In vitro evaluation of an agarose-alginate-based hydrogel for chondrocytes implantation 1Barnouin, L; +1Tan, N; 1Duverneuil, L; 1Gauduin, A; 1Laganier, L;

+1Tissue Bank of France, Mions, France laurence.barnouin@tbf-lab.com

INTRODUCTION Because of its avascularity and low cellularity, articular cartilage tissue has very poor self-repair capacity. Marrow stimulation, such as microfracture, and mosaicplasty are the most readily available techniques, although not completely satisfactory. That is why tissue engineering strategies are now employed with the goal of improving the quality and longevity of the repair tissue. Brittbergs technique, implantation of a chondrocytes suspension under a periosteal flap, proved long term cartilage repair but gave non-homogenous results due to cells leaking and chondrocytes differentiation. To avoid these complications and repair deeper osteochondral defects, current strategies aim at introducing chondrocytes into a biological scaffold. The biomaterial must fulfill both the mechanical capability to withstand the high contact stresses and strains of articular joint environment as well as the functional property allowing tissue growth. To this end, an agarose-alginate hydrogel has been developed for chondrocytes implantation. METHODS Hydrogel is composed of 1.5% agarose and 1% alginate in Earles saline solution. Biocompatibility tests were carried out according to ISO 10993 standard : genotoxicity (Ames test), acute systemic toxicity on mice and maximized sensitization test on guinea pig. Pyrogen test was performed on rabbits according to European pharmacopoeia. In order to increase the sensitivity of all tests, an hydrogel containing 2% agarose and 2.5% alginate was used. Cartilage was harvested from the non-weight-bearing area of the femoral condyle of living donors. It was washed several times in EDTA and trypsin and finely minced and then digested with 0.2% collagenase. The chondrocytes were cultured in monolayer and then suspended in the hydrogel at a final concentration of 10 x 106 cells/mL and moulded. The cell-scaffold combination was gelled by cooling down and incubation in calcium chloride solution. Then, the hydrogel containing chondrocytes was incubated at 37C under orbital agitation in expansion medium. Cell distribution assay was performed on 90 areas distributed in 6 sections per sample. Statistical difference (Student t test) between top and bottom and between middle and edge of the hydrogel was evaluated. The level of significance was set at 0.01. Viability assay was performed by incubation of cell-scaffold combinations in a propidium iodide solution. Living versus dead cells were quantified on 100 areas distributed in 2 sections per sample. Redifferentiation of chondrocytes following incubation in the hydrogel was analyzed through cell phenotype using immuno-histochemistry (type II collagen and aggrecan) and quantitative RT-PCR (types I and II collagens, and aggrecan) techniques. RESULTS Agarose-alginate hydrogel gelation occurs through both thermal and chemical process, leading to three-dimensional network formation. At low temperature, agarose molecules association results in a reversible hydrogel, while in calcium chloride solution, alginate forms stable cross-linked junctions. Association of agarose and alginate allows gel molding to manufacture grafts adapted to the defect thanks to their defined shape and size. This agarose-alginate hydrogel is not pyrogenic and not genotoxic. It did not induce any acute systemic toxicity or delayed hypersensitization. Cell distribution assay showed no statistical difference between top and bottom and between middle and edge of the scaffold (t test, p0.01) (n=7). Cells were homogeneously distributed within the scaffold. Chondrocytes viability following incubation in agarose-alginate hydrogel was between 72 and 96% after 1 month (n=8), and over 85% after 3 months (n=5), 6 months (n=2) and 17 months (n=1). Histological sections prepared for immunohistochemistry analysis of type II collagen and aggrecan expression showed chondrocytes characterized with a spherical morphology close to their native aspect in cartilage, whereas a fibrobastic-like morphology was observed in monolayer culture. The chondrocytes differentiated phenotype was defined by medium to strong staining intensity in more than 25% of the cells reflecting type II collagen and aggrecan expression. After a two-

