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INTRODUCTION: Endochondral ossification is a complex biological phenomenon involving a variety of factors and cells. Cyclin-dependent kinase inhibitor 1 (p21) inhibits cell cycle progression and is affected by external stress. We recently reported that embryonic endochondral ossification is unaffected by endogenous p21 deficiency. In this study, we evaluated whether p21 expression affects endochondral ossification during fracture healing. METHODS: Tibial fractures were introduced into p21 knockout (p21-/-) (n = 24) and wild-type C57BL/6 (p21+/+) (n = 24) mice at age 10 weeks. Fracture healing was evaluated using radiological, histological, and immunohistochemical (IHC) analyses. The effect of p21 small interfering RNA (siRNA) on ATDC5 cells was assessed in vitro. RESULTS: The Allen score for fracture healing was lower in p21-/- mice than in p21+/+ mice. In addition, p21-/- mice exhibited larger calluses and lower bone mineral density. IHC analyses showed that p21-/- mice exhibited delayed endochondral ossification via the Ihh-Runx2-Osterix pathway in vivo. Down-regulation of p21 expression in ATDC5 cells delayed endochondral ossification in vitro. CONCLUSIONS: p21 deficiency leads to delayed endochondral ossification by attenuating the Ihh-Runx2-Osterix pathway in vivo, and p21 deficiency in hypertrophic chondrocytes causes delayed differentiation of hypertrophic chondrocytes in vitro. p21 plays a role in endochondral ossification during fracture healing.
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Subunidade alfa 1 de Fator de Ligação ao Core , Consolidação da Fratura , Camundongos , Animais , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Osteogênese/fisiologia , RNA Interferente Pequeno/metabolismo , Camundongos Endogâmicos C57BL , Condrócitos/metabolismo , Diferenciação Celular/fisiologia , Quinases Ciclina-Dependentes/metabolismoRESUMO
BACKGROUND: Femoroacetabular impingement (FAI) is a leading cause of hip pain in young adults and often leads to degenerative osteoarthritis (OA). A small animal model of hip deformities is crucial for unraveling the pathophysiology of hip OA secondary to FAI. PURPOSES: To (1) characterize a new minimally invasive surgical technique to create a proximal femoral head-neck deformity in a skeletally immature rabbit model and (2) document the effect of an injury to the medial proximal femoral epiphysis on head-neck morphology at 28 days after the injury. STUDY DESIGN: Controlled laboratory study. METHODS: Six-week-old New Zealand White rabbits (n = 10) were subjected to right hip surgery, with the left hip used as a control. An epiphyseal injury in the medial femoral head was created using a 1.6-mm drill. Hips were harvested bilaterally at 28 days after surgery. Alpha and epiphyseal shaft angles were measured on radiographs. Alpha angles at the 1- and 3-o'clock positions were measured on the oblique axial plane of micro-computed tomography images. Bone bar formation secondary to growth plate injuries was confirmed using alcian blue hematoxylin staining. RESULTS: All hips in the study group showed a varus-type head-neck deformity, with lower epiphyseal shaft angles on anteroposterior radiographs versus those in the control group (133°± 8° vs 142°± 5°, respectively; P = .022) and higher epiphyseal shaft angles on lateral radiographs (27°± 12° vs 10°± 7°, respectively; P < .001). The mean alpha angles in the study group were higher at both the 1- (103°± 14° vs 46°± 7°, respectively; P < .002) and 3-o'clock (99°± 18° vs 35°± 11°, respectively; P < .002) positions than those in the control group. Alcian blue hematoxylin staining of all hips in the study group indicated that the injured physis developed a bony bar, leading to growth plate arrest on the medial femoral head. CONCLUSION: The proposed model led to growth arrest at the proximal femoral physis, resulting in a femoral head-neck deformity similar to human FAI. CLINICAL RELEVANCE: Our novel small animal model of a femoral head-neck deformity is a potential platform for research into the basic mechanisms of FAI disease progression and the development of disease-modifying therapies.
