147 (1999). https://doi.org/10.1126/science.284.5411.1432. A. I. Caplan, “ Review: Esenchymal stem cells: Cell-based reconstructive therapy in orthopedics,” Tissue Eng. 11(7–8), 1198–1211 (2005). https://doi.org/10.1089/ten.2005.11.11983. Y. Jiang, B. N. Jahagirdar, R. L. Reinhardt, R. E. Schwartz, C. D. Keene, X. R. Ortiz-Gonzalez et al., “ Pluripotency of mesenchymal stem cells derived from adult marrow,” Nature 418(6893), 41–49 (2002). https://doi.org/10.1038/nature008704. Z. Huang, P. G. Ren, T. Ma, R. L. Smith, and S. B. Goodman, “ Modulating osteogenesis of mesenchymal stem cells by modifying growth factor availability,” Cytokine 51(3), 305–310 (2010). https://doi.org/10.1016/j.cyto.2010.06.0025. T. R. Brazelton, F. M. Rossi, G. I. Keshet, and H. M. Blau, “ From marrow to brain: Expression of neuronal phenotypes in adult mice,” Science 290(5497), 1775–1779 (2000). https://doi.org/10.1126/science.290.5497.1775 By combining BMSCs with biomaterials such as hydrogels and implementing appropriate MSC-based tissue engineering methods, we can repair and rebuild damaged tissues more efficiently.6,76. E. M. Horwitz, D. J. Prockop, L. A. Fitzpatrick, W. W. K. Koo, P. L. Gordon, M. Neel et al., “ Transplantability and therapeutic effects of bone marrow-derived mesenchymal cells in children with osteogenesis imperfecta,” Nat. Med. 5(3), 309–313 (1999). https://doi.org/10.1038/65297. H. Lin, J. Sohn, H. Shen, M. T. Langhans, and R. S. Tuan, “ Bone marrow mesenchymal stem cells: Aging and tissue engineering applications to enhance bone healing,” Biomaterials 203, 96–110 (2019). https://doi.org/10.1016/j.biomaterials.2018.06.026 However, we can only effectively promote the formation of target tissue types by mastering the regulation of MSC differentiation in biomaterials to target phenotypes only.Recent studies have shown that MSCs are highly sensitive to their surrounding microenvironment, including matrix surface morphology, viscoelasticity, and anisotropy.8–118. A. M. Dulak, P. Stojanov, S. Peng, M. S. Lawrence, C. Fox, C. Stewart et al., “ Exome and whole-genome sequencing of esophageal adenocarcinoma identifies recurrent driver events and mutational complexity,” Nat. Genet. 45(5), 478–486 (2013). https://doi.org/10.1038/ng.25919. A. J. Engler, S. Sen, H. L. Sweeney, and D. E. Discher, “ Matrix elasticity directs stem cell lineage specification,” Cell 126(4), 677–689 (2006). https://doi.org/10.1016/j.cell.2006.06.04410. J. Fiedler, B. Ozdemir, J. Bartholomä, A. Plettl, R. E. Brenner, and P. Ziemann, “ The effect of substrate surface nanotopography on the behavior of multipotnent mesenchymal stromal cells and osteoblasts,” Biomaterials 34(35), 8851–8859 (2013). https://doi.org/10.1016/j.biomaterials.2013.08.01011. R. M. Salasznyk, W. A. Williams, A. Boskey, A. Batorsky, and G. E. Plopper, “ Adhesion to vitronectin and collagen I promotes osteogenic differentiation of human mesenchymal stem cells,” J. Biomed. Biotechnol. 2004(1), 24–34. https://doi.org/10.1155/S1110724304306017 In particular, the effect of matrix stiffness on MSC proliferation and phenotypic differentiation has attracted increasing attention.12–1412. A. Halim, A. D. Ariyanti, Q. Luo, and G. Song, “ Recent progress in engineering mesenchymal stem cell differentiation,” Stem Cell Rev. Rep. 16(4), 661–674 (2020). https://doi.org/10.1007/s12015-020-09979-413. A. J. Steward and D. J. Kelly, “ Mechanical regulation of mesenchymal stem cell differentiation,” J. Anat. 227(6), 717–731 (2015). https://doi.org/10.1111/joa.1224314. C. Huang, J. Dai, and X. A. Zhang, “ Environmental physical cues determine the lineage specification of mesenchymal stem cells,” Biochim. Biophys. Acta 1850(6), 1261–1266 (2015). https://doi.org/10.1016/j.bbagen.2015.02.011 Studies have shown that a stiff matrix promotes the osteogenic differentiation of MSCs, while a soft matrix promotes their differentiation to neurons or adipocytes.15–1815. A. S. Rowlands, P. A. George, and J. J. Cooper-White, “ Directing osteogenic and myogenic differentiation of MSCs: Interplay of stiffness and adhesive ligand presentation,” Am. J. Physiol. Cell Physiol. 