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Bone morphogenetic proteins (BMPs) have tremendous therapeutic potential regarding the treatment of bone and musculoskeletal disorders due to their osteo-inductive ability. More than twenty BMPs have been identified in the human body with various functions, such as embryonic development, skeleton genesis, hematopoiesis, and neurogenesis. BMPs can induce the differentiation of MSCs into the osteoblast lineage and promote the proliferation of osteoblasts and chondrocytes. BMP signaling is also involved in tissue remodeling and regeneration processes to maintain homeostasis in adults. In particular, growth factors, such as BMP-2 and BMP-7, have already been approved and are being used as treatments, but it is unclear as to whether they are the most potent BMPs that induce bone formation. According to recent studies, BMP-9 is known to be the most potent inducer of the osteogenic differentiation of mesenchymal stem cells, both in vitro and in vivo. However, its exact role in the skeletal system is still unclear. In addition, research results suggest that the molecular mechanism of BMP-9-mediated bone formation is also different from the previously known BMP family, suggesting that research on signaling pathways related to BMP-9-mediated bone formation is actively being conducted. In this study, we performed a phosphorylation array to investigate the signaling mechanism of BMP-9 compared with BMP-2, another influential bone-forming growth factor, and we compared the downstream signaling system. We present a mechanism for the signal transduction of BMP-9, focusing on the previously known pathway and the p53 factor, which is relatively upregulated compared with BMP-2.
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Fator 2 de Diferenciação de Crescimento , Osteogênese , Humanos , Proteína Morfogenética Óssea 2/farmacologia , Proteína Morfogenética Óssea 2/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Diferenciação Celular , Fator 2 de Diferenciação de Crescimento/metabolismo , Osteoblastos/metabolismo , Periósteo/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismoRESUMO
Tiron is a potent antioxidant that counters the pathological effects of reactive oxygen species (ROS) production due to oxidative stress in various cell types. We examined the effects of tiron on mitochondrial function and osteoblastic differentiation in human periosteum-derived cells (hPDCs). Tiron increased mitochondrial activity and decreased senescence-associated ß-galactosidase activity in hPDCs; however, it had a detrimental effect on osteoblastic differentiation by reducing alkaline phosphatase (ALP) activity and alizarin red-positive mineralization, regardless of H2O2 treatment. Osteoblast-differentiating hPDCs displayed increased ROS production compared with non-differentiating hPDCs, and treatment with tiron reduced ROS production in the differentiating cells. Antioxidants decreased the rates of oxygen consumption and ATP production, which are increased in hPDCs during osteoblastic differentiation. In addition, treatment with tiron reduced the levels of most mitochondrial proteins, which are increased in hPDCs during culture in osteogenic induction medium. These results suggest that tiron exerts negative effects on the osteoblastic differentiation of hPDCs by causing mitochondrial dysfunction.
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Osteogênese , Periósteo , Humanos , Sal Dissódico do Ácido 1,2-Di-Hidroxibenzeno-3,5 Dissulfônico , Espécies Reativas de Oxigênio , Peróxido de Hidrogênio/farmacologia , Mitocôndrias , AntioxidantesRESUMO
Periosteal stem cells are critical for bone regeneration, while the numbers will decrease with age. This study focused on whether Prx1+ cell, a kind of periosteal stem cell, could stimulate bone regeneration in aged mice. Four weeks and 12 months old Prx1CreER-GFP; Rosa26tdTomato mice were used to reveal the degree of Prx1+ cells participating in the femoral fracture healing procedure. One week, 8 weeks, 12 and 24 months old Prx1CreER-GFP mice were used to analyse the real-time distribution of Prx1+ cells. Twelve months old C57BL/6 male mice (n = 96) were used to create the bone defect model and, respectively, received hydrogel, hydrogel with Prx1- mesenchymal stem cells and hydrogel with Prx1+ cells. H&E staining, Synchrotron radiation-microcomputed tomography and mechanical test were used to analyse the healing results. The results showed that tdTomato+ cells were involved in bone regeneration, especially in young mice. At the same time, GFP+ cells decreased significantly with age. The Prx1+ cells group could significantly improve bone regeneration in the murine bone defect model via directly differentiating into osteoblasts and had better osteogenic differentiation ability than Prx1- mesenchymal stem cells. Our finding revealed that the quantity of Prx1+ cells might account for decreased bone regeneration ability in aged mice, and transplantation of Prx1+ cells could improve bone regeneration at the bone defect site.
