RESUMO
BACKGROUND: It has been reported that switching from daily (d) teriparatide (TPTD) to denosumab (DMAb) is effective for severe osteoporosis patients. However, there have been no reports about switching from weekly (w) TPTD to DMAb in patients with osteoporosis. Once-weekly 56.5-µg TPTD treatment increases bone mineral density (BMD) and reduces fracture events. The objective of the current retrospective study was to elucidate the impact of switching w-TPTD to DMAb in patients with osteoporosis. METHODS: In this study, 40 patients were treated with w-TPTD for 18 months and then switched to DMAb for 18 months. The sample included 2 men and 38 women with a mean age of 74.5 (60-85) years. Twenty-five subjects had primary osteoporosis, and 15 had secondary osteoporosis. The mean number of osteoporotic vertebral fractures was 4.1. Serum bone turnover markers and BMD were evaluated every 6 months. RESULTS: Bone alkaline phosphatase (BAP) and tartrate resistant acid phosphatase 5b (TRACP5b), markers of bone formation and resorption respectively, were not significantly different in w-TPTD subjects at 18 months compared with those at baseline (p > 0.05), but BAP and TRACP5b in subjects treated with DMAb were significantly lower at 36 months compared with those at baseline (p < 0.05). BMD of the lumbar spine (LS), femoral neck (FN), and total hip (TH) increased by 12.3%, 2.5%, and 2.2% by 36 months with DMAb treatment, significantly higher than at baseline (p < 0.05). Changes in BMD of FN and TH in primary osteoporosis patients were significantly higher than in secondary osteoporosis patients at 18 months (w-TPTD) and 36 months (DMAb, p < 0.05). CONCLUSION: BMD significantly increased in osteoporosis patients switched from w-TPTD to DMAb. However, the impact of switching from w-TPTD to DMAb in secondary osteoporosis patients was not as great as in primary osteoporosis patients at the view points of changes in BMD of FN and TH.
Assuntos
Remodelação Óssea/efeitos dos fármacos , Denosumab/uso terapêutico , Osteoporose/tratamento farmacológico , Teriparatida/uso terapêutico , Idoso , Idoso de 80 Anos ou mais , Densidade Óssea , Conservadores da Densidade Óssea/uso terapêutico , Substituição de Medicamentos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Osteoporose/diagnóstico , Estudos Retrospectivos , Resultado do TratamentoRESUMO
BACKGROUND: Although several types of culture medium have been used for preservation of osteochondral allografts, the viability of chondrocytes decreases with increasing storage duration. We previously showed the University of Wisconsin solution is more suitable for graft preservation than culture medium. QUESTIONS/PURPOSES: We determined whether the addition of allogenic serum to University of Wisconsin solution increases chondrocyte survival during prolonged storage of osteochondral allografts. METHODS: Osteochondral tissue samples harvested from the distal femora of rats were preserved in University of Wisconsin solution supplemented with 0%, 1%, 10%, and 50% allogenic serum at 4 °C for 14 days. Cell viability and chondrocyte degenerative changes of the samples then were assessed using a tetrazolium assay and histologic methods. We also evaluated time-dependent changes in cell viability and histologic findings of samples preserved for 7, 14, and 21 days in University of Wisconsin solution supplemented with or without 10% allogenic serum. RESULTS: After 14 days of preservation, osteochondral tissue samples maintained in University of Wisconsin solution containing 10% or greater allogenic serum exhibited the highest cell viability and lowest degenerative changes in chondrocytes. In the evaluation of time-dependent changes, we found the chondrocyte degenerative changes were greater in cartilage preserved in University of Wisconsin solution alone than in University of Wisconsin solution containing 10% allogenic serum after day 7 or later. CONCLUSIONS: Our results suggest the addition of 10% allogenic serum to University of Wisconsin solution enhances viability of osteochondral tissue samples. CLINICAL RELEVANCE: The use of allogenic serum-supplemented University of Wisconsin solution is expected to prolong the duration of osteochondral allograft storage and result in higher-quality grafts.
