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P.418 Dental pulp cells for facial nerve regeneration
Posters mucosa. The cerebrospinal fluid leak was identified and the dural defect oversewn. The encephalocele was reduced. The bone defect was closed with pericranium, a split calvarial graft, bone morphogenic protein (BMP-2) and fibrin gel (Tisseal). A followup CT demonstrated good reduction of the encephalocele. The transpalatal approach is safe and reliable for the treatment of transsphenoidal encephaloceles in young children.
Tissue engineering and cell therapy
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removing of the bone graft. Rapid bone regeneration demonstrated in radiographs was seen in patients treated with CG. 6 months after the procedure the bone structure in the place of the graft was no different than the surrounding tissue. In the control group the similar effect was achieved after 12 months. In patients with healing complications, the radiological bone regeneration was delayed. Conclusion: Composite grafts of frozen allogenic bone suplemented with autogenic bone marrow have exellent capacities accelerating bone healing.
Tissue engineering and cell therapy P.415 Bone formation with immobilized rhBMP-2 E. Yamachika, H. Tsujigiwa, M. Matsubara, Y. Kaneda, N. Shirasu, T. Ueno, N.I. Mizukawa. Okayama University, Okayama city, Japan Objectives: Bone morphogenetic protein (BMP)-2 plays an important role in bone growth and regeneration; however, BMP2 is easily lost by diffusion through body fluid and has some inhibitory pathways. To address this problem, we previously immobilized recombinant human BMP-2 (rhBMP-2) on succinylated type I atelocollagen. Here, we examined the effect of immobilized rhBMP-2 in vitro and vivo. Method: ST2, MC3T3-E1 and C2C12 cells were cultured for this study. For the preparation of immobilized rhBMP-2, rhBMP2 was mixed with succinylated type I atelocollagen including water-soluble carbodiimide (WSC) and stored for 24 h. Then the mixture was dialyzed for 24 h against pH 3.0 hydrochloric acid to remove unreacted WSC. Gene expression for alkaline phosphatase, type I collagen, osteopontin and osteocalcin was measured by quantitative reverse transcription polymerase chain reaction and cell signaling of phosphorylated receptor-activated Smads was detected by western blot analysis. In addition rhBMP2 implants were implanted into the backs of rats and removed implants were observed with a light microscope. Results: In ST2, MC3T3-E1, and C2C12 cells, alkaline phosphatase activity, which is a marker of osteoblast differentiation, was enhanced more by immobilized than nonimmobilized rhBMP-2. In addition, the phosphorylation of receptor-activated Smads, part of the signaling pathway activated by BMP-2, was prolonged by immobilized rhBMP-2 in these cells. Furthermore, implantation of immobilized rhBMP-2 into the backs of rats promoted the formation of mature bone-like structure. Conclusions: These results demonstrate that immobilized rhBMP-2 has higher bioactivity than nonimmobilized rhBMP2, and, therefore, immobilization of rhBMP-2 can prolong BMP signaling. P.416 Composite grafts in treatment of postcystic jaw bones lesion T. Pietka, W. Domanski, J. Przybysz, B. Biernacka, J. Dabrowski, G. Krzymanski. Military Institute of Health Services, Warsaw, Poland Objective: To assess the influence of composite grafts of frozen allogenic bone and autogenic bone marrow (CG) on the healing process rate of the jaw bones lesions. Methods: The study included 42 patients (9 women) with large postcystic bone defects treated with CG. The healing process rate assessed on the base of radiological examinations (mandibular pantomograms) taken immediately and 3, 6 and 12 months after the procedure. The results were compared with the spontaneous healing process rates of the historical group (45 patients, 15 women) from our previous study (control group). Results: 34 postoperative wounds healed without complications. There were transient incidents of wound dehiscence with partial excretion of the grafted bone in 8 patients. In such cases healing of the wounds was prolonged but in any case resulted in the
P.417 Culture of human osteoblasts on two different scaffolds L. Gallego, L. Junquera, A. Meana, I. Pe˜na, P. Rosado. University central Hospital, Oviedo, Spain Objectives: This study investigates the possibility of developing a bone-like tissue in vitro, by using mandibular osteoblasts that retained their osteoblastic phenotype and mineralization capacity in two different scaffolds. Histological, immunohistochemical and histomorphometric analysis of the cultures were made. Methods: Samples of mandibular bone were obtained during oral surgery. Osteoblast cells were cultured in two different scaffolds; a large particle mineralized cancellous allograft (scaffold 1) and a non calcified protein scaffold prepared with plasmatic albumin (scaffold 2). Measurement of the differentiation marker alkaline phosphatase and histologic examination was made after 30 days of incubation. The cultures were also evaluated with scanning electron microscopy (SEM) and histomorphometry. Results: Cultured osteoblasts showed comparable phenotypic profiles, and expressed alkaline phosphatase in both scaffolds. Hematoxylin-eosin staining revealed a bone-like extracellular matrix in scaffold 1 and mineralization of osteoblasts cultured in scaffold 2 was confirmed by von Kossa staining. In SEM examination cells formed multicellular layers around the graft material. Deposition of mineral and collagen contents was examined in different zones of cultured tissue of both scaffolds. Histomorphometry showed peaks of calcium in cultured material. Conclusion: Osteoblasts were able to proliferate in vitro and synthesize a bone-like extracellular matrix and mineralization. The results indicate that both scaffolds are a favorable substrate for the growth and differentiation of osteoblasts and a promising material for bone tissue engineering and repair of bone defects.
