Brigitte Tichy

Chondrogenesis of Aged Human Articular Cartilage in a Scaffold-Free Bioreactor

Chondrogenesis of aged human articular chondrocytes was evaluated under controlled in vitro conditions, using a rotating bioreactor vessel. Articular chondrocytes isolated from 10 aged patients (median age, 84 years) were increased in monolayer culture. A single-cell suspension of dedifferentiated chondrocytes was inoculated in a rotating wall vessel, without the use of any scaffold or supporting gel material. After 90 days of cultivation, a three-dimensional cartilage-like tissue was formed, encapsulated by fibrous tissue resembling a perichondrial membrane. Morphological examination revealed differentiated chondrocytes ordered in clusters within a continuous dense cartilaginous matrix demonstrating a strong positive staining with monoclonal antibodies against collagen type II and articular proteoglycan. The surrounding fibrous membrane consisted of fibroblast-like cells, and showed a clear distinction from the cartilaginous areas when stained against collagen type I. Transmission electron microscopy revealed differentiated and highly metabolically active chondrocytes, producing an extracellular matrix consisting of a fine network of randomly distributed cross-banded collagen fibrils. Chondrogenesis of aged human articular chondrocytes can be induced in vitro in a rotating bioreactor vessel using low shear and efficient mass transfer. Moreover, the tissue-engineered constructs may be used for further in vitro studies of differentiation, aging, and regeneration of human articular cartilage.

Influence of Cell Differentiation and IL-1β Expression on Clinical Outcomes After Matrix-Associated Chondrocyte Transplantation

Background: Several patient- and defect-specific factors influencing clinical outcomes after matrix-associated chondrocyte transplantation (MACT) have been identified, including the patient’s age, location of the defect, or duration of symptoms before surgery. Little is known, however, about the influence of cell-specific characteristics on clinical results after transplantation. Purpose: The aim of the present study was to investigate the influence of cell differentiation and interleukin-1 β (IL-1β) expression on clinical outcomes up to 5 years after MACT. Study Design: Case series; Level of evidence, 4. Methods: Twenty-seven patients who underwent MACT of the tibiofemoral joint area of the knee were included in this study. Clinical assessments were performed preoperatively as well as 6, 12, 24, and 60 months after transplantation by using the following scores: the Knee injury and Osteoarthritis Outcome Score (KOOS), the International Knee Documentation Committee (IKDC) Subjective Knee Form, the Noyes sports activity rating scale, the Brittberg clinical score, and a visual analog scale (VAS) for pain. The quality of repair tissue was assessed by magnetic resonance imaging using the magnetic resonance observation of cartilage repair tissue (MOCART) score at 1 and 5 years. Cell differentiation (defined as collagen type II:type I expression ratio), aggrecan, and IL-1β expression were determined by real-time polymerase chain reaction in transplant residuals and were correlated with clinical outcomes. Results: The largest improvements in clinical scores were found during the first year. Two years postoperatively, a stable improvement was reached until 5 years after transplantation, with a mean IKDC score of 34.4 ± 8.6 preoperatively to 77.9 ± 16 after 24 months (P < .001). Cell differentiation showed a significant positive correlation with nearly all clinical scores at different time points, especially after 12 months (P < .05). IL-1β expression negatively influenced clinical outcomes at 24 months (Brittberg score) and 60 months (Brittberg and VAS scores) after surgery (P < .05). No correlation was found between the MOCART score and clinical outcomes or gene expression. Conclusion: Our data demonstrate that cell differentiation and IL-1β expression influence clinical outcomes up to 5 years after MACT.

An in vivo mouse model for human cartilage regeneration.

Cartilage regeneration methods have been examined in various animal models. The major limitation of those studies is the biological difference between human and animal cartilage. We propose an in vivo model for human chondrocytes in a human cartilage defect environment. Human full‐thickness (2–4 mm) articular cartilage discs (diameter 10 mm) attached to 3–6 mm subchondral bone, were obtained from human femur heads. Chondral defects (diameter 4 mm) were set within the cartilage disc without violating the subchondral bone. Human chondrocytes were isolated, cultivated for three passages and then suspended at a concentration of 107 cells/ml. The defect was completely filled with the cell suspension (∼30 µl) and then covered with a thin sheet of human periosteum, fixed with fibrin sealant. Discs were implanted subcutaneously in the backs of nude mice for 5 and 8 weeks. Controls were uncovered discs filled with cell suspension and covered discs without cells. Histological evaluation revealed a gradient of differentiation from the cartilage lateral side to the centre of the defect. A proteoglycan‐rich matrix was formed with some chondron‐like structures at the border of native cartilage, whereas fibrous tissue was built in the centre of the defect. After 8 weeks the areas of differentiating cells enlarged compared to 5 weeks, indicating the progress of cartilage repair. The control discs without cells or cover showed no chondrogenesis. Interestingly, uncovered discs filled with cells showed comparable areas of differentiating cells at the defect surface but lack of fibrous tissue in the middle. The histological results were supported by MRI measurement. Copyright

Tibial cartilage hypertrophy due to matrix-associated autologous chondrocyte transplantation of the medial femoral condyle. A case report

