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BACKGROUNDS: Nonvascularized toe phalanx transfer is an accepted surgical option for short and hypoplastic digits in congenital hand abnormalities. However, one of the criticisms of this technique is the donor site morbidity. The purpose of this study was to evaluate donor foot morbidity after nonvascularized toe phalanx transfer using a new donor site reconstruction technique. METHODS: We retrospectively reviewed 116 nonvascularized toe phalanx transfers in 69 children between 2001 and 2020 in whom the donor foot was reconstructed with a new technique using iliac osteochondral bone graft with periosteum. Feet treated with an isolated donor proximal phalanx of the fourth toe were selected and morbidity was assessed both subjectively and objectively at a minimum of 2 years after surgery. Motion, stability, and alignment of the metatarsophalangeal joint were clinically evaluated. The relative length of the fourth toe to the third toe was measured on a roentgenogram. The satisfaction of the parents for overall function and appearance was evaluated using a visual analog scale. RESULTS: Ninety-four operated feet in 65 patients, including 43 boys and 22 girls, were included. The right foot was evaluated in 52 patients and the left foot in 42 patients. The mean age at operation was 2 years and the mean follow-up period was 7.6 years. Motion at the metatarsophalangeal joint was good at 69% with an average extension of 45 degrees and flexion of 25 degrees. Stability and alignment were good at 95% and 84%, respectively. Only 4 toes had gross instability and 4 toes with poor alignment required revision surgery. Sixty-two toes (66%) maintained proportional length and 9 toes were graded as short. Parental satisfaction was high for appearance as well as function. CONCLUSIONS: This newly described technique of using iliac osteochondral bone graft with periosteum to reconstruct toe phalanx donors provided satisfactory results. The function and appearance of the donor foot after a nonvascularized toe phalanx transfer was well preserved. LEVEL OF EVIDENCE: Level IV; therapeutic.
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Falanges dos Dedos da Mão , Deformidades Congênitas da Mão , Criança , Masculino , Feminino , Humanos , Estudos Retrospectivos , Dedos do Pé/cirurgia , Pé/cirurgia , Deformidades Congênitas da Mão/cirurgiaRESUMO
PURPOSE: Plain hip radiograph is commonly used for the diagnosis of infantile acetabular dysplasia. Many infants are unable to maintain adequate position during radiography. Besides, the infantile hip is much smaller and has a higher cartilage component in the acetabulum and proximal femur compared with the adult hip. In this study, we developed a digitally reconstructed radiograph synthesized from magnetic resonance imaging (MRI) and investigated errors of hip radiographic measurements in different pelvic positions. PATIENTS AND METHODS: MRI of both hips was performed in 10 patients (mean age 3.9 years). Three-dimensional (3D) bone models were created from MRI data. We tilted 3D pelvic bone models between 10° anteversion and retroversion and through 10° rotation on the affected and contralateral sides using 3D axes. Following this, we created digitally reconstructed radiographs in each pelvic position and calculated the acetabular index (AI), center-edge angle (CEA), migration percentage (MP), and teardrop distance (TDD). RESULTS: AI tended to increase with pelvic retroversion and did not change with pelvic rotation. CEA tended to decrease with pelvic retroversion and rotation on the contralateral side. MP increased with pelvic retroversion and rotation on the contralateral side. TDD did not change significantly with pelvic tilt and rotation. CONCLUSIONS: Radiographic measurements of hip in infants were highly influenced by pelvic movement. AI was influenced by pelvic tilt; CEA and MP were influenced by both pelvic tilt and rotation. We need to keep in mind that infantile hip radiographs could have about ±5° errors in AI and CEA.