P.418 Dental pulp cells for facial nerve regeneration R. Sasaki1,2 , S. Aoki1,3 , M. Yamato2 , H. Uchiyama1 , H. Ogiuchi1 , K. Wada4 , T. Okano2 , T. Ando1 . 1 Department of Oral and Maxillofacial Surgery, Tokyo Women’s Medical University, School of Medicine, Japan; 2 Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University, Japan; 3 Department of Bioscience and Bioinformatics, Faculty of Computer Science and Systems Engineering, Kyushu Institute of Technology, Japan; 4 Department of Degenerative Neurological Diseases, National Institute of Neuroscience, NCNP, Japan Objective: Nerve autografting is the most common surgical procedure currently used for repairing facial nerve gaps caused by traffic accidents or tumor resections. A number of recent studies have shown the effectiveness of tubulation as an alternative therapy to that of nerve autografting. For large peripheral nerve gaps, tubulation alone is limited to nerve regeneration. It has been reported that tubulation using brain-derived neural progenitor cells or Schwann cells promotes nerve regeneration. However, the use of these neural cells from other neural donor tissues has potentially serious clinical complications. Therefore, we focused on the use of dental pulp as a new cell source for such artificial conditions.
S272
Journal of Cranio-Maxillofacial Surgery 36(2008) Suppl. 1
Methods: A silicone tube containing collagen gel embedded with or without rat dental pulp cells was transplanted into the gap of the rat facial nerve, and functional nerve regeneration was examined. Results: Twelve days after transplantation, defective facial nerves connected with silicone tubes containing dental pulp cells were repaired more rapidly than control tubes containing the collagen gel alone. When a tube containing green fluorescent protein (GFP)-positive dental pulp cells was transplanted into a facial nerve gap in a GFP-negative rat, we observed regenerated nerves with GFP-positive cells at 2 weeks post-transplantation. The regenerated nerves included Tuj1-positive axons, RECA1 and GFP double-positive blood vessels and S100 and GFP doublepositive myelin-like structures. Conclusion: The transplanted dental pulp cells formed blood vessels and myelinating tissue and contributed to the promotion of nerve regeneration. P.419 Dura mater: anatomy, embryology and clinical implications M. Carstens, M.S. Curtis. Saint Louis University, Saint Louis, USA Objectives: The dura mater is implicated in a variety of disease processes, including migraine headaches. The aim of this article is to revisit the anatomy and embryology of the dura mater, and use neuromeric theory to explain the pathophysiology and symptomatology of related clinical entities. Methods: An extensive literature search from 1940 to the present was performed utilizing the MEDLINE database. Basic science and clinical reference texts were also used in data collection. Results: The dura mater is derived from neural crest cells and is composed of two layers with different functional purposes. Dural reflections signify transition points in the vascularity and nerve supply of the dura. The vascular supply of the dura originates from the internal and external carotid arteries. The innervation of the supratentorial dura is primarily served by the trigeminal nerve, while the posterior cranial fossa draws its nerve supply primarily from the upper cervical nerves. The neuromeric theory explains these differences and can be applied with our knowledge of embryology to explain clinical phenomena and rationales for treatment. Conclusions: This review of the dura mater brings to light the form and function of the dura and the relationship between its embryologic origins and clinical implications in numerous areas of medicine, including the fields of headache, congenital malformations, and craniofacial deformities. P.420 Ectopic chondroosteoid tissue formed by tricalcium phosphate O. Moztarzadeh, D. Hrusak, P. Andrle, L. Hauer, L. Hosticka. University Hospital and Medical Faculty Pilsen, Charles University Prague, Pilsen, Czech Republic Beta-Tricalcium Phosphate ceramics are synthetic bone graft substitutes, widely used for the treatment of bone defects due to their osteoconductive properties. Although, these materials are not considered as osteoinductive, there are suggestions that they are. The aim of this study was to evaluate in vivo the possible osteoinduction potential of two different bone augmentation materials and if ectopic bone formation could be induced when implanted subcutaneously to the extremities of the pigs. The experiment was performed under general anaesthesia, using aseptic techniques. Two bone augmentation materials: microporous granules (Ø 500–1000 mm) of pure phase b-tricalcium phosphate ceramic Cerasorb® and granular mix (Ø 0.5−1 mm) derived from equine bone BIO-GEN® were applied at the ulnar region between muscular and cutaneus tissue. We performed two intramuscular applications of the antibiotic Tetracycline at 16 and