Assuntos
Envelhecimento/patologia , Regeneração Óssea , Osso e Ossos/patologia , Osso e Ossos/fisiopatologia , Periósteo/patologia , Células-Tronco/patologia , Animais , Fenômenos Biomecânicos , Osso e Ossos/diagnóstico por imagem , Contagem de Células , Diferenciação Celular , Proliferação de Células , Separação Celular , Modelos Animais de Doenças , Consolidação da Fratura , Proteínas de Homeodomínio/metabolismo , Células-Tronco Mesenquimais/citologia , Camundongos Endogâmicos C57BL , Osteogênese , Células-Tronco/metabolismo , Microtomografia por Raio-XRESUMO
Nuclear factor kappa B (NF-κB) regulates inflammatory gene expression and represents a likely target for novel disease treatment approaches, including skeletal disorders. Several plant-derived sesquiterpene lactones can inhibit the activation of NF-κB. Parthenolide (PTL) is an abundant sesquiterpene lactone, found in Mexican Indian Asteraceae family plants, with reported anti-inflammatory activity, through the inhibition of a common step in the NF-κB activation pathway. This study examined the effects of PTL on the enhanced, in vitro, osteogenic phenotypes of human periosteum-derived cells (hPDCs), mediated by the inflammatory cytokine tumor necrosis factor (TNF)-α. PTL had no significant effects on hPDC viability or osteoblastic activities, whereas TNF-α had positive effects on the in vitro osteoblastic differentiation of hPDCs. c-Jun N-terminal kinase (JNK) signaling played an important role in the enhanced osteoblastic differentiation of TNF-α-treated hPDCs. Treatment with 1 µM PTL did not affect TNF-α-treated hPDCs; however, 5 and 10 µM PTL treatment decreased the histochemical detection and activity of alkaline phosphatase (ALP), alizarin red-positive mineralization, and the expression of ALP and osteocalcin mRNA. JNK phosphorylation decreased significantly in TNF-α-treated hPDCs pretreated with PTL. These results suggested that PTL exerts negative effects on the increased osteoblastic differentiation of TNF-α-treated hPDCs by inhibiting JNK signaling.
Assuntos
Asteraceae/química , Inflamação/tratamento farmacológico , Osteogênese/efeitos dos fármacos , Sesquiterpenos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Humanos , Hidrolases/genética , Inflamação/genética , Inflamação/patologia , Proteínas Quinases JNK Ativadas por Mitógeno , Lactonas/química , Lactonas/farmacologia , Sistema de Sinalização das MAP Quinases , NF-kappa B , Osteoblastos/efeitos dos fármacos , Osteogênese/genética , Periósteo/efeitos dos fármacos , Periósteo/crescimento & desenvolvimento , Fenótipo , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Sesquiterpenos/química , Fator de Necrose Tumoral alfa/genéticaRESUMO
Stem/progenitor cell-based regenerative medicine using the osteoblast differentiation of mesenchymal stem cells (MSCs) is regarded as a promising approach for the therapeutic treatment of various bone defects. The effects of the osteogenic differentiation of stem/progenitor cells on osteoclast differentiation may have important implications for use in therapy. However, there is little data regarding the expression of osteoclastogenic proteins during osteoblastic differentiation of human periosteum-derived cells (hPDCs) and whether factors expressed during this process can modulate osteoclastogenesis. In the present study, we measured expression of RANKL in hPDCs undergoing osteoblastic differentiation and found that expression of RANKL mRNA was markedly increased in these cells in a time-dependent manner. RANKL protein expression was also significantly enhanced in osteogenic-conditioned media from hPDCs undergoing osteoblastic differentiation. We then isolated and cultured CD34+ hematopoietic stem cells (HSCs) from umbilical cord blood (UCB) mononuclear cells (MNCs) and found that these cells were well differentiated into several hematopoietic lineages. Finally, we co-cultured human trabecular bone osteoblasts (hOBs) with CD34+ HSCs and used the conditioned medium, collected from hPDCs during osteoblastic differentiation, to investigate whether factors produced during osteoblast maturation can affect osteoclast differentiation. Specifically, we measured the effect of this osteogenic-conditioned media on expression of osteoclastogenic markers and osteoclast cell number. We found that osteoclastic marker gene expression was highest in co-cultures incubated with the conditioned medium collected from hPDCs with the greatest level of osteogenic maturation. Although further study will be needed to clarify the precise mechanisms that underlie osteogenic-conditioned medium-regulated osteoclastogenesis, our results suggest that the osteogenic maturation of hPDCs could promote osteoclastic potential.
