Pierre Mainil-Varlet

Autologous osteochondral grafting in the knee: indication, results, and reflections.

The use of multiple autologous osteochondral plugs (mosaicplasty) for repair of articular cartilage defects is a well-accepted technique. Since 1995, the authors have used mosaicplasty to treat more than 110 patients with cartilage defects of the knee, hip, and ankle. The first 52 consecutive patients who had mosaicplasty of the knee and have an average followup of 37 months (range, 24–56 months) were examined. Indications for surgical treatment were osteochondritis dissecans, acute trauma, and posttraumatic lesions of the femorotibial joint, femoropatellar maltracking with recurrent episodes of patella dislocations, and distinct femoropatellar arthrosis. Preoperatively, cartilage defects were classified as International Cartilage Repair Society Grade III lesions in 23 patients and Grade IV lesions in 29 patients. Two years after surgery, an increased level of knee function was found in 86% of the patients. At the latest followup, improved knee function was observed in 92% of the patients. In four patients, reoperation was necessary because of graft failure. Complications and reoperation rate were related to large surface lesions. Autologous osteochondral transplantation is a valid option for the treatment of full-thickness osteochondral defects. However, the method is limited by the defect size and the number of plugs to be taken at the donor site.

Early osteoarthritic changes of human femoral head cartilage subsequent to femoro-acetabular impingement☆

OBJECTIVE To use the surgical samples of patients with femoro-acetabular impingement due to a nonspherical head to analyze tissue morphology and early cartilage changes in a mechanical model of hip osteoarthritis (OA). DESIGN An aberrant nonspherical shape of the femoral head has been assumed to cause an abutment conflict (impingement mechanism) of the hip with subsequent cartilage lesions of the acetabular rim and surface alterations of the nonspherical portion of the head. In this study, 22 samples of the nonspherical portions of the head have been obtained during hip surgery from young adults (mean 30.4 years, range 19-45 years) with an impingement conflict. The samples were first compared with tissue from the same area obtained from six age-matched deceased persons (control group) with normal hip morphology and second with cartilage from 14 older patients with advanced OA. All samples were characterized histologically and hyaline cartilage was graded according to the Mankin criteria. They were further subjected to examination on a molecular basis by immunohistology for cartilage oligomeric matrix protein (COMP), tenascin-C and a collagenase cleavage product (COL2-3/4C(long)) and by in situ hybridization for collagen type I and collagen type II. RESULTS All samples from the patient group revealed hyaline cartilage with degenerative signs. According to the Mankin criteria, the cartilage alterations were significantly different when compared with the control group (p=0.007) but were less distinct when compared with cartilage from patients with advanced OA (p=0.014). Positive staining and distribution pattern for COMP, tenascin-C and COL2-3/4C(long) showed similarities between the samples from the impingement group and osteoarthritic cartilage but they were distinctly different when compared with healthy cartilage. Levels of collagen I and II transcripts were upregulated in 6 and 10, respectively, of the 14 samples with OA and in 9 and 12, respectively, of the 22 samples from the impingement group. None of the samples from the control group showed upregulation of Collagen I and II mRNA. CONCLUSIONS The aberrant nonspherical portion of the femoral head in young patients with an impingement conflict consists of hyaline cartilage which shows clear degenerative signs similar to the findings in osteoarthritic cartilage. The tissue alterations are distinctly different when compared with a control group, which substantiates an impingement conflict as an early mechanism for degeneration at the hip joint periphery.

