S30 - Matrix strain modulates osteogenic-angiogenic coupling and development in a ligand density and time dependent manner within porous polymeric scaffolds

Introduction: : Pre-vascularization of biomaterial scaffolds is a promising strategy to enhance engraftment and cell survival in tissue engineering. This study investigates the influence of scaffold pore size and hydrogel matrix composition on angiogenesis and osteogenesis within soft, open porous polyurethane (PUR) scaffolds. Mesenchymal stem cells (MSCs) were seeded onto PUR scaffolds having pore sizes of 45, 60, and 80 pores per inch (ppi), while adipose-derived microvascular fragments (MVFs) were embedded within collagen:fibrin (col:fib) hydrogels at collagen concentrations of 2 mg/mL and 4 mg/mL, combined in co-gels in a 1:1 ratio with 20 mg/mL fibrin. Over 21-days in vitro culture, vessel formation and osteogenic differentiation were assessed using mRNA sequencing, lectin staining, and evaluating media calcium. Results indicated that scaffold pore size and hydrogel composition significantly influenced vascular network formation, with vessels either traversing pores or aligning along scaffold struts, guided by scaffold-induced strain fields. Higher collagen concentration hydrogels exhibited heterogeneous degradation, affecting sustained angiogenesis. Gene expression analysis revealed temporal progression as the primary source of variation, with hydrogel composition contributing additional differences. Calcium measurements indicated delayed osteogenic differentiation due to the absence of an inherent calcium source. These findings elucidate that hydrogel parameters more than scaffold pore size in polymeric scaffolds are central to developing stable vascular networks within engineered bone constructs, highlighting how scaffold architecture and hydrogel composition modulate angiogenic patterns and osteogenic differentiation through time-resolved changes in the matrix support.

Materials and

Methods: : Scaffold Preparation: PUR foams of 45, 60, and 80 pores per inch were prepared as cylinders (hxd, 3 mm x 8 mm) and sterilized by autoclave treatment. MSCs were first seeded on sterilized PUR scaffolds (200,000 MSCs/scaffold) and cultured for 24 hours in growth media prior to MVF seeding and hydrogel casting. MVFs were isolated from the fat pads of rats and resuspended in hydrogel components and cast over PUR scaffold at 100 µl/scaffold and an MVF seeding density of 20,000 MVFs/ml. Col:fib hydrogel blends were formed at one of two collagen concentrations (2 and 4 mg/mL) and a fibrinogen concentration of 20 mg/ml at a volume ratio of 50:50. Samples were collected at days 3, 7, 14, and 21 for immunohistochemistry, RNA sequencing, and calcium concentration. Samples for RNA sequencing were barcoded, pooled, and sequenced in an Illumina NovaSeq 6000.
Statistical Analysis: A two-way ANOVA followed by Tukey’s test for post hoc evaluation was used to determine significance at p< 0.05 for the calcium concentration levels (n=4, across time and scaffold pore size) (SigmaPlot). Statistical analysis of differential expression was performed using DESeq2 package on the R platform, starting from the read counts for each gene in each sample. First a PERMANOVA analysis determined main effects of the PUR pore size, matrix type and culture duration, at p< 0.05. Subsequent detailed interrogations were made and significant hits (adjusted P-value < 0.05) were extracted and arranged by log fold change.

Results, Conclusions, and Discussions:: Polyurethane porous scaffolds of various pore sizes were seeded with MVFs and MSCs in a col: fib hydrogel to evaluate the effect of scaffold stiffness, pore size, and hydrogel composition towards angiogenic-osteogenic coupling (Fig. 1a). The effect of pore size is evident in the progression of cellular growth over time (Fig. 2), where larger pores sizes are not as conducive for vessel and matrix development. One of the effects of gel composition is possible differential gel degradation over time, which may be possible due to incomplete percolation of mixed gels, resulting in islands of one component within the other. The combination of pore size and gel composition results in differential strain fields that develop within pores. Vessell growth is either stretching across the length of a pore ore they are looping around the struts of the scaffold (Fig. 2b).
The other effect of gel composition is seen in the PCA of gene expression, where at earlier time points, there is clear separation shown between matrix composition and time (Fig. 1b). Temporal differences within up- and down-regulated genes indicate angiogenic and osteogenic occurring, specifically in Hif1a and calcium signaling pathways (Fig. 3a). The temporal changes within calcium signaling are seen in supernatant calcium concentration (Fig. 3b), where at earlier timepoints there is significantly less calcium in solution than later timepoints. When examining significant genes of interest towards angiogenesis and osteogenesis (Fig. 3c), it is seen that there are differences in how each gel composition dictated vessel sprout and stabilization within the system. Additionally, there is evidence of delayed osteogenesis occurring by the upregulation of early markers of osteogenesis at later time points, which may be due to the lack of additional strong osteoinductive factors.
Overall, it was shown that overall scaffold stiffness impacts the resulting strain fields within the porous scaffold and that osteogenesis was delayed in comparison to previous studies due to differences in scaffold material.

Acknowledgements and/or References (Optional):: This research was supported in part by the National Science Foundation (NSF) CAREER Award (CBET#1847103) to TG and in part by the National Institutes of Health (R21 DE032179) to JLO.