RESUMO
BACKGROUND AIMS: Regenerative medicine strategies based on cell therapy are considered a promising approach to repair bone defects. The aims of this study were to evaluate the effect of subculturing on the osteogenic potential of osteoblasts derived from newborn rat calvaria and the effect of these osteoblasts on bone repair of rat calvaria defects. METHODS: Cells were obtained from 50 newborn rat calvaria, and primary osteoblasts (OB) were compared with first passage (OB-P1) in terms of osteogenic potential by assaying cell proliferation, alkaline phosphatase (ALP) activity, extracellular matrix mineralization and gene expression of the osteoblastic markers RUNX2, ALP, osteocalcin and bone sialoprotein. Then, 5-mm calvaria defects were created in 24 Wistar rats, and after 2 weeks, they were locally injected with 50 µL of phosphate-buffered saline containing either 5â¯×â¯106 osteoblasts (OB-P1, nâ¯=â¯12) or no cells (control, nâ¯=â¯12). Four weeks post-injection, the bone formation was evaluated by micro-computed tomography and histological analyses. Data were compared by analysis of variance, followed by the Student-Newman-Keuls's test or Student's t-test (P ≤ 0.05). RESULTS: OB-P1 showed high proliferation and ALP activity, and despite the reduced gene expression of osteoblastic markers and extracellular matrix mineralization compared with OB, they displayed osteogenic potential, being a good choice for injection into calvaria defects. The micro-tomographic and histological data showed that defects treated with OB-P1 presented higher bone formation compared with control defects. DISCUSSION: Our results indicate that cells derived from newborn rat calvaria retain osteoblastic characteristics after subculturing and that these osteoblasts stimulate bone repair in a rat calvaria defect model.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos/métodos , Osteoblastos/transplante , Crânio/lesões , Fosfatase Alcalina/metabolismo , Animais , Biomarcadores , Células Cultivadas , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Osteoblastos/metabolismo , Osteoblastos/fisiologia , Osteocalcina/biossíntese , Osteocalcina/genética , Osteogênese/fisiologia , Ratos Wistar , Crânio/citologia , Transplante Homólogo/métodos , Microtomografia por Raio-XRESUMO
OBJECTIVES: Autografts from mandibular symphysis and ramus are often used for bone reconstruction. Based on this, we hypothesized that these sites could be useful cell sources for bone tissue engineering approaches. Thus, our study aimed at evaluating the proliferation and osteoblast phenotype development of cells derived from mandibular symphysis and ramus. MATERIALS AND METHODS: Cells were isolated from bone fragments of four patients by enzymatic digestion and cultured under osteogenic condition for up to 17 days. Cultures were assayed for cell proliferation, gene expression of key bone markers runt-related transcription factor 2 (Runx2), distal-less homeobox 5 (DLX5), SATB homeobox 2 (SATB2), Osterix (OSX), family with sequence similarity 20, member C (FAM20C), bone sialoprotein (BSP), osteopontin (OPN) and osteocalcin (OC), alkaline phosphatase (ALP) expression and activity, and extracellular matrix mineralization. Data were compared by two-way ANOVA or t-test for independent samples when appropriate. RESULTS: Cells derived from ramus displayed lower proliferative activity and higher gene expression of Runx2, DLX5, SATB2, OSX, FAM20C, BSP, OPN and OC, ALP protein expression and activity and extracellular matrix mineralization compared with symphysis-derived cells. CONCLUSION: Symphysis and ramus may be considered as cell sources for bone tissue engineering approaches but due to the higher osteogenic potential, ramus-derived cells are more appealing for constructing cell-based biomaterials.
Assuntos
Osso e Ossos , Mandíbula/citologia , Osteoblastos/citologia , Engenharia Tecidual/métodos , Proliferação de Células , Células Cultivadas , Humanos , Osteoblastos/transplante , Osteogênese/genética , FenótipoRESUMO
PURPOSE: The present study investigated osteointegration of autogenous bone (AB) from calvaria graft associated with osteoblastic cells (OC) in bone defects in rats subjected to daily administration of caffeine. MATERIALS AND METHODS: Male rats received daily intraperitoneal injection of 1.5% caffeine (0.2 mL/100 g body weight) or saline solution for 30 days. Then they were anesthetized, submitted to the extraction of the upper right incisor, and implanted with AB only and AB + OC. The animals were killed on 7th, 21st, and 42nd days after surgery, and their maxilla were processed for obtaining semiserial sections (5 µm) stained with hematoxylin and eosin. Through image analysis system, the bone volume and the quality of graft in adjacent areas were estimated. RESULTS: The results showed that in caffeine treatment, the AB + OC graft showed no foreign body and acute inflammatory reactions inside the defect when compared to AB. The histometric results revealed that the association AB + OC produced significant increase (10%-15%) in bone volume in later experimental period (42 days) when compared with saline solution group (P ≤ 0.01). CONCLUSIONS: It was concluded that the association of AB from calvaria + OC demonstrated progressive osteointegration and accelerated the repair of bone defects in animals treated with daily caffeine.