Immunophenotypic analysis of human articular chondrocytes: Changes in surface markers associated with cell expansion in monolayer culture

Cartilage tissue engineering relies on in vitro expansion of primary chondrocytes. Monolayer is the chosen culture model for chondrocyte expansion because in this system the proliferative capacity of chondrocytes is substantially higher compared to non‐adherent systems. However, human articular chondrocytes (HACs) cultured as monolayers undergo changes in phenotype and gene expression known as “dedifferentiation.” To gain a better understanding of the cellular mechanisms involved in the dedifferentiation process, our research focused on the characterization of the surface molecule phenotype of HACs in monolayer culture. Adult HACs were isolated by enzymatic digestion of cartilage samples obtained post‐mortem. HACs cultured in monolayer for different time periods were analyzed by flow cytometry for the expression of cell surface markers with a panel of 52 antibodies. Our results show that HACs express surface molecules belonging to different categories: integrins and other adhesion molecules (CD49a, CD49b, CD49c, CD49e, CD49f, CD51/61, CD54, CD106, CD166, CD58, CD44), tetraspanins (CD9, CD63, CD81, CD82, CD151), receptors (CD105, CD119, CD130, CD140a, CD221, CD95, CD120a, CD71, CD14), ectoenzymes (CD10, CD26), and other surface molecules (CD90, CD99). Moreover, differential expression of certain markers in monolayer culture was identified. Up‐regulation of markers on HACs regarded as distinctive for mesenchymal stem cells (CD10, CD90, CD105, CD166) during monolayer culture suggested that dedifferentiation leads to reversion to a primitive phenotype. This study contributes to the definition of HAC phenotype, and provides new potential markers to characterize chondrocyte differentiation stage in the context of tissue engineering applications.

Equine Peripheral Blood-Derived Progenitors in Comparison to Bone Marrow-Derived Mesenchymal Stem Cells

Fibroblast‐like cells isolated from peripheral blood of human, canine, guinea pig, and rat have been demonstrated to possess the capacity to differentiate into several mesenchymal lineages. The aim of this work was to investigate the possibility of isolating pluripotent precursor cells from equine peripheral blood and compare them with equine bone marrow‐derived mesenchymal stem cells. Human mesenchymal stem cells (MSCs) were used as a control for cell multipotency assessment. Venous blood (n = 33) and bone marrow (n = 5) were obtained from adult horses. Mononuclear cells were obtained by Ficoll gradient centrifugation and cultured in monolayer, and adherent fibroblast‐like cells were tested for their differentiation potential. Chondrogenic differentiation was performed in serum‐free medium in pellet cultures as a three‐dimensional model, whereas osteogenic and adipogenic differentiation were induced in monolayer culture. Evidence for differentiation was made via biochemical, histological, and reverse transcription‐polymerase chain reaction evaluations. Fibroblast‐like cells were observed on day 10 in 12 out of 33 samples and were allowed to proliferate until confluence. Equine peripheral blood‐derived cells had osteogenic and adipogenic differentiation capacities comparable to cells derived from bone marrow. Both cell types showed a limited capacity to produce lipid droplets compared to human MSCs. This result may be due to the assay conditions, which are established for human MSCs from bone marrow and may not be optimal for equine progenitor cells. Bone marrow‐derived equine and human MSCs could be induced to develop cartilage, whereas equine peripheral blood progenitors did not show any capacity to produce cartilage at the histological level. In conclusion, equine peripheral blood‐derived fibroblast‐like cells can differentiate into distinct mesenchymal lineages but have less multipotency than bone marrow‐derived MSCs under the conditions used in this study.

Effect of in vivo and in vitro degradation on molecular and mechanical properties of various low‐molecular‐weight polylactides

The in vivo and in vitro degradation of low-molecular-weight poly(L-lactide), poly(L/D-lactide), and poly (L/DL-lactide) rods was investigated. The low-molecular-weight fast-degrading materials were used to accelerate the degradation process and make the test conditions more critical. In the in vivo study the rods were implanted in the soft tissue of sheep and explanted at 1, 3, 6, and 12 months. In the in vitro experiments the samples were subjected to aging at 37 degrees C in the phosphate buffer using two different modes. In the so-called pseudodynamic mode the aging buffer was regularly replaced if the pH dropped more than 0.5. In the static mode the buffer was not changed over the whole testing period of 52 weeks. The mechanical, molecular, and crystalline properties of the rods were measured and their appearance in the course of aging was evaluated using scanning electron microscopy. It was found that the changes in the mechanical properties of poly(L-lactide), poly(L/D-lactide), and poly(L/DL-lactide) samples subjected to in vitro degradation tests in both the static and pseudodynamic modes are in good approximation with data obtained from the in vivo study. The pH of the buffer solution had no evident effect on the mechanical properties or the rate of degradation as estimated from the drop in molecular weight of the aged samples. The replacement of the aging buffer to maintain a constant pH at 7.4 does not seem to be critical for the degradation of the polylactides. In vitro degradation tests can be used as a relevant procedure for predicting the in vivo functionality of implants from the polylactides used if the criteria for assessing such a functionality are the changes in mechanical properties and molecular weight.