Assuntos
Cartilagem/transplante , Condrócitos , Temperatura Baixa , Soluções para Preservação de Órgãos , Soro , Preservação de Tecido/métodos , Adenosina , Alopurinol , Animais , Sobrevivência Celular , Glutationa , Insulina , Masculino , Rafinose , Ratos , Ratos Sprague-DawleyRESUMO
Frozen bone-patellar tendon bone allografts are useful in anterior cruciate ligament reconstruction as the freezing procedure kills tissue cells, thereby reducing immunogenicity of the grafts. However, a small portion of cells in human femoral heads treated by standard bone-bank freezing procedures survive, thus limiting the effectiveness of allografts. Here, we characterized the survival rates and mechanisms of cells isolated from rat bones and tendons that were subjected to freeze-thaw treatments, and evaluated the influence of these treatments on the mechanical properties of tendons. After a single freeze-thaw cycle, most cells isolated from frozen bone appeared morphologically as osteocytes and expressed both osteoblast- and osteocyte-related genes. Transmission electron microscopic observation of frozen cells using freeze-substitution revealed that a small number of osteocytes maintained large nuclei with intact double membranes, indicating that these osteocytes in bone matrix were resistant to ice crystal formation. We found that tendon cells were completely killed by a single freeze-thaw cycle, whereas bone cells exhibited a relatively high survival rate, although survival was significantly reduced after three freeze-thaw cycles. In patella tendons, the ultimate stress, Young's modulus, and strain at failure showed no significant differences between untreated tendons and those subjected to five freeze-thaw cycles. In conclusion, we identified that cells surviving after freeze-thaw treatment of rat bones were predominantly osteocytes. We propose that repeated freeze-thaw cycles could be applied for processing bone-tendon constructs prior to grafting as the treatment did not affect the mechanical property of tendons and drastically reduced surviving osteocytes, thereby potentially decreasing allograft immunogenecity.
Assuntos
Transplante Ósseo , Osso e Ossos/citologia , Congelamento , Osteócitos/citologia , Tendões/citologia , Tendões/fisiologia , Animais , Biomarcadores/metabolismo , Fenômenos Biomecânicos/fisiologia , Separação Celular , Forma Celular , Sobrevivência Celular , Fêmur/citologia , Regulação da Expressão Gênica , Humanos , Masculino , Osteoblastos/citologia , Osteoblastos/ultraestrutura , Osteócitos/ultraestrutura , Ratos , Ratos Wistar , Tendões/transplante , Tíbia/citologiaRESUMO
To eliminate the potential for infection, many tissue banks routinely process and terminally sterilize allografts prior to transplantation. A number of techniques, including the use of scanning electron microscopy, bone graft models, and mechanical property tests, are used to evaluate the properties of allograft bone. However, as these methods are time consuming and often destroy the bone sample, the quality assessment of allograft bones are not routinely performed after processing and sterilization procedures. Raman spectroscopy is a non-destructive, rapid analysis technique that requires only small sample volumes and has recently been used to evaluate the mineral content, mineral crystallinity, acid phosphate and carbonate contents, and collagen maturity in human and animal bones. Here, to establish a quality assessment method of allograft bones using Raman spectroscopy, the effect of several common sterilization and preservation procedures on rat femoral bones were investigated. We found that freeze-thawing had no detectable effects on the composition of bone minerals or matrix, although heat treatment and gamma irradiation resulted in altered Raman spectra. Our findings suggest Raman spectroscopy may facilitate the quality control of allograft bone after processing and sterilization procedures.
Assuntos
Densidade Óssea , Matriz Óssea , Transplante Ósseo , Raios gama/efeitos adversos , Análise Espectral Raman , Animais , Densidade Óssea/efeitos da radiação , Matriz Óssea/efeitos da radiação , Fêmur/química , Fêmur/efeitos da radiação , Congelamento/efeitos adversos , Temperatura Alta/efeitos adversos , Masculino , Controle de Qualidade , Ratos , Ratos Wistar , Esterilização/métodosRESUMO
Our goal in this study was to determine to what extent the physiologic consequences of ovariectomy (OVX) in bones are exacerbated by a lack of daily activity such as walking. We forced 14-week-old female rats to be inactive for 15 weeks with a unique experimental system that prevents standing and walking while allowing other movements. Tibiae, femora, and 4th lumbar vertebrae were analyzed by peripheral quantitative computed tomography (pQCT), microfocused X-ray computed tomography (micro-CT), histology, histomorphometry, Raman spectroscopy, and the three-point bending test. Contrary to our expectation, the exacerbation was very much limited to the cancellous bone parameters. Parameters of femur and tibia cortical bone were affected by the forced inactivity but not by OVX: (1) cross-sectional moment of inertia was significantly smaller in Sham-Inactive rat bones than that of their walking counterparts; (2) the number of sclerostin-positive osteocytes per unit cross-sectional area was larger in Sham-Inactive rat bones than in Sham-Walking rat bones; and (3) material properties such as ultimate stress of inactive rat tibia was lower than that of their walking counterparts. Of note, the additive effect of inactivity and OVX was seen only in a few parameters, such as the cancellous bone mineral density of the lumbar vertebrae and the structural parameters of cancellous bone in the lumbar vertebrae/tibiae. It is concluded that the lack of daily activity is detrimental to the strength and quality of cortical bone in the femur and tibia of rats, while lack of estrogen is not. Our inactive rat model, with the older rats, will aid the study of postmenopausal osteoporosis, the etiology of which may be both hormonal and mechanical.
