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1.
J Orthop Sci ; 2020 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-32317146

RESUMO

BACKGROUND: Carpal Tunnel Syndrome (CTS) is an idiopathic fibrotic disorder. Fibrosis in the subsynovial connective tissues (SSCT) of CTS and many other fibrotic diseases is mediated by Transforming growth factor ß (TGF-ß). Recently monocyte chemoattractant protein-1 (MCP-1) a cytokine involved in cellular recruitment has been suggested to regulate TGF-ß activity. It is related to the onset of diseases which are caused by fibrosis, such as idiopathic pulmonary fibrosis, renal fibrosis, and systemic scleroderma. In this study, we evaluated the effect of the MCP-1 synthesis inhibitor, Bindarit, on primary cultures of fibroblasts from the SSCT of five CTS patients. METHODS: Fibroblasts were treated with Bindarit (10 µM, 50 µM, 100 µM, or 300 µM). Responses to inhibitors were evaluated by regulation of CTS fibrosis-associated genes, fibrosis gene array and Smad luciferase reporter assay. We also assessed the combination effect of Bindarit and SD208, a TGF-ß receptor type 1 inhibitor on TGF-ß signaling. RESULTS: Collagen type III A1 (Col3), connective tissue growth factor (CTGF), and SERPINE1 expression were significantly down-regulated by Bindarit (300 µM) compared to vehicle control. In the fibrosis array, expression of inhibin beta E chain precursor (INHBE), beta actin (ACTB), endothelin 1 (EDN1) and hypoxanthine phosphoribosyltransferase 1 (HPRT1) were significantly down-regulated, and integrin beta-3 (ITGB3) was significantly up-regulated by Bindarit (300 µM). Smad signal transduction activation was significantly down-regulated by Bindarit (300 µM) and/or SD208 (1 µM) with TGF-ß1 compared to vehicle control with TGF-ß1. CONCLUSIONS: These results suggest that Bindarit in combination with SD208 may be beneficial as medical therapy for the SSCT fibrosis associated with CTS.

2.
J Orthop Res ; 2020 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-31930553

RESUMO

Flexor tendon injuries and tendinopathy are very common but remain challenging in clinical treatment. Exosomes-based cell-free therapy appears to be a promising strategy for tendon healing, while limited studies have evaluated its impacts on tenocyte biology. The objective of this study was to characterize a novel purified exosome product (PEP) derived from plasma, as well as to explore its cellular effects on canine tenocyte biology. The transmission electron microscope revealed that exosomes of PEP present cup-shaped structures with the diameters ranged from 80 to 141 nm, and the NanoSight report presented that their size mainly concentrated around 100 nm. The enzyme-linked immunosorbent assay kits analysis showed that PEP was positive for CD63 and AChE expression, and the cellular uptake of exosomes internalized into tenocyte cytoplasm was observed. The cell growth assays displayed that tenocyte proliferation ability was enhanced by PEP solution in a dose-dependent manner. Tenogenic phenotype was preserved as is evident by that tendon-related genes expression (SCX, COL1A, COL3A1, TNMD, DCN, and MKX) were expressed insistently in a high level, while tenocytes were treated with 5% PEP solution. Furthermore, migration capability was maintained and total collagen deposition was increased. More interesting, dexamethasone-induced cellular apoptosis was attenuated during the incubation of tenocytes with a 5% PEP solution. These findings will provide the basic understandings about the PEP, and support the potential use of this biological strategy for tendon healing.

3.
J Cell Physiol ; 235(7-8): 5679-5688, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31975377

RESUMO

Deletion of TGFß inducible early gene-1 (TIEG) in mice results in an osteopenic phenotype that exists only in female animals. Molecular analyses on female TIEG knockout (KO) mouse bones identified increased expression of sclerostin, an effect that was confirmed at the protein level in serum. Sclerostin antibody (Scl-Ab) therapy has been shown to elicit bone beneficial effects in multiple animal model systems and human clinical trials. For these reasons, we hypothesized that Scl-Ab therapy would reverse the low bone mass phenotype of female TIEG KO mice. In this study, wildtype (WT) and TIEG KO female mice were randomized to either vehicle control (Veh, n = 12/group) or Scl-Ab therapy (10 mg/kg, 1×/wk, s.c.; n = 12/group) and treated for 6 weeks. Following treatment, bone imaging analyses revealed that Scl-Ab therapy significantly increased cancellous and cortical bone in the femur of both WT and TIEG KO mice. Similar effects also occurred in the vertebra of both WT and TIEG KO animals. Additionally, histomorphometric analyses revealed that Scl-Ab therapy resulted in increased osteoblast perimeter/bone perimeter in both WT and TIEG KO animals, with a concomitant increase in P1NP, a serum marker of bone formation. In contrast, osteoclast perimeter/bone perimeter and CTX-1 serum levels were unaffected by Scl-Ab therapy, irrespective of mouse genotype. Overall, our findings demonstrate that Scl-Ab therapy elicits potent bone-forming effects in both WT and TIEG KO mice and effectively increases bone mass in female TIEG KO mice.

