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1.
J Orthop Sci ; 25(1): 161-166, 2020 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-30902537

RESUMEN

PURPOSE: The purpose of this study was to investigate the biomechanical properties of load distribution following a centralization procedure for extruded lateral menisci with posterior root deficiency in a porcine model. METHODS: Six porcine knee joints were analyzed in a universal tester, as follows: 1) Intact; 2) Extrusion (meniscus extrusion was created by resecting the posterior root of the lateral meniscus, as well as the posterior synovial capsule); and 3) Centralization (two anchors were inserted at the lateral tibial plateau, and the meniscus was sutured to secure it close to the original position). Meniscus extrusion was evaluated using two markers put on the posterior cruciate ligament and the lateral meniscus, and the load distribution were assessed using a pressure mapping sensor system after applying a loading force of 200 N to the knee joint. RESULTS: Distance between two markers (mm, Average; 95% CI) was larger in the extrusion group (21.9; 17.8, 25.6) than in the intact (18.1; 15.1, 22.7) or the centralization (15.3; 12.9, 18.0) groups. The contact area (mm2) in the middle of the meniscus was significantly smaller in the extrusion group (45.8; 18.5, 73.2) than in the intact (85.7; 72.1, 99.2) or the centralization (98.3; 88.8, 107.8) groups. The maximum contact pressure (MPa) in the tibial plateau was significantly higher in the extrusion group (0.37; 0.35, 0.40) than in the intact (0.29; 0.21, 0.37) or the centralization (0.29; 0.22, 0.36) groups. CONCLUSIONS: The centralization procedure enabled a reduction of the meniscus extrusion in the lateral meniscus with posterior root deficiency and restored the maximum load and contact pressure to values close to those of the normal knee joint.

2.
Sci Rep ; 9(1): 16835, 2019 Nov 14.
Artículo en Inglés | MEDLINE | ID: mdl-31728017

RESUMEN

Mesenchymal stem cells from the synovium (synovial MSCs) are attractive for cartilage and meniscus regeneration therapy. We developed a software program that can distinguish individual colonies and automatically count the cell number per colony using time-lapse images. In this study, we investigated the usefulness of the software and analyzed colony formation in cultured synovial MSCs. Time-lapse image data were obtained for 14-day-expanded human synovial MSCs. The cell number per colony (for 145 colonies) was automatically counted from phase-contrast and nuclear-stained images. Colony growth curves from day 1 to day 14 (for 140 colonies) were classified using cluster analysis. Correlation analysis of the distribution of the cell number per colony at 14 days versus that number at 1-14 days revealed a correlation at 7 and 14 days. We obtained accurate cell number counts from phase-contrast images. Individual colony growth curves were classified into three main groups and subgroups. Our image analysis software has the potential to improve the evaluation of cell proliferation and to facilitate successful clinical applications using MSCs.

3.
Cell Transplant ; 28(11): 1445-1454, 2019 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-31313604

RESUMEN

Complex degenerative tears of the medial meniscus in the knee are usually treated using meniscectomy. However, this procedure increases the risk of osteoarthritis, while other treatments aimed at meniscal repair remain challenging due to the high possibility of failure. The use of synovial mesenchymal stem cells (MSCs) is an attractive additional approach for meniscal repair, as these cells have high proliferative and chondrogenic potential. In this case report, we surgically repaired a complex degenerative tear of the medial meniscus and then transplanted autologous synovial MSCs. We evaluated clinical outcomes at 2 years and assessed adverse events. We enrolled patients with clinical symptoms that included a feeling of instability in addition to pain caused by their complex degenerative tears of the medial meniscus. Two weeks after surgical repair of the torn meniscus, autologous synovial MSCs were transplanted onto the menisci of five patients. The total Lysholm knee score, the Knee Injury and Osteoarthritis Outcome Scale scores for "pain," "daily living," "sports activities," and the Numerical Rating Scale were significantly increased after 2 years. Three adverse events, an increase in c-reactive protein, joint effusion, and localized warmth of the knee were recorded, although these could have been due to the meniscal repair surgery. This first-in-human study confirmed that the combination of surgical repair and synovial MSC transplantation improved the clinical symptoms in patients with a complex degenerative tear of the medial meniscus. No adverse events occurred that necessitated treatment discontinuation. These findings will serve as pilot data for a future prospective study.

