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Bone repair by periodontal ligament stem cellseeded nanohydroxyapatite-chitosan scaffold.

ABSTRACT: BACKGROUND: A nanohydroxyapatite-coated chitosan scaffold has been developed in recent years, but the effect of this composite scaffold on the viability and differentiation of periodontal ligament stem cells (PDLSCs) and bone repair is still unknown. This study explored the behavior of PDLSCs on a new nanohydroxyapatite-coated genipin-chitosan conjunction scaffold (HGCCS) in vitro as compared with an uncoated genipin-chitosan framework, and evaluated the effect of PDLSC-seeded HGCCS on bone repair in vivo. METHODS: Human PDLSCs were cultured and identified, seeded on a HGCCS and on a genipin-chitosan framework, and assessed by scanning electron microscopy, confocal laser scanning microscopy, MTT, alkaline phosphatase activity, and quantitative real-time polymerase chain reaction at different time intervals. Moreover, PDLSC-seeded scaffolds were used in a rat calvarial defect model, and new bone formation was assessed by hematoxylin and eosin staining at 12 weeks postoperatively. RESULTS: PDLSCs were clonogenic and positive for STRO-1. They had the capacity to undergo osteogenic and adipogenic differentiation in vitro. When seeded on HGCCS, PDLSCs exhibited significantly greater viability, alkaline phosphatase activity, and upregulated the bone-related markers, bone sialoprotein, osteopontin, and osteocalcin to a greater extent compared with PDLSCs seeded on the genipin-chitosan framework. The use of PDLSC-seeded HGCCS promoted calvarial bone repair. CONCLUSION: This study demonstrates the potential of HGCCS combined with PDLSCs as a promising tool for bone regeneration.

SUBMITTER: Ge S

PROVIDER: S-EPMC3474464 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Bone repair by periodontal ligament stem cellseeded nanohydroxyapatite-chitosan scaffold.

Ge Shaohua S Zhao Ning N Wang Lu L Yu Meijiao M Liu Hong H Song Aimei A Huang Jing J Wang Guancong G Yang Pishan P

International journal of nanomedicine 20121010

<h4>Background</h4>A nanohydroxyapatite-coated chitosan scaffold has been developed in recent years, but the effect of this composite scaffold on the viability and differentiation of periodontal ligament stem cells (PDLSCs) and bone repair is still unknown. This study explored the behavior of PDLSCs on a new nanohydroxyapatite-coated genipin-chitosan conjunction scaffold (HGCCS) in vitro as compared with an uncoated genipin-chitosan framework, and evaluated the effect of PDLSC-seeded HGCCS on bo ...[more]

PMID: 23091383

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Project description:The structural and functional integrity of bone-periodontal ligament (PDL)-cementum complex stems from the load-bearing attachment sites (entheses) between soft (PDL) and hard (bone, cementum) tissues. These attachment sites are responsible for the maintenance of a bone-PDL-cementum complex biomechanical function. The objective was to investigate changes in spatiotemporal expression of key biomolecules in developing and functionally active entheses.Multilabeling technique was performed on hemimandibles of 3 wk and 3 mo-old scleraxis-GFP transgenic mice for CD146, CD31, NG2, osterix and bone sialoprotein. Regions of dominant stretch within the PDL were evaluated by identifying directionality of collagen fibrils, PDL fibroblasts and PDL cell cytoskeleton.CD146+ cells adjacent to CD31+ vasculature were identified at PDL-bone enthesis. NG2+ cells were located at coronal bone-PDL and apical cementum-PDL entheses in the 3-wk-old group, but at 3 mo, NG2 was positive at the entheses of the apical region and alveolar crest. NG2 and osterix were colocalized at the osteoid and cementoid regions of the PDL-bone and PDL-cementum entheses. Bone sialoprotein was prominent at the apical region of 3-wk-old mice. The directionality of collagen fibers, fibroblasts and their cytoskeleton overlapped, except in the apical region of 3 wk.Colocalization of biomolecules at zones of the PDL adjacent to attachment sites may be essential for the formation of precementum and osteoid interfaces at a load-bearing bone-PDL-tooth fibrous joint. Biophysical cues resulting from development and function can regulate recruitment and differentiation of stem cells potentially from a vascular origin toward osteo- and cemento-blastic lineages at the PDL-bone and PDL-cementum entheses. Investigating the coupled effect of biophysical and biochemical stimuli leading to cell differentiation at the functional attachment sites is critical for developing regeneration strategies to enable functional reconstruction of the periodontal complex.

