A dominant negative cadherin inhibits osteoblast differentiation.

We have previously indicated that human osteoblasts express a repertoire of cadherins and that perturbation of cadherin-mediated cell-cell interaction reduces bone morphogenetic protein 2 (BMP-2) stimulation of alkaline phosphatase activity. To test whether inhibition of cadherin function interferes with osteoblast function, we expressed a truncated N-cadherin mutant (NCaddeltaC) with dominant negative action in MC3T3-E1 osteoblastic cells. In stably transfected clones, calcium-dependent cell-cell adhesion was decreased by 50%. Analysis of matrix protein expression during a 4-week culture period revealed that bone sialoprotein, osteocalcin, and type I collagen were substantially inhibited with time in culture, whereas osteopontin transiently increased. Basal alkaline phosphatase activity declined in cells expressing NCaddeltaC, relative to control cells, after 3 weeks in culture, and their cell proliferation rate was reduced moderately (17%). Finally, 45Ca uptake, an index of matrix mineralization, was decreased by 35% in NCaddeltaC-expressing cells compared with control cultures after 4 weeks in medium containing ascorbic acid and beta-glycerophosphate. Similarly, BMP-2 stimulation of alkaline phosphatase activity and bone sialoprotein and osteopontin expression also were curtailed in NCaddeltaC cells. Therefore, expression of dominant negative cadherin results in decreased cell-cell adhesion associated with altered bone matrix protein expression and decreased matrix mineralization. Cadherin-mediated cell-cell adhesion is involved in regulating the function of bone-forming cells.     

Distinct proliferative and differentiated stages of murine MC3T3-E1 cells in culture: an in vitro model of osteoblast development.

We examine clonal murine calvarial MC3T3-E1 cells to determine if they exhibit a developmental sequence similar to osteoblasts in bone tissue, namely, proliferation of undifferentiated osteoblast precursors followed by postmitotic expression of differentiated osteoblast phenotype. During the initial phase of developmental (days 1-9 of culture), MC3T3-E1 cells actively replicate, as evidenced by the high rates of DNA synthesis and progressive increase in cell number, but maintain a fusiform appearance, fail to express alkaline phosphatase, and do not accumulate mineralized extracellular collagenous matrix, consistent with immature osteoblasts. By day 9 the cultures display cuboidal morphology, attain confluence, and undergo growth arrest. Downregulation of replication is associated with expression of osteoblast functions, including production of alkaline phosphatase, processing of procollagens to collagens, and incremental deposition of a collagenous extracellular matrix. Mineralization of extracellular matrix, which begins approximately 16 days after culture, marks the final phase of osteoblast phenotypic development. Expression of alkaline phosphatase and mineralization is time but not density dependent. Type I collagen synthesis and collagen accumulation are uncoupled in the developing osteoblast. Although collagen synthesis and message expression peaks at day 3 in immature cells, extracellular matrix accumulation is minimal. Instead, matrix accumulates maximally after 7 days of culture as collagen biosynthesis is diminishing. Thus, extracellular matrix formation is a function of mature osteoblasts. Ascorbate and beta-glycerol phosphate are both essential for the expression of osteoblast phenotype as assessed by alkaline phosphatase and mineralization of extracellular matrix. Ascorbate does not stimulate type I collagen gene expression in MC3T3-E1 cells, but it is absolutely required for deposition of collagen in the extracellular matrix. Ascorbate also induces alkaline phosphatase activity in mature cells but not in immature cells. beta-glycerol phosphate displays synergistic actions with ascorbate to further stimulate collagen accumulation and alkaline phosphatase activity in postmitotic, differentiated osteoblast-like cells. Mineralization of mature cultures requires the presence of beta-glycerol phosphate. Thus, MC3T3-E1 cells display a time-dependent and sequential expression of osteoblast characteristics analogous to in vivo bone formation. The developmental sequence associated with MC3T3-E1 differentiation should provide a useful model to study the signals that mediate the switch between proliferation and differentiation in bone cells, as well as provide a renewable culture system to examine the molecular mechanism of osteoblast maturation and the formation of bone-like extracellular matrix.     

