Peter Angele

Cyclic hydrostatic pressure enhances the chondrogenic phenotype of human mesenchymal progenitor cells differentiated in vitro

Much attention has been given to the influences of bioactive factors on mesenchymal progenitor cell differentiation and proliferation, but few studies have examined the effect of mechanical factors on these cells. This study examined the effects of cyclic hydrostatic pressure on human bone marrow‐derived mesenchymal progenitor cells undergoing chondrogenic differentiation. Aggregates of bone marrow‐derived mesenchymal progenitor cells were cultured in a defined chondrogenic medium and were subjected to cyclic hydrostatic pressure. Aggregates were loaded at various time points: single (day 1 or 3) or multiple (days 1–7). At 14 and 28 days, aggregates were harvested for histology, immunohistochemistry, and quantitative DNA and matrix macromolecule analysis. The aggregates loaded for a single day did not demonstrate significant changes in proteoglycan and collagen contents compared with the non‐loaded controls. In contrast, for the multi‐day loaded aggregates, statistically significant increases in proteoglycan and collagen contents were found on both day 14 and day 28. Aggregates loaded for seven days were larger and histological staining indicated a greater matrix/cell ratio. This study indicates that hydrostatic pressure enhances the cartilaginous matrix formation of mesenchymal progenitor cells differentiated in vitro, and suggests that mechanical forces may play an important role in cartilage repair and regeneration in vivo.

Development of new reconstructive techniques: use of Integra in combination with fibrin glue and negative-pressure therapy for reconstruction of acute and chronic wounds.

Large wounds resulting from severe injuries are generally treated with extended reconstructive operations (e.g., free flaps), which are accompanied by long hospitalizations and risks of infection, thrombosis, and flap loss. Integra is a collagen template that can be used for reconstruction of defects. The take rate and the rate of infection are essential for the successful use of Integra (Johnson and Johnson, Hamburg, Germany). Whether the take rate and integration of Integra could be improved with the use of fibrin glue and negative-pressure therapy was assessed. Between January of 2002 and December of 2002, patients with large defects who underwent Integra grafting for reconstruction were randomly divided into groups receiving either a new treatment with fibrin glue-anchored Integra and postoperative negative-pressure therapy or conventional treatment. Demographic features, cause of the wound, location of the wound, take rate, complications of Integra coverage, time from Integra coverage to skin transplantation, and functional and aesthetic results were assessed. Twelve patients (with similar group distributions with respect to sex, age, and location and cause of the injury) were included in the study. The take rate was 78 ± 8 percent in the conventional treatment group and 98 ± 2 percent in the fibrin/negative-pressure therapy group (p < 0.003). The mean period from Integra coverage to skin transplantation was 24 ± 3 days in the conventional treatment group but only 10 ± 1 days in the fibrin/negative-pressure therapy group (p < 0.002). The decrease in the interval between coverage with Integra and skin transplantation resulted in shorter hospital stays. The use of fibrin glue and negative-pressure therapy in combination with Integra could shorten the period from coverage to integration, which would be beneficial in terms of decreased risks of infection, thrombosis, and catabolism. Therefore, it is suggested that Integra be used in combination with fibrin glue and negative-pressure therapy to improve clinical outcomes and shorten hospital stays, with decreased risks of accompanying complications.

