Bone-like Nodule Formation Research Articles

Effect of 17β-estradiol on the in vitro differentiation of murine embryonic stem cells into the osteogenic lineage

Embryonic stem (ES) cells have the potential to differentiate into various cell types of the three germ layers. They are therefore a useful cell source for transplantation and tissue engineering. In the present paper, we studied the influences of ascorbic acid (AA), dexamethasone (Dex), and 17beta-estradiol (E(2)) on the osteogenic differentiation of ES cells. Differentiation into the osteoblastic phenotype was demonstrated by the appearance of osteoblastic markers such as alkaline phosphatase (ALP), the transcription factor core binding factor alpha 1 (Cbfa1), and osteocalcin, which were detected by immunohistochemistry. Bone nodule formation, including the deposition of collagen fibrils and matrix mineralization, was studied by transmission electron microscopy. In all our cultures, a progressive upregulation of ALP activity was observed, followed by a decline after 21 d of culture. Cbfa1 was first detected after 14 d in culture and increased during the culture time. The addition of E(2) resulted in a decrease in the formation of bone-like nodules in the embryoid bodies (EBs) compared with the EBs cultured in the presence of AA and AA supplemented with Dex. An increased osteocalcin concentration was observed in the EBs cultured with Dex and E(2) compared with the EBs cultured in a control medium. EBs cultured in the presence of E(2) resulted in a culture with a high amount of osteoblast-like cells not entrapped in bone-like nodules, creating the possibility to obtain a purified osteoblast population for bone tissue engineering.

Transglutaminase 2 Is Central to Induction of the Arterial Calcification Program by Smooth Muscle Cells

Arterial calcification is a phenotype of vascular repair in atherosclerosis, diabetes, hyperphosphatemic renal failure, and aging. Arterial calcification is modulated by transition of arterial smooth muscle cells (SMCs) from contractile to chondro-osseous differentiation programmed in response to increases in P(i), bone morphogenetic protein-2, and certain other stimuli. Transglutaminase (TG)2 release modulates tissue repair, partly by transamidation-catalyzed covalent crosslinking of extracellular matrix substrates. TG2 regulates cultured SMC differentiation, resistance artery remodeling to vasoconstriction, and atherosclerotic lesion size. Here, TG2 expression was required for the majority of TG activity in mouse and human aortic SMCs. TG2(-/-) SMCs lost the capacity for P(i) donor-induced formation of multicellular bone-like nodules and for increased expression of the type III sodium-dependent P(i) cotransporter Pit-1 and certain osteoblast and chondrocyte genes (tissue-nonspecific alkaline phosphatase, the osteoblast master transcription factor runx2, and chondrocyte-restricted aggrecan), and for P(i) donor- and bone morphogenetic protein-2-induced calcification. Uniquely in TG2(-/-) SMCs, P(i) donor treatment increased expression of the physiological SMC chondro-osseous differentiation and calcification inhibitors osteoprotegerin, matrix Gla protein, and osteopontin. Conversely, TG2(-/-) SMCs, unlike wild-type SMCs, failed to maintain contractile differentiation on laminin. Exogenous catalytically active TG2 augmented calcification by TG2(-/-) SMC in response to P(i) donor treatment. TG2 expression also drove P(i)-stimulated calcification of mouse aortic ring organ cultures, which was suppressed by the TG2 catalytic site-specific inhibitor Boc-DON-Gln-Ile-Val-OMe (10 micromol/L). Our results suggest that TG2 release in injured arteries is critical for programming chondro-osseous SMC differentiation and calcification in response to increased P(i) and bone morphogenetic protein-2.

