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OBJECTIVES: To determine death occurrences of Puerto Ricans on the mainland USA following the arrival of Hurricane Maria in Puerto Rico in September 2017. DESIGN: Cross-sectional study. PARTICIPANTS: Persons of Puerto Rican origin on the mainland USA. EXPOSURES: Hurricane Maria. MAIN OUTCOME: We use an interrupted time series design to analyse all-cause mortality of Puerto Ricans in the USA following the hurricane. Hispanic origin data from the National Vital Statistics System and from the Public Use Microdata Sample of the American Community Survey are used to estimate monthly origin-specific mortality rates for the period 2012-2018. We estimated log-linear regressions of monthly deaths of persons of Puerto Rican origin by age group, gender, and educational attainment. RESULTS: We found an increase in mortality for persons of Puerto Rican origin during the 6-month period following the hurricane (October 2017 through March 2018), suggesting that deaths among these persons were 3.7% (95% CI 0.025 to 0.049) higher than would have otherwise been expected. In absolute terms, we estimated 514 excess deaths (95% CI 346 to 681) of persons of Puerto Rican origin that occurred on the mainland USA, concentrated in those aged 65 years or older. CONCLUSIONS: Our findings suggest an undercounting of previous deaths as a result of the hurricane due to the systematic effects on the displaced and resident populations in the mainland USA. Displaced populations are frequently overlooked in disaster relief and subsequent research. Ignoring these populations provides an incomplete understanding of the damages and loss of life.
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Tempestades Ciclônicas , Estudos Transversais , Hispânico ou Latino , Humanos , Análise de Séries Temporais Interrompida , Porto Rico/epidemiologia , Estados Unidos/epidemiologiaRESUMO
Hard tissue regeneration represents a challenge for the Regenerative Medicine and Mesenchymal stem cells (MSCs) could be a successful therapeutic strategy. T-LysYal® (T-Lys), a new derivative of Hyaluronic Acid (HA) possessing a superior stability, has already been proved efficient in repairing corneal epithelial cells damaged by dry conditions in vitro. We investigated the regenerative potential of T-Lys in the hard tissues bone and cartilage. We have previously demonstrated that cells isolated from the tooth germ, Dental Bud Stem Cells (DBSCs), differentiate into osteoblast-like cells, representing a promising source of MSCs for bone regeneration. Herewith, we show that T-Lys treatment stimulates the expression of typical osteoblastic markers, such as Runx-2, Collagen I (Col1) and Alkaline Phosphatase (ALP), determining a higher production of mineralized matrix nodules. In addition, we found that T-Lys treatment positively affects αVß3 integrin expression, key integrin in the osteoblastic commitment, leading to the formation of focal adhesions (FAs). The efficacy of T-Lys was also tested on chondrogenic differentiation starting from human articular chondrocytes (HACs) resulting in an increase of differentiation markers and cell number.
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Cartilagem Articular/citologia , Diferenciação Celular , Condrócitos/citologia , Condrogênese , Lisina/farmacologia , Células-Tronco Mesenquimais/citologia , Osteogênese , Cloreto de Sódio/farmacologia , Timina/farmacologia , Cartilagem Articular/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Humanos , Ácido Hialurônico/química , Lisina/química , Células-Tronco Mesenquimais/efeitos dos fármacos , Cloreto de Sódio/química , Timina/química , Engenharia TecidualRESUMO
In this paper, a pilot production process for mesenchymal stem/stromal freeze-dried secretome was performed in a validated good manufacturing practice (GMP)-compliant cell factory. Secretome was purified from culture supernatants by ultrafiltration, added to cryoprotectant, lyophilized and characterized. We obtained a freeze-dried, "ready-off-the-shelf" and free soluble powder containing extracellular vesicles and proteins. In the freeze-dried product, a not-aggregated population of extracellular vesicles was detected by nanoparticle tracking analysis; Fourier transform infrared spectra showed the simultaneous presence of protein and lipids, while differential scanning calorimetry demonstrated that lyophilization process successfully occurred. A proteomic characterization allowed the identification of proteins involved in immune response, response to stress, cytoskeleton and metabolism. Moreover, the product was not cytotoxic up to concentrations of 25 mg/mL (on human fibroblasts, chondrocytes and nucleus pulposus cells by MTT assay) and was blood compatible up to 150 mg/mL. Finally, at concentrations between 5 and 50 mg/mL, freeze-dried secretome showed to in vitro counteract the oxidative stress damage induced by H2O2 on nucleus pulposus cells by MTT assay.
