Effect of bioactive ceramic dissolution on the mechanism of bone mineralization and guided tissue growth in vitro.

A major objective of this research work was to evaluate the effect of bone cells on the dissolution-precipitation reaction in vitro. Rat bone marrow stem cells were seeded on silica-calcium phosphate nano composite (SCPC) with different chemical compositions and crystalline structures. Measurements of the Ca, P, Si, and Na concentrations in the tissue culture media using inductively coupled plasma indicated that bone marrow stem cells attached to the surface of SCPC did not affect the dissolution behavior of the material. However, bone marrow stem cells interfered with the back precipitation reaction and inhibited the formation of a calcium phosphate (Ca-P) layer on the material surface. Scanning electron microscope-energy-dispersive X-ray analyses showed that, in the absence of cells, a Ca-P layer formed on the material surface because of the dissolution-precipitation reaction. Bone cells attached to SCPC that contains high silica content absorbed significantly higher concentrations of medium Ca than cells attached to SCPC that contains low silica content. In conjunction with the absorption of high Ca concentration, attached bone marrow stem cells produced calcified nodules and mineralized extracellular matrix, indicating osteoblastic differentiation. Results of the study strongly suggest that the mechanism of bone mineralization at the interface with bioactive ceramics is mainly cell mediated and is enhanced by the absorption of critical concentrations of dissolved Ca and P. The silicon-rich phase also provided a guided cell adhesion and tissue growth in vitro. The enhanced bioactivity reactions and strong stimulatory effect on bone cell function are attributed to the modified crystalline structure of the SCPC material.

Effects of silica on the bioactivity of calcium phosphate composites in vitro.

In the present study, silica-calcium phosphate composites (SiO(2)-CaP composites) were developed by mixing the starting materials (SiO(2) and CaHPO(4)) in different ratios with the addition of 0.1% w/v NaOH solution. The phase composition of the SiO(2)-CaP composites was determined by XRD and FTIR. After thermal treatment at 350 degrees C/1 h and at 1000 degrees C/3.5 h; all SiO(2)-CaP composites composed of beta-quartz, alpha-cristobalite and beta-Ca2P2O7. The presence of calcium phosphate enhanced the transformation of beta-quartz into alpha-cristobalite at 1000 degrees C. SEM observation indicated favorable attachment and spreading of neonatal rat calvaria osteoblasts onto the surface of silica-rich SiO(2)-CaP composites. After attachment, these cells produced significantly higher amount of protein and expressed higher AP activity than cells attached to silica-poor samples. Results of the study suggested that the silica-based composites are more bioactive than calcium phosphate-based composites. Silica promoted the expression of osteoblast phenotype by both solution-mediated effect and direct interaction with the surface of the substrate.

Cyclosilicate nanocomposite: a novel resorbable bioactive tissue engineering scaffold for BMP and bone-marrow cell delivery.

Porous bioactive resorbable silica-calcium phosphate nanocomposite (SCPC) was prepared by a sintering technique. XRD analyses showed that the main crystalline phases of the SCPC are Na(3)CaPSiO(7) (clinophosinaite), beta-NaCaPO(4) (rhenanite), Na(2)CaSiO(4), and beta-quartz (SiO(2)). The clinophosinaite is a novel cyclosilicate bioactive mineral that enhanced the mechanical and bioactivity properties of the SCPC. TEM analysis showed that the grain sizes of the multiphase SCPC are in the nanometer scale. Moreover, the SCPC was engineered with nano- and microscale porosity. The SCPC had significantly higher compressive strength than porous hydroxyapatite (HA). FTIR analyses revealed the formation of biological hydroxyapatite layer on the SCPC surface after 4 days of immersion in SBF. When SCPC was loaded with rhBMP-2, it provided a superior release profile of biologically active rhBMP-2 compared to porous HA. Bone-marrow cells incubated with medium treated with the rhBMP-2 released from the SCPC-rhBMP-2 hybrid expressed significantly higher alkaline phosphatase activity than that expressed by cells incubated with media treated with rhBMP-2 released from HA-rhBMP-2. In addition, cells attached to the SCPC-rhBMP-2 hybrid produced mineralized extracellular matrix (ECM) and bone-like tissue that covered the material surface and filled pores in the entire thickness of the template after 3 weeks in culture. In contrary, cells attached to the HA-rhBMP-2 produced limited amount of unmineralized ECM after the same time period. Results of the study strongly suggest that the porous bioactive silica-calcium phosphate nanocomposite can serve as a delivery system for cells and biological molecules. The SCPC-rhBMP-2-marrow cell hybrid may serve as an alternative to autologous bone grafting.