Culture of osteogenic cells from human alveolar bone: A useful source of alkaline phosphatase

The aim of this study was to obtain membrane-bound alkaline phosphatase from osteoblastic-like cells of human alveolar bone. Cells were obtained by enzymatic digestion and maintained in primary culture in osteogenic medium until subconfluence. First passage cells were cultured in the same medium and at 7, 14, and 21 days, total protein content, collagen content, and alkaline phosphatase activity were evaluated. Bone-like nodule formation was evaluated at 21 days. Cells in primary culture at day 14 were washed with Tris-HCl buffer, and used to extract the membrane-bound alkaline phosphatase. Cells expressed osteoblastic phenotype. The apparent optimum pH for PNPP hydrolysis by the enzyme was pH 10.0. This enzyme also hydrolyzes ATP, ADP, fructose-1-phosphate, fructose-6-phosphate, pyrophosphate and beta-glycerophosphate. PNPPase activity was reduced by typical inhibitors of alkaline phosphatase. SDS-PAGE of membrane fraction showed a single band with activity of approximately 120 kDa that could be solubilized by phospholipase C or Polidocanol.

In vitro osteogenesis induced by cells derived from sites submitted to sinus grafting with anorganic bovine bone

This study evaluated key parameters of the in vitro osteogenesis induced by osteoblastic cells obtained from sites submitted to sinus grafting with anorganic bovine bone (ABB) in comparison with cells derived from bone sites of the same patients. In three patients, the augmentation of maxillary sinus was carried out using ABB (Bio-Oss). After at least 6 months, during the surgical intervention for titanium implants placement, biopsies were taken from these areas using trephine burs (grafted group). Bone fragments, of the same patients, from sites that had not received graft were also obtained with trephine burs and used as a control group. Osteoblastic cells were obtained from grafted and control groups by enzymatic digestion and cultured under standard osteogenic condition until subconfluence. First passaged cells were cultured in 24-well culture plates. Cell adhesion was evaluated at 24 h. For proliferation and viability assay, cells were cultured for 1, 3, 7, and 10 days. Total protein content and alkaline phosphatase (ALP) activity were measured at 3, 7, 10, 14, 17, and 21 days. Cultures were stained with Alizarin red S at 21 days, for detection of mineralized matrix. Data were compared by Student's t-test. Cell adhesion and viability were not affected by cell source (P>0.05). Total protein content was greater (P<0.05) for grafted group. Cell proliferation, ALP activity, and bone-like nodule formation were all greater (P<0.05) for the control group. Taken together, these results indicate that the in vivo long-term contact of cells with ABB downregulates the expression of osteoblast phenotype and consequently the in vitro osteogenesis.

Bone cell responses to the composite of Ricinus communis polyurethane and alkaline phosphatase

The aim of this study was to evaluate the response of osteoblastic cells to the composite of Ricinus communis polyurethane (RCP) and alkaline phosphatase (ALP) incubated in synthetic body fluid (SBF). RCP pure (RCPp) and RCP blended with ALP 6 mg/mL polymer (RCP+ALP) were incubated in SBF for 17 days. Four groups of RCP were tested: RCPp, RCP+ALP, and RCPp and RCP+ALP incubated in SBF (RCPp/SBF and RCP+ALP/SBF). Stem cells from rat bone marrow were cultured in conditions that allowed osteoblastic differentiation on RCP discs and were evaluated: cell adhesion, culture growth, cell viability, total protein content, ALP activity, and bone-like nodule formation. Data were compared by ANOVA or Kruskal-Wallis test. The group RCP+ALP was highly cytotoxic and, therefore, was not considered here. Cell adhesion (p = 0.14), culture growth (p = 0.39), viability (p = 0.46) and total protein content (p = 0.12) were not affected by either RCP composition or incubation in SBF. ALP activity was affected (p = 0.0001) as follows: RCPp < RCPp/SBF < RCP+ALP/SBF. Bone-like nodule formation was not observed on all evaluated groups. The composite RCP+ALP prior to SBF incubation is cytotoxic and must not be considered as biomaterial, but the incorporation of ALP to the RCP followed by SBF incubation could be a useful alternative to improve the biological properties of the RCP.

