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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. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 30:37–46, 2012
Tendons regenerate and repair slowly and inefficiently after injury. The crimp pattern of collagen fibers and fibrils was smaller than that of the control1 and the regenerated fibrotic scar tissue could not return to its original mechanical strength for a long time after injury.1–3 It was known that collagens and proteoglycans in the extracellular matrix (ECM) have roles in modulating the activities of tenocytes in addition to their structural roles.4 Despite studies about the tendon healing process, there is still limited and inconsistent understanding about the change in biochemical composition of ECM after tendon injury5–7 and how does the ECM regulate cellular functions. On the other hand, ectopic ossification after midpoint tenotomy of rat or mouse Achilles tendon has been reported.8–12 Clinical studies have occasionally reported ectopic calcification after Achilles tendon rupture13, 14 as well as calcification and tendinopathic-like changes in patellar tendon donor site after anterior cruciate ligament (ACL) reconstruction.15–21 We hypothesized that chondro-osteogenesis and change in the ECM composition of the repair tissue after acute tendon injury might contribute to the poor tissue quality. This study therefore aimed to examine the presence of chondrocyte phenotype and ossification inside the window wound of the patellar tendon. The spatial-temporal changes of major collagens including collagen types I and III and major proteoglycans including decorin, biglycan, fibromodulin, and aggrecan in the window wound after tendon injury were also investigated.
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Despite the vast amount of research on tendon healing, much remained unknown about the process. Our result showed that chondrocyte-like cells were observed in 33% samples starting at week 4 after injury while ectopic ossification surrounded by chondrocyte-like cells was observed in 50% of samples at week 12. The ectopic chondrogenesis and ossification observed were not due to technical errors due to harvesting of the tendon–bone junction as the window wound was created without damaging the tendon–bone injunction. We tried to avoid the tendon–bone junction during sample collection. Although the normal tendon–bone junction could be observed partly in a few samples, they were not included in all analyses. The chondrocyte-like cells and the ossified deposits were observed only in the mid-substances of window wound of patellar tendon and there was no specific localization of the chondrocyte-like cells and ossified deposits in the tendon mid-substances, supporting that they were unlikely to be originated from the intact tendon–bone junction. The chondrocyte-like cells and ossified deposits inside wound were confirmed to be in the tendon mid-substance at low magnification of the histological images. There was no difference in the generation of defect in the animals that showed ectopic chondrogenesis and ossification. The ossified deposits inside wound were formed by endochondral ossification as shown by the expression of collagen type X. Ectopic ossification after midpoint tenotomy of rodent Achilles tendon has been reported in previous studies.8–12 We have not performed this surgery in other species of animal. Whether ectopic chondrogenesis and ossification occur only in rodent tendons after injury is not clear and this study should be repeated in other species of animal to confirm the present findings. However, calcification was also reported clinically in the follow-up of some, but not all, patients with Achilles tendon rupture13 and open Achilles tendon repair.14 The erroneous deposition of ECM as a result of the presence of chondrocyte-like cells and ossification might negatively impact the material property of patellar tendon after acute injury. The presence of ossified deposits in tendon could theoretically increase the stiffness. Both could decrease the tensile strength of the repair tissue. We observed more ossification after 6 months post-injury (Supplementary S3) though the sample size was small, suggesting that chondrocyte phenotype and ossification persisted for at least 6 months after injury.
Review of the literatures showed that calcification of patellar tendon, in fact, has been reported in some patients with ACL reconstruction.15, 16, 21 Other tendinopathic-like features such as mild degenerative changes,18 increase in cellularity and vascularity,17 hypoechogeneity, and hyperechogeneity,15, 20, 21 increase in tendon width15, 17–20, 21 as observed with MRI and ultrasonography have also been reported. Bayar et al.15 further described their patients having changes similar to tendinopathy in 6 out of 20 patients. This suggested that acute injury as created by the removal of the central one-third of patellar tendon could develop tendinopathic-like changes. Acquisition of chondrocyte phenotypes and ectopic ossification has been reported in clinical samples and animal models of tendinopathy.23, 25 Given unfavorable factors, acute injury might develop tendinopathic-like changes.
