Publications (9)12.47 Total impact
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Article: Cryopreservation effect on proliferative and chondrogenic potential of human chondrocytes isolated from superficial and deep cartilage.
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ABSTRACT: To compare the proliferative and chondrogenic potential of fresh and frozen chondrocytes isolated from superficial and deep articular cartilage biopsies. The study included 12 samples of fresh and frozen healthy human knee articular cartilage. Cell proliferation was tested at 3, 6 and 9 days. Studies of mRNA quantification, protein expression and immunofluorescence for proliferation and chondrogenic markers were performed. Stimulation of fresh and frozen chondrocytes from both superficial and deep cartilage with fetal bovine serum produced an increase in the proliferative capacity compared to the non-stimulated control group. In the stimulated fresh cells group, the proliferative capacity of cells from the deep biopsy was greater than that from cells from the superficial biopsy (0.046 vs 0.028, respectively, p<0.05). There was also a significant difference between the proliferative capacity of superficial zone fresh (0.028) and frozen (0.051) chondrocytes (p<0.05). CCND1 mRNA and protein expression levels, and immunopositivity for Ki67 revealed a higher proliferative capacity for fresh articular chondrocytes from deep cartilage. Regarding the chondrogenic potential, stimulated fresh cells showed higher SOX9 and Col II expression in chondrocytes from deep than from superficial zone (p<0.05, T student test). The highest rate of cell proliferation and chondrogenic potential of fresh chondrocytes was found in cells obtained from deep cartilage biopsies, whereas there were no statistically significant differences in proliferative and chondrogenic capacity between biopsy origins with frozen chondrocytes. These results indicate that both origin and cryopreservation affect the proliferative and chondrogenic potential of chondrocytes.The Open Orthopaedics Journal 01/2012; 6:150-9. -
Article: Human amniotic membrane as an alternative source of stem cells for regenerative medicine.
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ABSTRACT: The human amniotic membrane (HAM) is a highly abundant and readily available tissue. This amniotic tissue has considerable advantageous characteristics to be considered as an attractive material in the field of regenerative medicine. It has low immunogenicity, anti-inflammatory properties and their cells can be isolated without the sacrifice of human embryos. Since it is discarded post-partum it may be useful for regenerative medicine and cell therapy. Amniotic membranes have already been used extensively as biologic dressings in ophthalmic, abdominal and plastic surgery. HAM contains two cell types, from different embryological origins, which display some characteristic properties of stem cells. Human amnion epithelial cells (hAECs) are derived from the embryonic ectoderm, while human amnion mesenchymal stromal cells (hAMSCs) are derived from the embryonic mesoderm. Both populations have similar immunophenotype and multipotential for in vitro differentiation into the major mesodermal lineages, however they differ in cell yield. Therefore, HAM has been proposed as a good candidate to be used in cell therapy or regenerative medicine to treat damaged or diseased tissues.Differentiation 02/2011; 81(3):162-71. · 2.81 Impact Factor -
Article: Potential use of the human amniotic membrane as a scaffold in human articular cartilage repair.
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ABSTRACT: The human amniotic membrane (HAM) is an abundant and readily obtained tissue that may be an important source of scaffold for transplanted chondrocytes in cartilage regeneration in vivo. To evaluate the potential use of cryopreserved HAMs as a support system for human chondrocytes in human articular cartilage repair. Chondrocytes were isolated from human articular cartilage, cultured and grown on the chorionic basement membrane side of HAMs. HAMs with chondrocytes were then used in 44 in vitro human osteoarthritis cartilage repair trials. Repair was evaluated at 4, 8 and 16 weeks by histological analysis. Chondrocytes cultured on the HAM revealed that cells grew on the chorionic basement membrane layer, but not on the epithelial side. Chondrocytes grown on the chorionic side of the HAM express type II collagen but not type I, indicating that after being in culture for 3-4 weeks they had not de-differentiated into fibroblasts. In vitro repair experiments showed formation on OA cartilage of new tissue expressing type II collagen. Integration of the new tissue with OA cartilage was excellent. The results indicate that cryopreserved HAMs can be used to support chondrocyte proliferation for transplantation therapy to repair OA cartilage.Cell and Tissue Banking 04/2010; 11(2):183-95. · 0.96 Impact Factor -
Article: Apoptosis in fresh and cryopreserved cardiac valves of pig samples.
