RESUMEN
PURPOSE: This cadaver study investigated the temperature profile in the wrist joint and distal radioulnar joint (DRUJ) during radiofrequency energy (RFE) application for triangular fibrocartilage complex resection. METHODS: An arthroscopic partial resection of the triangular fibrocartilage complex using monopolar and bipolar RFE was simulated in 14 cadaver limbs. The temperature was recorded simultaneously in the DRUJ and at 6 other anatomic locations of the wrist during RFE application. RESULTS: The mean temperature in the DRUJ was 43.3 ± 8.2°C for the bipolar system in the ablation mode (60 W) and 30.4 ± 3.4°C for the monopolar system in the cut mode (20 W) after 30 seconds. The highest measured temperature in the DRUJ was 54.3°C for the bipolar system and 68.1°C for the monopolar system. CONCLUSIONS: The application of RFE for debridement or resection of the triangular fibrocartilage complex in a clinical setting can induce peak temperatures that might cause damage to the cartilage of the DRUJ. Bipolar systems produce higher mean temperatures than monopolar devices. CLINICAL RELEVANCE: RFE application increases the mean temperature in the DRUJ after 30 seconds to a level that may jeopardize cartilage tissue.
Asunto(s)
Temperatura Corporal , Ablación por Catéter/efectos adversos , Fibrocartílago Triangular/lesiones , Traumatismos de la Muñeca/cirugía , Articulación de la Muñeca/fisiopatología , Humanos , Radio (Anatomía)/fisiopatología , Fibrocartílago Triangular/cirugía , Cúbito/fisiopatologíaRESUMEN
BACKGROUND: The application of radiofrequency energy (RFE) has become widespread for surgical performed chondroplasty especially due to the anticipated sealing effect, however the safety of this procedure in the wrist remains unclear. The purpose of this study was to investigate the subchondral temperature during radiofrequency energy (RFE) application simulating chondroplasty in an arthroscopic setting of the wrist. METHODS: A chondroplasty of the lunate fossa was performed during an arthroscopy setting on 14 cadaver arms using monopolar or bipolar RFE. The temperature was recorded simultaneously from 7 predefined anatomical landmarks. RESULTS: The mean temperature for both application modes did not exceed more than 30°C at all measured points, except for the lunate fossa. The highest subchondral measured peak temperature was 49.35°C (monopolar) and 69.21°C (bipolar) in the lunate fossa. In addition, the temperature decreased for both radiofrequency (RF) devices depending on the distance of the sensors to the RF-probe. CONCLUSION: It remains to be questionable how safe RFE can be used for chondroplasty in wrist arthroscopy under continuous irrigation and constant movement to obtain the desired sealing effect. However, the bipolar device should be applied with more caution since peak temperature in the lunate fossa almost reached 70°C even under continuous irrigation.
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Artroscopía/efectos adversos , Artroscopía/métodos , Cartílago Articular/cirugía , Ablación por Catéter/efectos adversos , Ablación por Catéter/métodos , Condrocitos/patología , Articulación de la Muñeca/cirugía , Artroscopía/instrumentación , Cadáver , Cartílago Articular/patología , Ablación por Catéter/instrumentación , Humanos , Factores de Riesgo , Temperatura , Irrigación Terapéutica , Articulación de la Muñeca/patologíaRESUMEN
PURPOSE: This study was to investigate the cytotoxic potency of local anesthetics on human mesenchymal stem cells during chondrogenesis. METHODS: Aggregates were created from density-gradient centrifugation-separated bone marrow-derived mesenchymal stem cells. After 7, 14, and 21 days, aggregates were analyzed histologically and immunohistochemically and exposed to equipotent concentrations of bupivacaine, ropivacaine, and mepivacaine for 1 h. Cell viability, apoptosis, and necrosis were determined using live-dead and caspase staining. Additionally, following a 1-h exposure on day 7, aggregates were cultured under chondrogenic conditions until day 21 to assess the effects of local anesthetics on differentiation potency of mesenchymal stem cells. RESULTS: In the course of chondrogenesis, mesenchymal stem cells were embedded in varying amount and structure of cartilage-specific extracellular matrix. Contents of sulfated glycosaminoglycan, type I and II collagen increased from day 7 to day 21. Compared to control, death rates of mesenchymal stem cells were significantly elevated 1 day after treatment at 7 and 14 days. Four days after exposure, death rates were 13-15 % at 7 and 11-17 % at 14 days. Mesenchymal stem cell viability in aggregates at 21 days was unchanged to controls. The width of the superficial aggregate zone containing stem cell necrosis decreased with elongated differentiation time. Apoptosis rates were elevated in the edge regions of aggregates, reaching maximum values 4 days after treatment. Local anesthetic exposure on day 7 reduced Collagen II, but not DNA contents in aggregates at 21 days. Bupivacaine, ropivacaine, and mepivacaine did not differ in mesenchymal stem cell cytotoxicity in aggregates. CONCLUSION: Local anesthetic exposure results in cytotoxicity of mesenchymal stem cells undergoing chondrogenesis, especially in superficial layers. Therefore, induced cell damage should be avoided during chondrogenesis of mesenchymal stem cells, particularly early after cartilage repair.