成骨细胞在珍珠层复合人工骨表面的粘附和增殖

目的　评价人成骨细胞在珍珠层聚乳酸人工骨上的粘附能力。方法　将珍珠层聚乳酸人工骨与人成骨细胞体外复合培养 ,采用倒置相差显微镜、扫描电镜、透射电镜及流式细胞仪检测 ,观察人成骨细胞在珍珠层聚乳酸人工骨上的粘附能力。结果　珍珠层聚乳酸人工骨与人成骨细胞体外复合培养 1周后 ,细胞通过伪足贴附于人工骨表面。透射电镜下成骨细胞形态正常 ,胞浆内有丰富的粗面内质网和线粒体 ,并可观察到细胞之间形成的连接。流式细胞仪检测结果显示 ,附着在珍珠层复合人工骨的成骨细胞增殖指数增高。结论　珍珠层复合人工骨材料有利于成骨细胞的粘附、增殖和分化     

Effect of surface roughness and composition on costochondral chondrocytes is dependent on cell maturation state.

During endochondral bone formation, as occurs in fracture healing, chondrocytes are one of the first cells to see an implant surface. We tested the hypothesis that chemical composition and surface roughness affect chondrocyte differentiation, matrix synthesis, and local factor production and that the nature of the response is dependent on the state of maturation of the cells. To do this, we harvested rat growth zone and resting zone chondrocytes and examined their response to smooth and rough disk surfaces manufactured from either commercially pure titanium or titanium alloy. Profilometry, scanning electron microscopy, Auger spectroscopy, and Fourier transform infrared spectroscopy were used to characterize the surfaces. Average roughness values were 0.22 microm for smooth titanium surfaces, 0.23 microm for smooth titanium alloy surfaces, 4.24 microm for rough titanium surfaces, and 3.20 microm for rough titanium alloy surfaces. Cells were grown on the different disk surfaces until the cultures had reached confluence on plastic. The effect of the surfaces was determined by assaying cell number and [3H]thymidine incorporation as measures of cell proliferation, cell layer and cell alkaline phosphatase specific activity as markers of differentiation, and collagen production and [35S]sulfate incorporation as indicators of extracellular matrix production. In addition, the synthesis of prostaglandin E2 and transforming growth factor-beta were examined to measure changes in local factor synthesis. In growth zone and resting zone cultures, cell number and [3H]thymidine incorporation were decreased on rough surfaces; however, this effect was greater on commercially pure titanium surfaces. Cell layer and cell alkaline phosphatase specific activity were decreased in resting zone cells grown on rough surfaces. Cell alkaline phosphatase specific activity in growth zone cells was decreased on rough surfaces, whereas cell layer alkaline phosphatase specific activity was increased only in growth zone cells grown on rough commercially pure titanium surfaces. Resting zone cell collagen production was decreased only on rough commercially pure titanium, whereas in growth zone cells, collagen production was increased. Increased prostaglandin E2 release into the media was found for growth zone and resting zone cell cultures on the disks with rough surfaces. The observed effect was greater on rough commercially pure titanium. Production of transforming growth factor-beta by resting zones was similarly affected, whereas an increase in its production by growth zone cells was measured only on rough commercially pure titanium. These results indicate that surface roughness affects chondrocyte proliferation, differentiation, matrix synthesis, and local factor production and that these parameters are also affected by chemical composition. Furthermore, the nature and extent of the cell response is dependent on cell maturation. The overriding variable in response to an implant material, however, appears to be roughness of the surface.     

Role of surface roughness of titanium versus hydroxyapatite on human bone marrow cells response

This study was designed to assess human bone marrow cell response and particularly cell adhesion, proliferation, and differentiation, when cultured in vitro on titanium alloy and hydroxyapatite with different values of surface roughness. A further aim was to compare the cell response on the two materials, currently used in spinal surgery. Cell adhesion was determined after 0.5, 2, 4, and 18 hours of incubation; proliferation after 8, 11, 14, and 16 days of culture; and differentiation was evaluated with the expression of alkaline phosphatase activity after 8 and 16 days of culture. This study showed that bone marrow cells grew faster on titanium alloy than on hydroxyapatite, although fewer cells attached to titanium, compared to those attached to hydroxyapatite. No statistically significant difference was observed as the expression of alkaline phosphatase activity on hydroxyapatite and titanium alloy of the same roughness. Cell adhesion, proliferation, and differentiation are dependent on surface roughness of the biomaterial, and all three increased as the roughness of titanium alloy increased. Conclusively, surface roughness of titanium and hydroxyapatite significantly influences bone marrow cell response, and therefore these biomaterials should be used with rough outer surface, if a significant cell response on them is desired. These advantages of titanium and hydroxyapatite theoretically seem to be of particular importance in the following situations: long fusions, lumbosacral fusion, revision surgery with poor bone bank, neuropathic scoliosis associated with few bone graft reserves, and adult patients with severe osteoporosis.     

