Expression of bone morphogenetic protein, vascular endothelial growth factor, and basic fibroblast growth factor in irradiated mandibles during distraction osteogenesis.

PURPOSE: The present study evaluated the expression of bone morphogenetic proteins (BMPs)-2, -4, -7, vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF) in irradiated mandibles during distraction osteogenesis. MATERIALS AND METHODS: A total of 24 rabbits were randomly assigned to the control and experimental groups. Each rabbit in the experimental group underwent preoperative radiation to 9 Gy in 5 fractions. After 1 month, all rabbits underwent osteotomy and distraction osteogenesis with 7 days of latency. Three rabbits in the control and experimental groups were killed at day 7 (end of the latency period), day 12 (middle of active distraction), day 18 (end of active distraction), and day 25 (1 week after consolidation). The specimens were used for immunohistochemical staining and real-time polymerase chain reaction analysis. RESULTS: Histologically, at day 25, cortical bone formation was much better in the control group than in the radiotherapy group. In the radiotherapy group, the bone spicules were aligned in the direction of tension stress. At day 12, the expression of BMP-2, -4, and -7 was elevated in the radiotherapy group compared with the control group. At day 25, the expression of BMP-2 was significantly greater in the radiotherapy group. At day 7, the expression of bFGF was significantly suppressed in the radiotherapy group. At day 12, the expression of bFGF and VEGF was significantly elevated in the radiotherapy group compared with the control group. At day 25, the expression of VEGF was significantly greater in the radiotherapy group. CONCLUSIONS: The results of our study have shown that radiotherapy changes the expression pattern of BMPs, VEGF, and bFGF.

Histological and radiographic evaluation of the muscle tissue of rats after implantation of bone morphogenic protein (rhBMP-2) in a scaffold of inorganic bone and after stimulation with low-power laser light.

OBJECTIVE: The present study histologically and radiologically evaluates the muscle tissue of rats after implantation of bone morphogenic protein (rhBMP-2) in a natural inorganic bone mineral scaffold from a bull calf femur and irradiation with low-power light laser. MATERIALS AND METHODS: The right and left hind limbs of 16 rats were shaved and an incision was made in the muscle on the face corresponding to the median portion of the tibia, into which rhBMP-2 in a scaffold of inorganic bone was implanted. Two groups of limbs were formed: control (G1) and laser irradiation (G2). G2 received diode laser light applied in the direction of the implant, at a dose of 8 J/cm2 for three minutes. On the 7th, 21st, 40th and 112th days after implantation, hind limbs of 4 animals were radiographed and their implants removed together with the surrounding tissue for study under the microscope. The histological results were graded as 0=absence, 1=slight presence, 2=representative and 3=very representative, with regard to the following events: formation of osteoid structure, acute inflammation, chronic inflammation, fibrin deposition, neovascularization, foreign-body granuloma and fibrosis. RESULTS: There were no statistically significant differences in these events at each evaluation times, between the two groups (P > 0.05; Mann-Whitney test). Nevertheless, it could be concluded that the natural inorganic bone matrix with rhBMP-2, from the femur of a bull calf, is a biocompatible combination. CONCLUSIONS: Under these conditions, the inductive capacity of rhBMP-2 for cell differentiation was inhibited. There was a slight acceleration in tissue healing in the group that received irradiation with low-power laser light.

Superpulsed laser irradiation increases osteoblast activity via modulation of bone morphogenetic factors.