Assuntos
Envelhecimento/patologia , Modelos Biológicos , Ovariectomia , Tíbia/patologia , Animais , Biomarcadores/metabolismo , Fenômenos Biomecânicos/fisiologia , Peso Corporal/fisiologia , Densidade Óssea/fisiologia , Matriz Óssea/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Remodelação Óssea/fisiologia , Epífises/diagnóstico por imagem , Epífises/fisiopatologia , Feminino , Marcadores Genéticos , Imuno-Histoquímica , Vértebras Lombares/diagnóstico por imagem , Vértebras Lombares/fisiopatologia , Osteócitos/metabolismo , Osteócitos/patologia , Ratos , Ratos Wistar , Análise Espectral Raman , Tíbia/diagnóstico por imagem , Tíbia/metabolismo , Tíbia/fisiopatologia , Caminhada/fisiologia , Microtomografia por Raio-XRESUMO
Many investigators are currently studying the use of decellularized tissue allografts from human cadavers as scaffolds onto which patients' cells could be seeded, or as carriers for genetically engineered cells to aid cell transplantation. However, it is difficult to seed cells onto very dense regular connective tissue which has few interstitial spaces. Here, we discuss the development of a chemotactic cell seeding technique using solvent-preserved human meniscus. A chemokinetic response to recombinant human bone morphogenetic protein-2 (rhBMP-2) was observed in a monolayer culture of primary chondrocytes derived from femoral epiphyseal cartilage of 2-day-old rats. The rhBMP-2 significantly increased their migration upto 10 ng/ml in a dose-dependent manner. When tested with solvent-preserved human meniscus as a scaffold, which has few interstitial spaces, rhBMP-2 was able to induce chondrocytes to migrate into the meniscus. After a 3-week incubation, newly-formed cartilaginous extracellular matrix was synthesized by migrated chondrocytes throughout the meniscus, down to a depth of 3 mm. These findings demonstrate that rhBMP-2 may be a natural chemokinetic factor in vivo, which induces migration of proliferative chondrocytes into the narrow interfibrous spaces. Our results suggest a potential application of rhBMP-2 for the designed distribution of chondrocytes into a scaffold to be used for tissue engineering.
Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Condrócitos/citologia , Meniscos Tibiais/citologia , Engenharia Tecidual/métodos , Fator de Crescimento Transformador beta/metabolismo , Animais , Movimento Celular , Células Cultivadas , Fêmur/citologia , Lâmina de Crescimento/citologia , Humanos , Meniscos Tibiais/ultraestrutura , Ratos , Ratos Wistar , Proteínas Recombinantes/metabolismo , SolventesRESUMO
Low intensity pulsed ultrasound (LIPUS) is an extremely useful noninvasive treatment which halves the duration of fracture healing when the bone is exposed once a day for 20 min. To elucidate the direct reactions of bone and cartilage, dissected rat femora were immobilized in culture dish wells, exposed to LIPUS from a certain angle every day, and the local pattern of ossification was analyzed in relation to the ultrasound. Daily 20-min exposures were started 24 hr after isolation of the femora, and at days 5, 10, and 15, samples were harvested for measurements, morphological, and histochemical analyses. While the gross features of the samples were identical to the untreated controls, extended mineralization of the periosteum was observed with alizarin red staining, antiosteocalcin immunohistochemical staining, and micro-three dimensional computed tomography. Interestingly, the newly deposited mineral was found perpendicular to the ultrasound path, strongly suggesting that LIPUS accelerates periosteal bone formation. Zones of epiphyseal cartilage and hypertrophic and calcified cartilage did not exhibit any differences with and without this exposure. LIPUS also did not influence the secreted proteoglycan components or amounts in the culture medium. The absence of any additional longitudinal growth of the femur demonstrated that LIPUS did not accelerate endochondral bone formation. We conclude that cartilage alone does not directly respond to therapeutic ultrasound, whereas the periosteum does.