Osteochondral graft hypertrophy is commonly seen after autologous chondrocyte transplantation with use of a periosteal flap1,2. Reoperation may be performed because of a high grade of hypertrophy causing persistent clinical symptoms. The use of a collagen membrane to cover the chondrocyte suspension can reduce the risk of graft hypertrophy3,4. Further technological advances have led to a third generation of autologous chondrocyte transplantation with use of biomaterials seeded with chondrocytes as so-called all-in-one scaffolds that generate new articular cartilage5. Nevertheless, graft hypertrophy continues to occur, although the prevalence has been reduced with matrix-associated autologous chondrocyte transplantation6,7. We present the case of a patient who complained of a reduced range of motion after matrix-associated autologous chondrocyte transplantation of the medial femoral condyle. The patient was found to have tibial cartilage hypertrophy opposite the site of the matrix-associated autologous chondrocyte transplantation. The patient was informed that data concerning the case would be submitted for publication, and he consented. A forty-nine-year-old man was treated with matrix-associated autologous chondrocyte transplantation for a single full-thickness cartilage defect of the medial femoral condyle. The nature of the cartilage defect was traumatic, and the time interval between the initial symptoms and diagnosis was approximately six months. Surgical treatment followed after another six months, as nonsurgical therapy did not provide substantial clinical improvement. During the two-step surgical procedure, in a first arthroscopic step (Fig. 1), the isolated nature of the cartilage defect was documented, the borders of the defect were debrided, approximately 200 mg of normal cartilage tissue was harvested for culture, and the chondrocytes were cultured for three weeks. In the second step, with use of a mini-arthrotomy, a hyaluronan-based scaffold (Hyalograft C; Fidia Advanced Biopolymers, Abano Terme, Italy), seeded with the cultured chondrocytes, …

Matrix Production Affects MRI Outcomes After Matrix-Associated Autologous Chondrocyte Transplantation in the Knee

Background: Matrix-associated autologous chondrocyte transplantation (MACT) has been an effective therapy for large, full-thickness cartilage lesions for years. However, little is known about how graft maturation is affected by characteristics of transplanted chondrocytes. Purpose: To investigate the influence of gene expression of chondrocytes at the time of transplantation on MRI outcomes up to 2 years after MACT. Study Design: Case series; Level of evidence, 4. Methods: This study included 25 patients with 27 symptomatic traumatic defects of articular cartilage, who had undergone MACT in the knee. Postoperative MRI examinations were conducted at 3, 6, 12, and 24 months after surgery. Biochemical graft maturation was assessed by measuring T2 relaxation time values of the transplant and healthy native cartilage areas. The MOCART (magnetic resonance observation of cartilage repair tissue) score was used to evaluate the morphological quality of regeneration tissue. Gene expression (collagen type I, collagen type II, aggrecan, versican, and interleukin-1β) was determined by real-time polymerase chain reaction (PCR) in transplant residuals at the time point of transplantation and was correlated with MRI outcomes using Spearman’s rank correlation coefficient. A Friedman test with post hoc analysis (Wilcoxon signed rank test) conducted with a Bonferroni correction was applied to compare scores at different time points. Results: T2 relaxation time of regeneration tissue improved from a mean ± SD of 74.6 ± 20.1 milliseconds at 3 months to 47.9 ±13.3 milliseconds at 24 months (P < .003). These values were similar to the T2 relaxation times of the native surrounding cartilage (50.9 ± 15 ms). The calculated T2 index (ratio of regeneration tissue to native cartilage) improved from 1.63 ± 0.76 at 3 months to 1.0 ± 0.4 at 24 months (P < .011). The MOCART score increased from 51.6 ± 15 points to 72.4 ± 12.2 points (P < .001). Improvement of the T2 index over time significantly correlated with aggrecan, COL1A1, COL2A1, and versican expression (rs = 0.9, P < .001; rs = 0.674, P < .012; rs = 0.553, P < .05; and rs = 0.575, P < .04, respectively). No correlation was found for IL-1β. Conclusion: These data demonstrate that matrix production in transplanted chondrocytes affects maturation of MACT grafts in MRI 2 years after surgery.

Redifferentiation of aged human articular chondrocytes by combining bone morphogenetic protein-2 and melanoma inhibitory activity protein in 3D-culture

Melanoma inhibitory activity (MIA) affects the differentiation to hyaline cartilage and can inhibit the osteogenic potential of bone morphogenetic protein (BMP)-2 in mesenchymal stem cells (MSC). The aim of this study was to investigate if MIA also inhibits the osteogenic potential of BMP-2 in human articular chondrocytes during redifferentiation, which may lead to a higher grade of differentiation without calcification. HAC of four female patients (mean age: 73.75 ±6.98) were seeded into 3D culture for 28 days; after adding the recombinant proteins, four groups were formed (Control, BMP-2, MIA, BMP-2+MIA). Samples were analysed for gene expression, glycosaminoglycan (GAG) content and histology on day 0, 14 and 28. Collagen type 2 (COL2A1) was significantly increased in the BMP-2 containing groups on day 28; BMP-2 (100-fold, p = 0.001), BMP-2+MIA (65-fold, p = 0.009) and similar to the level of native cartilage. Higher aggrecan (Agg) levels were present in the BMP-2 (3-fold, p = 0.007) and BMP-2+MIA (4-fold, p = 0.002) group after 14 days and in the BMP-2 (9-fold, p = 0.001) group after 28 days. Collagen type 10 (COL10A1) was increased in the BMP-2 containing groups (6-fold, p = 0.006) but these levels were significantly below native cartilage. Alkaline phosphatase (ALP), collagen type 1 (COL1A1) and the glycosaminoglycan (GAG) content did not reveal any relevant differences between groups. BMP-2 is a potent inducer for differentiation of HAC. A significant enhancement of this effect in combination with MIA could not be observed. Furthermore no significant reduction of osteogenic markers during re-differentiation of chondrocytes was present combining BMP-2 and MIA.