Project description:BackgroundBMP9-stimulated DPSCs, SCAPs and PDLSCs are effective candidates for repairing maxillofacial bone defects in tissue engineering, while the most suitable seed cell source among these three hDMSCs and the optimal combination of most suitable type of hDMSCs and BMP9 have rarely been explored. Moreover, the orthotopic maxillofacial bone defect model should be valuable but laborious and time-consuming to evaluate various candidates for bone regeneration. Thus, inspired from the maxillofacial bone defects and the traditional in vivo ectopic systems, we developed an intrabony defect repair model to recapitulate the healing events of orthotopic maxillofacial bone defect repair and further explore the optimized combinations of most suitable hDMSCs and BMP9 for bone defect repair based on this modified ectopic system.MethodsIntrabony defect repair model was developed by using decellularized bone matrix (DBM) constructs prepared from the cancellous part of porcine lumbar vertebral body. We implanted DBM constructs subcutaneously on the flank of each male NU/NU athymic nude mouse, followed by directly injecting the cell suspension of different combinations of hDMSCs and BMP9 into the central hollow area of the constructs 7 days later. Then, the quality of the bony mass, including bone volume fraction (BV/TV), radiographic density (in Hounsfield units (HU)) and the height of newly formed bone, was measured by micro-CT. Furthermore, the H&E staining and immunohistochemical staining were performed to exam new bone and new blood vessel formation in DBM constructs.ResultsBMP9-stimulated periodontal ligament stem cells (PDLSCs) exhibited the most effective bone regeneration among the three types of hDMSCs in DBM constructs. Furthermore, an optimal dose of PDLSCs with a specific extent of BMP9 stimulation was confirmed for efficacious new bone and new blood vessel formation in DBM constructs.ConclusionsThe reported intrabony defect repair model can be used to identify optimized combinations of suitable seed cells and biological factors for bone defect repair and subsequent development of efficacious bone tissue engineering therapies.

Project description:Incorporation of nanohydroxyapatite (nHAP) within a chitosan (CS)/silk fibroin (SF) nanofibrous membrane scaffold (NMS) may provide a favorable microenvironment that more closely mimics the natural bone tissue physiology and facilitates enhanced osteogensis of the implanted cell population. In this study, we prepared pristine CS/SF NMS, composite CS/SF/nHAP NMS containing intrafibrillar nHAP by in situ blending of 10% or 30% nHAP before the electrospinning step, and composite CS/SF/nHAP NMS containing extrafibrillar nHAP by depositing 30% nHAP through alternative soaking surface mineralization. We investigated the effect of the incorporation of HAP nanoparticles on the physicochemical properties of pristine and composite NMS. We confirmed the presence of ~30 nm nHAP in the composite nanofibrous membranes by thermogravimetry analysis (TGA), X-ray diffraction (XRD), and scanning electron microscopy (SEM), either embedded in or exposed on the nanofiber. Nonetheless, the alternative soaking surface mineralization method drastically influenced the mechanical properties of the NMS with 88% and 94% drop in Young's modulus and ultimate maximum stress. Using in vitro cell culture experiments, we investigated the effects of nHAP content and location on proliferation and osteogenic differentiation of human bone marrow mesenchymal stem cells (hMSCs). The proliferation of hMSCs showed no significant difference among pristine and composite NMS. However, the extent of osteogenic differentiation of hMSCs was found to be positively correlated with the content of nHAP in the NMS, while its location within the nanofiber played a less significant role. In vivo experiments were carried out with hMSCs seeded in CS/SF/30%nHAP NMS prepared by in situ blending and subcutaneous implantation in nude mice. Micro-computed tomography images as well as histological and immunohistochemical analysis of the retrieved hMSCs/NMS construct 1 and 2 months postimplantation indicated that NMS had the potential for bone regeneration and can be suggested as a promising scaffold for bone tissue engineering.

Project description:BackgroundThe invasive and complicated procedures involving the use of traditional stem cells limit their application in bone tissue engineering. Cell-free, tissue-engineered bones often have complex scaffold structures and are usually engineered using several growth factors (GFs), thus leading to costly and difficult preparations. Urine-derived stem cells (USCs), a type of autologous stem cell isolated noninvasively and with minimum cost, are expected to solve the typical problems of using traditional stem cells to engineer bones. In this study, a graphene oxide (GO)-modified silk fibroin (SF)/nanohydroxyapatite (nHA) scaffold loaded with USCs was developed for immunomodulation and bone regeneration.MethodsThe SF/nHA scaffolds were prepared via lyophilization and cross-linked with GO using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxy succinimide (NHS). Scaffolds containing various concentrations of GO were characterized using scanning electron microscopy (SEM), the elastic modulus test, Fourier transform infrared spectroscopy (FTIR), and X-ray photoelectron spectrometer (XPS). Examinations of cell adhesion, proliferation, viability, morphology, alkaline phosphatase activity, and osteogenesis-related gene expression were performed to compare the osteogenesis-related biological behaviors of USCs cultured on the scaffolds. The effect of USC-laden scaffolds on the differentiation of macrophages was tested using ELISA, qRT-PCR, and immunofluorescence staining. Subcutaneous implantations in rats were performed to evaluate the inflammatory response of the USC-laden scaffolds after implantation. The scaffolds loaded with USCs were implanted into a cranial defect model in rats to repair bone defects. Micro-computed tomography (μCT) analyses and histological evaluation were performed to evaluate the bone repair effects.ResultsGO modification enhanced the mechanical properties of the scaffolds. Scaffolds containing less than 0.5% GO had good biocompatibility and promoted USC proliferation and osteogenesis. The scaffolds loaded with USCs induced the M2-type differentiation and inhibited the M1-type differentiation of macrophages. The USC-laden scaffolds containing 0.1% GO exhibited the best capacity for promoting the M2-type differentiation of macrophages and accelerating bone regeneration and almost bridged the site of the rat cranial defects at 12 weeks after surgery.ConclusionsThis composite system has the capacity for immunomodulation and the promotion of bone regeneration and shows promising potential for clinical applications of USC-based, tissue-engineered bones.

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Bone repair by periodontal ligament stem cellseeded nanohydroxyapatite-chitosan scaffold.

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Bone repair by periodontal ligament stem cellseeded nanohydroxyapatite-chitosan scaffold.

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