4.
BMC Musculoskelet Disord ; 20(1): 316, 2019 Jul 06.
Artículo en Inglés | MEDLINE | ID: mdl-31279341

RESUMEN

BACKGROUND: Synovial mesenchymal stem cells (MSCs) are an attractive cell source for cartilage and meniscus regeneration. The optimum cryopreservation medium has not been determined, but dimethylsulfoxide (DMSO) should be excluded, if possible, because of its toxicity. The purposes of our study were to examine the possible benefits of higher concentrations of serum and the effectiveness of 100% serum (without DMSO) for the cryopreservation of synovial MSCs. METHODS: Human synovium was harvested from the knees of four donors with osteoarthritis during total knee arthroplasty. Synovial MSCs (8 × 105 cells) were suspended in 400 µL medium and used as a Time 0 control. The same number of synovial MSCs was also suspended in 400 µL α-MEM medium containing 10% fetal bovine serum (FBS) (5% DMSO, and 1% antibiotic), 95% FBS (and 5% DMSO), or 100% FBS (no DMSO) and cryopreserved at - 80 °C for 7 days. After thawing, the cell suspensions (1.5 µL; 3 × 103 cells) were cultured in 60 cm2 dishes for 14 days for colony formation assays. Additional 62.5 µL samples of cell suspensions (1.25 × 105 cells) were added to tubes and cultured for 21 days for chondrogenesis assays. RESULTS: Colony numbers were significantly higher in the Time 0 and 95% FBS groups than in the 10% FBS group (n = 24). Colony numbers were much lower in the 100% FBS group than in the other three groups. The cell numbers per dish reflected the colony numbers. Cartilage pellet weights were significantly heavier in the 95% FBS group than in the 10% FBS group, whereas no difference was observed between the Time 0 and the 95% FBS groups (n = 24). No cartilage pellets formed at all in the 100% FBS group. CONCLUSION: Synovial MSCs cryopreserved in 95% FBS with 5% DMSO maintained their colony formation and chondrogenic abilities to the same levels as observed in the cells before cryopreservation. Synovial MSCs cryopreserved in 100% FBS lost their colony formation and chondrogenic abilities.


Asunto(s)
Condrogénesis/efectos de los fármacos , Criopreservación/métodos , Crioprotectores/farmacología , Células Madre Mesenquimatosas , Membrana Sinovial/citología , Anciano , Técnicas de Cultivo de Célula , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Crioprotectores/química , Dimetilsulfóxido/química , Dimetilsulfóxido/farmacología , Femenino , Humanos , Articulación de la Rodilla/citología , Trasplante de Células Madre Mesenquimatosas , Osteoartritis/terapia , Suero/química
5.
J Orthop Res ; 37(11): 2466-2475, 2019 11.
Artículo en Inglés | MEDLINE | ID: mdl-31115925

RESUMEN

The meniscus functions as a load distributor and secondary stabilizer in the knee, and the loss of the meniscus increases the risk of osteoarthritis. Freeze-thawed menisci are used in clinical practice to replace defective menisci; however, the disadvantages of freeze-thawed tissues include disease transmission and immune rejection. In this study, we decellularized menisci using high hydrostatic pressure (HHP) and compared the decellularized menisci with freeze-thawed menisci. Porcine menisci were either pressurized at 1,000 MPa for 10 min and then washed with DNase solution or frozen at -80°C for 2 days and thawed. These menisci then underwent in vitro histological, biochemical, and biomechanical comparisons with native menisci. The HHP-treated and freeze-thawed menisci were also subcutaneously implanted in a pig, and later harvested for histological analysis. The numbers of histologically detected cells were significantly lower and the amount of biochemically detected DNA was approximately 100-fold lower in HHP-treated than in native and freeze-thawed menisci. The compression strength of the HHP-decellularized menisci was decreased after 1 and 50 cycles at 20% strain but was unchanged in the freeze-thawed menisci. After implantation, the numbers of multinucleated giant cells were significantly lower around the HHP-treated menisci than around the freeze-thawed menisci. Recellularization of the HHP-decellularized menisci was confirmed. Thus, although the HHP-decellularized menisci were mechanically inferior to the freeze-thawed meniscus in vitro, they were immunologically superior. Our study is the first to demonstrate the use of HHP for decellularization of the meniscus. © 2019 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 37:2466-2475, 2019.


Asunto(s)
Aloinjertos , Meniscos Tibiales/trasplante , Animales , Congelación , Presión Hidrostática , Porcinos
6.
J Orthop Res ; 37(6): 1350-1357, 2019 06.
Artículo en Inglés | MEDLINE | ID: mdl-29737046

RESUMEN

In vitro chondrogenesis of mesenchymal stem cells (MSCs) mimics in vivo chondrogenesis of MSCs. However, the size of the cartilage pellets that can be attained in vitro is limited by current methods; therefore, some modifications are required to obtain larger pellets. Petaloid pieces of recombinant peptide (petaloid RCP) have the advantage of creating spaces between cells in culture. The RCP used here is based on the alpha-1 sequence of human collagen type I and contains 12 Arg-Gly-Asp motifs. We examined the effect and mechanisms of adding petaloid RCP on the in vitro chondrogenesis of human synovial MSCs by culturing 125k cells with or without 0.125 mg petaloid RCP in chondrogenic medium for 21 days. The cartilage pellets were sequentially analyzed by weight, sulfated glycosaminoglycan content, DNA retention, and histology. Petaloid RCP significantly increased the weight of the cartilage pellets: The petaloid RCP group weighed 7.7 ± 1.2 mg (n = 108), whereas the control group weighed 5.3 ± 1.6 mg. Sulfated glycosaminoglycan and DNA contents were significantly higher in the petaloid RCP group than in the control group. Light and transmission electron microscopy images showed that the petaloid RCP formed the framework of the pellet at day 1, the framework was broken by production of cartilage matrix by the synovial MSCs at day 7, and the cartilage pellet grew larger, with diffuse petaloid RCP remaining, at day 21. Therefore, petaloid RCP formed a framework for the pellet, maintained a higher cell number, and promoted in vitro cartilage formation of synovial MSCs. © 2018 The Authors. Journal of Orthopaedic Research® Published by Wiley Periodicals, Inc. J Orthop Res 37:1350-1357, 2019.


