A Self-Setting Hydrogel of Silylated Chitosan and Cellulose for the Repair of Osteochondral Defects in Canin Model

Purpose: Articular cartilage may be affected by many injuries including traumatic lesions. Because of the increase in life expectancy, such osteoarthritis-associated cartilaginous disorders are nowadays a serious public health issue. Currently there is no long-term clinically efficient cure for cartilage lesions and total joint replacement remains often the ultimate procedure to alleviate painful symptoms. In this context, new strategies for regenerating articular cartilage are contemplated with a growing interest. Among these innovative strategies, tissue engineering (TE) is probably one of the most promising. TE is based on the association of cells with a biomaterial capable of supporting cell growth and differentiation. To address the clinical issue of cartilage TE, we have recently developed an injectable, self-hardening and mechanically reinforced hydrogel (Si-HPCH) composed of silanised hydroxypropylmethyl cellulose (Si-HPMC) mixed with silanised chitosan. Methods: The in vitro cytocompatibility of Si-HPCH was tested using human nasal chondrocytes (hNC) or human adipose stromal cells (hASC) with a live and dead assay kit. The in vivo biofunctionality of our hydrogel (250 μL) was then determined by implantation in nude mice subcutis. Six different conditions were implanted: Si-HPMC or Si-HPCH alone, Si-HPMC or Si-HPCH mixed with hNC (1 million/ml) and Si-HPMC or Si-HPCH mixed with hASC (1 million/ml). Samples were collected 6 weeks after implantation and characterized by immunohistochemistry with antibodies against type I and II collagen and aggrecan. Si-HPCH was then tested for the repair of calibrated 6 mm-diameter and 5 mm-depth osteochondral defects performed on the medial femoral condyle of twelve 4-year old beagles. 6 defects were filled with Si-HPCH (150 μL) alone or mixed with 1 million/ml of autologous ASC. As negative control, 2 additional defects were left empty. Four months after implantation, histological (safranin O and Movat pentachrome stainings) and immunohistological (type I and II collagen and aggrecan) analyses were performed. Results: Our data demonstrated that Si-HPCH supports hNC and hASC viability in 3D culture. Si-HPMC or Si-HPCH also allowed the maintenance of hNC and hASC viability in the subcutis of nude mice. Subcutaneous explants of hNC with Si-HPMC or Si-HPCH showed the formation of cell clusters surrounded by a cartilage-like extracellular matrix (ECM). ECM was more abundant with Si-HPCH than with Si-HPMC. Interestingly, the explants containing hASC mixed with Si-HPMC or Si-HPCH showed the presence of a low but significant number of cells expressing chondrogenic markers. In the canine osteochondral defect model, all the tested hybrid constructs provided with satisfactory clinical results. While the empty defects were only partially filled with a fibrous tissue, defects filled with Si-HPCH with or without autologous ASC, revealed a significant osteochondral regeneration. In empty defects, the repair tissue was thus mainly composed of fibroblast-like cells expressing type I collagen and no chondrogenic marker had been detected. On the contrary, in the defects filled with Si-HPCH, whatever the presence of autologous ASC, the ECM of the repair tissue was positively stained for type II collagen and aggrecan. Conclusions: Si-HPCH is an injectable, self-setting and cytocompatible hydrogel able to support the formation of a cartilage-like tissue in nude mice subcutis when implanted with chondrocytes. Interestingly, this hydrogel also supports the regeneration of osteochondral defects in dogs when implanted alone or with ASC. Taken together, these data make Si-HPCH a promising candidate for the cell-free regeneration of articular cartilage. To address whether Si-HPCH may support the regeneration of articular cartilage in a preclinical model closely mimicking the human situation, further experiments in equine model of osteochondral defects are now under investigation.