Effect of polymer molecular weight and addition of calcium stearate on response of MG63 osteoblast-like cells to UHMWPE particles.

Periprosthetic osteolysis and implant loosening is associated with the presence of ultrahigh molecular weight polyethylene (UHMWPE) wear debris particles. Osteoblast phenotypic expression in vitro is affected by UHMWPE particles, suggesting that bone formation may also be affected by wear debris. Here we tested the hypothesis that the response of osteoblasts to UHMWPE can be modified by changes in UHMWPE particle chemistry. We used four different commercially available preparations of GUR UHMWPE particles to determine if chemical composition (+/- Ca-stearate) or polymer molecular weight (3.1-4.2 million or 5.4-6.5 million g/mol) modulates osteoblast response. Particles were characterized by size distribution, morphology, and number of particles added to the culture medium. They had an average equivalent circle diameter ranging from 0.46-1.26 microm. MG63 cell response was assessed by measuring cell number, cellular and cell layer alkaline phosphatase, and prostaglandin E2 (PGE2) production. There were dose-dependent effects of the particles on cell response. Cell number and PGE, production were increased, while alkaline phosphatase specific activity was decreased. In addition, there was a marked difference between cultures treated with particles containing Ca-stearate and as a function of polymer molecular weight. Particles of higher molecular weight caused a greater stimulation of proliferation and inhibition of alkaline phosphatase than particles of lower molecular weight. The presence of Castearate exerted a more pronounced depression of osteoblast phenotype as well as a significantly greater increase in PGE2 release by the cells. The present study shows that chemical composition and polymer molecular weight of UHMWPE are capable of modulating osteoblast response to particles. The results suggest that osteoblast differentiation is inhibited by UHMWPE particles, whereas cell proliferation and PGE2 production are stimulated. This may have direct effects on osteoblasts and bone formation, but also paracrine effects on cells of the monocytic lineage inducing bone resorption and promoting inflammation which may lead to aseptic loosening. The present results suggest that the cellular events in aseptic loosening may be modulated or even accelerated by changes in the composition of the UHMWPE used to fabricate implants.     

Pretreatment of bone with osteoclasts affects phenotypic expression of osteoblast-like cells.

Implant surface morphology regulates osteoblast phenotypic expression. Osteoblast sensitivity to non-biologic surfaces suggests that native bone surface features may also affect osteoblast response. To test this, MG63 osteoblast-like cells were grown for 7 days on bovine cortical bone wafers pretreated with rat bone marrow osteoclasts for 0, 10 or 20 days. Response to osteoclast-treated surfaces was compared to the response of MG63 cells to titanium surfaces with smooth and rough microtopographies. Cell number, differentiation (alkaline phosphatase activity and osteocalcin levels), and local factors (PGE(2) and TGF-beta1) were measured in confluent cultures. Compared to culture on plastic, cell number was reduced on all three types of bone wafers; this effect was dose-dependent with increasing resorption of the surface. Alkaline phosphatase specific activity was increased (P相似文献   

The pericyte as a possible osteoblast progenitor cell.

Bovine capillary and microvessel pericytes were grown in monolayer in standard tissue culture medium supplemented with 10% newborn calf serum at various oxygen tensions for up to ten weeks. The pericytes synthesized alkaline phosphatase and formed colonies that mineralized. Energy dispersive X-ray spectrometry revealed the presence of calcium and phosphate, showed positive staining for collagen and glycosaminoglycan, and, most importantly, demonstrated the synthesis of osteocalcin. Cell proliferation, hydroxyproline production, and alkaline phosphatase synthesis were greatest in 3% oxygen, whereas osteocalcin production was least in 3% oxygen. These findings demonstrate that the capillary or microvessel pericyte exhibits phenotypic expressions in vitro that are similar to that of in vitro bone cells, and these expressions may be somewhat oxygen dependent. It is suggested from these findings that the capillary or microvessel pericyte may be an osteoblast precursor cell.     