week incubation period in agarose-alginate hydrogel, chondrocytes of all samples (n=9) showed a differentiated phenotype. In 5 out of these 9 samples, more than 75% of the chondrocytes were redifferentiated. After long-term incubation, either 1 month (n=9), 3 months (n=6), 6 months (n=2) or 17 months (n=1), most of the samples showed chondrocytes with redifferentiated phenotype. Extracellular staining was found in few samples. A kinetics study allowed to analyze data after 1 month, 3 months and either 6 months (n=2) or 17 months (n=1). In 2 samples, the number of differentiated chondrocytes increased with time whereas it decreased in 1 out of the 3 samples. In order to study the effect of incubation in agarose-alginate hydrogel on the phenotype of monolayer-expanded chondrocytes, aggrecan, type II collagen and type I collagen expressions were quantified by RT-PCR. During monolayer expansion, chondrocytes phenotype was modified. Type II collagen rate decreased and was no longer detectable after 3 to 4 passages (n=21), while type I collagen rate increased by 3 fold (n=27). Aggrecan expression was quite stable during monolayer expansion (n=26). Incubation in agarose-alginate hydrogel led to chondrocytes redifferentiation. Type II collagen expression was detectable in 7 samples out of 21 after 2 weeks and in 20 out of 21 after 3 months with a 10,000 fold increase. Type I collagen median expression decreased gradually down of about 10 fold in 3 months. Aggrecan median expression was not modified by incubation in agarose-alginate hydrogel. Catabolic mediators such as MMP-1, MMP-13 and ADAMTS-5 were then studied. MMP-1 expression decreased of about 3 fold during monolayer expansion (n=11) and increased of about 7 fold after a two-week incubation period (n=21). Then it increased very slightly at 3 months (n=8). MMP-13 expression followed the same pattern but with a higher increase of about 16 fold after 2 weeks of incubation (n=21) and a slight decrease during the period between 2 weeks and 3 months (n=9). On the contrary, ADAMTS-5 expression made a 3-fold increase during monolayer expansion (n=16) and decreased of more than 100 fold (n=16) after 2 weeks of incubation. It was increased of 15 fold after a 3-month incubation period (n=7). Incubation in agarose-alginate hydrogel reversed chondrocytes monolayer expression pattern of catabolic mediators. DISCUSSION This agarose-alginate-based hydrogel shows interesting properties for cartilage repair. It is very easy to handle for the surgeon with a press-fit fixation, requiring no periosteal flap or suturing. It can support chondrocytes transplantation and do not hinder tissue repair. Cells are homogeneously distributed inside the scaffold with a good viability until a 17-month period of incubation. This agarose-alginate hydrogel allows redifferentiation of chondrocytes, which have lost their phenotype due to monolayer expansion. Matrix metalloproteinases increased expression in the scaffold may be due to hydrogel degradation by chondrocytes that would replace it gradually by their own matrix proteins. This hypothesis is supported by the results of the phase II clinical study, showing that the scaffold was completely substituted by a neomatrix [1]. Chondrocytes embedded in extracellular matrix are protected against immunogenic reactions [2-4]. As incubation in agarose-alginate allows chondrocytes to produce their own matrix proteins, the agarose-alginate hydrogel could be used for allogenic chondrocytes implantation without rejection of the graft, in the same way as osteochondral allografts. REFERENCES 1. Selmi TA, et al. Autologous chondrocyte implantation in a novel alginate-agarose hydrogel: outcome at two years. J Bone Joint Surg Br 2008 May;90(5):597-604. 2. Yao X, et al. Chondrocyte allografts for repair of full-thickness defects in the condylar articular cartilage of rabbits. Chin J Dent Res 2000 Nov;3(3):24-30. 3. Fragonas E, et al. Articular cartilage repair in rabbits by using suspensions of allogenic chondrocytes in alginate. Biomaterials 2000 2000 Apr;21(8):795-801. 4. Rahfoth B, et al. Transplantation of allograft chondrocytes embedded in agarose gel into cartilage defects of rabbits. Osteoarthritis Cartilage 1998;6(1):50-65.

Poster No. 1195 56th Annual Meeting of the Orthopaedic Research Society