Differential MMP 1 and MMP 13 expression in proliferation and ligamentization phases of graft remodeling in anterior cruciate ligament reconstruction.

Purpose: Graft remodeling in anterior cruciate ligament reconstruction (ACLR) demonstrates three distinct phases: necrosis, proliferation and ligamentization. Biological enhancement involves modulating these processes, but the cellular activities related to extracellular matrix remodeling have not been investigated. We hypothesized that changes in matrix metalloproteinases (MMPs) 1 and 13 expression are involved in the transition of proliferation phase to ligamentization phase of graft remodeling.Materials and methods: Thirty-three rats underwent ACLR. Tendon grafts were harvested at week 1 (necrosis), 2 (proliferation), or 12 (ligamentization) post-operation for histological examination (n = 3), or for isolation of graft-derived cells (n = 8) for flow cytometry, proliferation assay, cell invasion assay, measurement of gene expression related to matrix remodeling (Col1A1, Col3A1, MMP1, tissue inhibitor of marix metalloproteinase 1 (TIMP1), and MMP13) and total MMP activities.Results: Increased cellularity in tendon graft was contributed by active cell proliferation and migration at week 2 post-operation, while decreased cellularity were paralleled by increased apoptosis at week 12. All genes measured (Col1A1, Col3A1, MMP1, TIMP1, and MMP13) increased significantly in week 2 cells compared to week 1 cells. MMP1 expression subsided at week 12, while MMP13 expression kept increasing till 12 weeks post-operation. Total MMP activities was 3-fold higher in cultured graft-derived cells from week 2 as compared to cells from week 12. Two distinct processes of graft remodeling were identified, characterized by increased MMP1 expression with cell proliferation and increased MMP13 expression with cell apoptosis.Conclusions: Unfavorable matrix remodeling during the proliferation phase is found with increased MMP1, while remodeling leading to ligamentization is associated with increased MMP13 expression.

Transplantation of tendon-derived stem cells pre-treated with connective tissue growth factor and ascorbic acid in vitro promoted better tendon repair in a patellar tendon window injury rat model.

BACKGROUND AIMS: Treatment of tendon-derived stem cells (TDSCs) with connective tissue growth factor (CTGF) and ascorbic acid promoted their tenogenic differentiation. We investigated the effects of TDSCs pre-treated with CTGF and ascorbic acid on tendon repair in a patellar tendon window injury rat model. METHODS: Green fluorescent protein-TDSCs (GFP-TDSCs) were pre-treated with or without CTGF and ascorbic acid for 2 weeks before transplantation. The patellar tendons of rats were injured and divided into three groups: fibrin glue-only group (control group), untreated and treated TDSC group. The rats were followed up until week 16. RESULTS: The treated TDSCs accelerated and enhanced the quality of tendon repair compared with untreated TDSCs up to week 8, which was better than that in the controls up to week 16 as shown by histology, ultrasound imaging and biomechanical test. The fibrils in the treated TDSC group showed better alignment and larger size compared with those in the control group at week 8 (P = 0.004). There was lower risk of ectopic mineralization after transplantation of treated or untreated TDSCs (all P ≤ 0.050). The transplanted cells proliferated and could be detected in the window wound up to weeks 2 to 4 and week 8 for the untreated and treated TDSC groups, respectively. CONCLUSIONS: The transplantation of TDSCs promoted tendon repair up to week 16, with CTGF and ascorbic acid pre-treatment showing the best results up to week 8. Pre-treatment of TDSCs with CTGF and ascorbic acid may be used to further enhance the rate and quality of tendon repair after injury.

Peri-tunnel bone loss: does it affect early tendon graft to bone tunnel healing after ACL reconstruction?