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Anestésicos Locales/efectos adversos , Traumatismos en Atletas/terapia , Cartílago Articular/patología , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Condrogénesis/efectos de los fármacos , Células Madre Mesenquimatosas/efectos de los fármacos , Adulto , Apoptosis/efectos de los fármacos , Traumatismos en Atletas/patología , Cartílago Articular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Estudios de Seguimiento , Humanos , Masculino , Adulto JovenRESUMEN
PURPOSE: To investigate whether applied radiofrequency energy (RFE) for shrinkage of the scapholunate interosseus ligament reaches temperatures required for ligament shrinkage while leaving adjacent structures unaffected. METHODS: Standard wrist arthroscopy was performed on 7 pairs of cadaveric limbs with continuous saline irrigation and gravity-assisted outflow through an 18-gauge needle. We subjected 14 scapholunate ligaments to treatment with monopolar (n = 7) or bipolar (n = 7) RFE for ligament shrinkage. Temperature was recorded simultaneously inside the dorsal part of the scapholunate interosseus ligament at a depth of 0.9 ± 0.1 mm and at 6 other sites in and around the wrist because thermal shrinkage starts at 60°C to 65°C. RESULTS: We observed an increase in temperature corresponding to the time of energy application. The highest measured peak temperatures at the scapholunate ligament were 43°C (monopolar) and 32°C (bipolar). Mean temperatures at 30 seconds of application were 29°C ± 7°C (monopolar) and 28°C ± 3°C (bipolar). CONCLUSIONS: Temperatures sufficiently high to induce ligament shrinkage were not reached with either monopolar or bipolar RFE. We did not monitor temperature levels responsible for damage on adjacent cartilage or immediately adjacent capsular tissue in this setting. CLINICAL RELEVANCE: This study suggests that RFE for capsular shrinkage in the wrist is safe but ineffective.
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Artroscopía/métodos , Temperatura Corporal/fisiología , Ligamentos Articulares/fisiopatología , Ligamentos Articulares/cirugía , Hueso Semilunar/fisiopatología , Hueso Semilunar/cirugía , Tratamiento de Radiofrecuencia Pulsada/instrumentación , Tratamiento de Radiofrecuencia Pulsada/métodos , Hueso Escafoides/fisiopatología , Hueso Escafoides/cirugía , Humanos , Modelos Biológicos , TermometríaRESUMEN
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.
Asunto(s)
Amidas/toxicidad , Anestésicos Locales/toxicidad , Bupivacaína/toxicidad , Cartílago/efectos de los fármacos , Condrocitos/efectos de los fármacos , Mepivacaína/toxicidad , Adulto , Amidas/uso terapéutico , Anestésicos Locales/uso terapéutico , Apoptosis/efectos de los fármacos , Bupivacaína/uso terapéutico , Cartílago/enzimología , Cartílago/patología , Caspasas/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Condrocitos/enzimología , Condrocitos/patología , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Humanos , Mepivacaína/uso terapéutico , Microscopía Fluorescente , Persona de Mediana Edad , Necrosis , Osteoartritis/tratamiento farmacológico , Osteoartritis/patología , Ropivacaína , Factores de TiempoRESUMEN
BACKGROUND: Matrix-guided autologous chondrocyte transplantation (MACT) has been proposed as an option for treating large full-thickness cartilage defects. However, little is known about the chondrogenic potential of transplants for MACT at the time of implantation, although cell quality and chondrogenic differentiation of the implants are crucial for restoration of function after MACT. QUESTIONS/PURPOSES: We therefore asked: (1) Do MACT implants allow deposition of extracellular cartilage matrix in an in vitro culture model? (2) Are these implants associated with improved knee function 1 year after MACT in large cartilage defects? METHODS: We retrospectively reviewed all 125 patients with large localized cartilage defects (mean defect size 5 cm(2)) of the knee who were treated with MACT from 2005 to 2010. The mean age was 31 years (range, 16-53 years). Portions of the cell-matrix constructs (n = 50) that were not implanted in the cartilage defects were further cultured and tested for their potential to form articular cartilage. Knee function of all patients was analyzed preoperatively, 3 months, and 1 year postoperatively with the International Knee Documentation Committee (IKDC) score. RESULTS: In vitro assessment of the cell-matrix implants showed chondrogenic differentiation with positive staining for glycosaminoglycans and collagen II in all cultures. Enzyme-linked immunosorbent assay showed an increase of collagen II production. We observed an improvement in median IKDC score from 41 to 67 points at last followup. CONCLUSIONS: Cartilage extracellular matrix deposition shows adequate implant quality for MACT at the time of implantation and justifies the use for treatment of large cartilage defects.
