# Biodegradable and Biocompatible Systems Based on Hydroxapatite Nanoparticles
## 4 Functional HAp Biocomposites through Modification/Encapsulation Techniques
### Protein Adsorption and Release from HAp Nanoparticles  
Adsorption of peptides and proteins onto HAp surfaces may favor cell attachment and cell differentiation events and consequently may play a fundamental role in bone remodeling. Great efforts have been focused onto the adsorption of growth factors (e.g., the arginine-glycine-aspartic acid peptide, RGD) and collagen proteins. This adsorption process does not depend on peptide/protein characteristics, such as conformation and functional groups, and HAp surface characteristics, such as roughness, porosity, pore size, charge and growth face. Ionic interactions are easily established through calcium and phosphate ions of HAp and the charged groups of the organic molecules (NH3+, COO-). Lysozyme (LSM) is a small globular protein containing 129 amino acid residues that is stabilized by four internal disulfide bridges and has a net charge that varies with pH [136]. LSM has a wide interest in biomedicine since it catalyzes the hydrolysis of the \(\beta\)-(1, 4) glycosidic bond of peptidoglycan that constitute the cytoplasmic membrane of Gram-positive bacteria [137]. Therefore, multiple applications can be derived from the design of LSM delivery systems [138]. The adsorption of LSM onto HAp surfaces has been proposed in order to avoid problems caused by the relative instability of the protein. Adsorption could be favored trough the strong interactions that could be established between phosphate groups and LSM functional groups. The adsorbed protein can moreover be easily delivered by competition with other cations of the buffer release medium [139]. Selenium substituted HAps have been revealed appropriate to increase the LSM upload (e.g., 41.7% versus the 34.1% determined for non-substituted HAp) and slow the protein release. The results are also interesting since selenium substituted HAp nanoparticles can induce both apoptosis of bone tumor cells and enhance osteointegration [140]. Nano-sized HAp particles with needle-like morphology were prepared by using carboxymethyl cellulose as the template. These particles derived from spherical amorphous CaP particles after aging (14 days) under ambient conditions. A high LSM adsorption capacity (i.e., up to 930 mg/g) was observed as well as a pH-sensitive release of adsorbed LSM [141]. Surface charge of HAp nanoparticles can be modified by immobilization of amino acids by means of a precipitation process. Acidic amino acids (e.g., aspartic or glutamic acids) lead to a negatively charged surface that is ideal for the selective uptake of positive charged LSM. By contrast, basic amino acids (e.g., arginine) enhance the adsorption of negative charged proteins such as bovine serum albumin [142]. In the same way, organic compounds like mercaptosuccinic acid and citric acid were employed to increase the uptake of cytochrome C [143] and LSM [144], respectively. In the same way, the load of bone morphogenetic proteis could be enhanced (i.e., more than 50% increase in BMP-2 adsorption was observed when HA was prepared in the presence of 0.1 M of CA) and its release increased [144]. By contrast, zwitterionic amino acids, which contain two counter-charged groups and maintain overall charge neutrality, can prevent HAp surface from nonspecific protein adsorption and even provide clear antifouling properties [145]. Growth of HAp crystals is often inhibited by the adsorption of organic compounds (e.g., proteins and amino acids). This feature is highly interesting since a decrease on the degree of crystallinity should affect the mineralization of HAp and even could make feasible the attainment of a controlled solubility as recently proposed [146] when amino acids such as glycine, serine, aspartic acid and glutamic acid were considered. Alpha(2)-HS glycoprotein/fetuin A (Ahsg) is a well-known inhibitor of precipitation of CaPs that prevents calcification processes. The effect given by fetuin A is a consequence of the formation of colloidal spheres capable of incorporating CaPs.