Assuntos
Transplante Ósseo/patologia , Cafeína/farmacologia , Osseointegração/efeitos dos fármacos , Osteoblastos/transplante , Antagonistas de Receptores Purinérgicos P1/farmacologia , Processo Alveolar/efeitos dos fármacos , Processo Alveolar/patologia , Animais , Células da Medula Óssea/efeitos dos fármacos , Matriz Óssea/efeitos dos fármacos , Matriz Óssea/patologia , Técnicas de Cultura de Células , Reação a Corpo Estranho/induzido quimicamente , Masculino , Maxila/cirurgia , Osseointegração/fisiologia , Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Osteogênese/fisiologia , Osso Parietal/cirurgia , Ratos , Ratos Wistar , Cloreto de Sódio , Fatores de Tempo , Coleta de Tecidos e Órgãos/métodosRESUMO
PURPOSE: Because of limitations of autogenous grafts, allografts, xenografts, alloplasts, and hydroxyapatite as graft materials, researchers have been using bone tissue engineering as a strategy for bone regeneration. The aim of this work was to study the effect of bone tissue engineering, associating bone marrow osteoblastic cells, and autogenous bone in defects created by dental extraction in rats. MATERIALS AND METHODS: Eighty male rats from 250 to 300 g were anesthetized, submitted to the extraction of the superior incisor, and divided in control group (C), implanted with osteoblastic cells (OC), autogenous bone (AB), and osteoblastic cells + autogenous bone (OC + AB). The animals were killed on 10th and 20th days after surgery and their maxilla were processed for obtaining fine semiserial sections (5 mum), and then stained with hematoxylin-eosin. Through image analysis system, bone volume in areas adjacent to the implants was estimated. RESULTS: The histometric results revealed that the association OC + AB produced significant increase (10%-15%) of bone in both experimental periods when compared with the control group (P < or = 0.01). CONCLUSIONS: Osteoblastic cells associated with autogenous bone accelerated the repair of bone defect, and the action of the osteoblastic cells was more effective until the 10th day and of the autogenous bone after this period.
Assuntos
Transplante de Medula Óssea/patologia , Transplante Ósseo/patologia , Doenças Maxilares/cirurgia , Osteoblastos/transplante , Osteogênese/fisiologia , Animais , Matriz Óssea/patologia , Regeneração Óssea/fisiologia , Células Cultivadas , Tecido Conjuntivo/patologia , Masculino , Ratos , Ratos Wistar , Fatores de Tempo , Extração Dentária/efeitos adversos , Cicatrização/fisiologiaRESUMO
Mesenchymal stem cells (MSCs) can show osteogenic differentiation capability when implanted in vivo, as well as cultured in vitro; therefore we attempted to use allogeneic MSCs for an 8-month-old patient with hypophosphatasia. MSCs were obtained by culture expansion of fresh marrow from the patient's father. Some of the MSCs were further cultured under osteogenic conditions on a culture dish or porous hydroxyapatite ceramics, resulting in cultured osteoblasts and osteogenic constructs, respectively. The MSCs and osteoblasts were injected into the patient, and the constructs were implanted locally. After traditional bone marrow transplantation, the MSCs, osteoblasts, and osteogenic constructs were used for treatment and to improve the patient's respiratory condition and skeletal abnormality. The condition worsened again, and an MSC booster shot was administered. At the same time, the construct was retrieved. The respiratory condition improved, and the retrieved construct showed de novo bone derived from both donor and patient cells. We demonstrated the importance of allogeneic MSC transplantation for hypophosphatasia and the constructs as an alternative to bone fragments that provided further osteogenic capability in the patient.
Assuntos
Hipofosfatasia/terapia , Transplante de Células-Tronco Mesenquimais , Osteogênese , Fosfatase Alcalina/sangue , Densidade Óssea , Diferenciação Celular , Células Cultivadas , Feminino , Humanos , Hipofosfatasia/metabolismo , Hipofosfatasia/fisiopatologia , Lactente , Infusões Intraósseas , Infusões Intravenosas , Osteoblastos/transplante , Respiração Artificial , Engenharia Tecidual , Transplante HomólogoRESUMO
La rehabilitación implantológica en sectores posteriores del maxilar superior dispone de escasa cantidad de hueso a consecuencia de la neumatización de los senos maxilares. La técnica de elevación del seno maxilar significó un importante aporte para la solución de este problema. Desde su reporte inicial a principios de los 80, se utilizó como material de injerto autólogo, cadavérico y heterólogo, materiales sintéticos y combinaciones de todos los anteriores. El desafío actual es lograr una óptima calidad de injerto y acortar el período de formación ósea. En el prsente trabajo se presenta un caso clínico en el que se resolvió la elevación de seno maxilar, usando como material de relleno fosfato tricálcico, hueso autólogo y plasma rico en plaquetas (PRP), con la incorporación en el mismo de células mesenquimales y osteoblastos autólogos diferenciados, in vitro. Se obuvieron biopsias para su estudio histológico y se colocaron implantes 2 y 5 meses después de la cirugía. Los resultados observados sugieren un sustancial acortamiento de los tiempos de la formación de hueso apto para implantar, siguiendo el protocolo de trabajo propuesto en este reporte