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Cartílago Articular/metabolismo , Cartílago/trasplante , Condrogénesis/fisiología , Articulación de la Rodilla/cirugía , Adolescente , Adulto , Cartílago/metabolismo , Colágeno Tipo II/metabolismo , Matriz Extracelular/metabolismo , Femenino , Glicosaminoglicanos/metabolismo , Humanos , Articulación de la Rodilla/metabolismo , Masculino , Persona de Mediana Edad , Proyectos Piloto , Estudios Retrospectivos , Trasplante AutólogoRESUMEN
PURPOSE: The purpose of this study was to investigate the cytotoxic potency of local anesthetics on human mesenchymal stem cells (MSCs) before and after chondrogenic differentiation. METHODS: MSCs were exposed to equal and equipotent concentrations of bupivacaine, ropivacaine, and mepivacaine for 1 hour. Cell viability, apoptosis, and necrosis were determined using flow cytometry and live/dead staining. After chondrogenic differentiation, MSC viability was determined in aggregates exposed to equipotent concentrations of the named agents, applying fluorescence microscopy. RESULTS: All local anesthetics showed detrimental cytotoxic effects on MSC monolayer cultures in a concentration- and time-specific manner. Minimum viability rates were found 96 hours after a 1-hour exposure. Bupivacaine 0.5% caused a reduction of vital MSCs to 5% ± 1%. Sixteen percent ± 2% viable cells were detected after treatment with 0.75% ropivacaine. Exposure to 2% mepivacaine decreased vitality rates to 1% ± 0%. Ropivacaine was significantly less cytotoxic than were bupivacaine and mepivacaine. Immediate cell death was mainly caused by necrosis followed by apoptosis afterward. Viability rates of MSCs embedded in cartilaginous tissue after chondrogenic differentiation were not reduced by local anesthetic treatment. CONCLUSIONS: Local anesthetics are cytotoxic to MSCs in a concentration-, time-, and agent-dependent manner in monolayer cultures but not in whole-tissue probes. CLINICAL RELEVANCE: MSCs are applied for treatment of cartilage defects. Intra-articular application of local anesthesia is a common procedure in pain management and has shown chondrotoxic effects. Therefore, it is crucial to evaluate the impact of local anesthetics on human MSCs and regenerative cartilage tissue engineering.
Asunto(s)
Anestésicos Locales/toxicidad , Células Madre Mesenquimatosas/efectos de los fármacos , Adolescente , Adulto , Amidas/toxicidad , Apoptosis/efectos de los fármacos , Bupivacaína/toxicidad , Cartílago , Diferenciación Celular , Supervivencia Celular/efectos de los fármacos , Condrocitos/citología , Condrocitos/patología , Citometría de Flujo , Humanos , Mepivacaína/toxicidad , Células Madre Mesenquimatosas/patología , Microscopía Fluorescente , Necrosis , Ropivacaína , Adulto JovenRESUMEN
PURPOSE: The purpose of this study was to investigate the changes in temperature during wrist arthroscopy comparing monopolar and bipolar radiofrequency energy (RFE). METHODS: A standard wrist arthroscopy was performed on 14 arms of 7 cadavers without irrigation or with continuous irrigation with 0.9% saline solution and gravity-assisted outflow through an 18-gauge needle. We treated 7 wrists with a bipolar device (VAPR II with 2.3-mm side effect electrodes; DePuy Mitek, Westwood, MA) and 7 wrists with a monopolar device (OPES Ablator for small joints, 45°; Arthrex, Naples, FL). The temperature was recorded simultaneously from 7 predefined anatomic landmarks. RESULTS: We observed an increase in the temperature corresponding to the time of energy application. The highest measured peak temperatures were 52°C (monopolar) and 49.5°C (bipolar) without irrigation. Continuous irrigation led to a significant reduction in the temperature at the site of the energy application. The mean temperature decreased by 7°C for the monopolar system and 5°C for the bipolar system when irrigation was used. For both radiofrequency devices, we found a decrease in the temperature proportional to the distance of the sensors to the radiofrequency probe. CONCLUSIONS: Monopolar and bipolar RFE can be safely used in wrist arthroscopy if a continuous irrigation system is applied and the energy impulse does not exceed 5 to 10 seconds. However, it should be used with great care to avoid local heat damage especially at the cartilage. CLINICAL RELEVANCE: This basic science study was performed to gain data concerning the temperature in wrist arthroscopy and to broaden the knowledge about the risks when using RFE. Furthermore, we sought to control side effects of RFE by finding the best applied form of RFE regarding duration and pulsation (monopolar/bipolar).
