In these experiments, hydrogels were fabricated using two established MMP-cleavable peptides with different MMP sensitivities and used to examine how degradability of the surrounding matrix affects MSC response to tensile strain.14,15
These sequences were specifically selected to maintain a similar hydrogel structure while tuning the overall degradability of the matrix, and have not previously been compared directly. As expected, all constructs remained intact over the culture period, allowing continued loading over 2 weeks in vitro
Fabricated gels showed no statistically significant differences in fold swelling, implying that no differences exist in initial mesh size (and mechanical properties) between the two hydrogel types.27,28
However, gels rapidly degraded after exposure to collagenase. Moreover, enzyme-sensitive gels remained unchanged in culture medium over 1 week, demonstrating that active collagenase enzyme, and not other factors in medium, was the main effector of changes in hydrogel integrity. The implications of these results are twofold: (1) cells should be able to locally degrade the gel by releasing MMPs, and (2) cells encapsulated within an MMP-sensitive hydrogel could be recovered, via addition of exogenous enzyme, for further analysis or implantation into tissue defects. Thus, this system allows cells to be exposed to highly controlled 3D environments, with precise biochemical and mechanical stimulation, to “prime” cells prior to retrieval for further therapeutic applications.
Confocal microscopy and image analysis were used in tandem to verify cell viability within the construct as well as evaluate changes in MSC morphology during culture. At 14 days, cell spreading was visually noted in both types of gels, with image analysis revealing that significantly more cells were spread in fast-degrading gels relative to slow-degrading gels, regardless of strain condition (). Although the exact proteases secreted by these MSCs were not characterized, enough active enzyme was produced to cleave local matrix and allow spreading to occur, as clearly evidenced in the LIVE/DEAD images (). The combination of the degradation and cell morphology results readily demonstrates that these synthetic hydrogel formulations enabled precise presentation of bioactive factors to encapsulated MSCs to control changes in cell shape over the culture period.
Although cells were able to spread in these scaffolds, no alignment was visually detected over time in dynamically or statically strained samples in any hydrogel type ( and ). Previously, fibroblastic differentiation of stem cells was primarily examined in gels made of collagen (or other fibrous materials with comparatively large pore sizes), in which cells align in the direction of strain.22
In our studies, the presence of a covalently crosslinked, nonaligned scaffold may have deterred cell reorientation over this time period. However, it is possible that a PEG-based gel with larger pore sizes and sufficient proteolytic degradation will enable cell reorientation in response to strain.
PicoGreen data indicated that cell number in all groups decreased over time (). However, these results correlate with earlier work in our laboratory showing similar effects with MSCs encapsulated in nondegradable hydrogels.5
Importantly, no differences were noted in DNA content between gel types or strain conditions in this study, and confocal microscopy images demonstrated that cells were viable at all time points. Thus, cell number is likely not a driving factor behind differences seen among different gel types or strain conditions. Nevertheless, a decreasing cell number suggests that proliferation continues to be limited by a small hydrogel mesh size.29
Future tailoring of these gels may include increasing pore size to provide space for cell proliferation in these materials while using other crosslinkers or reinforcement to maintain sufficient mechanical properties to allow for tensile loading of the constructs.
After loading for up to 14 days, MSCs embedded in both gel types were evaluated for expression of genes related to the tendon/ligament fibroblast phenotype ( and Supplementary Fig. S1
). Time points in this study were selected based on previous work in our laboratory, and others, indicating that changes in gene expression and matrix deposition in response to tensile culture were observed after 7–14 days of tensile loading.5,30
Scleraxis, a transcription factor expressed by tendon progenitor cells in developing mesenchymal tissues, is often considered a tendon fibroblast marker.6,22
In this study, both scleraxis and tenomodulin, another tendon fibroblast marker,23
was minimally expressed and was maintained or downregulated in all groups. In contrast, scleraxis expression was upregulated in a study using rat MSCs in a collagen gel using the same strain amplitude and rate, but with no rest period over 7 days.22
Another study utilizing primary human MSCs in collagen gels showed maintenance of scleraxis expression under 1% strain at 1
Hz for 30
min/day over 7 days.6
Also, in a recent study, cells from a C3H10T1/2 MSC line in collagen gels upregulated scleraxis expression under 10% strain at 0.1
Hz with 10
s rest periods.31
Other work suggests that the binding substrate (in a 2D system) may affect differentiation as indicated by increases in scleraxis, tenomodulin, tenascin C, and collagen III expression by human MSCs on collagen-containing substrates over fibronectin-containing substrates.32
Tenomodulin expression appears to be also dependent on scleraxis expression.33
Variations in scleraxis and tenomodulin expression from what was observed in this study may thus be attributable to differences in type of scaffold (collagen), cells, or strain regimen employed by other groups.
