RESUMO
Type 1 (T1DM) and type 2 (T2DM) diabetes mellitus are characterized by changes in glucose metabolism and cause bone damage via a variety of mechanisms, including effects on osteoblasts. We aimed to evaluate the osteoblast differentiation of mesenchymal stem cells (MSCs) from rats with T1DM or T2DM and the effects of removing the hyperglycemic stimulus on the osteogenic potential of these cells. MSCs from healthy rats were cultured in normoglycemic medium, whereas MSCs from rats with T1DM or T2DM were cultured in hyperglycemic or normoglycemic medium. T1DM and T2DM reduced osteoblast differentiation of MSCs grown in hyperglycemic media, with T1DM having a more pronounced effect, as evidenced by alkaline phosphatase activity, RUNX2 protein expression, and extracellular matrix mineralization, and modulated the gene expression of several components of the bone morphogenetic protein signaling pathway. The restoration of the normoglycemic environment partially recovers the osteogenic potential of MSCs from rats with T1DM but not with T2DM. Our findings highlight the need for specific therapies to treat T1DM- or T2DM-induced bone loss, as both disrupt osteoblast differentiation at distinct levels and likely through different mechanisms.
Assuntos
Diabetes Mellitus Tipo 1 , Diabetes Mellitus Tipo 2 , Células-Tronco Mesenquimais , Ratos , Animais , Diabetes Mellitus Tipo 1/metabolismo , Células Cultivadas , Osteogênese/genética , Diferenciação Celular , Osteoblastos/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Células-Tronco Mesenquimais/metabolismoRESUMO
Cell therapy is a valuable strategy for the replacement of bone grafts and repair bone defects, and mesenchymal stem cells (MSCs) are the most frequently used cells. This study was designed to genetically edit MSCs to overexpress bone morphogenetic protein 9 (BMP-9) using Clustered Regularly Interspaced Short Palindromic Repeats/associated nuclease Cas9 (CRISPR-Cas9) technique to generate iMSCs-VPRBMP-9+, followed by in vitro evaluation of osteogenic potential and in vivo enhancement of bone formation in rat calvaria defects. Overexpression of BMP-9 was confirmed by its gene expression and protein expression, as well as its targets Hey-1, Bmpr1a, and Bmpr1b, Dlx-5, and Runx2 and protein expression of SMAD1/5/8 and pSMAD1/5/8. iMSCs-VPRBMP-9+ displayed significant changes in the expression of a panel of genes involved in TGF-ß/BMP signaling pathway. As expected, overexpression of BMP-9 increased the osteogenic potential of MSCs indicated by increased gene expression of osteoblastic markers Runx2, Sp7, Alp, and Oc, higher ALP activity, and matrix mineralization. Rat calvarial bone defects treated with injection of iMSCs-VPRBMP-9+ exhibited increased bone formation and bone mineral density when compared with iMSCs-VPR- and phosphate buffered saline (PBS)-injected defects. This is the first study to confirm that CRISPR-edited MSCs overexpressing BMP-9 effectively enhance bone formation, providing novel options for exploring the capability of genetically edited cells to repair bone defects.
Assuntos
Fator 2 de Diferenciação de Crescimento , Células-Tronco Mesenquimais , Osteogênese , Animais , Sistemas CRISPR-Cas , Diferenciação Celular , Células Cultivadas , Fator 2 de Diferenciação de Crescimento/genética , Células-Tronco Mesenquimais/citologia , Osteogênese/genética , RatosRESUMO
Quantitative real-time polymerase chain reaction (qRT-PCR) is a powerful tool to evaluate gene expression, but its accuracy depends on the choice and stability of the reference genes used for normalization. In this study, we aimed to identify reference genes for studies on osteoblasts derived from rat bone marrow mesenchymal stem cells (bone marrow osteoblasts), osteoblasts derived from newborn rat calvarial (calvarial osteoblasts), and rat osteosarcoma cell line UMR-106. The osteoblast phenotype was characterized by ALP activity and extracellular matrix mineralization. Thirty-one candidates for reference genes from a Taqman® array were assessed by qRT-PCR, and their expressions were analyzed by five different approaches. The data showed that several of the most traditional reference genes, such as Actb and Gapdh, were inadequate for normalization and that the experimental conditions may affect gene stability. Eif2b1 was frequently identified among the best reference genes in bone marrow osteoblasts, calvarial osteoblasts, and UMR-106 osteoblasts. Selected stable and unstable reference genes were used to normalize the gene expression of Runx2, Alp, and Oc. The data showed statistically significant differences in the expression of these genes depending on the stability of the reference gene used for normalization, creating a bias that may induce incorrect assumptions in terms of osteoblast characterization of these cells. In conclusion, our study indicates that a rigorous selection of reference genes is a key step in qRT-PCR studies in osteoblasts to generate precise and reliable data.
