Mineralization Of Human Osteoblasts Research Articles

Investigating the Biological Efficacy of Albumin-Enriched Platelet-Rich Fibrin (Alb-PRF): A Study on Cytokine Dynamics and Osteoblast Behavior.

The development of effective biomaterials for tissue regeneration has led to the exploration of blood derivatives such as leucocyte- and platelet-rich fibrin (L-PRF). A novel variant, Albumin-Enriched Platelet-Rich Fibrin (Alb-PRF), has been introduced to improve structural stability and bioactivity, making it a promising candidate for bone regeneration. This study aimed to evaluate Alb-PRF's capacity for cytokine and growth factor release, along with its effects on the proliferation, differentiation, and mineralization of human osteoblasts in vitro. Alb-PRF membranes were analyzed using histological, scanning electron microscopy, and fluorescence microscopy techniques. Cytokine and growth factor release was quantified over seven days, and osteoinductive potential was evaluated with MG-63 osteoblast-like cells. Structural analysis showed Alb-PRF as a biphasic, highly cellularized material that releases lower levels of inflammatory cytokines and higher concentrations of platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) compared to L-PRF. Alb-PRF exhibited higher early alkaline phosphatase activity and in vitro mineralization (p < 0.05) and significantly increased the OPG/RANKL mRNA ratio (p < 0.05). These results indicate that Alb-PRF has promising potential as a scaffold for bone repair, warranting further in vivo and clinical assessments to confirm its suitability for clinical applications.

The Effects of Titanium Dioxide Nanoparticles on Osteoblasts Mineralization: A Comparison between 2D and 3D Cell Culture Models.

Although several studies assess the biological effects of micro and titanium dioxide nanoparticles (TiO2 NPs), the literature shows controversial results regarding their effect on bone cell behavior. Studies on the effects of nanoparticles on mammalian cells on two-dimensional (2D) cell cultures display several disadvantages, such as changes in cell morphology, function, and metabolism and fewer cell-cell contacts. This highlights the need to explore the effects of TiO2 NPs in more complex 3D environments, to better mimic the bone microenvironment. This study aims to compare the differentiation and mineralized matrix production of human osteoblasts SAOS-2 in a monolayer or 3D models after exposure to different concentrations of TiO2 NPs. Nanoparticles were characterized, and their internalization and effects on the SAOS-2 monolayer and 3D spheroid cells were evaluated with morphological analysis. The mineralization of human osteoblasts upon exposure to TiO2 NPs was evaluated by alizarin red staining, demonstrating a dose-dependent increase in mineralized matrix in human primary osteoblasts and SAOS-2 both in the monolayer and 3D models. Furthermore, our results reveal that, after high exposure to TiO2 NPs, the dose-dependent increase in the bone mineralized matrix in the 3D cells model is higher than in the 2D culture, showing a promising model to test the effect on bone osteointegration.

Apigenin Promotes Proliferation and Mineralization of Human Osteoblasts and Up-Regulates Osteogenic Markers

Apigenin (APG), a natural flavonoid compound with anti-inflammatory and antioxidative properties, was found to promote in vitro osteogenic differentiation and to accelerate in vivo bone formation, indicating APG as a promising molecule in bone repair, with potential clinical application in bone-deficient conditions. In particular, in dentistry, it is fundamental to increase the available bone volume for implant placement in the maxilla. Therefore, this study aims to investigate the APG effects on osteoblasts (hOBs) obtained from a human jaw. hOBs were incubated with increasing concentrations of APG (5, 10, 20 µM) to assess cell viability and morphology at 24 h and proliferation at 48 and 72 h. Upon establishing the absence of cytotoxicity and any morphological changes, APG showed a stimulating effect on cell growth, with significative results using 5 µM (5-APG) at 48 h. Thus, 5-APG was chosen for further investigations in order to assess alkaline phosphate (ALP) at 7 days, mineralization at 14 days and expression of ALP, Osteocalcin (OCN) and Collagen 1 (COL1) genes at 7 days. Our results showed that 5-APG accelerated osteoblast mineralization activities and significantly upregulated ALP and COL1 gene expression. Hence, this study demonstrated that APG is able to promote human oral osteoblasts proliferation and mineralization, suggesting its potential usefulness in dentistry.