Chronic ethanol intake inhibits in vitro osteogenesis induced by osteoblasts differentiated from stem cells

The study investigated whether chronic ethanol (ETH) intake and subsequent ETH exposure of cell cultures affects osteoblast differentiation by evaluating key parameters of in vitro osteogenesis. Rats were treated with 5-20% (0.85-3.43 mm) ETH, increasing by 5% per week for a period of 4 weeks (habituation), after which the 20% level was maintained for 15 days (chronic intake). Bone-marrow stem cells from control (CONT) or ETH-treated rats were cultured in osteogenic medium which was either supplemented (ETH) or not supplemented (CONT) with 1.3 mm ethanol. Thus, four groups relating to rat treatment/culture supplementation were evaluated: (1) CONT/CONT, (2) ETH/CONT, (3) CONT/ETH and (4) ETH/ETH. Cell morphology, proliferation and viability, total protein content, alkaline phosphatase (ALP) activity and bone-like nodule formation were evaluated. Chronic ethanol intake significantly reduced both food and liquid consumption and body weight gain. No difference was seen in cell morphology among treatments. Cell number was affected at 7 and 10 days as follows: CONT/CONT = CONT/ETH < ETH/CONT = ETH/ETH. Doubling time between 3 and 10 days was greater in groups of CONT animals: ETH/ETH = ETH/CONT < CONT/ETH = CONT/CONT. Cell viability and ALP activity were not affected by either animal treatment or culture exposure to ethanol. At day 21, the total protein content was affected as follows: ETH/ETH = CONT/ETH < ETH/CONT = CONT/CONT. Bone-like nodule formation was affected as follows: ETH/ETH < CONT/ETH < ETH/CONT < CONT/CONT. These results show that chronic ethanol intake, followed by the exposure of osteoblasts to ethanol, inhibited the differentiation of osteoblasts, as indicated by an increased proliferation rate and reduced bone-like nodule formation.

Apolipoprotein E Stimulates Bone Formation on Titanium in Vitro

Objective: To examine the effects of apolipoprotein E2, a strongly expressed molecule during titanium implant healing, on bone formation by rat primary osteoblasts over titanium.Materials and Methods: Primary rat osteoblasts were cultured on titanium in the presence of 0.011.0 μg/mL apolipoprotein E2. After 2 or 3 weeks of culture, bone formation was analysed by staining the bone-like nodules with calcein blue. The mRNAs of the osteogenic genes type I collagen, osteopontin, and osteocalcin, were analysed using semi-quantitative reverse transcriptase polymerase chain reaction.Results: Apolipoprotein E2 enhanced bone-like nodule formation on titanium in a dose-dependent manner. Apolipoprotein E2 strongly upregulated osteopontin and osteocalcin mRNA expression, but had only a small effect on type I collagen mRNA expression.Conclusion: Apolipoprotein E2 stimulated bone formation on titanium disks in vitro. This may lead to a new approach to accelerating implant healing.

Effects of enamel matrix derivative and transforming growth factor‐β1 on human periodontal ligament fibroblasts

The objective of this study was to evaluate the effects of enamel matrix derivative (EMD), transforming growth factor-beta1 (TGF-beta1), and a combination of both factors (EMD+TGF-beta1) on periodontal ligament (PDL) fibroblasts. Human PDL fibroblasts were obtained from three adult patients with a clinically healthy periodontium, using the explant technique. The effects of EMD, TGF-beta1, or a combination of both were analysed on PDL cell proliferation, adhesion, wound healing, and total protein synthesis, and on alkaline phosphatase (ALP) activity and bone-like nodule formation. Treatment with EMD for 4, 7, and 10 days increased cell proliferation significantly compared with the negative control (p<0.05). At day 10, EMD and EMD+TGF-beta1 showed a higher cell proliferation compared with TGF-beta1 (p<0.01). Cell adhesion was significantly up-regulated by TGF-beta1 compared with EMD and EMD+TGF-beta1 (p<0.01). EMD enhanced in vitro wound healing of PDL cells compared with the other treatments. Total protein synthesis was significantly increased in PDL cells cultured with EMD compared with PDL cells treated with TGF-beta1 or EMD+TGF-beta1 (p<0.05). EMD induced ALP activity in PDL fibroblasts, which was associated with an increase of bone-like nodules. These findings support the hypothesis that EMD and TGF-beta1 may play an important role in periodontal regeneration. EMD induced PDL fibroblast proliferation and migration, total protein synthesis, ALP activity, and mineralization, while TGF-beta1 increased cellular adhesion. However, the combination of both factors did not positively alter PDL fibroblast behaviour.