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Impacto Femoroacetabular , Azul Alciano , Animais , Impacto Femoroacetabular/diagnóstico por imagem , Impacto Femoroacetabular/cirurgia , Hematoxilina , Articulação do Quadril/cirurgia , Humanos , Coelhos , Microtomografia por Raio-XRESUMO
PURPOSE: To evaluate the relationship between morphological differences in labral tears and clinical features of the hip joint in patients who underwent hip arthroscopy. MATERIALS AND METHODS: We retrospectively analyzed data from patients who underwent arthroscopic surgery for the treatment of labral tears. Hip labral tears were morphologically classified as longitudinal peripheral tears (group L), radial fibrillated tears (group FI), radial flaps (group FL), and an unstable labrum (group U). Radiographically, the center-edge angle, acetabular roof obliquity, vertical-center-anterior angle, alpha angle, femoral head-neck offset ratio, and crossover sign were evaluated and compared among the groups. The relationship between labral morphology and these radiographic findings, as well as clinical findings, such as age, gender, preoperative range of hip motion, and the clinical outcomes using modified Harris Hip Score (mHHS) were also examined. RESULTS: This study included fifty patients. Groups L and FI were often observed in late middle-aged patients with relatively shallow acetabular coverage. Group FL tears were frequently observed in young males with radiographic features, such as femoroacetabular impingement (FAI), compared to the other groups. Group U comprised mostly young females with relatively shallow acetabular coverage compared to the other groups. For the postoperative mHHS, group FL showed the best score among all groups, with a significant difference between groups FL and FI (p = 0.034). CONCLUSIONS: Our study revealed that morphologically, different labral tears were associated with different clinical features and radiological findings. Especially, our study can provide predictive findings for hip arthroscopists that younger males with FAI show better clinical outcomes when compared to middle-aged females with shallow acetabulum, which is indicative of degenerative hip labral tears. LEVEL OF EVIDENCE: IV case series.
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BACKGROUND: The molecular mechanism of how femoroacetabular impingement (FAI) morphology leads to hip osteoarthritis (OA) is yet to be determined. The expression and location of inflammation-related molecules during early- and late-stage FAI have not been previously described. Moreover, the characterization of intra-articular inflammation away from the cam deformity as well as the nature of adjacent synovial tissue have also not been extensively reported. HYPOTHESIS: Early-stage FAI has a similar expression of inflammation-related markers in the head-neck and acetabular cartilage but less synovitis than late-stage FAI. STUDY DESIGN: Controlled laboratory study. METHODS: Head-neck cartilage, acetabular cartilage, and synovial samples were obtained from patients undergoing hip preservation surgery for the treatment of symptomatic cam FAI (early FAI group; n = 15) and advanced OA secondary to cam FAI (late FAI group; n = 15). Samples procured from healthy young adult donors served as the control group (n = 7). Cartilage degeneration was assessed by histology, and the expression of inflammation-related proteins (interleukin-1 beta [IL-1ß], matrix metalloproteinase-13 [MMP-13], a disintegrin and metalloproteinase with thrombospondin motifs-4 [ADAMTS-4], type II collagen [COL2], and aggrecan neoepitope [NITEGE]) was measured by immunostaining. Synovial samples in the early and late FAI groups were examined for synovitis and the expression of IL-1ß. RESULTS: Head-neck cartilage in the early FAI group showed significantly more degeneration than the control group and an increased expression of inflammation-related proteins (IL-1ß: 69.7% ± 18.1% vs 20.2% ± 4.9%, respectively; MMP-13: 79.6% ± 12.6% vs 25.3% ± 9.5%; ADAMTS-4: 83.9% ± 12.2% vs 24.3% ± 11.1%; NITEGE: 89.7% ± 7.7% vs 39.8% ± 20.5%) (P < .001). Head-neck and acetabular cartilage in the early and late FAI groups showed a similar degree of degeneration. Moreover, a similar expression of inflammation-related proteins was observed between the early and late FAI groups for head-neck cartilage (IL-1ß: 69.7% ± 18.1% vs 72.5% ± 13.2%; MMP-13: 79.6% ± 12.6% vs 71.4% ± 18.8%; ADAMTS-4: 83.9% ± 12.2% vs 82.6% ± 12.5%; COL2: 93.6% ± 3.9% vs 92.5% ± 5.8%; NITEGE: 89.7% ± 7.7% vs 95.7% ± 4.7%) and acetabular cartilage (IL-1ß: 83.3% ± 24.8% vs 80.7% ± 15.6%; MMP-13: 94.3% ± 9.7% vs 85.2% ± 12.3%; ADAMTS-4: 98.5% ± 2.3% vs 98.4% ± 3.4%; COL2: 99.8% ± 0.7% vs 99.7% ± 1.1%; NITEGE: 96.7% ± 6.7% vs 99.2% ± 2.2%). In contrast, synovitis was minimal with a low expression of IL-1ß in the early FAI group compared with the late FAI group. CONCLUSION: Hip cartilage exhibited an OA phenotype in patients with early-stage FAI, similar to what was observed in hip OA secondary to FAI. Severe synovitis was only evident with late-stage FAI. CLINICAL RELEVANCE: This study supports the concept that early hip impingement is associated with cartilage degeneration and catabolism.