Assuntos
Diferenciação Celular/genética , Meios de Cultivo Condicionados/farmacologia , Osteogênese/efeitos dos fármacos , Ligante RANK/genética , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Linhagem da Célula/genética , Meios de Cultivo Condicionados/metabolismo , Sangue Fetal/citologia , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteoclastos/citologia , Osteoclastos/metabolismo , Osteogênese/genética , Periósteo/citologia , Periósteo/crescimento & desenvolvimentoRESUMO
The differentiation of mesenchymal stem cells towards an osteoblastic fate depends on numerous signaling pathways, including activation of bone morphogenetic protein (BMP) signaling components. Commitment to osteogenesis is associated with activation of osteoblast-related signal transduction, whereas inactivation of this signal transduction favors adipogenesis. BMP signaling also has a critical role in the processes by which mesenchymal stem cells undergo commitment to the adipocyte lineage. In our previous study, we demonstrated that an agonist of the perioxisome proliferator-activated receptor γ (PPARγ), a master regulator of adipocyte differentiation, stimulates osteoblastic differentiation of cultured human periosteum-derived cells. In this study, we used dorsomorphin, a selective small molecule inhibitor of BMP signaling, to investigate whether BMP signaling is involved in the positive effects of PPARγ agonists on osteogenic phenotypes of cultured human periosteum-derived cells. Both histochemical detection and bioactivity of ALP were clearly increased in the periosteum-derived cells treated with the PPARγ agonist at day 10 of culture. Treatment with the PPARγ agonist also caused an increase in alizarin red S staining and calcium content in the periosteum-derived osteoblasts at 2 and 3 weeks of culture. In contrast, dorsomorphin markedly decreased ALP activity, alizarin red S staining and calcium content in both the cells treated with PPARγ agonist and the cells cultured in osteogenic induction media without PPARγ agonist during the culture period. In addition, the PPARγ agonist clearly increased osteogenic differentiation medium-induced BMP-2 upregulation in the periosteum-derived osteoblastic cells at 2 weeks of culture as determined by quantitative reverse transcriptase polymerase chain reaction (RT-PCR), immunoblotting, and immunocytochemical analyses. Although further study will be needed to clarify the mechanisms of PPARγ-regulated osteogenesis, our results suggest that the positive effects of a PPARγ agonist on the osteogenic phenotypes of cultured human periosteum-derived cells seem to be dependent on BMP signaling.
Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Diferenciação Celular , Células-Tronco Mesenquimais/fisiologia , Osteoblastos/metabolismo , Osteogênese , PPAR gama/metabolismo , Periósteo/citologia , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Adipócitos/metabolismo , Adipogenia , Fosfatase Alcalina/metabolismo , Benzamidas/farmacologia , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Regulação da Expressão Gênica , Humanos , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/fisiologia , PPAR gama/agonistas , PPAR gama/antagonistas & inibidores , Pioglitazona , Cultura Primária de Células , Pirazóis/farmacologia , Piridinas/farmacologia , Pirimidinas/farmacologia , Transdução de Sinais , Tiazolidinedionas/farmacologiaRESUMO
Cigarette smoke is associated with delayed fracture healing, alterations in mineral content, and osteoporosis, however, its effects on osteoblastic differentiation of osteoprogenitor cells are not fully understood. In the present study, we examined the effects of cigarette smoke extract (CSE) on osteoblastic differentiation of cultured human periosteum-derived cells. We found that CSE inhibited alkaline phosphatase (ALP) activity, mineralization and Runx2 transactivation of the periosteum-derived cells. Nucleofection of RUNX2 into the periosteum-derived cells increased expression of endogenous osteocalcin (OC) and ALP genes in osteogenic induction medium and increased OC expression in non-osteogenic medium. Treatment of the periosteum-derived cells with CSE resulted in decreased phosphorylation of AKT and forkhead box protein O1 (FOXO1). The AKT phosphorylation-resistant mutant, FOXO1-A3, inhibited transcriptional activity of RUNX2 in the periosteum-derived cells. The current study suggests one mechanism by which CSE exposure leads to inhibition of osteoblastic differentiation of cultured human periosteum-derived cells.
Assuntos
Fatores de Transcrição Forkhead/fisiologia , Nicotiana/efeitos adversos , Osteoblastos/citologia , Periósteo/citologia , Fumaça/efeitos adversos , Fosfatase Alcalina/genética , Diferenciação Celular , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Proteína Forkhead Box O1 , Humanos , Osteocalcina/genética , Fosforilação , RNA Mensageiro/análiseRESUMO
The aim of this study is to validate a minimally invasive surgical procedure to harvest palate periosteum as a source of tissue for mesenchymal stromal/stem cells. We performed a standardized procedure to harvest the palate periosteum in ten subjects, which consisted of a 3 mm disposable punch and a Molt periosteal elevator to harvest a small full-thickness fragment of soft tissue at the hard palate area, between the upper bicuspids, 3 to 4 mm apical to the cement enamel junction. The one-third inner portion was fragmented, and following standard cell culture procedures, the adherent cells were cultured for three passages, after obtaining 70-90% confluence. Cell morphology analysis, flow cytometry analysis, and viability and osteogenic differentiation assays were performed. In all 10 cases, uneventful healing was observed, with no need for analgesic intake. The evaluation of cell morphology showed elongated spindle-shaped cells distributed in woven patterns. A high viability range was verified as well as an immunophenotype compatible with mesenchymal stem cell lineage. The differentiation assay showed the potential of the cells to differentiate into the osteogenic lineage. These results demonstrate that the minimally invasive proposed surgical technique is capable of supplying enough periosteum source tissue for stem cell culture and bone tissue engineering.