Assuntos
Cartilagem/fisiologia , Fêmur/fisiologia , Osteogênese/fisiologia , Periósteo/fisiologia , Terapia por Ultrassom , Animais , Pesos e Medidas Corporais , Sulfatos de Condroitina/metabolismo , Fêmur/patologia , Histocitoquímica , Ácido Hialurônico/metabolismo , Ratos , Ratos Wistar , Tomografia Computadorizada por Raios XRESUMO
STR/ort mice develop a naturally occurring osteoarthritis (OA) of the knee joints. However, the evaluation of early OA changes has been difficult due to variability caused by gender, individual differences, and differences between the right and left lower limbs. The objective of this study was to analyze the variability of the early OA changes with age in STR/ort mice and to identify the cause of onset. A total of 115 STR/OrtCrlj mice aged 10-45 weeks were examined. In addition to conventional radiological and histological evaluation of the knee joints, histological sections were used to examine the patellofemoral, femorotibial, and growth plate cartilage under similar conditions. A morphological evaluation of tibiae, including micro-3-dimensional computed tomography, was performed. Radiological evaluation showed OA changes in the joints of mice over 35 weeks old and histological evaluation showed early OA changes in the femorotibial joints of mice over 26 weeks old. However, these changes were not common in all individuals. In contrast, most common and reproducible OA changes were observed in the bilateral patellofemoral joints of all individuals, and even in subjects ranging from 10 to 20 weeks of age. Morphological evaluations also demonstrated an abnormal tibial internal torsion that increased with age and was associated with medial patellar dislocation. In conclusion, the earliest histological OA change was observed in the patellofemoral joint prior to similar observations in the femorotibial joint. Internal tibial torsion may be a cause of OA in the patellofemoral joints, which leads to the development of medial femorotibial OA.
Assuntos
Artrite Experimental/patologia , Articulação do Joelho/citologia , Osteoartrite do Joelho/patologia , Tíbia/patologia , Anormalidade Torcional/patologia , Fatores Etários , Animais , Artrite Experimental/diagnóstico por imagem , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Mutantes , Osteoartrite do Joelho/diagnóstico por imagem , Radiografia , Tíbia/diagnóstico por imagemRESUMO
BACKGROUND: Calcium phosphate cement [CPC (Biopex)] has been used as the drug delivery system of choice for treatment of infected joint replacement because of its good elution efficiency. The influence of CPC polymerization on the bactericidal activity of vancomycin (VCM) impregnated into CPC has not been investigated. We compared VCM concentration, bactericidal activity, and profile of eluates between CPC and polymethylmethacrylate (PMMA; Cemex RX). METHODS: Test specimens consisted of a powder composite of CPC or PMMA, VCM and solvent (10:0.25:3.3 g). Each test specimen was immersed in sterile phosphate-buffered saline. Eluates obtained on days 1, 3, 7, and 14 and weeks 4, 8, and 12 were evaluated by high performance liquid chromatography (HPLC) and by microbiological assay (MBA). RESULTS: The elution level of VCM from CPC/VCM on day 1 was 8.1 fold greater than that from PMMA/VCM. The detection periods of VCM from CPC/VCM and PMMA/VCM were 8 weeks and 14 days, respectively. The values of eluates from CPC/VCM and PMMA/VCM obtained by HPLC were comparable to those obtained by MBA. HPLC chromatogram showed that the elution profiles of VCM from CPC/VCM and PMMA/VCM on day 1 were very close to those of standard solutions. CONCLUSIONS: CPC could release more VCM over a longer period than PMMA. The polymerization of CPC and PMMA did not alter the inhibitory activity of VCM and did not denature VCM.
Assuntos
Antibacterianos , Fosfatos de Cálcio/síntese química , Sistemas de Liberação de Medicamentos , Polimetil Metacrilato/síntese química , Vancomicina , Cimentos Ósseos , Cromatografia Líquida de Alta Pressão , Testes de Sensibilidade a Antimicrobianos por Disco-Difusão , Técnicas In Vitro , Infecções Relacionadas à Prótese/prevenção & controleRESUMO
We compared Dulbecco's modified Eagle's medium (DMEM), saline, Euro-Collins (EC) solution and University of Wisconsin (UW) solution to determine which was best for cold preservation of rat osteochondral tissues (OCTs). After 7 days' cold preservation, OCTs kept in UW solution had the highest relative viable cell number by the tetrazolium assay and the lowest activity of lactate dehydrogenase released from damaged cells. Histological evaluation revealed chondrocyte deformity, such as shrunken cytoplasm and pyknotic nuclei, particularly in the deeper layer of articular cartilage after preservation in saline and EC solution and predominantly in all layers if preserved in DMEM. In contrast, chondrocyte morphology in all layers of the articular cartilage preserved in UW solution was relatively unchanged and remained similar to fresh OCTs. It is therefore concluded that UW solution is the most suitable for cold preservation of rat OCTs as well as solid organs.
