UC Davis

Tendinopathies in equine athletes pose as a major musculoskeletal challenge due to the slow and incomplete repair process in tendons. Ongoing exploration into novel therapeutics to aid tendon repair into its pre-injury capacity spur doctors and researchers in multiple disciplines and across various species. Tendinopathies amount to one of the most common musculoskeletal injuries costing owners and breeders time and resources with no guarantee of return competition. As such, elucidation into the effects of current and novel therapeutics on tendon in vitro, in addition to, understanding the native tendon profile across age is crucial for improving tendon repair therapeutics. Chapter one reviews the impact of tendinopathies, the molecular and cellular challenges causing incomplete tendon repair, and key components affecting tendon repair. Additionally, this chapter also covers the structure, assembly, maturation, and regulation of collagen in tendon. Regional cellular differences also exist within the tendon; the intrinsic (tendon proper; TP) and extrinsic (peritenon; PERI) cells play differing roles in tendon development and repair causing the response to injury must be assessed separately. Finally, aging also impacts the tendon repair outcome through changes in gene expression and epigenetics which ultimately may affect effective therapeutics for successful return to competition. Chapter two investigates the in vitro effect of supplementation of biglycan and decorin, small leucine-rich repeat proteoglycans (SLRPs) that play critical roles in tendon development, growth, and maturation. Equine tendon proper and peritenon cell from the superficial digital flexor tendon were seeded in three-dimensional constructs from five horses and supplemented with 5 nM or 25 nM of bovine biglycan or bovine decorin. Functionality and ultrastructural morphology using biomechanics, collagen content analysis, transmission electron microscopy (TEM), and gene expression assessed the effect of supplementation. The peritenon supplemented cells, compared to the tendon proper cells, produced constructs with better mechanical (or material) properties, in addition to, a tenogenic-like phenotype with decorin supplementation (5 nM decorin: increased BGN and SCX expression, ultimate tensile strength, Young’s modulus, decreased CSPG4 expression; 25 nM decorin: increased BGN, COL1A1, FMOD, and SCX expression, and increased collagen content, decreased CSPG4 expression). Biglycan supplementation also produced positive results with trending improvements to biomechanics and increased gene expression of tenogenic and extracellular markers (BGN, DCN, SCX for 5 nM biglycan; BGN, SCX, CSPG4 for 25 nM biglycan) but overall were not as effective as the decorin supplementation. Chapter three explores the transcriptome and methylome of the superficial digital flexor tendon to assess the regional differences between the tendon proper and peritenon in adolescent (0-5 yrs), midlife (6-14 yrs), and geriatric (15-27 yrs) horses using RNA sequencing and DNA methylation techniques. RNA from 9 non-breed or sex specific horses were sequenced before RNASeq analysis to identify differentially expressed genes, gene ontology, and pathway analysis. DNA from 10 horses were sequenced by reduced representation bisulfate sequencing before methylation analysis for methyl calls, CpG island identification, and genomic annotation to identify differentially methylated regions from adolescent to geriatric horses. Across age, regional differences between the native tendon proper and peritenon cell populations persist in the transcriptome and methylome. Additionally, increased transcriptional activity is present in the adolescent and geriatric groups most likely due to ongoing growth and maturation in the adolescent group with subsequent degradation of the tendon in the geriatric population. Chapter four aims to understand the in vitro effect of co-culturing adipose-derived mesenchymal stem cells (ADMSCs), a common equine tendinopathy therapeutic, with tendon proper and peritenon cells. Regional differences between the intrinsic (tendon proper) and extrinsic (peritenon) cells exist and identifying the influence of ADMSCs on the two tendon cell populations is vital for gaining insight into their effect as therapeutic. Tendon proper and peritenon cells from 5 non-breed or sex specific horses were co-cultured with adipose-derived mesenchymal stem cells over 120 hrs and assessed by expression of tenogenic, perivascular, and extracellular assembly markers in addition to assaying cellular proliferation. ADMSCs co-cultured with either peritenon or tendon proper cells stimulates increased expression of a tenogenic phenotype by LOX and SCX and decreased CSPG4 in the two tendon cell populations with further tenogenic specificity in the peritenon cells with increased MKX expression. Lastly, chapter five covers the effect of decorin supplementation in tendon proper and peritenon cells in two-dimension, the challenges of adipose-derived mesenchymal stem cell transfection with the small leucine-rich repeat proteoglycan decorin, and the future trajectory of enhanced cell therapeutics in tackling tendinopathies. Keywords: Tendon, SLRP, Equine, RNASeq, DNA methylation, ADMSC