ABSTRACT
BACKGROUND: For large, locally restricted cartilage defects in young patients, third-generation matrix-supported autologous chondrocyte implantation (ACI) with a variety of scaffolds has shown good mid- to long-term results. PURPOSE/HYPOTHESIS: This study aimed to monitor the clinical and radiological outcomes of patients who received ACI at the knee joint using the Igor scaffold (IGOR-Institute for Tissue and Organ Reconstruction) at 2-year follow-up. Our hypothesis was that there would be improvements in postoperative subjective scores and cartilage repair tissue quality. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: A total of 21 patients (12 male and 9 female) were available for 2-year follow-up after third-generation ACI using the Igor scaffold. All were clinically assessed using the Knee injury and Osteoarthritis Outcome Score (KOOS), Tegner Activity Scale, Brittberg score, International Knee Documentation Committee (IKDC) Subjective Knee Form, Noyes Sports Activity Rating Scale, and visual analog scale for pain. For morphological evaluation, the magnetic resonance observation of cartilage repair tissue (MOCART) and MOCART 2.0 scores were calculated using 3-T magnetic resonance imaging performed at 3, 6, 12, and 24 months postoperatively. Results were compared between baseline and 24 months postoperatively. RESULTS: After 2 years, the clinical and radiological scores showed good to excellent results in the majority of patients. On the IKDC, 10 patients were graded as excellent, 4 as good, 5 as fair, and 2 as severe; on the KOOS, 7 patients were graded as excellent, 8 as good, 4 as fair, and 2 as severe. From baseline to latest follow-up, visual analog scale pain scores decreased from 5.6 ± 3.2 (mean ± SD) to 1.5 ± 2; KOOS results increased from 51 ± 20.7 to 75.2 ± 15.4; and the Tegner score improved from 2.2 ± 1.8 to 4.3 ± 1.3. The MOCART and MOCART 2.0 scores were comparable at 2-year follow-up, with mean values of 74 ± 10 and 78 ± 13, respectively. Satisfactory filling and integration were found in 90.5%. Overall, 16 of 21 patients (76.1%) were satisfied with the surgery and would undergo the procedure again. CONCLUSION: Third-generation ACI using the Igor scaffold showed improvements in clinical and radiological results that were comparable with other scaffolds for patients with large traumatic or degenerative cartilage defects. Patients reported a decrease in pain and an increase in activity, with the majority reporting good results.
ABSTRACT
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.
Subject(s)
Bone Morphogenetic Protein 2/pharmacology , Cell Culture Techniques/methods , Chondrocytes/cytology , Collagen Type II/genetics , Extracellular Matrix Proteins/pharmacology , Aged , Aged, 80 and over , Cell Differentiation , Cells, Cultured , Chondrocytes/drug effects , Chondrocytes/metabolism , Female , Gene Expression Profiling , Gene Expression Regulation/drug effects , Glycosaminoglycans/metabolism , HumansABSTRACT
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-1ß) 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-1ß. CONCLUSION: These data demonstrate that matrix production in transplanted chondrocytes affects maturation of MACT grafts in MRI 2 years after surgery.
Subject(s)
Chondrocytes/transplantation , Knee Injuries/surgery , Transplantation, Autologous , Adult , Cartilage, Articular/injuries , Chondrocytes/metabolism , Gene Expression , Humans , Knee Joint/surgery , Magnetic Resonance Imaging , Middle Aged , Wound Healing/physiology , Young AdultABSTRACT
Lentiviral transduction is a highly efficient DNA delivery method for RNA interference applications. However, obtaining high lentiviral titers of shRNA and miRNA encoding vectors is challenging, since shRNA and miRNA cassettes have been shown to reduce lentiviral titers. In this study, we compare four commercially available packaging mixes and two producer cell lines in order to optimize lentiviral production for gene silencing experiments. Lentiviral vectors encoding a miRNA sequence and emerald green fluorescence protein were co-transfected with ViraPower™, Lenti-X™ HTX, MISSION(®) Lentiviral or Trans-Lentiviral™ packaging mix in HEK-293T or 293FT cells. After transducing HeLa cells with virus-containing supernatant, lentiviral titers were determined by flow cytomerty. In both cell lines, the highest lentiviral titer was obtained with MISSION(®) Lentiviral packaging mix, followed by ViraPower™, Lenti-X™ HTX, and Trans-Lentiviral™. On average, HEK-293T cells produced 6.2-fold higher lentiviral titers than 293FT cells (p < 0.001). With the combination of MISSION(®) Lentiviral packaging mix and HEK-293T cells, an up to 48.5-fold higher lentiviral titer was reached compared to other packaging mixes and producer cell lines. The optimized selection of packaging mix and cell line described in this work should facilitate the production of high-titer lentiviruses for gene silencing experiments.
Subject(s)
Lentivirus/physiology , RNA Interference , Virus Assembly , HEK293 Cells , HumansABSTRACT
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 ß (IL-1ß) 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-1ß 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-1ß 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-1ß expression influence clinical outcomes up to 5 years after MACT.