RESUMO
As a thin fibrous layer covering the bone surface, the periosteum plays a significant role in bone physiology during growth, development and remodeling. Over the past several decades, the periosteum has received considerable scientific attention as a source of mesenchymal stem cells (MSCs). Periosteum-derived cells (PDCs) have emerged as a promising strategy for tissue engineering due to their chondrogenic, osteogenic and adipogenic differentiation capacities. Starting from the history of PDCs, the present review provides an overview of their characterization and the procedures used for their isolation. This study also summarizes the chondrogenic, osteogenic, and adipogenic abilities of PDCs, serving as a reference about their potential therapeutic applications in various clinical scenarios, with particular emphasis on the comparison with other common sources of MSCs. As techniques continue to develop, a comprehensive analysis of the characterization and regulation of PDCs can be conducted, further demonstrating their role in tissue engineering. PDCs present promising potentials in terms of their osteogenic, chondrogenic, and adipogenic capacities. Further studies should focus on exploring their utility under multiple clinical scenarios to confirm their comparative benefit over other commonly used sources of MSCs.
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Periosteum is expected for bone repairing due to excellent regenerative potential. PDCs are the main source of cells for promoting bone repair. However, PDCs from different sites have been confirmed to be site specific due to their distinct embryonic origin and the methods of bone formation. Hippo-YAP pathway is proved to play a critical role in fate decision of mesenchymal stem cells. The effect of Hippo-YAP on PDCs has not been reported so far. Hence, we aim to explore the differences of PDCs from mandible and femur along with their possible responses to YAP signaling. mPDCs and fPDCs were obtained and tested through flow cytometry for identification. Follow-up results illustrated mPDCs was cubic shape and with better proliferation while fPDCs preferred slender cell shape with worse cell viability compared with mPDCs. mPDCs was superior to fPDCs in ALP activity, related mRNA expression and calcium deposits in late stage. Interestingly, downregulation of YAP promoted the ALP activity, related mRNA expression and calcium deposits of fPDCs while hindered that of mPDCs in vitro. Moreover, implant animal model in mandible and femur were constructed for evaluation in vivo. Histological results were similar to the results in vitro. We speculate this may result from their different embryonic origin and the way of bone formation. Taken together, results available suggested that mPDCs may serve as more optimal seed cells for tissue engineering compared with fPDCs; however, considering their different response to YAP signaling, to ensure sufficient YAP expression in mPDCs and appropriate declining YAP expression in fPDCs may establish better osteogenesis.
Assuntos
Osteogênese , Periósteo , Animais , Cálcio , Transdução de Sinais , RNA Mensageiro , Proliferação de CélulasRESUMO
BACKGROUND: Periosteum plays a significant role in bone formation and regeneration by storing progenitor cells, and also acts as a source of local growth factors and a scaffold for recruiting cells and other growth factors. Recently, tissue-engineered periosteum has been studied extensively and shown to be important for osteogenesis and chondrogenesis. Using biomimetic methods for artificial periosteum synthesis, membranous tissues with similar function and structure to native periosteum are produced that significantly improve the efficacy of bone grafting and scaffold engineering, and can serve as direct replacements for native periosteum. Many problems involving bone defects can be solved by preparation of idealized periosteum from materials with different properties using various techniques. METHODS: This review summarizes the significance of periosteum for osteogenesis and chondrogenesis from the aspects of periosteum tissue structure, osteogenesis performance, clinical application, and development of periosteum tissue engineering. The advantages and disadvantages of different tissue engineering methods are also summarized. RESULTS: The fast-developing field of periosteum tissue engineering is aimed toward synthesis of bionic periosteum that can ensure or accelerate the repair of bone defects. Artificial periosteum materials can be similar to natural periosteum in both structure and function, and have good therapeutic potential. Induction of periosteum tissue regeneration and bone regeneration by biomimetic periosteum is the ideal process for bone repair. CONCLUSIONS: Periosteum is essential for bone formation and regeneration, and it is indispensable in bone repair. Achieving personalized structure and composition in the construction of tissue engineering periosteum is in accordance with the design concept of both universality and emphasis on individual differences and ensures the combination of commonness and individuality, which are expected to meet the clinical needs of bone repair more effectively. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: To better understand the role of periosteum in bone repair, clarify the present research situation of periosteum and tissue engineering periosteum, and determine the development and optimization direction of tissue engineering periosteum in the future. It is hoped that periosteum tissue engineering will play a greater role in meeting the clinical needs of bone repair in the future, and makes it possible to achieve optimization of bone tissue therapy.