人骨髓基质干细胞与表面置换珊瑚羟基磷灰石培养的定向诱导分化

目的观察人骨髓基质干细胞（hMSC）与表面置换的珊瑚羟基磷灰石（SCHA）体外培养的细胞粘附、增殖和分化,寻找理想的骨修复材料。方法海南天然滨珊瑚在特定温度和压力下部分水热反应,制成表面置换的珊瑚羟基磷灰石,将SCHA薄片与人骨髓基质干细胞体外培养,经诱导后于4、8、12、16d分别用荧光显微镜和扫描电镜（SEM）观察细胞活性、粘附和分化过程。结果SCHA保留原有的珊瑚贯穿多孔的三维结构。荧光显微镜可见hMSC在SCHA的表面和孔道内生长良好,第16d达最高水平;电镜显示细胞粘附良好,分化为成骨细胞,分泌大量胶原纤维,可见合成的钙结节。结论hMSC在表面置换的珊瑚羟基磷灰石内粘附、增殖、分化良好,两者具有较好的生物相容性,SCHA是一种良好的骨组织工程支架材料。     

Enhanced Cell Integration to Titanium Alloy by Surface Treatment with Microarc Oxidation: A Pilot Study

Microarc oxidation (MAO) is a surface treatment that provides nanoporous pits, and thick oxide layers, and incorporates calcium and phosphorus into the coating layer of titanium alloy. We presumed such modification on the surface of titanium alloy by MAO would improve the ability of cementless stems to osseointegrate. We therefore compared the in vitro ability of cells to adhere to MAOed titanium alloy to that of two different types of surface modifications: machined and grit-blasted. We performed energy-dispersive x-ray spectroscopy and scanned electron microscopy investigations to assess the structure and morphology of the surfaces. Biologic and morphologic responses to osteoblast cell lines (SaOS-2) were then examined by measuring cell proliferation, cell differentiation (alkaline phosphatase activity), and αvβ3 integrin. The cell proliferation rate, alkaline phosphatase activity, and cell adhesion in the MAO group increased in comparison to those in the machined and grit-blasted groups. The osteoblast cell lines of the MAO group were also homogeneously spread on the surface, strongly adhered, and well differentiated when compared to the other groups. This method could be a reasonable option for treating the surfaces of titanium alloy for better osseointegration. Each author certifies that he has no commercial associations (eg, consultancies, stock ownership, equity interest, patent/licensing arrangements, etc) that might pose a conflict of interest in connection with the submitted article. This work was performed at Seoul St. Mary’s Hospital and St. Mary’s Hospital, Seoul, Korea.     

Osteoblast Function Is Compromised at Sites of Focal Bone Erosion in Inflammatory Arthritis

In rheumatoid arthritis (RA), synovial inflammation results in focal erosion of articular bone. Despite treatment attenuating inflammation, repair of erosions with adequate formation of new bone is uncommon in RA, suggesting that bone formation may be compromised at these sites. Dynamic bone histomorphometry was used in a murine model of RA to determine the impact of inflammation on osteoblast function within eroded arthritic bone. Bone formation rates at bone surfaces adjacent to inflammation were similar to those observed in nonarthritic bone; therefore, osteoblast activity is unlikely to compensate for the increased bone resorption at these sites. Within arthritic bone, the extent of actively mineralizing surface was reduced at bone surfaces adjacent to inflammation compared with bone surfaces adjacent to normal marrow. Consistent with the reduction in mineralized bone formation, there was a notable paucity of cells expressing the mid‐ to late stage osteoblast lineage marker alkaline phosphatase, despite a clear presence of cells expressing the early osteoblast lineage marker Runx2. In addition, several members of the Dickkopf and secreted Frizzled‐related protein families of Wnt signaling antagonists were upregulated in arthritic synovial tissues, suggesting that inhibition of Wnt signaling could be one mechanism contributing to impaired osteoblast function within arthritic bone. Together, these data indicate that the presence of inflammation within arthritic bone impairs osteoblast capacity to form adequate mineralized bone, thus contributing to the net loss of bone and failure of bone repair at sites of focal bone erosion in RA.     