Asunto(s)
Artroscopía , Temperatura Corporal , Ablación por Catéter , Articulación de la Muñeca/cirugía , Cadáver , Humanos , Irrigación TerapéuticaRESUMEN
PURPOSE: Mesenchymal stem cells (MSCs) express markers of hypertrophic chondrocytes during chondrogenic differentiation. We tested the suitability of parathyroid hormone-related protein (PTHrP), a regulator of chondrocyte hypertrophy in embryonic cartilage development, for the suppression of hypertrophy in an in vitro hypertrophy model of chondrifying MSCs. METHODS: Chondrogenesis was induced in human MSCs in pellet culture for two weeks and for an additional two weeks cultures were either maintained in standard chondrogenic medium or transferred to a hypertrophy-enhancing medium. PTHrP(1-40) was added to the medium throughout the culture period at concentrations from 1 to 1,000 pM. Pellets were harvested on days one, 14 and 28 for biochemical and histological analysis. RESULTS: Hypertrophic medium clearly enhanced the hypertrophic phenotype, with increased cell size, and strong alkaline phosphatase (ALP) and type X collagen staining. In chondrogenic medium, 1-100 pM PTHrP(1-40) did not inhibit chondrogenic differentiation, whereas 1,000 pM PTHrP(1-40) significantly reduced chondrogenesis. ALP activity was dose-dependently reduced by PTHrP(1-40) at 10-1,000 pM in chondrogenic conditions. Under hypertrophy-enhancing conditions, PTHrP(1-40) did not inhibit the induction of the hypertrophy. At the highest concentration (1,000 pM) in the hypertrophic group, aggregates were partially dedifferentiated and differentiated areas of these aggregates maintained their hypertrophic appearance. CONCLUSIONS: PTHrP(1-40) treatment dose-dependently reduced ALP expression in MSC pellets cultured under standard chondrogenic conditions and is thus beneficial for the maintenance of the chondrogenic phenotype in this medium condition. When cultured under hypertrophy-enhancing conditions, PTHrP(1-40) could not diminish the induced enhancement of hypertrophy in the MSC pellets.
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Aumento de la Célula/efectos de los fármacos , Condrogénesis/efectos de los fármacos , Células Madre Mesenquimatosas/efectos de los fármacos , Proteína Relacionada con la Hormona Paratiroidea/farmacología , Adulto , Fosfatasa Alcalina/metabolismo , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , ADN/análisis , ADN/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/patologíaRESUMEN
PURPOSE: Insulin is a commonly used additive in chondrogenic media for differentiating mesenchymal stem cells (MSCs). The indispensability of other bioactive factors like TGF-ß or dexamethasone in these medium formulations has been shown, but the role of insulin is unclear. The purpose of this study was to investigate whether insulin is essential for MSC chondrogenesis and if there is a dose-dependent effect of insulin on MSC chondrogenesis. METHODS: We cultivated human MSCs in pellet culture in serum-free chondrogenic medium with insulin concentrations between 0 and 50 µg/ml and assessed the grade of chondrogenic differentiation by histological evaluation and determination of glycosaminoglycan (GAG), total collagen and DNA content. We further tested whether insulin can be delivered in an amount sufficient for MSC chondrogenesis via a drug delivery system in insulin-free medium. RESULTS: Chondrogenesis was not induced by standard chondrogenic medium without insulin and the expression of cartilage differentiation markers was dose-dependent at insulin concentrations between 0 and 10 µg/ml. An insulin concentration of 50 µg/ml had no additional effect compared with 10 µg/ml. Insulin was delivered by a release system into the cell culture under insulin-free conditions in an amount sufficient to induce chondrogenesis. CONCLUSIONS: Insulin is essential for MSC chondrogenesis in this system and chondrogenic differentiation is influenced by insulin in a dose-dependent manner. Insulin can be provided in a sufficient amount by a drug delivery system. Therefore, insulin is a suitable and inexpensive indicator substance for testing drug release systems in vitro.