Abstracts, EACMFS XIX Congress 40 days after operation. Tetracycline is deposited where bone or cartilage matrix is mineralizing and can therefore demonstrate regions of active bone formation and mineralization. After 52 days the pigs were terminated. At the site of application of Cerasorb some hard tissue material was found (none at BIOGEN® ). The specimens were observed by optical and confocal laser microscopes after histological processing. The observation revealed incompletely mineralized osteoid material and layers of chondroid tissue mass. Bright fluorescent zones showed the deposition of tetracycline-induced fluorescence limited to area of active hard tissues formation. Thus, it is possible to use this methodology in soft tissues, with the objective to verify the osteoinduction potential of different bone augmentation materials. P.421 Effect of Nd:YAG laser on the in vitro osteogenesis S. Hwang, S.S. Lim, I.S. Kim, T.H. Cho, Y.M. Song, K.S. Kim. Dept. of Oral and Maxillofacial Surgery, Seoul National University Dental Hospital, Seoul, Republic of Korea Background: Recently, lasers have been studied for their effects on bone fracture healing, bone nodule formation, and osteoblast differentiation. This study aimed to investigate the effect of Nd:YAG laser on the in vitro osteogenesis of human bone marrow mesenchymal stromal cells (hBMSCs) and the bone regeneration using rat calvarial defects. Methods: hBMSCs and rat calvarial defects were irradiated with a Nd:YAG laser which has a wavelength of 1064 nm, a pulse energy of 0.75 W, and a pulse repetition rate of 15 Hz. Energy settings were varied with power densities of 3.5, 7, and 15 J/cm2 for hBMSCs, and a power density of 344 J/cm2 was applied for rat calvarial defects. After laser irradiation, cell proliferation was evaluated using MTT assay at day 3 and 6. The in vitro osteogenesis of hBMSCs was investigated by asessing alkaline phosphatase (ALP) activity, the mineral deposition, and the secreted amount of cytokines such as VEGF and nitic oxide (NO) using ELISA. For animal experiments, irradiation was applied one times (90 seconds) every two days for 2 weeks and bone formation was evaluated at day 14 after irradiation using soft X-ray. Results: ND:YAG laser stimulated cell proliferation, ALP activity and the mineral deposition in osteoblast differentiation of hBMSCs. This stimulation also induced VEGF and NO production. The laser effect was maximized at a power density of 15 J/cm2 and less effective at lower levels under 10 J/cm2 . In vivo bone formation was significantly increased in the irradiated group of rat calvarial defects. Conclusions: This study demonstrated that Nd:YAG laser is very efficient in osteoblast differentiation of hBMSCs and bone regeneration of rat calvarial defect, reflecting a prominent alternative for osteoinductive therapy. P.422 Impact of DMSO on osteoblast viability and differentiation T. Reuther, U. Muller-Richter, U. Klammert, I. Reuther, M. Kochel, H. Grimaldi, A.C. Kubler. Oral and Maxillofacial Surgery, University of Wuerzburg, Wuerzburg, Germany The aim of this study was to compare the viability of human osteoblasts cryopreserved with DMSO to those of fresh human iliac cancellous bone using cell culture techniques. Osteoblasts were obtained by spontaneous outgrowth of human iliac cancellous bone specimens in experiment I. In experiment II, human iliac cancellous bone was frozen with 10% DMSO at −80ºC for 2 weeks and osteoblasts grew spontaneously after thawing at 37ºC by removing DMSO with sucrose. The cells were grown in culture flasks containing DMEM as a culture medium, supplemented with 10% fetal calf serum. They were kept at 37ºC in a humidified atmosphere of 95% air and 5% CO2. Cells from the second passage were plated at a density of 5×10 cells/cm2
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removing of the bone graft. Rapid bone regeneration demonstrated in radiographs was seen in patients treated with CG. 6 months after the procedure the bone structure in the place of the graft was no different than the surrounding tissue. In the control group the similar effect was achieved after 12 months. In patients with healing complications, the radiological bone regeneration was delayed. Conclusion: Composite grafts of frozen allogenic bone suplemented with autogenic bone marrow have exellent capacities accelerating bone healing.
