Enhanced differentiation and mineralization of human fetal osteoblasts on PDLLA containing Bioglass composite films in the absence of osteogenic supplements.

This study investigates the cellular response of fetal osteoblasts to bioactive resorbable composite films consisting of a poly-D,L-lactide (PDLLA) matrix and bioactive glass 45S5 Bioglass (BG) particles at three different concentrations (0% (PDLLA), 5% (P/BG5), and 40% (P/BG40)). Using scanning electron microscopy (SEM) we observed that cells were less spread and elongated on PDLLA and P/BG5, whereas cells on P/BG40 were elongated but with multiple protrusions spreading over the BG particles. Vinculin immunostaining revealed similar distribution of focal adhesion contacts on all cells independent of substratum, indicating that all materials permitted cell adhesion. However, when differentiation and maturation of fetal osteoblasts was examined, incorporation of 45S5 BG within the PDLLA matrix was found to significantly (p < 0.05) enhance alkaline phosphatase enzymatic activity and osteocalcin protein synthesis compared to tissue culture polystyrene controls and PDLLA alone. Alizarin red staining indicated extracellular matrix mineralization on both P/BG5 and P/BG40, with significantly more bone nodules formed than on PDLLA. Real time RT-PCR revealed that expression of bone sialoprotein was also affected by the BG containing films compared to controls, whereas expression of Collagen Type I was not influenced. By performing these investigations in the absence of osteogenic factors it appears that the incorporation of BG stimulates osteoblast differentiation and mineralization of the extracellular matrix, demonstrating the osteoinductive capacity of the composite.

Osteoblast attachment and mineralized nodule formation on rough and smooth 45S5 bioactive glass monoliths.

Human primary osteoblast responses to smooth and roughened bioactive glass of 45S5 (Bioglass trade mark ) composition (46.1% SiO(2), 26.9% CaO, 2.6% P(2)O(5), 24.4% Na(2)O) were analysed in vitro. The smooth and rough surfaces had R(a) values and peak to valley distances of 0.04, 4.397, 2.027, and 21.328 microm, respectively. Cell attachment and morphology was observed using phalloidin staining of the actin cytoskeleton and revealed significant differences between smooth and rough surfaces. Cells that were spiky in appearance on the rough compared to the smooth surface formed an organized actin matrix much later on the rough surface. Scanning electron microscopy revealed many cell filipodia extending from more rounded cell bodies on the rough surface. A significantly greater number of nodules on the rough surface was observed, and these were shown to mineralize when supplemented with beta-glycerophosphate and dexamethasone. Raman spectroscopy confirmed the presence of hydroxyapatite in the mineralized cultures showing a definite peak at 964 cm(-1). FTIR analysis showed hydroxyapatite formation occurred more rapidly on the rough surface. This study demonstrates that although initial cell morphology was less advanced on the roughened surface, the cells were able to form mineralized nodules in greater numbers. This may have implications to bone tissue engineering using bioactive glasses.

Comparative bone growth behavior in granules of bioceramic materials of various sizes.

Various bioceramic materials were implanted into 6-mm-diameter holes made in the femoral condyles of mature Japanese white rabbits using different-sized granules to find an optimal material and granule diameter for use as a bone graft. Bioceramics include a bioinert ceramic (Alumina), surface-bioactive ceramics [hydroxyapatite (HAp) and Bioglass(R)], and resorbable bioactive ceramics [alphatricalcium phosphate (alpha-TCP), beta-TCP, tetracalcium phosphate (TeCP), Te. DCPD, Te. DCPA, and low-crystalline HAp]. Granule sizes were 100-300, 10, and 1-3 microm. Bone growth behavior varied with the kind of bioceramic and the size used. For surface-bioactive ceramics, 45S5 Bioglass(R) led to more rapid bone proliferation than synthetic HAp. In resorbable bioactive ceramics, the order of resorption was: low-crystalline HAp and OCP > TeCP, Te DCPD, Te DCPA > alpha-TCP, beta-TCP. In terms of biocompatibility, alpha-TCP was better than beta-TCP.

Toxicology and biocompatibility of bioglasses.

Evidence for the lack of toxicity of various bioglass formulations has been deduced from studies carried out, both in vivo and in vitro, in several different centers. Recent studies of the authors, described here, include testing of solid bioglass implants in the soft tissues of rats and rabbits for time periods of up to eight weeks. Two new techniques are described for the toxicological testing of particulate biomaterials. These tests, which involve rat peritoneal macrophages in culture and a mouse pulmonary biomaterial embolus model, indicate the biocompatibility of bioglass powders. Thus, the surface activity so critical in bone adhesion is without toxic effect in non-osseous tissues in contact with solid bioglass implants. Should wear occur and produce particulate bioglass, the material should be eliminated without consequence.

Compositional dependence of the formation of calcium phosphate films on bioglass.

Bioglass, which has a composition of sodium carbonate, calcium carbonate, phosphorous pentoxide and silica, has been shown to bond to living bone. This ability is dependent on controlled surface reactions. Investigators with 45S5 bioglass have demonstrated that the formation of a SiO2-rich layer and a calcium phosphate film on its surface in an aqueous environment is associated with the film bonding the bioglass to bone. The objects of this research were: 1. To study SiO2 dependence on the formation of a silica-rich layer and calcium phosphate films on a bioglass surface in a simulated physiological solution, and 2. To establish a correlation between in vitro surface reactions and in vivo bonding ability. It was discovered that three types of reactions occur in a simulated physiological solution depending on bioglass composition: 1. A calcium phosphate film and SiO2-rich layer form simultaneously and the reaction rate is fast for bioglasses which have a lower content of SiO2 (approximately 46 mol% SiO2). 2. A SiO2-rich layer forms first and a calcium phosphate film develops later between the aqueous environment and the SiO2-rich layer for bioglasses whose SiO2 content is between 46--55 mol %. 3. A calcium phosphate film does not form for glasses whose SiO2 content is more than 60 mol %.

Use of fracture mechanics theory in lifetime predictions for alumina and bioglass-coated alumina.

The fatigue behavior of alumina and bioglass-coated alumina was determined in air and biological test environments by the dynamic fatigue test technique in which strength is measured as a function of stressing rate. The good correlation found between the test data and fracture mechanics theory indicates that fatigue failure is controlled by the slow crack growth of preexisting flaws and that fracture mechanics theory can be used in making failure predictions for alumina and bioglass-coated alumina in biological environments. Thus, it is believed that lifetime predictions can be made for ceramic implants on the basis of short-term test data utilizing fracture mechanics principles.

Future developments and applications of biomaterials: an overview.

It is recommended that the emphasis of biomaterials research and development for the future should be to achieve improved reliability. Use of increasing numbers of implants per year coupled with decreasing long term (greater than 5 years) success rates are resulting in progressively larger numbers of reparative implant operations. This trend can be altered by emphasizing three areas of R&D: 1) Studies of composite biomaterial systems offering unique combinations of biological surface behavior and substrate mechanical performance; 2) Investigate mechanisms of interfacial reactions so that long term responses of the host-implant can be predicted; 3) Develop long term predictive relationships for biomaterials reliability based upon interfacial reactions, biomechanics, fracture mechanics, fatigue testing, and retrieval analysis. Brief examples of efforts to develop undrestanding in these three areas are described using bioglass coated metal and bioglass coated alumina implants.