4.
J Orthop Res ; 37(6): 1419-1428, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30548886

RESUMO

Flexor tendon injury is often associated with suboptimal outcomes and results in substantial digit dysfunction. Stem cells have been isolated from several experimental animals for the growing interest and needs of utilizing cell-based therapies. Recently, turkey has been developed as a new large animal model for flexor tendon research. In the present study, we reported the isolation and characterization of bone marrow-derived mesenchymal stem cells (BMSCs) from 8- to 12-month-old heritage-breed turkeys. The isolated cells demonstrated fibroblast-like morphology, clonogenic capacity, and high proliferation rate. These cells were positive for surface antigens CD90, CD105, and CD44, but were negative for CD45. The multipotency of turkey BMSCs was determined by differentiating cells into osteogenic, adipogenic, chondrogenic, and tenogenic lineages. There was upregulated gene expression of tenogenic markers, including mohawk, tenomodulin, and EGR1 as well as increased collagen synthesis in BMP12 induced cells. The successful isolation and verification of bone marrow-derived MSCs from turkey would provide opportunities of studying cell-based therapies and developing new treatments for tendon injuries using this novel preclinical large animal model. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1419-1428, 2019.


Assuntos
Separação Celular/métodos , Células-Tronco Mesenquimais/citologia , Adipogenia , Animais , Células da Medula Óssea/citologia , Diferenciação Celular , Proliferação de Células , Condrogênese , Osteogênese , Tendões/citologia , Perus
5.
Biomaterials ; 192: 189-198, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30453215

RESUMO

Reducing rotator cuff failure after repair remains a challenge due to suboptimal tendon-to-bone healing. In this study we report a novel biomaterial with engineered tendon-fibrocartilage-bone composite (TFBC) and bone marrow-derived mesenchymal stem cell sheet (BMSCS); this construct was tested for augmentation of rotator cuff repair using a canine non-weight-bearing (NWB) model. A total of 42 mixed-breed dogs were randomly allocated to 3 groups (n = 14 each). Unilateral infraspinatus tendon underwent suture repair only (control); augmentation with engineered TFBC alone (TFBC), or augmentation with engineered TFBC and BMSCS (TFBC + BMSCS). Histomorphometric analysis and biomechanical testing were performed at 6 weeks after surgery. The TFBC + BMSCS augmented repairs demonstrated superior histological scores, greater new fibrocartilage formation and collagen fiber organization at the tendon-bone interface compared with the controls. The ultimate failure load and ultimate stress were 286.80 ± 45.02 N and 4.50 ± 1.11 MPa for TFBC + BMSCS group, 163.20 ± 61.21 N and 2.60 ± 0.97 MPa for control group (TFBC + BMSCS vs control, P = 1.12E-04 and 0.003, respectively), 206.10 ± 60.99 N and 3.20 ± 1.31 MPa for TFBC group (TFBC + BMSCS vs TFBC, P = 0.009 and 0.045, respectively). In conclusion, application of an engineered TFBC and BMSCS can enhance rotator cuff healing in terms of anatomic structure, collagen organization and biomechanical strength in a canine NWB model. Combined TFBC and BMSCS augmentation is a promising strategy for rotator cuff tears and has a high potential impact on clinical practice.