BACKGROUND AND OBJECTIVE: Laser therapy is a new approach applicable in different medical fields when bone loss occurs, including orthopedics and dentistry. It has also been used to induce soft-tissue healing, for pain relief, bone, and nerve regeneration. With regard to bone synthesis, laser exposure has been shown to increase osteoblast activity and decrease osteoclast number, by inducing alkaline phosphatase (ALP), osteopontin, and bone sialoprotein expression. Studies have investigated the effects of continuous or pulsed laser irradiation, but no data are yet available on the properties of superpulsed laser irradiation. This study thus aimed to investigate the effect of superpulsed laser irradiation on osteogenic activity of human osteoblast-like cells, paying particular attention to investigating the molecular mechanisms underlying the effects of this type of laser radiation. STUDY DESIGN/MATERIALS AND METHODS: Human osteoblast-like MG-63 cells were exposed to 3, 7, or 10 superpulsed laser irradiation (pulse width 200 nanoseconds, minimum peak power 45 W, frequency 30 kHz, total energy 60 J, exposure time 5 minutes). The following parameters were evaluated: cell growth and viability (light microscopy, lactate dehydrogenase release), calcium deposits (Alizarin Red S staining), expression of bone morphogenetic factors (real-time PCR). RESULTS: Superpulsed laser irradiation decreases cell growth, induces expression of TGF-beta2, BMP-4, and BMP-7, type I collagen, ALP, and osteocalcin, and increases the size and the number of calcium deposits. The stimulatory effect is maximum on day 10, that is, after seven applications. CONCLUSIONS: Reported results show that superpulsed laser irradiation, like the continuous and pulsed counterparts, possesses osteogenic properties, inducing the expression of molecules known to be important mediators of bone formation and, as a consequence, increasing calcium deposits in human MG-63 cells. Moreover, the data suggest a new potential role for PPARgamma as a regulator of osteoblast proliferation.

Effects of Smads and BMPs induced by Ga-Al-As laser irradiation on calcification ability of human dental pulp cells.

We investigated the effects of Ga-Al-As laser irradiation on the mineralization ability of human dental pulp (HDP) cells and on Smads and bone morphogenetic protein (BMP) production as one mechanism for the transmission of laser photochemical energy to cells. HDP cells in vitro were irradiated once with a Ga-Al-As laser at 1.0 W for 500 s, and calcified nodule formation was assessed by Alizarin red S staining. The laser irradiation was greater in the laser-irradiated group than in the non-irradiated group. Both calcium production and alkaline phosphatase (ALP) activity were higher after laser irradiation. Expression of mRNAs for Smad1, Smad7, BMPs, ALP, and osteocalcin was greater after laser irradiation, whereas expression of Smad6 mRNA was inhibited. Production of BMP-2 and BMP-4 in conditioned medium was also higher after laser irradiation. These results suggest that Smads and BMPs play important roles in ALP activity and calcification upon laser irradiation of HDP cells.

BMP-7 and CBFA1 in allograft bone in vivo bone formation and the influence of gamma-irradiation.

An initial study showed that morselized human bone grafts were osteoconductive and osteoinductive when implanted in nude rat tibial window defects, and 25 kGy of gamma-irradiation significantly reduced those properties. The mechanism of the osteoinductivity and the influence of gamma-irradiation required further investigation. In this study we assessed the paraffin sections of seven morselized human bone grafts implanted into rat tibial defects for 3 weeks after being treated with 0, 15, or 25 kGy gamma-radiation respectively. Osteoclast-like cell counting and protein expressions of bone morphogenetic protein-7 (BMP-7), core binding factor alpha1 (CBFA1), and proliferating cell nuclear antigen (PCNA) were investigated and the positive signals were quantitatively analyzed. More new bone formation was observed in the 0 and 15 kGy groups compared with 25 kGy groups. The newly formed bones were found mainly from the intact cortex into the defects bridged by the implanted grafts. A dense staining of BMP-7 and CBFA1 was noted in the osteoblast-like cells in those areas. The BMP-7 and CBFA1 staining was also seen in the cells surrounding the implanted grafts in the centre areas of the defects in distance from the intact cortex. Quantitative analysis of immunohistochemical staining of the centre areas of the defects showed that gamma-irradiation (15 and 25 kGy) significantly reduced the expression of CBFA1 and BMP-7. In conclusion, morselized human bone grafts may contain some factors, which induced osteoblast lineage differentiation and bone formation and gamma-irradiation damages those bone inducing factors.

Effect of 650 nm low-power laser on bone morphogenetic protein in bone defects induced in rat femors.