Asunto(s)
Condrogénesis/efectos de los fármacos , Células Madre Mesenquimatosas/citología , Péptidos/farmacología , Membrana Sinovial/citología , Anciano , Anciano de 80 o más Años , Diferenciación Celular , Femenino , Glicosaminoglicanos/metabolismo , Humanos , Proteínas Recombinantes/farmacología
7.
PLoS One ; 13(8): e0202922, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30138399

RESUMEN

Osteoarthritis (OA), a common chronic joint disorder in both humans and canines, is characterized by a progressive loss of articular cartilage. Canines can serve as an animal model of OA for human medicine, and this research can simultaneously establish effective veterinary treatments for canine OA. One attractive treatment that can lead to cartilage regeneration is the use of mesenchymal stem cells (MSCs). However, for canine OA, little information is available regarding the best source of MSCs. The purpose of this study was to identify a promising MSC source for canine cartilage regeneration. We collected synovial, infrapatellar fat pad, inguinal adipose, and bone marrow tissues from six canines and then conducted a donor-matched comparison of the properties of MSCs derived from these four tissues. We examined the surface epitope expression, proliferation capacity, and trilineage differentiation potential of all four populations. Adherent cells derived from all four tissue sources exhibited positivity for CD90 and CD44 and negativity for CD45 and CD11b. The positive rate for CD90 was higher for synovium-derived than for adipose-derived and bone marrow-derived MSCs. Synovium-derived and infrapatellar fat pad-derived MSCs displayed substantial proliferation ability, and all four populations underwent trilineage differentiation. During chondrogenesis, the wet weight was heavier for cartilage pellets derived from synovium MSCs than from the other three sources. The synovium is therefore a promising source for MSCs for canine cartilage regeneration. Our findings provide useful information about canine MSCs that may be applicable to regenerative medicine for treatment of OA.


Asunto(s)
Tejido Adiposo/metabolismo , Cartílago/metabolismo , Condrogénesis/fisiología , Células Madre Mesenquimatosas/metabolismo , Membrana Sinovial/metabolismo , Tejido Adiposo/citología , Tejido Adiposo/fisiología , Animales , Técnicas de Cultivo de Célula/veterinaria , Diferenciación Celular , Proliferación Celular , Tratamiento Basado en Trasplante de Células y Tejidos/veterinaria , Perros , Femenino , Masculino , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/fisiología , Osteoartritis/terapia , Osteoartritis/veterinaria , Regeneración , Medicina Regenerativa , Membrana Sinovial/citología
8.
Stem Cell Res Ther ; 9(1): 123, 2018 05 02.
Artículo en Inglés | MEDLINE | ID: mdl-29720268

RESUMEN

BACKGROUND: Synovial mesenchymal stem cells (MSCs) are an attractive cell source for cartilage and meniscus regeneration. Synovial tissue can be histologically classified into three regions; surface, stromal and perivascular region, but the localization of synovial MSCs has not been fully investigated. We identified markers specific for each region, and compared properties of MSCs derived from each region in the synovium. METHODS: The intensity of immunostaining with 19 antibodies was examined for surface, stromal, and perivascular regions of human synovium from six osteoarthritis patients. Specific markers were identified and synovial cells derived from each region were sorted. Proliferation, surface marker expression, chondrogenesis, calcification and adipogenesis potentials were compared in synovial MSCs derived from the three regions. RESULTS: We selected CD55+ CD271- for synovial cells in the surface region, CD55- CD271- in the stromal region, and CD55- CD271+ in the perivascular region. The ratio of the sorted cells to non-hematopoietic lineage cells was 5% in the surface region, 70% in the stromal region and 15% in the perivascular region. Synovial cells in the perivascular fraction had the greatest proliferation potential. After expansion, surface marker expression profiles and adipogenesis potentials were similar but chondrogenic and calcification potentials were higher in synovial MSCs derived from the perivascular region than in those derived from the surface and stromal regions. CONCLUSIONS: We identified specific markers to isolate synovial cells from the surface, stromal, and perivascular regions of the synovium. Synovial MSCs in the perivascular region had the highest proliferative and chondrogenic potentials among the three regions.