6.
BMC Musculoskelet Disord ; 19(1): 342, 2018 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-30243295

RESUMO

BACKGROUND: Fibroblast behavior and cell-matrix interactions of cells from normal and idiopathic carpal tunnel syndrome (CTS) subsynovial connective tissue (SSCT) with and without Triamcinolone Acetonide (TA) were compared in this study. A cell-seeded gel contraction model was applied to investigate the effect of steroid treatment on SSCT fibroblast gene expression and function. METHODS: SSCT cells were obtained from CTS patients and fresh cadavers. Cells were isolated by mechanical and collagenase digestion. Collagen gels (1 mg/ml) were prepared with SSCT cells (1 × 106/mL). A sterile Petri dish with a cloning ring in the center was prepared. The area between the ring and outer dish was filled with cell-seeded collagen solution and gelled for 1 h. The gel was released from the outer way of the petri dish to allow gel contraction. Cell seeded gels were treated with 10 M triamcinolone acetonide (TA) or vehicle (DMSO) in modified MEM. Every 4 h for 3 days the contracting gels were photographed and areas calculated. Duplicate contraction tests were performed with each specimen, and the averages were used in the analyses, which were conducted using two-factor analysis of variance in a generalized linear model framework utilizing generalized estimating equations (GEE) to account for the correlation between samples. The contraction rate was determined by the area change over time, and the decay time constant was calculated. A customized mechanical test system was used to determine gel stiffness and tensile strength. Gene expression was assessed using Human Fibrosis and Cell Motility PCR arrays. RESULTS: TA-treated gels had a significantly higher contraction rate, tensile strength and stiffness than the untreated gels. Proteinases involved in remodeling had increased expression in TA-treated gels of the patient group. Pro-fibrotic genes and ECM regulators, such as TGF-ß, collagens and integrins, were down-regulated by TA, indicating that TA may work in part by decreasing fibrotic gene expression. CONCLUSIONS: This study showed that TA affects cell-matrix interaction and suppresses fibrotic gene expression in the SSCT cells of CTS patients.


Assuntos
Síndrome do Túnel Carpal/tratamento farmacológico , Fibroblastos/efeitos dos fármacos , Glucocorticoides/farmacologia , Triancinolona Acetonida/farmacologia , Síndrome do Túnel Carpal/metabolismo , Colágeno/metabolismo , Feminino , Fibroblastos/metabolismo , Glucocorticoides/uso terapêutico , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Cultura Primária de Células , Fator de Crescimento Transformador beta/metabolismo , Triancinolona Acetonida/uso terapêutico
7.
Stem Cells Int ; 2018: 3697971, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29977306

RESUMO

Tendon injuries are among the most common and severe hand injuries with a high demand for functional recovery. Stem cells have been identified and isolated from different species and a variety of tissues for the sake of regenerative medicine. Recently, turkey has been suggested as a potential new large animal model for flexor tendon-related research. However, turkey tissue-specific stem cells have not been investigated. Here, we presented the isolation and verification of tendon-derived stem cells (TDSCs) from 6- to 8-month-old heritage-breed turkey. TDSCs were isolated from turkey flexor tendon by plating nucleated cells at the determined optimal density. Approximately 4% of the nucleated cells demonstrated clonogenicity, high proliferation rate, and trilineage differentiation potential after induction culturing. These cells expressed surface antigens CD90, CD105, and CD44, but did not express CD45. There was a high level of gene expression of tenogenic markers in TDSCs, including mohawk, collagen type I, tenascin C, and elastin. Turkey TDSCs also expressed transcription factors PouV, Nanog, and Sox2, which are critically involved in the regulation of stemness. The successful isolation of tendon-derived stem cells from turkey was beneficial for future studies in tendon tissue engineering and would help in the development of new treatment for tendon diseases using this novel animal model.

8.
J Tissue Eng Regen Med ; 12(7): 1690-1701, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29763517

RESUMO

Surgical repair of rotator cuff tears presents a significant clinical challenge with high failure rates and inferior functional outcomes. Graft augmentation improves repair outcomes; however, currently available grafting materials have limitations. Although cell-seeded decellularized tendon slices may facilitate cell infiltration, promote tendon incorporation, and preserve original mechanical strength, the unique fibrocartilage zone is yet to be successfully reestablished. In this study, we investigated the biological and mechanical properties of an engineered tendon-fibrocartilage-bone composite (TFBC) with cyclic tension (3% strain; 0.2 Hz). Decellularized TFBCs seeded with bone marrow-derived mesenchymal stem cell (BMSCs) sheets and subjected to mechanical stimulation for up to 7 days were characterised by histology, immunohistochemistry, scanning electron microscopy, mechanical testing, and transcriptional regulation. The decellularized TFBC maintained native enthesis structure and properties. Mechanically stimulated TFBC-BMSC constructs displayed increased cell migration after 7 days of culture compared with static groups. The seeded cell sheet not only integrated well with tendon scaffold but also distributed homogeneously and aligned to the direction of stretch under dynamic culture. Developmental genes were regulated including scleraxis, which was significantly upregulated with mechanical stimulation. The Young's modulus of the cell-seeded constructs was significantly higher compared with the noncell-seeded controls. In conclusion, the results of this study reveal that the TFBC-BMSC composite provides an ideal multilayer construct for cell seeding and growth, with mechanical preconditioning further enhances cell penetration and differentiation. The BMSC cell sheet revitalised TFBC in conjunction with mechanical stimulation could serve as a novel and primed biological patch to improve rotator cuff repair.