Assuntos
Perfilação da Expressão Gênica/métodos , Expressão Gênica/genética , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/citologia , Animais , Animais Recém-Nascidos , Linhagem Celular , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Endopeptidases/genética , Endopeptidases/metabolismo , Células-Tronco Mesenquimais/citologia , Osteoblastos/metabolismo , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Padrões de ReferênciaRESUMO
Among bone morphogenetic proteins (BMPs), BMP-9 has been described as one with higher osteogenic potential. Here, we aimed at evaluating the effect of BMP-9 on the osteoblast differentiation of cells grown on titanium (Ti) with nanotopography, a well-known osseoinductive surface. MC3T3-E1 cells were grown either in absence or presence of BMP-9 (20 nM) on Ti with nanotopography (Ti-Nano) or machined Ti (Ti-Machined) for up to 21 days to evaluate the gene expression of RUNX2, osterix, osteocalcin, bone sialoprotein, SMAD6 and SMAD4, protein expression of SMAD4, ALP activity and extracellular matrix mineralization. As expected BMP-9 increased osteoblast differentiation irrespective of Ti surface topography; however, the cells grown on Ti-Nano were more responsible to BMP-9 compared with cells grown on Ti-machined. This could be, at least in part, due to the fact that Ti-Nano may act on both ways, by increasing the activation (SMAD4) and decreasing the inhibition (SMAD6) of the signaling pathway triggered by BMP-9, while Ti-Machined only decrease the inhibition (SMAD6) of this pathway. In conclusion, the combination of the osteogenic potential of BMP-9 with the osseoinductive capacity of Ti-Nano could be a promising strategy to favor the osseointegration of Ti implants.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Fator 2 de Diferenciação de Crescimento/farmacologia , Nanoporos/ultraestrutura , Osteoblastos/citologia , Titânio/química , Titânio/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Análise de Variância , Animais , Adesão Celular/fisiologia , Linhagem Celular , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Expressão Gênica , Proteínas de Membrana/genética , Camundongos , Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Proteína Smad4/metabolismo , Proteína Smad6/metabolismo , Propriedades de SuperfícieRESUMO
One of the tissue engineering strategies to promote bone regeneration is the association of cells and biomaterials. In this context, the aim of this study was to evaluate if cell source, either from bone marrow or adipose tissue, affects bone repair induced by osteoblastic cells associated with a membrane of poly(vinylidene-trifluoroethylene)/barium titanate (PVDF-TrFE/BT). Mesenchymal stem cells (MSC) were isolated from rat bone marrow and adipose tissue and characterized by detection of several surface markers. Also, both cell populations were cultured under osteogenic conditions and it was observed that MSC from bone marrow were more osteogenic than MSC from adipose tissue. The bone repair was evaluated in rat calvarial defects implanted with PVDF-TrFE/BT membrane and locally injected with (1) osteoblastic cells differentiated from MSC from bone marrow, (2) osteoblastic cells differentiated from MSC from adipose tissue or (3) phosphate-buffered saline. Luciferase-expressing osteoblastic cells derived from bone marrow and adipose tissue were detected in bone defects after cell injection during 25 days without difference in luciferin signal between cells from both sources. Corroborating the in vitro findings, osteoblastic cells from bone marrow combined with the PVDF-TrFE/BT membrane increased the bone formation, whereas osteoblastic cells from adipose tissue did not enhance the bone repair induced by the membrane itself. Based on these findings, it is possible to conclude that, by combining a membrane with cells in this rat model, cell source matters and that bone marrow could be a more suitable source of cells for therapies to engineer bone.
