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Tendinopathy includes cases with chronic tendon pain and spontaneous tendon ruptures, which is putatively resulted from failed tendon healing. Overuse is a major risk factor of tendinopathy, which can impose mechanical and oxidative stress to tendons. Previous studies investigated the influences of mechanical stress, but the direct impact of oxidative stress on tendon healing remains unclear. We hypothesized that imposed oxidative stress can impair tendon healing and lead to tendinopathic changes. Thirty-nine rats were operated for patellar tendon window injury. From weeks 3-5 post-operation, the rats received three weekly subcutaneous injections of saline, 50 or 500 μM H

After anterior cruciate ligament reconstruction (ACLR), the biological healing of the graft is a rate-limiting step which can contribute to graft failure. The tripeptide-copper complex glycyl-L-histidyl-L-lysine-Cu(II) (GHK-Cu) is a well-known activator of tissue remodeling. We investigated whether GHK-Cu can improve graft healing following ACLR. Seventy-two rats underwent unilateral ACLR were randomized to saline, 0.3 or 3 mg/ml GHK-Cu groups (n = 24). Post-operational intra-articular injections were given from week 2, once a week, for 4 weeks. Gait analysis was performed pre-injury and at harvesting time. At 6 or 12 weeks post-operation, knee specimens were harvested for knee laxity test, graft pull-out test, and histology. At 6 weeks post-ACLR, GHK-Cu groups resulted in a smaller side-to-side difference in knee laxity as compared to the saline group (p = 0.009), but there was no significant difference at 12 weeks post-operation. The graft complex in the 0.3 mg/ml GHK-Cu group had higher stiffness than saline group at 6 weeks post-operation (p = 0.026), but there was no significant difference in ultimate load, gait parameters, and histological scores among treatment groups. All grafts failed mid-substance during pull-out test. Intra-articular supplementation with a bioactive small molecule GHK-Cu improved graft healing following ACLR in rat, but the beneficial effects could not last as treatment discontinued.

We hypothesized that BMP-2 might induce non-tenocyte differentiation and increase production of proteoglycans of tendon-derived stem cells (TDSCs). This study investigated the effects of BMP-2 on the differentiation and production of proteoglycans in TDSCs in vitro. Rat patellar TDSCs were treated without or with BMP-2. The osteogenic, adipogenic, chondrogenic, and tenogenic differentiation of TDSCs were assessed by (1) Alizarin red-S staining assay; (2) Oil Red-O staining assay; (3) haematoxylin-eosin staining, Safranin-O staining, immunohistochemical staining of Sox9, and collagen type II; and (4) qRT-PCR analysis of lineage-specific markers. The production of glycoaminoglycans (GAG) in the BMP-2-treated TDSCs was assessed by alcian blue staining. The mRNA expression of aggrecan (Acan), decorin (Dcn), biglycan (Bgn), and fibromodulin (Fmod) in TDSCs after BMP-2 treatment was assessed by qRT-PCR. BMP-2 promoted the osteogenic, adipogenic, and chondrogenic differentiation but inhibited tenogenic marker expression of TDSCs. GAG production and Acan increased while Dcn, Bgn, and Fmod decreased in TDSCs after BMP-2 stimulation. In conclusion, BMP-2 promoted GAG deposition, aggrecan expression, and enhanced non-tenocyte differentiation of TDSCs in vitro. The effect of BMP-2 on TDSCs might provide insights into the histopathological changes of tendinopathy.