PURPOSE: The clinical relevance and mechanisms of local bone loss early post-anterior cruciate ligament (ACL) reconstruction remain unclear. The early spatial and temporal changes of peri-tunnel bone, its molecular mechanisms and its relationships with graft-bone tunnel healing were investigated in a 12-week-old rat model. METHODS: At various times, the reconstructed ACL complex was harvested for vivaCT imaging, biomechanical test, histology and immunohistochemical staining of CD68+ cells (a monocyte-macrophage lineage marker), MMP1 and MMP13. RESULTS: The peri-tunnel bone resorbed simultaneously with improvement of graft-bone tunnel healing. There were 30.1 ± 17.4, 46.8 ± 10.5 and 81.5 ± 12.3 % loss of peri-tunnel BMD as well as 43.2 ± 21.7, 78.7 ± 8.5 and 92.4 ± 17.7 % loss of peri-tunnel bone volume/total volume (BV/TV) at week 6 at the distal femur, epiphysis and metaphysis of tibia, respectively. MMP1, MMP13 and CD68+ cells were expressed at the graft-bone tunnel interface and peri-tunnel bone and increased with time post-reconstruction at the tibia. The ultimate load and stiffness of the healing complex positively correlated with tibial tunnel bone formation and negatively correlated with tibial peri-tunnel bone. Tunnel BV/TV at the tibial metaphysis and epiphysis showed the highest correlation with ultimate load (ρ = 0.591; p = 0.001) and stiffness (ρ = 0.427; p = 0.026) of the complex, respectively. CONCLUSION: There was time-dependent loss of peri-tunnel bone early post-reconstruction, with the greatest loss occurring at the tibial metaphysis. This was consistent with high expression of MMP1, MMP13 and CD68+ cells at the graft-bone tunnel interface and the peri-tunnel region. The significant loss of peri-tunnel bone, though not critically affecting early tunnel healing, suggested the need to protect the knee joint early post-reconstruction.

Expression of Wnt pathway mediators in metaplasic tissue in animal model and clinical samples of tendinopathy.

OBJECTIVE: Tissue metaplasia is observed in both ossified failed healing animal model and clinical samples of tendinopathy. The Wnt signalling pathway plays a vital role in pathological calcification. We hypothesized that the Wnt signalling pathway might contribute to tissue metaplasia and failed healing in tendinopathy. This study aimed to examine the spatial-temporal expression of Wnt pathway mediators in an ossified failed tendon healing animal model and clinical samples of tendinopathy. The effect of Wnt3a on the osteogenic differentiation of tendon-derived stem cells (TDSCs) was also examined. METHODS: Ossified failed tendon healing was induced by the injection of collagenase into the patellar tendon of rats. At various times the tendons were harvested for immunohistochemical staining of Wnt3a, ß-catenin, Lrp5 and Tcf1. Patellar tendon samples were obtained from 13 patients with patellar tendinopathy (11 unossified and 2 ossified) and 10 controls. Immunohistochemical staining of Wnt3a, ß-catenin, Lrp5 and Tcf1 was similarly performed. Rat patellar TDSCs were treated with Wnt3a. The osteogenic differentiation of TDSCs was examined by ALP activity, alizarin red S staining and mRNA expression of osteogenic markers. RESULTS: There was increased expression of Wnt3a, ß-catenin, Lrp5 and Tcf1 in the healing fibroblast-like cells, chondrocyte-like cells and ossified deposits in the animal model and in some clinical samples of tendinopathy. Wnt3a increased ALP activity, calcium nodule formation and expression of osteogenic markers in TDSCs. CONCLUSION: Activation of the Wnt signalling pathway and its effect on TDSCs might contribute to tissue metaplasia and failed healing in some cases of tendinopathy.

Bioactive Decellularized Tendon-Derived Stem Cell Sheet for Promoting Graft Healing After Anterior Cruciate Ligament Reconstruction.

BACKGROUND: Stem cell sheets provide a scaffold-free option for the promotion of graft healing after anterior cruciate ligament reconstruction (ACLR). However, cell viability, stability, and potential uncontrolled actions create challenges for clinical translation. The decellularization of cell sheets may overcome these problems as studies have shown that the natural extracellular matrix of stem cells is bioactive and can promote tissue repair. HYPOTHESIS: The decellularized tendon-derived stem cell (dTDSC) sheet can promote graft healing after ACLR. STUDY DESIGN: Controlled laboratory study. METHODS: An optimized decellularization protocol was developed to decellularize the TDSC sheets. A total of 64 Sprague-Dawley rats underwent ACLR with or without the dTDSC sheet wrapping the tendon graft (n = 32/group). At 2 and 6 weeks after surgery, graft healing was assessed by micro-computed tomography, histology, and biomechanical testing. The accumulation of iNOS+ and CD206+ cells and the expression of metalloproteinase 1 (MMP-1), MMP-13, and tissue inhibitor of metalloprotease 1 (TIMP-1) were assessed by immunohistochemistry. RESULTS: The decellularization was successful, with the removal of 98.4% nucleic acid while preserving the collagenous proteins and bioactive factors. The expression of bone morphogenetic protein 2 (BMP-2) and VEGF in the dTDSC sheet was comparable with the TDSC sheet (P > .05). Micro-computed tomography showed significantly more tunnel bone formation in the dTDSC sheet group. The dTDSC sheet group demonstrated better graft osteointegration and higher integrity of graft midsubstance with significantly higher ultimate failure load (16.58 ± 7.24 vs 8.93 ± 2.45 N; P = .002) and stiffness (11.97 ± 5.21 vs 6.73 ± 2.20 N/mm; P = .027). Significantly fewer iNOS+ cells but more CD206+ cells, as well as lower MMP-1 and MMP-13 but higher TIMP-1 expression, were detected at the tendon-bone interface and graft midsubstance in the dTDSC sheet group. CONCLUSION: An optimized decellularization protocol for producing bioactive dTDSC sheets was developed. Wrapping tendon graft with a dTDSC sheet promoted graft healing after ACLR, likely via enhancing bone formation and angiogenesis by BMP-2 and VEGF, modulating macrophage polarization and MMP/TIMP expression, and physically protecting the tendon graft. CLINICAL RELEVANCE: dTDSC sheets alleviate the quality control and safety concerns of cell transplantation and can be used as a cell-free alternative for the promotion of graft healing in ACLR.