Subject(s)
Cell Differentiation/physiology , Chondrocytes/transplantation , Interleukin-1beta/metabolism , Knee Injuries/surgery , Adult , Aggrecans/metabolism , Arthroscopy , Cartilage, Articular/surgery , Chondrocytes/cytology , Collagen Type II/metabolism , Female , Humans , Knee Injuries/rehabilitation , Magnetic Resonance Imaging , Male , Pain Measurement , Real-Time Polymerase Chain Reaction , Transplantation, Autologous/methods , Treatment Outcome , Wound HealingABSTRACT
PURPOSE: Our aim was to evaluate the impact of cryopreservation, cultivation time and patient's age on the expression of specific chondrogenic markers in Hyalograft® C transplants. METHODS: Gene expression of chondrocyte markers [collagen type I (COL1A1), COL2A1, aggrecan, versican, melanoma inhibitory activity (MIA) and interleukin (IL)-1ß] was analysed in cartilage biopsies (n = 17) and Hyalograft® C transplant samples (non-cryopreserved = 78, cryopreserved = 13) by quantitative real-time polymerase chain reaction (PCR). Correlation analyses were performed to evaluate the influence of the above-named parameters on the level of gene expression. RESULTS: Cryopreservation of cells was found to decrease COL2A1 and MIA significantly (4.6-fold, p < 0.01 and 2-fold, p < 0.045, respectively). The duration of cryopreservation had no further influence on the expression of these factors. No correlation was detected between cultivation time (75 ± 31 days) and the expression level of any gene. Cartilage transplants from older patients (>35 years) exhibited a significantly higher IL-1ß expression (3.7-fold, p < 0.039) than transplants from younger patients (≤ 35 years). CONCLUSIONS: Our data demonstrate that cryopreservation has a profound impact on chondrocyte metabolic activity by decreasing the expression of COL2A1 and MIA in Hyalograft® C transplants, independent of the duration of cryopreservation.
Subject(s)
Bone Transplantation/methods , Cartilage, Articular/metabolism , Chondrocytes/transplantation , Chondrogenesis/genetics , Chondrogenesis/physiology , Cryopreservation/methods , Gene Expression/physiology , Adult , Age Factors , Aggrecans/genetics , Aggrecans/metabolism , Cartilage, Articular/cytology , Chondrocytes/cytology , Chondrocytes/metabolism , Collagen Type I/genetics , Collagen Type I/metabolism , Extracellular Matrix Proteins/genetics , Extracellular Matrix Proteins/metabolism , Female , Gene Expression/genetics , Humans , Interleukin-1beta/genetics , Interleukin-1beta/metabolism , Male , Middle Aged , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Time Factors , Tissue Engineering , Versicans/genetics , Versicans/metabolismABSTRACT
In this study we compared the expression of hormone receptors and markers of differentiation in growth plate chondrocytes (GPC) and articular chondrocytes (AC) under different culture conditions by real-time PCR and immunofluorescence. After one week of monolayer culture, porcine GPC and AC of 6-8-week-old piglets were either seeded in alginate beads or further grown in monolayer culture up to 5 weeks. During monolayer culture the expression of Col2, Col1, Col10, aggrecan, ERalpha and ERbeta decreased dramatically, whereas culture in alginate beads restored an expression pattern similar to native tissue. The receptors IGF-1R, IGF-2R and GHR were, however, increased in monolayer culture compared to alginate culture or native tissue. The relative proportion of ERalpha and ERbeta expression was different in GPC when compared to AC. All of our results on mRNA expression during culture in alginate beads were also verified to be translated to the protein level. Thus, the expression of hormone rereptors and differentiation markers were found to be highly influenced by culture conditions. Moreover, the porcine model is promising as defined by tissue availability, expression of relevant hormonal receptors and comparability to human conditions, in particular in the area of basic growth research.
Subject(s)
Antigens, Differentiation/biosynthesis , Cartilage, Articular/metabolism , Chondrocytes/metabolism , Growth Plate/metabolism , Receptors, Cell Surface/biosynthesis , Animals , Cartilage, Articular/cytology , Cell Culture Techniques , Cells, Cultured , Chondrocytes/cytology , Growth Plate/cytology , Organ Specificity/physiology , Swine , Time FactorsABSTRACT
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 10(7) cells/ml. The defect was completely filled with the cell suspension (approximately 30 microl) 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.
Subject(s)
Cartilage/physiology , Regeneration , Alcian Blue , Animals , Cartilage/cytology , Collagen Type I/metabolism , Collagen Type II/metabolism , Diffusion , Female , Humans , Immunohistochemistry , Magnetic Resonance Imaging , Male , Mice , Mice, Inbred BALB C , Models, Animal , Periosteum/cytologyABSTRACT
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.
Subject(s)
Aging/physiology , Bioreactors , Cartilage, Articular/cytology , Cartilage, Articular/physiology , Cell Culture Techniques/instrumentation , Chondrocytes/physiology , Chondrogenesis/physiology , Tissue Engineering/methods , Aged , Aged, 80 and over , Cell Culture Techniques/methods , Cell Differentiation/physiology , Cell Division/physiology , Cells, Cultured , Equipment Design , Equipment Failure Analysis , Feasibility Studies , HumansABSTRACT
The activation of Myc induces apoptosis of human ovarian adenocarcinoma N.1 cells when serum factors are limited. However, the downstream mechanism that is triggered by Myc is unknown. Myc-activation and treatment with the proapoptotic ligands TNFalpha, FasL, and TRAIL induced H-ferritin expression under serum-deprived conditions. H-ferritin chelates intracellular iron and also intracellular iron sequestration by deferoxamine-induced apoptosis of N.1 cells. Supplementation of serum-free medium with holo-transferrin blocked apoptosis of N.1 cells that was induced by Myc-activation or by treatment with TNFalpha, FasL, and TRAIL, whereas apotransferrin did not prevent apoptosis. This suggests that intracellular iron depletion was a trigger for apoptosis and that transferrin-bound iron rescued N.1 cells. Furthermore, apoptosis of primary human ovarian carcinoma cells, which was induced by TNFalpha, FasL, and TRAIL, was also inhibited by holo-transferrin. The data suggest that Myc-activation, FasL, TNFalpha, and TRAIL disturbed cellular iron homeostasis, which triggered apoptosis of ovarian carcinoma cells and that transferrin iron ensured survival by re-establishing this homeostasis.