Bone Matrix Formation in Osteogenic Cultures Derived from Human Embryonic Stem Cells in Vitro

Bone matrix production and mineralization involves sophisticated mechanisms, including the initial formation of an organic extracellular matrix into which inorganic hydroxyapatite crystals are later deposited. Human embryonic stem (hES) cells offer a potential to study early developmental processes and provide an unlimited source of cells. In this study, four different hES cell lines were used, and two different approaches to differentiate hES cells into the osteogenic lineage were taken. Undifferentiated cells were cultured either in suspension, facilitating the formation of embryoid bodies (EBs), or in monolayer, and both methods were in the presence of osteogenic supplements. Novel to our osteogenic differentiation study was the use of commercially available human foreskin fibroblasts to support the undifferentiated growth of the hES cell colonies, and their propagation in serum replacement-containing medium. Characterization of the osteogenic phenotype revealed that all hES cell lines differentiated toward the mesenchymal lineage, because T-Brachyury, Flt-1, and bone morphogenetic protein-4 could be detected. Main osteoblastic marker genes Runx2, osterix, bone sialoprotein, and osteocalcin were up-regulated. Alizarin Red S staining demonstrated the formation of bone-like nodules, and bone sialoprotein and osteocalcin were localized to these foci by immunohistochemistry. Cells differentiated in monolayer conditions exhibited greater osteogenic potential compared to those from EB-derived cells. We conclude that in vitro hES cells can produce a mineralized matrix possessing all the major bone markers, the differentiation of pluripotent hES cells to an osteogenic lineage does not require initiation via EB formation, and that lineage potential is not dependent on the mode of differentiation induction but on a cell line itself.

In vitro biocompatibility of a novel membrane of the composite poly(vinylidene‐trifluoroethylene)/barium titanate

This study was aimed at investigating the in vitro biocompatibility of a novel membrane of the composite poly(vinylidene-trifluoroethylene)/barium titanate (P(VDF-TrFE)/BT). Osteoblastic cells were obtained from human alveolar bone fragments and cultured under standard osteogenic condition until subconfluence. First passaged cells were cultured on P(VDF-TrFE)/BT and expanded polytetrafluoroethylene (e-PTFE--control) membranes in 24-well plates. Cell adhesion and spreading were evaluated at 30 min, and 4 and 24 h. For proliferation assay, cells were cultured for 1, 7, and 10 days. Cell viability was detected by trypan blue at 7 and 10 days. Total protein content and alkaline phosphatase (ALP) activity were measured at 7, 14, and 21 days. Cultures were stained with Alizarin red at 21 days, for detection of mineralized matrix. Data were compared by ANOVA and Student t test. Cell attachment (p = 0.001), cell number (p = 0.001), and ALP activity (p = 0.0001) were greater on P(VDF-TrFE)/BT. Additionally, doubling time was greater on P(VDF-TrFE)/BT (p = 0.03), indicating a decreased proliferation rate. Bone-like nodule formation took place only on P(VDF-TrFE)/BT. The present results showed that both membranes are biocompatible. However, P(VDF-TrFE)/BT presented a better in vitro biocompatibility and allowed bone-like nodule formation. Therefore, P(VDF-TrFE)/BT could be an alternative membrane to be used in guided tissue regeneration.

Purmorphamine enhances osteogenic activity of human osteoblasts derived from bone marrow mesenchymal cells

Purmorphamine is a novel small molecule with osteogenesis-inducing activity in multipotent mesenchymal progenitor cells, but there has been no evaluation of its effect on human cells to date. The aim of this study was to investigate the induction of osteogenic activity by purmorphamine in human osteoblasts differentiated from bone marrow mesenchymal cells. Cells were cultured in 24-well plates at a density of 2x10(4)/well in medium containing 1, 2 or 3 microM purmorphamine, or vehicle. At 7, 14 and 21 days, cell proliferation, viability, and alkaline phosphatase (ALP) activity were evaluated. Bone-like nodule formation was evaluated at 21 days. Purmorphamine did not affect cell proliferation or viability, but increased ALP activity and bone-like nodule formation. These results indicate that events related to osteoblast differentiation, including increased ALP activity and bone-like nodule formation, are enhanced by purmorphamine.

