Abstract

INTRODUCTION: Suboptimal healing of the tendon bone interface (TBI) remains an unsolved problem. One solution is a biocompatible tissue engineered construct that promotes augmented healing. We hypothesisze that 1.) the presence of Platelet Rich Plasma (PRP) and prolonged in vitro incubation produces tissue engineered tendon bone interface scaffolds with greater reseeding of viable Adipo-derived Stem Cells (ADSC) and 2.) when implanted with extracellular matrix hydrogel, constructs will display superior in vivo strength repair and biocompatibility.MATERIALS AND METHODS: 1. Achilles-calcaneal composite TBI scaffold grafts were harvested from thirty Wistar rats. After physicochemical decellularization and lyophilization, paired scaffolds were revitalized in either rat plasma or 100% activated rat PRP and reseeded with 1x10ADSCs/mL for 3, 7 and 14 days. Reseeding efficacy was quantified by live cell and total cell assays and qualified histologically. Statistical analysis of live cell and total cell assay data was performed using a paired Students t-test. 2. Ninety Sprague Dawley rats underwent Wistar tissue engineered TBI scaffold allograft reconstruction with one of five scaffold revitalization/reseeding conditions: (1) Decellularized, lyophilized, phosphate buffered saline , (2) Decellularized, Lyophilized, 100% activated PRP, (3) Decellularized, lyophilized, 100% activated PRP and extracellular matrix hydrogel, (4) Decellularized, lyophilized, 100% activated PRP and 14 day reseeding with ASC-luc2-eGFP cells and (5) Decellularized, lyophilized, 100% activated PRP, 14 day reseeding with ADSC-luc2-eGFP cells and extracellular matrix hydrogel. Daily, real-time in vivo bioluminescence imaging of all rats containing reseeded grafts was performed to follow cell viability. Subjects were sacrificed at weeks 2, 4, and 8 and constructs underwent biomechanical and histological evaluation. Comparisons across groups for biomechanical testing data was performed by one- way analysis of variance (ANOVA). Significance was set at p<0.05.RESULTS: 1. PRP revitalized grafts demonstrated greater live ADSC loads at 3 (p< 0.01), 7 (p< 0.001) and 14 days (p< 0.001), total ADSC loads at 7 (p< 0.001) and 14 days (p<0.001) with visibly greater live surface cellularity, layering, migration and penetration on histology. 2. Real-time in vivo bioluminescence imaging confirmed cell viability to day 22 post-implantation. Biomechanical strength testing demonstrated a significant increase in Ultimate failure load for reseeded groups compared to all other groups at week 2 (p<0.001), whereas only reseeded grafts supplemented with hydrogel remained significantly stronger compared to all other groups at weeks 4 (p<0.001) and 8 (p<0.01). Histology demonstrated most increased tendinous cellular invasion and fibrocartilage repopulation at 8 weeks in the reseeded group supplemented with hydrogel. Massons Trichrome staining demonstrated persistence of the scaffold structure at week 8 and blinded Image J analysis of Picosirius red staining specimens demonstrated significantly more Type III collagen in the ADSC reseeded hydrogel group at 2 (p< 0.01), 4 (p< 0.01) and 8 weeks (p< 0.01).CONCLUSIONS: Decellularized lyophilized allogeneic tendon-bone interface scaffolds can be optimized by revitalization in PRP, reseeding with ADSCs and supplemented by an extracellular matrix tendon hydrogel at the time of implantation. When done so, they display greater repair strength and biocompatibility.

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