Introduction: Biodegradable segmented polyurethanes (SPUs) represent a promising class of polymers for bone tissue reconstruction due to their controlled degradation, wide range of mechanical properties and acceptable biotolerance[1][2] . In the present study, we synthesized polyurethanes with dexamethasone or β-glycerol phosphate as chain extenders. These new polymers were physicochemically characterized and cultured with human mesenchymal stem cells (hMSCs) to assess their potential in bone tissue engineering.
Materials and Methods:
Synthesis of SPUs. Prepolymers were obtained by the reaction of poly(ε-caprolactone) diol with 4,4’-(methylene-bis-cyclohexyl isocyanate)[3]. Then, chain extension was conducted with dexamethasone (SPUDEXA) or sodium β-glycerol phosphate (SPUBETA).
Physicochemical characterization. SPUs were characterized by Fourier transform infrared spectroscopy (FTIR), 1H-Nuclear magnetic resonance analysis (1H NMR), Thermogravimetric analysis (TGA), Differential scanning calorimetry (DSC), Dynamic mechanical analysis (DMA), Gel permeation chromatography (GPC), Scanning electron microscopy (SEM), X ray diffraction (XRD) and mechanical testing.
Biological characterization. Human mesenchymal stem cells (hMSCs, from Lonza) were cultured on Mesenchymal Stem Cell Growth Medium (MSCGM, Lonza). Cells were seeded on polymer samples and tissue culture polystyrene (TCP).Cell number was analyzed using the CyQUANT Cell Proliferation Assay and a cell standard curve. Osteogenic differentiation assays hMSCs were performed as reported before[4]. Alkaline phosphatase (ALP) activity assay, was determined using the Pierce BCA Protein Kit. For mineralization studies, cultures at 21 days were stained with Alizarin red.
Results and Discussion: FTIR peaks at 3373 cm-1 (N-H), 1522 cm-1 (urethane N-H + C-N), 1166 cm-1 (C-O-C) confirmed urethane formation on SPUBETA and SPUDEXA. Additionally, SPUDEXA showed at 1666 cm-1 carbonyl absorptions corresponding to dexamethasone A-ring. The physicochemical and mechanical properties of these SPUs are summarized in Table 1.
SPUs were semicrystalline polymers as demonstrated by DSC and XRD. Also SPUs showed elastomeric properties, According this kind of polymers and molecular weight reached.
These SPUs promoted cell adhesion but SPUDEXA showed reduced viability at day 7 (see Figure1), while SPUBETA showed a significant increase in cell number. In addition, SPUBETA exhibited higher mineralization when compared to a non-induced hMSC control. This increase in mineralization is in agreement with the enhanced cell numbers and ALP activity. In contrast, SPUDEXA exhibited low levels of ALP and mineralization.
Conclusion: SPUs based on either β-glycerol phosphate or dexamethasone exhibited an interesting range of physicochemical properties for biomedical use. However, only SPUBETA allows hMSC adhesion and proliferation, high levels of ALP expression and high mineralization potential suggesting it as promising polymeric biomaterial for bone tissue regeneration.
Georgia Institute of Technology President’s Undergraduate Research Award National Institute of Health (NIH R01 AR062368).; FOMIX-CONACYT (170132); CONACYT (169398)
References:
[1] Gogolewski S. Selected topics in biomedical polyurethanes. A review. Colloid Polym Sci. 1989; 267(9):757-85.
[2] Middleton J.C., Tipton AJ. Synthetic biodegradable polymers as orthopedic devices. Biomaterials. 2000; 21(23):2335-46.
[3] Cetina-Diaz S., Chan-Chan L., Vargas-Coronado R., Cervantes-Uc J., Quintana-Owen P., Paakinaho K., et al. Physicochemical characterization of segmented polyurethanes prepared with glutamine or ascorbic acid as chain extenders and their hydroxyapatite composites. Journal of Materials Chemistry B. 2014; 2(14):1966-76.
[4] Phillips J.E., Petrie T.A., Creighton F.P., García A.J. Human mesenchymal stem cell differentiation on self-assembled monolayers presenting different surface chemistries. Acta Biomater. 2010; 6(1):12-20.