Collagen gels were seeded with rabbit bone marrow-derived mesenchymal stem cells (MSCs) and contracted onto sutures at initial cell densities of I , 4, and 8 million cellslml. These MSC-collagen composites were then implanted into full thickness, full length, central defects created in the patellar tendons of the animals providing the cells. These autologous repairs were compared to natural repair of identical defects on the contralateral side. Biomechanical, histological, and morphometric analyses were performed on both repair tissue types at 6, 12, and 26 weeks after surgery. Repair tissues containing the MSC-collagen composites showed significantly higher maximum stresses and moduli than natural repair tissues at 12 and 26 weeks postsurgery. However, no significant differences were observed in any dimensional or mechanical properties of the repair tissues across seeding densities at each evaluation time. By 26 weeks, the repairs grafted with MSC-collagen composites were one-fourth of the maximum stress of the normal central portion of the patellar tendon with bone ends. The modulus and maximum stress of the repair tissues grafted with MSCLcollagen composites increased at significantly faster rates than did natural repairs over time. Unexpectedly, 28% of the MSC ~ collagen grafted tendons formed bone in the regenerating repair site. Except for increased repair tissue volume, no significant differences in cellular organization or histological appearance were observed between the natural repairs and MSC-collagen grafted repairs. Overall, these results show that surgically implanting tissue engineered MSC-collagen composites significantly improves the biomechanical properties of tendon repair tissues, although greater MSC concentrations produced no additional significant histological or biomechanical improvement.

Despite various attempts to repair and replace injured tendon, an understanding of the repair processes and a systematic approach to achieving functional efficacy remain elusive. In this review the epidemiology of tendon injury and repair is first examined. Using a traditional paradigm for repair assessment, the biology and biomechanics of normal tendon, natural healing, and repair are then explored. New treatment strategies such as functional tissue engineering are discussed, including a functional approach to treatment that involves the development of in vivo functional design parameters to judge the acceptability of a repair outcome. The paper concludes with future directions.

Injuries to soft tissues such as tendons are becoming ever more frequent among the elderly. While increasing levels of activity likely contribute to these injuries, age-related declines in tendon strength may also be important. Whether these declines in biomechanical properties are associated with changes in fibril diameter or collagen type remains in question. In this study, age-related changes were investigated in patellar tendons from young adult rabbits (l-year old. n = 17) and from rabbits at the onset of senescence (4-year old, n = 33). Patellar tendon biomechanics was correlated with both collagen fibril diameter and with the presence of type V collagen, a known regulator of collagen fibril diameter. We hypothesize that (a) aging from 1 to 4 years results in significant reductions in patellar tendon biomechanical properties, and (b) these age-related declines are associated with smaller fibril diameters and with the presence of type V collagen. Maximum stress declined 25Yn between 1 and 4 years of age (100.7 f 5.6 MPa and 74.3 i 3.4 MPa, respectively, p < 0.0003) (mean & SEM) and strain energy density declined 40% (p < 0.001). The distribution of collagen fibrils from 4-year old rabbits was skewed significantly towards smaller diameters compared to fibrils from I-year old rabbits (p < 0.001). Type V collagen was observed only in the 4-year old rabbit tendons. These correlations suggest that with increasing age after skeletal maturity, type V collagen may help to regulate the assembly and thus diameter of collagen fibrils and thereby adversely affect patellar tendon strength.

Successful tissue engineered repair in the aging adult requires an abundant source of autologous, multipotent mesenchymal stem cells (MSCs). Although the number of bone marrow-derived MSCs declines dramatically with aging, their effectiveness in repair with increasing age has not been studied. We tested the hypothesis that MSCs harvested from geriatric rabbits would not repair patellar tendon defects as well as MSCs harvested from younger adult rabbits. In a novel within-subjects experiment, autologous MSCs were isolated from I-year old rabbits, culture expanded, and cryogenically preserved. After housing the rabbits for 3 years, MSCs were re-harvested from the 4-year old rabbits and expanded. Five hundred thousand thawed and fresh MSCs were each separately mixed with type I collagen gel (333.3 x lo3 cells/mg collagen) 24 h before surgery, and the resulting constructs implanted in bilateral full-length central third tendon defects. Twelve weeks post-surgery, the bone-tendon repair-bone units were failed in tension. Intra-animal (paired) comparisons between repair tissue treated with 1-year old MSCs and repair tissue treated with 4-year old MSCs resulted in no significant differences (ct = 0.051 in material properties including maximum stress (10.8 MPa vs. 9.9 MPa; p = 0.762), modulus (139.8 MPa vs. 146.2 MPa; p = 0.914), and strain energy density (0.52 N d m m ' vs. 0.53 Nmndmm'; p = 0.966). Despite an age-related trend, there were also no significant differences in structural properties including maximum force (62.9 N vs. 27.0 N; p = 0.070), stiffness (24.9 Nlmm vs. 12.0 N/mm; p = 0.11 l), and strain energy (87.2 Nmm vs. 31.4 Nmm; p = 0.061). A subset of the rabbits (n = 4 1 yrMSC, n = 2 4 yrMSC) showed the presence of ectopic bone in the repair region and were not included in the mechanical analyses. We conclude that in the rabbit model MSCs do not lose their benefit as a tendon repair therapy with aging and that MSCs can be cryogenically stored for 3 years and still effectively repair soft tissue injuries.

Our group has shown that numerous factors can influence how tissue engineered tendon constructs respond to in vitro mechanical stimulation. Although one study showed that stimulating mesenchymal stem cell (MSC)-collagen sponge constructs significantly increased construct linear stiffness and repair biomechanics, a second study showed no such effect when a collagen gel replaced the sponge. While these results suggest that scaffold material impacts the response of MSCs to mechanical stimulation, a well-designed intra-animal study was needed to directly compare the effects of type-I collagen gel versus type-I collagen sponge in regulating MSC response to a mechanical stimulus. Eight constructs from each cell line (n=8 cell lines) were created in specially designed silicone dishes. Four constructs were created by seeding MSCs on a type-I bovine collagen sponge, and the other four were formed by seeding MSCs in a purified bovine collagen gel. In each dish, two cell-sponge and two cell-gel constructs from each line were then mechanically stimulated once every 5 min to a peak strain of 2.4%, for 8 h/day for 2 weeks. The other dish remained in an incubator without stimulation for 2 weeks. After 14 days, all constructs were failed to determine mechanical properties. Mechanical stimulation significantly improved the linear stiffness (0.048+/-0.009 versus 0.015+/-0.004; mean+/-SEM (standard error of the mean ) N/mm) and linear modulus (0.016+/-0.004 versus 0.005+/-0.001; mean+/-SEM MPa) of cell-sponge constructs. However, the same stimulus produced no such improvement in cell-gel construct properties. These results confirm that collagen sponge rather than collagen gel facilitates how cells respond to a mechanical stimulus and may be the scaffold of choice in mechanical stimulation studies to produce functional tissue engineered structures.

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