Asunto(s)
Biomarcadores/metabolismo , Células Madre Mesenquimatosas/metabolismo , Microscopía Electrónica de Transmisión/métodos , Membrana Sinovial/metabolismo , Anciano , Anciano de 80 o más Años , Diferenciación Celular , Citometría de Flujo , Humanos , Persona de Mediana Edad
9.
J Orthop Sci ; 23(4): 676-681, 2018 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-29724468

RESUMEN

BACKGROUND: Meniscus surgery is the most commonly performed orthopedic surgery, and despite recent emphasis on saving the meniscus, the current status of meniscus surgeries is little known in many countries, including Japan. The National Database of Health Insurance Claims and Specific Health Checkups of Japan and the Statistics of Medical Care Activities in Public Health Insurance track meniscus surgeries through health insurance claims. The National Database provides the numbers for 2014 and 2015, and the Statistics of Medical Care Activities provides the numbers from June 2011 to June 2016. Our aim was to analyze isolated meniscus surgery numbers and meniscus repair ratios by age group based on the National Database and evaluate trends of meniscus repair ratios for the latest six years from the Statistics of Medical Care Activities. METHODS: Meniscus surgeries by age group were counted from the National Database for 2014-2015, and meniscus repair ratios (meniscus repairs/meniscus surgeries) were calculated. The numbers were also counted from the Statistics of Medical Care Activities in 2011-2016. For statistical analysis of annual trends of meniscus repair ratios, the Cochran-Armitage trend test was used. Meniscus surgeries with concomitant knee ligament surgeries were excluded. RESULTS: According to the National Database, isolated meniscus surgeries totaled 34,966 in 2015, with peak ages of patients in their late teens and 60s. The meniscus repair ratio was 19% in 2014 and 24% in 2015. According to the Statistics of Medical Care Activities, the meniscus repair ratio was 9% in 2011 and significantly increased to 25% in 2016 (p = 0.0008). The ratio also increased significantly in each age group between the early 20s and late 70s. CONCLUSIONS: Approximately 35,000 meniscus surgeries are performed in Japan annually, with peak ages in the late teens and 60s. The number of meniscus repairs has increased over the past six years.


Asunto(s)
Meniscectomía/tendencias , Meniscos Tibiales/cirugía , Lesiones de Menisco Tibial/cirugía , Adolescente , Adulto , Distribución por Edad , Anciano , Anciano de 80 o más Años , Estudios de Cohortes , Bases de Datos Factuales , Femenino , Humanos , Incidencia , Japón/epidemiología , Masculino , Meniscectomía/métodos , Meniscos Tibiales/fisiopatología , Persona de Mediana Edad , Complicaciones Posoperatorias/epidemiología , Complicaciones Posoperatorias/fisiopatología , Pronóstico , Estudios Retrospectivos , Medición de Riesgo , Distribución por Sexo , Lesiones de Menisco Tibial/diagnóstico por imagen , Lesiones de Menisco Tibial/epidemiología , Resultado del Tratamiento , Adulto Joven
10.
Stem Cell Res Ther ; 9(1): 80, 2018 03 27.
Artículo en Inglés | MEDLINE | ID: mdl-29587847

RESUMEN

BACKGROUND: Latent microorganism infection is a safety concern for the clinical application of mesenchymal stem cells (MSCs). The aim of this study is to investigate the frequencies and sensitivities of the latent virus and mycoplasma infections in synovium, bone marrow, peripheral blood cells, and blood plasma and cultured synovial MSCs. METHODS: Total DNA and RNA of the synovium (n = 124), bone marrow (n = 123), peripheral blood cells (n = 121), plasma (n = 121), and 14-day cultured synovial MSCs (n = 63) were collected from patients who underwent total knee arthroplasty or anterior ligament reconstruction after written informed consents were obtained. The multiplex polymerase chain reaction (PCR) primers were designed to quantitatively measure the representative genomes of 13 DNA viruses, 6 RNA viruses, and 9 mycoplasmas. Multi-spliced mRNA detection and virus spike test were also performed to demonstrate the sensitivity of synovial MSCs to the candidate pathogens. RESULTS: In synovium and bone marrow, the positive rates of parvovirus B19 genome were significantly higher than in peripheral blood cells (18.7% and 22% vs. 0.8%, respectively). Multi-alignment analysis of amplified and sequenced viral target genes showed the proximity of the parvovirus B19 gene from different tissue in the same patients. Synovial MSCs cultured for 14 days were positive for virus infection only in two patients (2/62 = 3%). Parvovirus B19 multi-spliced mRNAs were not detected in these two samples. Virus spike test demonstrated the sensitivity of synovial MSCs to herpes simplex virus (HSV)1 and cytomegalovirus (CMV), but not to parvovirus B19. CONCLUSION: This study revealed a relatively high incidence of latent parvovirus B19 in synovium and bone marrow tissue.


