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Stable biofunctionalization of hydroxyapatite (HA) surfaces by HA-binding/osteogenic modular peptides for inducing osteogenic differentiation of mesenchymal stem cells.

ABSTRACT: Hydroxyapatite (HA), the principal component of bone mineral, shows osteoconductive properties when employed for coating metal implants as well as scaffold materials in synthetic bone grafts. With the goal of providing this material with osteoinductive capabilities to promote faster bone regeneration, we show an easy approach to functionalize HA implant surfaces and enrich them with osteoinductive properties by the use of HA-binding modular peptides. The modular peptides are designed as a combination of two domains, an HA-binding peptide motif and an osteogenic peptide motif derived from the osteogenic growth peptide (OGP) or bone morphometric protein 7 (BMP-7). To identify the best HA-binding peptide, several nature-inspired peptides derived from natural bone extracellular matrix proteins (bone sialoprotein, osteonectin, osteocalcin, and salivarin statherin) were compared for HA-binding activity, revealing concentration-dependent and incubation-time-dependent behaviours. We discovered that a Poly-E heptamer (E7) is the best HA-binding peptide, and thus combined it with a second osteogenic peptidic domain to create an osteoinductive modular peptide. After binding/release characterization, we found that the addition of the second osteogenic peptide domain did not change the binding profile of the modular peptides and caused only a slight change in their release kinetics. Mesenchymal stem cells (MSCs) were cultured on the HA substrates functionalized with modular peptides, and cell adhesion, proliferation, and differentiation in a basal medium (i.e., without any osteogenic supplements) were investigated. Gene expression data clearly showed that MSCs were committed to differentiate into osteoblasts in the presence of the modular peptides. HA discs functionalized with the E7 BMP-7 modular peptide showed the best capability in inducing the osteogenic differentiation of MSCs among all modular peptides studied. The modular peptides can easily be used to functionalize the HA implants through its constituent HA-binding motif, leaving the osteogenic peptide motif protruding from the surface for inducing osteogenesis. Our work opens up a new approach to the formulation of new bioactive HA coatings and implants for bone and dental repair.

SUBMITTER: Polini A

PROVIDER: S-EPMC4307953 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Stable biofunctionalization of hydroxyapatite (HA) surfaces by HA-binding/osteogenic modular peptides for inducing osteogenic differentiation of mesenchymal stem cells.

Polini Alessandro A Wang Jianglin J Bai Hao H Zhu Ye Y Tomsia Antoni P AP Mao Chuanbin C

Biomaterials science 20140101

Hydroxyapatite (HA), the principal component of bone mineral, shows osteoconductive properties when employed for coating metal implants as well as scaffold materials in synthetic bone grafts. With the goal of providing this material with osteoinductive capabilities to promote faster bone regeneration, we show an easy approach to functionalize HA implant surfaces and enrich them with osteoinductive properties by the use of HA-binding modular peptides. The modular peptides are designed as a combin ...[more]

PMID: 25642327

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Project description:BackgroundThe selection of appropriate scaffold plays an important role in ensuring the success of bone regeneration. The use of scaffolds with different materials and their effect on the osteogenic performance of cells is not well studied and this can affect the selection of suitable scaffolds for transplantation. Hence, this study aimed to investigate the comparative ability of two different synthetic scaffolds, mainly hydroxyapatite (HA) and polycaprolactone (PCL) scaffolds in promoting in vitro and in vivo bone regeneration.MethodIn vitro cell viability, morphology, and alkaline phosphatase (ALP) activity of MC3T3-E1 cells on HA and PCL scaffolds were determined in comparison to the accepted model outlined for two-dimensional systems. An in vivo study involving the transplantation of MC3T3-E1 cells with scaffolds into an artificial bone defect of 4 mm length and 1.5 mm depth in the rat's left maxilla was conducted. Three-dimensional analysis using micro-computed tomography (micro-CT), hematoxylin and eosin (H&E), and immunohistochemistry analyses evaluation were performed after six weeks of transplantation.ResultsMC3T3-E1 cells on the HA scaffold showed the highest cell viability. The cell viability on both scaffolds decreased after 14 days of culture, which reflects the dominant occurrence of osteoblast differentiation. An early sign of osteoblast differentiation can be detected on the PCL scaffold. However, cells on the HA scaffold showed more prominent results with intense mineralized nodules and significantly (p < 0.05) high levels of ALP activity with prolonged osteoblast induction. Micro-CT and H&E analyses confirmed the in vitro results with bone formation were significantly (p < 0.05) greater in HA scaffold and was supported by IHC analysis which confirmed stronger expression of osteogenic markers ALP and osteocalcin.ConclusionDifferent scaffold materials of HA and PCL might have influenced the bone regeneration ability of MC3T3-E1. Regardless, in vitro and in vivo bone regeneration was better in the HA scaffold which indicates its great potential for application in bone regeneration.

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.

Dataset Information

Stable biofunctionalization of hydroxyapatite (HA) surfaces by HA-binding/osteogenic modular peptides for inducing osteogenic differentiation of mesenchymal stem cells.

Publications

Stable biofunctionalization of hydroxyapatite (HA) surfaces by HA-binding/osteogenic modular peptides for inducing osteogenic differentiation of mesenchymal stem cells.

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