Rat Plantar Fascia Stem/Progenitor Cells Showed Lower Expression of Ligament Markers and Higher Pro-Inflammatory Cytokines after Intensive Mechanical Loading or Interleukin-1ß Treatment In Vitro.

The pathogenesis of plantar fasciitis is unclear, which hampers the development of an effective treatment. The altered fate of plantar fascia stem/progenitor cells (PFSCs) under overuse-induced inflammation might contribute to the pathogenesis. This study aimed to isolate rat PFSCs and compared their stem cell-related properties with bone marrow stromal cells (BMSCs). The effects of inflammation and intensive mechanical loading on PFSCs' functions were also examined. We showed that plantar fascia-derived cells (PFCs) expressed common MSC surface markers and embryonic stemness markers. They expressed lower Nanog but higher Oct4 and Sox2, proliferated faster and formed more colonies compared to BMSCs. Although PFCs showed higher chondrogenic differentiation potential, they showed low osteogenic and adipogenic differentiation potential upon induction compared to BMSCs. The expression of ligament markers was higher in PFCs than in BMSCs. The isolated PFCs were hence PFSCs. Both IL-1ß and intensive mechanical loading suppressed the mRNA expression of ligament markers but increased the expression of inflammatory cytokines and matrix-degrading enzymes in PFSCs. In summary, rat PFSCs were successfully isolated. They had poor multi-lineage differentiation potential compared to BMSCs. Inflammation after overuse altered the fate and inflammatory status of PFSCs, which might lead to poor ligament differentiation of PFSCs and extracellular matrix degeneration. Rat PFSCs can be used as an in vitro model for studying the effects of intensive mechanical loading-induced inflammation on matrix degeneration and erroneous stem/progenitor cell differentiation in plantar fasciitis.

Expression of chondro-osteogenic BMPs in clinical samples of patellar tendinopathy.

PURPOSE: The pathogenesis of patellar tendinopathy remains unclear. Expression of BMP-2/-4/-7 was reported in an ossified failed tendon healing animal model of patellar tendinopathy. This study aimed to investigate the expression of these chondro-osteogenic BMPs in clinical samples of patellar tendinopathy. METHODS: Patellar tendon samples were collected from 16 consecutive patients with patellar tendinopathy and 16 consecutive controls undergoing anterior cruciate ligament reconstruction with bone-patellar tendon-bone autograft in the authors' hospital after getting their consent. The expression of BMP-2/-4/-7 was examined in all samples using immunohistochemistry. Ossification observed in two tendinopathy samples was characterized by histology, alizarin red S staining, alcian blue staining, TRAP staining and immunohistochemical staining of Sox9, osteopontin (OPN) and osteocalcin (OCN). RESULTS: Regions of hypo- and hyper-cellularity and vascularity, with loss of crimp structure of collagen matrix, were observed in patellar tendinopathy samples. Round cells and in some cases, cells with typical chondrocyte phenotype were observed. For the ossified tendinopathy samples with positive alizarin red S staining, OPN-positive and Sox9-positive chondrocyte-like cells in alcian blue-stained extracellular matrix, OCN-positive osteoblast-like cells and TRAP-positive multi-nucleated cells were observed around the ossified deposits. No expression of BMP-2/-4/-7 was observed in healthy patellar tendons. However, the expression of BMP-2/-4/-7 was observed in all patellar tendinopathy samples with or without ossification. CONCLUSIONS: Clinical samples of patellar tendinopathy showed ectopic expression of BMP-2/-4/-7. This was not evident in control samples from healthy patellar tendons. LEVEL OF EVIDENCE: Prognostic studies, Level III.