Assuntos
Transplante de Células-Tronco Mesenquimais/métodos , Osteoblastos/citologia , Crânio , Engenharia Tecidual/métodos , Tecido Adiposo/citologia , Animais , Compostos de Bário , Materiais Biocompatíveis , Células da Medula Óssea/citologia , Diferenciação Celular , Masculino , Células-Tronco Mesenquimais/citologia , Polivinil , Ratos , Ratos Wistar , TitânioRESUMO
The treatment of polytrauma patients represents a great challenge in the maxillofacial and orthopedic surgery fields. Therefore, this study tested the hypothesis that the use of a bioactive coating (by plasma electrolytic oxidation, PEO) on titanium microplates could improve the fracture healing of low bone mineral density (BMD) rats. Thirty female rats underwent bilateral ovariectomy surgery (OVX), and 35 rats underwent fake surgery (SHAM). Three months later, animals were subjected to femoral fracture simulation and were fixed with either non-coated (CONV) or coated (PEO) titanium miniplates. Eight weeks postoperatively, microplate/bone complexes were analyzed through computed microtomography, histometric, confocal microscopy, molecular, and biomechanical analysis. Bioactive elements (Ca and P) were incorporated on the PEO microplate and the surface was modified in a volcano-like structure. In the microCT analysis the OVX/PEO group had greater values for Tb.Th (bone trabecular thickness), Tb.Sp (separation of bone trabeculae) and Tb.N (number of trabeculae) parameters compared to the OVX/CONV group. According to histometric analysis, the OVX/PEO group showed significantly higher new bone formation than the OVX/CONV group (P < 0.05). For the fluorochrome area, the OVX groups (PEO and CONV) showed greater values for calcein precipitation (old bone) than alizarin red (new bone). Molecular results showed greater values for proteins related to the final phase of bone formation (P < 0.05) in the OVX/PEO group. The OVX/PEO group showed higher bone/miniplate system resilience compared to the others (P < 0.05). It was concluded that PEO coating optimizes bone healing on simulated femoral fractures in low bone mineral density rats. This sheds new light in the treatment of osteoporotic patients with bone fractures.
Assuntos
Doenças Ósseas Metabólicas , Fraturas do Fêmur , Osteoporose , Animais , Densidade Óssea , Feminino , Fraturas do Fêmur/diagnóstico por imagem , Fraturas do Fêmur/terapia , Humanos , Ovariectomia , RatosRESUMO
Since their discovery, mesenchymal stromal cells (MSCs) have received a lot of attention, mainly due to their self-renewal potential and multilineage differentiation capacity. For these reasons, MSCs are a useful tool in cell biology and regenerative medicine. In this article, we describe protocols to isolate MSCs from bone marrow (BM-MSCs) and adipose tissues (AT-MSCs), and methods to culture, characterize, and differentiate MSCs into osteoblasts, adipocytes, and chondrocytes. After the harvesting of cells from bone marrow by flushing the femoral diaphysis and enzymatic digestion of abdominal and inguinal adipose tissues, MSCs are selected by their adherence to the plastic tissue culture dish. Within 7 days, MSCs reach 70% confluence and are ready to be used in subsequent experiments. The protocols described here are easy to perform, cost-efficient, require minimal time, and yield a cell population rich in MSCs.
RESUMO
The major role of integrins is to mediate cell adhesion but some of them are involved in the osteoblasts-titanium (Ti) interactions. In this study, we investigated the participation of integrins in osteoblast differentiation induced by Ti with nanotopography (Ti-Nano) and with microtopography (Ti-Micro). By using a PCR array, we observed that, compared with Ti-Micro, Ti-Nano upregulated the expression of five integrins in mesenchymal stem cells, including integrin ß3, which increases osteoblast differentiation. Silencing integrin ß3, using CRISPR-Cas9, in MC3T3-E1 cells significantly reduced the osteoblast differentiation induced by Ti-Nano in contrast to the effect on T-Micro. Concomitantly, integrin ß3 silencing downregulated the expression of integrin αv, the parent chain that combines with other integrins and several components of the Wnt/ß-catenin and BMP/Smad signaling pathways, all involved in osteoblast differentiation, only in cells cultured on Ti-Nano. Taken together, our results showed the key role of integrin ß3 in the osteogenic potential of Ti-Nano but not of Ti-Micro. Additionally, we propose a novel mechanism to explain the higher osteoblast differentiation induced by Ti-Nano that involves an intricate regulatory network triggered by integrin ß3 upregulation, which activates the Wnt and BMP signal transductions. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1303-1313, 2019.