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OBJECTIVE: Bone regeneration is a potential technique for treating osteoporosis. A previous study reported that F-box and WD-40 domain-containing protein 2 (FBXW2) localized with osteocalcin in bovine periosteum after 5 weeks of explant culture. However, the osteoblastic functions of FBXW2 remain unclear. In this study, double-fluorescent immunostaining was used to investigate the potential role of FBXW2 and its relationship with osteocalcin. RESULTS: At day 0, FBXW2 was expressed in the cambium layer between the bone and periosteum, while osteocalcin was expressed in bone. After explant culture, changes in the periosteum were observed from weeks 1 to 7. At week 1, partial FBXW2 expression was seen with a small amount of osteocalcin. At week 2, a layer of FBXW2 was observed. From weeks 3 to 7, tube-like structures of FBXW and osteocalcin were observed, and periosteum-derived cells were released from the periosteum in areas where no FBXW2 was observed. Bovine periosteum-derived cells can form a three-dimensional cell pellet, because multilayered cell sheets are formed inside of the periosteum in vitro. It is shown that in results FBXW2 is produced in periosteal explants near sites where initial osteogenic activity is observed, suggesting that it may be involved in periosteal osteogenesis.
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Células-Tronco Mesenquimais , Periósteo , Animais , Regeneração Óssea , Bovinos , Osteocalcina/genética , OsteogêneseRESUMO
The impact of diabetes mellitus on bone fracture healing is clinically relevant as the patients experience delayed fracture healing. Even though efforts have been made to understand the detrimental effects of type 2 diabetes mellitus (T2DM) on the fracture healing process, the exact mechanisms causing the pathophysiological outcomes remain unclear. The aim of this study was to assess alterations in bone fracture healing (tibial fracture surgery, intramedullary pinning) of diet-induced obese (DIO) mice, and to investigate the in vitro properties of osteochondroprogenitors derived from the diabetic micro-environment. High-resolution contrast-enhanced microfocus X-ray computed tomography (CE-CT) enabled a simultaneous 3D assessment of the amount and spatial distribution of the regenerated soft and hard tissues during fracture healing and revealed that osteogenesis as well as chondrogenesis are altered in DIO mice. Compared to age-matched lean controls, DIO mice presented a decreased bone volume fraction and increased callus volume and adiposity at day 14 post-fracture. Of note, bone turnover was found altered in DIO mice relative to controls, evidenced by decreased blood serum osteocalcin and increased serum CTX levels. The in vitro data revealed that not only the osteogenic and adipogenic differentiation of periosteum-derived cells (PDCs) were altered by hyperglycemic (HG) conditions, but also the chondrogenic differentiation. Elevated PPARγ expression in HG conditions confirmed the observed increase in differentiated adipocytes in vitro. Finally, chondrogenesis-related genes COL2 and COL10 were downregulated for PDCs treated with HG medium, confirming that chondrogenic differentiation is compromised in vitro and suggesting that this may affect callus formation and maturation during the fracture healing process in vivo. Altogether, these results provide novel insights into the alterations of long bone fracture repair and suggest a link between HG-induced dysfunctionality of osteochondroprogenitor differentiation and fracture healing impairment under T2DM conditions.
Assuntos
Diabetes Mellitus Tipo 2 , Consolidação da Fratura , Animais , Calo Ósseo/diagnóstico por imagem , Dieta , Humanos , Camundongos , Camundongos Obesos , Osteogênese , Tomografia , Tomografia Computadorizada por Raios XRESUMO
Coupling between osteoblast-mediated bone formation and osteoclast-mediated bone resorption maintains both mechanical integrity and mineral homeostasis. Zinc is required for the formation, mineralization, growth, and maintenance of bones. We examined the effects of zinc sulfate on osteoblastic differentiation of human periosteum-derived cells (hPDCs) and osteoclastic differentiation of THP-1 cells. Zinc sulfate enhanced the osteoblastic differentiation of hPDCs; however, it did not affect the osteoclastic differentiation of THP-1 cells. The levels of extracellular signaling-related kinase (ERK) were strongly increased during osteoblastic differentiation in zinc sulfate-treated hPDCs, compared with other mitogen-activated protein kinases (MAPKs). Zinc sulfate also promoted osteogenesis in hPDCs and THP-1 cells co-cultured with the ratio of one osteoclast to one osteoblast, as indicated by alkaline phosphatase levels, mineralization, and cellular calcium contents. In addition, the receptor activator of nuclear factor kappa B ligand (RANKL)/osteoprotegerin (OPG) ratio was decreased in the zinc sulfate-treated co-cultures. Our results suggest that zinc sulfate enhances osteogenesis directly by promoting osteoblastic differentiation and osteogenic activities in osteoblasts and indirectly by inhibiting osteoclastic bone resorption through a reduced RANKL/OPG ratio in co-cultured osteoblasts and osteoclasts.