Sustained expression of proteoglycans and collagen type III/type I ratio in a calcified tendinopathy model.

OBJECTIVES: Alteration in the composition of extracellular matrix has been suggested as the major factor for the development of tendinopathy and calcified tendinopathy, which has poorer clinical manifestation. This study investigated the changes of major proteoglycans and collagens in a calcified tendinopathy model and correlated the expression with the acquisition of chondrocyte phenotype, ectopic ossification and loss of matrix organization in the same model. METHODS: Thirty-six rats were used. Collagenase or saline was injected into the patellar tendons of each rat. At Weeks 2, 4 and 12, samples were used for immunohistochemistry of major proteoglycans and collagens and mRNA quantification. RESULTS: An increase in collagen type III and I expression was observed after injury at Week 2. Although their levels diminished with time, the ratio of collagen type III to collagen type I remained higher than that in healthy tendon at Week 12. The expression of biglycan, fibromodulin and aggrecan increased with time, whereas the expression of decorin was sustained from Week 2 to Week 12. The expression of major proteoglycans and collagens was observed in the tendon cells and matrix at Week 2 and became localized at the chondrocyte-like cells around the calcific deposits at Week 12. CONCLUSION: Sustained expression of proteoglycans and a high collagen type III/collagen type I ratio might account for poor matrix organization in calcified tendinopathy. The localization of major proteoglycans around chondro-osseous region might indicate interference of collagen assembly, which favours ectopic chondrogenesis, ossification and predisposition to tendon rupture.

In vivo low-intensity pulsed ultrasound (LIPUS) following tendon injury promotes repair during granulation but suppresses decorin and biglycan expression during remodeling.

STUDY DESIGN: Bench research, cross-sectional. OBJECTIVE: To determine if the effects of low-intensity pulsed ultrasound (LIPUS) on matrix synthesis change at different stages of tendon healing. BACKGROUND: LIPUS is effective in promoting tendon healing by stimulation of matrix synthesis. The timing of initiation and duration of LIPUS treatment have been shown to affect its effectiveness to promote tendon healing, suggesting a change of tissue responses to LIPUS stimulation. Understanding how the cellular responses to LIPUS stimulation change during tendon healing is thus important. METHODS: In a rat model of patellar tendon donor site injury, a single sonication of LIPUS or mock sonication was delivered to the injured knee of the rats on the fourth, 14th or 28th day postinjury. Tendon samples were harvested at 4 hours and 24 hours after sonication and the mRNA expression of COL1A1, COL3A1, decorin, biglycan, and TGF-beta 1 was analyzed. RESULTS: The results showed that a single sonication of LIPUS increased COL1A1 and COL3A1 mRNA in healing patellar tendons when administered on the fourth or 14th day postinjury, but not when administered on the 28th day postinjury. Both decorin and biglycan mRNA were decreased by treatment with LIPUS on the 28th day postinjury. Our results showed that LIPUS enhanced collagen synthesis in vivo only during the granulation phase. Matrix remodeling may be affected by LIPUS with the suppressed expression of decorin and biglycan. CONCLUSION: Our findings suggest that LIPUS should be applied during the granulation phase but not during the remodeling phase, to promote tendon healing.

Inferior tendon graft to bone tunnel healing at the tibia compared to that at the femur after anterior cruciate ligament reconstruction.