Role of mesenchymal stem cells in tissue engineering of meniscus

Tissue engineering is a promising approach for the treatment of tissue defects. Mesenchymal stem cells are of potential use as a source of repair cells or of important growth factors for tissue engineering. The purpose of this study was to examine the role of mesenchymal stem cells in meniscal tissue repair. This was tested using several cell and biomaterial-based treatment options for repair of defects in the avascular zone of rabbit menisci. Circular meniscal punch defects (2 mm) were created in the avascular zone of rabbit menisci and left empty or filled with hyaluronan-collagen composite matrices without cells, loaded with platelet-rich plasma, autologous bone marrow, or autologous mesenchymal stem cells. In some experiments, matrices with stem cells were precultured in chondrogenic medium for 14 days before implantation. Rabbits were then allowed free cage movement after surgery for up to 12 weeks. Untreated defects and defects treated with cell-free implants had muted fibrous healing responses. Neither bone marrow nor platelet-rich plasma loaded in matrices produced improvement in healing compared with cell-free implants. The implantation of 14 days precultured chondrogenic stem cell-matrix constructs resulted in fibrocartilage-like repair tissue, which was only partially integrated with the native meniscus. Non-precultured mesenchymal stem cells in hyaluronan-collagen composite matrices stimulated the development of completely integrated meniscus-like repair tissue. The study shows the necessity of mesenchymal stem cells for the repair of meniscal defects in the avascular zone. Mesenchymal stem cells seem to fulfill additional repair qualities besides the delivery of growth factors.

Hypertrophy in Mesenchymal Stem Cell Chondrogenesis: Effect of TGF-β Isoforms and Chondrogenic Conditioning

Induction of chondrogenesis in mesenchymal stem cells (MSCs) with TGF-β leads to a hypertrophic phenotype. The hypertrophic maturation of the chondrocytes is dependent on the timed removal of TGF-β and sensitive to hypertrophy-promoting agents in vitro. In this study, we have investigated whether TGF-β3, which has been shown to be more prochondrogenic compared to TGF-β1, similarly enhances terminal differentiation in an in vitro hypertrophy model of chondrogenically differentiating MSCs. In addition, we tested the impact of the time of chondrogenic conditioning on the enhancement of hypertrophy. MSCs were chondrogenically differentiated in pellet culture in medium containing TGF-β1 or TGF-β3. After 2 or 4 weeks, chondrogenic medium was switched to hypertrophy-inducing medium for 2 weeks. Aggregates were analyzed histologically and biochemically on days 14, 28 and 42. The switch to hypertrophy medium after 14 days induced hypertrophic cell morphology and significant increase in alkaline phosphatase activity compared to the chondrogenesis only control using both TGF-β1 and TGF-β3. After 28 days predifferentiation, differences between hypertrophic and control groups diminished compared to 14 days predifferentiation. In conclusion, chondrogenic conditioning with both TGF-β isoforms similarly induced hypertrophy in our experiment and allowed the enhancement of the hypertrophic chondrocyte phenotype by hypertrophic medium. Enhancement of hypertrophy was seen more clearly after the shorter chondrogenic conditioning. Therefore, to utilize this experimental model as a tool to study hypertrophy in MSC chondrogenesis, a predifferentiation period of 14 days is recommended.

Oxidative stress induces senescence in human mesenchymal stem cells.

Mesenchymal stem cells (MSCs) contribute to tissue repair in vivo and form an attractive cell source for tissue engineering. Their regenerative potential is impaired by cellular senescence. The effects of oxidative stress on MSCs are still unknown. Our studies were to investigate into the proliferation potential, cytological features and the telomere linked stress response system of MSCs, subject to acute or prolonged oxidant challenge with hydrogen peroxide. Telomere length was measured using the telomere restriction fragment assay, gene expression was determined by rtPCR. Sub-lethal doses of oxidative stress reduced proliferation rates and induced senescent-morphological features and senescence-associated β-galactosidase positivity. Prolonged low dose treatment with hydrogen peroxide had no effects on cell proliferation or morphology. Sub-lethal and prolonged low doses of oxidative stress considerably accelerated telomere attrition. Following acute oxidant insult p21 was up-regulated prior to returning to initial levels. TRF1 was significantly reduced, TRF2 showed a slight up-regulation. SIRT1 and XRCC5 were up-regulated after oxidant insult and expression levels increased in aging cells. Compared to fibroblasts and chondrocytes, MSCs showed an increased tolerance to oxidative stress regarding proliferation, telomere biology and gene expression with an impaired stress tolerance in aged cells.