Asunto(s)
Médula Ósea/virología , Mycoplasma/patogenicidad , Infecciones por Parvoviridae/epidemiología , Parvovirus B19 Humano/patogenicidad , Membrana Sinovial/virología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Médula Ósea/microbiología , Células Cultivadas , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mycoplasma/genética , Infecciones por Mycoplasma/diagnóstico , Infecciones por Mycoplasma/epidemiología , Infecciones por Parvoviridae/diagnóstico , Parvovirus B19 Humano/genética , Membrana Sinovial/microbiología
11.
BMC Musculoskelet Disord ; 19(1): 78, 2018 03 09.
Artículo en Inglés | MEDLINE | ID: mdl-29523119

RESUMEN

BACKGROUND: Mobilization of mesenchymal stem cells (MSCs) from the synovium was revealed using a "suspended synovium culture model" of osteoarthritis (OA). The pathology of rheumatoid arthritis (RA) differs from that of OA. We investigated whether mobilization of MSCs from the synovium also occurred in RA, and we compared the properties of synovial MSCs collected from suspended synovium culture models of RA and OA. METHODS: Human synovium was harvested during total knee arthroplasty from the knee joints of patients with RA (n = 8) and OA (n = 6). The synovium was suspended in a bottle containing culture medium and a culture dish at the bottom. Cells were harvested from the dish and analyzed. RESULTS: No significant difference was observed between RA and OA in the harvested cell numbers per g of synovium. However, the variation in the number of cells harvested from each donor was greater for RA than for OA. The harvested cells were multipotent and no difference was observed in the cartilage pellet weight between RA and OA. The surface epitopes of the cells in RA and OA were similar to those of MSCs. CONCLUSION: Mobilization of MSCs from the synovium was demonstrated using a suspended synovium culture model for RA. The harvested cell numbers, chondrogenic potentials, and surface epitope profiles were comparable between the RA and OA models.


Asunto(s)
Artritis Reumatoide/patología , Técnicas de Cultivo de Célula/métodos , Células Madre Mesenquimatosas/fisiología , Osteoartritis/patología , Membrana Sinovial/citología , Membrana Sinovial/fisiología , Adulto , Anciano , Células Cultivadas , Femenino , Humanos , Masculino , Persona de Mediana Edad
12.
Stem Cell Res Ther ; 8(1): 144, 2017 06 13.
Artículo en Inglés | MEDLINE | ID: mdl-28610596

RESUMEN

BACKGROUND: In our clinical practice, we perform transplantations of autologous synovial mesenchymal stem cells (MSCs) for cartilage and meniscus regenerative medicine. One of the most important issues to ensuring clinical efficacy involves the transport of synovial MSCs from the processing facility to the clinic. Complete human serum (100% human serum) is an attractive candidate material in which to suspend synovial MSCs for their preservation during transport. The purpose of this study was to investigate whether complete human serum maintained MSC viability and chondrogenic potential and to examine the optimal temperature conditions for the preservation of human synovial MSCs. METHODS: Human synovium was harvested from the knees of 14 donors with osteoarthritis during total knee arthroplasty. Passage 2 synovial MSCs were suspended at 2 million cells/100 µL in Ringer's solution or complete human serum at 4, 13, and 37 °C for 48 h. These cells were analyzed for live cell rates, cell surface marker expression, metabolic activity, proliferation, and adipogenic, calcification, and chondrogenic differentiation potentials before and after preservation. RESULTS: After preservation, synovial MSCs maintained higher live cell rates in human serum than in Ringer's solution at 4 and 13 °C. Synovial MSCs preserved in human serum at 4 and 13 °C also maintained high ratios of propidium iodide- and annexin V- cells. MSC surface marker expression was not altered in cells preserved at 4 and 13 °C. The metabolic activities of cells preserved in human serum at 4 and 13 °C was maintained, while significantly reduced in other conditions. Replated MSCs retained their proliferation ability when preserved in human serum at 4 and 13 °C. Adipogenesis and calcification potential could be observed in cells preserved in each condition, whereas chondrogenic potential was retained only in cells preserved in human serum at 4 and 13 °C. CONCLUSION: The viability and chondrogenic potential of synovial MSCs were maintained when the cells were suspended in human serum at 4 and 13 °C.


Asunto(s)
Diferenciación Celular/genética , Trasplante de Células Madre Mesenquimatosas , Osteoartritis/genética , Membrana Sinovial/trasplante , Cartílago Articular/citología , Cartílago Articular/crecimiento & desarrollo , Condrocitos/citología , Condrocitos/metabolismo , Condrogénesis/genética , Humanos , Células Madre Mesenquimatosas/citología , Osteoartritis/patología , Osteoartritis/terapia , Medicina Regenerativa , Membrana Sinovial/citología
13.
Stem Cell Res Ther ; 8(1): 115, 2017 05 16.
Artículo en Inglés | MEDLINE | ID: mdl-28511664