BACKGROUND: Tunnel widening after anterior cruciate ligament (ACL) reconstruction (ACLR) is commonly reported without a clear understanding of the mechanism. This study aimed to quantify the spatiotemporal change of the newly formed bone mass, bone tunnel diameter, and area along both bone tunnels using micro-computed tomography (microCT) and correlated the result with histology. METHODS: ACLR was performed in 24 rabbits. At baseline and weeks 2, 6, and 12, the juxta-articular, middle, and exit segments of both tunnels were harvested for microCT and histological evaluation. RESULTS: microCT and histology revealed significant bone tunnel and graft-bone tunnel healing, respectively, only at week 6 after reconstruction. Despite this, the mean tunnel diameter and area remained relatively unchanged with time. The newly formed bone mass [new bone volume/total bone volume (BV/TV) ratio] and its bone mineral density (BMD) were both higher, whereas the mean tunnel diameter and area were significantly smaller at the femoral tunnel compared to those at the tibial tunnel at weeks 6 and 12 and at week 12, respectively. These were consistent with histological findings, which showed inferior graft remodeling and integration at the tibial tunnel at weeks 6 and 12. The BV/TV increased, whereas the mean tunnel diameter and area decreased toward the exit segment of both tunnels. However, whereas better histological healing occurred at the femoral exit segment, poorer graft remodeling and Sharpey's fiber formation occurred at the tibial exit segment. CONCLUSIONS: Poor healing was observed during the initial 6 weeks, particularly that of the tibia, after ACLR. Bone resorption was rapid during healing, resulting in unchanged tunnel diameter and area with time.

The role of ubiquitin linkages on alpha-synuclein induced-toxicity in a Drosophila model of Parkinson's disease.

Parkinson's disease (PD) is a common movement disorder marked by the loss of dopaminergic (DA) neurons in the brain stem and the presence of intraneuronal inclusions designated as Lewy bodies (LB). The cause of neurodegeneration in PD is not clear, but it has been suggested that protein misfolding and aggregation contribute significantly to the development of the disease. Misfolded and aggregated proteins are cleared by ubiquitin proteasomal system (UPS) and autophagy lysosomal pathway (ALP). Recent studies suggested that different types of ubiquitin linkages can modulate these two pathways in the process of protein degradation. In this study, we found that co-expression of ubiquitin can rescue neurons from alpha-syn-induced neurotoxicity in a Drosophila model of PD. This neuroprotection is dependent on the formation of lysine 48 polyubiquitin linkage which is known to target protein degradation via the proteasome. Consistent with our results that we observed in vivo, we found that ubiquitin co-expression in the cell can facilitate cellular protein degradation by the proteasome in a lysine 48 polyubiquitin-dependent manner. Taken together, these results suggest that facilitation of proteasomal protein degradation can be a potential therapeutic approach for PD.

Local administration of Trolox, a vitamin E analog, reduced tendon adhesion in a chicken model of flexor digitorum profundus tendon injury.

BACKGROUND: Hand flexor tendon injuries are compromised with tendon adhesion. Tendon adhesion forms between flexor tendon and tendon sheath, reduces the range of motion of fingers, and affects their function. Oxidative stress is increased in flexor tendon after injury and might play a role in tendon adhesion formation. Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), a water-soluble analog of vitamin E, is antioxidative. Trolox reduced oxidative stress and the expression of fibrotic cytokines in the bile gut ligation animal model. Vitamin C and Trolox are strong antioxidants, but they might also have prooxidant properties. The prooxidant properties of vitamin C and Trolox are different. In this study, our aim was to determine the effect of Trolox in reducing tendon adhesion formation. METHODS: Flexor digitorum profundus tendon injury was induced in 54 Kai-Mei Chicken according to a well-established protocol. After wound closure, an injection of 50 µL saline, 10mM Trolox, or 100mM Trolox was administered into the wound area. At 2 weeks or 6 weeks after the surgery, chicken feet were harvested for gliding test, high-resolution ultrasound measurement on a fibrotic area, and histology. RESULTS: At Week 2 after the surgery, Trolox has no effect on the flexion angle and gliding resistance, whereas a significant improvement was observed in the flexion angle and gliding resistance in the Trolox-treated groups at Week 6. However, no dose response was observed. In the ultrasound measurement, there was no significant difference in the fibrotic mass in the Trolox-treated group as compared to the saline group at Week 2. At Week 6, fibrotic mass was significantly reduced in both Trolox-treated groups. From the histological examination, the Trolox-treated groups presented a higher cellularity at Week 2 as compared to the saline group, and reduced fibrosis and adhesion at Week 6. CONCLUSION: Our results suggest that local administration of Trolox can reduce tendon adhesion, and a higher dose of Trolox did not have negative effects. CLINICAL SIGNIFICANCE: Trolox solution might be feasible to reduce tendon adhesion via intraoperative injection at the wound area during tendon repair.

Effects of Redox Modulation on Cell Proliferation, Viability, and Migration in Cultured Rat and Human Tendon Progenitor Cells.