PURPOSE: To investigate the influence of 650 nm GaAlAs laser on the action of bone morphogenetic protein (BMP) in bone defects produced in rat femurs. METHODS: The sample consisted of 12 male albino Wistar rats (Rattus norvegicus). The animals were randomly divided into four experimental groups. After undergoing anesthesia, the fur was removed from the lateral face of the right thigh and surgical dissection was performed to view the femur region. A bone defect was created using a spherical diamond-tipped drill bit. In groups 1 and 2, the defect was filled with a paste of Gen-Tech bone-inducing substance. The animals were treated with GaAlAs laser, at a predetermined dose of joules/cm(2) for 80 seconds, over an area of 1 cm(2). Groups 2 and 4 were used as controls. Bone samples were removed to perform histological procedures and morphometric analyses on the 7th, 14th and 21st days after the operation. The results obtained were subjected to statistical analysis using ANOVA variance according to two criteria, with four repetitions, followed by the post hoc t test. The rejection level for the nullity hypothesis was 0.05 or 5% (alpha < or = 0.05). RESULTS: In comparisons between G1, G2, G3 and G4, p = 0.024 was observed. In statistical comparisons using the t test for paired samples, only G1 vs. G4 presented a statistically significant result (p = 0.021). CONCLUSION: The association of low-power laser application and Gen-Tech bone-inducing substance achieved a better result than laser application alone or BMP use alone.

Effect of 650 nm low-power laser on bone morphogenetic protein in bone defects induced in rat femors

PURPOSE:To investigate the influence of 650 nm GaAlAs laser on the action of bone morphogenetic protein (BMP) in bone defects produced in rat femurs. METHODS: The sample consisted of 12 male albino Wistar rats (Rattus norvegicus). The animals were randomly divided into four experimental groups. After undergoing anesthesia, the fur was removed from the lateral face of the right thigh and surgical dissection was performed to view the femur region. A bone defect was created using a spherical diamond-tipped drill bit. In groups 1 and 2, the defect was filled with a paste of Gen-Tech bone-inducing substance. The animals were treated with GaAlAs laser, at a predetermined dose of joules/cm² for 80 seconds, over an area of 1 cm². Groups 2 and 4 were used as controls. Bone samples were removed to perform histological procedures and morphometric analyses on the 7th, 14th and 21st days after the operation. The results obtained were subjected to statistical analysis using ANOVA variance according to two criteria, with four repetitions, followed by the post hoc t test. The rejection level for the nullity hypothesis was 0.05 or 5 percent (alpha <= 0.05). RESULTS: In comparisons between G1, G2, G3 and G4, p = 0.024 was observed. In statistical comparisons using the t test for paired samples, only G1 vs. G4 presented a statistically significant result (p = 0.021). CONCLUSION: The association of low-power laser application and Gen-Tech bone-inducing substance achieved a better result than laser application alone or BMP use alone.

OBJETIVO: Este trabalho tem como objetivo estudar comparativamente a influência do laser AsGaAl de 650nm sobre a ação das proteínas morfogenéticas ósseas( BMP) em defeitos ósseos produzidas em fêmures de ratos. METODOLOGIA: Utilizamos uma amostra composta por 12 ratos machos (Rattus norvegicus), de linhagem Wistar albino, mantidos confinados em caixas com temperatura ambiente constante e iluminação adequada. Os animais foram divididos aleatoriamente em 4 grupos experimentais. Após o procedimento anestésico, foi realizada a retirada dos pelos da face lateral da coxa direita, seguida de procedimento cirúrgico para permitir a visualização da região. Foi realizado um defeito ósseo empregando brocas diamantadas do tipo esférica. Nos grupos 1 e 2 o defeito foi preenchido com uma pasta formada por substância osteoindutora Gen - Tech. Os animais foram tratados com o Laser GaAlAs, com dose pré-determinada de 4 joules/cm² e tempo de 80 segundos para uma área de 1cm². Os grupos 2 e 4 foram adotados como grupo-controle. As amostras do osso foram retiradas para realização de procedimento histológico e análise morfométrica nos 7°,14°e 21° dias de pós-operatório. Os resultados obtidos foram submetidos à análise estatística pela variância ANOVA segundo dois critérios, com quatro repetições. Seguido do post hoc test de t teste, com nível de rejeição da hipótese de nulidade de 0,05 ou 5 por cento (alfa <= 0,05). RESULTADOS: Na comparação entre G1, G2, G3 e G4, observou-se: (P = 0,024) e durante a comparação estatística através do teste "t" para amostras pareadas apenas o cruzamento entre Grupo 1 vs. o Grupo 4 apresentou, resultado estatisticamente significante com (p=0,021). CONCLUSÃO: Concluímos que a associação da aplicação do laser de baixa potência e a substância osteoindutora Gen Tech alcançou melhor resultado do que do que a aplicação de laser ou apenas o uso das BMPs.