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Islet transplantation has been reported to restore normoglycemia and the overall metabolic control in type 1 diabetes mellitus (DM). In the most experienced centers, islet transplantation clinical outcome is similar to that of the whole pancreas transplantation. Long-term islet transplantation function remains a very interesting matter worth discussing. A progressive islet function decrease was reported, probably due to islet exhaustion. In 5 islet-transplanted patients with at least 3-yr follow-up and still insulin independent, their glycemic control was characterized by a blinded retrospective continuous glucose monitoring system (CGMS). Islet transplantation restored glycemic control and glucose variability. Data were compared with patients in the waiting list. All the parameters of glycemic variability tested had improved significantly in patients who had islet transplantation compared with those patients who were on the waiting list. In conclusion, islet transplantation is able to maintain a proper glucose control and normalize glycemic variability in selected patients. A blinded retrospective CGMS is a useful method to characterize glucose homeostasis deeply in vivo in islet-transplanted patients.
Assuntos
Glicemia/metabolismo , Transplante das Ilhotas Pancreáticas , Adulto , Automonitorização da Glicemia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de TempoRESUMO
(1) Objective: to obtain a reproducible, robust, well-defined, and cost-affordable in vitro model of human cartilage degeneration, suitable for drug screening; (2) Methods: we proposed 3D models of engineered cartilage, considering two human chondrocyte sources (articular/nasal) and five culture methods (pellet, alginate beads, silk/alginate microcarriers, and decellularized cartilage). Engineered cartilages were treated with pro-inflammatory cytokine IL-1ß to promote cartilage degradation; (3) Results: articular chondrocytes have been rejected since they exhibit low cellular doubling with respect to nasal cells, with longer culture time for cell expansion; furthermore, pellet and alginate bead cultures lead to insufficient cartilage matrix production. Decellularized cartilage resulted as good support for degeneration model, but long culture time and high cell amount are required to obtain the adequate scaffold colonization. Here, we proposed, for the first time, the combined use of decellularized cartilage, as aggrecanase substrate, with pellet, alginate beads, or silk/alginate microcarriers, as polymeric scaffolds for chondrocyte cultures. This approach enables the development of suitable models of cartilaginous pathology. The results obtained after cryopreservation also demonstrated that beads and microcarriers are able to preserve chondrocyte functionality and metabolic activity; (4) Conclusions: alginate and silk/alginate-based scaffolds can be easily produced and cryopreserved to obtain a cost-affordable and ready-to-use polymer-based product for the subsequent screening of anti-inflammatory drugs for cartilage diseases.
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It has been demonstrated that the biological effector of mesenchymal stem/stromal cells (MSCs) is their secretome, which is composed of a heterogeneous pool of bioactive molecules, partially enclosed in extracellular vesicles (EVs). Therefore, the MSC secretome (including EVs) has been recently proposed as possible alternative to MSC therapy. The secretome can be considered as a protein-based biotechnological product, it is probably safer compared with living/cycling cells, it presents virtually lower tumorigenic risk, and it can be handled, stored and sterilized as an Active Pharmaceutical/Principle Ingredient (API). EVs retain some structural and technological analogies with synthetic drug delivery systems (DDS), even if their potential clinical application is also limited by the absence of reproducible/scalable isolation methods and Good Manufacturing Practice (GMP)-compliant procedures. Notably, EVs secreted by MSCs preserve some of their parental cell features such as homing, immunomodulatory and regenerative potential. This review focuses on MSCs and their EVs as APIs, as well as DDS, considering their ability to reach inflamed and damaged tissues and to prolong the release of encapsulated drugs. Special attention is devoted to the illustration of innovative therapeutic approaches in which nanomedicine is successfully combined with stem cell therapy, thus creating a novel class of "next generation drug delivery systems."