Tissue engineering and cell therapy P.415 Bone formation with immobilized rhBMP-2 E. Yamachika, H. Tsujigiwa, M. Matsubara, Y. Kaneda, N. Shirasu, T. Ueno, N.I. Mizukawa. Okayama University, Okayama city, Japan Objectives: Bone morphogenetic protein (BMP)-2 plays an important role in bone growth and regeneration; however, BMP2 is easily lost by diffusion through body fluid and has some inhibitory pathways. To address this problem, we previously immobilized recombinant human BMP-2 (rhBMP-2) on succinylated type I atelocollagen. Here, we examined the effect of immobilized rhBMP-2 in vitro and vivo. Method: ST2, MC3T3-E1 and C2C12 cells were cultured for this study. For the preparation of immobilized rhBMP-2, rhBMP2 was mixed with succinylated type I atelocollagen including water-soluble carbodiimide (WSC) and stored for 24 h. Then the mixture was dialyzed for 24 h against pH 3.0 hydrochloric acid to remove unreacted WSC. Gene expression for alkaline phosphatase, type I collagen, osteopontin and osteocalcin was measured by quantitative reverse transcription polymerase chain reaction and cell signaling of phosphorylated receptor-activated Smads was detected by western blot analysis. In addition rhBMP2 implants were implanted into the backs of rats and removed implants were observed with a light microscope. Results: In ST2, MC3T3-E1, and C2C12 cells, alkaline phosphatase activity, which is a marker of osteoblast differentiation, was enhanced more by immobilized than nonimmobilized rhBMP-2. In addition, the phosphorylation of receptor-activated Smads, part of the signaling pathway activated by BMP-2, was prolonged by immobilized rhBMP-2 in these cells. Furthermore, implantation of immobilized rhBMP-2 into the backs of rats promoted the formation of mature bone-like structure. Conclusions: These results demonstrate that immobilized rhBMP-2 has higher bioactivity than nonimmobilized rhBMP2, and, therefore, immobilization of rhBMP-2 can prolong BMP signaling. P.416 Composite grafts in treatment of postcystic jaw bones lesion T. Pietka, W. Domanski, J. Przybysz, B. Biernacka, J. Dabrowski, G. Krzymanski. Military Institute of Health Services, Warsaw, Poland Objective: To assess the influence of composite grafts of frozen allogenic bone and autogenic bone marrow (CG) on the healing process rate of the jaw bones lesions. Methods: The study included 42 patients (9 women) with large postcystic bone defects treated with CG. The healing process rate assessed on the base of radiological examinations (mandibular pantomograms) taken immediately and 3, 6 and 12 months after the procedure. The results were compared with the spontaneous healing process rates of the historical group (45 patients, 15 women) from our previous study (control group). Results: 34 postoperative wounds healed without complications. There were transient incidents of wound dehiscence with partial excretion of the grafted bone in 8 patients. In such cases healing of the wounds was prolonged but in any case resulted in the
P.417 Culture of human osteoblasts on two different scaffolds L. Gallego, L. Junquera, A. Meana, I. Pe˜na, P. Rosado. University central Hospital, Oviedo, Spain Objectives: This study investigates the possibility of developing a bone-like tissue in vitro, by using mandibular osteoblasts that retained their osteoblastic phenotype and mineralization capacity in two different scaffolds. Histological, immunohistochemical and histomorphometric analysis of the cultures were made. Methods: Samples of mandibular bone were obtained during oral surgery. Osteoblast cells were cultured in two different scaffolds; a large particle mineralized cancellous allograft (scaffold 1) and a non calcified protein scaffold prepared with plasmatic albumin (scaffold 2). Measurement of the differentiation marker alkaline phosphatase and histologic examination was made after 30 days of incubation. The cultures were also evaluated with scanning electron microscopy (SEM) and histomorphometry. Results: Cultured osteoblasts showed comparable phenotypic profiles, and expressed alkaline phosphatase in both scaffolds. Hematoxylin-eosin staining revealed a bone-like extracellular matrix in scaffold 1 and mineralization of osteoblasts cultured in scaffold 2 was confirmed by von Kossa staining. In SEM examination cells formed multicellular layers around the graft material. Deposition of mineral and collagen contents was examined in different zones of cultured tissue of both scaffolds. Histomorphometry showed peaks of calcium in cultured material. Conclusion: Osteoblasts were able to proliferate in vitro and synthesize a bone-like extracellular matrix and mineralization. The results indicate that both scaffolds are a favorable substrate for the growth and differentiation of osteoblasts and a promising material for bone tissue engineering and repair of bone defects.
