Bone-Like Mineral and Organically Modified Bone-Like Mineral Coatings - Biological and Biomedical Coatings: Processing and Characterization - page 23

et al. 2006). These inhibitory effects are stronger when the BSA is in solution as compared
to being preadsorbed onto the substrate (Areva et al. 2002). Also, the stage at which the
protein is included affects the extent of inhibition in a concentration-dependent manner.
Increasing BSA concentration causes an increase in induction periods for apatite nucle-
ation. Addition of low concentrations of BSA (<10 g/l) during the growth phase causes an
increase in the growth rate of the crystals, whereas higher concentrations (>10 g/l) inhibit
the growth rate (Combes, Rey, and Freche 1999). Although the exact mechanism by which
coprecipitation occurs is not completely understood and may differ from protein to pro-
tein, these results suggest that proteins may modulate apatite formation by adsorbing to
the initially formed nuclei and stabilizing them by causing a decrease in interfacial energy
between the crystal and solution. At low concentrations, there is no sufficient protein to
coat the entire mineral surface, allowing the nuclei to grow quickly. At higher concentra-
tions due to coverage of the mineral surface by the protein molecules, growth is prevented
(Combes and Rey 2002).
Mineral crystals nucleated in the presence of BSA are smaller in size and less crystalline
compared to mineral formed in the absence of BSA (Liu et al. 2001; Combes and Rey 2002).
The morphology of the mineral is also affected; protein-free SBF forms sharp platelike
mineral crystals that are rounded in the presence of BSA (Luong et al. 2006; Liu et al. 2001)
(Figure 1.2). Coprecipitating BSA onto a premineralized surface causes higher quantities
of BSA to be loaded, which has been attributed to ability of the negatively charged BSA
to interact with the positively charged Ca 2+ ions. The BSA is attracted to the Ca 2+ ions in
the preliminary mineral layer, causing it to be incorporated into the mineral, which then
attracts the Ca 2+ ions from solution, resulting in a cyclical growth process (Luong et al.
2006; Liu et al. 2001). This interaction of BSA with Ca 2+ ions is confirmed by slower release
of Ca 2+ ions from coatings formed by coprecipitation with BSA as compared to that in the
absence of BSA (Liu et al. 2003).
The above findings show that coprecipitation can be used to integrate proteins into the
mineral layer, with their subsequent release being dependent primarily on the rate of dis-
solution of the mineral. The interaction of each protein with different types of mineral is
dependent on several factors including the size, concentration, and charge of the protein,
and its influence on the mineral characteristics such as size and crystallinity.
Applications of Protein Coprecipitation in Bone Tissue Engineering
Coprecipitation has been used to incorporate ECM proteins, enzymes, and drugs into bio-
mimetic calcium phosphate coatings. Bone analogs have been produced by coprecipitation
of collagen I and mineral onto PLLA substrates using a highly concentrated SBF solution
such as 5XSBF. These coatings are capable of enhancing proliferation and differentiation
of Saos-2 cells (human osteosarcoma cell line) in vitro (Chen et al. 2008). Coprecipitation
has also been used to incorporate enzymes such as amylase and lysozyme into BLM coat-
ings on starch-based polymers (Leonor et al. 2003). These materials can be potentially
used as stimulus-responsive scaffolds, undergoing gradual degradation by the incorpo-
rated enzyme over time (Martins et al. 2009). Antibiotics such as tobramycin have also
been integrated into biomimetic Ca-P coatings on titanium implants and hinder bacterial
growth. Biomimetically coated implants that are coprecipitated with antibiotics not only
possess the osteoconductive properties of BLM coatings, but are also capable of preventing
postoperative infections (Stigter, de Groot, and Layrolle 2002).
Cell-matrix interactions in the natural bone environment orchestrate complex growth
factor release profiles that help control bone resorption and formation. TGF-β1 is present