Stimulating Effect of a Novel Synthesized Sulfonamido-Based Gallate ZXHA-TC on Primary Osteoblasts

PURPOSE: This study is intended to investigate the effects of plants or plant-derived antioxidants on prevention of osteoporosis through the maintenance of reactive oxygen species (ROS) at a favorable level. MATERIALS AND METHODS: In this study, a novel antioxidant, namely 3,4,5-Trihydroxy-N-[4-(5-hydroxy-6-methoxy-pyrimidin-4-ylsulfamoyl)-phenyl]-benzamide (ZXHA-TC) was synthesized from gallic acid and sulfadimoxine. Its effect on osteoblast metabolism was investigated via the detection of cell proliferation, cell viability, production of ROS, and expression of osteogenic-specific genes including runt-related transcription factor 2 (RUNX2), bone sialoprotein (BSP), osteocalcin (OCN), alpha-1 type I collagen (COL1A1), and osteogenic-related proteins after treatment for 2, 4, and 6 days respectively. RESULTS: The results showed that ZXHA-TC has a stimulating effect on the proliferation and osteogenic differentiation of primary osteoblasts by promoting cell proliferation, cell viability, and the expression of genes BSP and OCN. Productions of bone matrix and mineralization were also increased by ZXHA-TC treatment as a result of up-regulation of COL1A1 and alkaline phosphatase (ALP) at the early stage and down-regulation of both genes subsequently. A range of 6.25x10(-3) microg/mL to 6.25x10(-1) microg/mL is the recommended dose for ZXHA-TC, within which 6.25x10(-2) microg/mL showed the best performance. CONCLUSION: This study may hold promise for the development of a novel agent for the treatment of osteoporosis.

Effects of Polycaprolactone-Tricalcium Phosphate, Recombinant Human Bone Morphogenetic Protein-2 and Dog Mesenchymal Stem Cells on Bone Formation: Pilot Study in Dogs

PURPOSE: The aim of this study was to evaluate the survival, proliferation, and bone formation of dog mesenchymal stem cells (dMSCs) in the graft material by using Polycaprolactone-tricalcium phosphate (PCL-TCP), auto-fibrin glue (AFG), recombinant human bone morphogenetic protein-2 (rhBMP-2), and dMSCs after a transplantation to the scapula of adult beagle dogs. MATERIALS AND METHODS: The subjects were two beagle dogs. Total dose of rhBMP-2 on each block was 10 microg with 50 microg/mg concentration. The cortical bone of the scapula of the dog was removed which was the same size of PCL-TCP block (Osteopore International Pte, Singapore; 5.0x5.0x8.0 mm in size), and the following graft material then was fixed with orthodontic mini-implant, Dual-top(R) (Titanium alloy, Jeil Co. Seoul, Korea). Four experimental groups were prepared for this study, Group 1: PCL-TCP + aFG; Group 2: PCL-TCP + aFG + dMSCs; Group 3: PCL-TCP + aFG + dMSCs + rhBMP-2; Group 4: PCL-TCP + aFG + dMSCs + rhBMP-2 + PCL membrane. The survival or proliferation of dMSCs cells was identified with an extracted tissue through a fluorescence microscope, H-E staining and Von-Kossa staining in two weeks and four weeks after the transplantation. RESULTS: The survival and proliferation of dMSCs were identified through a fluorescence microscope from both Group 1 and Group 2 in two weeks and four weeks after the transplantation. Histological observation also found that the injected cells were proliferating well in the G2, G3, and G4 scaffolds. CONCLUSION: This study concluded that bone ingrowth occurred in PCL-TCP scaffold which was transplanted with rhBMP-2, and MSCs did not affect bone growth. More sufficient healing time would be needed to recognize effects of dMSCs on bone formation.

Bone morphogenetic proteins: its application in the process of repairing the dentin pulp complex

Evolution of knowledge related to molecular Biology has been applied in different areas of clinical Biology, Dentistry among them, allowing the use of these new advances in the treatment of offenses to the pulpal organ. The use of bone morphogenetic proteins (BMPs) represents another possibility in the treatment of exposed pulps with no contact with aggressive agents and free from undesirable effects to maintain pulp vitality.

