Evaluation of ostsogenic markers in the differentiation of murine preosteoblasts during the interaction with polyurethaneanohydroxyapatite blomaterials

摘要

In humans, the regenerative capacity of tissues is limited; consequently, there are bone structural damages for which it is appropriate to devise therapeutics that promote regeneration. The materials used currently as bone grafts have the main purpose of replacing the bone to fulfill a support function and not for tissue regeneration. The most common are organic, polymeric and metallic implants, each with advantages and disadvantages; among the latter, the main thing is that most are not biocompatible and resorbable, generating an immune rejection response in the body. Tissue engineering has the challenge of developing bone substitutes that, besides serving as a support, promote bone regeneration; for this, the material must promote cell adhesion, proliferation and differentiation, have a temporary function, and gradually be resorted and replaced by newly formed bone tissue. For this purpose, the use of polyurethaneano-hydroxyapatite hybrids (PUHAp) was proposed in this project. Objective: To determine in vitro the optimal composition of the PUHAp hybrid that presents the best cell differentiation and bone mineralization stimulus. Methodology: PUHAp hybrid with compositions of 5,10, and 20 weight%. nHAp were evaluated. A murine preosteoblast cell line, MC3T3-E1 subclone 4, was used as the cellular model. To evaluate cell viability, trypan blue and alamar blue assays were used. Scanning Electron Microscopy (SEM) was used to analyze the surface of biomaterial samples. Expression of osteogenic markers was evaluated by RT-PCR and gel electrophoresis in 1 % agarose. Results: None of the PUHAp biomaterials are cytotoxic, and neither have inhibitory effects on cell growth. Cell viability and the degree of mineralization are directly linked to the percentage of nHAp in the biomaterial composition. SEM micrographs at 0,7,10 and 14 days show cell adhesion and proliferation on the surface of biomaterial with PU-20%nHAp. The data suggests that the PU/20%nHAp composite might stimulate the osteogenic process in a greater degree, compared to the other composites tested. In control cultures, MC3T3-E1 in osteogenic medium and without the presence of biomaterials, the obtained osteocalcin, osterix and type Ⅰ collagen gene expression profiles agree with previous reports using these genes as osteogenic markers; in cell cultures contacting biomaterials, the osteoinductive capability and bioactivity of PUHAp biomaterials was evident, without the need for an external osteogenic stimulus. Conclusions: The working hypothesis is confirmed: Biomaterials compounds of polyurethane and nano-hydroxyapatite, with compositions 20 weight % or lower of hydroxyapatite, stimulate cell differentiation and mineralization of newly formed bone tissue.