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theses

Ligaments and tendons are dense collagenous tissues that assist the body during locomotion and mechanically stabilize joints by operating primarily in tension. During sports and daily use, they often sustain sub-failure injuries such as sprains and strains. Due to a lack of sufficient tissue vascularization and a tenuous healing response after injury, healing is often lengthy and incomplete. Current non-surgical treatment is conservative and includes drug therapy (non-steroidal anti-inflammatory drugs or NSAIDs, corticosteroid injections, etc.) and rest, ice, compression, and elevation or RICE. However, the repaired tissue can be prone to chronic instability and re-injury. To overcome these limitations, we investigated the potential of a series of tissue engineering (TE) approaches to specifically address the major obstacles associated with sub-failure ligament/tendon injury repair. Proliferative therapy (or prolotherapy) is an alternative treatment for damaged connective tissues that involves serial injections of an irritant solution into the injury site to initiate a localized healing response. Prior investigations have yielded inconclusive results and studies on its molecular mechanisms are limited. In our in vitro model, we demonstrated that prolotherapy, most notably with the popular irritant P2G, functions at the cellular level by significantly attenuating human tenocyte metabolic activity and cell migration in addition to greatly increasing prostaglandin activity. Prostaglandins, prominent inducers of tissue inflammation, were greatly increased at both mRNA (COX-2) and protein (PGE2) levels. Cell therapy, more specifically, the use of multipotent human mesenchymal stromal cells (hMSC) has shown great potential to serve as a suitable cell source for tenogenic regeneration and a source of trophic mediators to aid in tissue repair. Utilizing an in vitro bidirectional paracrine co-culture model, we assessed the tenogenic response of the interaction between primary hMSC and tenocytes across three distinct groups of human donors. Our findings showed that cross-talk led to no increases in metabolic activity in either cell type, but instead induced strong increases in collagen protein deposition. Secretome analysis using a TGF-β reporter cell line showed strong TGF-β bioactivity during co-culture. Furthermore, gene expression analysis confirmed changes in the expression patterns of a panel of anabolic and catabolic markers known to be downstream targets of TGF-β signaling and key regulators of tendon matrix maintenance. Finally, using a series of in vitro and in vivo experiments, we demonstrated the feasibility of utilizing high elastic modulus single-walled carbon nanohorns (CNH) as a novel therapy to modulate tendon biomechanics and cell response. Our findings revealed that CNH are capable of altering explanted ovine tendon elastic modulus without affecting the ultimate tensile strength (UTS) of the tissue. Next, cell studies showed that CNH immediately affect human tenocyte response in a manner dependent on the size of aggregates formed by CNH in solution. Lastly, functional in vivo evaluation using a stretch-injured rat Achilles tendon model indicated that injected CNH persist in the tissue and alter injured tendon elastic modulus most notably after 7 days of treatment, without altering UTS. In conclusion, the data presented here demonstrate the potential of three unique TE approaches for repairing sub-failure ligament/tendon injury.

ETD_6426

Ph.D.

Includes bibliographical references

by Emmanuel C. Ekwueme

doi:10.7282/T3VD7193

ETD doctoral

The author owns the copyright to this work.