Assuntos
Células da Medula Óssea/metabolismo , Fibrocartilagem , Células-Tronco Mesenquimais/metabolismo , Lesões do Manguito Rotador , Manguito Rotador , Engenharia Tecidual , Tecidos Suporte/química , Animais , Células da Medula Óssea/patologia , Movimento Celular , Cães , Fibrocartilagem/química , Fibrocartilagem/metabolismo , Fibrocartilagem/patologia , Células-Tronco Mesenquimais/patologia , Manguito Rotador/química , Manguito Rotador/metabolismo , Manguito Rotador/patologia , Lesões do Manguito Rotador/metabolismo , Lesões do Manguito Rotador/patologia , Lesões do Manguito Rotador/terapia
9.
J Orthop Res ; 2018 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-29575268

RESUMO

This study was to test our hypothesis that flexor tendon reconstruction with an allograft revitalized with bone marrow stromal cells (BMSCs) and synovialized with carbodiimide derivatized autologous synovial fluid (cd-SYN) would result in better digit functional restoration than the conventional allograft tendon. A total of 32 flexor digital profundus tendons from the second and fifth digit of 16 dogs were created a repair failure model first. Then, failed-repaired tendons were reconstructed with either a revitalized-synovialized allograft tendon or a clinical standard autograft tendon (control group). The allograft tendon was seeded with autologous BMSCs in multiple slits and the graft surface was coated with cd-SYN. A 6 weeks after tendon reconstruction, the digits were harvested and evaluated for digit function, adhesion status, tendon gliding resistance, attachment strength, cell viability, and histologic factors. The allograft group had significantly improved digit function compared with the control group through decreased work of flexion, increased digit range of motion under 2-Newton force, and less adhesion score (p < .05). However, the distal attachment-site strength and stiffness in the allograft tendon were significantly weaker than the autografts (p < .05). No significant difference was found for gliding resistance. Histologically, allograft tendons coated with allograft had smoother surfaces and showed tendon-to-bone and tendon-to-tendon incorporation. Viable BMSCs were found in the tendon slits 6 weeks after the graft. In conclusion, cellular lubricant-based modification of allograft tendons improved digit function and reduced the adhesions compared with autograft for flexor tendon reconstruction. However, improvement of graft-to-host tendon healing is still challenging. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

10.
J Cell Physiol ; 233(3): 2067-2074, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28294324

RESUMO

Fibrosis of the subsynovial connective tissue (SSCT) in carpal tunnel syndrome (CTS) patients is increasingly recognized as an important aspect of CTS pathophysiology. In this study, we evaluated the effect of blocking profibrotic pathways in fibroblasts from the SSCT in CTS patients. Fibroblasts were stimulated with transforming growth factor ß1 (TGF-ß1), and then treated either with a specific fibrosis pathway inhibitor targeting TGF-ß receptor type 1 (TßRI), platelet-derived growth factor receptor (PDGFR), epidermal growth factor receptor (EGFR), or vascular endothelial growth factor receptor (VEGFR). Fibrosis array and quantitative real-time polymerase chain reaction of fibrotic genes were evaluated. Array gene expression analysis revealed significant down-regulation of multiple fibrotic genes after treatment with TßRI, PDGFR, and VEGFR inhibitors. No array fibrotic genes were significantly down-regulated with EGFR inhibition. Further gene expression analysis of known CTS fibrosis markers collagen type I A2 (Col1), collagen type III A1 (Col3), connective tissue growth factor (CTGF), and SERPINE1 showed significantly down-regulation after TßRI inhibition. In contrast, VEGFR inhibition significantly down-regulated CTGF and SERPINE1, whereas, PDGFR and EGFR inhibition significantly down-regulated Col3. Taken together the inhibition of TßRI appears to be the primary mediator of fibrotic gene expression in fibroblasts from CTS patients. TGF-ß/Smad activity was further evaluated, and as expected inhibition of Smad activity was significantly down-regulated after inhibition of TßRI, but not with PDGFR, VEGFR, or EGFR inhibition. These results indicate that local therapies specifically targeting TGF-ß signaling alone or in combination offer the potential of a novel local antifibrosis therapy for patients with CTS.