Long-term in vivo degradation and bone reaction to various polylactides. 1. One-year results

Injection-moulded pins from poly(L-lactide), poly(L/DL-lactide) (95/5%) were implanted in the cortex of the tibiae of sheep. The bone-implant interface was evaluated to observe whether there is any bone resorption caused by the implants. The molecular weight and crystallinity changes upon implantation were also measured. There was no net bone loss around the implants or sterile cyst formation in any of the animals implanted with polylactides up to 1 year. The new bone formed around the poly(L-lactide) and poly(L/D-lactide) pins was separated from the implants with a thin layer of connective tissue. For the implants from poly(L/DL-lactide), there was direct apposition of bone on the polymeric material. At 1 year of implantation, the implants were not completely resorbed, although the molecular weight of polylactides was reduced from 40,000-50,000 to 500-300. The crystallinity at 1 year was about 45% for poly(L/DL-lactide) and poly(L/DL-lactide) and 65% for poly(L-lactide), respectively, indicating the presence in the degraded material of thermodynamically stable crystals.

Immunophenotypic changes of human articular chondrocytes during monolayer culture reflect bona fide dedifferentiation rather than amplification of progenitor cells

In this study, a time‐course comparison of human articular chondrocytes (HAC) and bone marrow‐derived mesenchymal stem cells (MSC) immunophenotype was performed in order to determine similarities/differences between both cell types during monolayer culture, and to identify HAC surface markers indicative of dedifferentiation. Our results show that dedifferentiated HAC can be distinguished from MSC by combining CD14, CD90, and CD105 expression, with dedifferentiated HAC being CD14+/CD90bright/CD105dim and MSC being CD14‐/CD90dim/CD105bright. Surface markers on MSC showed little variation during the culture, whereas HAC showed upregulation of CD90, CD166, CD49c, CD44, CD10, CD26, CD49e, CD151, CD51/61, and CD81, and downregulation of CD49a, CD54, and CD14. Thus, dedifferentiated HAC appear as a bona fide cell population rather than a small population of MSC amplified during monolayer culture. While most of the HAC surface markers showed major changes at the beginning of the culture period (Passage 1–2), CD26 was upregulated and CD49a downregulated at later stages of the culture (Passage 3–4). To correlate changes in HAC surface markers with changes in extracellular matrix gene expression during monolayer culture, CD14 and CD90 mRNA levels were combined into a new differentiation index and compared with the established differentiation indices based on the ratios of mRNA levels of collagen type II to I (COL2/COL1) and of aggrecan to versican (AGG/VER). A correlation of CD14/CD90 ratio at the mRNA and protein level with the AGG/VER ratio during HAC dedifferentiation in monolayer culture validated CD14/CD90 as a new membrane and mRNA based HAC differentiation index. J. Cell. Physiol. 214:75–83, 2008.

International Cartilage Repair Society (ICRS) Recommended Guidelines for Histological Endpoints for Cartilage Repair Studies in Animal Models and Clinical Trials.