The effect of purmorphamine on osteoblast phenotype expression of human bone marrow mesenchymal cells cultured on titanium

Purmorphamine is a new molecule with osteogenesis-inducing activity in multipotent progenitor cells. The aim of this study was to evaluate whether purmorphamine maintains its osteogenic potential on human bone marrow mesenchymal cells cultured on commercially pure titanium (cpTi). Cells were cultured either in the absence or presence of purmorphamine 3 μ m on cpTi in supplemented α-MEM. At 7, 14, and 21 days, cell proliferation, viability, total protein content, collagen content, and alkaline phosphatase (ALP) activity were evaluated. Bone-like nodule formation was evaluated at 21 days. All experiments were done in quintuplicate and data were compared by ANOVA or t-test. Purmorphamine did not affect cell proliferation ( p=0.619), viability ( p=0.831), and collagen content ( p=0.088). Total protein content ( p=0.047), ALP activity ( p=0.001), and bone-like nodule formation ( p=0.002) were increased by purmorphamine. The present results indicate that events related to osteoblast differentiation, including increased ALP activity and bone-like nodule formation, are enhanced by purmorphamine in the presence of cpTi. It means that this molecule could be useful as an adjunct therapy to improve the osseointegration of the implants in the fields of dentistry and orthopaedics.

Cytokine and chemokine response of bone cells after dentin challenge in vitro.

The aim of this study was to characterize the effects of dentin extracts on cytokine, chemokine and nitric oxide (NO) production by primary rat bone cells. Osteoblastic bone marrow cultures were exposed to particulate (D-part), non-particulate (D-n-part) and demineralized dentin extracts and evaluated for proliferative activity, cell morphology, alkaline phosphatase activity and bone-like nodule formation. Cytokine production was assessed by enzyme-linked immunosorbent assay and NO release by the Griess method. The dentin extracts did not affect osteoblast numbering. Conversely, they up regulated in a dose-dependent manner the production by the osteoblasts of the pro-inflammatory interleukin-1beta (IL-1beta), tumor necrosis factor-alpha, IL-6, cytokine-induced neutrophil chemoattractant-1, and of the anti-inflammatory cytokine, IL-10. The NO production was stimulated only by D-n-part. These results demonstrate that dentin induces the production of inflammatory cytokines by osteoblasts and suggest that pro-resorptive pathways might be stimulated when dentin molecules come into contact with bone cells during pathological processes associated with dentin and bone matrix dissolution.

Synergistic interaction of topographic features in the production of bone-like nodules on Ti surfaces by rat osteoblasts

The objective of this study was to study the responses of osteoblast-like cells to rough Titanium (Ti)-coated epoxy surfaces of differing topographic complexity. Four topographies were studied: polished (PO), coarse-blasted (CB), acid-etched (AE) and coarse-blasted+acid-etched (SLA). Rat osteoblasts were cultured on these surfaces and their morphology, thickness as well as the number and size of bone-like nodules measured. To determine cell shape and cell thickness, fluorescein-5-thiosemicarbazide was used to stain the cell components including the cell membrane, the stained cells were optically sectioned using epifluorescent microscopy and the optical sections were computationally reconstructed to obtain three-dimensional images in which cell volume and cell thickness could be determined. Similarly optical sections of bone-like nodules labeled with tetracycline were also reconstructed to determine their size. The different surface topographies were found to alter the thickness and morphology of osteoblasts cultured on these surfaces. Osteoblasts produced significantly more and larger nodules on SLA compared to other surfaces . Nevertheless and perhaps surprisingly, given the evidence in various cell populations that cell shape can affect cell differentiation, cell thickness was not directly correlated with an increase in bone-like nodule formation. Data were analyzed by factorial analysis of variance. In this way the primary effect of each surface treatment ( i.e. blasting and acid etching) could be assessed as well as their interaction. Both the acid etching and blasting processes significantly affected the number and size of bone-like nodules cultured on Ti surfaces. Moreover there were significant interaction effects indicating that surface topographic features can act synergistically to enhance bone formation. This result suggests that a useful approach to the optimization of surfaces for bone production could involve systematic investigation of combinations of processes each of which produces distinct surface topographical features.

