Tissue Extracellular Matrix-based Microribbon Scaffolds for Bone Regeneration via Immunomodulation

<b>Introduction:</b> The immune system plays a critical role in bone regeneration as the mediator of the body’s inflammatory response to injury. Previous biomaterials research on bone tissue engineering has largely focused on enhancing stem cell osteogenesis rather than modulating immune cell responses. Tissue-derived extracellular matrix (ECM) has been used in nanoporous hydrogels and demonstrated advantages over synthetic hydrogels in promoting regenerative immune responses when injected in muscle [1]. However, previous tissue ECM-based research has been limited to soft tissue models and nanoporous hydrogels. How ECM modulates immune cell response and bone regeneration in macroporous scaffolds have not been investigated before. Our lab has reported gelatin microribbon (μRB) scaffolds that contain macroporosity and enhance endogenous bone regeneration <i>in vivo</i> compared to nanoporous gelatin hydrogels [2]. We hypothesize that incorporating ECM into μRB scaffolds would modulate immune responses and bone regeneration. To test this hypothesis, the present study seeks to develop μRB scaffolds containing tissue-derived ECM, and assess the effect of varying ECM dosage and tissue type on immunomodulation and bone regeneration <i>in vitro </i>and<i> in vivo</i>.<br/><br/><b>Materials and Methods:</b> To derive ECM from multiple tissue types (bone, muscle, articular cartilage, and meniscus cartilage), juvenile bovine stifles were used. Bone was first demineralized, and all tissues were decellularized and digested following previous protocols. Gelatin μRBs were synthesized by wet spinning as we previously reported [2]. To incorporate ECM, two methods were evaluated including co-spinning a mixture of bone ECM (bECM) containing gelatin solution, or coating ECM from various tissue types onto gelatin μRBs through physical adsorption. To characterize ECM incorporation, immunostaining of various tissue markers was performed. For <i>in vitro </i>studies, mouse MSCs or macrophages were encapsulated in µRB scaffolds with varying ECM dosages or tissue types. To assess immunomodulation, macrophage phenotype was assessed using gene expression and ELISA. MSC osteogenesis was assessed using histology of bECM markers. Leading compositions that led to regenerative immune responses and enhanced osteogenesis were further validated <i>in vivo </i>using a mouse critical size cranial defect model. Samples were harvested at weeks 1 and 6. Immunomodulation and bone formation <i>in vivo </i>were assessed using histology and microCT imaging.<br/><br/><b>Results and Discussion:</b> Immunostaining confirmed both co-spinning and coating methods enable successful incorporation of tissue ECM into macroporous μRB scaffolds. The coating method induces more homogenous ECM presentation relative to the co-spinning method. Incorporating bECM promotes macrophage polarization towards the regenerative M2 phenotype while promoting MSC osteogenesis in a dose-dependent manner <i>in vitro</i>. We found intermediate bECM concentration led to enhanced MSC osteogenesis, yet further increase of bECM inhibited osteogenesis. Adding tricalcium phosphate particles synergized with a low dosage of bECM to accelerate bone formation <i>in vivo</i>. Varying tissue ECM further enhances MSC osteogenesis <i>in vitro</i>, with the most mineralized bone formation found in meniscus cartilage and muscle-derived ECM-coated groups. Together, these results support our hypothesis, and future studies will evaluate the effect of varying ECM type on immunomodulation <i>in vitro</i> and bone formation <i>in vivo</i>. While the present study focuses on bone as a target tissue for regeneration, the ECM-coated macroporous µRB scaffolds may be broadly applied to enhance regeneration of other tissue types.<br/><br/><b>Acknowledgments:</b> The authors would like to acknowledge NIH R01DE024772 (FY), R01AR074502 (FY), NSF GRFP (CV), EDGE fellowship (CV), Stanford MCHRI Postdoctoral fellowship (NS), and Stanford Bioengineering REU program (AY) for funding.<br/><br/>[1] <i>Biomaterials 192</i> (2019) 405–415.<br/>[2] <i>J. Biomed. Mat. Res. A 104</i>(6) (2016) 1321-31.