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1.
Polymers (Basel) ; 16(14)2024 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-39065286

RESUMO

Hyaline cartilage is a highly specialized tissue. When injured, its repair capacity is low, which results in the massive destruction of the articular surface. Using tissue engineering and genetic engineering techniques, it is possible to provide a suitable microenvironment providing chondrocyte growth factors involved in the development of hyaline cartilage proteins, as well as cell proliferation and differentiation. Our aim was to stimulate the synthesis of an extracellular matrix via the chondrocytes included in a fibrin matrix through the addition or overexpression of IGF1 and/or FGF2, while maintaining a constant agitation of the culture medium. Collagen type II and glycosaminoglycans increased during the entire incubation time. In contrast, collagen type I decreased its expression under the same culture conditions, transfecting or supplementing growth factors to chondrocytes. However, chondrocytes that were not transfected or supplemented showed a general increase in the proteins analyzed in this study. The presence of IGF1 and FGF2 increased the protein synthesis of the hyaline cartilage, regardless of which one was the source of growth factors. Continuous agitation using the spinner flask allows for the adequate nutrition of chondrocytes included in the fibrin matrix. However, they require growth factors to up-regulate or down-regulate collagenous proteins.

2.
Life (Basel) ; 13(8)2023 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-37629580

RESUMO

BACKGROUND: The presence of side effects and low bioavailability of rhein has limited its use in the treatment of osteoarthritis. We aimed to evaluate the in vitro response of human articular chondrocytes to the presence of the combination of platelet-rich plasma (PRP) and rhein. METHODS: Solutions of rhein were prepared to assess solubility and select a working concentration. A stimulus with interleukin-1ß (IL-ß, 10 ng/mL) was induced for 24 h on human chondrocytes. Five treatment groups were established: control, IL-ß control, PRP, rhein, and PRP + rhein. Cell viability, cell migration, nitric oxide (NO) production, tumor necrosis factor-α (TNF-α), and gene expression analyses were carried out. RESULTS: A concentration of 50 mg/L was selected after a dose-response curve assay. Both NO and tumor TNF-α production significantly decreased after PRP and PRP + rhein treatments at 24 and 48 h. The wound healing assay revealed a significant stimulation of migration after 72 h with the PRP and PRP + rhein treatments. Expression of IL-1ß, IL-6, MMP-13, and ADAMTS-5 was significantly downregulated, particularly after treatment with the combination of PRP + rhein. CONCLUSIONS: Much of the determinations denoted a better performance of the combination of PRP and rhein in decreasing the levels of the different targets evaluated; however, this was not great enough to detect a significant difference in comparison with the PRP treatment alone.

3.
Curr Issues Mol Biol ; 44(12): 5827-5838, 2022 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-36547057

RESUMO

Background: Achilles-tendon rupture prevails as a common tendon pathology. Adipose-derived mesenchymal stem cells (ADMSCs) are multipotent stem cells derived from adipose tissue with attractive regeneration properties; thus, their application in tendinopathies could be beneficial. Methods: Male rabbit ADMSCs were obtained from the falciform ligament according to previously established methods. After tenotomy and suture of the Achilles tendon, 1 × 106 flow-cytometry-characterized male ADMSCs were injected in four female New Zealand white rabbits in the experimental group (ADMSC group), whereas four rabbits were left untreated (lesion group). Confirmation of ADMSC presence in the injured site after 12 weeks was performed with quantitative sex-determining region Y (SRY)-gene RT-PCR. At Week 12, histochemical analysis was performed to evaluate tissue regeneration along with quantitative RT-PCR of collagen I and collagen III mRNA. Results: Presence of male ADMSCs was confirmed at Week 12. No statistically significant differences were found in the histochemical analysis; however, statistically significant differences between ADMSC and lesion group expression of collagen I and collagen III were evidenced, with 36.6% and 24.1% GAPDH-normalized mean expression, respectively, for collagen I (p < 0.05) and 26.3% and 11.9% GAPDH-normalized mean expression, respectively, for collagen III (p < 0.05). The expression ratio between the ADMSC and lesion group was 1.5 and 2.2 for collagen I and collagen III, respectively. Conclusion: Our results make an important contribution to the understanding and effect of ADMSCs in Achilles-tendon rupture.