Plasma hydroxyproline in uremia: Relationships with histologic and biochemical indices of bone turnover

Plasma hydroxyproline and alkaline phosphatase were measured in 52 patients with chronic renal failure and related to histologic indices of bone cell activity. Plasma hydroxyproline decreased after each dialysis treatment. When levels of hydroxyproline were measured immediately preceding dialysis treatments, we observed significant correlations between plasma hydroxyproline and alkaline phosphatase, and osteoblast and osteoclast surfaces on trabecular bone. In contrast, no significant correlations were noted between the biochemical measurements and the osteoid or rough crenated surface. When patients with hyperparathyroid bone disease were treated with 1,25-dihydroxyvitamin D₃, its synthetic analog 1α(OH)D₃, or with synthetic salmon calcitonin or subjected to bilateral nephrectomy, changes noted in plasma hydroxyproline were proportional to those noted in alkaline phosphatase. Following parathyroidectomy, plasma hydroxyproline fell abruptly without a decrease in alkaline phosphatase, suggesting an early effect of parathyroidectomy on bone resorption without affecting bone formation. In contrast, the early responses of patients to treatment with 24,25(OH)₂D₃ showed that plasma alkaline phosphatase increased without a change in plasma hydroxyproline. These observations suggest that plasma hydroxyproline is a useful biochemical marker of bone resorption in renal failure. The measurement of rough crenated surfaces and osteoid surfaces are inadequate indices of bone cell activity in uremia.     

源于脾细胞的破骨细胞样细胞体外培养及其对成骨细胞的影响

目的探讨破骨细胞及其亚细胞结构对成骨细胞生长的影响。方法C57雌性小鼠,经尾静脉注射5-FU后,取其脾脏细胞,在白介素(IL)-3,6和粒细胞-巨噬细胞集落刺激因子(GM-CSF)、1α,25-(OH)2D3的诱导下获得大量的破骨样细胞(OLC)。将破骨样细胞、NaF、离心去除OLC的培养基及其亚细胞结构——细胞核、线粒体与成骨细胞共培养5d后,检测成骨细胞增殖率和碱性磷酸酶含量以及成骨细胞Cbfα1的表达活性。结果OLC及离心去除OLC的50%培养基均可使成骨细胞增殖率显著增加(P<0.05)。NaF、OLC、OLC细胞核和离心去除OLC的25%培养基均可使成骨细胞的碱性磷酸酶活性增高(P<0.05);离心去除OLC的培养基对成骨细胞的碱性磷酸酶比活性具有显著的促进作用(P<0.05)。NaF、OLC细胞质和离心去除OLC的50%培养基均可使成骨细胞的Cbfα1的表达明显加强(P<0.05)。结论OLC对成骨细胞的生长和功能均有促进作用。     

Alphavbeta integrins play an essential role in BMP-2 induction of osteoblast differentiation.

Both integrins and BMP-2 exert similar effects on osteoblasts. We examined the relationship between the alphav-containing integrins (alphavbeta) and BMP-2 in osteoblast function. BMP-2 stimulates alphavbeta expression. BMP-2 receptors co-localize/overlap with alphavbeta integrins, and the intact function of alphavbeta is essential in BMP-2 activity. INTRODUCTION: Bone morphogenetic protein (BMP)-2 not only induces osteoblast differentiation and bone matrix mineralization, but also stimulates osteoblast migration on and adhesion to bone matrix proteins. The alphavbeta- and beta1- (alphabeta1) containing integrins mediate osteoblast interaction with many bone matrix proteins and play important roles in osteoblast adhesion, migration, and differentiation. Because alphavbeta integrins and BMP-2 share common effects on osteoblasts, we analyzed their relationship in osteoblast function. MATERIALS AND METHODS: The effects of BMP-2 on integrin expression were determined by surface labeling/immunoprecipitation and cell adhesion to matrix proteins. Confocal analysis of the immunostained cells and co-immunoprecipitation of cell extracts were used to study the spatial relationship between integrins and BMP-2 receptors. A function-blocking anti-alphavbeta integrin antibody (L230) was employed to investigate the roles of alphavbeta integrins in BMP-2 function. RESULTS: Human osteoblasts (HOBs) express alphabeta1, alphavbeta3, alphavbeta5, alphavbeta6, and alphavbeta8 integrins at focal adhesion sites. BMP-2 increases the levels of these integrins on osteoblast surface and enhances HOB adhesion to osteopontin and vitronectin. Immunoprecipitation and immunostaining analyses show that BMP-2 receptors co-localize or overlap with alphavbeta and alphabeta1 integrins. Incubation of HOBs with L230 abolishes the antiproliferative effect of BMP-2 and reduces the capacity of BMP-2 to stimulate alkaline phosphatase activity and the expression of osteocalcin, osteopontin, and bone sialoprotein. Furthermore, L230 prevents BMP-2 induction of matrix mineralization. Although BMP-2 retains its receptor-binding capability in the presence of L230, BMP-2 stimulation of Smad signaling is abolished by L230. CONCLUSION: BMP-2 upregulates the expression of alphavbeta integrins, and these integrins, in turn, play a critical role in BMP-2 function in osteoblasts.     