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ABSTRACT: To analyse the influence of cold ischemic time (CIT) (2-24 h) and of cryopreservation (liquid phase) on the viability of the valvular fibroblasts and in the presence of apoptosis. Cardiac valves from 10 pigs were evaluated by anatomo-pathological study of the wall, muscle and leaflet. At the same time, the presence of cellular death due to apoptosis was investigated in two ways; directly on tissue by Apodetec system and by two-colour flow cytometry assay analyzing a suspension of fibroblast from valve leaflets using Anexina V and propidium iodure (PI). We established three groups of samples to compare different experimental conditions: 2 h of ischemia (group 1), 24 h of ischemia (group 2), and a programme of cryopreservation (-1 degrees C/min) after 2 h of ischemia, followed by storage in liquid nitrogen during a week and thawing was performed (group 3). The analysis of viabilities showed slight differences between all three groups. The results indicated CIT of 24 h undergoing more structural affectation than CIT of 2 h. Flow cytometry analysis did not show important differences between groups; however cryopreserved samples (group 3) slightly less viability and a higher percentage of death by apoptosis than group 1 and 2 using flow cytometry. Apoptosis was confirmed on tissue from all valves but mainly in samples of group 2 and group 3. In summary, the viability of the valves in the case of ischemic times of 2 h, 24 h or after cryopreservation/thawing differs slightly. The death of the cells is mainly mediated by necrosis and not by apoptosis.Cell and Tissue Banking 07/2008; 9(2):101-7. · 0.96 Impact Factor -
Article: Cellular cardiomyoplasty: development of a technique to culture human myoblasts for clinical transplantation
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ABSTRACT: Some recent studies have demonstrated that epicardial injection of autologous myoblasts, obtained from satellite cells of skeletal muscle, in association to coronary artery bypass graft surgery (CABG) in patients with decreased left ventricular function secondary to ischaemic disease could be of some utility to get a better recovery of ventricular function due to the ability of these cells to grow and generate new muscle fibers over the previous fibrotic scar. The aims are the setting up of a process for the collection of the cellular cardiomyoplasty in samples of multiorganic donations and to carry out this technique in the same surgical moment as the revascularisation is performed in two patients. For this purpose we obtained muscle through biopsy of 15 human multiorgan donors and of two patients. Separation of fatty tissue, minced, and further digestion with collagenase type I (1.5 mgr/ml/2 gr by weight) and trypsin 1 . Filtration of the cellular suspension, centrifugation and sowing of this suspension in culture medium, with 20% of human serum. Culture for three weeks until obtainment of between 200–300 million cells. Inmunohistochemistry and flow cytometry for the identification of the myoblasts was carried out. The results were obtained through flow cytometry, using CD56 as an indicator of the presence of myoblasts, between 70 and 80% of these types of cells were obtained after three weeks of culture. By inmunohistochemistry analyses, different markers were analyzed: desmin and myogenin. The results indicated the presence of a great number of positive cells with these markers, possibly myoblasts. Skeletal myoblast implant was not associated with adverse effects. The culture of autologous myoblasts is a rapid and simple technique where after three weeks of culture a great number of cells for implantation are obtained. In patients with old myocardial infarction, treatment with skeletal myoblast in conjunction with coronary artery bypass is safe and feasible. and it is easy to obtain myoblasts from muscle tissue for transplant into patients.Cell and Tissue Banking 05/2005; 6(2):117-124. · 0.96 Impact Factor -
Article: Cellular cardiomyoplasty: development of a technique to culture human myoblasts for clinical transplantation.
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ABSTRACT: Some recent studies have demonstrated that epicardial injection of autologous myoblasts, obtained from satellite cells of skeletal muscle, in association to coronary artery bypass graft surgery (CABG) in patients with decreased left ventricular function secondary to ischaemic disease could be of some utility to get a better recovery of ventricular function due to the ability of these cells to grow and generate new muscle fibers over the previous fibrotic scar. The aims are the setting up of a process for the collection of the cellular cardiomyoplasty in samples of multiorganic donations and to carry out this technique in the same surgical moment as the revascularisation is performed in two patients. For this purpose we obtained muscle through biopsy of 15 human multiorgan donors and of two patients. Separation of fatty tissue, minced, and further digestion with collagenase type I (1.5 mgr/ml/2 gr by weight) and trypsin 1 x. Filtration of the cellular suspension, centrifugation and sowing of this suspension in culture medium, with 20% of human serum. Culture for three weeks until obtainment of between 200-300 million cells. Inmunohistochemistry and flow cytometry for the identification of the myoblasts was carried out. The results were obtained through flow cytometry, using CD56 as an indicator of the presence of myoblasts, between 70 and 80% of these types of cells were obtained after three weeks of culture. By inmunohistochemistry analyses, different markers were analyzed: desmin and myogenin. The results indicated the presence of a great number of positive cells with these markers, possibly myoblasts. Skeletal myoblast implant was not associated with adverse effects. The culture of autologous myoblasts is a rapid and simple technique where after three weeks of culture a great number of cells for implantation are obtained. In patients with old myocardial infarction, treatment with skeletal myoblast in conjunction with coronary artery bypass is safe and feasible. and it is easy to obtain myoblasts from muscle tissue for transplant into patients.Cell and Tissue Banking 02/2005; 6(2):117-24. · 0.96 Impact Factor -
Article: Functional assessment of human femoral arteries after cryopreservation.