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Condrogénesis/efectos de los fármacos , Insulina/farmacología , Células Madre Mesenquimatosas/efectos de los fármacos , Análisis de Varianza , Diferenciación Celular , Células Cultivadas , Colágeno/metabolismo , ADN/metabolismo , Relación Dosis-Respuesta a Droga , Sistemas de Liberación de Medicamentos , Ensayo de Inmunoadsorción Enzimática , Glicosaminoglicanos/metabolismo , Humanos , Técnicas In Vitro , Insulina/administración & dosificación , Coloración y EtiquetadoRESUMEN
OBJECTIVE: We undertook this study to examine the effects of estradiol on chondrogenesis of human bone marrow-derived mesenchymal stem cells (MSCs), with consideration of sex-dependent differences in cartilage repair. METHODS: Bone marrow was obtained from the iliac crest of young men. Density-gradient centrifugation-separated human MSCs proliferated as a monolayer in serum-containing medium. After confluence was achieved, aggregates were created and cultured in a serum-free differentiation medium. We added different concentrations of 17beta-estradiol (E2) with or without the specific estrogen receptor inhibitor ICI 182.780, membrane-impermeable E2-bovine serum albumin (E2-BSA), ICI 182.780 alone, G-1 (an agonist of G protein-coupled receptor 30 [GPR-30]), and G15 (a GPR-30 antagonist). After 21 days, the aggregates were analyzed histologically and immunohistochemically; we quantified synthesized type II collagen, DNA content, sulfated glycosaminoglycan (sGAG) concentrations, and type X collagen and matrix metalloproteinase 13 (MMP-13) expression. RESULTS: The existence of intracellular and membrane-associated E2 receptors was shown at various stages of chondrogenesis. Smaller aggregates and significantly lower type II collagen and sGAG content were detected after treatment with E2 and E2-BSA in a dose-dependent manner. Furthermore, E2 enhanced type X collagen and MMP-13 expression. Compared with estradiol alone, the coincubation of ICI 182.780 with estradiol enhanced suppression of chondrogenesis. Treatment with specific GPR-30 agonists alone (G-1 and ICI 182.780) resulted in a considerable inhibition of chondrogenesis. In addition, we found an enhancement of hypertrophy by G-1. Furthermore, the specific GPR-30 antagonist G15 reversed the GPR-30-mediated inhibition of chondrogenesis and up-regulation of hypertrophic gene expression. CONCLUSION: The experiments revealed a suppression of chondrogenesis by estradiol via membrane receptors (GPR-30). The study opens new perspectives for influencing chondrogenesis on the basis of classic and nonclassic estradiol signaling.
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Cartílago/citología , Diferenciación Celular/efectos de los fármacos , Estradiol/farmacología , Células Madre Mesenquimatosas/citología , Transducción de Señal/fisiología , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Cartílago/efectos de los fármacos , Estradiol/análogos & derivados , Antagonistas de Estrógenos/farmacología , Receptor alfa de Estrógeno/fisiología , Receptor beta de Estrógeno/fisiología , Fulvestrant , Humanos , Masculino , Células Madre Mesenquimatosas/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Adulto JovenRESUMEN
Mesenchymal stromal cells have the potential to differentiate into a variety of mesenchymal tissues such as bone, cartilage and ligaments. The potential for the regeneration of bone with cartilage coverage has still not been achieved. We evaluated the ability of bone marrow mesenchymal stromal cells to regenerate osteochondral defects in the cavity of the lunate in an animal model. Autologous mesenchymal stromal cells were harvested from the iliac crest of New Zealand white rabbits and expanded in vitro. Total lunate excision was performed in 24 animals and the isolated cells were loaded onto scaffolds. Cell-free scaffolds were implanted in the lunate space of the right wrists of all animals, and the left lunate spaces were filled with predifferentiated, cell-loaded scaffolds. Radiographic and histological analyses were performed after two, six and 12 weeks. In addition, the animals were injected with a fluorescent agent every five days, starting at day 30. After two and six weeks there was no radiographic evidence of ossification, whereas after 12 weeks all animals showed radiographic evidence of ossification. Histological sections showed increasing evidence of cartilage-like cell formation at the edges and new bone tissue in the centre of the newly formed tissue in all groups. The histological examinations showed that bone tissue was located around the newly incorporated vascularisation. This study demonstrated that newly formed vascularisation is necessary for the regeneration of bone tissue with cell-loaded scaffolds.