Model surfaces engineered with nanoscale roughness and RGD tripeptides promote osteoblast activity.

Cell adhesion to biomaterials is a prerequisite for tissue integration with the implant surface. Herein, we show that we can generate a model silica surface that contains a minimal-length arginine-glycine-aspartic acid (RGD) peptide that maintains its biological activity. In the first part of this study, attachment of MC3T3-E1 osteoblast-like cells was investigated on silicon oxide, amine terminated substrates [i.e., 3-aminopropyl triethoxysilane (APTS)], grafted RGD, and physisorbed RGD control. The APTS layer exhibited nanoscale roughness and presented amine functional groups for grafting a minimal RGD tripeptide devoid of any flanking groups or spacers. Contact angle measurements indicated that the hydrophobicity of the APTS surface was significantly lower than that of the surface with grafted RGD (RGD-APTS). Atomic force microscopy showed that surfaces covered with RGD-APTS were smoother (Ra = 0.71 nm) than those covered with APTS alone (Ra = 1.59 nm). Focusing mainly on cell morphology, experiments showed that the RGD-APTS hybrid provided an optimum surface for cell adhesion, spreading, and cytoskeletal organization. Discrete focal adhesion plaques were also observed consistent with successful cell signaling events. In a second set of experiments, smooth, monolayers of APTS (Ra = 0.1 nm) were used to prepare arginine-glycine-aspartic acid-serine (RGDS)-APTS and arginine-glycine-glutamic acid-serine (RGES)-APTS (control) substrates. Focusing mainly on cell function, integrin and gene expression were all enhanced for rate osteosarcoma cells on surfaces containing grafted RGDS. Both sets of studies demonstrated that grafted molecules of RGD(S) enhance both osteoblast-like cell adhesion and function.

Crystallization behavior of silica-calcium phosphate biocomposites: XRD and FTIR studies.

Silica and calcium phosphates (CaP) are the most important ingredients in bioactive materials that bond to bone and enhance bone tissue formation. In this study, silica-calcium phosphate (SiO2-CaP) composites were developed by powder metallurgy method, using silica (SiO2) and anhydrous dicalcium phosphate (CaHPO4) powders (CaP) in the ratios (wt%): 20/80, 40/60, 60/40 and 80/20. The effects of temperature and chemical composition on crystallization and phase transformation of the SiO2-CaP composites were evaluated by XRD and FTIR. Thermal treatment of the starting material suggested that CaHPO4 transforms into: gamma-Ca2P2O7 at 800 degrees C; beta-Ca2P2O7 at 1000 degrees C and alpha-Ca2P2O7 at 1200 degrees C. On the other hand, beta-quartz was the only detected phase after thermal treatment of silica in the temperature range 800-1200 degrees C. For all SiO2-CaP composites, SiO2 and CaP did not modify the crystallization behavior of each other when sintered in the temperature range 800-1000 degrees C. However, at 1200 degrees C, CaP promoted the transformation of gamma-quartz into alpha-cristobalite. Moreover, SiO2 stabilized beta-Ca2P2O7. The modifications in the crystallization behavior were related to ion substitution and formation of solid solutions.

Effect of thermal treatment on bioactive glass microstructure, corrosion behavior, zeta potential, and protein adsorption.

Bioactive glass ceramic is characterized by high mechanical strength and a slow rate of bone bonding. To understand the factors contributing to a decrease in the rate of bone bonding to bioactive glass ceramic, we evaluated the effect of different percentages of bioactive glass crystallization on corrosion behavior, zeta potential, and serum protein adsorption. X-ray diffraction analysis showed that heat treatment of bioactive glass in the temperature range 550 degrees -700 degrees C resulted in the precipitation of Na(2)Ca(2)Si(3)O(9) crystals in the glass matrix. The percentage of crystallization increased in the order: 5%, 8%, 45%, and 83% after thermal treatment at 550 degrees, 600 degrees, 650 degrees, and 700 degrees C/1 h, respectively. Scanning electron microscopic analyses of bioactive glass treated at 550 degrees C showed major glass in glass-phase separation. Moreover, energy-dispersive X-ray analyses indicated that during crystallization P is concentrated in the glassy phase. Induced-coupled plasma analyses showed that after 24 h immersion in simulated body fluid, the concentration of the released P ion increased as the crystallization percentage of bioactive glass increased. zeta potential of bioactive glass samples containing 5% crystallization had a statistically significant higher negative value than control untreated bioactive glass (p <.02). Control untreated bioactive glass adsorbed a statistically significant higher amount of serum protein than bioactive glass samples containing 5% crystallization (p <.02). Results of our study suggest that inhibition of protein adsorption might be responsible for the slow rate of bone bonding to bioactive glass ceramic. It is also possible that conformation changes inhibit the activity of the protein adsorbed onto thermally treated bioactive glass.