Assuntos
Síndrome do Túnel Carpal/tratamento farmacológico , Receptores ErbB/antagonistas & inibidores , Fibrose/patologia , Receptores do Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Membrana Sinovial/patologia , Fator de Crescimento Transformador beta/metabolismo , Síndrome do Túnel Carpal/patologia , Células Cultivadas , Colágeno Tipo I/biossíntese , Colágeno Tipo I/genética , Colágeno Tipo III/biossíntese , Colágeno Tipo III/genética , Tecido Conjuntivo/patologia , Células do Tecido Conjuntivo/citologia , Fator de Crescimento do Tecido Conjuntivo/biossíntese , Fator de Crescimento do Tecido Conjuntivo/genética , Fibroblastos/metabolismo , Fibrose/tratamento farmacológico , Humanos , Inibidor 1 de Ativador de Plasminogênio/biossíntese , Inibidor 1 de Ativador de Plasminogênio/genética , Membrana Sinovial/citologia
11.
Sci Rep ; 7(1): 16192, 2017 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-29170419

RESUMO

Fibrosis of the subsynovial connective tissue (SSCT) is a pathognomonic change in carpal tunnel syndrome (CTS). Identification of molecular targets and anti-fibrotic therapies could provide new treatment strategies for CTS. The contribution of SSCT cells to fibrosis and the signaling pathways that initiate and aggravate fibrosis in CTS remain unknown. Here we report that platelet-derived growth factor receptor alpha (PDGFRα) positive ( + ) cells accumulate in CTS SSCT and that the presence of fibrotic growth factor, PDGF-AA, results in increased proliferation of PDGFRα+ cells via PI3K/Akt signaling pathway. Although PI3K inhibition decreased proliferation, there was no change in fibrosis-related gene expression. Indeed, protein levels of fibrosis signaling mediator TGF-ß remained the same and the second messenger, Smad2/3, accumulated in the nucleus. In contrast AMP-activated protein kinase (AMPK) activation, which can be induced with metformin and AICAR inhibited proliferation, TGF-ß expression, and altered cell morphology in SSCT cells. Further we show that AMPK activation by metformin reduced collagen III levels and the ratio of Collagen I to Collagen III. Both AICAR and metformin reduced F-actin and significantly reduced the fiber cross alignment. Our results suggest that PDGFRa signaling may be an important fibrosis target and that activators of AMPK, may be an important therapeutic approach for treating CTS.


Assuntos
Síndrome do Túnel Carpal/metabolismo , Tecido Conjuntivo/metabolismo , Tecido Conjuntivo/patologia , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Síndrome do Túnel Carpal/genética , Proliferação de Células/genética , Proliferação de Células/fisiologia , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Proteína Oncogênica v-akt/genética , Proteína Oncogênica v-akt/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fator de Crescimento Derivado de Plaquetas/genética , Fator de Crescimento Derivado de Plaquetas/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia
12.
Endocrinology ; 158(10): 3354-3368, 2017 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-28977607

RESUMO

Endoxifen, the primary active metabolite of tamoxifen, is currently being investigated as a novel endocrine therapy for the treatment of breast cancer. Tamoxifen is a selective estrogen receptor modulator that elicits potent anti-breast cancer effects. However, long-term use of tamoxifen also induces bone loss in premenopausal women and is associated with an increased risk of endometrial cancer in postmenopausal women. For these reasons, we have used a rat model system to comprehensively characterize the impact of endoxifen on the skeleton and uterus. Our results demonstrate that endoxifen elicits beneficial effects on bone in ovary-intact rats and protects against bone loss following ovariectomy. Endoxifen is also shown to reduce bone turnover in both ovary-intact and ovariectomized rats at the cellular and biochemical levels. With regard to the uterus, endoxifen decreased uterine weight but maintained luminal epithelial cell height in ovariectomized animals. Within luminal epithelial cells, endoxifen resulted in differential effects on the expression levels of estrogen receptors α and ß as well as multiple other genes previously implicated in regulating epithelial cell proliferation and hypertrophy. These studies analyze the impact of extended endoxifen exposure on both bone and uterus using a Food and Drug Administration-recommended animal model. Although endoxifen is a more potent breast cancer agent than tamoxifen, the results of the present study demonstrate that endoxifen does not induce bone loss in ovary-intact rats and that it elicits partial agonistic effects on the uterus and skeleton in ovariectomized animals.