La evolución del conocimiento relacionado a la Biología Molecular ha sido aplicado en diferentes áreas de la Biología Clínica, entre ellas la Odontología, permitiendo el uso de esos nuevos adelantos en el tratamiento de agresiones al órgano pulpar. El uso de proteínas óseas morfogenéticas (BMPs), representa otra posibilidad en el tratamiento de pulas expuestas sin la presencia de agentes agresores, libre principalmente de los efectos indeseables a la mantención de la vitalidad pulpar.

Chondrogenic differentiation of mouse bone marrow mesenchymal stem cells induced by cartilage-derived morphogenetic protein-2 in vitro / 华中科技大学学报（医学）（英德文版）

To study the cartilage differentiation of mouse mesenchymal stem cells (MSCs) induced by cartilage-derived morphogenetic proteins-2 in vitro, the MSCs were isolated from mouse bone marrow and cultured in vitro. The cells in passage 3 were induced into chondrogenic differentiation with different concentrations of recombinant human cartilage-derived morphogenetic proteins-2 (0, 10, 20, 50 and 100 ng/mL). After 14 days of induction, morphology of cells was observed under phase-contrast microscope. Collagen II mRNA and protein were examined with RT-PCR, Western blotting and immunocytochemistry respectively and the sulfate glycosaminoglycan was measured by Alcian blue staining. RT-PCR showed that CDMP-2 could promote expression of collagen II mRNA in an dose-dependant manner, especially at the concentration of 50 ng/mL and 100 ng/mL. Immunocytochemistry and Western blotting revealed a similar change. Alcian blue staining exhibited deposition of typical cartilage extracellular matrix. Our results suggest that mouse bone marrow mesenchymal stem cells can differentiate into chondrogenic phonotype with the induction of CDMP-2 in vitro, which provides a basis for further research on the role of CDMP-2 in chondrogenesis.

Assessment of bovine biomaterials containing bone morphogenetic proteins bound to absorbable hydroxyapatite in rabbit segmental bone defects

PURPOSE: To evaluate the osteo-regenerative capacity of two proprietary bone grafting materials, using a segmental defect model in both radial diaphyses of rabbits. METHODS: The right defect was filled with pooled bone morphogenetic proteins (pBMPs) bound to absorbable ultrathin powdered hydroxyapatite (HA) mixed with inorganic and demineralized bone matrix and bone-derived collagen, derived from bovine bone (Group A). The left defect was filled with bovine demineralized bone matrix and pBMPs bound to absorbable ultrathin powdered HA (Group B). In both groups, an absorbable membrane of demineralized bovine cortical was used to retain the biomaterials in the bone defects, and to guide the tissue regeneration. The rabbits were euthanized 30, 90 and 150 days after surgery. Radiographic, tomographic and histologic evaluations were carried out on all specimens. RESULTS: At 30 days, the demineralized cortical bone cover was totally resorbed in both groups. HA was totally resorbed from Group A defects, whereas HA persisted in Group B defects. A prominent foreign body reaction was evident with both products, more pronounced in sections from Group B. At 90 days, the defects in Group B exhibited more new bone than Group A. However, at 150 days after surgery, neither treatment had stimulated complete repair of the defect. CONCLUSION: The partial bone healing of the segmental defect occurred with low or none performance of the biomaterials tested.

OBJETIVO: Avaliar a capacidade osteo-regenerativa de dois biomateriais utilizando um modelo de defeito segmentar efetuado nas diáfises do rádio de coelhos. MÉTODOS: O defeito direito foi preenchido com pool de proteínas morfogenéticas ósseas (pBMPs) e hidroxiapatita em pó ultrafina absorvível (HA) combinada com matriz óssea inorgânica desmineralizada e colágeno, derivados do osso bovino (Grupo A). O defeito esquerdo foi preenchido com matriz óssea desmineralizada bovina com pBMPs e hidroxiapatita em pó ultrafina absorvível (Grupo B). Em ambos os defeitos utilizou-se membrana reabsorvível de cortical bovina desmineralizada para reter os biomateriais no defeito ósseo e guiar a regeneração tecidual. Os coelhos foram submetidos à eutanásia aos 30, 90 e 150 dias após a cirurgia. Foram efetuados exames radiográficos, tomográficos e histológicos em todos os espécimes. RESULTADOS: Aos 30 dias de pós-cirúrgico, o osso cortical desmineralizado foi totalmente reabsorvido em ambos os grupos. A HA tinha reabsorvido nos defeitos do Grupo A, mas persistiu nos do Grupo B. Uma reação de corpo estranho foi evidente com ambos os produtos, porém mais pronunciada no Grupo B. Aos 90 dias os defeitos do grupo B tinham mais formação óssea que os do Grupo A. Entretanto, aos 150 dias após a cirurgia, nenhum tratamento havia promovido o completo reparo do defeito. CONCLUSÃO: Os biomateriais testados contribuíram pouco ou quase nada para a reconstituição do defeito segmentar.