Osteoblastic differentiation of cultured rat bone marrow cells on hydroxyapatite with different surface topography

Hydroxyapatite (HA) has been used in orthopedic, dental, and maxillofacial surgery as a bone substitute. Objective. The aim of this investigation was to study the effect of surface topography produced by the presence of microporosity on the response of the rat bone marrow cells, evaluating: cell attachment, proliferation, total protein content, alkaline phosphatase (ALP) activity, and bone-like nodule formation. Methods. Cells were cultured on HA discs manufactured by a combination of uniaxial powder pressing and different sintering conditions, with different percentage of microporosity (<5%-HA5, 15%-HA15, and 30%-HA30). For attachment evaluation, cells were cultured for 2 h. Proliferation was evaluated after 7 and 14 days. After 14 days, total protein content and ALP activity were measured. For bone-like nodule formation, cells were cultured for 21 days. Data were compared by ANOVA and Duncan's multiple range test when appropriate. Results. Cell attachment was not affected by surface topography ( p=0.37). Proliferation ( p=0.001), total protein content ( p=0.039), ALP activity ( p=0.050), and bone-like nodule formation ( p=0.00001) were all significantly decreased by the most irregular surface (HA30). Significance. These results suggest that initial cell events were not affected by the surface topography of the HA. However, intermediary and final events such as proliferation, protein synthesis, ALP activity, and bone-like nodule formation favored surfaces with a more regular topography, such as that presents in HA with 15% or less of microporosity.

Response of rat bone marrow cells to commercially pure titanium submitted to different surface treatments

Objectives. Alterations in the commercially pure titanium (cpTi) surface may be undertaken to improve its biological properties. The aim of this study is to investigate the biocompatibility of cpTi submitted to different surface treatments. Methods. The cpTi surfaces were prepared so that machined and blasted surfaces, either acid etched or not, were compared using rat bone marrow cells cultured to differentiated into osteoblast. For attachment evaluation, cells were cultured for 4 and 24 h. Cell morphology was evaluated after 3 days. After 7, 14, and 21 days cell proliferation was evaluated. Total protein content and alkaline phosphatase (ALP) activity were evaluated after 14 and 21 days. For bone-like nodule formation, cells were cultured for 21 days. Data were compared by analysis of variance. Results. Cell attachment, cell morphology, cell proliferation, and ALP activity were not affected by surface treatments. Total protein content was reduced by blasted and acid etched surface. Bone-like nodule formation was significantly reduced by blasted, acid etched, and a combination of both blasted and acid etched surfaces. Conclusions. Based on these results, it can be suggested that cpTi surfaces that were submitted only to machining treatment favor the final event of osteoblastic differentiation of the rat bone marrow cells, evidenced by increased bone-like nodule formation.

Rat bone marrow cell response to titanium and titanium alloy with different surface roughness.

In general, cell response is affected by both chemical composition and surface roughness of implant materials. The aim of this study was to evaluate the effect of titanium (Ti) chemical composition and surface roughness on the response of rat bone marrow cells, examining cell attachment, cell proliferation, total protein content, alkaline phosphatase (ALP) activity, and bone-like nodule formation. Cells were cultured on both commercially pure titanium (cpTi) and titanium-6-aluminum-4-vanadium alloy (Ti-A) discs with four different average roughnesses (Ra). For attachment evaluation, cells were cultured for 2 h. After 14 days, cell proliferation, total protein content, and ALP activity were evaluated. Bone-like nodule formation was evaluated after 21 days. Data were compared by anova and Duncan's multiple range test when appropriate. Cell attachment and total protein content were affected by neither Ti chemical composition (P = 0.201, and P = 0.639, respectively) or surface roughness (P = 0.972, and P = 0.660, respectively). Proliferation, ALP activity, and bone-like nodule formation were affected only by Ti chemical composition (P = 0.0001, P = 0.064, and P = 0.0001, respectively). These results suggest that cpTi would optimize osteoblastic differentiation by rat bone marrow cells, including reduced cell proliferation, and increased ALP activity and bone-like nodule formation, while surface roughness, within the Ra parameters used, would not affect significantly the rat bone marrow cell response.