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Adequate bone volume is required for osseointegrated implants to restore lost teeth and oral function. Several studies have demonstrated potential advantage of stem cells in regenerative medicine using osteoblasts. The periosteum is composed of osteoblasts, fibroblasts, and osteoprogenitor cells. It may be an alternative source for bone tissue engineering because of easy isolation and rapid proliferation in vivo and in vitro. Low-intensity pulsed ultrasound (LIPUS) has proved successful in recoveries from nonunions, delayed unions, and fracture of the bone in both animal experiments and clinical treatments. The study was to investigate the influence of LIPUS on the osteogenic differentiation in murine periosteum-derived cells (PDCs) and the underlying mechanism of LIPUS. PDCs were treated daily with LIPUS for 20 min up to 21 days with 3 MHz frequency, 30 mW/cm2 intensity, and pulse repetition frequency of 1 kHz. The effects of LIPUS on cell proliferation and viability were investigated. Osteogenic differentiation was analyzed by alkaline phosphatase (ALP)-positive cell staining, ALP activity assay, mineralized nodule formation, real-time reverse transcription-polymerase chain reaction, as well as western blotting. The results indicated that ultrasound stimulation did not significantly affect the proliferation of PDCs. But LIPUS significantly increased ALP activity on day 7 and markedly promoted formation of mineralized nodules on day 21. mRNA expression of ALP and osteocalcin was significantly upregulated by stimulation with LIPUS. LIPUS enhanced gene expression of both bone morphogenetic protein-2 (BMP-2) and osterix only in the presence of osteogenic medium. LIPUS stimulation did not affect Smad 1 and Smad 5 protein expression, but significantly upregulated protein levels of BMP-2 and phosphor-Smad 1/5/9 in PDCs. Thus, LIPUS stimulation increased early osteogenic differentiation in a normal medium and further enhanced expression of BMP-2 and subsequent osterix expression through the canonical Smad-signaling pathway in an osteogenic medium, leading to mineral apposition. Therefore, LIPUS might have potential to promote osteogenesis in PDCs. Impact statement There are few studies on periosteum-derived cells (PDCs) because conventional methods of their isolation are relatively difficult to procure abundant cells for cell culture and the total cell numbers are limited. In this study, a modified isolation technique of murine calvarial PDCs using gelatin is described. PDCs were initiated to emerge as early as day 3 and showed increased proliferation, which can be used for further studies. Low-intensity pulsed ultrasound stimulation increased early osteogenic differentiation in a normal medium and further enhanced expression of bone morphogenic protein-2 and subsequent osterix expression through the canonical Smad-signaling pathway in an osteogenic medium, leading to mineral apposition.
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Osteoblastos , Osteogênese , Animais , Diferenciação Celular , Células Cultivadas , Camundongos , Células-Tronco , Ondas UltrassônicasRESUMO
Regenerative cell-based implants using periosteum-derived stem cells were developed for the treatment of large 3 cm fresh and 4.5 centimeter biological compromised bone gaps in a tibial sheep model and compared with an acellular ceramic-collagen void filler. It was hypothesized that the latter is insufficient to heal large skeletal defects due to reduced endogenous biological potency. To this purpose a comparison was made between the ceramic dicalciumphosphate scaffold (CopiOs®) as such, the same ceramic coated with clinical grade Bone Morphogenetic Protein 2 and 6 (BMP) only or a BMP coated cell-seeded combination product. These implants were evaluated in 2 sheep models, a fresh 3 cm critical size tibial defect and a 4.5 cm biologically exhausted tibial defect. For the groups in which growth factors were applied, BMP-6 was chosen at a dose of 344 µg for 3 cm and 1.500 µg or 3.800 µg for 4.5 cm defects. An additional group in the 4.5 cm defect was tested using BMP-2 in a dose of 1.500 µg. For all the cell based implants autologous periosteum-derived cells were used which were cultured in monolayer during 6 weeks. For the fresh defect 408 million cells and for the biologically exhausted tibial defect 612 million cells were drop-seeded on the BMP coated scaffolds. Bone healing was studied during 16 weeks postimplantation, using standard radiographs. While fresh defects responded to all treatments, regardless the use of cells, the biologically hampered defects responded in half of the cases and only if the BMP-cell combination product was used, supporting the concept that cell-based therapies may become attractive in treating defects with a compromised biological status.