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Sistemas de Liberação de Medicamentos , Vesículas Extracelulares , Células-Tronco Mesenquimais , Animais , HumanosRESUMO
The aim of this study is to assess whether stromal vascular fraction (SVF)-soaked silk fibroin nonwoven mats (silk-SVF) can preserve the functionality of encapsulated pancreatic endocrine cells (alginate-PECs) after transplantation in the subcutaneous tissue of diabetic mice. Silk scaffolds are selected to create an effective 3D microenvironment for SVF delivery in the subcutaneous tissue before diabetes induction: silk-SVF is subcutaneously implanted in the dorsal area of five healthy animals; after 15 d, mice are treated with streptozotocin to induce diabetes and then alginate-PECs are implanted on the silk-SVF. All animals appear in good health, increasing weight during time, and among them, one presents euglycemia until the end of experiments. On the contrary, when PECs are simultaneously implanted with SVF after diabetes induction, mice are euthanized due to suffering. This work clearly demonstrates that silk-SVF creates a functional niche in subcutaneous tissue and preserves endocrine cell survival and engraftment.
Assuntos
Alginatos , Diabetes Mellitus Experimental/cirurgia , Fibroínas , Transplante das Ilhotas Pancreáticas/métodos , Animais , Sobrevivência Celular , Ácido Glucurônico , Ácidos Hexurônicos , Masculino , CamundongosAssuntos
Diabetes Mellitus Tipo 1/cirurgia , Everolimo/uso terapêutico , Hipoglicemiantes/uso terapêutico , Imunossupressores/uso terapêutico , Transplante das Ilhotas Pancreáticas , Peptídeos/uso terapêutico , Peçonhas/uso terapêutico , Aloenxertos , Peptídeo C/sangue , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/diagnóstico , Everolimo/efeitos adversos , Exenatida , Hemoglobinas Glicadas/metabolismo , Sobrevivência de Enxerto/efeitos dos fármacos , Humanos , Hipoglicemiantes/efeitos adversos , Imunossupressores/efeitos adversos , Peptídeos/efeitos adversos , Projetos Piloto , Fatores de Tempo , Resultado do Tratamento , Peçonhas/efeitos adversosRESUMO
With the aim of establishing the formulation of a new hydrophilic auto-gelling medical device for biomedical applications, fibroin-based microspheres were prepared. The proposed microspheres were produced by a cost-effective and industrially scalable technique, such as the spray-drying. Spray-dried silk fibroin microspheres were obtained and the effects of different hydrophilic polymer on the process yield, microsphere morphology and conformation transition of fibroin were evaluated. The final auto-gelling formulations were obtained by adding calcium gluconate (as a calcium source for alginate crosslinking) to the prepared microspheres and tested by an in vitro gelling test. This study showed that the combination of fibroin with sodium alginate and poloxamer produced the most promising auto-gelling formulation for specific biomedical applications, such as the treatment of pressure ulcers.
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Alginatos/química , Bombyx/química , Gluconato de Cálcio/química , Fibroínas/química , Géis/química , Poloxâmero/química , Polietilenoglicóis/química , Animais , Dessecação , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Microesferas , Tamanho da Partícula , Pós/químicaRESUMO
Chronic wounds are an expression of underlying complex pathologies and have a high incidence. Skin substitutes may represent an alternative approach to treat chronic ulcers. The aim of this retrospective observational study was to evaluate the wound reduction using skin substitutes based on allogenic fibroblasts or keratinocytes in 30 patients not responding to conventional therapy. Wound bed was prepared, then keratinocytes on Laserskin(®) to treat superficial wounds or fibroblasts on Hyalograft 3D(R) to treat deep leg ulcers were applied, and finally wounds were treated with a secondary dressing composed of nanocrystalline silver. Once a week constructs were removed and new bioengineered products were applied, as well as nanocrystalline silver medication. In none of the cases under examination did any complications arise relating to the treatment. We also achieved a reduction in wound dimension and exudates, and an increase in wound bed score. Postoperative assessment shows a degree of healing that is statistically higher in the group treated with keratinocytes as compared with the fibroblast group. This retrospective study improves our understanding and defines the clinical indications for the various uses of the two types of skin substitutes.