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BACKGROUND: Various deformities appear in hereditary multiple exostoses (HMEs). Deformities around the knee or ankle joints are easy to detect in this disease because such deformities are visible in appearance. However, deformities in the hip joints of skeletally mature patients are not well understood because their tumors are invisible. METHODS: To understand deformities around the hip joint in HMEs, we investigated 36 hip joints in 19 skeletally mature patients (12 males, 7 females). The mean age at last X-ray imaging investigation was 29.2 years (14.5-66.5 years). We evaluated the lesions of exostoses around the acetabulum and proximal femur, Wiberg's center-edge angle (CEA), neck-shaft angle (NSA), acetabular depth-width ratio (ADR), and Sharp's acetabular angle. RESULTS: No exostoses were present in four hips of three cases. Thirty-one hip joints had exostoses on the medial side of the femoral neck. Exostoses existed on the lateral side of the femoral neck in 16 hips. None of the patients had acetabuluar tumors. One patient experienced pain because of impingement between the acetabular rim and medial tumors of the femoral neck. The increase in NSA, which is an index of proximal femoral deformity, was common with a mean NSA of 147.3 °. Two indices of acetabular deformity, Sharp's angle and ADR, were within normal limits with a mean Sharp's angle of 41.3 ° and mean ADR of 269. The average CEA was 29.9 °. CONCLUSIONS: Hip dysplasia is not necessarily common in skeletally mature patients with HMEs. To determine the possibility of hip dysplasia in skeletally immature patients with HMEs, ADR may be a useful reference index.
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Coxa Valga/diagnóstico por imagem , Coxa Valga/epidemiologia , Exostose Múltipla Hereditária/diagnóstico por imagem , Exostose Múltipla Hereditária/epidemiologia , Luxação do Quadril/diagnóstico por imagem , Luxação do Quadril/epidemiologia , Adolescente , Adulto , Distribuição por Idade , Idoso , Estudos de Coortes , Comorbidade , Feminino , Humanos , Japão/epidemiologia , Masculino , Pessoa de Meia-Idade , Prevalência , Radiografia/métodos , Índice de Gravidade de Doença , Distribuição por Sexo , Tomografia Computadorizada por Raios X/métodos , Adulto JovemRESUMO
Fibrous dysplasia (FD) of bone is a rare skeletal disease often associated with bone pain, deformities and fractures. The bisphosphonate therapies are reported to be useful for bone pain, but seem to be not suitable for fracture repairs of extremities. This is the first report of zoledronate-induced radiological improvement and long bone fracture union in polyostotic FD. A 30-year-old Japanese female had bilateral shepherd's crook deformities typical to FD and right pathological femoral fracture and left humeral fracture nonunion. These fractures occurred without major traumas and the humeral fracture was not united for 1 year with conservative therapy. Laboratory blood test results were notable for elevated serum alkaline phosphatase and urine N-terminal cross-linking telopeptide of type I collagen. Her subtrochanteric femoral fracture was percutaneously fixed using Kirschner wires. After surgery, a hip spica cast was applied for 2 months and the orthosis for the next 2 months. Bony union of the femoral fracture was observed 5 months after surgery. Increased bone turnover and typical radiological features suggested that the constant elbow pain was due to both FD itself and humeral nonunion. Considering the possible side effects of zoledronate delaying acute fracture healing, we initiated zoledronate (Zometa(®); Novartis, Tokyo, Japan) therapy after femoral fracture union. Intravenous zoledronate acid was administered at a dose of 2 mg, along with supplementation of calcium (600 mg/day) and vitamin D (alfacalcidol 0.5 µg/day) to limit the risk of osteomalacia and improve the efficacy of bisphosphonate therapy. The patient's elbow pain rapidly resolved 1 week after treatment. Second therapy with same dose was performed after 6 months. No recurrence of elbow pain was reported and bony union was diagnosed after 1 year from the first administration. This patient is currently doing well without recurrence of bone pain. She can also walk for a short distance with crutches. We presented the case of an FD patient with persistent elbow pain due to FD itself and nonunion of humeral fracture, which was ameliorated promptly by intravenous zoledronate therapies. This case illustrated the benefit of zoledronate treatment in patients with extensive polyostotic FD and pathological fractures of extremities.
