Introduction: Stem cells can be induced towards an osteogenic lineage by a number of factors, not the least of which are the mechanical properties of their surrounding environment[1],[2]. The most common method of altering the stability and mechanics of tissue engineering scaffolds is through the use of cross-linking. This can take the form of either a chemical agent or physical process, and many are reported in the literature for use with biological polymers[3]. However, cross-linking methods are often tuned to give scaffolds the greatest mechanical performance, neglecting the influence of cross-linking on cell behaviour, such as attachment and proliferation. In the case of stem cells, the altered mechanical and chemical environment can lead to differences in cell fate. It was therefore hypothesized that osteogenic differentiation of human stem cells could be modulated by altering the cross-linking method.
Materials and Methods: Linear scaffolds were fabricated via ice-templating from synthetic collagen-like peptide (SCP), in a process outlined by van Boxtel[4]. Ice was nucleated at the base of a cylindrical mold and grown linearly, resulting in channels of between 350–400 μm in width, as measured from scanning electron microscopy (SEM). Scaffolds were cross-linked by one physical (dehydrothermal cross-linking (DHT), 24 hrs, 160°C) and two chemical cross-linking methods: genipin (0.125wt%), hexamethylene diisocyanate (HMDIC, 0.025g/gSCP). Human mesenchymal stem cells (MSCs) were seeded onto the scaffolds and the proliferation and mineralization was evaluated over four weeks in vitro.
Results and Discussion: The structure of the linear scaffolds remained unchanged, regardless of the cross-linking method used. In contrast, large differences in the mechanical properties were noted. Scaffold swelling was also strongly dependent on the cross-linking method, with DHT cross-linking giving the lowest percentage of swelling, and HMDIC the highest.
With large channels in the structure, stem cells were able to penetrate the entire pore volume of the scaffolds. While the rate of proliferation was dependent on the cross-linking method, MSCs attached and grew on all scaffold types, Figure 1. In addition, the cell response was sensitive to the cross-linking, showing changes in markers for osteogenic differentiation such as osteocalcin and alkaline phosphatase. While little attention is generally given to the cross-linker once cytocompatability is demonstrated, these results show that it is an important factor in the final outcome of tissue engineering scaffolds.
Figure 1. Mesenchymal stem cells cultured for 7 days on SCP scaffolds stabilized with (a) DHT cross-linking and (b-c) chemical cross-linking: (b) genipin and (c) HMDIC. Scale bar is 50 μm.
Conclusion: The mechanical properties of linear SCP scaffolds were highly dependent on the cross-linking method used. By changing the mechanical environment of the scaffolds, the osteogenic differentiation of human MSCs was altered. Thus, cross-linking can be used as a modulator of cell behaviour and can drive cell fate within tissue engineering scaffolds.
European Union Seventh Framework Programme FP7/2007-2013 (no 607051)
References:
[1] Wu S. et al., Mater. Sci.Eng. R. 80:1-36, 2014.
[2] Engler A.J. et al., Cell 126:677–689, 2006.
[3] Rault I. et al, J Mater Sci: Mater Med.7:215-21, 1996.
[4] van Boxtel, H WO2013/068722