295(4), C1037–C1044 (2008). https://doi.org/10.1152/ajpcell.67.200816. J. P. Winer, P. A. Janmey, M. E. McCormick, and M. Funaki, “ Bone marrow-derived human mesenchymal stem cells become quiescent on soft substrates but remain responsive to chemical or mechanical stimuli,” Tissue Eng., Part A 15(1), 147–154 (2009). https://doi.org/10.1089/ten.tea.2007.038817. R. McBeath, D. M. Pirone, C. M. Nelson, K. Bhadriraju, and C. S. Chen, “ Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment,” Dev. Cell 6(4), 483–495 (2004). https://doi.org/10.1016/S1534-5807(04)00075-918. J. S. Park, J. S. Chu, A. D. Tsou, R. Diop, Z. Tang, A. Wang et al., “ The effect of matrix stiffness on the differentiation of mesenchymal stem cells in response to TGF-β,” Biomaterials 32(16), 3921–3930 (2011). https://doi.org/10.1016/j.biomaterials.2011.02.019 These results suggest that biomaterial properties significantly affect the differentiation of MSCs in tissue engineering materials.Mcbeath et al.1717. R. McBeath, D. M. Pirone, C. M. Nelson, K. Bhadriraju, and C. S. Chen, “ Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment,” Dev. Cell 6(4), 483–495 (2004). https://doi.org/10.1016/S1534-5807(04)00075-9 first reported the effect of mechanical cues on MCS differentiation. They demonstrated that MSCs that adhered, spread, and expanded showed osteogenic differentiation, while cells that were not fully spread and appeared round were more likely to differentiate into adipocytes. This cellular phenotype switch is mediated by a change in RhoA activity.The regulation of mechanical cues serves as an effective method to guide the directional differentiation of MSCs.19,2019. D. R. Critchley, “ Focal adhesions: The cytoskeletal connection,” Curr. Opin. Cell Biol. 12(1), 133–139 (2000). https://doi.org/10.1016/S0955-0674(99)00067-820. Y. R. Shih, K. F. Tseng, H. Y. Lai, C. H. Lin, and O. K. Lee, “ Matrix stiffness regulation of integrin-mediated mechanotransduction during osteogenic differentiation of human mesenchymal stem cells,” J. Bone Miner. Res. 26(4), 730–738 (2011). https://doi.org/10.1002/jbmr.278 However, traditional regulatory methods, such as the use of small-molecule inhibitors or growth factors, lack stability and specificity to target cells, so their translational applications in clinical development are relatively limited. Therefore, the use of microRNA is a promising method of regulating mechanotransduction. miRNAs are a class of short noncoding RNA molecules that negatively regulate the expression of genes by inducing translational repression and mRNA degradation.21,2221. D. P. Bartel, “ MicroRNAs: Target recognition and regulatory functions,” Cell 136(2), 215–233 (2009). https://doi.org/10.1016/j.cell.2009.01.00222. K. N. Ivey and D. Srivastava, “ microRNAs as developmental regulators,” Cold Spring Harbor Perspect. Biol. 7(7), a008144 (2015). https://doi.org/10.1101/cshperspect.a008144 Regulating cellular activity with miRNA is relatively simple and has relatively high regulation efficiency and low cost. miRNAs have been shown to play an important role in the proliferation and phenotypic differentiation of MSCs.23–2723. T. Eguchi, K. Watanabe, E. S. Hara, M. Ono, T. Kuboki, and S. K. Calderwood, “ OstemiR: A novel panel of microRNA biomarkers in osteoblastic and osteocytic differentiation from mesencymal stem cells,” PLoS One 8(3), e58796 (2013). https://doi.org/10.1371/journal.pone.005879624. S. Bork, P. Horn, M. Castoldi, I. Hellwig, A. D. Ho, and W. Wagner, “ Adipogenic differentiation of human mesenchymal stromal cells is down-regulated by microRNA-369-5p and up-regulated by microRNA-371,” J. Cell. Physiol. 226(9), 2226–2234 (2011). https://doi.org/10.1002/jcp.2255725. D. Guérit, D. Philipot, P. Chuchana, K. Toupet, J. M. Brondello, M. Mathieu et al., “ Sox9-regulated miRNA-574-3p inhibits chondrogenic differentiation of mesenchymal stem cells,” PLoS One 8(4), e62582 (2013). https://doi.org/10.1371/journal.pone.006258226. A. Schoolmeesters, T. Eklund, D. Leake, A. Vermeulen, Q. Smith, S. Force Aldred et al., “ Functional profiling reveals critical role for miRNA in differentiation of human mesenchymal stem cells,” PLoS One 4(5), e5605 (2009). https://doi.org/10.1371/journal.pone.000560527. T. Eskildsen, H. Taipaleenmäki, J. Stenvang, B. M. Abdallah, N. Ditzel, A. Y. Nossent et al., “ MicroRNA-138 regulates osteogenic differentiation of human stromal (mesenchymal) stem cells in vivo,” Proc. Natl. Acad. Sci. U. S. A. 108(15), 6139–6144 (2011). https://doi.org/10.1073/pnas.1016758108 In addition, studies have found that miRNAs regulate the interfacial behavior of MSCs cultured on microgrooved substrates. Through miRNA sequencing, Wang et al.2828. Y. Wang, X. L. Jiang, S. C. Yang, X. Lin, Y. He, C. Yan et al., “ MicroRNAs in the regulation of interfacial behaviors of MSCs cultured on microgrooved surface pattern,” Biomaterials 32(35), 9207–9217 (2011). https://doi.org/10.1016/j.biomaterials.2011.08.058 found that the expression levels of miR-140, miR-210, miR-214, miR-351, and miR-99b were significantly increased in MSCs cultured on microgrooved substrates compared with MSCs cultured on smooth substrates. Several of these miRNA mimic combinations were transfected into MSCs cultured on smooth substrates, significantly increasing osteogenesis-related genes' expression. This suggests that miRNAs may play an important role in mechanosignaling and MSC differentiation. However, it is unclear whether these miRNAs are regulated by matrix stiffness; their possible regulatory mechanism is also unknown.

