Improvement of the biological performance of CaP coatings by using the PILP-mineralized collagen method

Abstract

Mineralization of collagen via a polymer-induced liquid-precursor (PILP) method was used to mimic the intrafibrillar mineralization of collagen. This method was optimized and adapted to achieve a coating of PILP-mineralized collagen in microplates for cell culture experiments. Prior to mineralization, optimization experiments were performed, to find the best conditions to obtain a homogeneous coating of collagen fibres. The first optimization step was tuning buffer conditions (pH, ionic strength, col-I concentration) that allow collagen fibril self- assembly, showing the characteristic D-spacing, from an acid-dissolved stock solution. It was found that the best conditions to obtain this kind of collagen fibrils were 100 μg/mL of collagen in FFB with 200 mM NaCl, 100 μg/mL of collagen in FFB with 400 mM NaCl and 500 μg/mL of collagen in FFB with 400 mM NaCl. The second optimization step was regarding the coverage of the tissue culture plastic substrate by fibrils, and two methods were tested. In the first method, quite homogeneous coatings were attempted by deposition of sequential layers of fibril suspensions. The second was a two-step method: first a thin, non-fibrillar layer of collagen was deposited, followed by fibril formation in the second step. The later method proved to be most effective, as observed by immunostaining and SEM. Observation of collagen banding pattern and confirmation of intrafibrillar mineralization of collagen was done by TEM. Furthermore, the effectiveness of the PILP solution was tested by mineralizing bovine type-I commercial MatrixMEM collagen membranes. Quantification of mineral content was done by TGA. The PILP-mineralized collagen coating is hypothesized to have better biological performance than current biomimetic calcium phosphate coatings. To study so, five different substrates (TCPS, hydroxyapatite, collagen, PILP-mineralized collagen and collagen-coated hydroxyapatite coatings) were prepared for a resorption experiment with osteoclasts. Surface morphology of the samples was analysed by SEM, and calcium phosphate phases were identified by XRD. Osteoclast cells were obtained by differentiating murine RAW 264.7 macrophages under the influence of RANKL. Cell density and RANKL concentration optimization experiments were also performed. A seeding density of 4000 cells/cm2 and 100 ng/mL of RANKL were found as optimal conditions. Osteoclast formation on the different substrates was assessed by TRAP staining, and quantification of TRAP activity and DNA amount. The morphology and the number of osteoclasts present in each substrates were analysed by SEM, as well as the resorption of the different calcium phosphate coatings. Results from TRAP staining and SEM indicated that PILP- mineralized collagen coatings performed the best, showing a greater amount of osteoclast-like cells than the rest of the coatings. Although resorption was not observed in any of the coatings, some degradation of collagen was found, which could indicate the beginning of the resorption process, but no clear conclusions could be done. Following the resorption study, collagen micropatterning was attempted and it was also successful by using μCP with PVA. However, its mineralization by PILP method was not possible due to the limitation of time. Overall, PILP-mineralized collagen coatings showed promising clues to improve the biological performance of the biomimetic coatings used nowadays in the clinic.