Assuntos
Osso e Ossos/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Criopreservação/métodos , Meios de Cultura/farmacologia , Soluções para Preservação de Órgãos/farmacologia , Adenosina/farmacologia , Alopurinol/farmacologia , Animais , Osso e Ossos/citologia , Condrócitos/citologia , Crioprotetores/farmacologia , Glutationa/farmacologia , Insulina/farmacologia , Masculino , Preservação de Órgãos , Rafinose/farmacologia , Ratos , Ratos Sprague-Dawley , SalinidadeRESUMO
The effects of cryopreservation on tendon allograft have been reported, but remain unclear, particularly the potential effects on mechanical properties and histological changes by ice crystal formation. There are also few studies about effects of heating for sterilization of tendon. We evaluated the effect of cryopreservation or heating on the mechanical properties and histomorphology of rat bone-patellar tendon-bones (BTBs). BTBs were processed by cryopreservation at -80 degrees C for 3 weeks, or heating at 80 degrees C for 10 min. Tensile testing and histomorphological examination were performed. The cryopreservation of tendons showed less influences on their mechanical properties. When cryopreserved BTBs in frozen state were fixed by freeze-substitution method, many spaces were observed in interfibrillar substances. These results suggest that the collagen fibers of cryopreserved tendons were histomorphologically affected by ice crystals. The heating of tendons completely destroyed the collagen fibers of the tendons and is therefore thought to be inappropriate for the sterilization of BTBs.
Assuntos
Colágeno/fisiologia , Substituição ao Congelamento , Preservação de Órgãos , Ligamento Patelar/fisiologia , Esterilização , Animais , Colágeno/ultraestrutura , Criopreservação , Temperatura Alta , Masculino , Microscopia Eletrônica de Transmissão , Ligamento Patelar/transplante , Ligamento Patelar/ultraestrutura , Ratos , Ratos Wistar , Resistência à Tração/fisiologiaRESUMO
Demand for banked bone allografts is increasing in Japan; however, there are too few bone banks and the bone bank network is not well-established. One reason for this was lack of funding for banks. Bone banks had to bear all material expenses of banked bone allografts themselves because this was not designated a covered expense. In December 2004, the Japanese government started a new "Advanced Medical Treatment" administration system which allowed an approved institution to charge the expense of authorized advanced medical treatments directly to patients. The treatment named "Cryopreserved allogenic bone and ligamentous tissue retrieved from cadaveric donor" was approved as an advanced medical treatment in March 2007. We present the calculation method and the expense per implantation of a banked bone allograft from a cadaveric donor under this treatment and raise issues which affect this advanced medical treatment and remain to be resolved in the Japanese orthopaedic field.
Assuntos
Bancos de Ossos/economia , Transplante Ósseo/economia , Doadores de Tecidos , Transplante Ósseo/diagnóstico por imagem , Cadáver , Criopreservação , Humanos , Japão , RadiografiaRESUMO
Low-intensity pulsed ultrasound, a form of mechanical energy transmitted as high-frequency acoustical pressure waves, provides noninvasive therapeutic treatment for accelerating fracture repair and distraction osteogenesis. Relatively young osteoblasts respond to ultrasound by transiently upregulating message levels of immediate-early genes as well as that of osteocalcin and insulin-like growth factor I (IGF-I). Osteocytes derived from newborn rat tibia and calvaria responded to a lesser extent only in c-fos and cyclooxygenase-2 (COX-2) messages. Compared with the stretched osteocytes, which use stretch-activated and parathyroid hormone (PTH)-potentiated Ca2+ influx as an entry route to the protein kinase A (PKA) signal transduction pathways, there was no evidence of Ca2+ internalization by any of the cells tested on exposure to the ultrasound. On the other hand, inhibitors of p38 mitogen-activated protein kinase (MAPK) and upstream phosphoinositide 3-kinase (PI3K) blocked COX-2 and osteocalcin upregulation by the ultrasound-exposed ST2, murine bone marrow-derived cells. This is distinct from the aforementioned osteocytic response to low-frequency stretching and implies the involvement of integrins. Our findings suggested that accelerated fracture repair and distraction osteogenesis by the low-intensity pulsed ultrasound depend, at least in part, on the stimulation of osteoblastic cells at relatively early stages of osteogenic lineage. Bone is under control of multiple regulatory mechanisms so that diverse physical forces can be reflected to the microenvironment of each cell, in turn, to the entire bone.
