Proliferation and differentiation of mesenchymal stem cells on scaffolds containing chitosan, calcium polyphosphate and pigeonite for bone tissue engineering

Objectives Treatment of critical‐sized bone defects with cells and biomaterials offers an efficient alternative to traditional bone grafts. Chitosan (CS) is a natural biopolymer that acts as a scaffold in bone tissue engineering (BTE). Polyphosphate (PolyP), recently identified as an inorganic polym...

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Published in:	Cell proliferation Vol. 51; no. 1
Main Authors:	Dhivya, S., Keshav Narayan, A., Logith Kumar, R., Viji Chandran, S., Vairamani, M., Selvamurugan, N.
Format:	Journal Article
Language:	English
Published:	England John Wiley & Sons, Inc 01-02-2018 John Wiley and Sons Inc
Subjects:	Adsorption Alizarin Alkaline phosphatase Animals Biocompatibility Biocompatible Materials - pharmacology Biological activity Biomaterials Biomedical materials Biopolymers Bone biomaterials Bone grafts Bone growth Bone Regeneration - drug effects Bone Regeneration - physiology Bones Calcification, Physiologic - drug effects Calcium Calcium - metabolism Calcium Phosphates - metabolism Cbfa-1 protein Cell Differentiation - drug effects Cell Differentiation - physiology Cell growth Cell proliferation Cell Proliferation - drug effects Cells, Cultured Chitosan Chitosan - metabolism Collagen (type I) Defects Differentiation Engineering Fluorescence Gene expression Genes Grafts Implantation In vivo methods and tests Iron Mechanical properties Mesenchymal stem cells Mesenchymal Stem Cells - cytology Mesenchyme Mice Mineralization Original Osteoblastogenesis Osteoblasts Osteoblasts - drug effects Osteogenesis Osteogenesis - drug effects Osteogenesis - physiology Particulates Phase transitions Polymerase chain reaction Protein adsorption Radiographs Radiography Regeneration Regeneration (physiology) Scaffolds Spectroscopy, Fourier Transform Infrared - methods Staining Stem cells Surgical implants Tissue engineering Tissue Engineering - methods Tissue Scaffolds
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Summary:	Objectives Treatment of critical‐sized bone defects with cells and biomaterials offers an efficient alternative to traditional bone grafts. Chitosan (CS) is a natural biopolymer that acts as a scaffold in bone tissue engineering (BTE). Polyphosphate (PolyP), recently identified as an inorganic polymer, acts as a potential bone morphogenetic material, whereas pigeonite (Pg) is a novel iron‐containing ceramic. In this study, we prepared and characterized scaffolds containing CS, calcium polyphosphate (CaPP) and Pg particles for bone formation in vitro and in vivo. Materials and methods Chitosan/CaPP scaffolds and CS/CaPP scaffolds containing varied concentrations of Pg particles (0.25%, 0.5%, 0.75% and 1%) were prepared and characterized by SEM, XRD, EDAX, FT‐IR, degradation, protein adsorption, mechanical strength and biomineralization studies. The cytocompatibility of these scaffolds with mouse mesenchymal stem cells (mMSCs, C3H10T1/2) was determined by MTT assay and fluorescence staining. Cell proliferation on scaffolds was assessed using MUSE™ (Merck‐Millipore, Germany) cell analyser. The effect of scaffolds on osteoblast differentiation at the cellular level was evaluated by Alizarin red (AR) and alkaline phosphatase (ALP) staining. At the molecular level, the expression of osteoblast differentiation marker genes such as Runt‐related transcription factor‐2 (Runx2), ALP, type I collagen‐1 (Col‐I) and osteocalcin (OC) was determined by real‐time reverse transcriptase (RT‐PCR) analysis. Bone regeneration was assessed by X‐ray radiographs, SEM and EDAX analyses, and histological staining such as haematoxylin and eosin staining and Masson's trichrome staining (MTS) in a rat critical‐sized tibial defect model system. Results The inclusion of iron‐containing Pg particles at 0.25% concentration in CS/CaPP scaffolds showed enhanced bioactivity by protein adsorption and biomineralization, compared with that shown by CS/CaPP scaffolds alone. Increased proliferation of mMSCs was observed with CS/CaPP/Pg scaffolds compared with control and CS/CaPP scaffolds. Increase in cell proliferation was accompanied by G0/G1 to G2/M phase transition with increased levels of cyclin(s) A, B and C. Pg particles in CS/CaPP scaffolds enhanced osteoblast differentiation at the cellular and molecular levels, as evidenced by increased calcium deposits, ALP activity and expression of osteoblast marker genes. In vivo implantation of scaffolds in rat critical‐sized tibial defects displayed accelerated bone formation after 8 weeks. Conclusion The current findings indicate that CS/CaPP scaffolds containing iron‐containing Pg particles serve as an appropriate template to support proliferation and differentiation of MSCs to osteoblasts in vitro and bone formation in vivo and thus support their candidature for BTE applications.
Bibliography:	Funding information This work was, in part, supported by the SRM University, the Council for Science and Industrial Research, India [grant no. 60(0110)/13/EMR‐II to N. S.], the Department of Science and Technology, India [grant no. SB/SO/HS‐0181/2013 to N. S] ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0960-7722 1365-2184
DOI:	10.1111/cpr.12408