Pool of bovine morphogenetic proteins and guided tissue regeneration in the treatment of intrabony periodontal defects: I- Clinical measurements

Este estudo teve como objetivo avaliar a aplicação do ''pool'' de BMPs bovinas no tratamento de defeitos intra-ósseos. A amostra constou de quinze indivíduos com idade entre 26 e 57 anos, apresentando um par de defeitos intra-ósseos comparáveis, localizados no mesmo arco em dentes do mesmo tipo (pré-molares ou molares) e com perda de inserção (PI) 35mm. Em cada indivíduo, o defeito teste foi tratado com a associação ''pool'' de BMPs- carreador Hidroxiapatita reabsorvível (BMPs-HA), matriz orgânica bovina desmineralizada liofilizada (MO) e barreira de colágeno bovino, enquanto os defeitos controle foram tratados com MO-HA e barreira de colágeno. Na avaliação clínica aos seis meses após a medida inicial, o grupo teste apresentou variação de profundidade de sondagem (PS) de -1,63mm ± 1,141 na vestibular (V) e -1,93mm ± 0,961 na lingual (L) e variação do nível de inserção (NI) de -1,60mm ± 1,168 na V e -1,46mm ± 0,972 na L. O grupo controle apresentou variação de PS de -1,93mm ± 1,347 (V) e -2,0mm ± 1,511 (L) e variação de NI de -1,03mm ± 1,245 (V) e -1,30mm ± 1,114 (L). A análise de variância indicou que a variação de PS e NI foi significativa em ambos os grupos (p<0,05). Entretanto, de acordo com o teste t não houve diferença significativa entre os grupos quanto à variação PS e NI (p<0,05). Em conclusão, a aplicação do ''pool'' de BMPs não oferece benefícios adicionais ao tratamento de defeitos intra-ósseos.

The Effects of Recombinant Human BMP-7, Prepared from a COS-7 Expression System, on the Proliferation and Differentiation of Rat Newborn Calvarial Osteoblasts

A family of proteins, the bone morphogenetic proteins (BMPs), which promote osteoblast differentiation and bone mineralization, have recently been identified. One, BMP-7, has shown the ability to induce cartilage and bone formation processes. In this report, the possibility that other cell lines, to CHO cells, may also be available as host cells for the expression of hBMP-7 was validated. Recombinant human BMP (rhBMP) -7 was produced in COS-7 cells, as a processed mature disulfide-linked homodimer, with an apparent molecular weight of 36, 000. Examination of the expressions of the markers characteristic of osteoblast phenotypes showed that the rhBMP-7 specifically stimulated the inductions of alkaline phosphatase (ALP) (5-fold increase at 100 ng of rhBMP-7/ml), parathyroid hormone (PTH) -mediated intracellular cAMP production (4-fold increase at 100 ng of rhBMP-7/ml) and osteocalcin synthesis (5-fold increase at 100 ng of rhBMP-7/ml). In summary, the in vitro mineralization assay results provide evidence that the rhBMP-7 peptide, produced by COS-7 expression system, possesses intact biological activity. A similar pattern of biological activity was observed for the BMP-7 in COS-7 cells compared to the corresponding CHO cell expression system. Thus, these findings can be experimentally utilized for the production of rhBMPs for in vitro or in vivo studies.

