Implante de condrócitos homólogos em defeitos osteocondrais de cães: padronização da técnica e avaliação histopatológica / Homologous articular chondrocytes implantation in osteochondral defects of dogs: technique and histopathological evaluation standardization

Padronizou-se a metodologia para cultura de condrócitos em cães e avaliou-se seu implante em lesões osteocondrais, utilizando-se a membrana biossintética de celulose (MBC) como revestimento. Dez cães, adultos e clinicamente sadios, foram submetidos à artrotomia das articulações fêmoro-tíbio-patelares. Defeitos de 4mm de diâmetro e profundidade foram induzidos no sulco troclear de ambos os membros. MBC foi aplicada na base e na superfície das lesões. Os defeitos do membro direito foram preenchidos com condrócitos homólogos cultivados formando o grupo-tratado (GT); os do membro esquerdo, sem implante celular, foram designados grupo-controle (GC). A evolução pós-operatória foi analisada com especial interesse nos processos de reparação da lesão, por meio de histomorfometria e imuno-histoquímica para colágeno tipo II e sulfato de condroitina. A cultura de condrócitos homólogos apresentou alta densidade e taxa de viabilidade. Observou-se integridade do tecido neoformado com a cartilagem adjacente na avaliação histológica, em ambos os grupos. Na imuno-histoquímica, verificou-se predomínio de colágeno tipo II no GT. Morfometricamente, não houve diferença significativa entre o tecido fibroso e o fibrocartilaginoso entre os grupos. A cultura de condrócitos homólogos de cães foi exequível. O tecido neoformado apresentou qualidade discretamente superior associado ao implante homólogo de condrócitos, contudo não promoveu reparação por cartilagem hialina.

The aim of the study is to standardize the methodology to achieve canine chondrocytes culture, and evaluate its implant on osteochondral defects made in the femoral trochlear sulcus of dogs, using the cellulose biosynthetic membrane (CBM) as coating. Ten healthy adult dogs without locomotor disorders were used. All animals were submitted to arthrotomy of stifle joints and defects of four millimeters in diameter x four millimeters deep were done in the femoral trochlear sulcus of both limbs. CBM were applied in the lesion base and surface of all limbs. In the treated group (TG), defects of the right limb were filled with cultivated homologous chondrocytes, and in control group (CG), defects of the left limb were left without cellular implant. Postoperative follow up was done by histomorphometry and Collagen type II and anti-chondroitin sulfate immunohistochemistry. The homologous chondrocytes culture showed high density and viability rate. Upon immunohistochemistry the predominance of type II collagen in extracellular matrix of TG was verified. However, no significant statistical difference was observed between the groups upon histomorphometry analysis of fibrous and fibrocartilaginous tissues. Canine homologous chondrocytes culture was practicable. Neoformed tissue showed slightly higher quality in TG, but without promoting repair by the hyaline cartilage.

Reparação de defeitos osteocondrais de cães com implante de cultura de condrócitos homólogos e membrana biossintética de celulose: avaliação clínica, ultrassonográfica e macroscópica / The repair of osteochondral defects in dogs with homologous articular chondrocytes and biosynthetic cellulose membrane: clinical, ultrasound and macroscopic evaluation

Avaliou-se o implante de condrócitos homólogos em lesões osteocondrais, utilizando a membrana biossintética à base de celulose (MBC) como revestimento. Dez cães adultos e clinicamente sadios foram submetidos à artrotomia das articulações fêmoro-tíbio-patelares. Defeitos de quatro milímetros de diâmetro por quatro milímetros de profundidade foram induzidos na tróclea femoral de ambos os membros. A MBC foi aplicada na base e superfície das lesões. Os defeitos do membro direito foram preenchidos com condrócitos homólogos cultivados e formaram o grupo tratado (GT); e os defeitos do membro esquerdo, sem implante celular, formaram o grupo controle (GC). Os animais foram avaliados clínica e ultrassonograficamente aos 30 e 60 dias. A evolução pós-operatória dos cães foi analisada com especial interesse nos processos de reparação da lesão, por meio de macroscopia. Não houve diferença clínica e ultrassonográfica entre os grupos. Entretanto, à macroscopia, ocorreu maior prevalência de formação de tecido cicatricial esbranquiçado no GT. O tecido neoformado apresentou melhor qualidade associado ao implante homólogo de condrócitos, mas não promoveu reparação por cartilagem hialina.

