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To compare 2 different graft preparation techniques to determine biomechanical strength and resultant tissue trauma evaluated by histology. Twelve common flexors of the finger's tendons were prepared with either tubulization (SpeedTrap™) or transtendon stiches (Orthocord™). The stiffness, resistance and energy at maximum load were tested for biomechanical assessment in both groups. After load testing, Samples were stained with hematoxylin and eosin (HE) to evaluate histological damage. We observe that the time to prepare tendons with SpeedTrap™ was 8.3 times faster (1:25 min) than traditional ones (15:02 min). In all cases, the mean values for SpeedTrap™ were higher in terms of strength, stiffness and energy at maximum load than for traditional suture but without significant difference (p > 0.05). The Krackow stitch produces greater structural damage to the collagen fibers while SpeedTrap™ maintains better organized arrangement of the fibers after tubulization preparation. With the results obtained, we can conclude that the tubulization technique allows faster graft preparation with less structural damage to the manipulated tissue without altering the biomechanical resistance provided by the transtendon suture technique.
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Técnicas de Sutura , Suturas , Tendões , Fenômenos Biomecânicos , Tendões/fisiologia , Humanos , Resistência à TraçãoRESUMO
We focus on this study in designing an alternative technique for obtaining mesenchymal stem cells (MSCs) from residual tissue, Hoffa fat, in arthroscopic procedures. Two males and two females were included, and underwent knee arthroscopy; a sample of infrapatellar adipose tissue was obtained with basket forceps. The primary culture was made using the explant method and the culture media: DMEM-high glucose, supplemented with 10% of inactivated human allogeneic serum. All the cellular cultures remained under culture conditions for three weeks, after that by flow cytometry the cells were characterized by MSCs antibody panel: CD105, CD73 and CD90. Subsequently, in the first pass, the MSCs were cultured in commercial human chondrogenic, osteogenic and adipogenic mediums, respectively. After primary culture, we obtained on average 95,600.00 ± 7,233.26 cells/cm2, and the duplication time of MSCs isolate from Hoffa fat pad was established in 39 hours. By flow cytometry, we found that surface markers percentage for expanded MSCs (CD105, CD73, CD90) in primary culture significantly increased and its morphology was fibroblastic-like. After differentiation culture which was made in the first pass, by immunofluorescence, we obtained positive cell markers for three lineages of differentiation, adipocytes: LPL protein, osteocytes: RUNX2, Osteopontin, chondrocytes: SOX9, Aggrecan and COL2A1. We managed to isolate a significant number of MSCs from this source using an easy method to implement and minimal nutrient supplementation, with high potential for differentiation to mature mesenchymal tissues and potential use in basic experimental, preclinical and even clinical research.
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Tecido Adiposo , Células-Tronco Mesenquimais , Masculino , Feminino , Humanos , Células Cultivadas , Diferenciação Celular , Meios de Cultura/farmacologia , Meios de Cultura/metabolismo , Células-Tronco Mesenquimais/metabolismo , Proliferação de CélulasRESUMO
Autologous chondrocyte implantation has shown optimal long-term outcomes in the treatment of cartilage lesions. The challenge for a single-stage approach lies in obtaining sufficient number of cells with high viability. The answer could lie in supplementing or replacing them with allogenic chondrocytes coming from cadaveric donors. In the present work, we aimed to compare the number of viable cells isolated from cartilage of live and cadaveric donors and to determine the suitable characteristics of the best donors. A total of 65 samples from donors aged from 17 to 55 years, either women or men, were enrolled in this study (33 living vs. 32 cadaveric). The mean time of hours from death to processing samples in cadaveric donors was higher compared to live donors (64.3 ± 17.7 vs. 4.6±6.4). The number of isolated chondrocytes per gram of cartilage was higher in cadaveric donors (5.389 × 106 compared to 3.067 × 106 in living donors), whereas the average of cell viability was comparable in both groups (84.16% cadaveric, 87.8% alive). It is possible to obtain viable chondrocytes from cartilage harvested from cadaveric donors, reaching a similar cell number and viability to that obtained from the cartilage of living donors.
