To improve understanding of the mechanism of calcification of hydrogel implants, the calcification process for PVA/VP and PHEMA-based hydrogels both in-vitro and in-vivo have been studied.
Water in hydrogels plays two important roles during the calcification process. First, bound water facilitates the chelation of calcium ions onto the polymer, and second intermediate water mediates the diffusion of calcium ions. The diffusion coefficients of calcium ions in the PHEMA and PVA/PVP hydrogels were found to be approximately 1.31 X 10-11 and 4.14 x 10-10 m2/s, respectively.
The salting-out effect suggests that PHEMA hydrogels are the most attractive for chelation of calcium ions, whilst P(HEMA-co-NVP) hydrogels are the least susceptible. As evidenced by NMR and infrared analyses, the interactions between calcium ions and the polymer occur preferentially onto the hydroxyl and ester oxygen atoms of the PHEMA. The complexation was identified to proceed via a weak interaction, possibly by electrostatic or ion-dipole interactions.
Most of the calcium phosphate (CaP) deposits were formed on the surface of the hydrogels exposed to SBF-1 solution. For the PHEMA hydrogels, the deposits formed inside the hydrogels were only up to about 40 µm in depth. Whilst for the PVA/PVP hydrogels, the deposits were found throughout the hydrogels, including on the surface not directly contacted with SBF-1 solution. It appears that the formation of CaP deposits inside the hydrogels depend largely on the mesh/pore size of the network and its water content. The deposition of CaP was favoured in a steady-batch system without stirring the solution.
The morphology of the CaP deposits was spherical aggregates in the PHEMA-based hydrogels and spherical to flake-like deposits in the PVA/PVP hydrogels. For the PHEMA-based hydrogels, the deposits were comprised mainly of whitlockite [Ca9MgH(PO4)7] type apatite and brushite (CaHPO4.2H2O) as the precursors of hydroxyapatite [Ca10(P04)6(OH)2]. Whilst for the PVA/PVP hydrogels, the deposits contained mainly Ca2+ and OH- deficient hydroxyapatite with CI- as the most common substituting species in the OH- site. All deposits were found to be poorly crystalline or to have nanosize crystals or even to occur as an amorphous phase. Carbonate was also detected both in the SBF-1 calcified PVA/PVP and PHEMA-based hydrogels.
Incorporation of 10 mol% of phosphate groups in the PHEMA copolymer structure was found to inhibit the calcification significantly. A similar trend or even lower degree of calcification was demonstrated by PHEMA hydrogels releasing citric acid (0.5 - 1.5 mM). The deposits formed have the same CaP phases as those formed in the samples without phosphate groups. On the other hand, due to the inhibition and dissolution effect of the citric acid, the deposits were mainly present as non-apatite phases, possibly MCPM [Ca(H2PO4)2.H2O] and brushite with a porous morphology of the outer surface of the spherical aggregates.
Organic compounds in SBF-1 solution (referred to as SBF-2 solution), particularly protein, was found to change the initial preferential chelation of the calcium ions from the polymer to the protein. Consequently, the morphology and the types of CaP deposits formed on the PHEMA-based hydrogels were significantly different. The deposits were exclusively formed on the surface in a much thicker layer with a morphology of much bigger spherical size and a porous outer surface. The deposits contained mainly calcium-deficient hydroxyapatite and octacalcium phosphate [Ca8H2(PO4)6.5H2O] with a minor phase of brushite.
In-vivo calcification revealed that the mechanism of calcification in a biological (subcutaneous) environment was very similar to that of the mechanism of calcification in SBF-2 solution. This includes the involvement of protein adsorption, which leads to the formation of CaP deposits exclusively on the surface, the types of CaP phases formed and the crystallinity of the deposits, and the Ca/P molar ratios of the apatite deposits. The only differences were in the morphology and the density of the CaP deposits. The deposits formed during subcutaneous implantation were comprised of much smaller sized and highly packed spherules with a much thicker deposit layer compared with the CaP deposits formed in SBF-2 calcification.
The inhibition effect of phosphate and carboxylic groups oh the calcification agrees very well between in-vitro and in-vivo. The presence of protein enhances the extent of calcification. However, an appropriate amount of phosphate or carboxylic groups in the PHEMA-based hydrogels reduces the adsorption of protein very significantly.