Oxidative stress induces senescence in chondrocytes

Cellular senescence is a program activated during diverse situations of cell stress. Chondrocytes differ from other somatic cells as articular cartilage is an avascular tissue. The effects of oxidative stress on chondrocytes are still unknown. Our studies were to investigate into the proliferation potential, cytological features and the telomere linked stress response system of human osteoarthritic chondrocytes, subjected to acute or prolonged oxidant challenge with hydrogen peroxide. Telomere length was measured using the telomere restriction fragment assay, gene expression was determined by RT‐PCR. Sub‐lethal doses of oxidative stress induced cell‐cycle arrest, senescent‐morphological features and senescence‐associated β‐galactosidase positivity. Prolonged oxidative treatment had no effects on cell proliferation or morphology. Sub‐lethal and prolonged low doses of oxidative stress considerably accelerated telomere attrition. The effects of sub‐lethal oxidative stress regarding proliferation and telomere biology were more distinct in senescent cells. Acute oxidant insult caused up‐regulation of p21 expression to levels comparable to senescent cells. TRF2 protects telomere ends and showed elevated expression levels. SIRT1 and XRCC5 enable cells to cope with unfavorable growing conditions. Both were up‐regulated after oxidant insult, but expression levels decreased in aging cells. Taken together, oxidative stress considerably accelerated telomere shortening and cellular aging in chondrocytes. Senescent cells showed a reduced tolerance to oxidative stress.

The cytotoxicity of bupivacaine, ropivacaine, and mepivacaine on human chondrocytes and cartilage.

BACKGROUND: Intraarticular injections of local anesthetics are frequently used as part of multimodal pain regimens. However, recent data suggest that local anesthetics affect chondrocyte viability. In this study, we assessed the chondrotoxic effects of mepivacaine, ropivacaine, and bupivacaine. We hypothesized that specific cytotoxic potencies directly correlate with analgesic potencies, and that cytotoxic effects in intact cartilage are different than in osteoarthritic tissue. METHODS: Human articular chondrocytes were exposed to equal and equipotent concentrations of bupivacaine, ropivacaine, and mepivacaine for 1 hour. Cell viability, apoptosis, and necrosis were determined at predefined time points using flow cytometry, live–dead staining, and caspase detection. Intact and osteoarthritic human cartilage explants were treated with equipotent concentrations of named drugs to determine cell viability applying fluorescence microscopy. RESULTS: Chondrotoxic effects increased from ropivacaine to mepivacaine to bupivacaine in a time-dependent and concentration-dependent manner. Compared with control, bupivacaine 0.5% decreased chondrocyte viability to 78% ± 9% (P = 0.0183) 1 hour and 16% ± 10% (P < 0.0001) 24 hours later, as determined by live–dead staining in monolayer cultures. Viability rates were reduced to 80% ± 7% (P = 0.0475) 1 hour and 80% ± 10% (P = 0.0095) 24 hours after treatment with ropivacaine 0.75%. After exposure to mepivacaine 2%, viable cells were scored 36% ± 6% (P < 0.0001) after 1 hour and 30% ± 11% (P < 0.0001) after 24 hours. Ropivacaine treatment was less chondrotoxic than bupivacaine (P = 0.0006) and mepivacaine exposure (P = 0.0059). Exposure to concentrations up to 0.25% of bupivacaine, 0.5% of ropivacaine, and 0.5% of mepivacaine did not reveal significant chondrotoxicity in flow cytometry. However, chondrotoxicity did not correlate with potency of local anesthetics. Immediate cell death was mainly due to necrosis followed by apoptosis. Cellular death rates were clearly higher in osteoarthritic compared with intact cartilage after bupivacaine, mepivacaine, and ropivacaine treatment in a decreasing order. CONCLUSION: Bupivacaine, ropivacaine, and mepivacaine are chondrotoxic in a time-dependent, concentration-dependent, and drug-dependent manner. Chondrotoxic and analgesic potencies do not directly correlate. Cellular death rates were higher in osteoarthritic compared with intact cartilage after local anesthetic treatment.