Tendon healing is slow and usually results in inferior fibrotic tissue formation. Recently, application of tendon derived stem cells (TDSCs) improved tendon healing in animal studies. In a chicken model, local injection of antioxidants reduced tendon adhesion after tendon injury. An in vitro study demonstrated that supplementation of H2O2 reduced tenogenic marker expression in TDSCs. These findings suggested that the possibility of TDSCs is involved in tendon healing and the cellular activities of TDSCs might be affected by oxidative stress of the local environment. After tendon injury, oxidative stress is increased. Redox modulation might affect healing outcomes via affecting cellular activities in TDSCs. To study the effect of oxidative stress on TDSCs, the cellular activities of rat/human TDSCs were measured under different dosages of vitamin C or H2O2 in this study. Lower dose of vitamin C increased cell proliferation, viability and migration; H2O2 affected colony formation and suppressed cell migration, cell viability, apoptosis, and proliferation. Consistent with previous studies, oxidative stresses (H2O2) affect both recruitment and survival of TDSCs, while the antioxidant vitamin C may exert beneficial effects at low doses. In conclusion, redox modulation affected cellular activities of TDSCs and might be a potential strategy for tendon healing treatment.

Application of tendon-derived stem cell sheet for the promotion of graft healing in anterior cruciate ligament reconstruction.

BACKGROUND: Both osteointegration and remodeling of graft midsubstance (collectively called graft healing) are slow processes after anterior cruciate ligament (ACL) reconstruction. Tendon-derived stem cells (TDSCs) form a cell sheet after treatment with connective tissue growth factor (CTGF) and ascorbic acid, which exhibits higher tenogenic and maintains high chondro-osteogenic gene expression of TDSCs. No external scaffold is required for cell delivery. HYPOTHESIS: Wrapping the TDSC sheet around the ACL graft would promote early graft healing in a rat model. STUDY DESIGN: Controlled laboratory study. METHODS: Green fluorescent protein (GFP) rat TDSCs were treated with connective tissue growth factor and ascorbic acid to promote cell sheet formation. Rats undergoing unilateral ACL reconstruction were divided into a control group and a TDSC group. The tendon graft was wrapped with the GFP-TDSC sheet before graft insertion in the TDSC group. At weeks 2, 6, and 12 after reconstruction, the samples were harvested for computed tomography imaging and histologic or biomechanical testing. The fate of the transplanted cell sheet was examined by immunohistochemical staining of GFP. RESULTS: There were significantly higher tunnel bone mineral density (BMD) (42.3% increase, P = .047) and bone volume/total volume (BV/TV) (625% increase, P = .009) at the metaphyseal region of the tibial tunnel at week 2 and at the femoral tunnel at week 6 (BMD: 30.8% increase, P = .014; BV/TV: 100% increase, P = .014) in the TDSC group compared with the control group. Only the TDSC group showed a time-dependent increase in tunnel BMD (overall P = .038) and BV/TV (overall P = .015) at the epiphyseal region of the tibial tunnel. Semiquantitative image analysis showed better graft osteointegration and higher intra-articular graft integrity with lower cellularity and vascularity, better cell alignment, and higher collagen birefringence in the TDSC group. The ultimate load at week 2 (52.5% increase, P = .027) and stiffness at week 6 (62% increase, P = .008) were significantly higher in the TDSC group. Cells positive for GFP were observed in all samples in the TDSC group at week 2 but became reduced with time after reconstruction. CONCLUSION: The TDSC sheet improved early graft healing after ACL reconstruction in the rat model. CLINICAL RELEVANCE: The TDSC sheet could potentially be used for the promotion of graft healing in ACL reconstruction.

Scx-transduced tendon-derived stem cells (tdscs) promoted better tendon repair compared to mock-transduced cells in a rat patellar tendon window injury model.

