We are focussed on understanding the interface between living and synthetic (resorbable/man-made/artificial etc.) tissues. Part of this focus is constrained by our belief that the ultimate resorbable substrate will be a combination of polymer and ceramic. In the most optimistic view of hard tissue replacements the monolith must be able to function mechanically within the body during slow and measured regeneration of natural tissue (along with the attendant need for modulus matching). Thus the patient will stress the implant; much of the literature demonstrates that this is necessary to promote the development of the appropriate bone structure.
Using HA particles produced utilizing the procedure of Lazic, HA-PMMA composites were formed using standard dental techniques. These composites were then used in studies of osteoblast-surface interaction. The degree of interaction was assessed using a well characterized model of osteogenesis, fetal rat calvarial osteoblasts. Osteoblasts were isolated from 21-day-old fetal rat calvariae. Following an initial treatment of calvariae for 10 minutes at 37°C with 570 U/ml collagenase (Worthington Biochemical Corp., Freehold, NJ), the cells released from calvariae by two 10-minute and two 20-minute sequential collagenase digestions were pooled and then filtered through both 100 mm and 37 mm Nitex filters. Cells were grown overnight at 25,000 cells/cm2 on plastic dishes (Corning, Corning, NY). The cells were then detached, using 0.05% trypsin and 0.53 mM EDTA in Hanks buffered salt solution (Gibco, Grand Island, NY). Cells were plated at a density of 36,000 cells/cm2 on both the experimental (HA-PMMA) and the control substrates. The cells were grown in alpha-Minimum Essential Medium supplemented with 10% heat-inactivated fetal calf serum (Gibco). After confluence (3 days) the medium was further supplemented with freshly prepared ascorbic acid (50 mg/ml) and b -glycerophosphate (3 mM) (Sigma Chemical Co., St. Louis, MO) to trigger differentiation (contact Amr Moursi, Moursi.email@example.com, if this level of detail is not exhaustive enough). The results are pictured in Figure 1.
Overall observations showed that proliferation and attachment were similar to the control. Matrix deposition appeared to be uniform, however the micrographs (Figure 1) show that cell-HA interactions were extensive. Osteoblast attachment to these composites was higher than that of PMMA or dense Ti substrates.
Figure 1a). SEM micrograph of an HA-PMMA composite surface following a 14 day exposure to calvarial osteoblast culture. The cell layer has been removed to allow for direct observation of the effects of exposure on the ceramic particles. The HA is visible as irregular solid pieces in the PMMA matrix. Fine parallel lines are an artifact of the cutting operation used to produce this surface.
Figure 1b). Micrograph of calvarial osteoblast cell layer cultured on the HA-PMMA composite surface for 14 days. The cell layer shows evidence of abundant attachment, proliferation and extra-cellular matrix deposition. Correlations between the characteristics of the overall cell layer and HA particle location have not yet been established.
Figure 1c). Higher magnification image of an HA particle embedded in the polymer matrix. Separation of the particle from the polymer matrix is evident. The surface of the particle is no longer visible due to deposition of collagen and other fibrillar and globular extra-cellular matrix components. The PMMA surrounding the particle shows much less evidence of matrix deposition.
Figure 1d). High magnification micrograph of the exposed particle surface covered with a network of fibrillar and globular extra-cellular matrix components. Clearly, matrix synthesis and deposition was very strong during the 14 day culture period. Cross-sectional TEM studies will be necessary to observe these cell-HA interactions at a more meaningful level.