4.
Exp Ther Med ; 22(5): 1282, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34630637

RESUMO

Joint cartilage damage affects 10-12% of the world's population. Medical treatments improve the short-term quality of life of affected individuals but lack a long-term effect due to injury progression into fibrocartilage. The use of mesenchymal stem cells (MSCs) is one of the most promising strategies for tissue regeneration due to their ability to be isolated, expanded and differentiated into metabolically active chondrocytes to achieve long-term restoration. For this purpose, human adipose-derived MSCs (Ad-MSCs) were isolated from lipectomy and grown in xeno-free conditions. To establish the best differentiation potential towards a stable chondrocyte phenotype, isolated Ad-MSCs were sequentially exposed to five differentiation schemes of growth factors in previously designed three-dimensional biphasic scaffolds with incorporation of a decellularized cartilage matrix as a bioactive ingredient, silk fibroin and bone matrix, to generate a system capable of being loaded with pre-differentiated Ad-MSCs, to be used as a clinical implant in cartilage lesions for tissue regeneration. Chondrogenic and osteogenic markers were analyzed by reverse transcription-quantitative PCR and cartilage matrix generation by histology techniques at different time points over 40 days. All groups had an increased expression of chondrogenic markers; however, the use of fibroblast growth factor 2 (10 ng/ml) followed by a combination of insulin-like growth factor 1 (100 ng/ml)/TGFß1 (10 ng/ml) and a final step of exposure to TGFß1 alone (10 ng/ml) resulted in the most optimal chondrogenic signature towards chondrocyte differentiation and the lowest levels of osteogenic expression, while maintaining stable collagen matrix deposition until day 33. This encourages their possible use in osteochondral lesions, with appropriate properties for use in clinical patients.

5.
Int J Mol Sci ; 20(20)2019 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-31627374

RESUMO

In cartilage tissue engineering, biphasic scaffolds (BSs) have been designed not only to influence the recapitulation of the osteochondral architecture but also to take advantage of the healing ability of bone, promoting the implant's integration with the surrounding tissue and then bone restoration and cartilage regeneration. This study reports the development and characterization of a BS based on the assembly of a cartilage phase constituted by fibroin biofunctionalyzed with a bovine cartilage matrix, cellularized with differentiated autologous pre-chondrocytes and well attached to a bone phase (decellularized bovine bone) to promote cartilage regeneration in a model of joint damage in pigs. BSs were assembled by fibroin crystallization with methanol, and the mechanical features and histological architectures were evaluated. The scaffolds were cellularized and matured for 12 days, then implanted into an osteochondral defect in a porcine model (n = 4). Three treatments were applied per knee: Group I, monophasic cellular scaffold (single chondral phase); group II (BS), cellularized only in the chondral phase; and in order to study the influence of the cellularization of the bone phase, Group III was cellularized in chondral phases and a bone phase, with autologous osteoblasts being included. After 8 weeks of surgery, the integration and regeneration tissues were analyzed via a histology and immunohistochemistry evaluation. The mechanical assessment showed that the acellular BSs reached a Young's modulus of 805.01 kPa, similar to native cartilage. In vitro biological studies revealed the chondroinductive ability of the BSs, evidenced by an increase in sulfated glycosaminoglycans and type II collagen, both secreted by the chondrocytes cultured on the scaffold during 28 days. No evidence of adverse or inflammatory reactions was observed in the in vivo trial; however, in Group I, the defects were not reconstructed. In Groups II and III, a good integration of the implant with the surrounding tissue was observed. Defects in group II were fulfilled via hyaline cartilage and normal bone. Group III defects showed fibrous repair tissue. In conclusion, our findings demonstrated the efficacy of a biphasic and bioactive scaffold based on silk fibroin and cellularized only in the chondral phase, which entwined chondroinductive features and a biomechanical capability with an appropriate integration with the surrounding tissue, representing a promising alternative for osteochondral tissue-engineering applications.