Injured tendons heal slowly and often result in the formation of mechanically and functionally inferior fibrotic scar tissue or fibrous adhesions. This study investigated the use of tendon-derived stem cells (TDSCs) for tendon repair in a rat patellar tendon window defect model. Fibrin glue constructs with or without GFP-TDSCs were transplanted into the window defect. The patellar tendons were harvested for histology, ex vivo fluorescent imaging and biomechanical test at various time points up to week 4. Our results showed that TDSCs significantly enhanced tendon healing as indicated by the increase in collagen production as shown by hematolxylin stain-ability of the tissue, improvement of cell alignment, collagen fiber alignment and collagen birefringence typical of tendon. The labeled cells were observed at weeks 1 and 2 and became almost undetectable at week 4. Both the ultimate stress and Young's modulus were significantly higher in the TDSCs group compared to those in the fibrin glue group at week 4. In conclusion, TDSCs promoted earlier and better repair in a rat patellar tendon window defect model. © 2011 Orthopaedic Research Society. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 30:613–619, 2012

The acquisition of chondro-osteogenic phenotypes and erroneous matrix deposition may account for poor tissue quality after acute tendon injury. We investigated the presence of chondrocyte phenotype, ossification, and the changes in the expression of major collagens and proteoglycans in the window wound in a rat patellar tendon window injury model using histology, von Kossa staining and immunohistochemistry of Sox 9, major collagens, and proteoglycans. Our results showed that the repair tissue did not restore to normal after acute injury. Ectopic chondrogenesis was observed in 33% of samples inside wound at week 4 while ectopic ossification surrounded by chondrocyte-like cells were observed in the window wound in 50% of samples at week 12. There was sustained expression of biglycan and reduced expression of aggrecan and decorin in the tendon matrix in the repair tissue. The erroneous deposition of extracellular matrix and ectopic chondro-ossification in the repair tissue, both might influence each other, might account for the poor tissue quality after acute injury. Higher expression of biglycan and aggrecan were observed in the ectopic chondro-ossification sites in the repair tissue, suggesting that they might have roles in ectopic chondro-osteogenesis.

We investigated the hypothesis that if the area, length, and mineralization of newly formed bone could be used to predict the healing quality of patella-patellar tendon (PPT) junction after partial patellectomy. Twenty-four rabbits underwent partial patellectomy and their PPT complexes of the operated limbs were harvested at weeks 6, 12, and 18 postoperatively. The area, length, and mineralization of newly formed bone at PPT junction healing interface was evaluated radiographically and peripheral quantitative computational tomographically. The healing quality of PPT complexes in terms of its tensile property was determined by biomechanical testing. The results showed that the area, length, and mineral content of newly formed bone, and its tensile strength increased significantly with follow-up time. The area of newly formed bone was strongly correlated with the failure load, ultimate strength and energy at failure (r = 0.75, 0.76, and 0.70, respectively, p 0.01 for all). The length of newly formed bone was also found to be correlated with failure load, ultimate strength and energy at failure (r = 0.61, 0.54, 0.67, respectively, p 0.01 for all). In addition, the bone mineral content of newly formed bone but not its bone mineral density was moderately correlated with failure load, ultimate strength and energy at failure (r = 0.44, 0.51, 0.42, respectively, p 0.05 for all). In conclusion, the area, length, and mineralization of newly formed bone at the PPT junction after partial patellectomy may serve as useful noninvasive indices in assessing the quality of the bone-tendon junction repair.

Chondrocytes phenotype/markers were expressed in clinical samples of tendinopathy and calcifying tendinopathy. This study examined the spatial-temporal expression of chondro-osteogenic Bone Morphogenetic Proteins (BMPs), which might contribute to ectopic chondro-osteogenesis and failed healing process in tendinopathy. Collagenase was injected into patellar tendon of rats to induce ossified failed tendon healing. At week 2, 4, 8, 12, and 16, the patella tendon was harvested for immunohistochemical staining and analysis of BMP-2/4/7. BMP-4/7 showed similar expression patterns, which was different from BMP-2. The expression of BMP-2 in the tendon matrix increased at week 2 and was reduced to nearly undetectable level afterwards except at the chondro-ossification sites. However, the expression of BMP-4/7 in the healing tendon fibroblast-like cells and matrix increased at week 2, reduced at week 4 and 8 and increased again at week 12 and 16, consistent with transient healing at week 8 in this animal model. There was increasing strong expression of BMP-4/7 in the chondrocyte-like cells in the un-ossified and ossified areas from week 8-16. BMP-4/7, besides BMP-2, might also contribute to ectopic chondro-osteogenesis and failed healing in tendon injuries. BMP-4/7, but not BMP-2, might be involved in regulating late events in ossified failed tendon healing.