In this study, we found that the expression of miR-99b increased with increasing hydrogel matrix stiffness. In addition to affecting the proliferation and migration of BMSCs, the regulation of miR-99b expression could also affect the osteogenic or adipogenic differentiation of BMSCs by targeting the mTOR signaling pathway. Finally, we demonstrated that increased expression of miR-99b promotes the osteogenic differentiation of BMSCs in soft 3D hydrogels and promotes bone regeneration in rat calvarial defect areas. These results demonstrate that miR-99b plays a key role in the mechanotransduction and phenotypic transformation of BMSCs, suggesting its value in MSC-based tissue engineering applications.

II. RESULTS

Section:

ChooseTop of pageABSTRACTI. INTRODUCTIONII. RESULTS <<III. DISCUSSION AND CONCL...IV. METHODSSUPPLEMENTARY MATERIAL

A. Substrate stiffness affects the morphology and phenotypic differentiation of BMSCs

We prepared three groups of polyacrylamide (PA) gels ranging from soft to hard with Young's moduli of approximately 0.8, 35, and 80 kPa and applied 25 μg/ml collagen I to the surface of the gels by cross-linking [Fig. 1(a)]. Actin fibers were stained with phalloidin after seeding BMSCs on gel surfaces of different stiffnesses for 24 h. The morphology of BMSCs showed marked differences, with BMSCs on relatively hard substrates (35 and 80 kPa) spread more widely with thick and aligned actin fibers, while BMSCs on soft substrates had a smaller spread area and randomly oriented actin fibers [Fig. 1(b)]. After measurement, it was found that the average areas of actin fibers and nuclei were 1339.25 and 150.65 μm2 on the surface of the soft gel (0.8 kPa), respectively, and 5377.50 and 219.75 μm2 on the surface of the hard gel (80 kPa) [Figs. 1(c) and 1(d)].To clarify the effects of soft and hard matrices on the osteogenic and adipogenic differentiation of BMSCs, we used a mixture of osteogenic and adipogenic induction medium (1:1) to culture cells for 15 days and found that BMSCs on hard matrices mainly differentiated into osteoblasts. However, with decreasing matrix stiffness, BMSCs gradually tended toward adipogenic differentiation [Figs. 1(e) and 1(f)].

B. miR-99b expression in response to changes in gel substrate stiffness

miRNAs have been shown to play a key role in the regulation of MSC fate, including proliferation, migration, and phenotypic differentiation. More interestingly, some miRNAs were found to regulate the interface behavior of MSCs cultured on microgrooved substrates. However, whether these miRNAs are regulated by matrix stiffness and their possible regulatory mechanisms are still unknown.