Assuntos
Regeneração Óssea , Engenharia Tecidual/métodos , Animais , Cartilagem , Diferenciação Celular , Condrócitos , Fibroínas , Suínos , Alicerces Teciduais
6.
Stem Cells Int ; 2019: 9792369, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31149016

RESUMO

Articular cartilage injuries remain as a therapeutic challenge due to the limited regeneration potential of this tissue. Cartilage engineering grafts combining chondrogenic cells, scaffold materials, and microenvironmental factors are emerging as promissory alternatives. The design of an adequate scaffold resembling the physicochemical features of natural cartilage and able to support chondrogenesis in the implants is a crucial topic to solve. This study reports the development of an implant constructed with IGF1-transduced adipose-derived mesenchymal stem cells (immunophenotypes: CD105+, CD90+, CD73+, CD14-, and CD34-) embedded in a scaffold composed of a mix of alginate/milled bovine decellularized knee material which was cultivated in vitro for 28 days (3CI). Histological analyses demonstrated the distribution into isogenous groups of chondrocytes surrounded by a de novo dense extracellular matrix with balanced proportions of collagens II and I and high amounts of sulfated proteoglycans which also evidenced adequate cell proliferation and differentiation. This graft also shoved mechanical properties resembling the natural knee cartilage. A modified Bern/O'Driscoll scale showed that the 3CI implants had a significantly higher score than the 2CI implants lacking cells transduced with IGF1 (16/18 vs. 14/18), representing high-quality engineering cartilage suitable for in vivo tests. This study suggests that this graft resembles several features of typical hyaline cartilage and will be promissory for preclinical studies for cartilage regeneration.

7.
Cir Cir ; 85(2): 127-134, 2017.
Artigo em Espanhol | MEDLINE | ID: mdl-27633464

RESUMO

BACKGROUND: A fracture repair involves complex cellular processes. However, despite optimal treatment, some fractures heal slowly or do not repair. These complications support the need for innovative therapies. Electromagnetic stimulation is a non-invasive technology that could have a direct impact on many cellular pathways. OBJECTIVE: To demonstrate the effectiveness of electro-stimulation by alternating current applied during bone elongation to accelerate the consolidation process for 30 days in an animal model. MATERIALS AND METHODS: A device with closed circuit and graduated voltage was designed and kept in contact with the external fixator. Group A was elongated without electro-stimulation and group B was electro-stimulated since the beginning of the distraction. Radiographs were taken at 15 and 30 days post-surgical. Haematoxylin and eosin staining and Masson's trichrome stain were performed. RESULTS: No significant difference were observed in bone density of group A (4.05±3.24, P=0.163). In group B there was a significant difference (61.06±20.17, P=0.03) in bone density. Group A maintained a fibrous tissue repair, with areas of cartilage and bone matrix. Group B had more organised tissue in the stages of bone repair. CONCLUSION: Because there is a significant difference in the growth and callus formation at 15 and 30 days between groups, electro-stimulation could be considered as an adjuvant during bone elongation.


Assuntos
Alongamento Ósseo/métodos , Terapia por Estimulação Elétrica , Fixação de Fratura , Animais , Osso e Ossos , Cães , Terapia por Estimulação Elétrica/métodos , Fixadores Externos , Modelos Animais
8.
Stem Cells Int ; 2016: 7403890, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27818692

RESUMO

Adipose-derived mesenchymal stem cells (ADMSCs) are inducible to an osteogenic phenotype by the bone morphogenetic proteins (BMPs). This facilitates the generation of implants for bone tissue regeneration. This study evaluated the in vitro osteogenic differentiation of ADMSCs transduced individually and in combination with adenoviral vectors expressing BMP2 and BMP7. Moreover, the effectiveness of the implant containing ADMSCs transduced with the adenoviral vectors AdBMP2/AdBMP7 and embedded in demineralized bone matrix (DBM) was tested in a model of tibial fracture in sheep. This graft was compared to ewes implanted with untransduced ADMSCs embedded in the same matrix and with injured but untreated animals. In vivo results showed accelerated osteogenesis in the group treated with the AdBMP2/AdBMP7 transduced ADMSC graft, which also showed improved restoration of the normal bone morphology.