RESUMEN

BACKGROUND: Mesenchymal stem cells derived from the synovial membrane (synovial MSCs) are a candidate cell source for regenerative medicine of cartilage and menisci due to their high chondrogenic ability. Regenerative medicine can be expected for RA patients with the inflammation well-controlled as well as OA patients and transplantation of synovial MSCs would also be a possible therapeutic treatment. Some properties of synovial MSCs vary dependent on the diseases patients have, and whether or not the pathological condition of RA affects the chondrogenesis of synovial MSCs remains controversial. The purpose of this study was to compare the properties of primary synovial MSCs between RA and OA patients. METHODS: Human synovial tissue was harvested during total knee arthroplasty from the knee joints of eight patients with RA and OA respectively. Synovial nucleated cells were cultured for 14 days. Total cell yields, surface markers, and differentiation potentials were analyzed for primary synovial MSCs. RESULTS: Nucleated cell number per 1 mg synovium was 8.4 ± 3.9 thousand in RA and 8.0 ± 0.9 thousand in OA. Total cell number after 14-day culture/1 mg synovium was 0.7 ± 0.4 million in RA and 0.5 ± 0.3 million in OA, showing no significant difference between in RA and OA. Cells after 14-day culture were mostly positive for CD44, CD73, CD90, CD105, negative for CD45 both in RA and OA. There was no significant difference for the cartilage pellet weight and sGAG content per pellet between in RA and OA. Both oil red O-positive colony rate and alizarin red-positive colony rate were similar in RA and OA. CONCLUSIONS: Yields, surface markers and chondrogenic potential of primary synovial MSCs in RA were comparable to those in OA. Synovium derived from RA patients can be the cell source of MSCs for cartilage and meniscus regeneration.


Asunto(s)
Artritis Reumatoide/patología , Condrogénesis , Células Madre Mesenquimatosas/patología , Osteoartritis/patología , Membrana Sinovial/patología , Adipogénesis , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/metabolismo , Calcificación Fisiológica , Recuento de Células , Núcleo Celular/metabolismo , Forma de la Célula , Ensayo de Unidades Formadoras de Colonias , Demografía , Femenino , Humanos , Masculino , Persona de Mediana Edad , Tamaño de los Órganos
14.
Arthroscopy ; 33(4): 800-810, 2017 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-28043752

RESUMEN

PURPOSE: To develop an in vitro model, the "suspended synovium culture model," to demonstrate the mobilization of mesenchymal stem cells (MSCs) from the synovium into a noncontacted culture dish through culture medium. In addition, to examine which synovium, fibrous synovium or adipose synovium, released more MSCs in the knee with osteoarthritis. METHODS: Human synovial tissue was harvested during total knee arthroplasty from knee joints of 34 patients with osteoarthritis (28 patients: only fibrous synovium, 6 patients: fibrous and adipose synovium). One gram of synovium was suspended with a thread in a bottle containing 40 mL of culture medium and a 3.5-cm-diameter culture dish at the bottom. After 7 days, the culture dish in the bottle was examined. For the cells harvested, multipotentiality and surface epitopes were analyzed. The numbers of colonies derived from fibrous synovium and adipose synovium were also compared. RESULTS: Colonies of spindle-shaped cells were observed in the culture dish in all 28 donors. Colonies numbered 26 on average, and the cells derived from colony-forming cells had multipotentiality for chondrogenesis, adipogenesis, calcification, and surface epitopes similar to MSCs. The number was colonies was significantly higher in fibrous synovium than in adipose synovium (P < .05, n = 6). CONCLUSIONS: We developed a suspended synovium culture model. Suspended synovium was able to release MSCs into a noncontacted culture dish through medium in a bottle. Fibrous synovium was found to release greater numbers of MSCs than adipose synovium in our culture model. CLINICAL RELEVANCE: This model could be a valuable tool to screen drugs capable of releasing MSCs from the synovium into synovial fluid.


Asunto(s)
Tejido Adiposo/patología , Células Madre Mesenquimatosas/patología , Osteoartritis de la Rodilla/patología , Membrana Sinovial/patología , Adipogénesis/fisiología , Anciano , Anciano de 80 o más Años , Diferenciación Celular , Células Cultivadas , Condrogénesis/fisiología , Ensayo de Unidades Formadoras de Colonias , Medios de Cultivo , Femenino , Fibrosis , Humanos , Articulación de la Rodilla/patología , Masculino , Células Madre Mesenquimatosas/fisiología , Persona de Mediana Edad , Osteoartritis de la Rodilla/metabolismo , Osteoartritis de la Rodilla/cirugía , Técnicas de Cultivo de Tejidos
15.
Stem Cell Res Ther ; 6: 243, 2015 Dec 10.
Artículo en Inglés | MEDLINE | ID: mdl-26652649