Stem cell-based tissue-engineering for treatment of meniscal tears in the avascular zone.

Meniscal tears in the avascular zone have a poor self-healing potential, however partial meniscectomy predisposes the knee for early osteoarthritis. Tissue engineering with mesenchymal stem cells and a hyaluronan collagen based scaffold is a promising approach to repair meniscal tears in the avascular zone. 4 mm longitudinal meniscal tears in the avascular zone of lateral menisci of New Zealand White Rabbits were performed. The defect was left empty, sutured with a 5-0 suture or filled with a hyaluronan/collagen composite matrix without cells, with platelet rich plasma or with autologous mesenchymal stem cells. Matrices with stem cells were in part precultured in chondrogenic medium for 14 days prior to the implantation. Menisci were harvested at 6 and 12 weeks. The developed repair tissue was analyzed macroscopically, histologically and biomechanically. Untreated defects, defects treated with suture alone, with cell-free or with platelet rich plasma seeded implants showed a muted fibrous healing response. The implantation of stem cell-matrix constructs initiated fibrocartilage-like repair tissue, with better integration and biomechanical properties in the precultured stem cell-matrix group. A hyaluronan-collagen based composite scaffold seeded with mesenchymal stem cells is more effective in the repair avascular meniscal tear with stable meniscus-like tissue and to restore the native meniscus.

Chondrogenitor cells and gene therapy.

The development of isolation and culture techniques for mesenchymal progenitor cells from various tissues has promoted interest in the use of these cells for repair and regeneration of musculoskeletal tissues. The chondrogenic differentiation of these pluripotential cells seems to be mediated by numerous cytokines most of which belong to the transforming growth factor-beta superfamily. Strategies to repair articular cartilage have focused on delivery of these cytokines or progenitor cells to the area of damage. More recently, with the development of gene transfer techniques, these cells have become the target of in vivo gene therapy, which involves direct injection of viral and nonviral vectors carrying transgenes. Furthermore, they are viewed as potential carriers of the transgenes for ex vivo gene therapy, in which the gene transfer is done in vitro with culture-expanded cells that then are implanted or injected. In vitro data suggest that the chondrogenic potential of these cells is maintained with virally mediated ex vivo gene transfer. By transducing these cells with chondroinductive factors, the bioactive factors and the target cells are delivered to the repair site.

Influence of the growth factors PDGF‐BB, TGF‐β1 and bFGF on the replicative aging of human articular chondrocytes during in vitro expansion

Decreasing replicative potential and dedifferentiation of articular chondrocytes during expansion in cell culture are essential limitations for tissue engineering and cell therapy approaches. Telomeres and telomerase play a key role in cell development, aging, and tumorigenesis. There is evidence that growth factors are involved in regulating telomerase activity. Therefore, the objective was to evaluate the effect of selected growth factors on telomere biology of serially passaged chondrocytes. Human articular chondrocytes were isolated from cartilage of three patients undergoing total knee arthroplasty. The chondrocytes were cultured in monolayer with the growth factors PDGF‐BB, TGF‐β1, and bFGF. Telomere length was measured by telomere restriction fragment length assay, and telomerase activity was determined by quantifying the gene expression of its catalytic subunit hTERT by rtPCR. Chondrocytes cultured with PDGF‐BB and TGF‐β1 showed a significantly higher proliferation rate than control cells. None of the growth factor cultures revealed an accelerated rate of telomere shortening. Telomerase was not expressed in significant amounts in any of the chondrocyte cultures. Growth factor treatment of chondrocyte cell cultures for cell therapy purposes can be regarded as safe in terms of telomere biology.