RESUMO
In our quest to standardize our formula for a clinical trial, transforming growth factor-beta3 (TGF-ß3) alone and in combination with bone morphogenetic protein-6 (BMP-6) were evaluated for their effectiveness in cartilage differentiation. Bone Marrow Stem Cells (BMSCs) and Adipose Derived Stem Cells (ADSCs) were induced to chondrogenic lineage using two different media. Native chondrocytes served as positive control. ADSCs and BMSCs proved multipotency by tri-lineage differentiations. ADSC has significantly higher growth kinetics compare to Chondrocyte only p ≤ 0.05. Using TGF-ß3 alone, BMSC revealed higher expressions for hyaline cartilage genes compare to ADSCs. Chondrocyte has significantly higher early chondrogenic markers expression to ADSCs and BMSCs, while BMSCs was only higher to ADSC at chondroadherin, p ≤ 0.0001. On mature chondrogenic markers, chondrocytes were significantly higher to ADSCs and BMSCs for aggrecan, collagen IX, sry (sex determining region y)-box9, collagen II and fibromodullin; and only to ADSC for collagen XI. BMSC was higher to ADSC for aggrecan and collagen IX, p ≤ 0.0001. The combination of TGF-ß3 + BMP-6 revealed increased gene expressions on both BMSCs and ADSCs for early and mature chondrogenic markers, but no significance difference. For dedifferentiation markers, ADSC was significantly higher to chondrocyte for collagen I. Glycosaminoglycan evaluations with both formulas revealed that chondrocytes were significantly higher to ADSCs and BMSCs, but none was significant to each other, p ≤ 0.0001. Combination of 10 ng TGF-ß3 with 10 ng of BMP-6 enhanced chondrogenic potentials of BMSCs and ADSCs compare to TGF-ß3 alone. This could be the ideal cocktail for either cell's chondrogenic induction.
Assuntos
Células-Tronco Adultas/citologia , Células da Medula Óssea/citologia , Proteína Morfogenética Óssea 6/metabolismo , Condrogênese , Fator de Crescimento Transformador beta3/metabolismo , Tecido Adiposo/citologia , Células-Tronco Adultas/metabolismo , Animais , Células da Medula Óssea/metabolismo , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Masculino , Ovinos , Engenharia TecidualRESUMO
This study was to determine if autologous bone marrow mesenchymal stem cells (BMSCs) cultured in chondrogenic medium could repair surgically induced osteoarthritis. Sheep BMSCs were cultured in medium containing 5ng/ml TGFbeta3 + 50ng/ml IGF-1 for three weeks. The cultured cells were then suspended at density of 2x10(6) cell/ml and injected intraarticularly into the osteoarthritic knee joint. After six weeks, the distal head of the femur and the proximal tibial plateau were removed and stained with H&E. The results indicated that knee joints treated with autologous BMSCs cultured in chondrogenic medium showed clear evidence of articular cartilage regeneration in comparison with other groups.
Assuntos
Medula Óssea , Terapia Baseada em Transplante de Células e Tecidos , Condrócitos/transplante , Articulação do Joelho/patologia , Transplante de Células-Tronco Mesenquimais , Osteoartrite/terapia , Transplante Autólogo , Animais , Células Cultivadas , Fêmur/patologia , Masculino , Modelos Animais , Osteoartrite/patologia , Medicina Regenerativa , Carneiro Doméstico , Tíbia/patologiaRESUMO
BACKGROUND: Hyaline articular cartilage, which protects the bones of diarthrodial joints from forces associated with load bearing, frictions, and impacts has very limited capacities for self-repair. Over the years, the trend of treatments has shifted to regenerations and researchers have been on the quest for a lasting regeneration. We evaluated the treatment of osteoarthritis by chondrogenically induced ADSCs and BMSCs for a long time functional recovery. METHODS: Osteoarthritis was induced at the right knee of sheep by complete resection of ACL and medial meniscus. Stem cells from sheep were induced to chondrogenic lineage. Test sheep received 5â¯mls single doses of 2â¯×â¯107 autologous PKH26-labelled ADSCs or BMSCs, while controls received basal medium. Functional recovery of the knees was evaluated via electromyography. RESULTS: Induced ADSCs had 625, 255, 393, 908, 409, 157 and 1062 folds increases of collagen I, collagen II, aggrecan, SOX9, cartilage oligomeric protein, chondroadherin and fibromodullin compare to uninduced cells, while BMSCs had 702, 657, 321, 276, 337, 233 and 1163 respectively; pâ¯=â¯.001. Immunocytochemistry was positive for these chondrogenic markers. 12â¯months post-treatment, controls scored 4 in most regions using ICRS, while the treated had 8; Pâ¯=â¯.001. Regenerated cartilages were positive to PKH26 and demonstrated the presence of condensing cartilages on haematoxylin and eosin; and Safranin O. OA degenerations caused significant amplitude shift from right to left hind limb. After treatments, controls persisted with significant decreases; while treated samples regained balance. CONCLUSIONS: Both ADSCs and BMSCs had increased chondrogenic gene expressions using TGF-ß3 and BMP-6. The treated knees had improved cartilage scores; PKH26 can provide elongated tracking, while EMG results revealed improved joint recoveries. These could be suitable therapies for osteoarthritis.
