Bioactive glass stimulates in vitro osteoblast differentiation and creates a favorable template for bone tissue formation.

In this study, we have investigated the behavior of fetal rat osteoblasts cultured on bioactive glasses with 55 wt% silica content (55S) and on a bioinert glass (60S) used either in the form of granules or in the form of disks. In the presence of Bioglass granules (55 wt% silica content), phase contrast microscopy permitted step-by-step visualization of the formation of bone nodules in contact with the particles. Ultrastructural observations of undecalcified sections revealed the presence of an electron-dense layer composed of needle-shaped crystals at the periphery of the material that seemed to act as a nucleating surface for biological crystals. Furthermore, energy dispersive X-ray (EDX) analysis and electron diffraction patterns showed that this interface contains calcium (Ca) and phosphorus (P) and was highly crystalline. When rat bone cells were cultured on 55S disks, scanning electron microscopic (SEM) observations revealed that cells attached, spread to all substrata, and formed multilayered nodular structures by day 10 in culture. Furthermore, cytoenzymatic localization of alkaline phosphatase (ALP) and immunolabeling with bone sialoprotein antibody revealed a positive staining for the bone nodules formed in cultures on 55S. In addition, the specific activity of ALP determined biochemically was significantly higher in 55S cultures than in the controls. SEM observations of the material surfaces after scraping off the cell layers showed that mineralized bone nodules remained attached on 55S surfaces but not on 60S. X-ray microanalysis indicated the presence of Ca and P in this bone tissue. The 55S/bone interfaces also were analyzed on transverse sections. The interfacial analysis showed a firm bone bonding to the 55S surface through an intervening apatite layer, confirmed by the X-ray mappings. All these results indicate the importance of the surface composition in supporting differentiation of osteogenic cells and the subsequent apposition of bone matrix allowing a strong bond of the bioactive materials to bone.

In vitro bone formation on a bone-like apatite layer prepared by a biomimetic process on a bioactive glass-ceramic.

In this study we have investigated the behavior of fetal rat osteoblasts, cultured up to 23 days, on a bioactive apatite-wollastonite (AW) glass-ceramic and on the same material on which a carbonated apatite layer had been formed by a biomimetic process (AWa). At the last day of culture, the specific activity of alkaline phosphatase activity, as determined biochemically, was about 30% greater on AWa compared with AW disks. After the cell layers had been scraped off, scanning electron microscopic (SEM) observations of the materials' surfaces revealed that mineralized bone nodules remained attached to both surfaces but in larger amounts on AWa. X-ray microanalysis indicated the presence of calcium (Ca) and phosphorus (P) in the bone tissue throughout the AWa surface and Ca, P, and silicon (Si) on the AW surface. The AW/ and AWa/bone interfaces also were analyzed after fracturing of the disks. The interfacial analysis showed firm bone bonding to the AW and AWa surfaces, confirmed by the X-ray microanalytic mappings. These results indicate the importance of surface composition in supporting differentiation of osteogenic cells and the subsequent apposition of bone matrix, which allows a strong bond of the bioactive materials to the bone. Furthermore, prefabrication of a biologic apatite layer by a method that mimics biomineralization could find application to bone-repairing materials.

Cartilage formation by fetal rat chondrocytes cultured in alginate beads: a proposed model for investigating tissue-biomaterial interactions.

Chondrocytes from 21-day-old rat fetal nasal cartilage were cultured in alginate beads for up to 20 days. It was found that chondrocytes retained their spherical shape and typical chondrocytic appearance. During the culture time, chondrocytes underwent differentiation, as demonstrated by the alkaline phosphatase-specific activity and rate of proteoglycan synthesis. Morphological data confirmed chondrocyte differentiation with the appearance of hypertrophic chondrocytes scattered in the alginate gel and a dense extracellular matrix containing filamentous structures and matrix vesicles. In addition, Northern blot analysis performed on day 8 of culture showed that chondrocytes cultured in alginate beads expressed type II collagen mRNA. The alginate bead method also appeared to be suitable for testing biomaterials, and the ready dissolution of the alginate beads by chelating agents provided a simple means for the rapid recovery of encapsulated chondrocytes. Powdered glass-ceramic particles entrapped in the alginate gel were colonized by chondrocytes, which then proliferated and formed a tissue similar to a true calcified cartilaginous structure. These results indicate that the alginate system represents a relevant model for studies of chondrogenesis and endochondral ossification. Furthermore, the encapsulation method could prove useful for studies of tissue-biomaterial interactions in an in vitro environment which more closely mirrors the cartilage matrix than other culture methods.

Behavior of fetal rat chondrocytes cultured on a bioactive glass-ceramic.

We examined the behavior of fetal rat chondrocytes cultured on a bioactive glass-ceramic containing apatite and wollastonite (A.W.G.C.). Biomaterial surface topography and profiles were evaluated by bidimensional profilometry and revealed a rough surface for the glass-ceramic compared to the plastic coverslips used as controls. Chondrocyte attachment was evaluated by measuring the number of attached cells after one day of culture and by morphological observations. Chondrocytes attached in great numbers to the material surface by means of focal contacts containing vinculin and beta1-integrin. Fluorescent labeling of actin and vimentin revealed a poor spreading of chondrocytes on the bioactive glass-ceramic compared to the plastic coverslips, where the cells appeared to adhere intimately to the surface and exhibited polygonal arrays of stress fibers. During the following days of culture, chondrocytes proliferated, colonized the surface of the material, and, finally, on day 10, formed nodular structures composed of round cells separated by a dense extracellular matrix. Furthermore, these clusters of round cells were positive for type II collagen and chondroitin sulfate, both hard markers of the chondrocyte phenotype. In addition, protein synthesis, alkaline phosphatase activity, and proteoglycan production were found to increase gradually during the culture period with a pattern similar to that observed on control cultures. These results demonstrate that the bioactive glass-ceramic tested in this study appears to be a suitable substrate for in vitro chondrocyte attachment, differentiation, and matrix production.

Bioactive glass-ceramic containing crystalline apatite and wollastonite initiates biomineralization in bone cell cultures.

Rat bone cells were cultured in the presence of bioactive glass-ceramic containing crystalline apatite and wollastonite. Scanning electron microscopy observations of the surface of the seeded ceramic disks revealed that cells attached, spread, and proliferated on the material surface. Soaking in cell-free culture medium showed that no change occurred in the surface structure. However, when cultured with bone cells and observed under a transmission electron microscope, an electron-dense layer was noted initially at the surface of the material, before bone formation occurred. In addition, energy-dispersive X-ray microanalysis demonstrated the presence of calcium and phosphorus in this layer. Progressively, during the following days of culture, active osteoblasts synthetized and laid down an osteoid matrix composed of numerous collagen fibrils arranged either parallel or perpendicularly to the first-formed electron-dense layer. Mineralization initiated on the ceramic surface dispersed then along the collagenous fibrils, leading to a mineralized matrix which surrounded the ceramic particles. These results demonstrate the capacity of apatite-wollastonite glass ceramic to initiate biomineralization in osteoblast cultures and to achieve a direct bond between the surface apatite layer of the bioactive glass-ceramic and the mineralized bone matrix.