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Extracellular signal-regulated kinase 5 (ERK5) is a member of the mitogen-activated protein kinase (MAPK) family and is activated by its upstream kinase, MAPK kinase 5 (MEK5), which is a member of the MEK family. Although the role of MEK5 has been investigated in several fields, little is known about its role in osteoblastic differentiation. In this study, we have demonstrated the role of MEK5 in osteoblastic differentiation in mouse preosteoblastic MC3T3-E1 cells and bone marrow stromal ST2 cells. We found that treatment with BIX02189, an inhibitor of MEK5, increased alkaline phosphatase (ALP) activity and the gene expression of ALP, osteocalcin (OCN) and osterix, as well as it enhanced the calcification of the extracellular matrix. Moreover, osteoblastic cell proliferation decreased at a concentration of greater than 0.5 µM. In addition, knockdown of MEK5 using siRNA induced an increase in ALP activity and in the gene expression of ALP, OCN, and osterix. In contrast, overexpression of wild-type MEK5 decreased ALP activity and attenuated osteoblastic differentiation markers including ALP, OCN and osterix, but promoted cell proliferation. In summary, our results indicated that MEK5 suppressed the osteoblastic differentiation, but promoted osteoblastic cell proliferation. These results implied that MEK5 may play a pivotal role in cell signaling to modulate the differentiation and proliferation of osteoblasts. Thus, inhibition of MEK5 signaling in osteoblasts may be of potential use in the treatment of osteoporosis.
Assuntos
Diferenciação Celular , MAP Quinase Quinase 5/metabolismo , Osteoblastos/citologia , Compostos de Anilina/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Indóis/farmacologia , MAP Quinase Quinase 5/antagonistas & inibidores , MAP Quinase Quinase 5/genética , Camundongos , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Fosforilação/efeitos dos fármacos , Interferência de RNA , Regulação para CimaRESUMO
Hyaluronan (HA), one of the major structural extracellular components in cartilage, regulates cellular responses via receptors such as CD44. However, the direct effects of HA on osteoblastic differentiation has not been studied in detail. Here, we investigated the effects of HA (molecular weight: 900-1200 kDa) on osteoblastic differentiation that was induced by bone morphogenetic protein (BMP) in C2C12 cells (mouse myoblastic cells) and ST2 cells (mouse bone marrow cells). BMP-induced osteoblastic differentiation and Smad1/Smad5/Smad8 phosphorylation were downregulated by HA. Use of the CD44-blocking antibody restored HA-induced inhibition of osteoblastic differentiation and Smad1/Smad5/Smad8 phosphorylation. Our results indicate that HA inhibits BMP-induced osteoblastic differentiation through the CD44 receptor.
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Proteína Morfogenética Óssea 2/fisiologia , Diferenciação Celular , Ácido Hialurônico/farmacologia , Osteoblastos/fisiologia , Adulto , Idoso , Fosfatase Alcalina/metabolismo , Animais , Linhagem Celular , Feminino , Humanos , Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/fisiologia , Masculino , Camundongos , Pessoa de Meia-Idade , Osteoartrite/metabolismo , Osteoartrite/patologia , Mapeamento por Restrição , Proteínas rho de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTPRESUMO
The Tec family of nonreceptor tyrosine kinases has been shown to play a key role in inflammation and bone destruction. Bruton tyrosine kinase (Btk) has been the most widely studied because of its critical role in B cells. Furthermore, recent evidence has demonstrated that blocking Btk signaling is effective in ameliorating lymphoma progression and experimental arthritis. The role of Btk in osteoblastic differentiation has not been well elucidated. In this study, we demonstrated the role of Btk in osteoblastic differentiation and investigated the effects of a Btk inhibitor on osteoblastic differentiation in mouse preosteoblastic MC3T3-E1 cells, primary calvarial osteoblasts, and bone marrow stromal ST2 cells. Btk expression was detected in all three cell lines. Btk inhibition stimulated mRNA expression of osteoblastic markers (alkaline phosphatase, osteocalcin, and osterix) and promoted mineralization of the extracellular matrix. In addition, Btk knockdown caused increased mRNA expression of osteoblastic markers. Furthermore, Btk inhibition suppressed the phosphorylation of mitogen-activated protein kinase (MAPK), nuclear factor kappa B (NFκB), and protein kinase Cα (PKCα). Our results indicate that Btk may regulate osteoblastic differentiation through the MAPK, NFκB, and PKCα signaling pathways.