9.
Gac Med Mex ; 152(3): 381-5, 2016.
Artigo em Espanhol | MEDLINE | ID: mdl-27335195

RESUMO

Since the introduction of bone morphogenetic proteins, their use has become an invaluable ally for the treatment of bone defects. These proteins are potent growth factors, related to angiogenic and osteogenic activity. The osteoinductive capacity of recombinant bone morphogenetic protein (rhBMP) in the formation of bone and cartilage has been confirmed in in vitro studies and evaluated in clinical trials. To obtain a therapeutic effect, administration is systemic, by injection over the physiological dose. Among the disadvantages, ectopic bone formation or high morbidity in cases of spinal fusion is observed. In this review, the roles of bone morphogenetic proteins in bone repair and clinical applications are analyzed. These findings represent advances in the study of bone regeneration and application of growth factors for more predictable results.


Assuntos
Doenças Ósseas/terapia , Proteínas Morfogenéticas Ósseas/administração & dosagem , Regeneração Óssea/efeitos dos fármacos , Doenças Ósseas/patologia , Proteínas Morfogenéticas Ósseas/efeitos adversos , Humanos , Injeções , Ossificação Heterotópica/induzido quimicamente , Osteogênese/efeitos dos fármacos , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/efeitos adversos , Fusão Vertebral/métodos
10.
Int. j. morphol ; 32(4): 1199-1206, Dec. 2014. ilus
Artigo em Espanhol | LILACS | ID: lil-734659

RESUMO

La osteoartrosis es un padecimiento del aparato locomotor con una prevalencia elevada y en crecimiento, paralela al envejecimiento de la población. La infiltración intraarticular de sustancias para aliviar la sintomatología de la osteoartrosis es una práctica común en el consultorio médico de los especialistas que atienden esta enfermedad. Aunque la sintomatología mejora con la infiltración de anestésicos locales, corticoesteroides y suplementos viscosantes, es aún incierto el efecto que estas sustancias tienen sobre la integridad del cartílago articular. Este estudio explora a nivel macroscópico e histológico el efecto de la infiltración de ropivacaína, metilprednisolona y ácido hialurónico sobre el cartílago articular en un modelo de osteoartrosis química en conejos (n=24). Nuestros resultados indican que en los grupos infiltrados con metilprednisolona (n=8) y ropivacaína (n=8) la estructura del cartílago articular presento alteraciones más severas con respecto a su grupo control, además de una disminución importante en la síntesis de matriz extracelular. En el grupo infiltrado con ácido hialurónico (n=8), las alteraciones macroscópicas e histológicas del cartílago articular mejoraron con respecto a su grupo control, presentando una estructura integra y síntesis de matriz extracelular normal.


Osteoarthritis is a musculoskeletal condition with a high prevalence, increasing with the aging of population. The intraarticular infiltration of substances to relieve the symptoms of osteoarthritis is a common practice in medical practice. Although symptoms improved with the infiltration of local anesthetics, corticosteroids and supplements, it is still uncertain what effect these substances have on the integrity of articular cartilage. This study explores the macroscopic and histological effects of infiltration of Ropivacaine, Methylprednisolone and Hyaluronic Acid on articular cartilage in a model of chemical osteoarthritis in rabbits (n=24). Our results indicate that in the infiltrated groups with Methylprednisolone (n=8) and Ropivacaine (n=8) the structure of articular cartilage present more severe alterations with respect to its control group and an important decrease in the synthesis of extracellular matrix. In-group infiltrated with hyaluronic acid (n=8), macroscopic and histological changes of articular cartilage improved with respect to its control group, presenting a normal structure and normal extracellular matrix synthesis.


Assuntos
Animais , Masculino , Coelhos , Metilprednisolona/administração & dosagem , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/patologia , Osteoartrite do Joelho , Amidas/administração & dosagem , Ácido Hialurônico/administração & dosagem , Metilprednisolona/farmacologia , Modelos Animais de Doenças , Amidas/farmacologia , Ácido Hialurônico/farmacologia
11.
Cir Cir ; 82(2): 163-9, 2014.
Artigo em Espanhol | MEDLINE | ID: mdl-25312315

