Intrafibrillar Mineralization of Collagen is Controlled Through Steric Hindrance

Objectives: Mineralization of fibrillar collagen (c-fibril) with biomimetic process-directing agents provided new insights into the potential mechanisms involved in biomineralization. A relatively small number of molecules has been identified that facilitate the infiltration of c-fibrils by calcium and phosphate to induce intrafibrillar mineralization via the polymer-induced liquid precursor (PILP) method. To determine fundamental mechanisms of intrafibrillar mineralization we characterized several chelating molecules as well as proteins and peptides and tested their ability to produce oriented apatite crystals by in vitro experiments.
Methods: Dynamic light scattering, zeta-potential analysis and a calcium binding assays were used to determine the particle size, surface charges and calcium affinity of poly-Aspartic acid (pAsp) at different molecular weights (MW) (1.4 to 27 kD), poly-Glutamic acid (pGlu) (MW: 3 to 30kD), osteopontin (OPN-10) as well as EDTA and albumin (BSA). Treated collagen fibrils were analyzed by AFM and TEM.
Results: As expected, surface charge and Ca-binding were strongly related to each other but did not correlate with the ability to mineralize c-fibrils. For example, pAsp was able to produce intrafibrillar mineralization, but only at MW above 14kD. None of the pGlu molecules succeeded in mineralizing collagen fibrils, despite both pAsp and pGlu having similar charge distribution and calcium binding ability. OPN-10, on the other hand, showed low Ca-binding with reduced zeta-potential but was able to introduce apatite mineral in collagen fibrils. Interestingly, all molecules with demonstrated ability of intrafibrillar mineral induction (14 and 27kD pAsp, OPN-10) form nanoparticles between 5.9 and 9 nm in diameter under the conditions tested, while all other molecules aggregated to larger particles between 89 to 410 nm. Analysis of the structure of collagen showed that gap-zones on the fibril surface cannot be accessed if particles are above 20 nm in diameter due to steric hindrance.
Conclusions: According to collagen structural model, mineralization of c-fibrils by PILP is facilitated if process-directing agent forms particles smaller than 20 nm in diameter.