Nicotine of tobacco component has a controversial impact in the clinical outcome of dental implants. Although numerous nicotine effects on bone healing around implants have been presented, it is rarely reported in vitro study about normal human osteoblast(NHost) from oral and maxillofacial area at the surface of implants. The purpose of the present study was to evaluate the effect of nicotine on the proliferation and differentiation response of NHost to plasmatic and salivary levels of nicotine reported in smokers at the surface of screw-type plasma-sprayed titanium implants. NHosts were seeded on the surface of titanium implants and cultured for 21 days in α-MEM supplemented with 10% FBS, 50mg/ml ascorbic acid, 5mM β-glycerophosphate and 100nM dexamethasone. Seeded implants were exposed to various nicotine concentration(0.05-0.5mg/ml) from 1 to 21 days, and characterized for cell morphology, proliferation, differentiation, alkaline phosphatase(ALP) activity and ionized calcium concentration(Cai) of medium. Continuous exposure to higher nicotine concentration(above 0.3mg/ml) induced a dose- and time-dependent vacuolation of the cytoplasm, and a tendency to detach from the implant surface. 0.05mg/ml(lower nicotine concentration) did not cause significant effects in the cell proliferation and ALP activity. 0.1-0.2mg/ml caused evident dose-dependent effects in increased cell proliferation, ALP activity and earlier onset of matrix mineralization at levels up to 0.2mg/ml, while a dose-dependent inhibitory effect at 0.3-0.5mg/ml. Cai concentration of control group was decreased at 14 days. Increased Cai concentration at 0.1-0.2mg/ml, decreased Cai concentration at 0.3mg/ml and no change at 0.5mg/ml during the culture period were seen. It suggested that nicotine concentration could paly an role in modulating NHost activity as a contributing factor associated with proliferation and differentiation of NHost at the surface of implants.

Recently, extensive research has been performed in the field of orthopedic medicine to develop cell-based therapies for the restoration of injured bone tissue. But there has been rarely reported about rehabilitaton of oral and maxillofacial bone defect using self-derived osteoblasts. Normal human osteoblast cell(NHost) was previously established into marrow-derived human mesenchymal stem cells for their capacity to proliferate and differentiate into osteoblasts under various culture conditions. The purpose of this study was to examine proliferation and differentiation of NHosts effected by growth factors with ALP activity and RT-PCR. After NHosts were cultured under basal and osteogenic medium at 37℃ and 5% CO2, they were analyzed by ALP activity and RT-PCR. BMP-2 under osteogenic medium decreased growth rate of NHosts compared to under osteogenic medium. BMP-2 under osteogenic medium induced osteoblastic differentiation in NHosts by increased ALP activity. The differentiating capacity of NHosts under osteogenic medium showed that NHosts expressed higher mRNA expression levels of OSX and OCN, while that of RUNX2 decreased after BMP-2 treatment. It suggested that NHosts having characteristics of osteoprecursor cells might be more advanced in their osteogenesis development by BMP-2, making NHosts an interesting biological tool for treatment of skeletal defects and diseases of oral and maxillofacial bone.

Cyclosporin A(CsA) as immunosuppressive drug is used to prevent immune reactions after organ transplant. And also It is reported that the effect of CsA on osteoblast differentiation has been controversial according to dosage. The purpose of this study was to examine the effect of various CsA concentrations on osteoblast differentiation. According to different concentration o f CsA, growth curve, apoptosis index MTT assay, ALP activation and osteocalcin secretion, in cultured NHost were analyzed. Treating osteoblasts with low concentrations of CsA increased growth rate, MTT assay activity, ALP activation and osteocalcin protein levels in a dose-dependent manner, while high concentration showed opposite results. Therefore, these results showed that low concentrations of CsA increased osteoblast differentiation, while high concentrations elicited an opposite response, showing inhibition of CsA on osteoblast differentiation. It suggested that different CsA concentrations might play in regulating NHost differentiation, and its specific activation of lower concentration will represent a viable anabolic therapy for bone resorption disease in future.

Numerous bone cell culture models have been presented by the development of isolation and culturing techniques of cells. The culture of osteoblast-like cells of human origin with a specific osteoblastic phenotype has become an important experimental model in bone biology. Recently, it has become increasingly popular to utilize bone marrow cultures because these cultures are therefore thought to represent earlier stages of the osteoblast differentiation pathway. There is no report about culturing normal human osteoblast from oral and maxillofacial area. Primary cultured cells from oral and maxillofacial cancellous bone were analyzed by morphologic features, total DNA contents, ALP, osteocalcin and von Kossa staining positivity. The purpose of this study were to culture the cell population from oral and maxillofacial cancellous bone and to analyze the phenotypic expression of cultured normal human osteoblast by the bone marrow isolation technique. Growth curve of NHost showed about 45hrs of doubling time and about 70μ g/well of total DNA content. NHost showed spindle shaped cytoplasm with ovoid nucleus under preconfluency and after cellular differentiation, they formed irregular numerous nodules from stratified cellular layers under D medium. ALP activity was about 2 folds higher under control medium with 10nM 1,25(OH)2D3 than that under control medium. Osteocalcin expression was about seven folds higher under control medium with 100nM 1,25(OH)2D3 than that under control medium. Scattered mineralized nodules stained by von Kossa method were seen on the cellular layer under D medium. It suggested that NHost might be established from oral and maxillofacial area by characteristic cellular shape, ALP activity, osteocalcin expression and numerous mineralized nodules.

