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Chitosan microparticles based polyelectrolyte complex scaffolds for bone tissue engineering in vitro and effect of calcium phosphate.

ABSTRACT: Chitosan microparticles were mixed with chitosan and carboxymethyl cellulose solution to achieve a good binding between the microparticles. Three different compositions of scaffolds were made by varying the calcium phosphate (CaP) amount: 0%, 10%, and 20%. Potassium chloride was used as salt, to make pores inside the scaffolds after leaching out when immersed in phosphate buffer saline (PBS). Compressive strength and compressive modulus of both non-porous (before leaching out), and porous (after leaching out) scaffolds were measured according to the ASTM standards. The highest compressive strength of 27?MPa was reported on 10% CaP scaffolds while 20% CaP scaffolds showed the lowest. The increasing CaP content reduces the compressive strength of the scaffolds. The highest wet state compressive strength was reported on 0% CaP scaffolds with 0.36 MPs and 0.40?MPa at day 1 and day 3 respectively. In vitro cell culture studies showed good cell adhesion and cell proliferation on 10% CaP scaffolds.

SUBMITTER: Unagolla JM

PROVIDER: S-EPMC7433903 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Chitosan microparticles based polyelectrolyte complex scaffolds for bone tissue engineering in vitro and effect of calcium phosphate.

Unagolla Janitha M JM Alahmadi Turki E TE Jayasuriya Ambalangodage C AC

Carbohydrate polymers 20180719

Chitosan microparticles were mixed with chitosan and carboxymethyl cellulose solution to achieve a good binding between the microparticles. Three different compositions of scaffolds were made by varying the calcium phosphate (CaP) amount: 0%, 10%, and 20%. Potassium chloride was used as salt, to make pores inside the scaffolds after leaching out when immersed in phosphate buffer saline (PBS). Compressive strength and compressive modulus of both non-porous (before leaching out), and porous (after ...[more]

PMID: 30143148

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Project description: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. 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. 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. 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.

Project description:ObjectivesCalcium phosphate cements (CPCs) are promising for dental and craniofacial repairs. The objectives of this study were to: (1) develop an injectable cell delivery system based on encapsulation of induced pluripotent stem cell-derived mesenchymal stem cells (iPSMSCs) in microbeads; (2) develop a novel tissue engineered construct by dispersing iPSMSC-microbeads in CPC to investigate bone regeneration in an animal model for the first time.MethodsiPSMSCs were pre-osteoinduced for 2 weeks (OS-iPSMSCs), or transduced with bone morphogenetic protein-2 (BMP2-iPSMSCs). Cells were encapsulated in fast-degradable alginate microbeads. Microbeads were mixed with CPC paste and filled into cranial defects in nude rats. Four groups were tested: (1) CPC-microbeads without cells (CPC control); (2) CPC-microbeads-iPSMSCs (CPC-iPSMSCs); (3) CPC-microbeads-OS-iPSMSCs (CPC-OS-iPSMSCs); (4) CPC-microbeads-BMP2-iPSMSCs (CPC-BMP2-iPSMSCs).ResultsCells maintained good viability inside microbeads after injection. The microbeads were able to release the cells which had more than 10-fold increase in live cell density from 1 to 14 days. The cells exhibited up-regulation of osteogenic markers and deposition of minerals. In vivo, new bone area fraction (mean±SD; n=5) for CPC-iPSMSCs group was (22.5±7.6)%. New bone area fractions were (38.9±18.4)% and (44.7±22.8)% for CPC-OS-iPSMSCs group and CPC-BMP2-iPSMSCs group, respectively, 2-3 times the (15.6±11.2)% in CPC control at 12 weeks (p<0.05). Cell-CPC constructs accelerated scaffold resorption, with CPC-BMP2-iPSMSCs having remaining scaffold material that was 7-fold less than CPC control.SignificanceNovel injectable CPC-microbead-cell constructs promoted bone regeneration, with OS-iPSMSCs and BMP2-iPSMSCs having 2-3 fold the new bone of CPC control. Cell delivery accelerated scaffold resorption, with CPC-BMP2-iPSMSC having remaining scaffold material that was 7-fold less than CPC control. Therefore, CPC-microbead-iPSMSC is a promising injectable material for orthopedic, dental and craniofacial bone regenerations.

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Chitosan microparticles based polyelectrolyte complex scaffolds for bone tissue engineering in vitro and effect of calcium phosphate.

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Chitosan microparticles based polyelectrolyte complex scaffolds for bone tissue engineering in vitro and effect of calcium phosphate.

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