Therefore, we cultured BMSCs on gel substrates with elastic moduli of 80, 35, and 0.8 kPa for 24 h and detected the levels of miR-140, miR-210, miR-214, miR-351, and miR-99b by qPCR [Figs. 2(a)–2(e)]. The results showed that the expression levels of miR-99b and miR-140 gradually decreased with decreasing gel substrate stiffness [Figs. 2(a) and 2(b)]. Furthermore, we extended the culture time of the cells to 7 days and found that the above-mentioned miRNA expression trends were largely maintained, and differences were more significant [Figs. 2(a) and 2(b)].

C. Regulation of miR-99b can alter the phenotypic differentiation bias of BMSCs

Given the significant differences in the expression of miR-99b and miR-140 in response to changes in gel substrate stiffness, we next continued to investigate the effects of the regulation of these differentially expressed miRNAs on the osteogenic and adipogenic differentiation bias of BMSCs. BMSCs were seeded on the surface of both soft and hard gels, and a mixed induction medium was used. Then, cells were transfected with miRNA mimics or inhibitors, and the deviation of osteogenic and adipogenic differentiation of cells was detected after 15 days of culture.

On gels with matrix stiffnesses of 80 and 0.8 kPa, we found no consistent effect of miR-210 on the differentiation bias of BMSCs. However, miR-99b mimics significantly increased the proportion of osteoblasts compared with blank control or control oligonucleotide-treated cells [Figs. 3(a) and 3(c)], and inhibition of miR-99b expression had the opposite effect on the differentiation bias of BMSCs [Figs. 3(b) and 3(d)].

D. Regulation of miR-99b can affect BMSC proliferation and migration in response to matrix stiffness

By performing the CCK-8 assay, we found that a low stiffness gel matrix inhibited the proliferation of BMSCs while increasing the expression of miR-99b in BMSCs could significantly promote cell proliferation [Fig. 4(a)]. Western blot results showed that the overexpression of miR-99b significantly increased the expression of PCNA and cyclin D1, which are crucial regulators of G1 to S phase progression in many cell types [Figs. 4(b)–4(d)]. Consistently, by cell cycle analysis, we found that Ad-miR-99b-sponge promoted the accumulation of cells in the G0/G1 phase and a lower proportion of cells in the S phase [Fig. 4(e)].We then used transwell analysis to investigate whether miR-99b could mediate the regulation of matrix stiffness on BMSC migration under 3D conditions. First, BMSCs were transfected with Ad-control, Ad-miR-99b, or Ad-miR-99b-sponge, the BMSC-encapsulated hydrogels with different stiffnesses were cut into disks, and the gel disks were placed in transwells [Fig. 4(f)]. BMSC migration was induced by FBS in the lower chamber. Cell staining and counting showed that compared with the results in the high-stiffness gel matrix, the migration ability of cells in the low-stiffness gel matrix was significantly decreased, while overexpression of miR-99b could significantly promote the migration of BMSCs [Figs. 4(g) and 4(h)].

E. Matrix stiffness-sensitive miR-99b targets mTOR to regulate BMSC differentiation

Next, we verified that both the mRNA and protein expression levels of Runx2 and osteocalcin (OCN) in BMSCs transfected with Ad-pri-miR-99b were significantly increased. In contrast, Ad-miR-99b-sponge significantly decreased the expression of Runx2 and OCN [Figs. 5(c)–5(g)]. Furthermore, we found that increasing the expression of miR-99b significantly enhanced RhoA activity [Fig. 5(h) and 5(i)]. Studies have reported that the mTOR signaling pathway is involved in regulating the osteogenic or adipogenic differentiation of MSCs. We found that the expression of mTOR decreased with increasing matrix stiffness [Figs. 5(a) and 5(b)] and that increasing the expression of miR-99b significantly reduced the expression of mTOR mRNA and protein, while ad-miR-99b-sponge significantly increased the expression of mTOR mRNA and protein [Figs. 5(c)–5(e)]. Combined with TargetScan's algorithm to predict the binding site of the mTOR gene 3′-UTR and miR-99b, we preliminarily concluded that mTOR is a potential target of miR-99b [Fig. 5(j)].To experimentally validate mTOR as a miR-99b target gene, we first constructed luciferase reporters including the mTOR 3′-UTR harboring the wild-type or mutated miR-99b binding site, and then performed transfection experiments. Experimental data showed that the luciferase activity of the wild-type mTOR 3′-UTR luciferase reporter was markedly reduced with increasing doses of miR-99b mimic, whereas no significant changes were observed in the luciferase activity of the mutant constructs, suggesting that miR-99b directly downregulates mTOR by its 3′-UTR binding site [Fig. 5(k)].We then applied an inhibitor of mTOR, rapamycin, to BMSCs to determine whether it has a similar effect to miR-99b in targeting and downregulating mTOR expression. The results of phenotypic differentiation bias experiments on BMSCs showed that rapamycin had a similar effect to miR-99b mimics and significantly promoted the osteogenic differentiation of cells [Fig. 5(l)]. Western blotting further demonstrated that rapamycin inhibits the mTORC1 pathway and induces Akt phosphorylation [Fig. 5(m)]. These results are consistent with previous studies showing that MSCs from mTORC1 knockout mice tend to differentiate into osteogenic cells and that rapamycin promotes osteogenesis.Next, to further verify that mTOR signaling pathway transduction is regulated by miR-99b, BMSCs were transfected with Ad-miR-99b-sponge and treated with 100 nM rapamycin. The inhibition of miR-99b expression promoted the adipogenic differentiation bias of BMSCs, resulting in a significant decrease in the proportion of osteoblasts, which was consistent with our previous findings. However, the addition of 100 nM rapamycin significantly enhanced the osteogenic differentiation of BMSCs, and at the same time, no significant difference was observed between the groups treated with rapamycin, demonstrating that the inhibition of mTOR signaling is sufficient to overcome the cell phenotypic differentiation-biased differences induced by ad-miR-99b-sponge [Figs. 6(a) and 6(b)]. The above results further verify that miR-99b affects the differentiation fate of BMSCs by targeting mTOR.

