Balancing osteoblast/osteoclast ratio in vitro by means of chitosan scaffolds either surficial sulfated or modified by hemocyanines and calcium phosphate phases. A co-culture study

摘要

Introduction: Bone tissue engineering is still a field of interest. Due to a variety of material properties the natural polysaccharide chitosan is an excellent candidate for a scaffold material. Recently, development focuses on modification for improvement of the impact on the healing process of bone. Main point of application is the ratio of osteoblasts and osteoclasts in the bone multicellular unit. The present study reports on differently sulfated chitosan scaffolds and scaffolds modified by hemocyanines, collagen and calcium phosphate phases. Materials and Methods: (1) Solid-phase sulfation was achieved by treating embroidered chitosan scaffolds with sulfur trioxide dimethylformamide complex (DMF/SO_3) or chlorethan sulfonic acid (CLESA) or sulphur trioxide pyridine complex (Pyr/SO_3). (2) Net shape nonwoven-(NSN)-chitosan scaffolds were functionalized by collagen coating. The microfibrous scaffolds were coated with bovine type Ⅰ collagen by dipping the scaffolds into a solution of 2 mg/ml collagen dissolved in 0.1 M TRIS buffer solution with pH 7.4. Then the scaffolds were freeze-dried and subsequently chemically cross-linked. (3) For the macroporous scaffolds homogeneous clear solutions of 1 % chitosan in acetic acid were prepared. Chitosan solution was transferred to 48-well plates and freeze-dried. After neutralization with NaOH and several washing steps the scaffolds were lyophilized again. To get mineralized chitosan scaffolds brushite or hydroxyapatite were added to the same weight of chitosan, while it was dissolved acetic acid. Hemocyanin modification was carried out by dip-coating. (4) Single culture experiments with hMSC/ Osteoblasts and Monocytes/ Osteoclasts as well as co-culture experiments were carried out as described in our cited previous studies. Results and Discussion: (1) All sulfation methods resulted in enhanced proliferation and enhanced osteogenic differentiation. During cultivation, Pyr/SO_3 scaffolds turned out to provide the best conditions for both. On the contrary, ostoclastogenesis was most effective for CLESA-sulfation. That is a key-result because the use of differently sulfated scaffolds could be a tool to adjust the ratio of bone building and bone resorting activity. (2) Collagen coating of the NSN scaffolds enhances the adhesion and proliferation of hMSC and their differention to osteoblasts, as well. So, the bone building activity could be supported by NSN chitosan collagen-hybrid scaffolds. (3) Modification of chitosan scaffolds by hemocyanines enhances attachment and proliferation of hMSC/osteoblasts as well as the bioactivity of the scaffold material. Co-culture experiments on special hemocyanine-calcium phosphate-chitosan compositions show the enhancement of osteogenic hMSC differentiation in comparison with the osteoclastogenesis. Conclusion: The in vitro-findings give reason to expect that all three approaches are applicable to the manipulation of the ratio of bone resorption and bone formation during remodeling.