Effect of the chemical composition of Ricinus communis polyurethane on rat bone marrow cell attachment, proliferation, and differentiation.

Alterations in the chemical composition of a polymer may be undertaken to improve its biological properties. The aim of this study was to investigate the in vitro biocompatibility of Ricinus communis polyurethane (RCP) with three different chemical compositions: RCPp (pure RCP), RCP+CaCO(3), and RCP+Ca(3)(PO(4))(2). Rat bone marrow cells were cultivated under conditions that allowed osteoblastic differentiation and were evaluated for cell attachment, cell proliferation, cell morphology, total protein content, alkaline phosphatase (ALP) activity, and bonelike nodule formation. For the evaluation of attachment, cells were cultured for 4 h. After 3 days, cell morphology was evaluated. Cell proliferation was evaluated after 7 and 14 days. Total protein content and ALP activity were evaluated after 14 days. For bonelike nodule formation, cells were cultured for 21 days. Data were compared with an analysis of variance and Duncan's multiple range test when appropriate. Cell attachment and ALP activity were not affected by RCP chemical composition. Proliferation, total protein content, and bonelike nodule formation were all affected by RCP chemical composition. These results suggest that initial cell events are not affected by RCP chemical composition, whereas RCPs blended with calcium carbonate or, better yet, calcium phosphate, by favoring events that promote matrix mineralization, are more biocompatible materials.

Variation of oxide films on titanium induced by osteoblast-like cell culture and the influence of an H2O2 pretreatment.

Variations of titanium oxide films induced by osteoblast-like cells in a rat calvaria culture system and the influence of an H2O2 pretreatment have been investigated by using X-ray photoelectron spectroscopy and electrochemical impedance spectroscopy. For abraded titanium, the results revealed that phosphate and calcium ions may incorporate into the surface oxide film during the cell culture, forming a precipitate with a Ca/P ratio near that of hydroxyapatite. Oxidized carbon also was found in the surface layer, most likely precipitated hydroxylcarbonated apatite (HCA). The H2O2 pretreatment of titanium in a phosphate-buffered saline solution results in a 10-fold thickened porous oxide film and large amounts of surface hydroxyl groups as well as a certain amount of phosphate ions inside the oxide film. During the cell culture, the H2O2-treated titanium surface favors the ion incorporation and precipitation of the HCA-like compound, which probably is inlaid into the oxide film. Osteoblast-like cells on the H2O2-treated titanium showed a more active morphology during the initial stage compared with cells on abraded titanium. Moreover, bone-like nodule formation and mineralization appear to be related to the precipitation of the HCA-like compound on the surface. The results are discussed with respect to corrosion resistance, ion incorporation and precipitation of the HCA-like compound on the surface, osseointegration, and bioactivity of titanium implants.

Relationship between the expression of parathyroid hormone receptors and hormonal effect during rat osteoprogenitor cell differentiation

The number of parathyroid hormone (PTH) receptors in rat calvaria (RC) cells increased with culture duration, reached a maximum on day 13 when the cells had not yet differentiated into osteoblasts, and then gradually decreased by day 21. However, short exposure (48h) of RC cells to PTH-(1–34) before day 12, but not after day 15, did not decrease alkaline phosphatase (ALP) activity of the cells. Bone-like nodule (BN) formation was also suppressed only when PTH-(1–34) was present during the late culture period (days 14–21). Pretreating the cells with staurosporine, a protein kinase C inhibitor, on day 7 resulted in augmentation of PTH-(1–34)-induced cyclic adenosine monophosphate (cAMP) accumulation and inhibition of ALP activity. PTH-(7–34), which only activates PKC, however, had no effect on RC cell differentiation under any conditions used.