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ABSTRACT: An established method for the cryopreservation of human femoral arteries for subsequent transplantation as allografts has been studied with particular attention to preservation of smooth muscle and endothelium. Human femoral arteries (HFAs) were harvested from multi-organ donors. Two groups were established; a control group of unfrozen HFAs and a group of cryopreserved HFAs. Cryopreservation was performed using RPMI solution containing dimethyl sulfoxide and the rate of cooling was 1 degrees C/min to -40 degrees C and faster thereafter until -150 degrees C was reached. The contraction and relaxation responses of unfrozen and frozen/thawed arteries were assessed by measurement of the isometric force generated by the HFAs in an organ bath. After thawing (warming was at 15 degrees C/min) the maximal contractile response to noradrenaline was 43% of the response of unfrozen HFAs. The endothelium-independent response to sodium nitroprusside was not altered, whereas the endothelium-dependent relaxation response to acetylcholine was slightly altered. The cryopreservation method used provided limited preservation of the contractility of human femoral arteries, and good preservation of both endothelium-independent and endothelium-dependent relaxation responses.Cryobiology 09/2004; 49(1):83-9. · 2.06 Impact Factor -
Article: Viability and histologic structure of porcine valves after cryopreservation.
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ABSTRACT: Increased awareness of the limitations of current cardiac valve substitutes has generated a renewed interest in the use of allograft valves. The effects of currently used preservation techniques on the viability of the valve leaflets and the longevity of the implantation however remain controversial. The objective of this study is to analyze the influence of ischemic time, sterilization methods with or without fungicides, and storage procedures on the viability of the valve leaflets and on the histologic structure of the arterial wall, valve leaflet, and myocardium. The tissue sources were hearts from 40 pigs with 1 hour of warm ischemic time. The aortic and pulmonary valves were dissected after 2 or 24 hours of cold ischemic time. They were stored in antibiotic solution for 20 hours at 4 degrees C with or without an antifungal agent. The samples were cryopreserved using a programmed temperature decrease method. After 1 week of storage in a liquid nitrogen tank, either in a gas or a liquid phase, the cardiac valves were slowly thawed and examined. Pulmonary valves showed greater viability than aortic valves. Decreased cellular viability was observed independent of cold ischemic time, treatment with amphotericin B, or the storage method used. Treatment with or without amphotericin B had no influence on cellular viability. Conversely it was observed that there was greater cellular viability among those valves stored in a liquid phase. As far as the histologic structure of the valve is concerned we did not observe any influence either in the treatment with amphotericin B or the storage method used although it was observed that reduction of the cold ischemic time minimized histologic injury. Optimization of preservation methods may decrease the negative effects of cryopreservation on cell viability and histologic structure of the valve.The Annals of Thoracic Surgery 02/2004; 77(1):186-90. · 3.74 Impact Factor -
Article: Functional assessment of human femoral arteries after cryopreservation
[show abstract] [hide abstract]
ABSTRACT: An established method for the cryopreservation of human femoral arteries for subsequent transplantation as allografts has been studied with particular attention to preservation of smooth muscle and endothelium. Human femoral arteries (HFAs) were harvested from multi-organ donors. Two groups were established; a control group of unfrozen HFAs and a group of cryopreserved HFAs. Cryopreservation was performed using RPMI solution containing dimethyl sulfoxide and the rate of cooling was 1 °C/min to −40 °C and faster thereafter until −150 °C was reached. The contraction and relaxation responses of unfrozen and frozen/thawed arteries were assessed by measurement of the isometric force generated by the HFAs in an organ bath. After thawing (warming was at 15 °C/min) the maximal contractile response to noradrenaline was 43% of the response of unfrozen HFAs. The endothelium-independent response to sodium nitroprusside was not altered, whereas the endothelium-dependent relaxation response to acetylcholine was slightly altered. The cryopreservation method used provided limited preservation of the contractility of human femoral arteries, and good preservation of both endothelium-independent and endothelium-dependent relaxation responses.Cryobiology.
Top co-authors
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Institutions
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2010–2011
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University of A Coruña
- Department of Medicine
A Coruña, Galicia, Spain
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2004
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Centro de Supercomputación de Galicia
Santiago de Compostela, Galicia, Spain
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