Kai-Ming Chan

Mechanical loading increased BMP-2 expression which promoted osteogenic differentiation of tendon-derived stem cells†

This study aimed to investigate the effect of repetitive tensile loading on the expression of BMP‐2 and the effect of BMP‐2 on the osteogenic differentiation of tendon‐derived stem cells (TDSCs) in vitro. Repetitive stretching was applied to TDSCs isolated from rat patellar tendon at 0%, 4%, and 8%, 0.5 Hz. The expression of BMP‐2 was detected by Western blotting and qPCR. To study the osteogenic effects of BMP‐2 on TDSCs, BMP‐2 was added to the TDSC monolayer for the detection of ALP activity and calcium nodule formation in a separate experiment. TDSCs adhered, proliferated, and aligned along the direction of externally applied tensile force while they were randomly oriented in the control group. Western blotting showed increased expression of BMP‐2 in 4% and 8% stretching groups but not in the control group. Up‐regulation of BMP‐2 mRNA was also observed in the 4% stretching group. BMP‐2 increased the osteogenic differentiation of TDSCs as indicated by higher ALP cytochemical staining, ALP activity, and calcium nodule formation. Repetitive tensile loading increased the expression of BMP‐2 and addition of BMP‐2 enhanced osteogenic differentiation of TDSCs. Activation of BMP‐2 expression in TDSCs during tendon overuse might provide a possible explanation of ectopic calcification in calcifying tendinopathy.

Tendon-derived stem cells (TDSCs) promote tendon repair in a rat patellar tendon window defect model.

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 Youngs 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.

Chondrocyte Phenotype and Ectopic Ossification in Collagenase-induced Tendon Degeneration

We report chondrocyte phenotype and ectopic ossification in a collagenase-induced patellar tendon injury model. Collagenase or saline was injected intratendinously in one limb. The patella tendon was harvested for assessment at different times. There was an increase in cellularity, vascularity, and loss of matrix organization with time after collagenase injection. The tendon did not heal histologically until week 32. Ectopic mineralization as indicated by von Kossa staining started from week 8. Tendon calcification was mediated by endochondral ossification, as shown by expression of type X collagen. viva CT imaging and polarization microscopy showed characteristic bony porous structures and collagen fiber arrangement, respectively, in the calcific regions. Marrow-like cells and blood vessels were observed inside calcific deposits. Chondrocyte-like cells as indicated by morphology, expression of type II collagen, and sox 9 were seen around and embedded inside the calcific deposits. Fibroblast-like cells expressed type II collagen and sox 9 at earlier times, suggesting that erroneous differentiation of healing tendon fibroblasts may account for failed healing and ossification in collagenase-induced tendon degeneration. Because this animal model replicates key histopathological changes in calcific tendinopathy, it can be used as a model for the study of its pathogenesis at the patellar tendon.

Supplementation-time dependence of growth factors in promoting tendon healing

Growth factors potentially promote tendon healing. Understanding the right time to administer growth factors and the dosage of growth factors are prerequisites for designing effective cytokine therapy. We investigated the supplementation-time dependence of the effects of platelet-derived growth factor isoform B at various dosages on tendon healing, and the temporal responsiveness of healing tendon toward platelet-derived growth factor. Platelet-derived growth factor isoform B at various dosages (0, 10, 100, or 1000 ng) was delivered into the gap wound of rat patellar tendons via microsyringe injection on Day 3 or Day 7 after injury. Tendon specimens were harvested on Day 14 for measurement of cell proliferation, pyridinoline content, and mechanical properties. We found increased proliferative response only when the growth factor was supplemented on Day 3 after injury, whereas supplementation on Day 7 resulted in greater peak load, cross-sectional area, and pyridinoline content. The ultimate stress did not change. Our findings suggest supplementation of platelet-derived growth factor isoform B at Day 7 benefits the mechanical properties and maturation of healing tendons. We also found platelet-derived growth factor receptor β expressing cells at the remodeling site as much as 6 months after injury, suggesting healing tendon also may be responsive to long-term delivery of platelet-derived growth factor.