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Fibroblastos/transplante , Queratinócitos/transplante , Úlcera da Perna/terapia , Pele Artificial , Adulto , Idoso , Idoso de 80 Anos ou mais , Bandagens , Desbridamento , Feminino , Humanos , Masculino , Nanopartículas Metálicas/uso terapêutico , Pessoa de Meia-Idade , Estudos Retrospectivos , Compostos de Prata/uso terapêutico , Alicerces Teciduais , Transplante Homólogo , CicatrizaçãoRESUMO
The first islet transplantation in diabetes mellitus was performed more than 20 years ago. Since then, clinical results have progressively improved. Nowadays, islet transplantation can be considered a real therapeutic option after pancreatectomy for painful chronic pancreatitis (autotransplantation) and in selected adult patients affected by type 1 diabetes mellitus (allotransplantation). Better results are mainly due to the advances in the standardization of islet isolation and purification procedures as well as in the pharmacological treatment of recipients. Anti-inflammatory treatments facilitate islet engraftment and prevent metabolic exhaustion and functional ß-cell apoptosis; new strategies better control islet graft rejection. As a consequence, islet transplantation activities are no longer confined to few centers only, rather thousands of transplants are now performed all over the world. Many attempts are actually undertaken to find solutions to current problems of islets transplantation, from toxicity of immunosuppressive therapy to the limited engraftment, function and duration. There is general hope that these procedures will offer a safe and feasible therapeutic option for an increasing number of patients suffering from diabetes mellitus, including pediatric patients.
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Diabetes Mellitus Tipo 1/cirurgia , Transplante das Ilhotas Pancreáticas/métodos , Criança , Humanos , Transplante das Ilhotas Pancreáticas/normasRESUMO
This work reports on the formation of a carrier-in-carrier device for the systemic delivery and targeting of hydrophobic drugs mediated by micelle-loaded mesenchymal stromal cells (MSCs) (carrier-in-carrier) to be administered by intravenous injection. The innate ability of MSCs to reach injured tissues such as the central nervous system or other damaged tissues, is the key for the second order delivery and first order targeting. Inulin-D-alfa-tocopherol succinate micelles (INVITE M) are able to incorporate highly hydrophobic drugs and, due to their dimensions (≈7 nm diameter), to penetrate the cell membrane easily and quickly. This study demonstrates that the curcumin loaded micelles (INVITE MC), sterilized by filtration, reached the maximum loading in MSCs in few minutes and that the loading was concentration-dependent. When "naked" curcumin was used, an evident cytotoxicity on MSCs was detected, while INVITE micelles protected them from this effect. Moreover, MSCs loaded with INVITE MC are able to release the entrapped drug. This study strongly supports the feasibility of the carrier-in-carrier approach for the therapy of selected diseases, i.e., this innovative drug delivery system will be proposed for the treatment of the amyotrophic lateral sclerosis (ALS).