RESUMEN

INTRODUCTION: For expansion of human mesenchymal stem cells (MSCs), autologous human serum is safer than fetal bovine serum in clinical situations. One of the problems with the use of autologous human serum is that its proliferative effect on MSCs varies widely between donors. The threefold goals of this study were: (1) to demonstrate an improved method for preparing human serum; (2) to identify growth factors predictive of proliferative potential; and (3) to identify a cytokine to promote MSC proliferation in human serum. METHODS: Fresh blood was collected using a closed bag system containing glass beads. The bag was shaken at 20 °C for 30 minutes for rapid preparation, or kept stationary at 4 °C for 24 hours for slow preparation. Passage 0 synovial MSCs derived from four donors were cultured with 10 % conventional rapid preparation serum or modified slow preparation serum from four different donors. To perform the colony-forming unit assay, synovial MSCs were cultured in these serums. The protein expression profile in serum was analyzed using cytokine array. The candidate proteins were speculated from the correlation between the colony-forming ability and protein expression. As an evaluation of the candidate proteins, proliferation ability, surface marker phenotype and differentiation capability of synovial MSCs were examined. RESULTS: Compared with rapid preparation serum, slow preparation serum resulted in a significantly higher total colony number and twofold higher expression levels of nine proteins (angiopoietin-1, BDNF, EGF, ENA-78, IGFBP-2, platelet-derived growth factor (PDGF)-AA, PDGF-AB/BB, RANTES and TfR). Colony number was positively correlated with PDGF-AA/AB concentrations. Exogenous PDGF-AA significantly promoted proliferation of synovial MSCs, whereas PDGF receptor (PDGFR) inhibitor decreased it. Addition of PDGFs or PDGFR inhibitor did not affect surface epitopes of synovial MSCs. Pretreatment with PDGFs or PDGFR inhibitor did not affect chondrogenic, adipogenic, or calcification potentials of synovial MSCs. CONCLUSION: Slow preparation serum contained higher concentrations of PDGF-AA/AB and increased the colony formation number of synovial MSCs. PDGF-AA/AB were indicators of the proliferative potential of human serum. Exogenous PDGF-AA increased proliferation of synovial MSCs without alteration of surface epitopes and differentiation potentials.


Asunto(s)
Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Membrana Sinovial/citología , Membrana Sinovial/metabolismo , Adulto , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , Medios de Cultivo/metabolismo , Citocinas/sangre , Humanos , Técnicas In Vitro , Masculino
17.
Ann N Y Acad Sci ; 1095: 292-9, 2007 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-17404041

RESUMEN

Rat osteoblasts were cultured for 4 and 5 days aboard a space shuttle and solubilized after a 24-h treatment with 1alpha,25 dihydroxyvitamin D(3). The quantitative RT-PCR determined the mRNA levels of signaling molecules upstream and downstream Ras. The small GTPase is activated by guanine nucleotide exchange protein (GEF) and deactivated by GTPase-activating protein (GAP). When external stimuli are transduced into intracellular signals, various pathways are recruited: focal adhesion kinase (FAK) is associated with integrin-beta, and directs tyrosine phosphorylation of downstream substrates, including phospholipase C-gamma (PLC-gamma) and son of sevenless (SOS, a Ras GEF). The mRNA levels of FAK and PLC-gamma1 and -gamma2 in the flight cultures were increased 150% and 250% of the ground controls. The SOS mRNA levels in the flight cultures were increased 520% and 320% of the ground controls. Signals via G protein-coupled receptors are transmitted through PLC-beta and Ras GRF (another Ras GEF). Activated Ras then stimulates Raf, mitogen-activated protein kinase (MAPK) cascades. The mRNA levels of Raf, extracellular signal-regulated protein kinase of MAPK family (ERK-1 and -2), and PLC-beta were increased during spaceflight. Rho GAP expression in the flight cultures was increased twofold of the ground controls. Since Rho GAP deactivates Rho, microgravity may suppress Rho signals, regulating actin filament rearrangement. Microgravity signals may involve two pathways (G protein-coupled receptor-mediated pathway and tyrosine phosphorylation-mediated pathway) that activate Ras, Raf, and MAPK cascades in rat osteoblasts.


Asunto(s)
Osteoblastos/enzimología , Vuelo Espacial , Proteínas ras/biosíntesis , Proteínas ras/genética , Proteínas de Unión al GTP rho/biosíntesis , Proteínas de Unión al GTP rho/genética , Animales , Células Cultivadas , Ratas , Transducción de Señal/fisiología , Ingravidez
18.
Ann N Y Acad Sci ; 1090: 311-7, 2006 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-17384275

RESUMEN

Rat osteoblasts were cultured for 4 or 5 days aboard the Space Shuttle and solubilized during spaceflight. Post-flight analyses by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) determined the relative mRNA levels of matrix proteins, adhesion molecules, and cytoskeletal proteins including osteopontin (OP), osteonectin (ON), CD44, alpha-tubulin, actin, vimentin, fibronectin (FN), and beta1-integrin. The mRNA levels of OP and alpha-tubulin in the flight cultures were decreased by 30% and 50% on day 4 and day 5 of flight, as compared to the ground controls. In contrast, the CD44 mRNA levels in the flight cultures increased by 280% and 570% of the ground controls on day 4 and day 5. The mRNA levels of ON and FN in the flight cultures were slightly increased as compared to ground controls. The mRNA levels of actin, vimentin, or beta1-integrin did not change in spaceflight conditions. The matrix proteins, adhesion molecules, and cytoskeletal proteins may form dynamic network complexity with signaling molecules as an adaptive response to perturbation of mechanical stress under microgravity.


