Introduction: Restoration of nucleus pulposus (NP) using biomaterials capable of retaining cells and stimulating molecules is one of the new treatment strategies for intervertebral disc (IVD) degeneration. Hydorgels have been receiving great attention in this regard because they can be applied through minimally invasive surgery and they can retain cells, water and other substances such as growth factors[1]. Ideal hydrogels must rapidly gelify, be biocompatible, injectable and possess enough strength. However, most of the hydrogel systems proposed until now lack enough mechanical strength and/or cell compatibility[2]-[5]. Our group has recently synthesized injectable hydrogels with improved mechanical and biological properties using chitosan (CH)[6]. Chondroitin sulfate (CS) is a glycosaminoglycan found in IVD extracellular matrix (ECM). It inhibits the activity of ECM degrading enzymes, stimulates production of proteoglycans by chondrocytes and reduces apoptosis in these cells[7],[8]. Our objective is to develop an injectable and biocompatible CH/CS hydrogel with mechanical properties comparable to those of native NP.
Materials and Methods: Hydrogels were made by mixing purified CH (2% w/v) with gelation agents (GA) (BGP0.4M, SHC0.075M/BGP0.1M and SHC0.075M/PB0.4M, pH8) with and without CS (highest concentration: 1% w/v, dissolved in the GA). pH of the hydrogels was measured at room temperature (RT) after mixing and after 24h of gelation at 37°C. Rheological properties (within 1h at 37°C) were analyzed following preparation of the hydrogels. Elastic properties of the hydrogels were studied after 24h of gelation at 37°C using an ElastoSensTM system. Injectability of the hydrogels was evaluated by studying their initial viscosity at 37°C and also by measuring the maximum force applied to extrude the hydrogels after preparation. Direct cytotoxicity of the hydrogels was evaluated by encapsulating L929 fibroblasts in the hydrogels and performing alamar blue and LIVE/DEAD assays. Indirect cytotoxicity was evaluated by studying the effect of hydrogel extracts on cell viability.
Results and Discussion: All the formulations resulted in thermosensitive and injectable hydrogels with pH close to physiological levels and addition of CS did not disturb the pH balance. Rheological studies showed a higher storage modulus for the formulations containing SHC compared to BGP0.4M. The hydrogels containing PB showed the highest gelation rate. Addition of CS to the hydrogels prepared using SHC0.075M/PB0.4M, accelerated gelation and increased the storage modulus. Interestingly, addition of CS improved the growth of encapsulated cells.
Conclusion: The preliminary results obtained so far in this study suggest the promising potential of CH/CS hydrogels as a thermosensitive, biocompatible and injectable matrix with good mechanical properties. Additional experiments evaluating mechanical properties of these hydrogels with various CS concentrations under compression are being performed. The compatibility of the hydrogels with human mesenchymal stem cells and the effect of CS addition on the differentiation potential of these cells towards chondrocytes and NP cells will be studied as well.
Rheolution Inc. and C. Ceccaldi for the use of Elastosense; Funding by the Canada Research Chair program; YA also acknowledges FRQ-S scholarship
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