Increased Deposition of Sulfated Glycosaminoglycans in Human Patellar Tendinopathy

Objective:To investigate if the increased proteoglycans in patellar tendinopathy involves a qualitative change in the types of proteoglycans. Design:This is an observational study based on the biochemical analysis of proteoglycans. Setting:University Teaching Hospital. Patients:Patellar tendon samples from 12 patients with patellar tendinopathy and 12 healthy controls were collected and proteoglycans were extracted for biochemical analyses. All patients fulfilled the diagnostic criteria of having patellar tendinopathy with well-defined clinical features, more than 6 months of insufficient nonoperative treatment including physiotherapeutic modalities, and verification by ultrasound or magnetic resonance imaging. Twelve control subjects, 10 men and 2 women with an average age of 31 years (range 16 to 38 years), represented patients with anterior cruciate ligament deficiency who were operated on using the healthy patellar tendon as an autograft. The control subjects had no previous history or clinical signs of patellar tendon injury. Interventions:The independent variable is the presence of pathological conditions of patellar tendinopathy. Main Outcome Measurements:The dependent variables include the electromobility of proteoglycans, staining intensity of proteoglycan core proteins, and the tissue content of glycosaminoglycan disaccharides. Results:The results indicated that the increased proteoglycans in pathological tissues also exhibited qualitative changes as compared to those in healthy patellar tendons. Dermatan monosulfates were significantly increased in the proteoglycans extracted from the pathological tissues of patellar tendinopathy. Conclusions:Our results indicate that proteoglycans deposited in the pathological tissues of patellar tendinopathy were oversulfated as compared to healthy tendons, which may represent a new pathological attribute for the understanding of chronic pain in patellar tendinopathy.

Extracorporeal Shock Wave Therapy in Treatment of Delayed Bone-Tendon Healing

Background Extracorporeal shock wave therapy is indicated for treatment of chronic injuries of soft tissues and delayed fracture healing and nonunion. No investigation has been conducted to study the effect of shock wave on delayed healing at the bone-tendon junction. Hypothesis Shock wave promotes osteogenesis, regeneration of fibrocartilage zone, and remodeling of healing tissue in delayed healing of bone-tendon junction surgical repair. Study Design Controlled laboratory study. Methods Twenty-eight mature rabbits were used for establishing a delayed healing model at the patella–patellar tendon complex after partial patellectomy and then divided into control and shock wave groups. In the shock wave group, a single shock wave treatment was given at week 6 postoperatively to the patella–patellar tendon healing complex. Seven samples were harvested at week 8 and 7 samples at week 12 for radiologic, densitometric, histologic, and mechanical evaluations. Results Radiographic measurements showed 293.4% and 185.8% more new bone formation at the patella–patellar tendon healing junction in the shock wave group at weeks 8 and 12, respectively. Significantly better bone mineral status was found in the week 12 shock wave group. Histologically, the shock wave group showed more advanced remodeling in terms of better alignment of collagen fibers and thicker and more mature regenerated fibrocartilage zone at both weeks 8 and 12. Mechanical testing showed 167.7% and 145.1% higher tensile load and strength in the shock wave group at week 8 and week 12, respectively, compared with controls. Conclusion Extracorporeal shock wave promotes osteogenesis, regeneration of fibrocartilage zone, and remodeling in the delayed bone-to-tendon healing junction in rabbits. Clinical Relevance These results provide a foundation for future clinical studies toward establishment of clinical indication for treatment of delayed bone-to-tendon junction healing.

Ultrasound detection of trypsin-treated articular cartilage: its association with cartilaginous proteoglycans assessed by histological and biochemical methods

Abstract. We studied the correlation between histological imaging quantification and the biochemical assessment of proteoglycan (PG) content in articular cartilage in vitro, which served as a basis for the validation of ultrasound detection as a noninvasive tool in the assessment of PG changes in full-thickness articular cartilage. Articular cartilage of 14 intact fresh bovine femoral condyles was used for trypsin digestion. Full-thickness articular cartilage cylinders, 3 mm in diameter, were harvested at time intervals of 0.5, 1, 2, and 3 h after trypsin digestion. Each cartilage cylinder was then cut into two equal parts for either histomorphometric quantification of the PG area fraction stained with Safranine O or conventional biochemical assessment of uronic acid content. In addition, five fresh mature bovine patellae were used for the validation of an ultrasound compression system developed for testing the potential layered biomechanical properties of articular cartilage, including the equilibrium compressive modulus, i.e., the slope of the linear regression of the equilibrium stress-strain curve. Results showed that PG content in the articular cartilage was significantly decreased with increasing time of trypsin digestion, both histologically and biochemically, with a significant correlation of r= 0.502 (P < 0.001). Ultrasound measurements demonstrated differences in the equilibrium compressive moduli of the digested zone, the undigested zone, and the entire articular cartilage layer, as well as a characteristically large ultrasound reflection signal detected in the interface of the trypsin digestion front of articu-lar cartilage. The results of this study suggested that the histomorphometric quantification of PG content could be used to reflect not only PG quantity but also its spatial distribution; also, the ultrasound compression system might have potential for the non-invasive detection of pathological changes in articular cartilage.

Ectopic chondro-ossification and erroneous extracellular matrix deposition in a tendon window injury model.

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.

Area, length and mineralization content of new bone at bone-tendon junction predict its repair quality.

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.