Tissue engineering and the Hyalograft®C surgical technique
The Hyalograft
® C Autograft is a tissue graft consisting of autologous chondrocytes grown on a three-dimensional scaffold made of hyaluronic acid, used in clinical practice since 1999 for the treatment of full-thickness cartilage defects. It is obtained by seeding autologous chondrocytes into a three-dimensional biodegradable material derived from the total esterification of hyaluronan with benzyl alcohol and constructed with a non-woven configuration (Hyaff 11). Briefly, cells were obtained from the digestion of biopsies as described elsewhere [
11], were re-suspended in DMEM medium, and were seeded and cultured onto the Hyaff 11 scaffold for 14 days.
In the majority of cases, the Hyalograft® C Autograft was positioned at the lesion site by a mini-arthrotomy using a simple procedure. In the case of large uncontained defects, particularly in the patello-femoral compartment, fibrin glue and/or sutures were used to keep the graft in place. Subsequent immobilization was recommended for 12 to 24 hours post grafting.
Histological evaluation
Full-thickness cylindrical biopsies with a 2 mm diameter extending from the articular surface to the subchondral bone were obtained from the centre of the defect, were embedded in optimal cutting temperature (OCT) embedding medium, were snap-frozen and were cut into 7 μm sections. Cellular and histochemical characteristics of the repair tissue were evaluated histologically and immunohistochemically.
For routine histology, specimens were stained with H & E to visualize the cellularity, the morphology of cells and the matrix appearance of the repair tissue. Safranin-O stain was used to detect the presence of glycosaminoglycans. Specimens were also analysed using polarized light microscopy to examine collagen organization in the tissue extracellular matrix.
For immunohistological analysis with specific antibodies – monoclonal collagen I antibody (Sigma, St Louis, MO, USA) and monoclonal anti-collagen II antibody (Developmental Studies Hybridoma Bank, Iowa City, IA, USA) – frozen sections were fixed in acetone, were predigested with hyaluronidase (to expose collagen epitopes to antibodies) and were subsequently incubated in Tris buffer saline (10 mM Tris, 150 mM NaCl, pH 7.4) containing 10% normal rabbit serum (Dako, Glostrup, Denmark), primary antibody and secondary rabbit anti-mouse antibody (Dako), and were finally treated with an alkaline phosphatise-antialkaline phosphatise (APAAP) complex (Dako) and stained with fast red (Sigma).
Human placenta was used as a positive control for collagen type I, and healthy articular cartilage for collagen type II. Negative control slides were obtained by treating specimens with mouse monoclonal IgG1 or mouse monoclonal IgM (Dako). The positive red reaction for collagens obtained with immunohistochemistry was classified as very intense, intense, slight or absent.
Repair tissue was evaluated with specific histological criteria regarding cellularity, the cell distribution and the matrix composition in hyaline tissue, fibrocartilage and mixed tissues. Two investigators scored each biopsy under blind conditions, and classified them: as hyaline-like cartilage when round cells in lacunae were in clusters and columns, and collagen fibrils, mainly of type II collagen (very intense), were parallel to the surface; as fibrocartilage when cells with rounded morphology and collagen type I fibrils (very intense) were localized randomly; or as mixed tissues when hyaline-like tissue and fibrocartilage-like tissue were present, with a moderate content of collagen type II (intense).
The biopsies were divided into two groups considering the time from implantation: biopsies taken within 18 months of Hyalograft® C Autograft implantation, and biopsies taken longer than 18 months after surgery.
Analysis of tidemark
The degree of integration of implanted tissue to bone was analysed by the presence of a tidemark; that is, the calcified interface between cartilage and bone where the tissues have become mineralized. This analysis was only performed on samples containing subchondral bone (21 cases). In such cases, the bone was separated from the overlying cartilage, decalcified and placed in paraffin. The samples then underwent histological analysis (H & E) to assess the degree of integration between the newly formed cartilage and the underlying bone surface.
Stained sections were independently scored by three investigators, blinded to the treatment outcome, each describing the histological appearance under light microscopy (Zeiss, Oberkochen, Germany). The presence of a tidemark (yes or no) was also evaluated.