F. Regulation of miR-99b expression can promote the differentiation of BMSCs in soft 3D hydrogels

We then verified whether modulation of miR-99b expression could affect the differentiation of BMSCs in soft 3D GelMA hydrogel structures (elastic modulus ∼3.2 kPa, supplementary material Fig. 1). Briefly, BMSCs were transfected with ad-pri-miR-99b, encapsulated in photocrosslinkable GelMA hydrogels and then cultured with osteogenic induction medium for 15 days.After 15 days of culture, through microscopy, we found that numerous BMSCs in the hydrogel agglomerated and turned black and granular [Fig. 7(a)]. Macroscopic observation showed that with the extension of the culture period, the hydrogel gradually became opaque, and white deposits were visible to the naked eye after the fixation of some of the hydrogel samples, which we deduced to be calcium salt deposits.Afterward, the samples of each group were observed by alizarin red staining, treated with cetyl pyridine chloride reagent, and quantitatively analyzed by means of a microplate reader. The results showed that compared with the control group, the amount of calcium salt deposition in the Ad-pri-miR-99b treatment group was significantly increased [Figs. 7(b) and 7(c)].

G. Ad-miR-99b-BMSC-GelMA enhances rat calvarial regeneration

Finally, we constructed a rat skull defect model to test the bone regeneration ability of the Ad-miR-99b-BMSC-GelMA flexible hydrogel scaffold implanted in vivo [Fig. 8(a)]. Micro-CT scanning and three-dimensional reconstruction imaging were performed on the sixth week after the operation. The reconstructed images showed that the new bone tissue in the Ad-miR-99b-BMSC-GelMA treatment group was significantly greater than that in the Ad-control-BMSC-GelMA control group and the blank control group [Figs. 8(b) and 8(c)]. Bone mineral density (BMD) and bone volume to total bone volume (BV/TV) were quantitatively calculated by CTAn software. The results showed that the bone volume fraction (BV/TV) and BMD values of the Ad-miR-99b-BMSC-GelMA treatment group were significantly higher than those of the control group [Figs. 8(d) and 8(e)]. In addition, we performed immunofluorescence staining for Runx2 in the area of the skull defect, and the results showed that the Ad-miR-99b-BMSC-GelMA-treated group expressed significantly more Runx2.

III. DISCUSSION AND CONCLUSION

Section:

ChooseTop of pageABSTRACTI. INTRODUCTIONII. RESULTSIII. DISCUSSION AND CONCL... <<IV. METHODSSUPPLEMENTARY MATERIALBone marrow-derived mesenchymal stem cells have always been an important source of seed cells for tissue repair and regenerative medicine due to their multiple differentiation potentials. Therefore, it is particularly important to control the differentiation of MSCs.29,3029. G. D. Kusuma, J. Carthew, R. Lim, and J. E. Frith, “ Effect of the microenvironment on mesenchymal stem cell paracrine signaling: Opportunities to engineer the therapeutic effect,” Stem Cells Dev. 26(9), 617–631 (2017). https://doi.org/10.1089/scd.2016.034930. M. Ullah, D. D. Liu, and A. S. Thakor, “ Mesenchymal stromal cell homing: Mechanisms and strategies for improvement,” iScience 15, 421–438 (2019). https://doi.org/10.1016/j.isci.2019.05.004 Studies have shown that the regulation of MSC differentiation is not only affected by biochemical factors but also closely related to the physical stimulation of their surrounding microenvironment. In particular, matrix stiffness has been widely shown to be an important factor affecting the proliferation and differentiation of MSCs, but here, we demonstrate for the first time that-mechanosignaling involving miR-99b plays an important role in regulating the response of rat BMSCs to the surrounding physical environment. Furthermore, regulating substrate stiffness-sensitive miR-99b can be used to guide the directed differentiation of BMSCs.