P.418 Dental pulp cells for facial nerve regeneration R. Sasaki1,2 , S. Aoki1,3 , M. Yamato2 , H. Uchiyama1 , H. Ogiuchi1 , K. Wada4 , T. Okano2 , T. Ando1 . 1 Department of Oral and Maxillofacial Surgery, Tokyo Women’s Medical University, School of Medicine, Japan; 2 Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University, Japan; 3 Department of Bioscience and Bioinformatics, Faculty of Computer Science and Systems Engineering, Kyushu Institute of Technology, Japan; 4 Department of Degenerative Neurological Diseases, National Institute of Neuroscience, NCNP, Japan Objective: Nerve autografting is the most common surgical procedure currently used for repairing facial nerve gaps caused by traffic accidents or tumor resections. A number of recent studies have shown the effectiveness of tubulation as an alternative therapy to that of nerve autografting. For large peripheral nerve gaps, tubulation alone is limited to nerve regeneration. It has been reported that tubulation using brain-derived neural progenitor cells or Schwann cells promotes nerve regeneration. However, the use of these neural cells from other neural donor tissues has potentially serious clinical complications. Therefore, we focused on the use of dental pulp as a new cell source for such artificial conditions.
S272
Journal of Cranio-Maxillofacial Surgery 36(2008) Suppl. 1
Methods: A silicone tube containing collagen gel embedded with or without rat dental pulp cells was transplanted into the gap of the rat facial nerve, and functional nerve regeneration was examined. Results: Twelve days after transplantation, defective facial nerves connected with silicone tubes containing dental pulp cells were repaired more rapidly than control tubes containing the collagen gel alone. When a tube containing green fluorescent protein (GFP)-positive dental pulp cells was transplanted into a facial nerve gap in a GFP-negative rat, we observed regenerated nerves with GFP-positive cells at 2 weeks post-transplantation. The regenerated nerves included Tuj1-positive axons, RECA1 and GFP double-positive blood vessels and S100 and GFP doublepositive myelin-like structures. Conclusion: The transplanted dental pulp cells formed blood vessels and myelinating tissue and contributed to the promotion of nerve regeneration. P.419 Dura mater: anatomy, embryology and clinical implications M. Carstens, M.S. Curtis. Saint Louis University, Saint Louis, USA Objectives: The dura mater is implicated in a variety of disease processes, including migraine headaches. The aim of this article is to revisit the anatomy and embryology of the dura mater, and use neuromeric theory to explain the pathophysiology and symptomatology of related clinical entities. Methods: An extensive literature search from 1940 to the present was performed utilizing the MEDLINE database. Basic science and clinical reference texts were also used in data collection. Results: The dura mater is derived from neural crest cells and is composed of two layers with different functional purposes. Dural reflections signify transition points in the vascularity and nerve supply of the dura. The vascular supply of the dura originates from the internal and external carotid arteries. The innervation of the supratentorial dura is primarily served by the trigeminal nerve, while the posterior cranial fossa draws its nerve supply primarily from the upper cervical nerves. The neuromeric theory explains these differences and can be applied with our knowledge of embryology to explain clinical phenomena and rationales for treatment. Conclusions: This review of the dura mater brings to light the form and function of the dura and the relationship between its embryologic origins and clinical implications in numerous areas of medicine, including the fields of headache, congenital malformations, and craniofacial deformities. P.420 Ectopic chondroosteoid tissue formed by tricalcium phosphate O. Moztarzadeh, D. Hrusak, P. Andrle, L. Hauer, L. Hosticka. University Hospital and Medical Faculty Pilsen, Charles University Prague, Pilsen, Czech Republic Beta-Tricalcium Phosphate ceramics are synthetic bone graft substitutes, widely used for the treatment of bone defects due to their osteoconductive properties. Although, these materials are not considered as osteoinductive, there are suggestions that they are. The aim of this study was to evaluate in vivo the possible osteoinduction potential of two different bone augmentation materials and if ectopic bone formation could be induced when implanted subcutaneously to the extremities of the pigs. The experiment was performed under general anaesthesia, using aseptic techniques. Two bone augmentation materials: microporous granules (Ø 500–1000 mm) of pure phase b-tricalcium phosphate ceramic Cerasorb® and granular mix (Ø 0.5−1 mm) derived from equine bone BIO-GEN® were applied at the ulnar region between muscular and cutaneus tissue. We performed two intramuscular applications of the antibiotic Tetracycline at 16 and