Assuntos
Osteoblastos/diagnóstico por imagem , Osteoblastos/patologia , Ultrassom , Animais , Animais Recém-Nascidos , Antineoplásicos Hormonais/farmacologia , Western Blotting , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Cálcio/metabolismo , Diferenciação Celular , Linhagem da Célula , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ciclo-Oxigenase 2 , Dexametasona/farmacologia , Dinoprostona/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Isoenzimas/metabolismo , Camundongos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Osteoblastos/metabolismo , Osteocalcina/metabolismo , Osteócitos/diagnóstico por imagem , Osteócitos/metabolismo , Osteócitos/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Prostaglandina-Endoperóxido Sintases/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Crânio/metabolismo , Tíbia/metabolismo , Fatores de Tempo , Ultrassonografia , Regulação para Cima , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Mesenchymal stem cells (MSCs) are thought to be multipotential, capable of differentiating into multiple lineages. We attempted to characterize rat cells derived from fetal circulating blood (FCBCs) that displayed a fibroblastic morphology and differentiated into osteoblastic and chondrocytic lineages. Notably, they differentiated into a chondrocyte-specific phenotype on plastic culture dishes in medium supplemented only with 10% fetal bovine serum (FBS) without the use of a three-dimensional culture substrate. Bone marrow-derived cells did not convey such phenotypic expression under the same conditions. The characteristic features of these cells were analyzed by reverse transcription polymerase chain reaction, immunohistological and von Kossa staining, and by immuno-dot blotting. In one population, expression of collagen types II and X was detected in differentiated cells at the same levels as observed in chondrocytes derived from rat rib cartilage. In another population, parathyroid hormone receptor, alkaline phosphatase, and osteocalcin were also expressed at levels almost equal to those observed in long bone-derived osteoblasts. After 3 weeks in culture, extensively condensed cell masses, stained with anti-type II collagen antibody, could be distinguished histologically from small, multilayered, von Kossa-positive nodules, which stained with anti-osteocalcin, but not with anti-type II collagen antibody. In addition, the FCBCs differentiated into adipogenic cells in the presence of methyl-isobutyl xanthine, dexamethasone, insulin, and indomethacin. These cells expressed PPARgamma2 mRNA and accumulated lipid vesicles detectable by Oil red-O staining. Our findings suggest that FCBCs have the potential to readily differentiate into multiple lineages and that they are distinct from mesenchymal stem cells derived from bone marrow or circulating blood from more mature and adults in their spontaneous differentiation in the absence of specific factors such as transforming growth factor-beta (TGF-beta) or dexamethasone, or a three-dimensional culture environment.
Assuntos
Diferenciação Celular , Sangue Fetal/citologia , Células-Tronco Mesenquimais/citologia , Animais , Proliferação de Células , Células Cultivadas , Colágeno Tipo II/metabolismo , Sangue Fetal/metabolismo , Imuno-Histoquímica , Células-Tronco Mesenquimais/metabolismo , Osteocalcina/metabolismo , RNA Mensageiro/genética , Ratos , Fatores de TempoRESUMO
Basic fibroblast growth factor 2 (bFGF) is a potent mitogen for mesenchymal cells, and the local application of recombinant bFGF accelerates bone union and defect repair. However, repeated dosing is required for sustained therapeutic effect as the efficacy of bFGF decreases rapidly following its diffusion from bone defect sites. Here, we attempted to develop a collagen-based bone formation system using a fusion protein (collagen binding-bFGF, CB-bFGF) consisting of bFGF and the collagen-binding domain (CBD) of Clostridium histolyticum collagenase. The addition of the CBD to bFGF did not modify its native biological activity, as shown by the capacity of the fusion protein to promote the in vitro proliferation of periosteal mesenchymal cells. The affinity of the fusion protein towards collagen and demineralized bone matrix (DBM) was also confirmed by collagen-binding assays. Moreover, in vivo periosteal bone formation assays showed that the combination of CB-bFGF with a collagen sheet induced periosteal bone formation at protein concentrations lower than those required for bFGF alone. In addition, grafts of DBM loaded with CB-bFGF accelerated new bone formation in rat femurs compared to the same concentration of bFGF administered alone. Taken together, these properties suggest that the CB-bFGF/collagen composite is a promising material for bone repair in the clinical setting.
