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Hydroxypropylmethyl cellulose (HPMC) crosslinked keratin/hydroxyapatite (HA) scaffold fabrication, characterization and in vitro biocompatibility assessment as a bone graft for alveolar bone regeneration

ABSTRACT: Wool derived keratin has garnered significant advancements in the field of biomaterials for hard tissue regeneration. The main limitation of keratin-based biomaterials for bone tissue engineering is their fragile nature. This paper proposes the development of a novel hydroxypropyl methylcellulose (HPMC) crosslinked keratin scaffold, containing hydroxyapatite as a major inorganic component by freeze drying technique for alveolar bone regeneration. The prepared keratin/hydroxyapatite/HPMC (K/HA/HPMC) scaffold was characterized to study its chemical, physical, and mechanical properties by Scanning electron microscope (SEM), Fourier transform infrared spectroscopy (FTIR), Energy dispersive X-ray spectroscopy (EDX), X-Ray diffractometric (XRD) analysis. The SEM images of the scaffolds showed highly porous interconnected architecture with average pore size of 108.36 ± 22.56 while microcomputed tomographic analysis measured total porosity as 79.65 %±. Energy dispersive X-ray spectroscopic (EDX) analysis confirmed that inorganic component of scaffold was mainly composed of calcium and phosphorous ions having Ca/P molar ration of 1.6. The maximum compressive strength was found to be in the range of 0.841 ± 0.37 MPa. Furthermore, the K/HA/HPMC scaffold was structurally stable and weight loss of about 26% was observed when soaked in phosphate buffered solution (PBS) for 28 days. In vitro biocompatibility testing showed that K/HA/HPMC scaffold was cytocompatible and supported the attachment, proliferation of osteoblast (Saos-2) cells. Thus, the development of a non-toxic chemical cross-linking system with HPMC was investigated to fabricate K/HA/HPMC scaffold and our results showed great potential of these scaffolds to regenerate alveolar bone due to their structural similarity and excellent in vitro biocompatibility. Keratin; (Hydroxypropyl) methylcellulose; Hydroxyapatite; Alveolar bone; Dental implants; Tissue engineering.

SUBMITTER: Feroz S

PROVIDER: S-EPMC8571510 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The specific objective of this study was to stabilize a simple valid method to prepare pure nanorod hydroxyapatite (HA) mixed with berberine chloride (BER) and Moghat water extract (ME) as composites for incorporation into cellulose acetate (CA) nanofibers to be used as novel bone scaffolds and to determine their efficacy in bone regeneration process In Vitro. Preparation of HA/BER and HA/ME composites were performed by mixing powders using the ball-milling machine. The HA, HA/BER, and HA/ME composites at a concentration of 6.25, 12.5, 25, 50, 100, and 200 mg were mixed with CA solution (13%), then the fiber was formed using electrospinning technique. The properties of the obtained CA fibers were investigated (SEM, TEM, EDX, FTIR, TGA, water uptake, porosity, and mechanical tests). The efficacy of HA and HA composites loaded into CA nanofiber on osteoblast and osteoclast differentiation were measured by tacking ALP, osteocalcin, TRAcP, calcium, and total protein concentration. Moreover, their effects on cell differentiation (CD90 and PARP- ɣ) and death markers (GSK3b, MAPK, Wnt-5 and β-catenin) were evaluated by using ELISA and qPCR. The obtained TEM results indicated that the continuous CA and CA/HA composites electrospun fibers have ultrafine fiber diameters of about 200 nm and uniform distribution of discrete n-HA clusters throughout. In addition, hydrocortisone (HCT) was found to increase the formation of adipocytes and osteoclastic markers CD90 and p38-MAPK which indicated the bone lose process take placed. Treatment with CA loaded with HA, HA/BER or HA/ME decreased CD90, Wnt-5, PARP- ɣ, GSK3b and p38-MAPK associated elevation of osteogenic markers: ALP and osteocalcin. Moreover, HCT overexpressed RANKL and down expressed Osterix gene. Treatment with CA/HA/BER or CA/HA/ME downregulated RANKL and upregulated Osterix associated with a reduction in RANKL/OPG ratio, at p < 0.05. In conclusion, novel CA composite nanofibers (CA/HA/BER and CA/HA/ME) reversed the HCT adverse effect on osteoblast cell death through canonical and non-canonical pathways regulated by Wnt/β-catenin and Wnt/Ca(2+) pathways. Furthermore, our data confirmed that the novel scaffolds create a crosstalk between RUNX-2, RANKL, p38-MAPK, and Wnt signals which positively impact bone regeneration process. Treatment with CA/HA/BER is better compared to the treatment with CA/HA/ME. Nevertheless, both are considered as alternative biomaterial scaffolds with a potential for biomedical applications in the field of bone tissue engineering.

Project description:BackgroundTissue engineering has been shown to exhibit great potential for the creation of biomaterials capable of developing into functional tissues. Cellular expansion and integration depends on the quality and surface-determinant factors of the scaffold, which are required for successful biological implants. The objective of this research was to characterize and evaluate the in vitro characteristics of rabbit bone marrow mesenchymal stem cells (BM-MSCs) associated with a bacterial cellulose membrane (BCM). We assessed the adhesion, expansion, and integration of the biomaterial as well as its ability to induce macrophage activation. Finally, we evaluated the cytotoxicity and toxicity of the BCM.MethodsSamples of rabbit bone marrow were collected. Mesenchymal stem cells were isolated from medullary aspirates to establish fibroblast colony-forming unit assay. Osteogenic, chondrogenic, and adipogenic differentiation was performed. Integration with the BCM was assessed by scanning electron microscopy at 1, 7, and 14 days. Cytotoxicity was assessed via the production of nitric oxide, and BCM toxicity was assessed with the MTT assay; phagocytic activity was also determined.ResultsThe fibroblastoid colony-forming unit (CFU-F) assay showed cells with a fibroblastoid morphology organized into colonies, and distributed across the culture area surface. In the growth curve, two distinct phases, lag and log phase, were observed at 15 days. Multipotentiality of the cells was evident after induction of osteogenic, chondrogenic, and adipogenic lineages. Regarding the BM-MSCs' bioelectrical integration with the BCM, BM-MSCs were anchored in the BCM in the first 24 h. On day 7 of culture, the cytoplasm was scattered, and on day 14, the cells were fully integrated with the biomaterial. We also observed significant macrophage activation; analysis of the MTT assay and the concentration of nitric oxide revealed no cytotoxicity of the biomaterial.ConclusionThe BCM allowed the expansion and biointegration of bone marrow progenitor cells with a stable cytotoxic profile, thus presenting itself as a biomaterial with potential for tissue engineering.

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Hydroxypropylmethyl cellulose (HPMC) crosslinked keratin/hydroxyapatite (HA) scaffold fabrication, characterization and in vitro biocompatibility assessment as a bone graft for alveolar bone regeneration

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