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Artroplastia/métodos , Células de la Médula Ósea/citología , Hueso Semilunar/cirugía , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Animales , Células de la Médula Ósea/fisiología , Regeneración Ósea/fisiología , Diferenciación Celular , Células Cultivadas , Articulaciones/cirugía , Hueso Semilunar/irrigación sanguínea , Células Madre Mesenquimatosas/fisiología , Modelos Animales , Neovascularización Fisiológica , Osteogénesis/fisiología , Conejos , Células del Estroma/citología , Células del Estroma/fisiología , Ingeniería de Tejidos , Andamios del TejidoRESUMEN
Induction of chondrogenesis in mesenchymal stem cells (MSCs) with TGF-beta leads to a hypertrophic phenotype. The hypertrophic maturation of the chondrocytes is dependent on the timed removal of TGF-beta and sensitive to hypertrophy-promoting agents in vitro. In this study, we have investigated whether TGF-beta3, which has been shown to be more prochondrogenic compared to TGF-beta1, 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-beta1 or TGF-beta3. 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-beta1 and TGF-beta3. After 28 days predifferentiation, differences between hypertrophic and control groups diminished compared to 14 days predifferentiation. In conclusion, chondrogenic conditioning with both TGF-beta 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.
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Diferenciación Celular/fisiología , Aumento de la Célula/efectos de los fármacos , Condrocitos/citología , Condrogénesis/fisiología , Células Madre Mesenquimatosas/citología , Factor de Crecimiento Transformador beta/farmacología , Adulto , Fosfatasa Alcalina/metabolismo , Calcificación Fisiológica/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Condrocitos/metabolismo , Condrogénesis/efectos de los fármacos , Colágeno Tipo I/metabolismo , Colágeno Tipo II/metabolismo , Colágeno Tipo X/metabolismo , Medios de Cultivo Condicionados/metabolismo , ADN/metabolismo , Matriz Extracelular/metabolismo , Femenino , Glicerofosfatos/farmacología , Glicosaminoglicanos/metabolismo , Humanos , Masculino , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Isoformas de Proteínas/farmacología , Factor de Crecimiento Transformador beta1/farmacología , Factor de Crecimiento Transformador beta3/farmacología , Adulto JovenRESUMEN
The purpose of this study was to evaluate the quality of surface contouring of chondromalacic cartilage by bipolar radio frequency energy using different treatment patterns in an animal model, as well as examining the impact of the treatment onto chondrocyte viability by two different methods. Our experiments were conducted on 36 fresh osteochondral sections from the tibia plateau of slaughtered 6-month-old pigs, where the thickness of the cartilage is similar to that of human wrist cartilage. An area of 1 cm2 was first treated with emery paper to simulate the chondromalacic cartilage. Then, the treatment with RFE followed in 6 different patterns. The osteochondral sections were assessed for cellular viability (live/dead assay, caspase (cell apoptosis marker) staining, and quantitative analysed images obtained by fluorescent microscopy). For a quantitative characterization of none or treated cartilage surfaces, various roughness parameters were measured using confocal laser scanning microscopy (Olympus LEXT OLS 4000 3D). To describe the roughness, the Root-Mean-Square parameter (Sq) was calculated. A smoothing effect of the cartilage surface was detectable upon each pattern of RFE treatment. The Sq for native cartilage was Sq = 3.8 ± 1.1 µm. The best smoothing pattern was seen for two RFE passes and a 2-second pulsed mode (B2p2) with an Sq = 27.3 ± 4.9 µm. However, with increased smoothing, an augmentation in chondrocyte death up to 95% was detected. Using bipolar RFE treatment in arthroscopy for small joints like the wrist or MCP joints should be used with caution. In the case of chondroplasty, there is a high chance to destroy the joint cartilage.