We hypothesized that the transplantation of Scx-transduced tendon-derived stem cells (TDSCs) promoted better tendon repair compared to the transplantation of mock-transduced cells. This study thus aimed to investigate the effect of Scx transduction on the expression of lineage markers in TDSCs and the effect of the resulting cell line in the promotion of tendon repair. Rat non-GFP or GFP-TDSCs were transduced with Scx or empty lentiviral vector (Mock) and selected by blasticidin. The mRNA expressions of Scx and different lineage markers were examined by qRT-PCR. The effect of the transplantation of GFP-TDSC-Scx on tendon repair was then tested in a rat unilateral patellar tendon window injury model. The transplantation of GFP-TDSC-Mock and scaffold-only served as controls. At week 2, 4 and 8 post-transplantation, the repaired patellar tendon was harvested for ex vivo fluorescent imaging, vivaCT imaging, histology, immunohistochemistry and biomechanical test. GFP-TDSC-Scx consistently showed higher expressions of most of tendon- and cartilage- related markers compared to the GFP-TDSC-Mock. However, the effect of Scx transduction on the expressions of bone-related markers was inconclusive. The transplanted GFP-TDSCs could be detected in the window wound at week 2 but not at week 4. Ectopic mineralization was detected in some samples at week 8 but there was no difference among different groups. The GFP-TDSC-Scx group only statistically significantly improved tendon repair histologically and biomechanically compared to the Scaffold-only group and the GFP-TDSC-Mock group at the early stage of tendon repair. There was significant higher expression of collagen type I in the window wound in the GFP-TDSC-Scx group compared to the other two groups at week 2. The transplantation of GFP-TDSC-Scx promoted healing at the early stage of tendon repair in a rat patellar tendon window injury model.

Immunogenicity and escape mechanisms of allogeneic tendon-derived stem cells.

The immunogenicity of tendon-derived stem cells (TDSCs) has implications for their clinical use for the promotion of tendon repair. The immunogenicity and escape mechanisms of rat patellar TDSCs were examined after allogeneic transplantation. Our results showed that TDSCs exhibited low immunogenicity as evidenced by the following: (i) the incubation of target TDSCs with immunized serum did not show antibody recognition and did not induce the complement-dependent cytotoxicity; (ii) target TDSCs elicited a very low level of lymphocyte proliferation and did not exhibit host lymphocyte-mediated cytotoxicity; and (iii) target TDSCs dose dependently suppressed the phorbol 12-myristate 13-acetate (PMA)- and ionomycin-induced host lymphocyte proliferation. For the mechanistic studies, TDSCs expressed major histocompatibility complex (MHC)-I but a very low level of MHC-II, CD86 and CD80 for the induction of T-cell response. Also, TDSCs were found to express intracellular Fas and FasL. γ-IFN pretreatment did not increase the level of MHC-II and CD86 for the upregulation of immune response. Moreover, the immunosuppressive mediators indoleamine 2,3-dioxygenase (IDO) and transforming growth factor-beta 1 (TGF-ß1) were found not to be involved in the escape mechanism of target TDSCs from host lymphocyte attack. In conclusion, allogeneic TDSCs exhibited low immunogenicity. Allogeneic TDSCs might be used for transplantation.

Allogeneic tendon-derived stem cells promote tendon healing and suppress immunoreactions in hosts: in vivo model.

The medium- to long-term healing effect and infiltration of inflammatory cells, after transplantation of allogeneic tendon-derived stem cell (TDSC) to the rat patellar tendon window wound, were examined. Allogeneic patellar TDSCs derived from a green fluorescent protein rat were used. The outcome of tendon healing and the infiltration of inflammatory cells were examined by histology and immunohistochemistry up to week 16 postinjury. The fate of the transplanted cells was examined by ex vivo fluorescent imaging and immunohistochemistry. Our results showed that the transplantation of allogeneic TDSCs promoted tendon healing with no increased risk of ectopic chondro-ossification up to week 16. A low infiltration of T cells, ED1 macrophages, ED2 macrophages, and mast cells in the window wound was obtained. The transplanted TDSCs were found in the window wound at week 1 and 2, but were absent after week 4 postinjury. In conclusion, allogeneic TDSCs promoted tendon repair in the medium to long term and exhibited weak immunoreactions and anti-inflammatory effects in the hosts after transplantation in a rat model. There was no increased risk of ectopic chondro-ossification after TDSC transplantation. The decrease in the number of transplanted cells with time suggested that allogeneic TDSCs did not promote tendon repair through direct differentiation.

In vivo identity of tendon stem cells and the roles of stem cells in tendon healing.