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Proteína Morfogenética Óssea 2 , Periósteo , Animais , Regeneração Óssea , Ovinos , Células-Tronco , Alicerces TeciduaisRESUMO
OBJECTIVE: The aim of this research is to investigate the effects of concentrated growth factor (CGF) on the proliferation, osteogenic differentiation, and angiogenic potential of rabbit periosteum-derived cells (PDCs) in vitro. METHODS: PDCs were isolated from the femoral and tibial periosteum of rabbits and cultured with or without CGF membranes or CGF conditioned media. Scanning electron microscopy (SEM) was used for the structural characterization. Cell Counting Kit-8 assay was used to measure cell proliferation. Alkaline phosphatase (ALP) activity of PDCs was also measured. Immunohistochemistry was used to detect the expression of CD34. Enzyme-linked immunosorbent assay (ELISA), quantitative real-time PCR (qPCR), and Western blot were used to evaluate the secretion and expression levels of osteogenic differentiation markers (bone morphogenetic protein-2, type I collagen, osteocalcin) and angiogenesis markers (vascular endothelial growth factor, basic fibroblast growth factor) in supernatants and PDCs at days 3, 7, 14, and 21. RESULTS: The SEM analysis showed a dense three-dimensional fibrin network in CGF, and CGF membranes were covered by PDCs with elongated or polygonal morphological features. Compared with the control group, CGF significantly promoted the proliferation of PDCs during the experimental period (p < 0.05). Immunohistochemistry revealed that PDCs were dispersedly distributed among the CGF substrates, and CD34-positive cells were also present. Moreover, CGF significantly increased the ALP activity and upregulated the expression and secretion of osteogenic differentiation and angiogenesis markers in PDCs at days 3, 7, 14, and 21 (p < 0.05). CONCLUSION: CGF can increase the proliferation and promote the osteogenic differentiation and angiogenic potential of PDCs in vitro. These results indicate that CGF can be used as a new therapeutic means for biotechnological and clinical applications.
Assuntos
Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Neovascularização Fisiológica/fisiologia , Osteogênese/fisiologia , Periósteo/fisiologia , Animais , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Peptídeos e Proteínas de Sinalização Intercelular/isolamento & purificação , Masculino , Neovascularização Fisiológica/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Periósteo/citologia , Periósteo/efeitos dos fármacos , CoelhosRESUMO
BACKGROUND CONTEXT: Bone marrow derived mesenchymal stem cells (BMSCs) and periosteum-derived cells (PDCs) have shown great viability in terms of osteogenic potential and have been considered the major cellular source for skeletal tissue engineering. Using a PDCs-impregnated cell sheet to surround a BMSCs-impregnated tricalcium phosphate (TCP) scaffold might create a periosteum-bone biomimetic bone graft substitute to enhance spine fusion. PURPOSE: The purpose of this study was to determine the feasibility of using this newly tissue-engineered biomimetic bone graft for posterolateral spine fusion. STUDY DESIGN/SETTING: This study design was based on an animal model using adult male New Zealand White rabbits. METHODS: New Zealand White rabbits underwent operation and were divided into three groups based on the experimental material implanted in the bilateral L4-L5 intertransverse space. Group 1 was BMSCs-free TCP wrapped in a PDCs-free cell sheet. Group 2 was BMSCs-loaded-TCP wrapped in a PDCs-free cell sheet. Group 3 was BMSCs-loaded-TCP wrapped in a PDCs-loaded cell sheet. After 12 weeks, six rabbits from each group were euthanized for computed tomography scanning, manual palpation, biomechanical testing, and histology. Each group had 12 radiographic fusion areas for analysis because the right and left intertransverse fusion areas were collected separately. RESULTS: Radiographic union of 12 fusion areas for groups 1, 2, and 3 was 0, 3, and 9, respectively. Group 3 had significantly higher fusion success than groups 1 and 2 (p<.001). Solid fusion of six fusion segments in each group by manual palpation was 0, 1, and 5, accordingly. Group 3 had a higher successful solid fusion rate than groups 1 and 2 (p=.005). The average maximal torques at failure were 727±136 N mm, 627±91 N mm, and 882±195 N mm for groups 1, 2, and 3, accordingly. The maximal torque was significantly higher in group 3 than in group 2 (p=.028). Histological evaluation verified that new bone regeneration were greater in the group 3 samples. CONCLUSIONS: The results indicated the potential of using a PDCs-impregnated cell sheet to surround the BMSCs-impregnated TCP scaffold for creating a periosteum-bone biomimetic bone graft substitute to enhance bone regeneration and posterolateral fusion success.