Assuntos
Remodelação Óssea/efeitos dos fármacos , Osso e Ossos/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Tamoxifeno/análogos & derivados , Útero/efeitos dos fármacos , Animais , Endométrio/efeitos dos fármacos , Endométrio/metabolismo , Endométrio/patologia , Receptor alfa de Estrogênio/efeitos dos fármacos , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/efeitos dos fármacos , Receptor beta de Estrogênio/metabolismo , Feminino , Tamanho do Órgão , Osteoporose/induzido quimicamente , Ovariectomia , Ratos , Moduladores Seletivos de Receptor Estrogênico/efeitos adversos , Tamoxifeno/efeitos adversos , Tamoxifeno/farmacologia , Útero/metabolismo , Útero/patologia
15.
JCI Insight ; 2(7): e90517, 2017 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-28405613

RESUMO

Osteolytic bone diseases, such as osteoporosis, are characterized by diminished bone quality and increased fracture risk. The therapeutic challenge remains to maintain bone homeostasis with a balance between osteoclast-mediated resorption and osteoblast-mediated formation. Osteoclasts are formed by the fusion of monocyte/macrophage-derived precursors. Here we report, to our knowledge for the first time, that receptor-interacting protein 140 (RIP140) expression in osteoclast precursors and its protein regulation are crucial for osteoclast differentiation, activity, and coupled bone formation. In mice, monocyte/macrophage-specific knockdown of RIP140 (mϕRIP140KD) resulted in a cancellous osteopenic phenotype with significantly increased bone resorption and reduced bone formation. Osteoclast precursors isolated from mϕRIP140KD mice had significantly increased differentiation potential. Furthermore, conditioned media from mϕRIP140KD primary osteoclast cultures significantly suppressed osteoblast differentiation. This suppressive activity was effectively and rapidly terminated by specific Syk-stimulated RIP140 protein degradation. Mechanistic analysis revealed that RIP140 functions primarily by inhibiting osteoclast differentiation through forming a transcription-suppressor complex with testicular receptor 4 (TR4) to repress osteoclastogenic genes. These data reveal that monocyte/macrophage RIP140/TR4 complexes may serve as a critical transcription regulatory complex maintaining homeostasis of osteoclast differentiation, activity, and coupling with osteoblast formation. Accordingly, we propose a potentially novel therapeutic strategy, specifically targeting osteoclast precursor RIP140 protein in osteolytic bone diseases.


Assuntos
Diferenciação Celular , Macrófagos/metabolismo , Proteína 1 de Interação com Receptor Nuclear/metabolismo , Osteoclastos/citologia , Osteogênese , Receptores de Esteroides/metabolismo , Receptores dos Hormônios Tireóideos/metabolismo , Animais , Células Cultivadas , Homeostase , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 1 de Interação com Receptor Nuclear/genética , Receptores de Esteroides/genética , Receptores dos Hormônios Tireóideos/genética
16.
J Shoulder Elbow Surg ; 25(3): 469-77, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26387915

RESUMO

HYPOTHESIS: A composite of multilayer tendon slices (COMTS) seeded with bone marrow stromal cells (BMSCs) may impart mechanical and biologic augmentation effects on supraspinatus tendon repair under tension, thereby improving the healing process after surgery in rats. METHODS: Adult female Lewis rats (n = 39) underwent transection of the supraspinatus tendon and a 2-mm tendon resection at the distal end, followed by immediate repair to its bony insertion site under tension. Animals received 1 of 3 treatments at the repair site: (1) no augmentation, (2) COMTS augmentation alone, or (3) BMSC-seeded COMTS augmentation. BMSCs were labeled with a fluorescent cell marker. Animals were euthanized 6 weeks after surgery, and the extent of healing of the repaired supraspinatus tendon was evaluated with biomechanical testing and histologic analysis. RESULTS: Histologic analysis showed gap formation between the repaired tendon and bone in all specimens, regardless of treatment. Robust fibrous tissue was observed in rats with BMSC-seeded COMTS augmentation; however, fibrous tissue was scarce within the gap in rats with no augmentation or COMTS-only augmentation. Labeled transplanted BMSCs were observed throughout the repair site. Biomechanical analysis showed that the repairs augmented with BMSC-seeded COMTS had significantly greater ultimate load to failure and stiffness compared with other treatments. However, baseline (time 0) data showed that COMTS-only augmentation did not increase mechanical strength of the repair site. CONCLUSION: Although the COMTS scaffold did not increase the initial repair strength, the BMSC-seeded scaffold increased healing strength and stiffness 6 weeks after rotator cuff repair in a rat model.