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Diferenciação Celular/fisiologia , Osteoblastos/metabolismo , Osteoblastos/fisiologia , Proteínas Tirosina Quinases/metabolismo , Tirosina Quinase da Agamaglobulinemia , Fosfatase Alcalina/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Osteocalcina/metabolismo , Fosforilação/fisiologia , Proteína Quinase C-alfa/metabolismo , RNA Mensageiro/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Introduction. Pachydermodactyly is a rare disease with asymptomatic swelling of proximal interphalangeal joints. This disorder should be considered in the differential diagnosis of juvenile idiopathic arthritis or rheumatoid arthritis. However, pachydermodactyly is not well recognized by many orthopaedic surgeons and rheumatologists. Case Presentation. We report herein a case of a 13-year-old girl with pachydermodactyly. She presented to our clinic with symmetrical swelling of digits II through V without functional loss for the last 4 years. X-ray examination demonstrated no bone or joint destruction and magnetic resonance images showed only thickened skin tissues. No inflammatory signs were seen with laboratory blood tests. We reached a diagnosis of pachydermodactyly by exclusion. We had administered tranilast to her for 6 months and her symptom slightly improved. Conclusion. It is important to recognize pachydermodactyly and be able to differentiate it from other causes of PIP joint swelling such as rheumatoid arthritis, although pachydermodactyly is rare and benign. Physicians including orthopaedists and rheumatologists should make a prompt diagnosis to avoid unnecessary investigations and prevent the patient from receiving inappropriate treatment with steroids or cytotoxic agents. On the other hand, tranilast might be an effective drug to pachydermodactyly.
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Schwannoma is a benign peripheral nerve sheath tumor derived from Schwann cells. Bone scalloping, including spinal foramen enlargement, develops when the tumor is located adjacent to a bone and is a characteristic radiological feature. In the present study, to investigate the pathomechanism of bone resorption, the expression of noggin (a potent antagonist of bone morphogenetic protein) was analyzed in schwannoma tissues and compared with that observed in other soft tissue tumors. Quantitative polymerase chain reaction analysis revealed that the mRNA levels of noggin in schwannomas were significantly increased compared with the levels in other tumors. The gene product of noggin was only detected in a subset of schwannomas using immunohistochemistry and western blot analysis. Furthermore, the tissue extract from a noggin-producing schwannoma was found to inhibit osteoblastic differentiation in MC3T3 mouse osteoblastic cells in a dose-dependent manner. These findings indicate that bone scalloping in radiology may be induced by schwannoma-secreted noggin. In addition, noggin may have potential as a novel molecular and diagnostic marker for identifying certain types of schwannoma.
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It has been suggested that interleukin-6 (IL-6)plays a key role in the pathogenesis of rheumatoid arthritis(RA), including osteoporosis not only in inflamed joints but also in the whole body. However, previous in vitro studies regarding the effects of IL-6 on osteoblast differentiation are inconsistent. The aim of this study was to examine the effects and signal transduction of IL-6 on osteoblast differentiation in MC3T3-E1 cells and primary murine calvarial osteoblasts. IL-6 and its soluble receptor significantly reduced alkaline phosphatase (ALP) activity, the expression of osteoblastic genes (Runx2, osterix, and osteocalcin), and mineralization in a dose-dependent manner, which indicates negative effects of IL-6 on osteoblast differentiation. Signal transduction studies demonstrated that IL-6 activated not only two major signaling pathways, SHP2/MEK/ERK and JAK/STAT3, but also the SHP2/PI3K/Akt2 signaling pathway. The negative effect of IL-6 on osteoblast differentiation was restored by inhibition of MEK as well as PI3K, while it was enhanced by inhibition of STAT3. Knockdown of MEK2 and Akt2 transfected with siRNA enhanced ALP activity and gene expression of Runx2. These results indicate that IL-6 negatively regulates osteoblast differentiation through SHP2/MEK2/ERK and SHP2/PI3K/Akt2 pathways, while affecting it positively through JAK/STAT3. Inhibition of MEK2 and Akt2 signaling in osteoblasts might be of potential use in the treatment of osteoporosis in RA.