By miRNA sequencing, Wang et al. found that the expression levels of miR-140, miR-210, miR-214, miR-351, and miR-99b were significantly increased in MSCs cultured on microgrooved substrates compared with MSCs cultured on smooth substrates. Several of these miRNA mimic combinations were transfected into MSCs cultured on smooth substrates, and the expression of osteogenesis-related genes was significantly increased. However, they did not conduct in-depth research on the connection and mechanism of action. This attracted our interest, and we speculated that miRNAs might play an important role in mediating between mechanosignaling and MSC differentiation. Thus, we sought to further verify whether the above target miRNAs could affect MSC fate in response to changes in substrate stiffness.

We constructed three groups of hydrogel substrates with an increasing Young's modulus gradient and found by qRT-PCR that the expression of miR-99b increased with increasing hydrogel stiffness. To make substrates with different stiffnesses, we chose PA gels with good biocompatibility and controllable surface stiffness.9,20,31,329. A. J. Engler, S. Sen, H. L. Sweeney, and D. E. Discher, “ Matrix elasticity directs stem cell lineage specification,” Cell 126(4), 677–689 (2006). https://doi.org/10.1016/j.cell.2006.06.04420. Y. R. Shih, K. F. Tseng, H. Y. Lai, C. H. Lin, and O. K. Lee, “ Matrix stiffness regulation of integrin-mediated mechanotransduction during osteogenic differentiation of human mesenchymal stem cells,” J. Bone Miner. Res. 26(4), 730–738 (2011). https://doi.org/10.1002/jbmr.27831. J. R. Tse and A. J. Engler, “ Preparation of hydrogel substrates with tunable mechanical properties,” Curr. Protoc. Cell Biol. 47, 10.16.1 (2010). https://doi.org/10.1002/0471143030.cb1016s4732. B. Tian, X. Ding, Y. Song, W. Chen, J. Liang, L. Yang et al., “ Matrix stiffness regulates SMC functions via TGF-β signaling pathway,” Biomaterials 221, 119407 (2019). https://doi.org/10.1016/j.biomaterials.2019.119407 By adjusting the ratio of acrylamide and bis-acrylamide, the gel stiffness can be controlled to within several 100 Pa. The experiment had good repeatability up to 100 kPa and was able to simulate the stiffness of different tissues of the body. However, because the PA glue is prepared on the surface of the cover glass, the stiffness of the cover glass may have an impact on the surface stiffness of the PA glue. When the PA glue base is too thin, cells may pass through the PA glue base and sense the stiffness of the coverslip, affecting the experimental results. Therefore, to exclude this effect, the PA glue base must be thick enough to completely exclude the effect of the stiffness of the coverslip. Tian et al.3232. B. Tian, X. Ding, Y. Song, W. Chen, J. Liang, L. Yang et al., “ Matrix stiffness regulates SMC functions via TGF-β signaling pathway,” Biomaterials 221, 119407 (2019). https://doi.org/10.1016/j.biomaterials.2019.119407 found that the differences in gene expression in smooth muscle cells cultured on PA gels with different thicknesses were affected by the stiffness of the coverslip, while the differences in gene expression in SMCs decreased significantly with increasing substrate thickness. When the thickness was greater than 75 μm, there was no significant difference in gene expression in SMCs, indicating that when the thickness of the substrate was greater than 75 μm, SMCs could no longer sense the stiffness of the coverslip, and the difference in gene expression in SMCs arose from the stiffness of the PA glue. Based on this, we selected a PA glue substrate with a thickness of approximately 100 μm for subsequent experiments.We then used adenoviral vector tools to amplify and inhibit the expression of miR-99b in rat BMSCs cultured on soft and hard substrates, and we found that overexpression of miR-99b promoted the osteogenic differentiation of BMSCs. Through analysis of the miRNA target cell database, we preliminarily proposed mTOR as a potential target of miR-99b because the mTOR signaling pathway is known for regulating cell fate, and miR-99 has been reported to be associated with mTOR