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INTRODUCTION: Since the 1980s, adipose-derived stem cells (ASCs) have become a powerful and potential source for stem cell-based therapy, regenerative medicine, and even drug delivery in cancer treatment. The development of off-the-shelf mesenchymal stem cells (MSCs), including ASCs, has rapidly advanced in recent years with several clinical trials and approved products. In this technology, ASCs should be expanded long term in order to harvest higher cell number. In this study, senescence of ASCs after long-term expansion was evaluated. METHODS: Human ASCs (hASCs) were isolated and cultured continuously at a density of 103 cells/cm2 up to passage 15. The cells were assessed for aging via changes in the following: characteristics of MSCs, mitochondrial activity, accumulation of beta-galactosidase, and expression of tumor suppressor genes. RESULTS: The results showed that following in vitro expansion to the 15th passage, ASCs did not show changes in immunophenotype, except for decreased expression of CD105. However, the cells increased in size and in shape and complexity (toward the "fried egg" morphology). They also almost ceased to proliferate in passage 15. Nonetheless, they maintained in vitro differentiation potential toward osteoblasts, chondrocytes, and adipocytes. Expression of tumor suppressor genes p53 and p16 did not significantly change, while p27 was significantly downregulated. Mitochondrial activities also decreased slightly in culture from passage 5 to passage 10 and remained stable to passage 15. ASCs also showed increased accumulation of beta-galactosidase in culture, but it was negligible. CONCLUSION: In conclusion, hASCs exhibited some particular characteristics of aged stem cells when the number of subculture cells increased. However, up to passage 10, ASCs also retained almost all of the characteristics of MSCs.
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Adipócitos , Tecido Adiposo , Senescência Celular , Células-Tronco Mesenquimais , Adipócitos/citologia , Tecido Adiposo/citologia , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Humanos , Células-TroncoRESUMO
In vitro production of tissues or tissue engineering is a promising approach to produce artificial tissues for regenerative medicine. There are at least three important components of tissue engineering, including stem cells, scaffolds and growth factors. This study aimed to produce cartilage tissues in vitro from culture and chondrogenic differentiation of rabbit bone marrow-derived mesenchymal stem cells (BMMSCs), induced by chondrogenesis medium, on biodegradable polycaprolactone (PCL) scaffolds. BMMSCs were isolated from rabbit bone marrow according to the standard protocol. The adherence, proliferation and differentiation of BMMSCs on scaffolds were investigated using two scaffold systems: PCL scaffolds and collagen-coated PCL (PCL/col) scaffolds. The results showed that BMMSCs could attach and grow on both PCL and PCL/col scaffolds. However, the adhesion efficacy of BMMSCs on the PCL/col scaffolds was significantly better than on PCL scaffolds. Under induced conditions, BMMSCs on PLC/col scaffolds showed increased aggrecan accumulation and upregulated expression of chondrogenesis-associated genes (e.g. collagen type II, collagen type I, aggrecan and collagen type X) after 3, 7, 21 and 28 days of induction. These in vitro cartilage tissues could form mature chondrocyte-like cells after they were grafted into rabbits. The results suggest that use of BMMSCs in combination with polycaprolactone scaffolds and chondrogenesis medium can be a way to form in vitro cartilage tissue.
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Medula Óssea , Condrogênese , Células-Tronco Mesenquimais , Poliésteres , Alicerces Teciduais , Animais , Cartilagem/citologia , Células Cultivadas , Células-Tronco Mesenquimais/citologia , Poliésteres/química , Coelhos , Engenharia Tecidual , Alicerces Teciduais/químicaRESUMO
Umbilical cord (UC) is a rich source of rapidly proliferating mesenchymal stem cells (MSCs) that are easily cultured on a large-scale. Clinical applications of UC-MSCs include graft-versus-host disease, and diabetes mellitus types 1 and 2. UC-MSCs should be isolated and proliferated according to good manufacturing practice (GMP) with animal component-free medium, quality assurance, and quality control for their use in clinical applications. This study developed a GMP standard protocol for UC-MSC isolation and culture. UC blood and UC were collected from the same donors. Blood vasculature was removed from UC. UC blood was used as a source of activated platelet rich plasma (aPRP). Small fragments (1-2 mm(2)) of UC membrane and Wharton's jelly were cut and cultured in DMEM/F12 medium containing 1 % antibiotic-antimycotic, aPRP (2.5, 5, 7.5 and 10 %) at 37 °C in 5 % CO2. The MSC properties of UC-MSCs at passage 5 such as osteoblast, chondroblast and adipocyte differentiation, and markers including CD13, CD14, CD29, CD34, CD44, CD45, CD73, CD90, CD105, and HLA-DR were confirmed. UC-MSCs also were analyzed for karyotype, expression of tumorigenesis related genes, cell cycle, doubling time as well as in vivo tumor formation in NOD/SCID mice. Control cells consisted of UC-MSCs cultured in DMEM/F12 plus 1 % antibiotic-antimycotic, and 10 % fetal bovine serum (FBS). All UC-MSC (n = 30) samples were successfully cultured in medium containing 7.5 and 10 % aPRP, 92 % of samples grew in 5.0 % aPRP, 86 % of samples in 2.5 % aPRP, and 72 % grew in 10 % FBS. UC-MSCs in these four groups exhibited similar marker profiles. Moreover, the proliferation rates in medium with PRP, especially 7.5 and 10 %, were significantly quicker compared with 2.5 and 5 % aPRP or 10 % FBS. These cells maintained a normal karyotype for 15 sub-cultures, and differentiated into osteoblasts, chondroblasts, and adipocytes. The analysis of pluripotent cell markers showed UC-MSCs maintained the expression of the oncogenes Nanog and Oct4 after long term culture but failed to transfer tumors in NOD/SCID mice. Replacing FBS with aPRP in the culture medium for UC tissues allowed the successful isolation of UC-MSCs that satisfy the minimum standards for clinical applications.
