FACULTY OF MEDICINE University of CRETE Chitosan-Gelatin porous scaffolds support the osteogenesis of human ex-vivo expanded Bone Marrow-derived Mesenchymal Stem Cells F. Papadogiannis 1, 2, A.K. Batsali 1, 3, A. Georgopoulou 3, 4, K. Alpantaki 5, M. Chatzinikolaidou 3, 4, C. Pontikoglou 1   1Department of Hematology, University of Crete School of Medicine, Heraklion, Crete 2Department of Materials Science and Technology, University of Crete 3Graduate Program, University of Crete School of Medicine, Heraklion, Crete 4IESL-FORTH, Heraklion, Crete 5Department of Orthopaedics and Traumatology, University of Crete School of Medicine, Heraklion, Greece, Greece INTRODUCTION Background Bone Marrow-derived Mesenchymal Stem Cells (BM-MSCs) are regarded as an attractive cell source for bone tissue engineering because of their self-renewal capacity and osteogenic differentiation potential. These properties, have led to the development of tissue engineering protocols consisting of integration of MSCs within biocompatible, biodegradable and osteo-conductive scaffolds with the ultimate goal of inducing de novo bone formation [1,2]. Such scaffolds have been fabricated by several classes of materials, including natural polymers like chitosan (CS) and gelatin (Gel). Chitosan shares similar properties with glycosaminoglycans that are a major component of bone, whereas gelatin, as a natural biopolymer and hydrolyzed form of collagen, shows excellent biocompatibilty and biodegradability and promotes cell adhesion, proliferation and migration [3]. In the present study, we have explored the interactions of BM-MSCs with a custom-made CS-Gel scaffold. To this end, we have assessed the adhesion and proliferation of BM-MSCs seeded on scaffolds and in addition we have investigated the effect of CS-Gel constructs on the osteogenic differentiation of BM-MSCs RESULTS Cells were successfully expanded from all BM samples and fulfilled the minimal criteria for MSC definition (spindle-shape morphology, expression of CD90, CD105, CD29, CD73 and lack of CD45, CD14, CD34 (1a) and ability to differentiate into osteocytes (1b,c) and adipocytes (1d)). 2. As shown by SEM: CS:Gel scaffolds exhibit a highly porous morphology with good interconnectivity (2a). BM-MSCs acquired flattened fibroblast-like morphology and completely covered the scaffold with a dense layer. Furthermore, adhered cells formed cytoplasmic connections, thereby enabling their organization into tissue (2b). EXPERIMENTAL METHODS Preparation of chitosan-gelatin (CS-Gel) scaffolds Scaffolds were prepared by dissolving 2% (w/v) CS in a 1% (v/v) acetic acid solution and 2% (w/v) Gel in mineralized water at 50oC. The two solutions were poured together with the ratio of 40:60 wt% CS:Gel. A solution of 0.1% (w/v) glutaraldehyde (GTA) was added as crosslinker. After mixture in appropriate culture plates, a step of lyophilization was performed for 24 h at -20 oC. 2. Evaluation of proliferation and differentiation potential of in vitro expanded human BM-MSCs BM-MSCs were isolated from (n=6) consenting healthy donors’ BM aspirates, in vitro expanded until passage (P) 2 and phenotypically characterized by flow cytometry. BM-MSCs were also induced to differentiate in vitro to adipocytes and osteoblasts using appropriate culture media. Differentiation was assessed with specific cytochemical stains and by the expression of specific adipocytic and osteogenic genes. P2 BM-MSCs were seeded on a CS-Gel scaffold and subsequently their adhesion and proliferation potential were assessed via electron microscopy (SEM) and a methyl- triazolyl-tetrazolium assay (MTT), respectively. The osteogenic potential of BM-MSCs on CS-Gel scaffolds was estimated at specific time points during differentiation (days 0,7,14) by RUNX-2, OSC and ALP gene expression. 3. The growth BM-MSCs seeded on CS-Gel scaffolds was significantly increased, during a 12-day culture period, as compared to cells cultured in polysterene (TCPS control surface) (P < 0.0001). 4. CS-Gel scaffolds efficiently support the osteogenic differentiation of BM-MSCs cultured in the presence of appropriate inducers, as evidenced by the up-regulation of the osteogenesis-related genes RUNX2, OSC and ALP. RUNX2 expression did not differ between BM-MSCs cultured on CS:Gel scaffolds as compared to those cultured on TCPS at neither day 7 nor day 14. ALP gene expression was significantly up-regulated in BM-MSCs cultured on scaffolds, as compared to those cultured on TCPS at days 7 (P=0.0268) and day 14 (P<0.0001). OSC gene expression was significantly higher in BM-MSCs cultured on scaffolds, as compared to those cultured on TCPS at, both day 7 (P<0.0001) and day 14 (P=0.0002). CONCLUSION A custom-made 40%-60% CS-Gel hybrid scaffold was successfully fabricated, using glutaraldehyde as a crosslinker. The porous size and architecture of the prepared scaffolds provided a favorable support for BM-MSC growth. The scaffold enhanced BM-MSC osteogenic differentiation in vitro. Our results provide evidence that the fabricated CS:Gel scaffold may hold promise for bone tissue engineering applications and provide the theoretical background for evaluating the in vivo bone repair ability of the construct in appropriate animal models. REFERENCES [1] Bastiaan J.H. Jansen et al. Functional Differences between Mesenchymal Stem Cell Populations Are Reflected by Their Transcriptome. 2010. 19(4):481-89. [2] V.Chiono, et al. Genipin-crosslinked chitosan/gelatin blends for biomedical applications. J Mater Sci Mater Med, 2008. 19(2):889-98. [3] Chatzinikolaidou M, et al. Adhesion and growth of human bone marrow mesenchymal stem cells on precise-geometry 3D organic-inorganic composite scaffolds for bone repair, Mater Sci and Eng Part C 2015.48,301-309.