Assuntos
Diferenciação Celular , Integrina beta3/metabolismo , Nanoestruturas/química , Osteoblastos/metabolismo , Titânio/química , Via de Sinalização Wnt , Animais , Linhagem Celular , Masculino , Camundongos , Osteoblastos/citologia , Ratos , Ratos WistarRESUMO
Wnt/ß-catenin signal transduction is involved in the homeostatic control of bone mass. It is well established that a titanium surface with nanotopography (Ti-Nano) favors osteoblast differentiation by modulating different signaling pathways. However, few studies have investigated the participation of the Wnt/ß-catenin pathway in the osteogenic effect of nanoscale topographies. In this study, we aimed to determine whether the Wnt/ß-catenin signaling pathway is involved in the elevated osteogenic potential of Ti-Nano. MC3T3-E1 cells were cultured on Ti-Nano and machined Ti (Ti-Control) for evaluation of the expression of Wnt/ß-catenin signaling pathway-related genes. Based on the results to real-time PCR, the Wnt receptor Fzd4 was selected and silenced by CRISPRi. The resulting cells were cultured on both Ti surfaces, and several events involved in osteoblast differentiation were evaluated. The results revealed that Fzd4 gene silencing, corresponding to negative modulation of Wnt/ß-catenin, inhibits expression of the osteoblast phenotype. It is worthy of note that this inhibitory effect on osteoblast differentiation was more pronounced in cells grown on Ti-Nano compared with those grown on Ti-Control. By disrupting Fzd4 gene expression, we have shown that the elevated osteogenic potential of Ti-Nano is due to activation of the Wnt/ß-catenin signaling pathway, which reveals a new mechanism to explain osteoblast differentiation induced by nanotopography. Such an understanding of the intracellular machinery involved in surface guiding of osteoblast fate may contribute to the development of smart biomaterials to modulate the process of implant osseointegration.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Nanopartículas/química , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Titânio/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , Células 3T3 , Animais , Células Cultivadas , Camundongos , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
Treatment of large bone defects is a challenging clinical situation that may be benefited from cell therapies based on regenerative medicine. This study was conducted to evaluate the effect of local injection of bone marrow-derived mesenchymal stromal cells (BM-MSCs) or adipose tissue-derived MSCs (AT-MSCs) on the regeneration of rat calvarial defects. BM-MSCs and AT-MSCs were characterized based on their expression of specific surface markers; cell viability was evaluated after injection with a 21-G needle. Defects measuring 5 mm that were created in rat calvaria were injected with BM-MSCs, AT-MSCs, or vehicle-phosphate-buffered saline (Control) 2 weeks post-defect creation. Cells were tracked by bioluminescence, and 4 weeks post-injection, the newly formed bone was evaluated by µCT, histology, nanoindentation, and gene expression of bone markers. BM-MSCs and AT-MSCs exhibited the characteristics of MSCs and maintained their viability after passing through the 21-G needle. Injection of both BM-MSCs and AT-MSCs resulted in increased bone formation compared to that in Control and with similar mechanical properties as those of native bone. The expression of genes associated with bone formation was higher in the newly formed bone induced by BM-MSCs, whereas the expression of genes involved in bone resorption was higher in the AT-MSC group. Cell therapy based on local injection of BM-MSCs or AT-MSCs is effective in delivering cells that induced a significant improvement in bone healing. Despite differences observed in molecular cues between BM-MSCs and AT-MSCs, both cells had the ability to induce bone tissue formation at comparable amounts and properties. These results may drive new cell therapy approaches toward complete bone regeneration.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Animais , Biomarcadores , Regeneração Óssea , Diferenciação Celular , Sobrevivência Celular , Rastreamento de Células , Terapia Baseada em Transplante de Células e Tecidos/métodos , Imuno-Histoquímica , Imunofenotipagem , Masculino , Transplante de Células-Tronco Mesenquimais/métodos , Imagem Molecular , Osteogênese , Ratos , Resultado do Tratamento , Microtomografia por Raio-XRESUMO
Aim: The aim of this study was to investigate the effect of local injection of osteoblasts differentiated from bone marrow (BM-OB) or adipose tissue (AT-OB) mesenchymal stromal cells on bone tissue formation. Materials & methods: Defects were created in rat calvaria and injected with BM-OB or AT-OB and phosphate-buffered saline without cells were injected as control. Bone formation was evaluated 4 weeks postinjection. Results: Injection of BM-OB or AT-OB resulted in higher bone formation than that obtained with control. The bone tissue induced by cell injections exhibited similar mechanical properties as those of pristine calvarial bone, and its molecular cues suggested the occurrence of a remodeling process. Conclusion: Results of this study demonstrated that cell therapy with osteoblasts induced significant bone formation that exhibited the same quality as that of pre-existent bone.