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Anti-Inflamatórios não Esteroides/farmacologia , Curcumina/farmacologia , Portadores de Fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Micelas , Fármacos Neuroprotetores/farmacologia , Tecido Adiposo/citologia , Tecido Adiposo/cirurgia , Anti-Inflamatórios não Esteroides/química , Sobrevivência Celular/efeitos dos fármacos , Curcumina/química , Liberação Controlada de Fármacos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Inulina/química , Cinética , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Fármacos Neuroprotetores/química , Cultura Primária de Células , Vitamina E/química , alfa-Tocoferol/químicaRESUMO
This study aims to evaluate the effect of combined use of the racemic flavanone Naringenin (NRG) and the protein sericin as TNF-α blockers. Sericin (SMs) and (R/S) NRG-loaded Sericin (SNRGMs) microparticles were prepared by spray-drying, characterized in terms of morphology and particle size distribution, and encapsulation efficiency was determined. Concerning morphology and particle size distribution of microparticles, results indicated that they were not affected by the presence of NRG. The encapsulation efficiency was almost quantitative (93%), thus proving that sericin can be advantageously loaded with (R/S) NRG. Biological evaluation of (R/S) NRG, SMs and SNRGMs was then performed in lipopolysaccharide (LPS)-stimulated human peripheral blood mononuclear cells (hPBMC). SNRGMs resulted cytotoxic at the higher dose used (200 µg/mL) and the effect was greater than (R/S) NRG alone. Moreover, even if sericin alone was not effective in suppressing LPS-induced serum TNF-α levels, SNRGMs loaded with 9.3% of (R/S) NRG were significantly more potent than (R/S) NRG alone. In summary, this study provides the proof of concept that sericin-based microspheres loaded with TNF-α-blockers could contribute to the down regulation of the cytokine and represents the starting point for the development of new topical formulations for the treatment of middle-stage psoriasis.
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Flavanonas/uso terapêutico , Microesferas , Psoríase/tratamento farmacológico , Sericinas/química , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Células Cultivadas , Química Farmacêutica , Citocinas/metabolismo , Regulação para Baixo/efeitos dos fármacos , Flavanonas/química , Flavanonas/farmacologia , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Lipopolissacarídeos/toxicidade , Tamanho da Partícula , Sericinas/farmacologia , Sericinas/uso terapêutico , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Some biological properties of Bombyx mori sericins from twenty strains were investigated, fourteen fed with artificial diet, two with fresh mulberry leaves and four with both diets. Sericin exhibited ROS-scavenging, anti-tyrosinase and anti-elastase properties, the strain significantly influenced these properties, while diet only influenced the anti-tyrosinase activity. Sericins were clustered into 5 groups and one sericin from each group was further studied: sericins showed anti-proliferative activity on in vitro stimulated peripheral blood mononuclear cells; some strains decreased in vitro secretion of IFNγ, while no effects were observed on TNFα and IL10 release. Therefore, a mixture of sericins extracted from the most promising strains may be useful for dermatological and cosmetic use.
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Sequestradores de Radicais Livres/química , Fatores Imunológicos/química , Elastase Pancreática/antagonistas & inibidores , Espécies Reativas de Oxigênio/química , Sericinas/química , Animais , Bombyx/metabolismo , Proliferação de Células/efeitos dos fármacos , Citocinas/metabolismo , Dieta , Sequestradores de Radicais Livres/farmacologia , Proteínas Fúngicas/antagonistas & inibidores , Proteínas Fúngicas/química , Humanos , Fatores Imunológicos/biossíntese , Fatores Imunológicos/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/fisiologia , Monofenol Mono-Oxigenase/antagonistas & inibidores , Monofenol Mono-Oxigenase/química , Elastase Pancreática/química , Tamanho da Partícula , Pós/química , Sericinas/biossíntese , Sericinas/farmacologia , Especificidade da Espécie , Espectroscopia de Infravermelho com Transformada de Fourier , Sus scrofaRESUMO
Mesenchymal stem cells (MSCs) were first isolated more than 50 years ago from the bone marrow. Currently MSCs may also be isolated from several alternative sources and they have been used in more than a hundred clinical trials worldwide to treat a wide variety of diseases. The MSCs mechanism of action is undefined and currently under investigation. For in vivo purposes MSCs must be produced in compliance with good manufacturing practices and this has stimulated research on MSCs characterization and safety. The objective of this review is to describe recent developments regarding MSCs properties, physiological effects, delivery, clinical applications and possible side effects.