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Cartilagem Articular , Transplante de Células-Tronco Hematopoéticas , Masculino , Humanos , Feminino , Condrócitos , Doadores Vivos , Cadáver , Transplante AutólogoRESUMO
Background and Objectives: Deposits of monosodium urate (MSU) crystals due to increased levels of uric acid (UA) have been associated with bone formation and erosion, mainly in patients with chronic gout. The synovial membrane (SM) comprises several types of cells, including mesenchymal stem cells (SM-MSCs); however, it is unknown whether UA and MSU induce osteogenesis through SM-MSCs. Materials and Methods: Cultures of SM were immunotyped with CD44, CD69, CD90, CD166, CD105, CD34, and CD45 to identify MSCs. CD90+ cells were isolated by immunomagnetic separation (MACS), colony-forming units (CFU) were identified, and the cells were exposed to UA (3, 6.8, and 9 mg/dL) and MSU crystals (1, 5, and 10 µg/mL) for 3 weeks, and cellular morphological changes were evaluated. IL-1ß and IL-6 were determined by ELISA, mineralization was assessed by alizarin red, and the expression of Runx2 was assessed by Western blot. Results: Cells derived from SM and after immunomagnetic separation were positive for CD90 (53 ± 8%) and CD105 (52 ± 18%) antigens, with 53 ± 5 CFU identified. Long-term exposure to SM-MSCs by UA and MSU crystals did not cause morphological damage or affect cell viability, nor were indicators of inflammation detected. Mineralization was observed at doses of 6.8 mg/dL UA and 5 µg/mL MSU crystals; however, the differences were not significant with respect to the control. The highest dose of MSU crystals (10 µg/mL) induced significant Runx2 expression with respect to the control (1.4 times greater) and SM-MSCs cultured in the osteogenic medium. Conclusions: MSU crystals may modulate osteogenic differentiation of SM-MSCs through an increase in Runx2.
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Gota , Células-Tronco Mesenquimais , Humanos , Ácido Úrico/farmacologia , Osteogênese , Subunidade alfa 1 de Fator de Ligação ao Core , ProteínasRESUMO
The preservation of the chondrogenic phenotype and hypoxia-related physiological microenvironment are major challenges in the 2D culture of primary human chondrocytes. To address this problem, we develop a 3D culture system generating scaffold-free spheroids from human chondrocytes. Our results highlight the chondrogenic potential of cultured human articular chondrocytes in a 3D system combined with hypoxia independently of the cartilage source. After 14 days of culture, we developed spheroids with homogenous diameter and shape from hyaline cartilage donors. Spheroids generated in hypoxia showed a significantly increased glycosaminoglycans synthesis and up-regulated the expression of SOX9, ACAN, COL2A1, COMP, and SNAI1 compared to those obtained under normoxic conditions. Therefore, we conclude that spheroids developed under hypoxic conditions modulate the expression of chondrogenesis-related genes and native tissue features better than 2D cultures. Thus, this scaffold-free 3D culture system represents a novel in vitro model that can be used for cartilage biology research.
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Cartilagem Articular , Condrócitos , Cartilagem Articular/metabolismo , Condrócitos/metabolismo , Condrogênese , Humanos , Hipóxia/metabolismoRESUMO
BACKGROUND: The present review is focused on general aspects of the synovial membrane as well as specialized aspects of its cellular constituents, particularly the composition and location of synovial membrane mesenchymal stem cells (S-MSCs). S-MSC multipotency properties are currently at the center of translational medicine for the repair of multiple joint tissues, such as articular cartilage and meniscus lesions. METHODS AND RESULTS: We reviewed the results of in vitro and in vivo research on the current clinical applications of S-MSCs, surface markers, cell culture techniques, regenerative properties, and immunomodulatory mechanisms of S-MSCs as well as the practical limitations of the last twenty-five years (1996 to 2021). CONCLUSIONS: Despite the poor interest in the development of new clinical trials for the application of S-MSCs in joint tissue repair, we found evidence to support the clinical use of S-MSCs for cartilage repair. S-MSCs can be considered a valuable therapy for the treatment of repairing joint lesions.
