Agili-C induced cartilage regeneration: New insights in to repair of human cartilage

Purpose: Regeneration of hyaline cartilage is the ultimate goal of orthopedics. Current approaches for treating focal cartilage defects each present several drawbacks. Acellular scaffold, capable of attracting stem cells, that guides a regenerative process culminating in the formation of a hyaline-like cartilage could be an ideal solution. The Agili-C (CartiHeal, Israel) is a first-in-class acellular implant that has been designed to allow simultaneous regeneration of hyaline cartilage and subchondral bone. The purpose of the study was to investigate the ability of human adult articular chondrocytes to repair cartilage defects by forming hyaline-like cartilage within the Agili-C scaffold and to understand the underlying mechanisms. Methods: Cartilage was collected from the knee and ankle joints of 13 asymptomatic human donors obtained from the Gift of Hope Organ & Tissue Donor Network, (Itasca, IL). The chondral phase of the implant was placed inside cartilage plugs prepared in a donut-shape and cultured in 20% fetal bovine serum for up to 60 days. Chondrocyte survival, distance of migration, proteoglycan (PG) synthesis and content, expression of collagen I, II, X and aggrecan genes, histology with Safranin O and H&E were used to characterize cellular responses and confirm hyaline-like structure. The contribution of progenitor cells to observed repair was assessed by immunohistochemistry with CD-13, VCAM1, and Notch1 antibodies.The data were analyzed with two-tailed t-test comparing the mean of samples cultured with Agili-C construct to the mean of control. The P values <0.05 were considered statistically significant. Results: Chondrocyte migration into the Agili-C constructs was independent of the joint (knee or ankle), gender or donor’s age. An average distance that chondrocytes traveled towards and inside Agili-C scaffold was at least 60-fold longer than within empty defect control ((3359μn±398μn vs 51μn±32μn, p<0.001; Fig.1A). Cells remained viable inside 3D construct. They maintained their phenotype and deposited extracellular matrix enriched in collagen II and aggrecan (Fig.1B). By the end of culture, PG synthesis was more than 2-fold higher (p<0.05; Fig.1C), aggrecan gene expression was 4-fold higher (p<0.001) and collagen type II gene expression was 8-fold higher (p<0.001) respectively in explants containing Agili-C implant in comparison with empty defect control (Fig.1B). Gene expression of type I collagen was higher in the presence of Agili-C construct than in the control (p<0.001). Type X collagen expression was barely detectable and was similar between experimental groups (p=0.122). The defect space within the scaffold was filled with loosely assembled newly synthesized extracellular matrix. The surface of this matrix contained flat elongated cells (black arrows, Fig.1D). To get some insights in to the origin of flat cells, tissue sections were stained with anti-CD-13, VCAM1, and Notch1 antibodies. Positive staining with these three antibodies suggested that identified cells might represent progenitor cells originated from the superficial layer of host cartilage. Conclusions: The Agili-C implant is capable of inducing hyaline cartilage evidenced by the expression of specific markers and appropriate structural organization. Dual processes are suggested: a) migration and proliferation of chondrocytes from the periphery of the tissue with the formation of the extracellular matrix; and b) migration of progenitor cells and their differentiation inside the scaffold.