Endocrine disruption of vitamin D activity by perfluoro-octanoic acid (PFOA)

Perfluoroalkyl substances (PFAS) are a class of compounds used in industry and consumer products. Perfluorooctanoic acid (PFOA) is the predominant form in human samples and has been shown to induce severe health consequences, such as neonatal mortality, neurotoxicity, and immunotoxicity. Toxicological studies indicate that PFAS accumulate in bone tissues and cause altered bone development. Epidemiological studies have reported an inverse relationship between PFAS and bone health, however the associated mechanisms are still unexplored. Here, we present computational, in silico and in vitro evidence supporting the interference of PFOA on vitamin D (VD). First, PFOA competes with calcitriol on the same binding site of the VD receptor, leading to an alteration of the structural flexibility and a 10% reduction by surface plasmon resonance analysis. Second, this interference leads to an altered response of VD-responsive genes in two cellular targets of this hormone, osteoblasts and epithelial cells of the colorectal tract. Third, mineralization in human osteoblasts is reduced upon coincubation of PFOA with VD. Finally, in a small cohort of young healthy men, PTH levels were higher in the exposed group, but VD levels were comparable. Altogether these results provide the first evidence of endocrine disruption by PFOA on VD pathway by competition on its receptor and subsequent inhibition of VD-responsive genes in target cells.

Two-day-treatment of Activin-A leads to transient change in SV-HFO osteoblast gene expression and reduction in matrix mineralization.

Activins regulate bone formation by controlling osteoclasts and osteoblasts. We investigated Activin‐A mechanism of action on human osteoblast mineralization, RNA and microRNA (miRNA) expression profile. A single 2‐day treatment of Activin‐A at Day 5 of osteoblast differentiation significantly reduced matrix mineralization. Activin A‐treated osteoblasts responded with transient change in gene expression, in a 2‐wave‐fashion. The 38 genes differentially regulated during the first wave (within 8 hr after Activin A start) were involved in transcription regulation. In the second wave (1–2 days after Activin A start), 65 genes were differentially regulated and related to extracellular matrix. Differentially expressed genes in both waves were associated to transforming growth factor beta signaling. We identified which microRNAs modulating osteoblast differentiation were regulated by Activin‐A. In summary, 2‐day treatment with Activin‐A in premineralization period of osteoblast cultures influenced miRNAs, gene transcription, and reduced matrix mineralization. Modulation of Activin A signaling might be useful to control bone quality for therapeutic purposes.

An extract from Myracrodruon urundeuva inhibits matrix mineralization in human osteoblasts

Ethnopharmacological relevancePhytotherapy based on plant-derived compounds is an alternative medicinal strategy for the relief of symptoms and the curing of diseases. The leaves of Myracrodruon urundeuva a medicinal plant also known as “aroeira”, has been used in traditional medicine as healing, antiulcer and anti-inflammatory to treat skeletal diseases in Brazil, but its role in bone cell toxicity, as well as in bone formation, remains to be established. Aim of the studyWe sought to determine the in vitro osteogenic effects of a hydroalcoholic M. urundeuva leaves extract in primary human osteoblasts. Materials and methodsCell viability, reactive oxygen species (ROS) production, alkaline phosphatase (ALP) activity and matrix mineralization were evaluated by MTT assay, DCFH-DA probe, colorimetric-based enzymatic assay and Alizarin Red-staining, respectively. Besides, the matrix metalloproteinase (MMP)-2 and progressive ankylosis protein homolog (ANKH) gene expression were determined by real-time RT-qPCR and MMP-2 activity by zymography. ResultsExposure of osteoblasts to M. urundeuva extract significantly decreased viability and increased reactive oxygen species (ROS) production, regardless of the extract concentration. The M. urundeuva extract at 10 μg/mL also downregulated matrix metalloproteinase (MMP)-2, while upregulating progressive ankylosis protein homolog (ANKH) gene expression. By contrast, the MMP-2 activity was unchanged. The M. urundeuva extract at 10 μg/mL also reduced alkaline phosphatase (ALP) activity and mineralization. ConclusionsOverall, our findings suggest that the inhibition of osteogenic differentiation and matrix mineralization promoted by M. urundeuva may be due more to an increase in oxidative stress than to the modulation of MMP-2 and ANKH expression.