Assuntos
Regeneração Óssea , Substitutos Ósseos/química , Fusão Vertebral/métodos , Engenharia Tecidual/métodos , Animais , Substitutos Ósseos/uso terapêutico , Fosfatos de Cálcio/química , Vértebras Lombares/cirurgia , Masculino , Células-Tronco Mesenquimais/citologia , Osteogênese , Periósteo/citologia , CoelhosRESUMO
BACKGROUND: Cultured bovine-periosteum-derived cells can form three-dimensional structures on tissue culture dishes without artificial scaffolding material, can induce bone regeneration in vivo. The utility of cultured bovine-periosteum-derived cells for bone tissue regeneration after their transplantation into nude mice has been reported, the precise F-box molecular mechanism was unclear. OBJECTIVE: The aim of this study was to investigate the specific F-box proteins required for bone regeneration by cultured bovine-periosteum-derived cells in vitro. METHODS: In the present study, periosteum tissue and cultured periosteum-derived cells were cultured for 5 weeks in vitro and then embedded in collagen gel with a green tissue-marking dye. Electrophoresis and immunohistochemistry were used to identify the specific F-box proteins required for tissue bone regeneration. RESULTS: The bovine-periosteum-derived cells were observed to form bone shortly after the expression of F-box proteins. After the initial phase of bone formation, the expression of the F-box proteins ceased. FBXW2 was shown to be expressed in the periosteum, but not in cultured periosteum-derived cells. Furthermore, FBXL14 disappeared during bone formation. CONCLUSIONS: Bone regeneration requires progenitor cells, such as bovine-periosteum-derived cells and the activation of the F-box Proteins FBXW2 and FBXL14, over time the expression of these proteins ceases. Further scientific and clinical trials are needed to investigate how the F-box Proteins can be used therapeutically to treat osteoporosis and osteonecrosis.
RESUMO
The aim of this study was to evaluate the bone repair using autogenous periosteum-derived cells (PDC) and bovine anorganic apatite and collagen (HA-COL). PDC from Wistar rats (n=10) were seeded on HA-COL discs and subjected to osteoinduction during 6 days. Critical-size defects in rat calvarias were treated with blood clot (G1), autogenous bone (G2), HA-COL (G3) and HA-COL combined with PDC (G4) (n=40), and then analyzed 1 and 3 months after surgeries. Radiographic analysis exhibited no significant temporal change. G1 and G2 had discrete new marginal bone, but the radiopacity of graft materials in G2, G3 and G4 impaired the detection of osteogenesis. At 3 months, histopathological analysis showed the presence of ossification islets in G1, which was more evident in G2, homogeneous new bone around HA-COL in G3 and heterogeneous new bone around HA-COL in G4 in addition to moderate presence of foreign body cells in G3 and G4. Histomorphometric analysis showed no change in the volume density of xenograft (p>0.05) and bone volume density in G2 was twice greater than in G1 and G4 after 3 months (p<0.05), but similar to G3. The PDC did not increase bone formation in vivo, although the biomaterial alone showed biocompatibility and osteoconduction capacity.
O objetivo deste estudo foi avaliar o reparo ósseo usando células derivadas de periósteo (PDC) e apatita inorgânica e colágeno bovinos (HA-COL). PDC de ratos Wistar (n=10) foram semeadas sobre discos de HA-COL e osteoinduzidas por 6 dias. Defeitos de tamanho crítico em calvárias de ratos foram tratados com coágulo sanguíneo (G1), osso autógeno (G2), HA-COL (G3) ou HA-COL associado a PDC (G4) (n=40) e analisados em 1 e 3 meses após as cirurgias. Análise radiográfica não exibiu mudança temporal significante, G1 e G2 tiveram aumento discreto de novo osso marginal, entretanto a radiopacidade dos materiais de enxerto em G2, G3 e G4 prejudicou a detecção de osteogênese. Análise histopatológica mostrou em 3 meses ilhotas de ossificação em G1 que foi maior em G2, novo osso homogêneo ao redor de HA-COL em G3 e novo osso heterogêneo ao redor de HA-COL em G4 além da presença moderada de células gigantes de corpo estranho em G3 e G4. Análise histomorfométrica mostrou a densidade de volume inalterada do xenoenxerto (p>0,05) e a densidade de volume de novo osso em G2 duas vezes maior que G1 e G4 após 3 meses (p<0,05), mas similar a G3. PDC não aumentaram a formação óssea in vivo apesar do biomaterial sozinho ter apresentado biocompatibilidade e capacidade osteocondutora.