Assuntos
Interleucina-6/farmacologia , MAP Quinase Quinase 2/metabolismo , Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Camundongos , Osteoblastos/citologia , Osteoblastos/metabolismoRESUMO
We describe a three-generation family with tall stature, scoliosis and macrodactyly of the great toes and a heterozygous p.Val883Met mutation in Npr2, the gene that encodes the CNP receptor NPR2 (natriuretic peptide receptor 2). When expressed in HEK293A cells, the mutant Npr2 cDNA generated intracellular cGMP (cyclic guanosine monophosphate) in the absence of CNP ligand. In the presence of CNP, cGMP production was greater in cells that had been transfected with the mutant Npr2 cDNA compared to wild-type cDNA. Transgenic mice in which the mutant Npr2 was expressed in chondrocytes driven by the promoter and intronic enhancer of the Col11a2 gene exhibited an enhanced production of cGMP in cartilage, leading to a similar phenotype to that observed in the patients. In addition, blood cGMP concentrations were elevated in the patients. These results indicate that p.Val883Met is a constitutive active gain-of-function mutation and elevated levels of cGMP in growth plates lead to the elongation of long bones. Our findings reveal a critical role for NPR2 in skeletal growth in both humans and mice, and may provide a potential target for prevention and treatment of diseases caused by impaired production of cGMP.
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Condrócitos , Transtornos do Crescimento , Deformidades Congênitas dos Membros , Mutação de Sentido Incorreto , Receptores do Fator Natriurético Atrial , Adolescente , Substituição de Aminoácidos , Animais , Condrócitos/enzimologia , Condrócitos/patologia , GMP Cíclico , DNA Complementar , Dedos/anormalidades , Dedos/patologia , Expressão Gênica , Transtornos do Crescimento/enzimologia , Transtornos do Crescimento/genética , Transtornos do Crescimento/patologia , Células HEK293 , Humanos , Deformidades Congênitas dos Membros/enzimologia , Deformidades Congênitas dos Membros/genética , Deformidades Congênitas dos Membros/patologia , Masculino , Camundongos , Camundongos Transgênicos , Receptores do Fator Natriurético Atrial/genética , Receptores do Fator Natriurético Atrial/metabolismoRESUMO
BACKGROUND: Injuries to the epiphyseal plate sometimes result in partial growth arrest, which can lead to the development of angular deformities and limb length discrepancies in growing children. The aim of this study was to develop a new treatment for partial growth arrest of the physis. For this purpose, we investigated the feasibility of an in vitro-generated scaffold-free tissue-engineered construct (TEC) derived from synovial mesenchymal stem cells (MSCs) in a rabbit growth arrest model. METHODS: An experimental model for growth arrest was created by excising the medial half of the proximal growth plate of tibias from 6-week-old New Zealand White rabbits. Three experimental groups were set to evaluate TEC implantation: group 1, no implantation as controls; group 2, implantation of bone wax as additional controls; and group 3, implantation of TEC in the lesion. RESULTS: In group 1, all damaged growth plates were arrested and angular deformities appeared 4 weeks later. In groups 2 and 3, angular deformities were less than in the control group. Histologic images showed bone bridges developed at the damaged growth plate in group 1. Regeneration of growth plates was recognized in groups 2 and 3. Histologic examination showed greater regeneration of the growth plate in group 3 than in group 2. In addition, MSCs in the TEC differentiated into proliferative and prehypertrophic chondrocyte-like cells. CONCLUSIONS: A scaffold-free 3D TEC made using cultured synovium-derived MSCs differentiated into proliferative and prehypertrophic chondrocyte-like cells. CLINICAL RELEVANCE: The results of this experimental study suggest that scaffold-free 3D TEC made using cultured synovium-derived MSCs can be a new approach for the repair of epiphyseal injury. Clinical effectiveness of a scaffold-free 3D TEC for growth arrest remains to be determined.