Effect of gamma irradiation on the osteoinductivity of morphogenetic protein extract from reindeer bone.

BACKGROUND: Bone morphogenetic proteins (BMPs), which are capable of stimulating the production of new bone, must be sterilized before preclinical and clinical use to reduce the risk of infections and associated complications. In this study, we investigated the effects of gamma sterilization on the osteoinductivity of native reindeer BMP extract in the Balb/C mouse thigh muscle pouch model. METHODS: 5 mg of native reindeer BMP extract and 5 mg of bovine serum albumin were administered separately either in gelatine capsules or mixed with gelatine as injections. The dose of gamma irradiation was 4.1 Mrad. Unsterile capsules and injections served as controls. New bone formation was evaluated based on the incorporation of Ca45 and also radiographically 3 weeks after implantation. RESULTS: Albumin-containing implants and injections did not induce new bone formation, as monitored in radiographs. Gamma sterilization did not reduce the osteoinductivity of native BMP extract in capsules, but a significant decrease in osteoinductivity--measured as area (50%) and Ca45 incorporation of new bone (27%)--was seen after injection. Gamma sterilization had no effect on the optical density of new bone induced by native BMP extract administered in capsules or by injection. INTERPRETATION: We conclude that, as gamma irradiation did not reduce the osteoinductivity of reindeer BMP extract in gelatine capsules, this method appears to be suitable for sterilization of BMPs to be given in capsule form. Native reindeer BMP extract was more sensitive to irradiation in soluble collagen (gelatine) than BMP in gelatine capsules. This finding must be given serious consideration regarding treatment of patients, but the remaining activity may be sufficient for the induction of bone formation in preclinical and clinical situations.

Gamma irradiation and ethylene oxide in the sterilization of native reindeer bone morphogenetic protein extract.

BACKGROUND AND AIMS: For human use, it is necessary to sterilize bone morphogenetic proteins (BMPs), in order to reduce the risk of infections and associated complications. We compared the effects of ethylene oxide and gamma irradiation in the sterilization of native reindeer BMP extract with regard to bone induction in the Balb/C mouse thigh muscle pouch model. MATERIALS AND METHODS: BMP extract, sterilized with ethylene oxide gas (Steri-Vac 4XL, temperature 29 degrees C, exposure time 4 h, ethylene oxide concentration 860 mg/l), or gamma irradiation at doses of 3.15 MRad was administered in implants containing 5 or 10 mg of BMP extract with collagen carrier. Non-sterilized collagen implants served as controls. New bone formation was evaluated based on the incorporation of Ca45 and radiographically three weeks after implantation. RESULTS: The collagen was not able to induce new bone visible in radiographs. The mean Ca45 incorporation in the gamma sterilized group containing 5 mg of BMP extract was 30% (p = 0.04) and that containing 10 mg of BMP extract was 60% (p = 0.02) higher than seen in the corresponding ethylene oxide sterilized groups. The mean new bone areas were 45% higher in the gamma sterilized groups than in the corresponding ethylene oxide sterilized groups, but the differences were not significant. The mean optical density of new bone in the gamma sterilized group containing 5 mg of BMP extract was 75% (p = 0.00) and in that containing 10 mg of BMP extract was 70% (p = 0.00) higher than seen in the corresponding ethylene oxide sterilized groups. CONCLUSION: Native reindeer BMP extract is more sensitive to the effects of ethylene oxide gas sterilization than gamma irradiation. These results suggest that gamma irradiation is recommendable for the sterilization of BMP extracts.

[The effect of static magnetic field on bone morphogenetic protein-2 in periodontal membrane of the rat].