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Cartilagem Articular/patologia , Impacto Femoroacetabular/patologia , Inflamação/patologia , Osteoartrite do Quadril , Membrana Sinovial/patologia , Cartilagem Articular/cirurgia , Impacto Femoroacetabular/cirurgia , Articulação do Quadril/patologia , Articulação do Quadril/cirurgia , Humanos , Osteoartrite do Quadril/etiologia , Osteoartrite do Quadril/cirurgia , Adulto JovemRESUMO
BACKGROUND: Femoroacetabular impingement (FAI) has been proposed as an etiologic factor in up to 50% of hips with osteoarthritis (OA). Inflammation is thought to be one of the main initiators of OA, yet little is known about the origin of intra-articular inflammation in FAI hips. HYPOTHESIS: Articular cartilage from the impingement zone of patients with FAI has high levels of inflammation, reflecting initial inflammatory process in the hip. STUDY DESIGN: Controlled laboratory study. METHODS: Head-neck cartilage samples were obtained from patients with cam FAI (cam FAI, early FAI; n = 15), advanced OA secondary to cam FAI (FAI OA, late FAI; n = 15), and advanced OA secondary to developmental dysplasia of the hip (DDH OA, no impingement; n = 15). Cartilage procured from young adult donors (n = 7) served as control. Safranin O-stained sections were assessed for cartilage abnormality. Tissue viability was detected by TUNEL assay. Immunostaining of interleukin 1ß (IL-1ß), catabolic markers (matrix metalloproteinase 13 [MMP-13], a disintegrin and metalloproteinase with thrombospondin motif 4 [ADAMTS-4], aggrecan antibody to C-terminal neoepitope [NITEGE]), and an anabolic marker (type II collagen [COL2]) was performed to evaluate molecular inflammation and metabolic activity. The average percentage of immunopositive cells from the total cell count was calculated. Kruskal-Wallis test followed by Steel-Dwass post hoc test was used for multiple comparisons. RESULTS: Microscopic osteoarthritic changes were more prevalent in cartilage of cam FAI and FAI OA groups compared with DDH OA and control groups. Cartilage in cam FAI and FAI OA groups, versus the DDH group, had higher expression of inflammatory molecules IL-1ß (69.7% ± 18.1% and 72.5% ± 13.2% vs 32.7% ± 14.4%, respectively), MMP-13 (79.6% ± 12.6% and 71.4% ± 18.8% vs 38. 5% ± 13.3%), ADAMTS-4 (83.9% ± 12.2% and 82.6% ± 12.5% vs 45.7% ± 15.5%), and COL2 (93.6% ± 3.9% and 92.5% ± 5.8% vs 53.3% ± 21.0%) (P < .001). Expression of NITEGE was similar among groups (cam FAI, 89.7% ± 7.7%; FAI OA, 95.7% ± 4.7%; DDH OA, 93.9% ± 5.2%; P = .0742). The control group had minimal expression of inflammatory markers. Inflammatory markers were expressed in all cartilage zones of early and late FAI but only in the superficial zone of the no impingement group. CONCLUSION: Cartilage from the impingement zone in FAI is associated with a high expression of inflammatory markers, extending throughout all cartilage zones. CLINICAL RELEVANCE: Inflammation associated with FAI likely has a deleterious effect on joint homeostasis. Further clinical and translational studies are warranted to assess whether and how surgical treatment of FAI reduces molecular inflammation.