Effects of calcium ion on the migration and osteogenic differentiation of human osteoblasts

This study aimed to investigate the effect of calcium ion (Ca²⁺) on the migration and osteogenic differentiation of human osteoblasts and explore the proper concentration and correlation mechanism. A series of Ca²⁺ solutions with different concentrations was prepared. Osteoblast migration was assessed by Transwell assay, and proliferation was studied via the CCK-8 colorimetric assay. The mRNA expression of osteogenic genes was examined via reverse transcription-polymerase chain reaction (RT-PCR), and the mineralized nodule was examined by alizarin red-S method. After calcium sensitive receptor (CaSR) antagonism, Ca²⁺-induced migration and osteogenic differentiation were analyzed. In the migration experiment, 2, 4, and 6 mmol·L⁻¹ Ca²⁺ could promoted osteoblast migration at three timepoints (8, 16, and 24 h), whereas 10 mmol·L⁻¹ Ca²⁺ considerably inhibited migration at 8 h. The Ca²⁺ concentration range of 2-10 mmol·L⁻¹ could promote proliferation, osteogenic differentiation, and mineralization of human osteoblasts. Moreover, mineralization was predominantly induced by 8 and 10 mmol·L⁻¹ Ca²⁺. CaSR antagonism could reduce Ca²⁺-induced migration and osteogenic differentiation of human osteoblasts. Low Ca²⁺ concentration favored osteoblast migration, whereas high Ca²⁺ concentration favored osteogenic differentiation. The Ca²⁺ concentrations of 4 and 6 mmol·L⁻¹ could substantially induce osteoblast migration and osteogenic differentiation, and the Ca²⁺-CaSR pathway participated in signal transduction.

A novel gradated zirconia implant material embedding bioactive ceramics: Osteoblast behavior and physicochemical assessment

Bioactive ceramic coatings have been proposed to improve the bioactivity of zirconia although the coating detachment can occur during implant placement. The main aim of this study was to enhance bioactivity and strength of the implant surface by using a gradated bioactive zirconia structure. Zirconia discs (8 × 3 mm) embedding gradual content of hydroxyapatite (YTZP-HA) or beta-tricalcium phosphate (YTZP-βTCP) were produced by hot-pressing technique. Specimens were initially studied regarding hardness, roughness, wettability, and shear bond strength of the gradated zone. Functionally gradated ceramic discs and zirconia (control group) were placed in contact with human osteoblast culture for 1, 3, 7, and 14 days. Field emission guns scanning electron microscopy (FEGSEM) was used to assess the morphology and adhesion of osteoblasts while cell viability was assessed by fluorometric method. The mineralization on the test and control discs was evaluated by Alkaline phosphatase (ALP) activity and fluorescent microscopy. Shear strength mean values of the outer layer bioactive ceramic and zirconia bulk were recorded at 150 MPa. Mechanical assays demonstrated that the novel design and manufacturing approach proposed for producing gradated zirconia embedding bioactive ceramics resulted in significantly higher mechanical strength as compared to monolithic zirconia. Also, cell viability and ALP levels increased on gradated zirconia containing HA or βTCP over time. Gradated zirconia containing hydroxyapatite revealed an increased viability, bioactivity, and mineralization of human osteoblasts when compared to conventional zirconia surface, without substantial loss of strength.

