Chair of Department of Bioengineering University of Oregon, United States
Introduction:: To promote bone regeneration, avascular tissue engineering approaches must support host vascular recruitment via biochemical functionalization of engineered extracellular matrices (eECM) [1]. We have developed an eECM using matrix metalloproteinase (MMP)-degradable poly(ethylene glycol) (PEG) hydrogels with the adhesive peptide, RGD, to improve vascularization and bone regeneration [2]. Indeed, bone allografts modified with a tissue engineered periosteum comprised of the eECM resulted in a 4-fold increase in graft-localized vascular volume versus unmodified allografts [2]. To further investigate the combinatorial capability of hydrogel biochemical cues to recruit host vascular infiltration, adhesive peptide identity, concentration, and potential synergies between multiple epitopes, as well as cell-mediated degradation via MMPs was investigated herein. The overarching hypothesis is that unique combinations of adhesive peptides and MMP degradation kinetics will optimize vascularization and bone healing. In this study, an in vitro surrogate for vascularization was used to screen eECM using a human umbilical vein endothelial cell (HUVEC)-based vascular sprouting assay. The adhesive peptides RGD, GFOGER, and YIGSR, which mimic extracellular matrix proteins fibronectin, collagen, and laminin, respectively, were investigated. In addition, three MMP-degradable crosslinkers with different susceptibility to MMP-2, -9, and -14 were considered to obtain different hydrogel degradation rates. A design of experiments (DOE) approach was utilized to evaluate each adhesive peptide and MMP degradation rate individually and in combination to identify effects of hydrogel functionalization on sprouting. Finally, hydrogels are currently being evaluated in a murine femur defect model for vascularization and bone healing in bone allografts.
Materials and Methods:: Cell spheroids composed of HUVECs and human mesenchymal stem cells (hMSCs) were formed in 96-well round bottom plates with endothelial growth media and 0.24 wt% methyl cellulose. Spheroids included 3000 cells with HUVEC and hMSCs in a 1:1 ratio. Spheroids were encapsulated in hydrogels with RGD, GFOGER, and/or YIGSR, with RGE-functionalized eECM as a control (Fig 1A). Hydrogels without GFOGER (#1, 2, 3, and 5) were composed of 5 wt% PEG-norbornene (8-arm, 20 kDa) crosslinked via photo-initiated thiol-ene chemistry, with a 0.8:1 thiol:ene ratio, and 3 mM total peptide. To maintain a constant compressive modulus of 10 kPa with hydrogels containing GFOGER (#4, 6, 7, and 8), the hydrogel composition was 4.5 wt% PEG-norbornene with 0.7:1 thiol:ene ratio due to previously reported GFOGER mediated hydrogel stiffening [3]. Adhesive peptides were tested at 1 mM, with hydrogels including one to three peptides at a time and RGE used to maintain 3 mM peptide concentration. All hydrogels were enzymatically degradable via crosslinking with the MMP-degradable sequence GKKCGPQGIWGQCKKG. All hydrogels had an average mesh size of 18 ± 2 nm. Cell sprouting was observed over 7 days, using confocal and brightfield imaging, and quantified using the ImageJ “Sprout Morphology” plug-in. The statistical analysis software JMP Pro 17 was used to run a D-optimal DOE to analyze the individual and combinatorial effects of each adhesive peptide on cell infiltration and integration in the hydrogels.
Results, Conclusions, and Discussions:: In this study, 8 unique hydrogels were evaluated for their angiogenic potential using an in vitro sprouting assay. Figure 1A depicts representative confocal images of all groups on day 7, with sprouting quantification shown in Figure 1B. For all groups, total network length and average branches per sprout increased with time. Hydrogel groups 2, 5, and 8 showed the greatest total network length compared to all other groups, with ~1.5-fold greater network length than groups 4, 6, and 7, ~5-fold greater network length than hydrogel 3, and ~11.5-fold greater than the control (#1) at day 5. Due to the number of groups evaluated, a DOE was used to develop a model from the observed responses. Specifically, the number of sprouts on day 1, total network length at day 5, and average branches per sprout at day 5 were used. These responses were chosen due to the hypothesis that hydrogels that support early sprout development, later network formation, and continued remodeling via network branching will best support vascularization in vivo. The effect of each factor and interaction effects are displayed in Figure 1C. From this model, only RGD significantly promoted all cell sprouting responses, with GFOGER promoting network formation and early sprout number. Hydrogels with RGD and YIGSR inhibited early sprout formation, while composite GFOGER-YIGSR hydrogels significantly supported branching. Interestingly, hydrogels with both RGD and GFOGER had a significant negative effect on all sprouting outcomes. Hydrogels with all three adhesive peptides had a positive effect on cell sprouting, but this finding was not significant.
In conclusion, hydrogels with RGD alone best support all cell sprouting outcomes. However, some interaction effects were observed. For this experiment, the DOE enabled robust statistical analysis of the results, which would be difficult with traditional multiple comparison methods. Ongoing work investigates adhesive peptide effects in combination with MMP-mediated hydrogel degradation rate. Additionally, hydrogels that best support in vitro cell sprouting are being evaluated in vivo for improved bone allograft healing.
Acknowledgements (Optional): :
References (Optional): : [1] K. M. Park and S. Gerecht, “Harnessing developmental processes for vascular engineering and regeneration,” Development 2014; 141(14): 2760-2769
[2] Y. Li, et. al., “Matrix metalloproteinase (MMP)-degradable tissue engineered periosteum coordinates allograft healing via early stage recruitment and support of host neurovasculature,” Biomaterials 2021; 268: 120535
[3] D. Fraser and D. S. W. Benoit, “Dual peptide-functionalized hydrogels differentially control periodontal cell function and promote tissue regeneration,” Biomaterials Advances 2022; 141: 213093