Assuntos
Transplante de Células-Tronco Mesenquimais , Manguito Rotador/cirurgia , Tecidos Suporte , Animais , Modelos Animais de Doenças , Feminino , Xenoenxertos , Ratos , Ratos Endogâmicos Lew , Lesões do Manguito Rotador , Tendões/transplante , Cicatrização
17.
J Bone Joint Surg Am ; 97(21): 1792-8, 2015 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-26537167

RESUMO

BACKGROUND: Cell-based tissue engineering techniques have been introduced to improve tendon repair outcomes. The purpose of this study was to determine optimal concentrations of fibrinogen and thrombin for use as a scaffold to deliver stromal cells to the tendon repair site. METHODS: Lacerated flexor digitorum profundus tendons from forty canine forepaws underwent simulated repair with fibrin gel interposition. The tendons were divided into five groups with different ratios of fibrinogen (mg/mL) to thrombin (NIH units/mL) used to form the gels. These ratios, which ranged from those found in normal hemostasis to those used clinically as adhesives, were 5:25 (the physiological ratio, used as a control), 40:250 (a low adhesive concentration of fibrinogen and a low adhesive concentration of thrombin [low-low group]), 80:250 (high-low group), 40:500 (low-high group), and 80:500 (high-high group). The failure load and tensile stiffness at time zero, compressive stiffness of the fibrin gel, and cell viability and migration were evaluated. RESULTS: The failure loads of the high-low and high-high groups were significantly higher than that of the control group. The tensile stiffness of the high-high group was significantly higher than that of the control group. The high-low and high-high groups had significantly higher compressive stiffness than the other groups. While there was no significant difference among the groups regarding cell viability, the cells in the control, low-low, and low-high gels were spindle-shaped whereas those in the high-low and high-high groups were rounded. Cells migrated across scratch gaps within twenty-four hours in the control, low-low, and low-high groups, but not in the high-low and high-high groups. CONCLUSIONS: Higher concentrations of fibrinogen resulted in stronger and stiffer gels, but the strength was far less than that of a tendon suture and these gels were associated with a more rounded cell morphology and reduced cell migration. Therefore, lower concentrations of fibrinogen should be used if a fibrin gel is employed to deliver cells for tendon repair. CLINICAL RELEVANCE: Concentrations of fibrinogen lower than those used in fibrin glue may be more appropriate if fibrin is employed to create a cell delivery matrix for tendon repair.


Assuntos
Fibrinogênio , Regeneração Tecidual Guiada , Células-Tronco Mesenquimais/fisiologia , Traumatismos dos Tendões/cirurgia , Trombina , Tecidos Suporte , Animais , Movimento Celular , Sobrevivência Celular , Cães , Humanos , Masculino , Traumatismos dos Tendões/patologia , Técnicas de Cultura de Tecidos
18.
J Orthop Res ; 33(5): 668-74, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25626430

RESUMO

Carpal tunnel syndrome (CTS) is a peripheral neuropathy characterized by non-inflammatory fibrosis of the subsynovial connective tissues (SSCT). A rabbit model of CTS was developed to test the hypothesis that SSCT fibrosis causes the neuropathy. We used a cell-seeded collagen-gel contraction model to characterize the fibrosis in this model in terms of cellular mechanics, specifically to compare the ability of SSCT cells from the rabbit model and normal rabbits to contract the gel, and to assess the effect of transforming growth factor-ß1,which is upregulated in CTS, on these cells. SSCT fibrosis was induced in six retired breeder female rabbits which were sacrificed at 6 weeks (N = 3) and 12 weeks (n = 3). An additional two rabbits served as controls. SSCT was harvested according to a standard protocol. Gels seeded with SSCT cells from rabbits sacrificed at 6 weeks had significantly higher tensile strength (p < 0.001) and Young's modulus (p < 0.001) than gels seeded with cells from rabbits sacrificed at 12 weeks or control animals. TGF-ß1 significantly increased the decay time constant (p < 0.001), tensile strength (p < 0.001), and Young's modulus (p < 0.001) regardless of the cell source. This model may be useful in screening therapeutic agents that may block SSCT fibrosis, identifying possible candidates for CTS treatment.