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Lâmina de Crescimento/metabolismo , Transplante de Células-Tronco Mesenquimais/métodos , Tíbia/crescimento & desenvolvimento , Engenharia Tecidual/métodos , Animais , Regeneração Óssea , Diferenciação Celular , Condrócitos/metabolismo , Modelos Animais de Doenças , Lâmina de Crescimento/patologia , Masculino , Palmitatos/administração & dosagem , Coelhos , Membrana Sinovial/metabolismo , CerasRESUMO
Pigmented villonodular synovitis is a rare condition in children. We report a case of a 7-year-old girl with pigmented villonodular synovitis in her left hip. The diagnosis was confirmed with an arthroscopic hip biopsy. We completed a synovectomy twice for hip pain and were able to control her pain with these surgeries. There was, however, joint space narrowing in the affected hip. In addition, there was early closure of the proximal femoral epiphysis in the affected limb, leading to a discrepancy in leg length. This case report suggests that pigmented villonodular synovitis should be considered in children with a painful hip.
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Articulação do Quadril/patologia , Sinovite Pigmentada Vilonodular/diagnóstico , Feminino , Lâmina de Crescimento/patologia , Humanos , Desigualdade de Membros Inferiores , Osteoartrite do Quadril/etiologia , Osteoartrite do Quadril/patologia , Dor/etiologia , Dor/cirurgia , Complicações Pós-Operatórias , Sinovectomia , Sinovite Pigmentada Vilonodular/complicações , Sinovite Pigmentada Vilonodular/cirurgiaRESUMO
Spleen tyrosine kinase (Syk) is a non-receptor protein kinase present in abundance in a wide range of hematopoietic cells. Syk reportedly plays a crucial role in immune signaling in B cells and cells bearing Fcγ-activation receptors. The role of syk in osteoblastic differentiation has not been well elucidated. We report herein the role of syk in osteoblastic differentiation. We investigated the effects of two syk inhibitors on osteoblastic differentiation in mouse preosteoblastic MC3T3-E1 cells and bone marrow stromal ST2 cells. Expression of syk was detected in these two cell lines. Two syk inhibitors stimulated mRNA expression of osteoblastic markers (ALP, Runx2, Osterix). Mineralization of extracellular matrix was also promoted by treatment with syk inhibitors. Knockdown of Syk caused increased mRNA expression of osteoblastic markers. In addition, syk inhibitor and knockdown of Syk suppressed phosphorylation of mitogen-activated protein kinase (MAPK) and protein kinase Cα (PKCα). Our results indicate that syk might regulate osteoblastic differentiation through MAPK and PKCα.
Assuntos
Diferenciação Celular , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Osteoblastos/citologia , Osteogênese , Proteína Quinase C-alfa/metabolismo , Proteínas Tirosina Quinases/fisiologia , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/enzimologia , Técnicas de Silenciamento de Genes , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Osteoblastos/enzimologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/genética , Células Estromais/citologia , Células Estromais/enzimologia , Quinase SykRESUMO
Protein kinase C (PKC) plays an essential role in cellular signal transduction for mediating a variety of biological functions. There are 11 PKC isoforms and these isoforms are believed to play distinct roles in cells. Although the role of individual isoforms of PKC has been investigated in many fields, little is known about the role of PKC in osteoblastic differentiation. Here, we investigated which isoforms of PKC are involved in osteoblastic differentiation of the mouse preosteoblastic cell line MC3T3-E1. Treatment with Gö6976, an inhibitor of PKCα and PKCßI, increased alkaline phosphatase (ALP) activity as well as gene expression of ALP and Osteocalcin (OCN), and enhanced calcification of the extracellular matrix. Concurrently, osteoblastic cell proliferation decreased at a concentration of 1.0 µM. In contrast, a PKCß inhibitor, which inhibits PKCßI and PKCßII, did not significantly affect osteoblastic differentiation or cell proliferation. Knockdown of PKCα using MC3T3-E1 cells transfected with siRNA also induced an increase in ALP activity and in gene expression of ALP and OCN. In contrast, overexpression of wild-type PKCα decreased ALP activity and attenuated osteoblastic differentiation markers including ALP and OCN, but promoted cell proliferation. Taken together, our results indicate that PKCα suppresses osteoblastic differentiation, but promotes osteoblastic cell proliferation. These results imply that PKCα may have a pivotal role in cell signaling that modulates the differentiation and proliferation of osteoblasts.