We observed transient increases in the expression of collagen types III and I inside wound after injury, consistent with repair. The increase in the expression of collagen types I and III after tendon injury has also been reported in previous studies.5–7 We observed sustained expression of biglycan and reduced expression of aggrecan and decorin in the tendon matrix inside wound in our injury model. The decrease in the expression of decorin after injury was consistent with previous work studying the mRNA expression of decorin after tendon injury5, 6 but inconsistent with the observation of others.7 Our result was also consistent with the results of Berglund et al.5 whom reported sustained mRNA expression of biglycan in tendon and tendon sheath after injury but was inconsistent with them on the expression of aggrecan. The discrepancy might be due to different injury models, length of follow-up and the assessment method. The turnover of mRNA after injury might not follow exactly the changes of protein expression. Despite the differences, the change in the composition of proteoglycans was confirmed and the alteration in composition of the ECM might contribute to reduced mechanical property of the repair tissue as reported in our previous study.2
Biglycan and decorin both belong to class I of small leucine-rich proteoglycan (SLRP) family. They are highly homologous and co-expressed in various tissues. Young et al.26 reported that there was higher expression of decorin in tissues from the biglycan knock-out mouse model compared to that in wild-type animals, suggesting that these two related class I SLRPs could share common functions and, possibly, compensate for each other's functions when the other one was absent. The sustained expression of biglycan in the healing tissue in our study might be to compensate for the reduced expression of decorin in the healing tissue after injury. The reciprocal changes in decorin and biglycan was also reported in a transected unrepaired rabbit medial collateral ligament model, which the expression of decorin was barely detectable while the expression of biglycan accumulated and the post-translational modification of biglycan was altered in the repairing ligament over 2 years.27
Young et al.26 reported that knock-down of biglycan in a mouse model resulted in low bone mass and biglycan was essential for bone formation while the knock-down of decorin in a mouse model resulted in normal bone mass. The increase in biglycan in the repair tissue therefore might contribute to the formation of ossified deposits in our animal model. Young et al.26 also reported that biglycan knock-out mice displayed larger irregular fibrils while decorin knock-out mice displayed smaller fibrils in bone. This supported our hypothesis that the sustained expression of biglycan and reduced expression of decorin in the healing tendon as observed in our animal model might be associated with smaller fibrils and hence poor quantity of healing after injury. The accumulation of biglycan might interfere with proper collagen network remodeling as decorin and biglycan could compete for binding on collagen type I.28 We observed earlier expression of Sox 9 and collagen type II in healing tendon fibroblasts and this preceded their expression in the chondrocyte-like cells and ossified area. The alteration of ECM composition and growth factors after injury might favor erroneous chondrogenic and osteogenic differentiation of tendon progenitor cells to chondrocytes and osteoblasts, respectively, and negatively affect the mechanical property of the regenerated tissue.23, 29 Aggrecan and biglycan, the key ECM proteins of cartilaginous tissue, were highly expressed in the chondrocyte-like cells and ossified deposits at week 12 in our study. Apart from their roles in ECM organization, proteoglycans could have roles in modulating the activity of tendon cells.4 There was rapid upregulation of expression of proteoglycans during chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs).30 The expression of collagen types II and X occurred only at the late stage of the process, suggesting the importance of proteoglycans in modulating stem cell differentiation.30 The differentiation of marrow stromal cells was reported to depend on the expression of biglycan.26 We have reported increased expression of BMP-2 in the same patellar tendon window injury model, which might contribute to erroneous cell differentiaion.22 Regarding the possible source of tendon progenitor cells undergoing erroneous differentiation, it may be BMSCs which migrated into the wound or tendon-derived stem cells (TDSCs) identified recently.31, 32 BMP-2 has been reported to induce osteogenic32 and chondrogenic (unreported observation) differentiation of TDSCs in vitro. Tendon stem cells isolated from the biglycan and fibromodulin double knock-out mouse model were also reported to show increased sensitivity to BMP-2 signaling.31
Based on our observation, the expression of ECM in the intact region outside the window wound was similar to that in the intact contralateral control except that the expression of fibromodulin seemed to be slightly higher while the expression of aggrecan seemed to be slightly lower in the adjacent normal region compared to those in the intact contralateral control. However, we have not semi-quantified the expression levels in adjacent normal region and it would be interesting to look at it in future study.
In conclusion, ectopic chondrogenesis and ossification were observed inside wound in 50% samples at week 12. There was sustained expression of biglycan and reduced expression of aggrecan and decorin in the tendon matrix in the window wound. The erroneous deposition of ECM and ectopic chondro-ossification, both might influence each other, might account for the poor tissue quality after acute injury. Higher expression of biglycan and aggrecan at the ectopic chondro-ossification sites suggested that they might have roles in ectopic chondro-osteogenesis in the repair tissue.