RESUMO

BACKGROUND: in the treatment of early osteoartrosis, analgesics and non-steroidal anti-inflammatory drugs are frequently used to relieve pain. Currently, platelet rich plasma is used as an alternative in the treatment of osteoartrosis. The aim of this study was to evaluate the effect of platelet rich plasma compared to paracetamol as a treatment for patients with knee osteoartrosis grade I. METHODS: we evaluated 42 patients who were randomized into two groups. Group one was treated with 5 mL of platelet rich plasma in two applications, while group two was treated with 1 gr of oral paracetamol every 8 hours for 30 days. Both patient groups received supervised physical rehabilitation during the 6 month observation period. Peripheral blood samples were taken to measure plasma IL-1ß, TNF-a and TGF-ß1 levels at day 0 and at 6 months post-treatment. Clinical evaluation was conducted using the KOOS at the start of the study and for every subsequent month during the study period. RESULTS: the Knee injury and Osteoarthritis Outcome Score (KOOS) for group one at the start of the treatment was measured at 30.1 points, whereas at the end, it was measured at 48.2 points, showing a clinical improvement of 60%. There were no statistically significant differences in IL-1ß and TNF-a levels between groups treated either with platelet rich plasma or paracetamol. CONCLUSIONS: Our patients treated with platelet rich plasma showed a statistically significant increase in the serum levels of TGF-ß1, which was associated with an improvement in the clinical evaluation used (KOOS).


Antecedentes: la osteoartrosis se caracteriza por dolor, rigidez articular y crepitación, con datos radiológicos específicos. El dolor se controla con analgésicos y antiinflamatorios no esteroides. En la actualidad, el plasma rico en plaquetas es una alternativa de tratamiento de esta enfermedad. Objetivo: comparar el efecto del plasma rico en plaquetas versus paracetamol en el tratamiento de pacientes con osteoartrosis de rodilla grado I. Material y métodos: ensayo prospectivo y experimental en el que se estudiaron 42 pacientes que se asignaron al azar a dos grupos. Al grupo 1 (experimental) se le indicaron 5 mL de plasma rico en plaquetas, en dos aplicaciones; el grupo 2 (control) se trató con 1 g de paracetamol oral cada 8 horas durante 30 días. Ambos grupos recibieron terapia física supervisada, con un tiempo de seguimiento total de seis meses. Para determinar las concentraciones de sangre periférica se tomaron muestras de IL-1ß, TNF-a y TGF-ß en plasma el día 0 y a los 6 meses. La valoración clínica se realizó con el Knee injury and Osteoarthritis Outcome Score (KOOS) al inicio y mensualmente durante el estudio. Resultados: los resultados de IL-1ß y TNF-a no mostraron diferencia significativa entre los grupos tratados con plasma rico en plaquetas y paracetamol. La escala KOOS para el grupo 1 al inicio del tratamiento fue de 30.1 puntos y al finalizar el tratamiento 48.2 puntos, con mejoría clínica de 60%. Conclusiones: los pacientes tratados con plasma rico en plaquetas tuvieron un incremento significativo en las concentraciones séricas de TGF-ß1 que se asoció con la mejoría clínica relacionada con el KOOS.


Assuntos
Acetaminofen/uso terapêutico , Osteoartrite do Joelho/terapia , Plasma Rico em Plaquetas , Acetaminofen/administração & dosagem , Administração Oral , Adulto , Animais , Biomarcadores , Feminino , Humanos , Interleucina-1beta/sangue , Contagem de Leucócitos , Masculino , Pessoa de Meia-Idade , Osteoartrite do Joelho/sangue , Osteoartrite do Joelho/tratamento farmacológico , Osteoartrite do Joelho/reabilitação , Estudos Prospectivos , Fator de Crescimento Transformador beta1/sangue , Resultado do Tratamento , Fator de Necrose Tumoral alfa/análise
12.
Arch Plast Surg ; 39(6): 659-62, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23233894

RESUMO

Progressive facial hemiatrophy, also known as Parry-Romberg syndrome, is a progressive and self-limited deformation of the subcutaneous tissue volume on one side of the face that creates craniofacial asymmetry. We present the case of a patient with a five-year history of progressive right facial hemiatrophy, who underwent facial volumetric restoration using cell-assisted lipotransfer (CAL), which consists of an autologous fat graft enriched with adipose-derived stem cells (ASCs) extracted from the same patient. ASCs have the capacity to differentiate into adipocytes. They also promote angiogenesis, release angiogenic growth factors, and some can survive as stem cells. The use of autologous fat as a filler in soft tissue atrophy has been satisfactory in patients with mild and moderate Parry-Romberg syndrome. Currently, CAL has showed promising results in the long term by decreasing the rate of fat reabsorption. The permanence and stability of the graft in all the injected areas has showed that autologous fat grafts enriched with stem cells could be a promising technique for the correction of defects caused by this syndrome.