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Diferenciação Celular , Osteoblastos/citologia , Osteoblastos/enzimologia , Proteína Quinase C-alfa/metabolismo , Adenoviridae/genética , Animais , Carbazóis/farmacologia , Diferenciação Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Camundongos , Osteoblastos/efeitos dos fármacos , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Proteína Quinase C beta , Proteína Quinase C-alfa/antagonistas & inibidores , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Bone morphogenetic proteins (BMPs) regulate many mammalian physiologic and pathophysiologic processes. These proteins bind with the kinase receptors BMPR-I and BMPR-II, thereby activating Smad transcription factor. In this study, we demonstrate that neogenin, a receptor for netrins and proteins of the repulsive guidance molecule family, is a receptor for BMPs and modulates Smad signal transduction. Neogenin was found to bind directly with BMP-2, BMP-4, BMP-6, and BMP-7. Knockdown of neogenin in C2C12 cells resulted in the enhancement of the BMP-2-induced processes of osteoblastic differentiation and phosphorylation of Smad1, Smad5, and Smad8. Conversely, overexpression of neogenin in C2C12 cells suppressed these processes. Our results also indicated that BMP-induced activation of RhoA was mediated by neogenin. Inhibition of RhoA promoted BMP-2-induced processes of osteoblastic differentiation and phosphorylation of Smad1/5/8. However, treatment with Y-27632, an inhibitor of Rho-associated protein kinase, did not modulate BMP-induced phosphorylation of Smad1/5/8. Taken together, our findings suggest that neogenin negatively regulates the functions of BMP and that this effect of neogenin is mediated by the activation of RhoA.
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Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas de Membrana/metabolismo , Osteoblastos/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/genética , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Proteínas Morfogenéticas Ósseas/genética , Diferenciação Celular/fisiologia , Ativação Enzimática/fisiologia , Células HEK293 , Humanos , Proteínas de Membrana/genética , Osteoblastos/citologia , Fosforilação/fisiologia , Ligação Proteica/fisiologia , Proteínas Smad/genética , Proteínas Smad/metabolismo , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
The existence of inorganic polyphosphates [poly(P)] in human cells has been demonstrated. In osteoblasts, it is suggested that the concentration of cellular poly(P) is relatively high. In this study, we examined whether poly(P) accelerates the differentiation of human mesenchymal stem cells (hMSCs) from patients with osteoarthritis (OA) and rheumatoid arthritis (RA) into osteoblastic cells. Alkaline phosphatase (ALP) activity was induced by poly(P) in hMSCs from both OA and RA. In Alizarin Red S and osteocalcin EIA, there was a significant difference between the control and poly(P) group. In real-time PCR, there was a significant difference in ALP, collagen type 1A, osteocalcin, and bone sialoprotein between the control and poly(P) group. Our findings suggest that poly(P) have the potent role of differentiating hMSCs into osteoblastic cells at the early and later stages of osteoblastic differentiation.
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Diferenciação Celular/efeitos dos fármacos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Polifosfatos/farmacologia , Fosfatase Alcalina/metabolismo , Células Cultivadas , Feminino , Humanos , Técnicas Imunoenzimáticas , Osteocalcina/metabolismo , Reação em Cadeia da PolimeraseRESUMO
OBJECTIVE: To evaluate the osteoblastic differentiation of human mesenchymal stem cells (hMSCs) in patients with RA. METHODS: Heparinized bone marrow aspirate was obtained from patients with OA and RA. Mononuclear cells were cultured for 2 weeks and a colony-forming assay was performed. The phenotype of cells was analysed by flow cytometry. Passage 2 cells were cultured with beta-glycerophosphate (bGP) in the control group and bGP, ascorbic acid and dexamethasone in the differentiation group. After 2 weeks, ALP staining and activity were performed. After 3 weeks, Alizarin Red S assay was performed. Total RNA was extracted from cells cultured for 2 and 3 weeks. Gene expression of bone formation factor was examined by real-time PCR. RESULTS: The phenotype of cells was identical in both OA and RA and the content was thought to be hMSCs. The results of ALP activity and Alizarin Red S assay showed higher levels in the differentiation group for both OA and RA samples compared with the control group. The results of a colony-forming assay were identical in both OA and RA samples. Gene expression in the differentiation group was higher than in the control group in both OA and RA samples. There was no significant difference between OA and RA samples in all experiments. CONCLUSION: The function of osteoblastic differentiation of hMSCs is similar between OA and RA.