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Cartilagem Articular/patologia , Impacto Femoroacetabular/patologia , Osteoartrite do Quadril/etiologia , Biomarcadores/metabolismo , Cartilagem Articular/metabolismo , Feminino , Impacto Femoroacetabular/complicações , Impacto Femoroacetabular/metabolismo , Impacto Femoroacetabular/cirurgia , Humanos , Inflamação/patologia , Interleucina-1beta/metabolismo , Masculino , Metaloproteinase 13 da Matriz/metabolismo , Osteoartrite do Quadril/metabolismo , Osteoartrite do Quadril/patologiaRESUMO
Articular cartilage tissue has a poor healing potential, and when subjected to traumatic damage this tissue undergoes cartilage degeneration and osteoarthritis. The association between the regulation of cell cycle checkpoints and tissue regeneration has been previously investigated, and p21 was initially identified as a potent inhibitor of cell cycle progression. However, the effects of p21 defects on damaged tissue remain controversial. Therefore, the aim of the present study was to evaluate the effects of p21 deficiency on cartilage repair. A mouse model of articular cartilage repair was generated by inducing a patellar groove scratch in 8weekold p21knockout (KO) mice and C57Bl/6 wildtype (WT) mice. Mice were sacrificed at 4 and 8 weeks postsurgery. The present study also investigated the effect of p21 deficiency on cartilage differentiation in ATDC5 cells in vitro. Safranin O staining results indicated that cartilage repair initially occurred in p21 KO mice. In addition, immunohistochemical analysis demonstrated that p21 KO upregulated proliferating cell nuclear antigen and increased cell proliferation. However, type II collagen and Sox9 expression levels remained unchanged in p21 KO and WT mice. Moreover, it was identified that p21 downregulation did not affect Sox9 and type II collagen expression levels in vitro. Furthermore, p21 deficiency promoted healing of articular cartilage damage, which was associated with cell proliferation in vivo, and increased chondrocyte proliferation but not differentiation in vitro. Therefore, the present results suggested that p21 does not affect Sox9 or type II collagen expression levels during cartilage differentiation in the repair of cartilage defects.
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Cartilagem Articular/metabolismo , Proliferação de Células/fisiologia , Condrócitos/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Animais , Ciclo Celular , Diferenciação Celular , Colágeno Tipo II/metabolismo , Genótipo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteoartrite/metabolismo , Fatores de Transcrição SOX9/metabolismo , TranscriptomaRESUMO
Osteoarthritis (OA) is a multifactorial disease, and recent data suggested that cell cycle-related proteins play a role in OA pathology. Cyclin-dependent kinase (CDK) inhibitor 1 (p21) regulates activation of other CDKs, and recently, we reported that p21 deficiency induced susceptibility to OA induced by destabilization of the medial meniscus (DMM) surgery through STAT3-signaling activation. However, the mechanisms associated with why p21 deficiency led to susceptibility to OA by the STAT3 pathway remain unknown. Therefore, we focused on joint inflammation to determine the mechanisms associated with p21 function during in vitro and in vivo OA progression. p21-knockout (p21-/- ) mice were used to develop an in vivo OA model, and C57BL/6 (p21+/+ ) mice with the same background as the p21-/- mice were used as controls. Morphogenic changes were measured using micro-CT, IL-1ß serum levels were detected by ELISA, and histological or immunohistological analyses were performed. Our results indicated that p21-deficient DMM-model mice exhibited significant subchondral bone destruction and cartilage degradation compared with wild-type mice. Immunohistochemistry results revealed p21-/- mice susceptibility to OA changes accompanied by macrophage infiltration and enhanced MMP-3 and MMP-13 expression through IL-1ß-induced NF-κB signaling. p21-/- mice also showed subchondral bone destruction according to micro-CT analysis, and cathepsin K staining revealed increased numbers of osteoclasts. Furthermore, p21-/- mice displayed increased serum IL-1ß levels, and isolated chondrocytes from p21-/- mice indicated elevated MMP-3 and MMP-13 expression with phosphorylation of IκB kinase complex in response to IL-1ß stimulation, whereas treatment with a specific p-IκB kinase inhibitor attenuated MMP-3 and MMP-13 expression. Our results indicated that p21-deficient DMM mice were susceptible to alterations in OA phenotype, including enhanced osteoclast expression, macrophage infiltration, and MMP expression through IL-1ß-induced NF-κB signaling, suggesting that p21 regulation may constitute a possible therapeutic strategy for OA treatment. © 2017 American Society for Bone and Mineral Research.