Suppressive effects of tenofovir disoproxil fumarate, an antiretroviral prodrug, on mineralization and type II and type III sodium-dependent phosphate transporters expression in primary human osteoblasts.

Tenofovir disoproxil fumarate (TDF) is an antiretroviral drug commonly used for the management of Human Immunodeficiency Virus (HIV) in highly active antiretroviral therapy (HAART) and of chronic Hepatitis B Virus (HBV) infections. Long-term TDF-treated subjects present decrease of bone mineral density and rarely severe osteomalacia. Although these adverse effects have been attributed to the impaired proximal tubule function, a possible direct involvement of TDF on osteoblasts should be taken into account. The aim of this study was to evaluate whether sodium phosphate transporters NPT2A (sodium-dependent phosphate transport protein 2A), NPT2C (sodium-dependent phosphate transport protein 2C), PIT1 (sodium-dependent phosphate transporter 1), and PIT2 (sodium-dependent phosphate transporter 2) were expressed in primary human osteoblasts (HOBs), whether their expression was related to HOBs differentiation and whether TDF could affect mineralization and gene expression. PIT1 and PIT2 were expressed under proliferating conditions and increased after induction of mineralization, while NPT2A and NPT2C were almost undetectable. In HOBs TDF exposure induced a significant dose-dependent decrease in mineralization. Moreover, TDF caused a reduction of COL1A1 and of ATF4 expression in differentiated HOBs. In summary, HOBs do not express NPT2A and NPT2C and do express PIT1 and PIT2, suggesting a role of these two latter in human osteoblast mineralization. TDF impairs osteoblast mineralization, confirming a direct negative effect on bone. Therefore, in clinical practice, bone damage must be suspected and evaluated also in patients receiving TDF without kidney function alterations.

Increased interleukin (IL)-20 and IL-24 target osteoblasts and synovial monocytes in spondyloarthritis.

The pathogenesis of spondyloarthritis (SpA) involves activation of the innate immune system, inflammation and new bone formation. The two cytokines interleukin (IL)-20 and IL-24 have been shown to link innate immune activation and tissue homeostasis. We hypothesized that these two cytokines are secreted as part of activation of the innate immune system and affect bone homeostasis in SpA. IL-20 and IL-24 were measured in plasma from axial SpA patients (n = 83). Peripheral SpA patients (n = 16) were included for in-vitro cell culture studies. The plasma IL-20 and IL-24 levels were increased in SpA patients compared with healthy controls (HCs) by 57 and 83%, respectively (both P < 0·0001). The Toll-like receptor 4-induced secretion of the two cytokines was greater in SpA peripheral blood mononuclear cells (PBMCs) compared with HC PBMCs. IL-20 and IL-24 increased the production of monocyte chemoattractant protein-1 by activated SpA synovial fluid monocytes, decreased the production of Dickkopf-1 by SpA fibroblast-like synovial cells and induced mineralization in human osteoblasts. Taken together, our findings indicate disease-aggravating functions of IL-20 and IL-24 in SpA.

Synovial cell production of IL-26 induces bone mineralization in spondyloarthritis.

IL-26 levels are higher in synovial fluid compared to plasma in spondyloarthritis. IL-26 was identified in axial facet joints of spondyloarthritis patients. Myofibroblasts from the spondyloarthritis synovium produce large amounts of IL-26. IL-26 induces bone mineralization in human osteoblasts.

Rooibos flavonoids, orientin and luteolin, stimulate mineralization in human osteoblasts through the Wnt pathway.