Asunto(s)
Adhesión Celular , Proteínas del Citoesqueleto/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Osteoblastos/metabolismo , Ingravidez , Animales , Secuencia de Bases , Proteínas del Citoesqueleto/genética , Cartilla de ADN , Proteínas de la Matriz Extracelular/genética , Fibronectinas/genética , Fibronectinas/metabolismo , Receptores de Hialuranos/genética , Receptores de Hialuranos/metabolismo , Integrina beta1/genética , Integrina beta1/metabolismo , Osteoblastos/citología , Osteonectina/genética , Osteonectina/metabolismo , Osteopontina/genética , Osteopontina/metabolismo , ARN Mensajero/genética , Ratas , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Vimentina/genética , Vimentina/metabolismo
19.
J Gene Med ; 7(4): 432-41, 2005 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-15515118

RESUMEN

BACKGROUND: Gene transfer to salivary glands (SGs) can be accomplished in a minimally invasive manner, resulting in stable, long-term secretion of the transgene product. Therefore, SGs provide a novel target site for several potentially useful clinical gene therapeutics applications. Previous studies have indicated that intravenous, intramuscular and intranasal administration of recombinant adeno-associated virus serotype 2 (rAAV2) vectors induce host immune responses. There are no reported studies on immune responsiveness of rAAV2 vector administration to SGs. MATERIAL AND METHODS: Vectors were administered by retrograde infusion to the SGs of Balb/c mice in various combinations. Thereafter, transgene expression was determined, and evaluations of host innate and adaptive immune responsiveness performed over a 56-day period. RESULTS: Histological examination of SGs from vector-treated mice showed no significant changes in appearance from controls, including the frequency of activated macrophage detection. There were also no differences in salivary flow rates among experimental groups. In vitro stimulation of splenocytes from mice administered rAAV2 showed elevated interferon-gamma levels in culture media. Significant titers of neutralizing antibodies to rAAV2 were detected in serum of mice following rAAV2 vector administration. While SGs could be transduced with low doses of vector it was not possible to repeat the administration and detect transduction with the same serotype at low doses. However, repeat administration was possible with an alternative serotype (rAAV4). CONCLUSIONS: Following a single administration of rAAV2 vectors to SGs there is no significant innate immune response. However, rAAV2 vector administration to SGs results in both cellular and humoral immune responses. The latter may interfere with the efficacy of repeated rAAV2 vector administration.


Asunto(s)
Dependovirus/genética , Vectores Genéticos , Glándulas Salivales/inmunología , Animales , Línea Celular , Vectores Genéticos/administración & dosificación , Humanos , Inmunidad Innata , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos BALB C , Reacción en Cadena de la Polimerasa , Recombinación Genética
20.
J Virol ; 78(12): 6509-16, 2004 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-15163744

RESUMEN

To better understand the relationship between primate adeno-associated viruses (AAVs) and those of other mammals, we have cloned and sequenced the genome of an AAV found as a contaminant in two isolates of bovine adenovirus that was reported to be serologically distinct from primate AAVs. The bovine AAV (BAAV) genome has 4,693 bp, and its organization is similar to that of other AAV isolates. The left-hand open reading frame (ORF) and both inverted terminal repeats (ITRs) have the highest homology with the rep ORF and ITRs of AAV serotype 5 (AAV-5) (89 and 96%, respectively). However, the right-hand ORF was only 55% identical to the AAV-5 capsid ORF; it had the highest homology with the capsid ORF of AAV-4 (76%). By comparing the BAAV cap sequence with a model of an AAV-4 capsid, we mapped the regions of BAAV VP1 that are divergent from AAV-4. These regions are located on the outside of the capsid and are partially located in exposed loops. BAAV was not neutralized by antisera raised against recombinant AAV-2, AAV-4, or AAV-5, and it demonstrated a unique cell tropism profile in four human cancer cell lines, suggesting that BAAV might have transduction activity distinct from that of other isolates. A murine model of salivary gland gene transfer was used to evaluate the in vivo performance of recombinant BAAV. Recombinant BAAV-mediated gene transfer was 11 times more efficient than that with AAV-2. Overall, these data suggest that vectors based on BAAV could be useful for gene transfer applications.


Asunto(s)
Bovinos/virología , Clonación Molecular , Dependovirus/química , Dependovirus/clasificación , Análisis de Secuencia de ADN , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Dependovirus/genética , Dependovirus/metabolismo , Vectores Genéticos , Genoma Viral , Humanos , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Proteínas de Plantas , Recombinación Genética , Transactivadores , Factores de Transcripción/química , Factores de Transcripción/genética , Transducción Genética , Proteínas Virales/química , Proteínas Virales/genética
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