Assuntos
Doenças do Tecido Conjuntivo/fisiopatologia , Fibroblastos/fisiologia , Animais , Colágeno Tipo I , Doenças do Tecido Conjuntivo/patologia , Modelos Animais de Doenças , Feminino , Fibrose , Coelhos , Fatores de Tempo , Fator de Crescimento Transformador beta1
19.
J Biomed Mater Res A ; 103(2): 574-80, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24753289

RESUMO

Noninflammatory subsynovial connective tissue (SSCT) fibrosis with nerve compression is a prominent feature of carpal tunnel syndrome (CTS). Studies have shown that SSCT matrix synthesis and material property changes in CTS are associated with increased activity of transforming growth factor (TGF)-ß1. The aim of this study were to (1) investigate the ability of SSCT fibroblasts from CTS patients and unaffected individuals to contract a collagen gel ring and (2) determine how the addition of TGF-ß1 affects this ability. SSCT fibroblasts from three normal cadavers and three age-matched female patients who had undergone surgery for CTS were used. Results showed patient cell-seeded gels had a significantly higher contraction rate (p < 0.001) than control cells, and fully contracted gel rings possessed a significantly higher tensile strength (p = 0.003) and stiffness (p < 0.001). Furthermore, TGF-ß1 significantly intensified contraction rate (p < 0.001), tensile strength (p < 0.001), and stiffness (p < 0.001). In conclusion, SSCT cells from normal donors and CTS patients contract collagen gel rings differently, and this ability is affected by TGF-ß1 treatment. This cell-seeded collagen gel model may be useful for developing new methods of stopping or eliminating the effect of TGF-ß1 on the SSCT fibroblasts and surrounding matrix, which might aid in the identification of medical treatment for CTS.


Assuntos
Síndrome do Túnel Carpal/metabolismo , Colágeno/química , Fibroblastos/metabolismo , Fator de Crescimento Transformador beta1/biossíntese , Idoso , Síndrome do Túnel Carpal/patologia , Células Cultivadas , Feminino , Fibroblastos/patologia , Géis/química , Humanos , Pessoa de Meia-Idade , Resistência à Tração
20.
PLoS One ; 9(9): e108312, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25269071

RESUMO

Carpal tunnel syndrome (CTS) is an idiopathic disease that results from increased fibrosis of the subsynovial connective tissue (SSCT). A recent study found overexpression of both transforming growth factor-ß (TGF-ß) and connective tissue growth factor (CTGF) in the SSCT of CTS patients. This study investigated TGF-ß and CTGF expression in a rabbit model of CTS, in which SSCT fibrosis is induced by a surgical injury. Levels of TGF-ß1 and CTGF at 6, 12, 24 weeks after injury were determined by immunohistochemistry A significant increase in TGF-ß1 and a concomitant significant increase in CTGF were found at 6 weeks, in addition to higher cell density compared to normal (all p<0.05), Interestingly, CTGF expression was reduced at 12 and 24 weeks, suggesting that an initial insult results in a time limited response. We conclude that this rabbit model mimics the fibrosis found in human CTS, and may be useful to study pathogenetic mechanisms of CTS in vivo.


Assuntos
Síndrome do Túnel Carpal/genética , Fator de Crescimento do Tecido Conjuntivo/genética , Modelos Animais de Doenças , Coelhos , Fator de Crescimento Transformador beta1/genética , Animais , Síndrome do Túnel Carpal/metabolismo , Síndrome do Túnel Carpal/patologia , Contagem de Células , Tecido Conjuntivo/lesões , Tecido Conjuntivo/metabolismo , Tecido Conjuntivo/patologia , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose , Expressão Gênica , Humanos , Metacarpo/lesões , Metacarpo/metabolismo , Metacarpo/patologia , Fator de Crescimento Transformador beta1/metabolismo
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