Several epidemiological studies have shown that tea consumption is associated with higher bone mineral density in women. Flavonoids in tea are recognized as potential estrogen mimics and may positively influence bone metabolism in estrogen-deficient women. Luteolin and orientin, flavonoids from rooibos tea, are of particular interest as rooibos tea contains no caffeine that can be detrimental to bone health. This study analyzed changes in mineral content when luteolin or orientin was added to a human osteoblast cell line and the potential mechanisms involved. Measurements included alkaline phosphatase (ALP) activity, cell mitochondrial activity, toxicity, and changes in regulatory proteins involved in osteoblast metabolism. Mineral was significantly elevated in Saos2 cells treated with orientin (0.1-1.0 μM, 15-100 μM) or luteolin (5.0 μM) and was associated with increased ALP and mitochondrial activity, as determined by the production of p-nitrophenol and the reduction of 2-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, respectively. Greater mineral content was also associated with lower toxicity as determined by lactate dehydrogenase activity and lower expression of TNF-α, IL-6, sclerostin, osteopontin, and osteoprotegerin. Orientin and luteolin, flavonoids in rooibos tea, enhance mineral content in Saos2 cells. These findings provide guidance for doses to be studied in well-established animal models.

Acidosis Inhibits Mineralization in Human Osteoblasts

Osteoblasts and osteoclasts maintain bone volume. Acidosis affects the function of these cells including mineral metabolism. We examined the effect of acidosis on the expression of transcription factors and mineralization in human osteoblasts in vitro. Human osteoblasts (SaM-1 cells) derived from the ulnar periosteum were cultured with α-MEM containing 50 μg/ml ascorbic acid and 5 mM β-glycerophosphate (calcifying medium). Acidosis was induced by incubating the SaM-1 cells in 10 % CO₂ (pH approximately 7.0). Mineralization, which was augmented by the calcifying medium, was completely inhibited by acidosis. Acidosis depressed c-Jun mRNA and increased osteoprotegerin (OPG) production in a time-dependent manner. Depressing c-Jun mRNA expression using siRNA increased OPG production and inhibited mineralization. In addition, depressing OPG mRNA expression with siRNA enhanced mineralization in a dose-dependent manner. Acidosis or the OPG protein strongly inhibited mineralization in osteoblasts from neonatal mice. The present study was the first to demonstrate that acidosis inhibited mineralization, depressed c-Jun mRNA expression, and induced OPG production in human osteoblasts. These results suggest that OPG is involved in mineralization via c-Jun in human osteoblasts.

AB0602 Decreased serum CRP and MMP-3 levels in patients with rheumatoid arthritis treated with vitamin K2

BackgroundIt has been reported that Vitamin K2 (VitK2) is a key inducer of bone mineralization in human osteoblasts and also inhibits osteoclastogenesis by induction of the osteoclast apoptosis (1). Human...

A large-conductance (BK) potassium channel subtype affects both growth and mineralization of human osteoblasts

The pharmacology of the large-conductance K(+) (BK) channel in human osteoblasts is not well defined, and its role in bone is speculative. Here we assess BK channel properties in MG63 cells and primary human osteoblasts and determine whether pharmacological modulation affects cell function. We used RT-PCR and patch-clamp methods to determine the expression of BK channel subunits and cell number assays in the absence and presence of BK channel modulators. RT-PCR showed the presence of KCNMA1, KCNMB1, KCNMB2, KCNMB3, and KCNMB4 subunits. The BK channel was voltage dependent, with a mean unitary conductance of 228.8 pS (n = 10) in cell-attached patches (140 mM K(+)/140 mM K(+)) and a conductance of 142.5 pS (n = 16) in excised outside-out and 155 pS (n = 6) in inside-out patches in 3 mM K(+)/140 mM K(+). The selectivity ratio (ratio of K(+) to Na(+) permeability) was 15:1. The channel was blocked by tetraethylammonium (TEA, 0.3 mM), iberiotoxin (5-60 nM), tetrandrine (5-30 microM), and paxilline (10 microM) and activated by isopimaric acid (20 microM). BK channel modulators affected MG63 cell numbers: TEA and tetrandrine significantly increased cell numbers at low concentrations (3 mM and 3 microM, respectively) and reduced cell numbers at higher concentrations (>10 mM and >10 microM, respectively). Neither iberiotoxin (20-300 nM) nor slotoxin (300 nM) affected cell numbers. The increase in cell numbers by TEA was blocked by isopimaric acid. TEA (0.1-3.0 mM) significantly increased mineralization in primary osteoblasts. In conclusion, the BK channel has a distinctive pharmacology and is thus a target for therapeutic strategies aimed at modulating osteoblast proliferation and function.