Assuntos
Artrite Reumatoide/patologia , Células-Tronco Mesenquimais/patologia , Osteoartrite/patologia , Osteoblastos/patologia , Idoso , Idoso de 80 Anos ou mais , Fosfatase Alcalina/análise , Biomarcadores/análise , Estudos de Casos e Controles , Técnicas de Cultura de Células , Diferenciação Celular , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Feminino , Citometria de Fluxo , Expressão Gênica , Humanos , Pessoa de Meia-Idade , Osteocalcina/análise , Osteopontina/análise , Fenótipo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Coloração e Rotulagem , Estatísticas não ParamétricasRESUMO
Dynamic cytoskeletal changes appear to be one of intracellular signals that control cell differentiation. To test this hypothesis, we examined the effects of short-term actin cytoskeletal changes on osteoblastic differentiation. We found an actin polymerization interfering reagent, cytochalasin D, promoted osteoblastic differentiation in mouse preosteoblastic MC3T3-E1 cells. We also found that these effects were mediated by the protein kinase D (PKD) pathway. Short-term cytochalasin D treatment increased alkaline phosphatase (ALP) activity, osteocalcin (OCN) secretion, and mineralization of the extracellular matrix in MC3T3-E1 cells, with temporary changes in actin cytoskeleton. Furthermore, the disruption of actin cytoskeleton induced phosphorylation of 744/748 serine within the activation loop of PKD in a dose-dependent manner. The protein kinase C (PKC)/PKD inhibitor Go6976 suppressed cytochalasin D-induced acceleration of osteoblastic differentiation, whereas Go6983, a specific inhibitor of conventional PKCs, did not. Involvement of PKD signaling was confirmed by using small interfering RNA to knock down PKD. In addition, another actin polymerization interfering reagent, latrunculin B, also stimulated ALP activity and OCN secretion with PKD activation. On the other hand, the present data suggested that transient dynamic actin cytoskeletal reorganization could be a novel cellular signal that directly stimulated osteoblastic differentiation.
Assuntos
Actinas/metabolismo , Diferenciação Celular , Citoesqueleto/metabolismo , Osteoblastos/citologia , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Diferenciação Celular/efeitos dos fármacos , Citocalasina D/farmacologia , Citoesqueleto/efeitos dos fármacos , Camundongos , Osteoblastos/efeitos dos fármacos , Osteoblastos/enzimologia , Fosfosserina/metabolismo , Proteína Quinase C/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tiazolidinas/farmacologiaRESUMO
INTRODUCTION: Surgical results of correction of three-dimensional deformities with shortening of lower limbs by simple two-ring system of Taylor Spatial Frame have been reported. The deformities were caused by various kinds of skeletal disorders. METHODS: Ten cases were successfully corrected using distraction osteogenesis, being one case showed recurrence of deformity due to the nature of the disease. RESULTS: The ranges of corrected deformities were 5 degrees-55 degrees in coronal angulation, 2 degrees-47 degrees in sagittal angulation, and 5 degrees-40 degrees in rotational deformities. Amount of lengthening ranged from 1.2 to 6.0 cm. Pin-site infection occurred in five cases, being no other complications. External fixation index was longer in a group of rotational correction over 10 degrees. CONCLUSION: The current report seems the first series of cases with four-dimensional deformities treated by distraction osteogenesis by TSF and may help to determine the indication of use of TSF in case of treatment of patients suffering from deformities in the lower limbs.