PURPOSE: The purpose of this study is to evaluate the effect of static magnetic field on the expression of BMP-2 in the periodontal membrane of rat. METHODS: 35 female rats of wistar were randomly divided into three groups, which were the normal group, experimental control group and experimental group. The experimental rat's cheeks were put into magnet with the intensity of magnetic field being 0.12 tesla. The rats were sacrificed at 2 days, 4 days, 7 days, respectively. Immunohistochemical assays were used to evaluate the changes of BMP-2 expression in the periodontal membrane of the maxillary molars. RESULTS: BMP-2 was mainly found in the plasma of fibroblast, osteoblast, cementocyte, odontoblast separately. There were more BMP-2 in the experimental group treated with static magnetic field. There were significant differences between the experiment group of 4 day and 7 day and the control group. CONCLUSIONS: Static magnetic field stimulated BMP-2 secreted by the cell of periodontal membrane and played an important role in repairing and remodeling of periodontitis.

Long-term evaluation of bone formation by osteogenic protein 1 in the baboon and relative efficacy of bone-derived bone morphogenetic proteins delivered by irradiated xenogeneic collagenous matrices.

To investigate the long-term efficacy of irradiated recombinant human osteogenic protein 1 (hOP-1) in bone regeneration and morphogenesis, hOP-1 was combined with a bovine collagenous matrix carrier (0, 0.1, 0.5, and 2.5 mg hOP-1/g of matrix), sterilized with 2.5 Mrads of y-irradiation, and implanted in 80 calvarial defects in 20 adult baboons (Papio ursinus). The relative efficacy of partially purified bone-derived baboon bone morphogenetic proteins (BMPs), known to contain several osteogenic proteins, was compared with the recombinant hOP-1 device in an additional four baboons. Histology and histomorphometry on serial undecalcified sections prepared from the specimens harvested on day 90 and day 365 showed that gamma-irradiated hOP-1 devices induced regeneration of the calvarial defects by day 90, although with reduced bone area compared with a previous published series of calvarial defects treated with nonirradiated hOP-1 devices. One year after application of the irradiated hOP-1 devices, bone and osteoid volumes and generated bone tissue areas were comparable with nonirradiated hOP-1 specimens. Moreover, 365 days after healing regenerates induced by 0.5 mg and 2.5 mg of irradiated hOP-1 devices showed greater amounts of bone and osteoid volumes when compared with those induced by nonirradiated hOP-1 devices. On day 90, defects treated with 0.1 mg and 0.5 mg of bone-derived baboon BMPs, combined with irradiated matrix, showed significantly less bone compared with defects receiving irradiated devices containing 0.1 mg and 0.5 mg hOP-1; 2.5 mg of partially purified BMPs induced bone and osteoid volumes comparable with the 0.1-mg and 0.5-mg hOP-1 devices. Control specimens of y-irradiated collagenous matrix without hOP-1 displayed a nearly 2-fold reduction in osteoconductive bone repair when compared with nonirradiated controls. These findings suggest that the reduction in bone volume and bone tissue area on day 90 may be caused by a reduced performance of the irradiated collagenous matrix substratum rather than to a reduction in the biological activity of the irradiated recombinant osteogenic protein. This is supported by the results of in vitro and in vivo studies performed to determine the structural integrity of the recovered gamma-irradiated hOP-1 before application in the baboon. Recoveries by high-performance liquid chromatography (HPLC) and sodium dodecyl sulfate/ polyacrylamide gel electrophoresis (SDS/PAGE)/immunoblot analyses indicated that doses of 2.5-3 Mrads of gamma-irradiation did not significantly affect the structural integrity of the recovered hOP-1. Biological activity of the recovered hOP-1 was confirmed in vitro by showing induction of alkaline phosphatase activity in rat osteosarcoma cells (ROS) and in vivo by de novo endochondral bone formation in the subcutaneous space of the rat. These findings in the adult primate indicate that a single application of gamma-irradiated hOP-1 combined with the irradiated xenogeneic bovine collagenous matrix carrier is effective in regenerating and maintaining the architecture of the induced bone at doses of 0.5 mg/g and 2.5 mg/g of carrier matrix.