Asunto(s)
Enfermedades de los Cartílagos/terapia , Terapia por Radiofrecuencia , Animales , Artroplastia , Artroscopía , Contorneado Corporal , Enfermedades de los Cartílagos/diagnóstico por imagen , Enfermedades de los Cartílagos/cirugía , Cartílago Articular/diagnóstico por imagen , Cartílago Articular/patología , Cartílago Articular/cirugía , Muerte Celular , Condrocitos/patología , Modelos Animales de Enfermedad , Humanos , Microscopía Confocal , Fotomicrografía , Ondas de Radio , Porcinos , Tibia/diagnóstico por imagen , Tibia/cirugíaRESUMEN
The endogenous healing potential of avascular meniscal lesions is poor. Up to now, partial meniscectomy is still the treatment of choice for meniscal lesions within the avascular area. However, the large loss of meniscus substance predisposes the knee for osteoarthritic changes. Tissue engineering techniques for the replacement of such lesions could be a promising alternative treatment option. Thus, a polyurethane scaffold, which is already in clinical use, loaded with mesenchymal stromal cells, was analyzed for the repair of critical meniscus defects in the avascular zone. Large, approximately 7 mm broad meniscus lesions affecting both the avascular and vascular area of the lateral rabbit meniscus were treated with polyurethane scaffolds either loaded or unloaded with mesenchymal stromal cells. Menisci were harvested at 6 and 12 weeks after initial surgery. Both cell-free and cell-loaded approaches led to well-integrated and stable meniscus-like repair tissue. However, an accelerated healing was achieved by the application of mesenchymal stromal cells. Dense vascularization was detected throughout the repair tissue of both treatment groups. Overall, the polyurethane scaffold seems to promote the vessel ingrowth. The application of mesenchymal stromal cells has the potential to speed up the healing process.
RESUMEN
BACKGROUND: Treatment of meniscus tears within the avascular region represents a significant challenge, particularly in a situation of early osteoarthritis. Cell-based tissue engineering approaches have shown promising results. However, studies have not found a consensus on the appropriate autologous cell source in a clinical situation, specifically in a challenging degenerative environment. The present study sought to evaluate the appropriate cell source for autologous meniscal repair in a demanding setting of early osteoarthritis. METHODS: A rabbit model was used to test autologous meniscal repair. Bone marrow and medial menisci were harvested 4 weeks prior to surgery. Bone marrow-derived mesenchymal stem cells (MSCs) and meniscal cells were isolated, expanded, and seeded onto collagen-hyaluronan scaffolds before implantation. A punch defect model was performed on the lateral meniscus and then a cell-seeded scaffold was press-fit into the defect. Following 6 or 12 weeks, gross joint morphology and OARSI grade were assessed, and menisci were harvested for macroscopic, histological, and immunohistochemical evaluation using a validated meniscus scoring system. In conjunction, human meniscal cells isolated from non-repairable bucket handle tears and human MSCs were expanded and, using the pellet culture model, assessed for their meniscus-like potential in a translational setting through collagen type I and II immunostaining, collagen type II enzyme-linked immunosorbent assay (ELISA), and gene expression analysis. RESULTS: After resections of the medial menisci, all knees showed early osteoarthritic changes (average OARSI grade 3.1). However, successful repair of meniscus punch defects was performed using either meniscal cells or MSCs. Gross joint assessment demonstrated donor site morbidity for meniscal cell treatment. Furthermore, human MSCs had significantly increased collagen type II gene expression and production compared to meniscal cells (p < 0.05). CONCLUSIONS: The regenerative potential of the meniscus by an autologous cell-based tissue engineering approach was shown even in a challenging setting of early osteoarthritis. Autologous MSCs and meniscal cells were found to have improved meniscal healing in an animal model, thus demonstrating their feasibility in a clinical setting. However, donor site morbidity, reduced availability, and reduced chondrogenic differentiation of human meniscal cells from debris of meniscal tears favors autologous MSCs for clinical use for cell-based meniscus regeneration.
Asunto(s)
Menisco/citología , Trasplante de Células Madre Mesenquimatosas/métodos , Células Madre Mesenquimatosas/citología , Osteoartritis de la Rodilla/terapia , Ingeniería de Tejidos/métodos , Adulto , Animales , Células Cultivadas , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Humanos , Masculino , Menisco/metabolismo , Células Madre Mesenquimatosas/metabolismo , Conejos , Trasplante AutólogoRESUMEN
Collagen type-I is a major component of the extracellular matrix of most tissues and it is increasingly utilized for surface engineering of biomaterials to accelerate receptor-mediated cell adhesion. In the present study, coatings with layers of fibrillar type-I collagen were prepared on titanium, titanium alloy, and cobalt alloy to improve initial osteoblast adhesion and implant-tissue integration. To suppress the quick in vivo degradation rate of collagen the deposited layers were covalently immobilized at the metal surfaces as well as chemically cross-linked. The application of different oxidation techniques to the metallic substrates resulted in surfaces with varying hydroxyl group contents, which directly influenced the amount of immobilized silane coupling agents. It was found that a high density of surface-bound coupling agents increased the stability of the covalently linked collagen layers. After coating of metallic biomaterials with a cross-linked collagen layer, an improved cellular response of human osteoblast-like cells (MG-63) in vitro could be recognized.