Assuntos
Cartilagem Articular/fisiopatologia , Condrogênese , Transplante de Células-Tronco Mesenquimais , Osteoartrite do Joelho/terapia , Regeneração , Tecido Adiposo/citologia , Animais , Artroscopia , Células da Medula Óssea/citologia , Proteína Morfogenética Óssea 6/farmacologia , Cartilagem Articular/patologia , Cartilagem Articular/cirurgia , Técnicas de Cultura de Células , Diferenciação Celular , Proliferação de Células , Separação Celular , Sobrevivência Celular , Rastreamento de Células , Modelos Animais de Doenças , Expressão Gênica , Masculino , Células-Tronco Multipotentes/citologia , Osteoartrite do Joelho/fisiopatologia , Ovinos , Fator de Crescimento Transformador beta3/farmacologiaRESUMO
Antibiotic-loaded bone cement has been used as prophylaxis against infection in total joint replacement surgery. Its effect on the mechanical strength of cement is a major concern as high dose of antibiotic was associated with a significant reduction in mechanical strength of bone cement. However, the cut-off antibiotic that weakens the mechanical strength of cement remains to be determined. This study was undertaken to observe the changes in the mechanical properties of bone cement with gradual increments of Cefuroxime antibiotic. Cefuroxime at different doses: 0, 1.5, 3.0 and 4.5gm were added to a packet of 40gm bone cement (Simplex P) and study samples were prepared by using third generation cementing technique. Mechanical impact, flexural and tensile strength were tested on each sample. Significant impact and tensile strength reduction were observed after addition of 4.5 gm of Cefuroxime. However, flexural strength was significantly reduced at a lower dose of 3.0 gm. The maximum dose of Cefuroxime to be safely added to 40mg Surgical Simplex P is 1.5gm when third generation cementing technique is used. Further study is needed to determine whether it is an effective dose as regards to microbiological parameters.
Assuntos
Antibacterianos/farmacologia , Antibioticoprofilaxia/instrumentação , Cimentos Ósseos/análise , Cefuroxima/farmacologia , Portadores de Fármacos/análise , Estresse Mecânico , Resistência à Tração , Antibacterianos/administração & dosagem , Artroplastia de Quadril/efeitos adversos , Cefuroxima/administração & dosagem , Força Compressiva , Humanos , Teste de Materiais , Infecção da Ferida Cirúrgica/prevenção & controleRESUMO
Tracking of transplanted cells has become an important procedure in cell therapy. We studied the in vitro dye retention, survival and in vivo tracking of stem cells with PKH26 dye. Sheep BMSCs and ADSCs were labeled with 2, 4 and 8 µmol of PKH26 and monitored for six passages. Labeled BMSCs and ADSCs acquired mean cumulative population doubling of 12.7±0.4 and 14.6±0.5; unlabeled samples had 13.8±0.5 and 15.4±0.6 respectively. Upon staining with 2, 4 and 8 µmol PKH26, BMSCs had retentions of 40.0±5.8, 60.0±2.9 and 95.0±2.9%, while ADSCs had 92.0±1.2, 95.0±1.2 and 98.0±1.2%. ADSCs retentions were significantly higher at 2 and 4 µmol. On dye retention comparison at 8 µmol and 4 µmol for BMSCs and ADSCs; ADSCs were significantly higher at passages 2 and 3. The viability of BMSCs reduced from 94.0±1.2% to 90.0±0.6% and ADSCs from 94.0±1.2% to 52.0±1.2% (p<0.05) after 24h. BMSCs had significant up regulation of the cartilage genes for both the labeled and the unlabeled samples compared to ADSCs (p<0.05). PKH26 fluorescence was detected on the resected portions of the regenerated neo-cartilage. The recommended concentration of PKH26 for ADSCs is 2 µmol and BMSCs is 8 µmol, and they can be tracked up to 49 days.