Avaliaçäo clínica e radiográfica de lesöes periodontais intra-ósseas em humanos tratadas com "pool" de proteínas morfogenéticas bovinas e membrana de colágeno bovino / Radiographical and clinical evaluation of human intrabony periodontal lesions treated with combination bovine bone morphogenetics proteins and bovine collagen membrane

Este estudo teve como objetivo avaliar a aplicaçäo do "pool" de BMPs bovinas no tratamento de defeitos intra-ósseos. A amostra constou de quinze indivíduos com idade entre 26 e 57 anos, apresentando um par de defeitos intra-ósseos comparáveis, localizados no mesmo arco em dentes do mesmo tipo (pré-molares ou molares) e com perda de inserçäo (PI) 5mm. Em cada indivíduo, o defeito teste foi tratado com a associaçäo "pool" de BMPs- carreador Hidroxiapatita reabsorvível (BMPs-HA), matriz orgânica bovina desmineralizada liofilizada (MO) e barreira de colágeno bovino, enquanto os defeitos controle foram tratados com MO-HA e barreira de colágeno. Na avaliaçäo clínica aos seis meses após a medida inicial, o grupo teste apresentou variaçäo de profundidade de sondagem (PS) de -1,63mm 1,141 na vestibular (V) e -1,93mm 0,961 na lingual (L) e variaçäo do nível de inserçäo (NI) de -1,60mm 1,168 na V e -1,46mm 0,972 na L. O grupo controle apresentou variaçäo de PS de -1,93mm 1,347 (V) e -2,0mm 1,511 (L) e variaçäo de NI de -1,03mm 1,245 (V) e -1,30mm 1,114 (L). A análise de variância indicou que a variaçäo de PS e NI foi significativa em ambos os grupos (p < 0,05). Entretanto, de acordo com o teste t näo houve diferença significativa entre os grupos quanto à variaçäo PS e NI (p < 0,05). A radiografia de subtraçäo detectou mudança de densidade óssea de 0,034 0,423 no grupo teste e 0,105 0,423 no grupo controle, sendo as diferenças entre os grupos consideradas näo significativas (teste t p < 0,005). A análise de correlaçäo de Pearson indicou que näo houve correlaçäo entre mudança de densidade óssea e variaçäo do NI e PS. Tais resultados levaram às seguintes conclusöes: a aplicaçäo do "pool" de BMPs näo oferece benefícios adicionais ao tratamento de defeitos intra-ósseos. A näo correlaçäo entre mudança de densidade óssea e melhora dos parâmetros clínicos sugere a näo ocorrência de regeneraçäo periodontal propriamente dita, podendo esta melhora estar relacionada mais à qualidade de resposta dos tecidos moles

Avaliaçäo da osteogênese com proteínas ósseas morfogenéticas (BMPs): análise em defeitos na calvária e ao redor de implantes de titânio em coelhos / Osseointegration enhancement around titanium implants and calvarial reconstruction induced by bone morphogenetic proteins in rabbits

Objetivos. Avaliar a formaçäo óssea em defeitos ósseos criados em calvárias de coelhos, aplicando proteínas ósseas morfogenéticas (BMPs) carreadas pelo compósito carbonato de cálcio-colágeno. Analisar o percentual linear de contato osso-implante e o volume ósseo percentual no interior das roscas de implantes de titânio, inseridos em alvéolos cirúrgicos, preparados em tíbias de coelho e preenchidos pelo compósito carbonato de cálcio-colágeno combinado à BMPs. Material e métodos. Experimento I: Defeitos criados cirurgicamente em calvária de 45 coelhos (Oryctolagus cuniculus) brancos, raça Nova Zelândia, gênero masculino. Nos sítios teste, preencheu-se estes defeitos com rBMPs extraídas de ossos longos de renas (Rangifer tarandus) combinadas ao compósito carbonato de cálcio-colágeno, tanto em forma granular quanto em forma de bloco. Nos sítios controle, preencheu-se os defeitos com osso autógeno (controle positivo), com as duas formas do compósito. O controle negativo näo recebeu nenhum tipo de tratamento. Experimento II: Confeccionou-se alvéolos cirúrgicos nas tíbias de 30 coelhos. Previamente a inserçäo de implantes rosqueáveis de titânio, apresentando 3,75mm de diâmetro e 11,5mm de comprimento, preencheu-se os alvéolos dos sítios teste com compósito carbonato de cálcio-colágeno combinado a rBMP. Preencheram-se os sítios controle com carbonato de cálcio-colágeno, colágeno e; no controle negativo nenhum tipo de material foi inserido. Resultados. Experimento I: Após os períodos de quatro, seis e dez semanas os animais foram submetidos à eutanásia. A BMP extraída de rena e combinada ao compósito carbonato de cálcio-colágeno promoveu osteogênese semelhante à obtida quando utilizado osso autógeno e superior aos controles de compósito e coágulo sangüíneo. A análise tomográfica apresentou um quadro de resultados semelhantes aos obtidos com a histomorfometria, entretanto, superestimados para os períodos iniciais de observaçäo, nos grupos que receberam enxerto do compósito. O compósito carbonato de cálcio-colágeno isoladamente ou combinado a BMP näo induziu reaçöes inflamatórias desfavoráveis. Experimento II: A BMP extraída de rena e combinada ao compósito carbonato de cálcio-colágeno promoveu osteogênese superior aos controles do compósito, do colágeno e do coágulo sangüíneo, provendo à superfície do implante de titânio maior percentual de contato linear e maior volume percentual de osso no interior de suas roscas...