Assuntos
Clostridium histolyticum/enzimologia , Colágeno/metabolismo , Fator 2 de Crescimento de Fibroblastos/uso terapêutico , Colagenase Microbiana/metabolismo , Osteogênese/efeitos dos fármacos , Proteínas Recombinantes de Fusão/uso terapêutico , Animais , Células Cultivadas , Fator 2 de Crescimento de Fibroblastos/administração & dosagem , Fator 2 de Crescimento de Fibroblastos/química , Fator 2 de Crescimento de Fibroblastos/metabolismo , Humanos , Masculino , Colagenase Microbiana/química , Estrutura Terciária de Proteína , Ratos , Ratos Wistar , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Cicatrização/efeitos dos fármacosRESUMO
Although recent studies suggest that hyperlipidemia is a risk factor for osteoarthritis (OA), the link between OA and hyperlipidemia is not fully understood. As the number of activated, circulating myeloid cells is increased during hyperlipidemia, we speculate that myeloid cells contribute to the pathology of OA. Here, we characterized myeloid cells in STR/Ort mice, a murine osteoarthritis model, under hyperlipidemic conditions. Ratios of myeloid cells in bone marrow, the spleen, and peripheral blood were determined by flow cytometry. To examine the influence of the hematopoietic environment, including abnormal stem cells, on the hematopoietic profile of STR/Ort mice, bone marrow transplantations were performed. The relationship between hyperlipidemia and abnormal hematopoiesis was examined by evaluating biochemical parameters and spleen weight of F2 animals (STR/Ort x C57BL/6J). In STR/Ort mice, the ratio of CD11b(+)Gr1(+) cells in spleens and peripheral blood was increased, and CD11b(+)Gr1(+) cells were also present in synovial tissue. Splenomegaly was observed and correlated with the ratio of CD11b(+)Gr1(+) cells. When bone marrow from GFP-expressing mice was transplanted into STR/Ort mice, no difference in the percentage of CD11b(+)Gr1(+) cells was observed between transplanted and age-matched STR/Ort mice. Analysis of biochemical parameters in F2 mice showed that spleen weight correlated with serum total cholesterol. These results suggest that the increase in circulating and splenic CD11b(+)Gr1(+) cells in STR/Ort mice originates from hypercholesterolemia. Further investigation of the function of CD11b(+)Gr1(+) cells in synovial tissue may reveal the pathology of OA in STR/Ort mice.
Assuntos
Hiperlipidemias/patologia , Células Mieloides/imunologia , Células Mieloides/patologia , Osteoartrite/patologia , Membrana Sinovial/citologia , Membrana Sinovial/patologia , Animais , Antígenos Ly , Antígeno CD11b , Movimento Celular , Hiperlipidemias/complicações , Hiperlipidemias/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Osteoartrite/complicações , Osteoartrite/imunologiaRESUMO
As the in vivo function of bone marrow-engrafted umbilical cord blood (UCB)-derived mesenchymal cells (UCBCs) after UCB transplantation is unknown, we examined in vivo osteoblastic differentiation using mouse UCB transplantation and fracture models. UCBCs obtained from GFP transgenic mice were intravenously injected into irradiated C57BL/6 mice. After three months, the in vivo osteoblastic differentiation potential of bone marrow-engrafted UCBCs was examined histologically using a mouse fracture model. GFP-positive UCBCs were detected in the bone marrow of recipient mice. On day 7, UCBCs were observed in the fracture gap and surrounding the titanium screws of the fixation device. The UCBCs were also positive for alkaline phosphatase and von Kossa staining. By day 14, UCBCs were observed around and within a formed intramedullary callus. The newly formed woven bone consisted of ALP- and von Kossa-positive cells. Our findings suggest that UCBCs contribute to the fracture healing process after bone marrow engraftment and that UCBC transplantation can fully reconstruct not only hematopoietic cells but also mesenchymal cell lineages.
Assuntos
Células da Medula Óssea , Calo Ósseo/citologia , Calo Ósseo/fisiologia , Fraturas do Fêmur/fisiopatologia , Células-Tronco Fetais/citologia , Consolidação da Fratura , Osteogênese , Animais , Placas Ósseas , Parafusos Ósseos , Diferenciação Celular , Linhagem da Célula , Transplante de Células-Tronco de Sangue do Cordão Umbilical , Modelos Animais de Doenças , Feminino , Células-Tronco Fetais/química , Citometria de Fluxo , Proteínas de Fluorescência Verde/química , Humanos , Injeções Intravenosas , Camundongos , Camundongos Endogâmicos C57BL , Osteoblastos/citologia , Titânio , Quimeras de TransplanteRESUMO
The incidence of spontaneous osteoarthritis (OA) in female STR/Ort mice is much lower than that observed in male STR/Ort mice; however, the reason for the differential incidence of OA between sexes has not been elucidated. Here, we investigated and compared age- and sex-related bone mineral density and architectural changes in male and female STR/Ort mice. Bone architecture and bone mineral density (BMD) of femurs were examined in 5-, 10-, 15-, 20-, and 35-week-old male and female STR/Ort mice by microscopic computed tomography (µCT). Angular degrees of internal tibial torsion (ADITT) were also measured in mice at 5, 15, and 35 weeks of age. Earlier decreases of cancellous volume and BMD were found in male STR/Ort mice. Using µCT, an age-related decline of bone marrow space in femoral diaphysis was observed in both males and females but was more dramatic in females. In addition, an earlier increase of ADITT was observed in male STR/Ort mice, suggesting that internal rotation of the tibia may contribute to OA. Age- and sex-related bone architectural changes clearly differ between male and female STR/Ort mice. These differences in bone structure, particularly ADITT, may explain the differential incidence of OA in STR/Ort mice.
