Research Paper
Tissue Engineering
Properties of chitosan–collagen sponges and osteogenic differentiation of rat-bone-marrow stromal cells

https://doi.org/10.1016/j.ijom.2007.11.014Get rights and content

Abstract

The aim of this study was to further investigate effects of a combined chitosan and collagen matrix on osteogenic differentiation of rat-bone-marrow stromal cells (BMSCs), including analysis of the physical and mechanical properties of the sponges. There were 4 study groups: collagen, chitosan, 1:1 chitosan–collagen and 1:2 chitosan–collagen sponges. Chitosan–collagen sponges were fabricated using the freeze-drying technique. BMSCs were seeded on the sponges and cultivated in mineralized culture medium for 27 days. Attachment and growth of cells on the sponges were examined under a scanning electron microscope. Alkaline phosphatase activity and levels of osteocalcin were monitored. Tests of swelling, collagenase and lysozyme enzymatic degradation, and mechanical strength were performed. The BMSCs attached successfully to the structure of the sponges, and expression of ALP and osteocalcin on collagen and chitosan–collagen composite sponges was greater than on chitosan sponges. All sponges showed a high degree of water uptake. Chitosan and chitosan–collagen sponges showed a higher resistance to enzymatic degradation than collagen sponges. A 1:1 chitosan–collagen sponge demonstrated the highest compressive strength. Combined chitosan–collagen matrixes promoted osteoblastic differentiation of BMSCs, and improved the mechanical and physical properties of the sponges.

Introduction

One of the approaches in cell-based bone tissue engineering is to regenerate new bone by culturing living cells directly on three-dimensional scaffolds17. Mesenchymal stem cells presenting in bone marrow (BMSCs) are an excellent source of bone progenitor cells19, 39. BMSCs can be isolated from bone-marrow mononuclear cells by their tendency to adhere to plastic culture dishes, and induced to differentiate into many cell types in a mesenchymal lineage including osteoblasts15, 30. Progress of osteoblastic differentiation of BMSCs can be determined by analyzing expression of alkaline phosphatase (ALP) and osteocalcin, which are characteristic phenotypes of premature and mature osteoblasts, respectively3. Under appropriate culture conditions, BMSCs can be expanded and seeded onto the three-dimensional scaffold to form tissue-engineered constructs13. Autologous and allogenic BMSCs seeded directly onto porous biomaterial scaffolds have shown the capacity to regenerate bone within segmental and craniofacial defects7, 11, 44. The method of transferring BMSCs on an appropriate scaffold has potential as an alternative to autogenous bone grafting44.

Scaffolds influence the capacity of seeded cells to regenerate bone within the defect site6 by functioning as an extracellular matrix (ECM), providing surface contact and temporary mechanical support for functional cells and maintaining space for tissue development28. They should be biocompatible, porous and resorbable, and have suitable surface chemistry for cell attachment, proliferation and differentiation18. The mechanical strength, swelling property and degradation behavior of the scaffold play crucial roles in the long-term performance of a tissue-engineered cell/material construct, such as in cell growth, cell adhesion, nutrient perfusion and tissue regeneration18, 27, 31, 38, 44.

Natural biomaterials are widely used for scaffold fabrication in tissue engineering since they facilitate cell attachment and maintenance of differentiation function. Chitosan is a partially deacetylated derivative of chitin and is conducive to osteoblasts32. Collagen is a major component of ECM of bone and enhances proliferation, migration and differentiation of osteoblast-like cells26. To improve the mechanical and biological properties of scaffolds, collagen and chitosan were combined. It was previously reported that chitosan–collagen composite sponges are porous, biocompatible, and able to support growth and differentiation of osteoblasts to a greater extent than chitosan sponges2. To further evaluate the applicability of chitosan–collagen sponges in bone tissue engineering, the present study was designed to investigate the ability of the composite sponges to support osteoblastic differentiation of BMSCs, and the effects of combined chitosan–collagen on the mechanical strength and swelling and degradation properties of the sponges.

Section snippets

Study groups

The study was categorized into Group I: collagen, Group II: chitosan, Group III: 1:1 (by weight) chitosan–collagen and Group IV: 1:2 (by weight) chitosan–collagen sponges. In all groups, 5–15 samples were analyzed for each investigated parameter at given time points.

Preparation of chitosan, collagen and chitosan–collagen sponges

Porous matrices of chitosan, collagen and chitosan-collagen composite were fabricated using freezing and drying techniques reported by Arpornmaeklong and co-workers2. To prepare 1% chitosan suspension, chitosan powder (medium

Swelling test

All sponges absorbed and maintained a large volume of water within pore spaces. Average ratios of water uptake of all groups were 25–30 times higher than dry weight. The swelling ratios of all sponges were not significantly different. Chitosan–collagen sponges in a ratio of 1:1 (Group III, swelling ratio of 31.8 ± 7.7) demonstrated a tendency to preserve more water than 1:2 chitosan–collagen sponges (Group IV, 30.0 ± 8.8), chitosan sponges (Group II, 28.8 ± 6.5) and collagen sponges (Group I, 27.7 ± 

Discussion

This study demonstrated the effects of combining chitosan and collagen on the physical and mechanical properties of scaffolds, and the ability of these composite sponges to support osteoblastic differentiation of BMSCs. The results agree with those of previous reports that a high swelling ratio demonstrates ability of the scaffold to preserve a high volume of water within the porous structure27, 33, 36. All sponges swelled and had high ratios of water uptake (Fig. 1). This could be attributed

Acknowledgements

This study was supported by grants from the Prince of Songkla University and the Commission on Higher Education.

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