Effect of serum proteins on osteoblast adhesion to surface-modified bioactive glass and hydroxyapatite.

Previous studies indicate that modification of the surface of porous bioactive glass promotes osteoblast function. We hypothesize that bone formation on treated bioactive glass is due to the selective adsorption of serum attachment proteins. To test this hypothesis, we examined the profile of proteins adsorbed to treated bioactive glass and compared these proteins with those adsorbed to untreated bioactive glass and porous hydroxyapatite. Porous bioactive glass was treated with Tris-buffered electrolyte solution to generate a calcium phosphate-rich surface layer and then immersed in tissue-culture medium containing 10% serum. Proteins adsorbed to the ceramic surfaces were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. Porous hydroxyapatite bound a higher amount of total protein than did the other substrates. However, surface-modified porous bioactive glass adsorbed more fibronectin than did hydroxyapatite. The effect of serum-protein adsorption on osteoblast adhesion to bioactive glass and hydroxyapatite was also evaluated. Cell adhesion to porous bioactive glass that was surface-modified and serum-treated was significantly greater than to porous bioactive glass that was either surface-modified or serum-treated. Furthermore, cell adhesion to porous bioactive glass treated to form the dual layer of calcium phosphate and serum protein was significantly higher than adhesion to porous hydroxyapatite with adsorbed serum protein. Results of the study strongly suggest that adsorption of serum fibronectin to the surface of modified porous bioactive glass coated with calcium phosphate may be responsible for enhanced osteoblast adhesion.

Laminin-5 coating enhances epithelial cell attachment, spreading, and hemidesmosome assembly on Ti-6A1-4V implant material in vitro.

Enhancement of epithelial cell attachment to laminin-5-coated titanium alloy (Ti-6Al-4V) implant material was evaluated in vitro. Protein analysis showed that Ti-6Al-4V has a high affinity for laminin-5 and adsorbed significantly more laminin-5 than laminin-1. DNA analysis showed that laminin-5 enhanced attachment of normal human epidermal keratinocytes (NHEK) to Ti-6Al-4V significantly more than did laminin-1 or uncoated controls. The effect of passivation on laminin-5 adsorption and activity on Ti-6Al-4V also was evaluated. Passivation had no significant effect on the amount of protein adsorbed; however, AFM, ESCA, and ToF-SIMS analyses suggested that passivation affects the conformation of adsorbed laminin-5. Although laminin-5 coating significantly enhanced rapid attachment of epithelial cells to both passivated and unpassivated Ti-6Al-4V, surface area measurements showed that cells spread on laminin-5-coated passivated Ti-6Al-4V covered a significantly larger surface area than cells spread on laminin-5-coated unpassivated samples. TEM analysis showed that cells formed significantly more hemidesmosomes on the surface of laminin-5 coated passivated than on the surface of laminin-5 coated unpassivated titanium alloy. The enhancement of rapid cell attachment, spreading, and hemidesmosome assembly on laminin-5-coated passivated samples may reflect better integration between epithelial cells and titanium alloy and thus may be predictive of long-term implant stability.

Porous bioactive glass and hydroxyapatite ceramic affect bone cell function in vitro along different time lines.

We describe the effects on cell function of treating porous bioactive glass (BG) such that its surface is a composite of carbonated hydroxyapatite and serum protein. The effects on bone cell function of porous hydroxyapatite (HA) ceramic and porous glass treated to become amorphous calcium phosphate only also were studied subsequent to their having adsorbed a serum protein layer. Substrates treated for different durations were seeded with MC3T3-E1 cells and cultured for 3-17 days. Whereas cells seeded on any substrates, BG and HA produced collagen types I and III, bone sialoprotein, and osteopontin, there were significant differences between HA and BG, and among the various surface conditions created on BG. Covering the glass surface with hydroxyapatite and serum protein enhanced expression of high alkaline phosphatase activity, high rates of cell proliferation, and production of mineralized extracellular matrix. The enhancement may be due to the adsorption of a high quantity of fibronectin from the serum onto the reacted bioactive glass surface.

Formation of surface reaction products on bioactive glass and their effects on the expression of the osteoblastic phenotype and the deposition of mineralized extracellular matrix.