Isoform of Fibronectin Mediates Bone Loss in Patients With Primary Biliary Cirrhosis by Suppressing Bone Formation

Osteoporosis is a major cause of morbidity and decreased quality of life in patients with chronic cholestatic liver disease. It is established that this osteoporosis results from decreased bone formation, but the mechanisms for the interaction between liver and bone remain elusive. The aim of this study was to test the hypothesis that an increase in the production of cellular fibronectins during liver disease may result in decreased osteoblast-mediated mineralization and thus explain the decrease in bone formation. We performed a prospective cross-sectional study in patients with primary biliary cirrhosis and matched controls, followed by experiments on human and mouse osteoblasts in culture and injections in mice in vivo. In patients with primary biliary cirrhosis, the oncofetal domain of fibronectin correlated significantly with the decrease in osteocalcin, a marker of bone formation (r = -0.57, p < 0.05). In vitro, amniotic fluid fibronectin (aFN) containing mainly the oncofetal domain and EIIIA domain resulted in decreased osteoblast-mediated mineralization in human osteoblasts (69% decrease at 100 microg/ml; p < 0.01) and mouse osteoblasts (71% decrease; p < 0.05). Removing the EIIIA domain from aFN similarly suppressed mineralization by osteoblasts (78% decrease; p < 0.05). Injection of labeled aFN in mice showed that it infiltrates the bone, and its administration over 10 days resulted in decreased trabecular BMD (17% drop; p < 0.05), mineralizing surface (30% drop; p < 0.005), and number of osteoblasts (45% drop; p < 0.05). Increased production of a fibronectin isoform containing the oncofetal domain and its release in the circulation in patients with primary biliary cirrhosis is at least partially responsible for the decrease in bone formation seen in these patients. This establishes that a molecule that has thus far been viewed as an extracellular matrix protein exerts hormone-like actions.

Therapeutic touch affects DNA synthesis and mineralization of human osteoblasts in culture

Complementary and alternative medicine (CAM) techniques are commonly used in hospitals and private medical facilities; however, the effectiveness of many of these practices has not been thoroughly studied in a scientific manner. Developed by Dr. Dolores Krieger and Dora Kunz, Therapeutic Touch is one of these CAM practices and is a highly disciplined five-step process by which a practitioner can generate energy through their hands to promote healing. There are numerous clinical studies on the effects of TT but few in vitro studies. Our purpose was to determine if Therapeutic Touch had any effect on osteoblast proliferation, differentiation, and mineralization in vitro. TT was performed twice a week for 10 min each on human osteoblasts (HOBs) and on an osteosarcoma-derived cell line, SaOs-2. No significant differences were found in DNA synthesis, assayed by [(3)H]-thymidine incorporation at 1 or 2 weeks for SaOs-2 or 1 week for HOBs. However, after four TT treatments in 2 weeks, TT significantly (p = 0.03) increased HOB DNA synthesis compared to controls. Immunocytochemistry for Proliferating Cell Nuclear Antigen (PCNA) confirmed these data. At 2 weeks in differentiation medium, TT significantly increased mineralization in HOBs (p = 0.016) and decreased mineralization in SaOs-2 (p = 0.0007), compared to controls. Additionally, Northern blot analysis indicated a TT-induced increase in mRNA expression for Type I collagen, bone sialoprotein, and alkaline phosphatase in HOBs and a decrease of these bone markers in SaOs-2 cells. In conclusion, Therapeutic Touch appears to increase human osteoblast DNA synthesis, differentiation and mineralization, and decrease differentiation and mineralization in a human osteosarcoma-derived cell line.

