Phytochemical Loaded Functionally Graded Composite Regenerative Membrane for Potential Application of Osteochondral Regeneration

Abstract

Osteochondral defect is the fourth main reason of hospitalization round the world and remains a challenge. Different medical treatments are available to cure osteochondral disease but because of limitation in their effectiveness including shortage of source of chondrocytes, difficulty in fixation, long time for harvesting and less effectiveness for old people, it is still challenging to regenerate both subchondral bone and cartilage together. Generally, bone grafts contribute towards the treatment purpose but this repairmen technique has many limitations that it can only helpful in segmental bone repairmen. Therefore, it is the need to develop effective and economical treatment for regeneration of both cartilage and subchondral bone. Functional, physiological and structural differences of osteochondral defects required functionally and structurally graded biomaterial for effective regeneration. In this study, a functionally and structurally graded composite membrane was prepared. For osteochondral regeneration, polyvinyl alcohol-based composite membranes are frequently utilized. To improve the biological performance of membranes varying amounts of PVA and less explored protein source (oval albumin) were used. The layer that facilitates cartilage regeneration was formed by crosslinking oval albumin with polyvinyl alcohol (PVA). Four different compositions of membranes for cartilage repair were prepared including OA-PVA-100, OA-PVA-80, OA-PVA-60, and OA-PVA-50. And the other layer for bone regeneration was fabricated by using 60% OA with respect to PVA, hydroxyapatite (HA) and sodium alginate was used as cross linker. Hard layer was name as OA-PVA-60-HA Freeze thaw method was used install porosity in membrane and to promote its regeneration capacity. HA is best widely used bone graft bio-mineral. In its composition and morphology it resembles to the subchondral tissue of bone. HA was prepared by in-situ Co-precipitation process. The bilayer was prepared in two basics steps. The first step involved the synthesis of membrane for subchondral regeneration with the same method for single layer preparation and then the cartilage layer was poured on it and cured at 60 °C. To further improve the biological performance and to install antibacterial properties in bilayer membrane, samples were loaded with moringa plant extract. The extract loaded bilayer and hard layer membranes showed good antibacterial activity against Escherichia coli (E. coli) and S aureus. The impact of PVA and OA on water holding capacity (in PBS) and in vitro degradation (in PBS and PBS+lysozyme solutions) was studied that showed good % swelling. The rate of swelling increased with increase amount of PVA. The membrane with composition OA-PVA-50 showed the best swelling. Good % degradation was also shown in the membrane with composition OA-PVA-100 and results clearly show that by increasing the amount of PVA, the degradation rate decreased. FTIR and XRD results confirm the formation hydroxyapatite and membranes samples. Contact angle measurements showed by increasing concentration of PVA the hydrophobicity of membranes increases. . Thermogravimetric analysis showed less percentage loss was observed in subchondral regenerative membrane because of the presence of HA. Scanning electron microscopic examinations were used to characterize the prepared membranes that confirmed the homogeneous surface morphology. The NIH3T3 cells seeded in control (Tissue Culture) had percent cell viability with membrane samples, membrane with the composition OA-PVA-60 showed highest %viability that was 102.4866. Wound healing Assay showed good regeneration ability of different % of plant extract. Overall, a unique, regenerative, antibacterial, biocompatible, and bacterial resistant structurally and functionally graded membrane samples were synthesized.