gms | German Medical Science

Questionnaire: To avoid donor site morbidity material associated with autologous bone grafting and due to limited availability of bone autograft, the use of cancellous bone allografts (CBA) is an appealing strategy for the therapy of localized bone defects. In this context, functionalization with osteogenically active factors may enhance long-term osseointegration and thereby improve the clinical performance of CBA. The Layer-by-Layer (LbL) method involves the formation of polyelectrolyte multilayer (PEM) films whereby layers of oppositely-charged electrolytes are deposited on top of each other. This method has proven to be effective in modifying a variety of different types of surfaces. The aim of this study was to establish a protocol for optimized surface coating of 3D porous bone substitute materials applying alternating layers of hyaluronic acid (HA) and poly-L-lysine (PLL) by LbL method. The effects of LbL film thickness on the morphology, proliferation and osteogenic differentiation of mesenchymal stromal cells (MSC) were investigated.

Methods: Human MSC of N=3 donors were cultured in monolayer (2D) and on CBA scaffolds (3D) for up to 14 days. CBA scaffolds (DIZG, edge length ~5 mm) and cell culture plates were modified by coating with n=12 and n=24 alternating HA/PLL layers. Uncoated- plates and -CBA scaffolds, respectively, served as controls. Cellular proliferation was assessed by total DNA quantification. Osteogenic differentiation was evaluated by cell-specific alkaline phosphatase (ALP) activity assay. Coating density and cellular distribution were performed by fluorescence microscopy (FM) on day 14.

Results and Conclusions: The proliferation rate of MSCs with and without the use of the PLL-HA films showed no significant differences between uncoated-, n=12- and n=24- HA/PLL layers after 14 days. By 3D cultivation a decreased proliferation was observed. 2D cultivation of MSCs stimulated osteogenic differentiation as observed by increased cell-specific ALP activity compared with 3D cultivation on day 14. FM analysis demonstrated that the polymer layers were distributed homogenously throughout the CBA samples and were stable for up to 14 days. No intergroup differences in cellular distribution and proliferation within the interconnected pores of CBA scaffolds were observed.

PLL-HA coating with 24 layers is suitable for 2D and 3D static cultivation of MSCs on CBA scaffolds. Further studies will address the evaluation of LbL-mediated growth factor functionalized CBA on proliferation and osteogenic stem cell differentiation. These results denote the basis for the development of novel type of biofunctionalized bone substitute materials.