Contrary to some previously published reports,6,22
collagen I gene expression was downregulated over time in all gels, and although minor differences were shown between static and dynamic gels at day 7, this difference was not evident at day 14. These results differ from previous results in our laboratory using the same strain regimen, although those experiments used a scaffold that was not enzyme-degradable and MSCs from a different cell bank. The reason for the downregulation is unclear, although one report suggests that some changes in gene expression are dependent on scaffold and cell orientation.34
Since no orientation was observed in these gels, the lack of directional bias may have inhibited potential changes to collagen I expression. However, other results using bone-marrow-derived MSCs in a collagen scaffold have indicated that collagen I remains relatively unchanged under strain.6
From immunostained sections, collagen I deposition was detected at day 1 and more intense staining was observed at day 14, regardless of gel and strain parameters. The deposition observed may simply be a reflection of a high resident level of gene expression observed at day 1 relative to other tested genes (data not shown); therefore, additional gene upregulation might not be expected in this system.
Collagen III and tenascin-C are frequently used as markers of tendon/ligament fibroblast differentiation.4,5,35,36
Dynamically strained samples, regardless of gel type, demonstrated an increase in collagen III expression, both over time and relative to static samples. Correspondingly, immunostaining revealed collagen III staining only in dynamically strained gels. This correlates well with data from our laboratory and other studies, where collagen III has been identified as part of the native tendon repair process.5,37,38
Tenascin-C levels in all gels at days 7 and 14 were upregulated relative to day 1, with generally higher expression at day 7 relative to day 14. Importantly, however, dynamic samples exhibited significantly higher expression levels of tenascin-C relative to static samples on day 14. Tenascin-C has been previously shown to be regulated by mechanical strain39
but has also been speculated to play an “inhibitory for spreading” role that allows cells to modify adhesion contacts to avoid overstretching.40
Thus, restricting the ability to alter cell shape under strain may have triggered increased tenascin-C expression in slow-degrading dynamic gels relative to fast-degrading dynamic gels on days 7 and 14. Differences in gene expression levels, however, were not readily correlated with immunostaining. Tenascin-C was detected at days 1 and 14, with visually increased staining in all samples at day 14. The lack of differences between groups at day 14 may have been related to the consistent expression levels between groups on day 7. Longer culture times might provide tensile-strain-related upregulation of gene expression more time to emerge in the form of increased staining at time points beyond day 14.
Biglycan and decorin are also used as markers of tendon/ligament fibroblast differentiation and are constitutively found in tendon tissues.24,25,41
In our studies, decorin showed significant upregulation of all samples at day 14, with significantly higher expression in dynamic than in static samples at day 14 (Supplementary Fig. S1
). Biglycan also demonstrated significantly higher expression in the dynamic than static samples at day 14 only in fast-degrading samples. These results indicate expression of other tendon-related markers and further support the ability of this system to induce differentiation toward a tendon/ligament fibroblast phenotype.
In this work, synthetic, “blank slate” materials were used to present bioactive factors and modulate degradability and hydrogel properties in a precisely controlled manner to investigate the specific role of biomaterial degradation on changes in cell morphology and response to applied cyclic tensile strain. In particular, the responses of cells to tensile strain when encapsulated in gels of different MMP sensitivities have not been previously examined. Together, the results of this study suggest that, within the range of scaffold degradability that could be engineered into this system and still allow for long-term loading, hydrogel susceptibility to MMP degradation, with corresponding changes in morphology of embedded cells, did not affect MSC response to cyclic tensile strain over 14 days. While this result was unexpected, it should be noted that, in these experiments, significant cell spreading was seen only at 14 days, so a longer culture period may be needed to allow changes in spreading to affect gene expression levels accordingly. It is also possible that the inclusion of RGD peptides, which interact with integrins on MSCs in both gel types, might facilitate cytoskeletal interactions with the synthetic scaffold and enable sensing of mechanical strain.42
In addition, the specific integrin ligands bound may also play a role in the differentiation response,32
so future work may explore the role of engaging specific integrins in combination with changes in morphology on response to strain in this system. Finally, other aspects of the local mechanical environment, including changes in fluid flow, may change due to strain, the extent of local degradation, and possible pericellular matrix deposited by cells over time, subsequently affecting cytoskeletal organization and enabling MSCs in slow-degrading gels to respond to tensile strain even if their ability to spread is limited.43