A Novel PCL/Chitosan/TCP Composite Porous Scaffold for Bone Tissue Engineering

+1,2Chen, M; 2Hein, S; 1,2Le, D; 2Nygaard, J; 1Bünger, C +1Orthopaedic Research Lab, Aarhus University Hospital, Aarhus, DK, 2Interdisciplinary Nanoscience Center (iNANO), Aarhus University, DK

chen.muwan@ki.au.dk

INTRODUCTION: Tissue engineering strategies that combine porous biomaterial scaffolds with cells capable of osteogenesis have been shown promising as effective bone graft substitutes (1). However, one challenge is to design a porous structure, providing adequate mechanical support, allowing cell migration and nutrients diffusion, as well as mimicking the natural biophysical and biochemical properties for cell ingrowth and mineralization (2). The present study aimed to produce an osteopromotive porous composite scaffold by incorporating chitosan/tricalcium phosphate into a hierarchical polycaprolactone scaffold made by fused deposition method. METHODS: Polycaprolactone (PCL) scaffolds were plotted using rapid prototyping providing a uniform and ordered micro architecture throughout a cylindrical scaffold with Ø = 10 mm and h = 5 mm. The chitosan/tricalcium phosphate (TCP) sponge was incorporated into the PCL scaffold by freeze-drying. Telomere-elongated human bone marrow mesenchymal stem cells (hMSCs-TERT) were seeded into the PCL/chitosan/TCP scaffolds (1×106 cells/scaffold) and cultured in spinner flask with osteogenic stimulation medium for 21 days. Cell/scaffold constructs were harvested at day 1, day 7, day 14 and day 21. Cell viability, DNA amount, ALP activity, calcium contents, and histology within the scaffolds were determined accordingly. RESULTS SECTION: Cells and extracellular matrix deposition on the scaffold was observed by scanning electron microscope. (Fig.1) Cell viability showed that the composite scaffold is biocompatible. Cells growed into the macro and micro pores of the scaffold. Cell density in the scaffold increased with time. (Fig.2) DNA quantification and ALP activity: DNA amount increased during the culture period. ALP activity revealed decreased amounts of enzyme activity from day 7 and increased again from day 14. (Fig. 3) Mineralization: Von Kossa staining and calium contents showed that the scaffold was osteopromotive.(Fig.4) The quantification data of calium contents increased from day 7 to day 21. The amount was about 3 fold higher than PCL based scaffold on day 21. Histology: A highly cellular penetration depth (> 2 mm) was observed on day 7. A homogeneous cellular distribution in the composite scaffold on day 21 was observed with H&E, Toluidine blue and Hoechst staining. (Fig.5) DISCUSSION: In conclusion, the PCL/chitosan/TCP scaffold had a favorable environment for cell attachment, proliferation and osteogenic differentiation of hMSCs. These novel composite porous scaffold materials have the potential for a wide range of bone tissue engineering.

Fig. 1. Cells and extracellular matrix deposition on the scaffold was observed by scanning electron microscope (Left: day 7; right: day 21).

REFERENCES: [1] Dickson G, et al. Technol Health Care 2007; 15(1): 57-67. [2] Ikada Y. J.R.Soc.Interface 2006;3, 589-601. ACKNOWLEDGEMENTS The study was supported by Lundbeck Foundation. We thank Anette Baatrup and Lisa Feng for their technical assistance.

Fig.2. Confocal micrographs of cell viability. Green pixels: live cells; red pixels: nuclei; green pixels and red pixels overlap: chitosan structure.

Fig.3. DNA quantification (Left) and ALP activity (right). Data represent the mean ± SD (n=4).

Von Kossa Staining

Fig.4. Calcium contents per scaffold and von Kossa staining on day 1, day 7, day 14, and day 21. The amount of calcium is expressed as mean ± SD (n=4).

Fig.5. Representative histology micrographs showing cross-sections of the top part of cell/scaffold constructs after 7-day (up panel) and 21-day (bottom panel) culture (Left: H&E; middle: toluidine blue; right: Hoechst). Bars = 1 mm.

Poster No. 1839 • ORS 2011 Annual Meeting