Effect of caffeine on parameters of osteoblast growth and differentiation of a mineralized extracellular matrix in vitro.

The effects of caffeine exposure on bone formation were examined using a chick osteoblast culture system. Secondary cultures of normal diploid osteoblasts were exposed to chronic doses of 0, 0.1, 0.2, or 0.4 mM caffeine beginning on day 0 through day 28. Neither the rate of cell proliferation nor cell number, as measured by total DNA, was decreased for any of the doses examined. In contrast, osteocalcin levels, alkaline phosphatase activity, and total calcium levels showed a dose-related decrease in cultures treated with caffeine. These parameters were significantly decreased at the highest dose of 0.4 mM. The reduction in total protein levels ranged from 29 to 66% of control values and was independent of dose. In contrast, total collagen levels were more affected by the dose of caffeine used. Inhibition of collagen levels was most apparent on days 17 and 21, time points during the period of active formation of the matrix immediately preceding the deposition of mineral. By day 28 collagen levels in cultures exposed to the lower doses of caffeine had returned to control levels, and only the cultures exposed to the highest dose (0.4 mM) remained significantly inhibited with respect to both collagen and mineral. Histochemically, alkaline phosphatase and mineral staining of day 28 cultures mirrored the biochemical events with the 0.4 mM caffeine exposure. The results indicate that one of the effects of caffeine on bone development is to inhibit the formation of a competent extracellular matrix during the osteoblast differentiation sequence, which results in the inhibition of mineralization analogous to the delayed ossification observed in fetal animals after prenatal caffeine exposure.     

In vitro cell behavior of human osteoblasts after physiological dynamic stretching

The cell activity of human bone derived cell cultures was studied after mechanical stimulation by cyclic strain at a magnitude occurring in physiologically loaded bone tissue. Monolayers of subconfluently grown human bone derived cells were stretched in rectangular silicone dishes with cyclic uniaxial movement along their longitudinal axes. Strain was applied over two days for 30 min per day with a frequency of 1 Hz and a strain magnitude of 1000 mustrain. Cyclic stretching of the cells resulted in an increased proliferation (10-48%) and carboxyterminal collagen type I propeptide release (7-49%) of human cancellous bone derived osteoblasts while alkaline phosphatase activity and osteocalcin release were significantly reduced by 9-25% and 5-32% respectively. These results demonstrate that cyclic strain at physiologic magnitude leads to an increase of osteoblast activities related to matrix production while those activities which are characteristic for the differentiated osteoblast and relevant for matrix mineralization are decreased.     

Einfluß verschiedener Knochendesinfektions- und Sterilisationsverfahren auf die Osteoblastenfunktion Eine vergleichende In-vitro-Studie

All sterilization and disinfection procedures for bone grafts are different in regard to influence of bone graft features, which may influence the function of different cell types. We used an in-vitro approach to assess the influence of bone matrix, which was sterilized or disinfected, on osteoblastic activities in-vitro by simulating a cell-transplant-interface. Primary bovine osteoblast cell cultures were established from periosteum. Bone graft specimens made of bovine cortical bone (O 15 mm, 300 microns thickness) were treated in 5 different ways: autoclaved, ethylene-oxide-sterilized, demineralized and low-temperature-plasma-sterilized (DEM-LTP), chemically sterilized (modified Tutoplast method), and 80 degrees C-temperature disinfected. The following cell function parameters were assayed: plating efficiency proliferation by measuring the DNA-content, and MTT-activity, soluble protein and extracellular matrix synthesis, alkaline phosphatase, and osteocalcin expression. All disinfected bone grafts were biocompatible with primary periosteal osteoblasts. Measured cell activities upon bone specimens showed better results than cells of the plastic surface control. The DEM-LTP-bone showed better results in comparison to other groups, and stimulated the proliferation and differentiation.