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Terapia Baseada em Transplante de Células e Tecidos , Células-Tronco Mesenquimais/citologia , Humanos , Imunofenotipagem , Células-Tronco Mesenquimais/imunologiaRESUMO
OBJECTIVE: To test feasibility, safety, and efficacy of local transplant of stromal fraction of adipose tissue in the treatment of chronic headaches of cervical origin. BACKGROUND: Chronic headaches of cervical origin (chronic cervicogenic headache and occipital neuralgia) are characterized by persistent pain due to the involvement of the great occipital nerve, with concurrent myofascial spasm and the consequent nerve entrapment within the trapezoid tunnel. METHODS: Tolerability and effectiveness of treatment of chronic cervicogenic headaches refractory to conventional therapies were evaluated in 24 patients. The visual analog scale of pain and the medication use diary were used in the 3 months preceding treatment; moreover, in order to verify the quality of life, patients are required to fill before surgery the neck pain disability index, the headache disability index, migraine disability assessment scale questionnaire, and the short-form 12 standard v1 questionnaire. Follow-up examination was performed at 3 and 6 months. RESULTS: In 19 cases (79.2%), a good clinical response was recorded. At 6-month follow-up analysis, recurrence of occipital pain was recorded in 7 cases (29.2%); there is a significant reduction in disability and pain scores, and also a significant reduction of need for pharmacologic treatment and a fast return to previous work capacities. CONCLUSIONS: The key point of our therapeutic strategy might be the regenerative role of stromal fraction of adipose tissue transplanted in the area of the occipital nerve entrapment; the results of the present study are encouraging both in terms of reduction of pain scores and in terms of quality of life improvement. The technique is minimally invasive, and no complications were recorded; indeed, the procedure seems to be safe and effective, and thus, a randomized study with larger follow-up and in a large series will be started.
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Transtornos da Cefaleia/cirurgia , Lipectomia/métodos , Cefaleia Pós-Traumática/cirurgia , Adulto , Avaliação da Deficiência , Feminino , Seguimentos , Transtornos da Cefaleia/complicações , Humanos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Observação , Medição da Dor , Cefaleia Pós-Traumática/complicações , Nervos Espinhais/fisiologia , Resultado do TratamentoRESUMO
Human hair follicle cells, both bulge and dermal papilla cells, were isolated and cultured in a GMP cell factory, in order to obtain an in vitro hair follicle source for encapsulation end transplantation in alopecia regenerative cell therapy. An in vitro model, constituted by organotypic cultures of human skin sample, was set up to simulate the dermal-epidermal interaction between bulge cells and dermal papilla cells, evaluating the possible new follicles formation and the regenerative potentiality of these hair follicle cells. Both the bulge and dermal papilla cells show an excellent cellular proliferation as well as an abundant extracellular matrix production. The immunofluorescence investigation revealed the positivity of both cell lines to CK15 and CD200, whereas both cell lines were negative to CD71 and Oct-4. The pool of cultured bulge and dermal papilla cells was injected into the deep dermis; at day 28 of culture, some organized areas with a higher cell density can be observed: the cells self-organize into papilla-like lengthened aggregates. In samples in which the follicular cells have been seeded on the dermis surface, an epidermis-like homogeneous monolayer on the dermis surface can be seen, therefore showing a potentiality of these cells for epidermis regeneration. These data show the efficacy of a cellular isolation and amplification approach to obtain an in vitro human hair follicle regenerative source on industrial scale in a GMP cell factory. The results also proved an intrinsic potentiality of follicular cells to in vitro recreate the epidermis for tissue engineering purposes. Thus, it is feasible to produce bioengineered hair follicles in a GMP cell factory, for encapsulation and transplantation in alopecic patients.
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Alopecia/cirurgia , Transplante de Células/métodos , Folículo Piloso/citologia , Engenharia Tecidual , Técnicas de Cultura de Células , Linhagem Celular , Derme/citologia , Células Epidérmicas , Matriz Extracelular , Cabelo , HumanosRESUMO
Articular cartilage has limited repair and regeneration potential, and the scarcity of treatment modalities has motivated attempts to engineer cartilage tissue constructs. The use of chondrocytes in cartilage tissue engineering has been restricted by the limited availability of these cells, their intrinsic tendency to lose their phenotype during the expansion, as well as the difficulties during the first cell adhesion to the scaffold. Aim of this work was to evaluate the intra-articular adipose stromal vascular fraction attachment on silk fibroin scaffold to promote chondrocytes adhesion and proliferation. Physicochemical characterization has demonstrated that three-dimensionally organized silk fibroin scaffold is an ideal biopolymer for cartilage tissue engineering; it allows cell attachment, scaffold colonization, and physically cell holding in the area that must be repaired; the use of adipose-derived stem cells is a promising strategy to promote adhesion and proliferation of chondrocytes to the scaffold as an autologous human feeder layer.