Anterior cruciate ligament (ACL) reconstruction with placement of grafted tendon in bone tunnel is a common surgical procedure. Bone tunnel creation may result in stress shielding of postero-lateral regions of tibial tunnel. The present study was designed to characterize the changes of peri-graft bone and compare with tendon-to-bone (T-B) healing in spatial and temporal manners after ACL reconstruction in rabbit. Surgical reconstruction using digital extensor tendon in bone tunnel was performed on 48 rabbits. Twelve rabbits were sacrificed at 0, 2, 6, and 12 weeks postoperatively for radiological and histological examinations. Bone mass and microarchitecture at the anterior, posterior, medial, and lateral regions of tunnel wall at distal femur and proximal tibia were evaluated. Using peripheral quantitative computed tomography, a 26, 22, and 42% decrease in bone mineral density (BMD) relative to baseline was present in the medial region of the femoral tunnel and the posterior and lateral regions of the tibial tunnel, respectively, at week 12 postoperatively (p0.05). It was accompanied by a decrease in trabecular number and increase in trabecular spacing, the shift of platelike to rodlike trabeculae, and loss of anisotropy under micro-computed tomography evaluation. This finding was echoed by histology showing increased osteoclastic activities and poor T-B healing in these regions. In conclusion, the postoperative bone loss and associated poor T-B healing was region-dependent, which may result from adaptive changes after tunnel creation.

Cultured tendon fibroblasts (CTFs) from intact explants are widely used to study tendon healing in vitro. The significance of these findings may rely on similarities between CTFs and healing tendon fibroblasts in situ. Our purpose was to compare CTFs with fibroblasts cultured from healing tendons. We cultured CTFs from intact and healing tendons at day 7 and day 14 postinjury in a rat model of patellar donor site injury. The mRNA expression of COL1A1, COL3A1, decorin, and biglycan, with or without supplementation of 1 ng/mL TGF-beta1, was compared by quantitative real-time RT-PCR. The expression of proliferation cell nuclear antigen (PCNA) and alpha-smooth muscle actin (alpha-SMA) was determined by immunostain. COL3A1 and decorin mRNA in CTFs was lower as compared to day 7 healing fibroblasts, but its biglycan mRNA level was higher than day 14 healing fibroblasts. TGF-beta1 increased COL1A1 and decorin mRNA in CTFs, but decreased the mRNA of all four genes in day 7 healing tendon fibroblasts. CTFs exhibited lower PCNA immunopositivity as compared to day 7 and day 14 healing fibroblasts, but a higher alpha-SMA immunopositivity than cultured day 14 healing fibroblasts. These findings showed that CTFs did not resemble healing tendon cells with respect to major cellular activities related to tendon healing. Thus, fibroblasts from healing tendon may be a more appropriate model for studying cellular activities in tendon healing.

The ultimate stress of the central one-third of the patellar tendon was studied in a gap wound-healing model in the rat. The specimens were also analyzed for collagen and nonreducible crosslinks, as measured by hydroxyproline and pyridinoline content, respectively. Thirty days after injury, the ultimate stress of the healing patellar tendon was restored to an average of 71% of the control value and remained constant over time. The pyridinoline content of the healing tendon was twice the control value by 30 days after injury and reached a plateau; however, the hydroxyproline content did not change significantly over time. Stepwise regression analysis demonstrated that pyridinoline was a better biochemical marker for ultimate stress than was hydroxyproline. The current study provides insights into the functional behaviour of the healing patellar tendon by establishing the relationship between the two biochemical components and the ultimate stress of the healing patellar tendon. This study also suggests the possibility of using pyridinoline content as an indirect marker of the ultimate stress because in vivo assessment is impossible.