It is not yet clear to know how normal human osteoblasts(NHost) from oral and maxillofacial area deposit, stabilize, and configure their extracellular matrix on dental biomaterial surfaces. Therefore it is necessary to design biomaterials with improved biocompatibility that will promote earlier bone differentiation and mineralization. There is now increasing evidence that TGase 2 may play a role in the initiation and regulation of the mineralization processes and serves to cross-link osteocalcin and osteopontin, which are predominant substrates for TGase 2 expressed during bone mineralization. But it is still unclear as to which TGase 2 is expressed by NHost in vitro bone formation. The purpose of this s tudy was t o determine the level of TGase 2 expression associated with collagen I , osteopontin and osteocalcin in NHost cell lines from oral and maxillofacial area in vitro. We will investigate whether TGase 2 has an active role in the biocompatibility of dental biomaterials during differentiation and mineralization of NHost. NHost cell lines were cultured under DMEM with 10% FBS at 37ﾟC and 5% CO2. Collagen quantification, mineralization and calcium assay, ALP activity assay, and RT-PCR analysis during bone differentiation and mineralization were done. ALP levels showed higher activity under AA+hGP t han under AA. I nhibition o f T Gase 2 by cystamine showed d ecreased Ca++ concentration, c ollagen I deposition and ALP level during 11 days of differentiation. TGase 2 mRNA expression of NHost was constant during mineralization stage. TGase 2 enzyme activity was increased during differentiation. Osteopontin mRNA expression during mineralization was higher than that of osteocalcin and collagen I . It suggested that TGase 2 associated with collagen I, osteocalcin and osteonectin might play a role in the differentiation of NHost from oral and maxillofacial area, but a little involved in mineralization.

Laser is used to prevent the early dental caries in dental f ield and to apply for treatment of stomatitis and hyper sens it ivity , and laser mass Recently it is reported that laser i1'r adiation affect on soft tissue treatment and bone 1'emodelling after dental implantation. The purpose of this study was to examine laser irradia ti on effect on activity of normal human osteoblast on titanium plate in vitro by various laser wave length, and to observe morphologic change of NHost on LiLa nium plate and to a nalysis concentration of Ca"++ , IP and ALP NHost were cultured in DMEM containing 10% FBS, and observed by in verted microscope 1"or attatchment to the surface of titanium plate. Ca ++, I.P. , and a lkaline phosphat ase(ALP) concent ration in medium was calculated during 4 weeks, which was treated with Wilcoxon rank, Anova test and linear regression. The obtained results were as follows Morphologic changes showed rapid growth rate of NHost ++ at 3 days of laser lrradiatlOn ln spite of laser wave type, Ca" and P concentration was decreased at 2 weeks and was the hig hest at 3 weeks, but decreased at 4 weeks In spite of laser wave type, ALP concentration was decreased at 2 weeks but was increased at 3-4 weeks, From the aboving results, in spite of laser wave type, there were rapid growth rat e of NHost a nd no significant of Ca"++ , IP and P concen tr ation but these concentration showed predominant change than that of control

This experiment was performed to study the biocompatibility of xenograft materials (ABBM. coralline HA). Both autogenous bone grafts and allogenic banked bone were frequently and successfully used to promote regeneration of parts of skeleton. The use of these types of grafts were limited by the cost of donor site operation for autogenous boneor by fear of the risk of infection of allogenic materials. Another type of graft is xenograft which include ABBM and coralline HA. For investigating the biocompatibility, generally many investigators used cancer cell lines or animal cell lines. But cancer cell lines and animal cell lines had functioned different metabolism from normal human cell. So the experiment used normal human osteoblast for compare the biocompatibility of ABBM with coralline HA which were fixed in 24 well base contained culture medium. After 1st, 3rd, 7th, 14th, 28th days, the culture medium were taken out and checked the concentrations ofcalcium( Ca), inorganic phosphate(IP) and alkaline phosphatase(ALP). In another method, histologic samples were investigated after 8weeks of xenograft materials implantated on rabbit's tibia, the bone was cut and made undecalcified ground samples and checked with fluorecent microscope, polarizing microscope, reflection electron microscope and electron probe microanalysis. The statistical results of concentrations (Ca, IP, ALP) of materials in the culture medium have decreasedby day's, which meant that xenograft materials were effective for bone remodelling. The concentrations in the culture medium of ABBM were lower than that of coralline HA, that meant that biocompatibility of ABBM were superior than that of coralline HA. Histologic samples showed that ABBM had better bone remodelling effect than coralline HA. ABBM showed good alizarin red marking lines, more deposition of Ca, IP, and dense color of bone around newly formed osteon and bone trabeculae. it was concluded that ABBM was more biocompatible than corallineHA in vivo and in vitro test