in previous studies and has been shown to affect cell proliferation, migration, and phenotypic differentiation by targeting mTOR, especially in tumor cells.33–3533. D. Sun, Y. S. Lee, A. Malhotra, H. K. Kim, M. Matecic, C. Evans et al., “ miR-99 family of MicroRNAs suppresses the expression of prostate-specific antigen and prostate cancer cell proliferation,” Cancer Res. 71(4), 1313–1324 (2011). https://doi.org/10.1158/0008-5472.CAN-10-103134. J. Eniafe and S. Jiang, “ MicroRNA-99 family in cancer and immunity,” Wiley Interdiscip. Rev. RNA 12(3), e1635 (2021). https://doi.org/10.1002/wrna.163535. Y. Lin, W. Deng, J. Pang, T. Kemper, J. Hu, J. Yin et al., “ The microRNA-99 family modulates hepatitis B virus replication by promoting IGF-1R/PI3K/Akt/mTOR/ULK1 signaling-induced autophagy,” Cell. Microbiol. 19(5), e12709 (2017). https://doi.org/10.1111/cmi.12709 In addition, we noticed that the two complexes of mTOR can affect cell differentiation by regulating the expression of osteogenic and adipogenic differentiation-related marker proteins and can regulate RhoA to affect cell activity.36–3836. Y. Liu, X. Kou, C. Chen, W. Yu, Y. Su, Y. Kim et al., “ Chronic high dose alcohol induces osteopenia via activation of mTOR signaling in bone marrow mesenchymal stem cells,” Stem Cells 34(8), 2157–2168 (2016). https://doi.org/10.1002/stem.239237. E. Jacinto, R. Loewith, A. Schmidt, S. Lin, M. A. Rüegg, A. Hall et al., “ Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive,” Nat. Cell Biol. 6(11), 1122–1128 (2004). https://doi.org/10.1038/ncb118338. L. Liu, Y. Luo, L. Chen, T. Shen, B. Xu, W. Chen et al., “ Rapamycin inhibits cytoskeleton reorganization and cell motility by suppressing RhoA expression and activity,” J. Biol. Chem. 285(49), 38362–38373 (2010). https://doi.org/10.1074/jbc.M110.141168 This was consistent with our experimental results, confirming that miR-99b targets mTOR to regulate mechanotransduction and differentiation of rat BMSCs.Next, we demonstrated that treating BMSCs with low concentrations of rapamycin promoted osteogenic differentiation, which is consistent with the study by Gharibi et al.3939. B. Gharibi, S. Farzadi, M. Ghuman, and F. J. Hughes, “ Inhibition of Akt/mTOR attenuates age-related changes in mesenchymal stem cells,” Stem Cells 32(8), 2256–2266 (2014). https://doi.org/10.1002/stem.1709 In addition, it is consistent with the conclusion of Martin et al.4040. S. K. Martin, S. Fitter, A. K. Dutta, M. P. Matthews, C. R. Walkley, M. N. Hall et al., “ Brief report: The differential roles of mTORC1 and mTORC2 in mesenchymal stem cell differentiation,” Stem Cells 33(4), 1359–1365 (2015). https://doi.org/10.1002/stem.1931 that MSCs from mTORC1 knockout mice showed reduced osteogenic differentiation ability.The ability of rapamycin to override the action of the miR-99b sponge provides further evidence that the effect of basal stiffness on BMSC differentiation is mediated through the mTOR signaling pathway, and we demonstrate that miR-99b targets mTOR itself and, thus, may affect signaling through two complexes, mTORC1 and mTORC2. Our study found decreased expression of raptor and increased AKT-Ser473 phosphorylation in rapamycin-treated BMSCs, which agreed with the findings of Chen et al.4141. X. G. Chen, F. Liu, X. F. Song, Z. H. Wang, Z. Q. Dong, Z. Q. Hu et al., “ Rapamycin regulates Akt and ERK phosphorylation through mTORC1 and mTORC2 signaling pathways,” Mol. Carcinog. 49(6), 603–610 (2010). https://doi.org/10.1002/mc.20628

Based on the above findings, we further demonstrated that when the expression of miR-99b in BMSCs encapsulated in 3D GelMA hydrogels was upregulated, calcium deposition was significantly increased, suggesting that the regulation of miR-99b promotes the generation of osteoblast populations. Finally, we constructed a rat calvarial defect model and demonstrated the bone regeneration ability of the Ad-miR-99b-BMSC-GelMA flexible hydrogel scaffold. Therefore, combining MSCs with biomaterials to use this newly discovered regulatory technique in clinical applications may help to promote the differentiation of MSCs in the desired direction.