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Cartilagem Articular , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Diferenciação Celular , Transplante de Células-Tronco Mesenquimais/métodos , Membrana SinovialRESUMO
BACKGROUND: Few randomized controlled trials with a midterm follow-up have compared matrix-assisted autologous chondrocyte transplantation (MACT) with microfracture (MFx) for knee cartilage lesions. PURPOSE: To compare the structural, clinical, and safety outcomes at midterm follow-up of MACT versus MFx for treating symptomatic knee cartilage lesions. STUDY DESIGN: Randomized controlled trial; Level of evidence, 1. METHODS: A total of 48 patients aged between 18 and 50 years, with 1- to 4-cm2 International Cartilage Repair Society (ICRS) grade III to IV knee chondral lesions, were randomized in a 1:1 ratio to the MACT and MFx treatment groups. A sequential prospective evaluation was performed using magnetic resonance imaging (MRI) T2 mapping, the MOCART (magnetic resonance observation of cartilage repair tissue) score, second-look arthroscopic surgery, patient-reported outcome measures, the responder rate (based on achieving the minimal clinically important difference for the Knee injury and Osteoarthritis Outcome Score [KOOS] pain and KOOS Sport/Recreation), adverse events, and treatment failure (defined as a reoperation because of symptoms caused by the primary defect and the detachment or absence of >50% of the repaired tissue during revision surgery). RESULTS: Overall, 35 patients (18 MACT and 17 MFx) with a mean chondral lesion size of 1.8 ± 0.8 cm2 (range, 1-4 cm2) were followed up to a mean of 6 years postoperatively (range, 4-9 years). MACT demonstrated significantly better structural outcomes than MFx at 1 to 6 years postoperatively. At final follow-up, the MRI T2 mapping values of the repaired tissue were 37.7 ± 8.5 ms for MACT versus 46.4 ± 8.5 ms for MFx (P = .003), while the MOCART scores were 59.4 ± 17.3 and 42.4 ± 16.3, respectively (P = .006). More than 50% defect filling was seen in 95% of patients at 2 years and 82% at 6 years in the MACT group and in 67% at 2 years and 53% at 6 years in the MFx group. The second-look ICRS scores at 1 year were 10.7 ± 1.3 for MACT and 9.0 ± 1.8 for MFx (P = .001). Both groups showed significant clinical improvements at 6 years postoperatively compared with their preoperative status. Significant differences favoring the MACT group were observed at 2 years on the KOOS Activities of Daily Living (P = .043), at 4 years on all KOOS subscales (except Symptoms; P < .05) and the Tegner scale (P = .008), and at 6 years on the Tegner scale (P = .010). The responder rates at 6 years were 53% and 77% for MFx and MACT, respectively. There were no reported treatment failures after MACT; the failure rate was 8.3% in the MFx group. Neither group had serious adverse events related to treatment. CONCLUSION: Patients who underwent MACT had better structural outcomes than those who underwent MFx at 1 to 6 years postoperatively. Both groups of patients showed significant clinical improvements at final follow-up compared with their preoperative status. MACT showed superiority at 4 years for the majority of the KOOS subscales and for the Tegner scale at 4 to 6 years. The MACT group also had a higher responder rate and lower failure rate at final follow-up. REGISTRATION: NCT01947374 (ClinicalTrials.gov identifier).
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Cartilagem Articular , Fraturas de Estresse , Atividades Cotidianas , Adolescente , Adulto , Cartilagem Articular/cirurgia , Condrócitos , Seguimentos , Humanos , Articulação do Joelho/cirurgia , Imageamento por Ressonância Magnética , Pessoa de Meia-Idade , Estudos Prospectivos , Transplante Autólogo , Adulto JovemRESUMO
We hypothesized that the secretion of inflammatory mediators from synoviocytes affects the chondrocyte homeostasis of articular cartilage. This study was a preliminary attempt to elucidate the molecular mechanisms by which soluble mediators obtained from activated synoviocytes induce oxidative stress and inflammation in chondrocytes. We measured the concentrations of interleukin-6 (IL-6), interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1), nerve growth factor (NGF), superoxide anion (O2â¢-), hydrogen peroxide (H2O2), and nitric oxide (NOâ¢) from articular human cells. First, we created a conditional basal medium by exposing synoviocytes (HS) to monosodium urate crystals (CBM). The chondrocytes were exposed to either CBM (CCM), urate crystals directly (CMSU), or remained untreated (CC) as a negative control. Data were analyzed by ANOVA tests; Bonferroni test was performed for multiple comparisons between groups. Interestingly, we observed that mediators of inflammation and oxidative stress were significantly higher in CCM than CMSU and CC groups (P<0.01). The specific concentrations were as follows: 19.85 ng/mL of IL-6, 9.79 ng/mL of IL-8, 5.17 ng/mL of NGF, and 11.91 ng/mL of MCP-1. Of note, we observed the same trend for reactive oxygen and nitrogen species (P<0.001). Soluble mediators secreted by synoviocytes after being activated with MSU crystals (as observed in individuals who present gout attacks) trigger chondrocyte activation intensifying the articular inflammatory, oxidative, and pain states that damage cartilage in OA; this damage is more severe even when compared to HC directly exposed to monosodium urate crystals. Key Points ⢠The molecular relation between MSU depositions and cartilage damage could be mediated by pro-inflammatory soluble mediators and oxidative molecules. ⢠The secretion of pro-inflammatory mediators by activated synoviocytes is more harmful to chondrocytes than a direct activation in the chondrocyte culture. ⢠Under this model, there is an important imbalance in the matrix homeostasis due to changes in several chemokines, cytokines, and other factors such as NGF, as well as oxidative mediators.