We investigated the spatial distribution of stem cells in tendons and the roles of stem cells in early tendon repair. The relationship between tendon-derived stem cells (TDSCs) isolated in vitro and tendon stem cells in vivo was also explored. Iododeoxyuridine (IdU) label-retaining method was used for labeling stem cells in rat patellar tendons with and without injury. Co-localization of label-retaining cells (LRCs) with different markers was done by immunofluorescent staining. TDSCs were isolated from patellar tendon mid-substance after IdU pulsing, and the expression of different markers in fresh and expanded cells was done by immunofluorescent staining. More LRCs were found at the peritenon and tendon-bone junction compared with the mid-substance. Some LRCs at the peritenon were located at the perivascular niche. The LRC number and the expression of proliferative, tendon-related, pluripotency, and pericyte-related markers in LRCs in the window wound increased. Most of the freshly isolated TDSCs expressed IdU, and some TDSCs expressed pericyte-related markers, which were lost during expansion. Both freshly isolated and subcultured TDSCs expressed pluripotency markers, which were absent in LRCs in intact tendons. In conclusion, we identified LRCs at the peritenon, mid-substance, and tendon-bone junction. There were both vascular and non-vascular sources of LRCs at the peritenon, while the source of LRCs at the mid-substance was non-vascular. LRCs participated in tendon repair via migration, proliferation, activation for tenogenesis, and increased pluripotency. Some LRCs in the window wound were pericyte like. Most of the mid-substance TDSCs were LRCs. The pluripotency markers and pericyte-related marker in LRCs might be important for function after injury.

Local vitamin-C injection reduced tendon adhesion in a chicken model of flexor digitorum profundus tendon injury.

BACKGROUND: Adhesion formation is a complication of hand flexor tendon repair. Normal gliding function of flexor tendons can be impaired by an excessive fibrotic response, which may be caused by intraoperative and postoperative hemorrhage. As tissue damage and hemorrhage can disturb redox regulation, thereby favoring fibrotic responses, the purpose of this study was to investigate if antioxidants can reduce tendon adhesion by antagonizing oxidative stress. METHODS: Flexor digitorum profundus tendon injury was induced in fifty-seven chickens. In twelve chickens, oxidative stress preinjury, immediately after injury, and two and six weeks postinjury (n = 3 at each time period) was estimated by measuring tissue levels of the reduced form of glutathione (GSH) and oxidized glutathione (glutathione disulfide [GSSG]) in the proximal interphalangeal joint. In the remaining chickens, 50 µL of saline solution or vitamin-C solution (5 or 50 mg/mL) was injected into the wound immediately after closure of the tendon sheath. Samples were harvested at two weeks (n = 6 in each group) or six weeks (n = 6 in each group) postinjury for a gliding test, ultrasound imaging, and histological examination. Three chickens from each group were killed at two weeks postinjury for GSH and GSSG measurements to evaluate the treatment effects on postoperative oxidative stress. RESULTS: The GSH level was significantly decreased at two and six weeks postinjury, and the GSSG level was significantly increased at six weeks postinjury. Both 5 and 50-mg/mL vitamin C led to higher tissue levels of GSH at two weeks postinjury, as compared with that in the saline solution group, but no significant change in the GSSG level was detected. Chickens with vitamin-C supplementation showed no significant improvement in gliding resistance and no significant reduction of the fibrotic size at two weeks postinjury, but they did show significant improvement in gliding resistance at six weeks postinjury and the 5-mg/mL vitamin-C group showed a significant reduction of the fibrotic size at six weeks. Histological examination showed less peritendinous adhesion in the vitamin-C groups. CONCLUSIONS: Our results suggest that local injection of vitamin-C solution can reduce the extent of adhesion of healing tendons, probably by redox modulation, in a chicken model.

Local administration of alendronate reduced peri-tunnel bone loss and promoted graft-bone tunnel healing with minimal systemic effect on bone in contralateral knee.

Continued systemic administration of alendronate was reported to reduce peri-tunnel bone resorption and promoted graft-bone tunnel healing at the early stage post-anterior cruciate ligament (ACL) reconstruction. However, systemic increase in bone mineral density (BMD) in the contralateral intact knee was observed. We tested if single local administration of alendronate into the bone tunnel during ACL reconstruction could achieve similar benefits yet without the systemic effect on bone. Seventy-two rats with unilateral ACL reconstruction were divided into three groups: saline, low-dose (6 µg/kg) and mid-dose (60 µg/kg) alendronate. For local administration, alendronate was applied to the bone tunnels for 2 min before graft insertion and repair. At weeks 2 and 6, the reconstructed complex was harvested for high-resolution computed tomography (vivaCT) imaging followed by biomechanical test or histology. Our results showed that local administration of low-dose alendronate showed comparable benefits on the reduction of peri-tunnel bone loss, enhancement of bone tunnel mineralization, tunnel graft integrity, graft osteointegration and mechanical strength of the reconstructed complex at early stage post-reconstruction, yet with minimal systemic effect on mineralized tissue at the contralateral intact knee. A single local administration of alendronate at the low-dose therefore might be used to promote early tunnel graft healing post-reconstruction.