Optimization of Feline Adipose-derived Multipotent Stromal Cell Isolation and Canine Cranial Cruciate Ligament Regeneration with Intra-articular Adipose-derived Multipotent Stromal Cells

Abstract

Adult multipotent stromal cells (MSCs) have become popular within veterinary sciences for direct administration and tissue regeneration due to their differentiation, trophic and immunosuppressive properties. However, current isolation and expansion techniques provide a heterogeneous population with other types of cells, which may affect the efficiency and efficacy of treatments. The progenitor properties and function need to be further evaluated in vitro prior to in vivo application. The first part of this dissertation was to identify the optimal MSC source for general canine orthopedic applications such as intra-articular injection and joint tissue regeneration. Based on side-by-side comparisons of cell doublings, colony forming unit frequencies, target gene expression and immunophenotype, the infrapatellar fat pad (IFP) had MSCs with higher yield, expansion, multipotentiality and progenitor immunophenotypes than joint capsular and cranial cruciate ligament (CrCL) synovium. The second part of this dissertation was designed for optimization of isolation and in vitro expansion techniques for feline adipose-derived MSCs (ASCs). A new method was designed with which a therapeutic dose of ASCs (7 x 106 cells/kg) was available within 3 cell passages from epididymal adipose excised during castration. The isolated ASCs maintained their progenitor properties after cryopreservation. Feline-specific culture medium to induce adipogenesis, osteogenesis and chondrogenesis was also developed in the study for later application in feline tissue regeneration studies. The third part of this dissertation to apply canine IFP ASCs for CrCL regeneration. A custom perfusion bioreactor was designed and built to accommodate canine CrCL templates on which to grow viable grafts with IFP ASCs. Culture medium and template composition and design were optimized to induce the ASCs into fibroblasts to form a collagenous ligament. Compared to ASC templates cultured in stromal (basal) medium those cultured in induction medium had enhanced collagen deposition, procollagen synthesis and ligament specific gene mRNA levels. The results from this investigation confirm the feasibility of growing new canine CrCLs from ASCs in the laboratory for implantation to treat dogs with CrCL disruption. Overall, the investigations in this dissertation provide vital translational information for canine and feline MSC therapies, and may contribute to better efficiency and efficacy of in vivo treatment and tissue regeneration.