Assuntos
Artrite Experimental/patologia , Densidade Óssea , Osteoartrite do Joelho/patologia , Ovariectomia/efeitos adversos , Fatores Etários , Animais , Artrite Experimental/diagnóstico por imagem , Artrite Experimental/epidemiologia , Modelos Animais de Doenças , Feminino , Humanos , Articulação do Joelho/citologia , Masculino , Camundongos , Camundongos Endogâmicos , Osteoartrite do Joelho/diagnóstico por imagem , Osteoartrite do Joelho/epidemiologia , Radiografia , Fatores Sexuais , Tíbia/diagnóstico por imagem , Tíbia/patologia , Tomografia Computadorizada de Emissão , Anormalidade Torcional/patologiaRESUMO
The aim of this study is to clarify the effect of low intensity pulsed ultrasound (LIPUS) on shortening of the fracture healing period and endochondral ossification during the fracture healing process. We first established a model of aging-related delayed union fractures consisting of aged mouse (C57BL/6J; 40 weeks old) with closed femur fractures. We compared the healing process of 40-week-old mice to the healing process of 8-week-old (young) mice using radiological and histological analysis. In aged mice, some cartilage formation was observed 10 days after the fracture; however, endochondral ossification and hard callus bridging were observed 21 and 28 days after the fracture, respectively, whereas cartilage remained in the callus on day 28, suggesting delayed endochondral ossification following bone remodeling. Meanwhile, in aged mice with LIPUS treatment, cartilage formation was similar to that in aged mice without LIPUS; however, hard callus bridging and bone remodeling were observed 21 and 28 days after fracture, respectively, suggesting that LIPUS shortened the healing period due to promotion of endochondral ossification. Immunohistochemical analysis showed marked expression of vascular endothelial growth factor and neovascularization in the fibrous tissue comprising the periosteum that surrounded the whole callus. A cell migration test involving primary cultured human endothelial cells also showed promotion of cell migration by LIPUS. These results suggested that endothelial cell migration and neovascularization, which were observed around fracture sites, played a part in the mechanism of promotion of endochondral ossification by LIPUS.
Assuntos
Fraturas do Fêmur/terapia , Fêmur/patologia , Consolidação da Fratura , Fraturas Fechadas/terapia , Fraturas não Consolidadas/terapia , Osteogênese , Terapia por Ultrassom/métodos , Animais , Remodelação Óssea , Calo Ósseo/citologia , Calo Ósseo/diagnóstico por imagem , Movimento Celular , Fraturas do Fêmur/diagnóstico por imagem , Fraturas do Fêmur/patologia , Fêmur/diagnóstico por imagem , Fêmur/fisiopatologia , Fraturas Fechadas/diagnóstico por imagem , Fraturas Fechadas/patologia , Fraturas não Consolidadas/diagnóstico por imagem , Fraturas não Consolidadas/patologia , Humanos , Imageamento Tridimensional , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Neovascularização Fisiológica , Osteogênese/efeitos da radiação , Tomografia Computadorizada por Raios X , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
This study aimed to clarify the relationship between the mechanical environment at the fracture site and endogenous fibroblast growth factor-2 (FGF-2). We compared two types of fracture healing with different callus formations and cellular events using MouseFix(TM) plate fixation systems for murine fracture models. Left femoral fractures were induced in 72 ten-week-old mice and then fixed with a flexible (Group F) or rigid (Group R) Mouse Fix(TM) plate. Mice were sacrificed on days 3, 5, 7, 10, 14, and 21. The callus volumes were measured by 3D micro-CT and tissues were histologically stained with hematoxylin & eosin or safranin-O. Sections from days 3, 5, and 7 were immunostained for FGF-2 and Proliferating Cell Nuclear Antigen (PCNA). The callus in Group F was significantly larger than that in Group R. The rigid plate allowed bone union without a marked external callus or chondrogenesis. The flexible plate formed a large external callus as a result of endochondral ossification. Fibroblastic cells in the granulation tissue on days 5 and 7 in Group F showed marked FGF-2 expression compared with Group R. Fibroblastic cells showed ongoing proliferation in granulation tissue in group F, as indicated by PCNA expression, which explained the relative granulation tissue increase in group F. There were major differences in early phase endogenous FGF-2 expression between these two fracture healing processes, due to different mechanical environments.