The objective of the study was to examine the effect of alkali ion release, pH control and buffer capacity on the expression of the osteoblastic phenotype. In addition we determined the importance of modifications of the surface of porous bioactive glass (BG) on the activity of rat calvaria osteoblasts in vitro. We found that at a low tissue culture medium (TCM) volume to BG surface area (Vol/SA) ratio, the products of glass corrosion elevated the pH of the TCM to a value that adversely affected cellular activity; thus, the matrix synthesized by the cells was non-mineralized. On the other hand, when the Vol/SA was high and the buffer capacity of the medium was not exceeded, the cells generated a mineralized extracellular matrix. Addressing the second issue, we observed that modification of the composition of the BG surface markedly influenced osteoblast activity. BG that was coated with either a calcium phosphate-rich layer only or a serum protein layer changed the phenotypic characteristics of the osteoblasts. The presence of either of these surfaces lowered the alkaline phosphatase activity of the attached cells; this finding indicated that the osteoblast phenotype was not conserved. However, when the BG was coated with a bilayer of calcium phosphate and serum proteins, the alkaline phosphatase (AP) activity was elevated and the extracellular matrix contained characteristic bone markers. Our findings indicate that the calcium phosphate-rich layer promotes adsorption and concentration of proteins from the TCM, and it is utilized by the osteoblasts to form the mineralized extracellular matrix.

Genital Chlamydia trachomatis infection in Egyptian women: incidence among different clinical risk groups.

OBJECTIVE: To investigate genital Chlamydial infection incidence among high risk clinical conditions in Egyptian women. METHODS: A case control study in Ain Shams University Hospital involving 501 patients with cervicitis (n = 58), abnormal cervical smear (n = 256), tubal infertility (n = 85), ectopic pregnancy (n = 22), preterm labour (n = 80) and 192 controls. Active cervical Chlamydial infection was diagnosed using direct immunofluorescent technique. Data were analyzed by Chi-square (chi 2) and Z tests. RESULTS: Significant increase of Chlamydial infection among different clinical conditions compared to controls. The percentage of positive chlamydial infection was 79.3% among cervicitis group, 33.3% among subjects with inflammatory smear, 75.2% among those with cervical condyloma, 82.6% among those with cervical intraepithelial neoplasia, 51.8% among tubal infertility subjects, 77.2% among ectopic patients and 56.3% among subjects with preterm labour. CONCLUSION: The incidence of Chlamydial infection in these high risk Egyptian patients is relatively high. Emperical treatment is recommended as the diagnosis is costly and usually not available.

Bioactive material template for in vitro synthesis of bone.

We describe the synthesis of a new, porous, modified bioactive glass for use as a template for bone formation in vitro. The porosity of the glass was 36.4%; the pore size ranged from 10-160 mm, and there was no incipient devitrification. Prior to seeding the glass with cells, it was necessary to condition the disks. Optimum conditioning was achieved by immersing the templates in a tris buffer at pH 6.8 for 48 h and then treating the glass with tissue culture medium for 1 h at 37 degrees C. The conditioned glass disks were seeded with 10(6) neonatal rat calvaria osteoblast-like cells; cells on the substrate were maintained in culture for 3-7 days. To prevent pH shifts due to corrosion of the conditioned glass, the medium:glass ratio was maintained at 90 ml/g. We found that the templates were rapidly invaded by cells which maintained the osteoblast phenotype; thus, they exhibited high alkaline phosphatase activity and synthesized type I collagen and osteocalcin. SEM-EDAX showed that the cells elaborated substantial amounts of extracellular matrix and a bonelike tissue was present throughout the entire template thickness. FTIR analysis of material formed in the glass indicated that the mineral phase was a biologic hydroxyapatite. Controls (cells without substrate and substrate without cells) exhibited none of these features. Results of the study suggest that this porous glass can function as a template for generating bone in vitro.

Effect of bioactive glass templates on osteoblast proliferation and in vitro synthesis of bone-like tissue.

Using in vitro synthesized bone tissue with cells aspirated from the patient's marrow is an appealing idea to avoid the profound limitations of biological and synthetic grafts. Procedures to synthesize bone tissue in vitro primarily relied on seeding various substrates with cells that have osteogenic capacity in culture. It should be noted that in an in vitro system, osteoprogenitor cells, as well as bone cells themselves can rapidly change their phenotype, hence the substrate needs to promote the expression of the bone cell phenotype. Furthermore, it needs to provide a template for bone deposition while gradually resorbing once bone tissue has been laid down. This paper presents initial evidence that bioactive glass, a synthetic material with documented extensive bone bioactivity properties, represents a material that optimally combines the requirements of the ideal template for in vitro synthesis of bone tissue. When made in porous form, and conditioned to develop a bone-like surface prior to being seeded with pluripotential cells capable of expressing the osteoblastic phenotype, these templates lead to expeditious and abundant in vitro synthesis of extracellular matrix with most important characteristics of bone tissue.