Introduction: We studied the extracellular matrix (ECM) of 19 ruptured human Achilles tendons, comparing the tissue composition of specimens taken from area close to the rupture with specimens harvested from an apparently healthy area in the same tendon. The hypothesis was that the metabolism of these molecules is altered in patients with Achilles tendon rupture.

Materials and Methods: We compared the gene expression and the protein localization of the main ECM molecules (collagen type I, decorin and versican) including enzymes involved in their metabolism as matrix metallo-proteases (MMP2 and 9) and tissue inhibitory of metal-loproteinase (TIMP 1 and 2) using a Real Time PCR, zymography and FACE analysis.

Results: The gene expression of proteoglycans core protein, collagen type I, MMPs and TIMPs is more represented in the area close to the tendon rupture (p< 0.05). The expression of MMPs was confirmed by zymography analysis, showing a marked increase of gelatinolytic activity in area close to the tendon rupture (p< 0.05). The chemical composition of tendon changes showing that in the healthy area the carbohydrate content is higher than the ruptured area (p< 0.05).

Discussion/Conclusions: In the ruptured area, the tenocytes tried to restore the normal proteoglycan pattern increasing the core protein synthesis but without the normal glycosaminoglycan production. Our data support the hypothesis that, in human tendons, the tissue in the area of rupture undergoes marked rearrangement at molecular levels based on the MMP2 activity, and support the role of MMPs in the tendon pathology.

The purpose of this work was to create an in vitro model of tissue-engineered cartilage structure produced by isolated swine articular chondrocytes, expanded in culture and seeded onto a biological scaffold.

Swine articular chondrocytes were enzymatically isolated from pig joints and expanded in monolayer culture. When confluence was reached, cells were resuspended and seeded in vitro onto biological collagen scaffolds for 3, 4 and 6 weeks. Samples were retrieved from the culture and analysed macroscopically and biomechanically by compressive test. Gross evaluation was performed by simple probing, sizing and weighing the samples at all time periods. A baseline of the values was also recorded at time 0. Then, samples were biomechanically tested by unconfined compression and shear tests. Finally, the samples were fixed in 4% paraformaldehyde and processed for histological evaluation. Some samples were stained with Safranin-o, and some others subjected to immunostaining analysis for type II collagen.

Upon retrieval, samples showed dimensional enlargement and mass increase over time and gross mechanic integrity by simple probing. A biomechanical test demonstrated an initial reduction in the values of compressive and shear parameters, followed by a consistent increase throughout the tested time points. Histology showed cartilage-like tissue maturing over time within the biological scaffold.

The results from this study demonstrate that isolated chondrocytes could be seeded onto a biological collagen scaffold, producing cartilage-like matrix with tissue-specific morphology and biomechanical integrity. This tissue-engineered cartilage structure is easily reproducible and it could represent a valuable model for studying the behaviour of different variables on the newly formed cartilage.

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. Gene expression technique was also adopted to characterise the phenotype of the invading cells.

Light microscopy, immunohistochemistry (type I and II collagen), SEM and TEM analysis were performed on five biopsy specimens harvested from five different patients (range, 6 to 16 months after surgery). Fluorophore-assisted carbohydrate electrophoresis (FACE) and real-time PCR evaluation were carried out on two biopsy specimens harvested 6 and 16 months, respectively, after implantation. All these investigations were also applied on non-implanted scaffolds for comparison.

Scaffold sections appeared to be composed of parallel connective laminae, connected by smaller connective bundles surrounding elongated lacunae. In the biopsy specimens, the lacunae were filled by connective tissue with newly formed vessels and fibroblast-like cells. Immunohistochemistry revealed exclusively type I collagen in the scaffold, while type II collagen appeared in the biopsy specimens. FACE analysis carried out in the scaffold did not detect any GAG disaccharides. Conversely, disaccharides were detected in the implants. Real-time PCR showed a signal only for collagen type I. In the scaffolds no gene expression was recorded.

The morphological findings demonstrate that CMI is a biocompatible scaffold available for colonisation by connective cells and vessels. Biochemical data show a specific production of extracellular matrix after implantation. The absence of signal for type II collagen gene can be attributed to different maturation stages of the in-growing tissue.

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 10-30B5m, connected by smaller (5-10B5m) connective bundles, surrounding elongated lacunae of 40-60B5m 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.

Aims: Collagen meniscus implant (CMI) is a tissue engineering technique for the management of irreparable meniscal lesions. We report early clinical results achieved on 30 patients. The implant was also investigated by ultrastructural and biochemical analysis. Methods: Thirty patients, affected by irreparable meniscal lesions, were arthroscopically treated. Average age at the time of surgery was 38.6 years. Additional procedures included 8 ACL reconstruction, 2 high tibial osteotomy and 1 autologous chondrocyte implantation. All knees were evaluated according to the Lysholm II and Tegner activity scales. MRI was performed 6 and 12 months postoperatively. A biopsy of the implant was performed in occasion of a second arthroscopic look in two patients 6 months after surgery. The specimens, as well as the scaffold before implantation, were studied by light microscopy, TEM, SEM, EDAX microanalysis, HPLC and FACE analysis. Results: Follow up averaged 9.3 months. At 3 months, 27 patients showed an increase in the clinical scores. A progressive uniform signal was evident by MRI. Morphological analysis of the speciments showed hyaline tissue inþltrated by cells and vessels, surrounded by the scaffold þbers. At EDAX microanalysis no calciþcations were detected inside the speciments. Biochemical assays demonstrated the presence of GAG molecules of hyaluronic acid and chondroitinsulphate, that were not present in the scaffold before implantation. Conclusions: Early CMI results are promising and are supported by morphological and biochemical þndings, that indicate enhancement of new meniscal tissue by the scaffold.