Histomorphometric analysis of rat alveolar wound healing with hydroxyapatite alone or associated to BMPs

Several materials and techniques have been proposed to improve alveolar wound healing and decrease loss of bone height and thickness that normally follow dental extraction. The objective of this research was the histologic analysis of bone morphogenetic proteins implanted into dental alveoli of rats after extraction. A total of 45 adult male Wistar rats were divided into three groups of 15 animals each: control (no treatment), implanted with pure hydroxyapatite (HA, 3 mg) and implanted with hydroxyapatite plus bone morphogenetic proteins (HA/BMPs, 3 mg). Five animals from each group were sacrificed at 7, 21 and 42 days after extraction for the histometric analyses of the osteoconductive potential of hydroxyapatite associated or not with BMPs. After dissection, fixation, decalcification and serial microtomy of 6-µm thick sections, the samples were stained with hematoxylin-eosin for histologic and histometric analyses. Both HA and HA/BMPs caused a delay in wound healing compared to control animals, evaluated by the percentage of bone tissue in the alveoli. The treatment with HA/BMPs had the greatest delay at 21 days, even though it produced values similar to the control group at 42 days. The materials did not improve alveolar repair in the normal period of wound healing and the association of HA/BMPs did not have osteoconductive properties with granulated hydroxyapatite as the vehicle

Interaction of TGF-beta1 and rhBMP-2 on Human Bone Marrow Stromal Cells Cultured in Collagen Gel Matrix

Transforming growth factor-beta1 (TGF-beta1) and bone morphogenetic protein-2 (BMP-2) are abundant proteins in the bone matrix. However, their interaction in controlling osteoblast differentiation is not clearly understood. In this study, HBMSCs were cultured in collagen gel matrix with different condition of exogenous rhBMP-2 and TGF-beta1 in order to determine the interaction of BMP-2 and TGF-beta1 on human bone marrow stromal cells (HBMSCs) differentiation. The cultured cells were analyzed for cell proliferation, alkaline phophatase (ALP) activity and mineralization stainning with Von-Kossa. The cells treated with TGF-beta1 exhibited a higher rate of cell growth than those without. However, the cells cultured in collagen gel matrix showed a lower rate of cell growth than the cells cultured in a monolayer. To investigate the effects of both cytokines on osteoblast differentiation, the cells were treated with 0, 1, 5, 10 ng/ml of TGF-beta1 for 2 days. This was followed by culturing with 0, 1, 5, and 10 ng/ml of TGF-beta1 and 100 ng/ml of rhBMP-2 together for 3 days with the alkaline phosphatase (ALP) activity measured. The cells treated with 1 ng/ml of TGF-beta1 responded efficiently to rhBMP-2 and expressed ALP activity with a level equivalent to that exhibited by cells that were not treated with TGF-beta1. The cells treated with 5 and 10 ng/ml of TGF-beta1 showed a dramatic decrease in ALP activity. The cells treated with 10ng/ml of TGF-beta1 followed by rhBMP-2 alone exhibited an intermediate ALP activity. The cells treated with 100 ng/ml of rhBMP-2 demonstrated Von-Kossa positive solid deposits after 3 weeks, while there were few Von-Kossa positive solid deposits when the cells treated with 10 ng/ml of TGF-beta1. These results show that TGF-beta1 inhibits the effects of rhBMP-2 on the osteoblast differentiation of HBMSCs in a dose dependant manner. Furthermore, the effects of TGF-beta1 on HBMSCs are reversible. This suggest that TGF-beta1 and rhBMP-2 are coordinately controlled during the osteoblast differentiation of HMBSCs.