Cartilage repair strategies aim to resurface a lesion with osteochondral tissue resembling native cartilage, but a variety of repair tissues are usually observed. Histology is an important structural outcome that could serve as an interim measure of efficacy in randomized controlled clinical studies. The purpose of this article is to propose guidelines for standardized histoprocessing and unbiased evaluation of animal tissues and human biopsies. Methods were compiled from a literature review, and illustrative data were added. In animal models, treatments are usually administered to acute defects created in healthy tissues, and the entire joint can be analyzed at multiple postoperative time points. In human clinical therapy, treatments are applied to developed lesions, and biopsies are obtained, usually from a subset of patients, at a specific time point. In striving to standardize evaluation of structural endpoints in cartilage repair studies, 5 variables should be controlled: 1) location of biopsy/sample section, 2) timing of biopsy/sample recovery, 3) histoprocessing, 4) staining, and 5) blinded evaluation with a proper control group. Histological scores, quantitative histomorphometry of repair tissue thickness, percentage of tissue staining for collagens and glycosaminoglycan, polarized light microscopy for collagen fibril organization, and subchondral bone integration/structure are all relevant outcome measures that can be collected and used to assess the efficacy of novel therapeutics. Standardized histology methods could improve statistical analyses, help interpret and validate noninvasive imaging outcomes, and permit cross-comparison between studies. Currently, there are no suitable substitutes for histology in evaluating repair tissue quality and cartilaginous character.

Preclinical Studies for Cartilage Repair Recommendations from the International Cartilage Repair Society

Investigational devices for articular cartilage repair or replacement are considered to be significant risk devices by regulatory bodies. Therefore animal models are needed to provide proof of efficacy and safety prior to clinical testing. The financial commitment and regulatory steps needed to bring a new technology to clinical use can be major obstacles, so the implementation of highly predictive animal models is a pressing issue. Until recently, a reductionist approach using acute chondral defects in immature laboratory species, particularly the rabbit, was considered adequate; however, if successful and timely translation from animal models to regulatory approval and clinical use is the goal, a step-wise development using laboratory animals for screening and early development work followed by larger species such as the goat, sheep and horse for late development and pivotal studies is recommended. Such animals must have fully organized and mature cartilage. Both acute and chronic chondral defects can be used but the later are more like the lesions found in patients and may be more predictive. Quantitative and qualitative outcome measures such as macroscopic appearance, histology, biochemistry, functional imaging, and biomechanical testing of cartilage, provide reliable data to support investment decisions and subsequent applications to regulatory bodies for clinical trials. No one model or species can be considered ideal for pivotal studies, but the larger animal species are recommended for pivotal studies. Larger species such as the horse, goat and pig also allow arthroscopic delivery, and press-fit or sutured implant fixation in thick cartilage as well as second look arthroscopies and biopsy procedures.

In vitro model for the study of necrosis and apoptosis in native cartilage.

Apoptosis plays a role in everything from early development to ageing and in a host of disease states. Studying this important process in the in vivo state is critical, to understand its varied role and to open further avenues of therapeutic intervention. The present paper presents an ex vivo bovine articular cartilage model to study apoptotic and necrotic processes following acute injury. Ex vivo bovine articular cartilage was assessed 1, 3 and 6 days following holmium : YAG laser treatment (780 mJ). Markers to visualize cell viability, caspase‐3 activity, changes in mitochondrial membrane potential and the degree of DNA fragmentation (TUNEL assay) were used alone or in various combinations. Standard histology and transmission electron microscopy (TEM) were also performed for a more comprehensive assessment. A significant progression (p < 0.05) of ethidium/caspase‐3‐positive signal depth at day 3 preceded a significant increase (p < 0.05) in TUNEL signal depth by day 6. The mitochondrial matrix marker CMXRos was shown to provide an alternative to calcein‐AM for assessing cell viability. The identification of chondrocyte apoptosis morphology by TEM was not conclusive. Nevertheless, TEM revealed that cells which were clearly necrotic also stained positively for TUNEL, thus indicating the risk of using TUNEL alone for the assessment of apoptosis. The model described here allows the rapid, spatial and temporal determination of cell viability and of apoptotic and necrotic processes in whole‐tissue specimens after acute injury, and permits study of the balance between these events. The assessment of healthy and diseased cartilage and of the effects of surgical, pharmaceutical or in vitro intervention are immediate applications of these protocols. Moreover, this model may be useful for the study of key mechanisms involved in apoptosis or for the establishment of other markers of apoptosis. Copyright