Effect of different growth factors on human osteoblasts activities: A possible application in bone regeneration for tissue engineering

Cultured human primary osteoblasts reproduce the phenotypic differentiation and maturation of cells in vivo. We have investigated the influence of three isoforms of transforming growth factor beta (TGF-β1, TGF-β2 and TGF-β3), three fibroblast growth factors (FGF-2, FGF-4 and FGF-6) and the active metabolite of Vitamin D [1,25-(OH) 2D3] on proliferation, alkaline phosphatase activity and mineralization of human osteoblasts during a period of 24 days of culture. TGF-β isoforms and three FGFs examined have been proved to be inducers of osteoblasts proliferation (higher extent for TGF-β and FGF-2) and inhibitors of alkaline phosphatase activity and osteoblasts mineralization. Combination of these growth factors with the active form of Vitamin D induced osteodifferentiation. In fact Vitamin D showed an additive effect on alkaline phosphatase activity and calcium content, induced by FGF-2 and TGF-β in human osteoblast. These results highlight the potential of proliferating cytokines’ combination with mineralizing agents for in vitro bone growth induction in bone tissue engineering.

Topography-induced alterations in adhesion structures affect mineralization in human osteoblasts on titanium

The influence of the surface topography of titanium on adhesion proteins like tensin was investigated to reveal a correlation between cellular structures and function in human osteoblasts. We investigated the following pure titanium surfaces: polished, machined, glass blasted, and corundum blasted with R a values: 0.19, 0.54, 1.22, and 6.07 μm, respectively. Corundum-blasted titanium (CB) as the roughest surface with sharp edges inhibited the formation of fibrillar structures of tensin, impaired the colocalization of tensin and the β1 integrin, and affected the organization of the actin cytoskeleton in MG-63 cells. These effects correlated with a reduced mineralization. We suggest that distinct alterations in adhesion structures due to the surface topography are responsible for differences in cell signaling, which lead to changes in the cellular function like mineralization.

Growth behavior, matrix production, and gene expression of human osteoblasts in defined cylindrical titanium channels.

The purpose of the current study was to investigate the effect of different diameters of cylindrical titanium channels on human osteoblasts. Titanium samples having continuous drill channels with diameters of 300, 400, 500, 600, and 1000 microm were put into osteoblast cell cultures that were isolated from 12 adult human trauma patients. Cell migration into the drill channels was investigated by transmitted-light microscopy. The DNA content in the drill channels was measured photometrically, collagen type I production was analyzed by enzyme-linked immunosorbent assay (ELISA) and osteocalcin gene expression by reverse transcriptase-polymerase chain reaction (RT-PCR). Formation of mineralized tissue was assessed by microradiographs of histological sections. Within 20 days, cells grew an average of 838 microm (+/-128 microm) into the drill channels with a diameter of 600 microm and were significantly faster (p < 0.05) than in all other channels. Cells produced significantly more osteocalcin messenger RNA (mRNA) in 600-microm channels (p < 0.05) than they did in 1000-microm channels and demonstrated the highest osteogenic differentiation. The channel diameter did not influence collagen type I production. The highest cell density was found in 300-microm channels (p < 0.05). The DNA content of the channels linearly decreased with increasing channel diameters. After 40 days of culture, the proportion of mineralized tissue at the mouth section amounted to 6% in 300-microm channels and to 9-11% in 400-600-microm channels. In 1000-microm channels, only traces of mineralization were detected. Our data suggest that the diameter of cylindrical titanium channels has a significant effect on migration, gene expression, and mineralization of human osteoblasts.