In conclusion, we found for the first time that miR-99b expression responds to changes in hydrogel matrix stiffness and that the regulation of mechanosensitive miR-99b can affect the differentiation of BMSCs by targeting the mTOR signaling pathway. Furthermore, amplified expression of miR-99b can promote the osteogenic differentiation of BMSCs in soft 3D hydrogels. Consistently, the BMSC-GelMA flexible hydrogel transfected with miR-99b significantly promoted bone regeneration in the rat calvarial defect area (Fig. 9). These results suggest that miRNAs play a key role in the mechanotransduction and differentiation of BMSCs, suggesting their significant value in MSC-based tissue engineering and regenerative medicine applications.

IV. METHODS

Section:

ChooseTop of pageABSTRACTI. INTRODUCTIONII. RESULTSIII. DISCUSSION AND CONCL...IV. METHODS <<SUPPLEMENTARY MATERIAL

A. Primary culture of rat BMSCs

The bilateral femurs of three-week-old male SD rats were quickly removed in a sterile environment, taking care to keep the marrow cavity closed. Then, ophthalmic scissors were used to cut the bone marrow cavity at both ends on the ultraclean bench, and the marrow cavity was rinsed four to six times with α-MEM containing 10% fetal bovine serum into a 10 ml petri dish using a sterile syringe. Then, the cells were placed in an incubator for 36–48 h of static culture, the nonadherent cells were removed, and the medium was replaced. When the cells grew to 85%–90% confluence, they were passaged, and the third, or fourth generation was used for subsequent experiments.

B. Preparation of substrates with different stiffnesses

In this experiment, polyacrylamide (PA) hydrogel was used to make substrates with different stiffnesses. The relative concentrations of acrylamide, bisacrylamide, and other components were changed to adjust the stiffness of the hydrogel substrate. The specific ratio configurations are shown in Table I. Then, 0.5 μl of TEMED and 5 μl of 10% w/v ammonium persulfate were added per 500 μl of the prepared master mix, and the solution was shaken slowly, taking care to avoid bubbles. The polymerization reaction was carried out for approximately 5 min, and after the solution was completely solidified, a PA-based adhesive with a thickness of approximately 100 μm was prepared. The surface of the hydrogel was covered with 1 ml of 0.5 mg/ml sulfo SANPAH and then placed under a 365 nm UV lamp for approximately 10 min. After washing with PBS, 25 μg/ml collagen I solution was coated on the hydrogel. The plates were allowed to stand for approximately 5 h at room temperature away from light. After sterilization, the gel was used for cell seeding.

TABLE I. Composition of PA gels with different stiffnesses.

0.8 kPa35 kPa80 kPa40% acrylamide (μl)751842462% bis-acrylamide (μl)1070180.51 M HEPES buffer (4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid) (μl)555ddH2O (μl)41024168.5Total (μl)500500500

C. Measurement of the stiffness of PA hydrogels

Young's modulus of each group of hydrogel substrates was measured using atomic force microscopy. Briefly, the hydrogel substrate sample was immersed in PBS solution, 12 points on the sample were randomly selected for measurement, and each sample was randomly measured three times. Then, the data were collected for statistical analysis.

D. Osteogenic and adipogenic induction and staining of BMSCs

BMSCs were seeded on PA gels with different stiffnesses at a density of 8000 cells/cm2, cultured in α-MEM containing 10% FBS until the cell density reached approximately 70%, and then switched to mixed osteogenic and adipogenic induction culture (1:1 volume ratio of osteogenic and adipogenic medium), and the medium was changed every 3 days. The osteogenic induction medium was primarily DMEM with 100 ng/ml dexamethasone, 10 mM β-glycerophosphate, 50 μM ascorbic acid-2-phosphate, and 10% FBS. The adipogenic induction medium was primarily DMEM with 1 μg/ml dexamethasone, 0.2 mM indomethacin, 10 μg/ml insulin, 0.5 mM isobutyl-1-methylxanthine, and 10% FBS.

Alkaline phosphatase (ALP) and Oil Red O staining were performed on the 15th day of culture. Briefly, cells were gently washed three times with PBS, fixed with 4% paraformaldehyde at room temperature for 20 min, and then stained for 10 min in the dark using an ALP detection kit (Beyotime, Cat# P0321S). Next, the cells were stained with Oil Red O dye for 30 min in the dark and finally imaged with a microscope. we calculated the percentage of osteoblasts as the area of ALP-stained positive cells as a proportion of the total