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Separação Celular/métodos , Células-Tronco Mesenquimais/citologia , Cordão Umbilical/citologia , Animais , Biomarcadores/metabolismo , Diferenciação Celular , Linhagem da Célula , Proliferação de Células , Células Cultivadas , Cromossomos Humanos/metabolismo , Regulação da Expressão Gênica , Genes Supressores de Tumor , Humanos , Cariotipagem , Células-Tronco Mesenquimais/metabolismo , Mesoderma/citologia , Camundongos Nus , Camundongos SCID , OncogenesRESUMO
Background: Treating complex three- and four-part proximal humerus fractures, especially in the elderly, remains contentious, with internal fixation using locking plates and shoulder arthroplasty being primary options. Although proximal humerus locking plates are more commonly used than shoulder replacements, they have a high complication rate. Factors like low bone density, advanced age, multiple fragment fractures, and medial cortical support loss negatively impact treatment outcomes. This study evaluates the functional and radiographic outcomes of using locking plates for treating these fractures in patients aged 50 and older, and the factors influencing outcomes and complication rates at the Ho Chi Minh City Hospital for Trauma and Orthopedics. Method: A descriptive case series study was conducted on 58 patients aged 50 and older (15 males, 43 females) with three- and four-part proximal humerus fractures. These patients underwent open reduction and internal fixation with Locking - compression plate (LCP) periarticular proximal humerus plates at the Ho Chi Minh City Hospital for Traumatology and Orthopedics (HTO) from April 2020 to April 2022. The minimum postoperative follow-up period was 12 months. Results: The average age of the patients was 62.78 â± â7.73 years, with a mean follow-up of 26.24 â± â5.93 months. Among them, 41 had three-part fractures (70.68 â%) and 17 had four-part fractures (29.32 â%). At the final follow-up (≥12 months), the mean Constant-Murley score was 70.81 â± â9.15, and the mean QDASH score was 8.33 â± â2.77. Complications occurred in 6 cases (10.34 â%). Complex fractures, such as four-part fractures with displacement greater than 2 âmm, had lower Constant-Murley scores and higher QDASH scores (p â< â0.05). Age, gender, bone density by deltoid tuberosity index (DTI), bone grafting, and rotator cuff sutures showed similar trends, but the differences were not statistically significant. Conclusion: Open reduction and internal fixation (ORIF) provides good bone healing and functional outcomes for three- and four-part proximal humerus fractures. Factors such as age, gender, bone density, bone grafting, and rotator cuff sutures do not significantly affect outcomes. Therefore, osteoporosis should not be a contraindication for ORIF with locking plates in these cases. Complex fractures, however, often lead to poorer outcomes and higher complication rates post-surgery.
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INTRODUCTION: Adipose-derived stem cells (ADSCs) have been isolated, expanded, and applied in the treatment of many diseases. ADSCs have also been used to treat injured articular cartilage. However, there is controversy regarding the treatment efficiency. We considered that ADSC transplantation with activated platelet-rich plasma (PRP) may improve injured articular cartilage compared with that of ADSC transplantation alone. In this study, we determined the role of PRP in ADSC transplantation to improve the treatment efficiency. METHODS: ADSCs were isolated and expanded from human adipose tissue. PRP was collected and activated from human peripheral blood. The effects of PRP were evaluated in vitro and in ADSC transplantation in vivo. In vitro, the effects of PRP on ADSC proliferation, differentiation into chondrogenic cells, and inhibition of angiogenic factors were investigated at three concentrations of PRP (10%, 15% and 20%). In vivo, ADSCs pretreated with or without PRP were transplanted into murine models of injured articular cartilage. RESULTS: PRP promoted ADSC proliferation and differentiation into chondrogenic cells that strongly expressed collagen II, Sox9 and aggrecan. Moreover, PRP inhibited expression of the angiogenic factor vascular endothelial growth factor. As a result, PRP-pretreated ADSCs improved healing of injured articular cartilage in murine models compared with that of untreated ADSCs. CONCLUSION: Pretreatment of ADSCs with PRP is a simple method to efficiently apply ADSCs in cartilage regeneration. This study provides an important step toward the use of autologous ADSCs in the treatment of injured articular cartilage.