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Tecido Adiposo/irrigação sanguínea , Tecido Adiposo/citologia , Cartilagem/patologia , Fibroínas/química , Patela/citologia , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Varredura Diferencial de Calorimetria , Células Cultivadas , Condrócitos/citologia , Técnicas de Cocultura , Feminino , Imunofluorescência , Humanos , Masculino , Microscopia Eletrônica de Varredura , Pessoa de Meia-Idade , Espectroscopia de Infravermelho com Transformada de Fourier , Células Estromais/citologia , Adulto JovemRESUMO
The adipose-derived stromal vascular fraction (SVF) represents a rich source of mesenchymal cells, potentially able to differentiate into adipocytes, chondrocytes, osteoblasts, myocytes, cardiomyocytes, hepatocytes, and neuronal, epithelial, and endothelial cells. These cells are ideal candidates for use in regenerative medicine, tissue engineering, including gene therapy, and cell replacement cancer therapies. In this work, we aimed to the optimization of the adipose SVF-based therapy, and the effect of the collection site, surgical procedure, and tissue processing techniques on SVF yield was evaluated in terms of cell recovery and live cells, taking into account the effect of gender, age, and body mass index. Adipose tissue samples were recovered from 125 informed subjects (37 males and 88 females; mean age: 51.31 years; range: 15-87 years), and digested in different condition with collagenase. A multivariate linear model put in evidence that in males the best collection site in terms of yield is located in the abdomen, whereas in females the biopsy region do not influence cell recovery; the collection technique, the age, and the body mass index of donor seem not to influence the cell yield. The tissue-processing procedures strongly modify the yield and the vitality of cells: a collagenase concentration of 0.2% and a digestion time of 1 h could be chosen as the best operating conditions.
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Tecido Adiposo/citologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Medicina Regenerativa/métodos , Células Estromais/citologia , Tecido Adiposo/irrigação sanguínea , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Vasos Sanguíneos/citologia , Proliferação de Células , Separação Celular/métodos , Células Cultivadas , Eficiência , Feminino , Humanos , Masculino , Transplante de Células-Tronco Mesenquimais/métodos , Pessoa de Meia-Idade , Adulto JovemRESUMO
Techniques for medical tissue regeneration require an abundant source of human adult stem cells. There is increasing evidence that adipose stem cells contribute to restoration of tissue vascularization and organ function. The object of our study was to isolate and characterize adult adipose-derived stem cells from patients undergoing on lipoaspirate transplant with the aim to improve tissue regeneration. Adipose-derived stem cells were isolated and purified from the lipoaspirate of 15 patients and characterized for CD markers and the ability to differentiate toward the adipogenic lineage. We found that purified adipose stem cells express high level of CD49d, CD44, CD90, CD105, CD13, and CD71 and these markers of staminality were maintained at high level for at least 3 months and seven passages of in vitro culture. As expected, these cells resulted negative for the endothelial and hematopoietic-specific markers CD31, CD106, CD34, and CD45. Differentiation towards adipogenic lineage demonstrated that purified adipose-derived stem cells are still able to become adipocytes at least 3 months after in vitro culture. The analysis of Akt and MAPK phosphorylation confirmed a modulation of their activity during differentiation. Interestingly, we established for the first time that, among the p53 family members, a strong upregulation of p63 expression occurs in adipocytic differentiation, indicating a role for this transcription factor in adipocytic differentiation. Taken together, these data indicate that purified lipoaspirate-derived stem cells maintain their characteristic of staminality for a long period of in vitro culture, suggesting that they could be applied for cell-based therapy to improve autologous lipoaspirate transplant.