(L-480) Matrix mechanics trumps pore size in stromal cell biophysical control of tissue vascularization over time

Introduction::
Osteogenesis and the simultaneous development of vessel networks are foundational interconnected events necessary for bone regeneration that normally occur in a coupled fashion. With regards to the vascularization of bone, mesenchymal stem cells (MSCs) provide the stromal support necessary from both a biophysical perspective as well as biochemical signaling for the development of a robust vascular network. It has been hypothesized that factors such as temporal matrix mechanics, scaffold stiffnesses, porosity and mechanical environment that different pore sizes introduce, all come together to influence osteogenesis and successively vessel sprouting and growth over time. Hydroxyapatite (HA) porous scaffolds with porosity levels of either 45 pores per inch (ppi) or 60 ppi, that mimic trabecular bone, form the structural model system that we seed with MSCs to initiate osteogenic differentiation. 25:75 or 75:25 collagen: fibrin hydrogel blends seeded with microvascular fragments (MVFs), which are capillary fragments extracted from adipose tissue are used as the vessel network source within the scaffold’s porous interior. The objective of this study was to investigate both the magnitude and potential coordinated effects of stromal cell number, matrix mechanics and the changes in stiffness and mechanical strain fields created within different scaffold pore sizes, on the biophysical regulation of vessel network formation within developing bone matrix over time.




Materials and Methods::
Cylindrical 8x8 mm HA scaffolds were prepared by template coating and sintering, polyurethane scaffolds of varying porosities, 45 ppi and 60 ppi with an HA (CaP Biomaterials LLC, WI) based slurry. Bone marrow derived MSCs were harvested from femora of a male Lewis rat, cultured and seeded at passage 7 on sterilized HA scaffolds. 400,000 (50% confluency) or 800,000 (100% confluency) cells/scaffold were seeded on both pore sizes, and allowed to attach for 18 hours. MVFs were isolated from epididymal, subcutaneous and inguinal adipose pads of male Lewis rats. MVFs were then seeded within the porous scaffolds in either 25:75 or 75:25 fibrin:collagen (2 mg/mL) gels at 6,000 MVFs/scaffold (Figure 1). Scaffolds were cultured for 14 days in co-culture osteogenic (96% MEM α, 4% FBS, 1% PSA, 50 ug/mL L-ascorbic acid,10 nM dexamethasone and 10 mM ß-glycerophosphate) and vessel growth media (80%DMEM, 20% FBS, 1% PSA). Supernatant media was collected at days 1,3,7,10 and 14 and Alkaline Phosphatase (ALP) activity (n=6) was assessed using a fluorometric assay kit following manufacturer’s instructions (Abcam, Cambridge, UK). A three-way ANOVA was conducted across pore size, gel composition and time point, with Tukey’s test for post-hoc analysis. At days 7 and 14, scaffolds (n=3) were fixed overnight using 4% PFA and then decalcified at 25 °C using 10% formic acid (Thermo Fisher Scientific, MA). The gel portion remained and MVFs were stained with Griffonia (Banderaea) Simplicifolia Lectin I (GS-Lectin, Vector Labs, CA) and imaged using Operetta CLS (Perkin Elmer) to evaluate scaffold pore vascularization.




Results, Conclusions, and Discussions::
Independent of pore size and MSC confluency, ALP activity in 25:75 gels was immediate and sustained across time (Figure 2). Contrastingly and again independent of pore size and MSC seeding, ALP activity for 75:25 collagen:fibrin gel groups was consistently lower until 7 days, when a characteristic increase for 3 days and sustained activity was observed. ALP activity is indicative of osteogenic differentiation; thus, the 25:75 gels significantly supported osteogenesis in an early and sustained manner compared to the 75:25 groups. Greater vascularization at both MSC cell seeding densities and pore sizes was observed for the 25:75 group at 7 days and sustained until the end of study at 14 days, when compared to the 75:25 group at both pore sizes and MSC seeding densities (Figure 3). For both groups, however, greater vascularization was observed at lower MSC confluence, likely a result of less residual matrix contraction upon scaffold decalcification. Interestingly, 60 ppi scaffolds at 50% MSC seeding confluence allowed for sufficient matrix permissibility, even in the 75:25 group, which allowed the formation of a distributed vessel network by day 14. This indicates that there exists a close interplay between osteogenic enzymatic activity and pore size, regarding the effect matrix strain within different pore sizes has on vascular network formation. Osteogenic differentiation and the formation of a vascular network occurred in a coupled fashion over time for both gel groups. For the 25:75 gel group, osteogenic differentiation was immediate and sustained, and thus the vascular network formation response was strong at 7 days and sustained until completion of the study at 14 days. On the other hand, for the 75:25 gel group, osteogenesis was delayed, and the vessel network establishment was selective in the 60 ppi scaffold by day 14. Overall, these results indicate a clear and time dependent (temporal) coordination between osteogenesis, as demonstrated by ALP enzymatic activity, and vascular network formation, that is strongly determined by the extracellular matrix and the strain it sustains over time. In conclusion, matrix mechanics seems to overwhelm the effect of pore size, on osteogenic differentiation and vessel network formation over time.




Acknowledgements (Optional): :
This work is supported in part by NSF CBET#1847103 and the Jacobson, Lutcher Brown, and USAA Foundation endowments at the University of Texas at San Antonio. AI is supported by the UT Health SABER IRACDA Program (NIH 5K12GM111726).









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