13.
J Craniofac Surg ; 23(2): 392-6, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22421833

RESUMO

BACKGROUND: Bone morphogenetic proteins (BMPs) are actively involved in ossification, and BMP-2 participates throughout the entire process. Gene therapy for bone regeneration using adenovirus-expressing BMPs has been successful in small mammals, but it has not been satisfactory in large mammals. METHODS: We generated a 3-component implant (3C graft) comprising autologous mesenchymal stem cells (MSCs), ex vivo transduced with an adenovirus vector-expressing BMP-2 and embedded in a demineralized human bone matrix (DBM). RESULTS: In vitro studies demonstrated vector-induced osteogenesis; osteoblast population and mineralization of the extracellular matrix were greater in the vector-transduced cultures than in the controls (nontransduced MSCs stimulated with osteogenic media were used as positive controls, and nontransduced MSCs served as a negative control). The 3-component grafts were used to fill osteotomies created by bone distraction surgery in mongrel dogs. Control groups comprised dogs with bone distraction alone and dogs with nontransduced MSC grafts. The radiography follow-up, performed 10 weeks after distraction, demonstrated a remarkable reduction in the consolidation period compared with controls. Postmortem mandibles submitted for anatomic and histologic analyses showed improved remodeling and bone maturation in the 3C-grafted dogs. Inflammatory infiltrates were not observed in any of the treated areas, and no liver toxicity was detected. CONCLUSIONS: We demonstrated acceleration of osteogenesis in a dog model for bone distraction by using an implant of BMP-2 modified MSCs. These results are helpful for future clinical trials of mandible bone distraction.


Assuntos
Proteína Morfogenética Óssea 2/farmacologia , Regeneração Óssea/efeitos dos fármacos , Regeneração Óssea/genética , Mandíbula/cirurgia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteogênese por Distração/métodos , Adenoviridae/genética , Animais , Western Blotting , Estudos de Casos e Controles , Técnicas de Cultura de Células , Cães , Citometria de Fluxo , Vetores Genéticos/genética , Humanos , Técnicas Imunoenzimáticas , Modelos Animais , Osteoblastos/efeitos dos fármacos , Osteotomia , Transdução Genética
14.
Vet. Méx ; 39(2): 215-221, ene.-jun. 2008. tab
Artigo em Espanhol | LILACS-Express | LILACS | ID: lil-632872

RESUMO

A preliminary study was carried out to obtain serological evidence of the presence of the porcine respiratory and reproductive syndrome (PRRS) virus and the risk of infection in different areas and phases of production in five full cycle pig farms in the state of Nuevo Leon, Mexico. Sixty blood samples of each farm were obtained (10 for each phase of production: weaning, growing, finishing, pregnancy, lactation and mating-service). The detection of antibodies against the PRRS virus was carried out using a commercial kit. All farms were positives in all phases of production. The highest seroprevalences were found in the growing and finishing phases (36% y 56%, respectively).


Se realizó un estudio preliminar para obtener evidencia serológica de la presencia del virus del síndrome respiratorio y reproductivo porcino (PRRS, por sus siglas en inglés) y del riesgo de infección en las diferentes áreas y etapas de producción en cinco granjas porcinas de ciclo completo en Nuevo León, México. Se obtuvieron 60 muestras de sangre de cada granja (diez para cada etapa de producción: destete, iniciación, finalización, cerdas gestantes, lactando o en monta-servicio). La detección de anticuerpos antivirus del PRRS se realizó utilizando un equipo comercial. Todas las granjas resultaron positivas en las diferentes etapas de producción. La seroprevalencia fue mayor en las etapas de inicio y finalización (36% y 56%, respectivamente).

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