Collagen meniscus implant (CMI): ultrastructure, biochemistry and gene expression before and after implantation

Aim: Collagen meniscus implant (CMI) is a tissue engineering technique for the management of irreparable meniscal lesions. In this study we evaluate morphological and biochemical changes occurring in CMI after implantation, in order to better define tissue ingrowth inside the scaffold. Gene expression technique was also adopted to characterize the phenotype of the invading cells. Methods and materials: Morphological analysis was performed by light microscopy, immunohistochemistry (type I and II collagen), SEM and TEM on 5 biopsy specimens, harvested from 5 different patients (range, 6 to 16 months after surgery). Biochemical evaluation was carried out using Flurophore Assisted Carbohydrate Electrophoresis (FACE): this assay allowed to measure glycosaminoglycans (GAG) production in extracellular matrix of 2 biopsy specimens, harvested respectively 6 and 16 months after implantation. Real Time PCR was performed on the same 2 biopsy samples for detecting tissue-specific gene expression (collagen); RNAaseP gene expression was used as housekeeping gene. All these investigations were also applied on non implanted scaffolds for comparison. Results: Scaffold sections appeared composed by parallel connective laminae of 10-30m, connected by smaller (5-10m) connective bundles, surrounding elongated lacunae of 40-60m in diameter. In the biopsies specimens, the lacunae were filled by connective tissue with newly formed vessels and fibroblast-like cells. In the extracellular matrix, the collagen fibrils showed uniform diameters. The original structure of CMI was still recognizable and no inflammatory cells were detected inside the implant. A more organized architecture of the fibrillar network was evident in specimens with longer follow-up. Immunohistochemistry revealed exclusively type I collagen in the scaffold, while type II collagen appeared and was predominant in the biopsies specimens. FACE analysis carried out in the scaffold did not detect any GAG disaccharides. Conversely, high amount of disaccharides (unsulphated chondroitin, 4 and 6 sulphated chondroitin) were detected, together with hyaluronan, in the implants. Real Time PCR showed signal for Collagen type I alpha 1 and no signal for Collagen type II alpha 1. In the scaffolds used for comparison, no gene expression was recorded. Conclusions: The morphological findings of this study demonstrate that CMI acts as a biocompatible scaffold which provide a three-dimensional structure available for colonization by connective cells and vessels. Biochemical data are consistent with an active and specific production of extracellular matrix in the scaffold after implantation. The absence of signal for type II collagen gene in biopsies specimens can be attributed to different maturation stages of the ingrowing tissue.