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Inibidor de Quinase Dependente de Ciclina p21/deficiência , Predisposição Genética para Doença , Osteoartrite/metabolismo , Transdução de Sinais , Animais , Modelos Animais de Doenças , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Metaloproteinase 13 da Matriz/biossíntese , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 3 da Matriz/biossíntese , Metaloproteinase 3 da Matriz/genética , Camundongos , Camundongos Knockout , Osteoartrite/diagnóstico por imagem , Osteoartrite/genética , Osteoartrite/patologia , Microtomografia por Raio-XRESUMO
The potential relationship between cell cycle checkpoint control and tissue regeneration has been indicated. Despite considerable research being focused on the relationship between p21 and myogenesis, p21 function in skeletal muscle regeneration remains unclear. To clarify this, muscle injury model was recreated by intramuscular injection of bupivacaine hydrochloride in the soleus of p21 knockout (KO) mice and wild type (WT) mice. The mice were sacrificed at 3, 14, and 28 days post-operation. The results of hematoxylin-eosin staining and immunofluorescence of muscle membrane indicated that muscle regeneration was delayed in p21 KO mice. Cyclin D1 mRNA expression and both Ki-67 and PCNA immunohistochemistry suggested that p21 deficiency increased cell cycle and muscle cell proliferation. F4/80 immunohistochemistry also suggested the increase of immune response in p21 KO mice. On the other hand, both the mRNA expression and western blot analysis of MyoD, myogenin, and Pax7 indicated that muscular differentiation was delayed in p21KO mice. Considering these results, we confirmed that muscle injury causes an increase in cell proliferation. However, muscle differentiation in p21 KO mice was inhibited due to the low expression of muscular synthesis genes, leading to a delay in the muscular regeneration. Thus, we conclude that p21 plays an important role in the in vivo healing process in muscular injury.
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Inibidor de Quinase Dependente de Ciclina p21/deficiência , Músculo Esquelético/fisiologia , Regeneração/genética , Animais , Antígenos de Diferenciação/metabolismo , Membrana Celular/metabolismo , Ciclina D1/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Antígeno Ki-67/metabolismo , Camundongos , Camundongos Knockout , Desenvolvimento Muscular/genética , Músculo Esquelético/anatomia & histologia , Músculo Esquelético/citologia , Músculo Esquelético/lesões , Antígeno Nuclear de Célula em Proliferação/metabolismo , RNA Mensageiro/genética , Cicatrização/genéticaRESUMO
Eicosapentaenoic acid (EPA) is an antioxidant and n-3 polyunsaturated fatty acid that reduces the production of inflammatory cytokines. We evaluated the role of EPA in chondrocyte apoptosis and degeneration. Normal human chondrocytes were treated with EPA and sodium nitroprusside (SNP). Expression of metalloproteinases (MMPs) was detected by real-time polymerase chain reaction (PCR) and that of apoptosis-related proteins was detected by western blotting. Chondrocyte apoptosis was detected by flow cytometry. C57BL/6J mice were used for the detection of MMP expression by immunohistochemistry and for investigation of chondrocyte apoptosis. EPA inhibited SNP-induced chondrocyte apoptosis, caspase 3 and poly(ADP-ribose) polymerase cleavage, phosphorylation of p38 MAPK and p53, and expression of MMP3 and MMP13. Intra-articular injection of EPA prevented the progression of osteoarthritis (OA) by inhibiting MMP13 expression and chondrocyte apoptosis. EPA treatment can control oxidative stress-induced OA progression, and thus may be a new approach for OA therapy.