Abstracts, EACMFS XIX Congress 40 days after operation. Tetracycline is deposited where bone or cartilage matrix is mineralizing and can therefore demonstrate regions of active bone formation and mineralization. After 52 days the pigs were terminated. At the site of application of Cerasorb some hard tissue material was found (none at BIOGEN® ). The specimens were observed by optical and confocal laser microscopes after histological processing. The observation revealed incompletely mineralized osteoid material and layers of chondroid tissue mass. Bright fluorescent zones showed the deposition of tetracycline-induced fluorescence limited to area of active hard tissues formation. Thus, it is possible to use this methodology in soft tissues, with the objective to verify the osteoinduction potential of different bone augmentation materials. P.421 Effect of Nd:YAG laser on the in vitro osteogenesis S. Hwang, S.S. Lim, I.S. Kim, T.H. Cho, Y.M. Song, K.S. Kim. Dept. of Oral and Maxillofacial Surgery, Seoul National University Dental Hospital, Seoul, Republic of Korea Background: Recently, lasers have been studied for their effects on bone fracture healing, bone nodule formation, and osteoblast differentiation. This study aimed to investigate the effect of Nd:YAG laser on the in vitro osteogenesis of human bone marrow mesenchymal stromal cells (hBMSCs) and the bone regeneration using rat calvarial defects. Methods: hBMSCs and rat calvarial defects were irradiated with a Nd:YAG laser which has a wavelength of 1064 nm, a pulse energy of 0.75 W, and a pulse repetition rate of 15 Hz. Energy settings were varied with power densities of 3.5, 7, and 15 J/cm2 for hBMSCs, and a power density of 344 J/cm2 was applied for rat calvarial defects. After laser irradiation, cell proliferation was evaluated using MTT assay at day 3 and 6. The in vitro osteogenesis of hBMSCs was investigated by asessing alkaline phosphatase (ALP) activity, the mineral deposition, and the secreted amount of cytokines such as VEGF and nitic oxide (NO) using ELISA. For animal experiments, irradiation was applied one times (90 seconds) every two days for 2 weeks and bone formation was evaluated at day 14 after irradiation using soft X-ray. Results: ND:YAG laser stimulated cell proliferation, ALP activity and the mineral deposition in osteoblast differentiation of hBMSCs. This stimulation also induced VEGF and NO production. The laser effect was maximized at a power density of 15 J/cm2 and less effective at lower levels under 10 J/cm2 . In vivo bone formation was significantly increased in the irradiated group of rat calvarial defects. Conclusions: This study demonstrated that Nd:YAG laser is very efficient in osteoblast differentiation of hBMSCs and bone regeneration of rat calvarial defect, reflecting a prominent alternative for osteoinductive therapy. P.422 Impact of DMSO on osteoblast viability and differentiation T. Reuther, U. Muller-Richter, U. Klammert, I. Reuther, M. Kochel, H. Grimaldi, A.C. Kubler. Oral and Maxillofacial Surgery, University of Wuerzburg, Wuerzburg, Germany The aim of this study was to compare the viability of human osteoblasts cryopreserved with DMSO to those of fresh human iliac cancellous bone using cell culture techniques. Osteoblasts were obtained by spontaneous outgrowth of human iliac cancellous bone specimens in experiment I. In experiment II, human iliac cancellous bone was frozen with 10% DMSO at −80ºC for 2 weeks and osteoblasts grew spontaneously after thawing at 37ºC by removing DMSO with sucrose. The cells were grown in culture flasks containing DMEM as a culture medium, supplemented with 10% fetal calf serum. They were kept at 37ºC in a humidified atmosphere of 95% air and 5% CO2. Cells from the second passage were plated at a density of 5×10 cells/cm2