Asunto(s)
Materiales Biocompatibles/química , Cobalto/química , Colágeno/química , Colágenos Fibrilares/química , Titanio/química , Reactivos de Enlaces Cruzados/química , Humanos , Metales/química , Osteoblastos/metabolismo , Oxígeno/metabolismo , Silanos/química , Propiedades de SuperficieRESUMEN
A combination of mesenchymal stem cells (MSCs) and scaffolds seems to be a promising approach for meniscus repair. To facilitate the search for an appropriate scaffold material a reliable and objective in vitro testing system is essential. This paper introduces a new scoring for this purpose and analyzes a hyaluronic acid (HA) gelatin composite scaffold and a polyurethane scaffold in combination with MSCs for tissue engineering of meniscus. The pore quality and interconnectivity of pores of a HA gelatin composite scaffold and a polyurethane scaffold were analyzed by surface photography and Berliner-Blau-BSA-solution vacuum filling. Further the two scaffold materials were vacuum-filled with human MSCs and analyzed by histology and immunohistochemistry after 21 days in chondrogenic media to determine cell distribution and cell survival as well as proteoglycan production, collagen type I and II content. The polyurethane scaffold showed better results than the hyaluronic acid gelatin composite scaffold, with signs of central necrosis in the HA gelatin composite scaffolds. The polyurethane scaffold showed good porosity, excellent pore interconnectivity, good cell distribution and cell survival, as well as an extensive content of proteoglycans and collagen type II. The polyurethane scaffold seems to be a promising biomaterial for a mesenchymal stem cell-based tissue engineering approach for meniscal repair. The new score could be applied as a new standard for in vitro scaffold testing.
RESUMEN
Mesenchymal stem cells (MSCs) seeded on specific carrier materials are a promising source for the repair of traumatic cartilage injuries. The best supportive carrier material has not yet been determined. As natural components of cartilage's extracellular matrix, hyaluronic acid and collagen are the focus of biomaterial research. In order to optimize chondrogenic support, we investigated three different scaffold compositions of a hyaluronic acid (HA)-gelatin based biomaterial. METHODS: Human MSCs (hMSCs) were seeded under vacuum on composite scaffolds of three different HA-gelatin ratios and cultured in chondrogenic medium for 21 days. Cell-scaffold constructs were assessed at different time points for cell viability, gene expression patterns, production of cartilage-specific extracellular matrix (ECM) and for (immuno-)histological appearance. The intrinsic transforming growth factor beta (TGF-beta) uptake of empty scaffolds was evaluated by determination of the TGF-beta concentrations in the medium over time. RESULTS: No significant differences were found for cell seeding densities and cell viability. hMSCs seeded on scaffolds with higher ratios of HA showed better cartilage-like differentiation in all evaluated parameters. TGF-beta uptake did not differ between empty scaffolds. CONCLUSION: Higher ratios of HA support the chondrogenic differentiation of hMSCs seeded on a HA-gelatin composite scaffold.
RESUMEN
It was shown recently that the deposition of thin films of tantalum and tantalum oxide enhanced the long-term biocompatibility of stainless steel biomaterials due to an increase in their corrosion resistance. In this study, we used this tantalum oxide coating as a basis for covalent immobilization of a collagen layer, which should result in a further improvement of implant tissue integration. Because of the high degradation rate of natural collagen in vivo, covalent immobilization as well as carbodiimide induced cross-linking of the protein was performed. It was found that the combination of the silane-coupling agent aminopropyl triethoxysilane and the linker molecule N,N'-disulphosuccinimidyl suberate was a very effective system for collagen immobilizing. Mechanical and enzymatic stability testing revealed a higher stability of covalent bound collagen layers compared to physically adsorbed collagen layers. The biological response induced by the surface modifications was evaluated by in vitro cell culture with human mesenchymal stem cells as well as by in vivo subcutaneous implantation into nude mice. The presence of collagen clearly improved the cytocompatibility of the stainless steel implants which, nevertheless, significantly depended on the cross-linking degree of the collagen layer.