Extracellular matrix (ECM) is responsible for tendon strength and elasticity. Healed tendon ECM lacks structural integrity, leading to reinjury. Porcine urinary bladder matrix (UBM) provides a scaffold and source of bioactive proteins to improve tissue healing, but has received limited attention for treating tendon injuries. The objective of this study was to evaluate the ability of UBM to induce matrix organization and tenogenesis using a novel in vitro model. We hypothesized that addition of UBM to tendon ECM hydrogels would improve matrix organization and cell differentiation. Hydrogels seeded with bone marrow cells (n = 6 adult horses) were cast using rat tail tendon ECM ± UBM, fixed under static tension and harvested at 7 and 21 days for construct contraction, cell viability, histology, biochemistry, and gene expression. By day 7, UBM constructs contracted significantly from baseline, whereas control constructs did not. Both control and UBM constructs contracted significantly by day 21. In both groups, cells remained viable over time and changed from round and randomly oriented to elongated along lines of tension with visible compaction of the ECM. There were no differences over time or between treatments for nuclear aspect ratio, DNA, or glycosaminoglycan content. Decorin, matrix metalloproteinase 13, and scleraxis expression increased significantly over time, but not in response to UBM treatment. Mohawk expression was constant over time. Cartilage oligomeric matrix protein expression decreased over time in both groups. Using a novel ECM hydrogel model, substantial matrix organization and cell differentiation occurred; however, the addition of UBM failed to induce greater matrix organization than tendon ECM alone. / Master of Science / Tendon injuries are common in horses and are painful and can be career- and life-ending. Tendons have a special structure and organization that enables them to withstand high tensile forces without permanent deformation. Injured tendons heal by forming stiff, disorganized scar tissue that makes the tendon more prone to re-injury. The lining of urinary bladders from pigs (UBM) provides a physical mesh and signaling factors that help heal injuries in a variety of tissues to a more normal state. However, UBM has not been evaluated in a laboratory model of tendon tissue formation to determine how it can help heal tendon injuries. Three-dimensional models of new tendon tissue formation (neotendons) were made with rat tail tendon matrix and stem cells collected from horse bone marrow. The neotendons were placed under steady tension for 3 weeks. The models were collected after 1 and 3 weeks to measure their width, numbers of live cells, cell and matrix organization, levels of tendon matrix components and expression of genes found in tendons. Most cells in the neotendons remained alive during the study period. Over time, UBM-treated and untreated neotendons became narrower compared to their starting width. The width of UBM-treated neotendons decreased faster than non-treated neontendons in the first week of the study. Cells became longer, narrower, and oriented along lines of tension. Expression of genes important in tendon development and structure either increased or was constant over time. UBM treatment did not change cell shape or increase levels of tendon-associated genes, DNA, or tendon matrix components. Our novel tendon model successfully created organized tendon-like tissue when placed under tension. However, UBM treatment did not improve formation of tendon-like tissue to a greater extent than controls.