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Condrócitos , Sinoviócitos , Células Cultivadas , Humanos , Peróxido de Hidrogênio , Mediadores da Inflamação , Estresse Oxidativo , Dor , Ácido ÚricoRESUMO
Objective. To evaluate minimum biosecurity parameters (MBP) for arthroscopic matrix-encapsulated autologous chondrocyte implantation (AMECI) based on patients' clinical outcomes, magnetic resonance imaging (MRI) T2-mapping, Magnetic Resonance Observation of Cartilage Repair Tissue (MOCART) score, and International Cartilage Repair Society (ICRS) second-look arthroscopic evaluation, laying the basis for a future multicenter study. Design. Pilot clinical study. We analyzed the logistics to perform AMECI to treat focal chondral lesions in different hospitals following strict biosecurity parameters related to tissue and construct transportation, chondrocyte isolation, and cell expansion. Patient progress was analyzed with patient-reported outcome measures, MRI T2-mapping, MOCART, and ICRS arthroscopic second-look evaluation. Results. Thirty-five lesions in 30 patients treated in 7 different hospitals were evaluated. Cell viability before implantation was >90%. Cell viability in construct remnants was 87% ± 11% at 24 hours, 75% ± 17.1% at 48 hours, and 60% ± 8% at 72 hours after implantation. Mean final follow-up was 37 months (12-72 months). Patients showed statistically significant improvement in all clinical scores and MOCART evaluations. MRI T2-mapping evaluation showed significant decrease in relaxation time from 61.2 ± 14.3 to 42.9 ± 7.2 ms (P < 0.05). Arthroscopic second-look evaluation showed grade II "near normal" tissue in 83% of patients. Two treatment failures were documented. Conclusions. It was feasible to perform AMECI in 7 different institutions in a large metropolitan area following our biosecurity measures without any implant-related complication. Treated patients showed improvement in clinical, MRI T2-mapping, and MOCART scores, as well as a low failure rate and a favorable ICRS arthroscopic evaluation at a mid-term follow-up. Level of Evidence. 2b.
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Cartilagem Articular , Condrócitos , Cartilagem Articular/diagnóstico por imagem , Cartilagem Articular/cirurgia , Seguimentos , Humanos , América Latina , Transplante Autólogo/métodosRESUMO
METHODS: Seventeen patients aged 18 to 55 years with symptomatic full-thickness cartilage lesions on either patella or trochlea were treated with matrix autologous chondrocyte implantation (MACI) or microfracture (MF). Both procedures combined with unloading/realigning techniques. Clinical assessment and T2-mapping were evaluated at 48-months. RESULTS: Clinically results from pre-op to 48-months improved significantly in MACI and MF for Lysholm (p = 0.001, p = 0.001), IKDC-S (p = 0.001, p = 0.002), KOOS-P (p = 0.000, p = 0.002), KOOS-DLA (p = 0.002, p = 0.003), KOOS-Sports/Rec (p = 0.000, p = 0.004), KOOS-QoL (p = 0.000, p = 0.003), KOOS-symptoms (p = 0.001, p = 0.020), and Kujala (p = 0.000, p = 0.01), respectively. Tegner was significant between baseline and 48 months only for MACI (p < 0.008) compared with MF (p = 0.25). No significant difference was observed between groups for any score at 3, 12, 24, and 48-months (p > 0.05). T2-mapping values improved significantly over time in MACI compared with MF at 24 months (39.35 vs. 50.44, p = 0.007) and 48 months (36.54 vs. 48.37, p = 0.005). When comparing control values to MACI at 12-m (p = 0.714), 24-m (p = 0.175), and 48-m (p = 0.097), no significant difference was found. MOCART (Magnetic Resonance Observation of Cartilage Repair Tissue) score comparison gave no statistical difference between groups. CONCLUSIONS: Clinically both techniques improved significantly over time. However, quantitative assessment showed that only newly formed tissue with MACI technique improves significantly since 12-months and maintains stable values compared with native cartilage until 48-month follow-up. MF results were never comparable to those native values. Level of evidence II.