The aim of the study was to evaluate the repair of deep cartilaginous defects made in the femoral trochlear sulcus of dogs, using the cellulose biosynthetic membrane (CBM) as coating. Ten healthy adult dogs without locomotor disorders were used. All animals were submitted to arthrotomy of stifle joints and defects with four millimeters diameter x four millimeters deep were done in the femoral trochlear sulcus of both limbs. CBM was applied in the lesion's base and surface of all limbs. In the treated group (TG), the defects of the right limb were filled with cultivated homologous chondrocytes, and in control group (CG), the defects of the left limb were filled without cellular implant. The animals were evaluated by physical examination and ultrasound at 30 and 60 days. The postoperative follow up of the dogs was done by macroscopy with special interest in the healing process of the osteochondral defect. No clinical and ultrasonographic differences were observed in both groups. In the macroscopic evaluation higher prevalence of whitish scar tissue formation was noted in TG, but without statistical difference. The neoformed tissue showed slightly higher quality in TG, but without promoting repair by the hyaline cartilage.

Uso de células mononucleares da medula óssea no tratamento de tendinites induzidas experimentalmente em equinos / The use of mononuclear cells from bone marrow on the treatment of experimentally induced tendinitis in horses

Foram induzidas lesões no tendão flexor digital superficial (TFDS) de ambos os membros anteriores de seis equinos, seguidas de implante autólogo de células da fração mononuclear de medula óssea em apenas um membro de cada animal. Os animais foram avaliados por parâmetros clínicos, ultrassonográficos, histopatológico e imunoistoquímico. Paralelamente, realizou-se o cultivo de novas amostras para a caracterização das células utilizando-se marcadores CD34 e CD45 por meio da citometria de fluxo, confirmando a presença de células mesenquimais na fração mononuclear. A caracterização das fibras colágenas tipo I e tipo III no tecido neoformado mostrou melhora na qualidade da cicatrização tendínea dos membros tratados. A terapia com implante autólogo das células da fração mononuclear melhorou a organização tecidual e a sua qualidade, apresentando maior expressão significativa para colágeno tipo I.

The present study was developed inducing a lesion in the SDFT of both thoracic limbs of six horses followed by autologous implantation of mononuclear cells from bone marrow in only one affected limb of each horse. The horses were evaluated through clinical and ultrasonography exams, and through histopathology and immunohistochemistry patterns. Concomitantly, new samples were cultivated and characterized using CD34 and CD45 markers, proving the presence of mesenchymal cells in the mononuclear fraction. The characterization of collagen fibers type I and type III in the new tissue has showed an improvement in tendon healing in treated limbs. The therapy with autologous implant of the mononuclear fraction has improved tissue organization and its quality, having a significanlyt higher expression of collagen type I.

Characterization and transcriptional analysis of the promoter region of the Duffy blood group, chemokine receptor (DARC) gene in cattle.

The Duffy antigen is the only receptor for Plasmodium vivax, a hemoparasite of the phylum Apicomplexa and the cause of vivax malaria in humans. Resistance to this parasite in the majority of black African individuals and their descendents is due to a mutation in the gene promoter region, which blocks its transcription on erythrocytes. Regarding bovine babesiosis, it is known that taurine breeds are more susceptible to parasite infection than zebuine breeds. In order to verify whether the same human resistance occurs in bovine, the 5' flanking region of the DARC gene was isolated and characterized in Bos indicus and Bos taurus. Four single nucleotide polymorphisms were identified and genotyped (SNP1: EF_647729.1:g.91C>T; SNP2: EF_647729.1:g.405C>T; SNP3: EF_647729.1: g.433A>G and SNP4: EF_647729.1:g.588A>G), which showed significant frequency differences among 99 bovines of each species (n=198). Characterization of the isolated region revealed the presence of 6 putative haplotypes, 14 genotypes, which are formed by haplotypes, and numerous putative transcription factor binding sites. Only the thymine presence on SNPs 1 and 2, more common in B. indicus, was observed to alter some of the sites in this region. Despite this fact, analyses through real-time PCR on bovines that present the most common homozygote genotypes of each species, which contrast for all the polymorphism, revealed no difference on the DARC gene transcription. Thus, in principle, it was concluded that the polymorphisms identified would not be useful as molecular markers in an improvement program for resistance to babesiosis.