Assuntos
Células da Medula Óssea/citologia , Regeneração Óssea , Osso e Ossos/citologia , Terapia Baseada em Transplante de Células e Tecidos/métodos , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/citologia , Osteoblastos/citologia , Animais , Osso e Ossos/lesões , Diferenciação Celular , Masculino , Osteogênese , Ratos , Ratos Wistar , Engenharia Tecidual/métodosRESUMO
PURPOSE: To investigate the probability of survival of different implant-abutment connection designs in narrow versus standard-diameter implants supporting anterior crowns. MATERIALS AND METHODS: A total of 108 implants of either 3.5-mm or 4.0-mm diameter (narrow and standard, respectively) (10 mm in length, Implacil de Bortoli) were divided into six groups (n=18 each) as follows: external hexagon 3.5 mm or 4.0 mm (EH3.5 or EH4.0), internal hexagon 3.5 mm or 4.0 mm (IH3.5 or IH4.0), and Morse taper 3.5 mm or 4.0 mm (MT3.5 or MT4.0). The corresponding abutments were screwed to the implants, and standardized maxillary central incisor metal crowns were cemented and subjected to step-stress accelerated life testing in water. Use-level probability Weibull curves and reliability for a mission of 50,000 and 100,000 cycles at 100 N and 150 N (90% 2-sided confidence intervals [CI]) were calculated. Polarized-light and scanning electron microscopes were used to assess the failure modes. RESULTS: The calculated reliability with 90% CI for a mission of 50,000 cycles at 100 N and 150 N showed that cumulative damage from the respective loads would lead to ~93% and ~18% implant-supported restoration survival in group EH3.5, ~99% and ~1% in group IH3.5, ~97% and ~89% in the MT3.5, ~100% and ~99% in the group EH4.0, ~100% and ~100% in group IH4.0, and ~99% and ~99% in group MT4.0. For the 100,000-cycle mission, the probability of survival estimated at 100 N and 150 N was, respectively: 0% for EH3.5 and IH3.5 at both load levels, ~96% and ~87% for the MT3.5, 100% and ~99% for EH4.0, 100% and ~99% for IH4.0, and 98% and ~92% for the MT4.0. CONCLUSION: A significant decrease in the probability of survival as a function of elapsed fatigue cycles and load increase was observed for narrow implants only with EH and IH implant-abutment connections, but not on Morse taper.
Assuntos
Projeto do Implante Dentário-Pivô , Implantes Dentários , Cimentação/métodos , Coroas , Ligas Dentárias/química , Prótese Dentária Fixada por Implante , Falha de Restauração Dentária , Análise do Estresse Dentário/instrumentação , Humanos , Teste de Materiais , Microscopia Eletrônica de Varredura , Microscopia de Polarização , Probabilidade , Reprodutibilidade dos Testes , Estresse Mecânico , Propriedades de Superfície , Análise de Sobrevida , Água/químicaRESUMO
Clinical success of implant therapy is directly related to titanium (Ti) surface properties and the quality of bone tissue. The treatment of Ti implants with H2SO4/H2O2 is a feasible, reproducible, and low-cost technique to create surface nanotopography (Ti-Nano). As this nanotopography induces osteoblast differentiation, we hypothesized that it may affect bone response to Ti. Thus, this study was designed to evaluate the bone response to a machined Ti implant treated with H2SO4/H2O2 to generate Ti-Nano and to compare it with a commercially available microtopographic Ti implant (Ti-Porous). Implants were placed in rabbit tibias and evaluated after 2 and 6 weeks, and the bone tissue formed around them was assessed by microtomography to record bone volume, bone surface, specific bone surface, trabecular number, trabecular thickness, and trabecular separation. Undecalcified histological sections were used to determine the percentages of bone-to-implant contact, bone area formed between threads, and bone area formed in the mirror area. At the end of 6 weeks, the removal torque was evaluated using a digital torque gauge. The results showed bone formation in close contact with both Ti-Nano and Ti-Porous implants without relevant morphological and morphometric differences, in addition to a similar removal torque irrespective of surface topography. In conclusion, our results have shown that a simple and low-cost method using H2SO4/H2O2 is highly efficient for creating nanotopography on Ti surfaces, which elicits a similar bone response compared with microtopography presented in a commercially available Ti implant.
Assuntos
Implantes Dentários , Osseointegração , Titânio , Animais , Peróxido de Hidrogênio , Microscopia Eletrônica de Varredura , Coelhos , Propriedades de Superfície , TorqueRESUMO
In this study, we evaluated the effect of poly(vinylidene fluoride-trifluoroethylene)/barium titanate (P(VDF-TrFE)/BT) membrane on in vivo bone formation. Rat calvarial bone defects were implanted with P(VDF-TrFE)/BT and polytetrafluoroethylene (PTFE) membranes, and at 4 and 8 weeks, histomorphometric and gene expression analyses were performed. A higher amount of bone formation was noticed on P(VDF-TrFE)/BT compared with PTFE. The gene expression of RUNX2, bone sialoprotein, osteocalcin, receptor activator of nuclear factor-kappa B ligand, and osteoprotegerin indicates that P(VDF-TrFE)/BT favored the osteoblast differentiation compared with PTFE. These results evidenced the benefits of using P(VDF-TrFE)/BT to promote new bone formation, which may represent a promising alternative to be employed in guided bone regeneration.