Craig Willers

Gene expression profiles of human chondrocytes during passaged monolayer cultivation

Chondrocyte phenotype has been shown to dedifferentiate during passaged monolayer cultivation. Hence, we have investigated the expression profile of 27 chondrocyte‐associated genes from both osteoarthritic cartilage tissue and healthy passaged human articular chondrocytes by quantitative real‐time PCR. Our results indicate that the gene expression levels of matrix proteins and proteases in chondrocytes from monolayer culture decrease compared with those from cartilage tissue, while monolayer cultured chondrocytes from normal and osteoarthritic cartilage exhibit similar gene expression patterns. However, chondrocytic gene expression profiles were differentially altered at various stages of passage. The expression of the matrix proteins aggrecan, type II collagen, and fibromodulin inversely correlated with increasing passage number, while fibronectin and link protein exhibited a marked increase with passage. The expression of matrix proteinases MMP‐3/9/13 and ADAMTS‐4/5 decreased with passage, whereas proteinase inhibitors TIMP‐2/3 were elevated. The cytokine IL‐1 also showed increased expression with monolayer chondrocyte culture, while IGF‐1 expression levels were diminished. No significant changes in TGF‐β, or the chondrogenic transcription factors Sox‐9, c‐fos, or c‐jun were observed. Our data indicates that cultured chondrocytes undergo dedifferentiation during monolayer culture, although the gene expression level of transcription factors necessary for chondrogenesis remains unchanged. This data may prove important for the future development of more specific and efficacious cultivation techniques for human articular chondrocyte‐based therapies.

A CURRENT REVIEW ON THE BIOLOGY AND TREATMENT OF ARTICULAR CARTILAGE DEFECTS (PART I & PART II)

Osteochondral injury occurs predominantly in physically active young adult males. Injury to the articular cartilage and/or subchondral bone may not only cause acute joint disease resulting in osseous intracapsular (synovitis) or extracapsular pain, but may also act to spawn arthritic conditions in later life. Since the 18th century, such injury has proven difficult to treat clinically, and much therapy has been essentially palliative. Past treatments such as abrasion arthroplasty, drilling, microfracture and arthroscopic lavage have been useful in removing articular debris and promoting the formation of the fibrin clot used in most native repair mechanisms. However, the limitation of these techniques is their inability to restore the damaged cartilage and subchondral bone to their normal tissue architecture. Recent developments in tissue engineering have concentrated on the utilization of autologous chondrocyte implantation, biomaterials and growth factors to promote the regeneration of biomechanically su...

Articular cartilage repair: procedures versus products

This review discusses the current perspectives and practices regarding the treatment of articular cartilage injury. Specifically, the authors have delineated and examined articular cartilage repair techniques as either surgical procedures or manufactured products. Although both methodologies are used to treat articular cartilage injury, there are obvious advantages and disadvantages to the application of both, with the literature providing few recommendations on the most suitable regimen for the patient and surgeon. In recent times, cell-based tissue engineering products, predominantly autologous chondrocyte implantation, have been the subject of much research and have become clinically popular. Herein, we review the most used procedures and products in cartilage repair, compare and contrast their outcomes, and evaluate the issues that must be overcome in order to improve patient efficacy in the future.

Quantification of Chondrocyte Morphology by Confocal Arthroscopy

The purpose of this study was to determine the effectiveness of a novel Laser Scanning Confocal Arthroscope (LSCA) for the morphological quantification of articular cartilage chondrocytes. Healthy and debrided regions of the knee articular cartilage of six (6) New Zealand White rabbits were imaged during open follow-up surgery. Quantitative morphological analysis of chondrocyte cell populations was performed and compared to known parameters. Optical histology images were compared to conventional histology of similar sites. Optical histology revealed viable cells in normal hyaline cartilage tissue and enabled the visualization of fibro-cartilage in defect tissue. Morphological analysis was able to characterize the in vivo two-dimensional equivalent-area-diameter of chondrocytes. Significant differences (P<0.05) were seen between the morphology of chondrocytes observed in optical and conventional histology. This study concludes that the LSCA is capable of illustrating the surface and sub-surface appearance of healthy and defect articular cartilage, thereby providing a non-destructive method for assessing cartilage condition in vivo. In this role the LSCA may find application in the investigation of cartilage pathologies or repair techniques.

22.3 Matrix-induced Autologous Chondrocyte Implantation (MACI®): Biological and Histological Assessment.

Matrix-induced autologous chondrocyte implantation (MACI) has been a treatment of cartilage injury since 2000, but little is known of the histological paradigm of tissue regeneration after implantation. MACI is a stable cell-based delivery system that enables the regeneration of hyaline-like cartilage. From a cohort of 56 MACI patients, we examined the phenotype of chondrocytes seeded on type I/III collagen scaffold, and conducted progressive histologic assessment over a period of 6 months. Chondrocyte-seeded collagen scaffolds from patient implants were analyzed by electron microscopy, immunohistochemistry (type II collagen and S-100), and reverse transcription polymerase chain reaction (RT-PCR) (aggrecan and type II collagen). Coincidental cartilage biopsies were obtained at 48 hours, 21 days, 6 months, 8 months, 12 months, 18 months, and 24 months. Our data showed that chondrocytes on the collagen scaffold appeared spherical, well integrated into the matrix, and maintained the chondrocyte phenotype as evidenced by aggrecan, type II collagen, and S-100 expression. Progressive histologic evaluation of the biopsies showed the formation of cartilage-like tissue as early as 21 days, and 75% hyaline-like cartilage regeneration after 6 months. This preliminary study has suggested that MACI may offer an improved alternative to traditional treatments for cartilage injury by regenerating hyaline-like cartilage as early as 6 months after surgery.

Evaluation of Intraoperative Retention of Autologous Chondrocytes on Type I/III Collagen Scaffold (Ortho-ACI™) for Cartilage Repair

Methods and Materials We tested cell retention on the scaffold by confocal microscopy at 7, 15, 20, 40, 60, 90 and 120 minutes after seeding, and the molecular profile (collagen II, aggrecan, Sox9, HAPLN1) of chondrocytes seeded at 20 minutes and 4 days (preoperative seeding method). Fifteen OrthoACITM patients with 25 cartilage defects were assessed by arthroscopic or magnetic resonance imaging (MRI). Graft repair was graded as excellent, good, poor or no infill. Associations between repair outcome and case variables were also investigated.