BackgroundThe possibility for isolating bovine mesenchymal multipotent cells (MSCs) from fetal adnexa is an interesting prospect because of the potential for these cells to be used for biotechnological applications. Bone marrow and adipose tissue are the most common sources of MSCs derived from adult animals. However, little knowledge exists about the characteristics of these progenitors cells in the bovine species. Traditionally most cell cultures are developed in two dimensional (2D) environments. In mammalian tissue, cells connect not only to each other, but also support structures called the extracellular matrix (ECM). The three-dimensional (3D) cultures may play a potential role in cell biotechnology, especially in tissue therapy. In this study, bovine-derived umbilical cord Wharton’s jelly (UC-WJ) cells were isolated, characterized and maintained under 3D-free serum condition as an alternative of stem cell source for future cell banking.ResultsBovine-derived UC-WJ cells, collected individually from 5 different umbilical cords sources, were successfully cultured under serum-free conditions and were capable to support 60 consecutive passages using commercial Stemline® mesenchymal stem cells expansion medium. Moreover, the UC-WJ cells were differentiated into osteocytes, chondrocytes, adipocytes and neural-like cells and cultured separately. Additionally, the genes that are considered important embryonic, POU5F1 and ITSN1, and mesenchymal cell markers, CD105+, CD29+, CD73+ and CD90+ in MSCs were also expressed in five bovine-derived UC-WJ cultures. Morphology of proliferating cells typically appeared fibroblast-like spindle shape presenting the same viability and number. These characteristics were not affected during passages. There were 60 chromosomes at the metaphase, with acrocentric morphology and intense telomerase activity. Moreover, the proliferative capacity of T cells in response to a mitogen stimulus was suppressed when bovine-derived UC-WJ cells was included in the culture which demonstrated the immunossupression profile typically observed among isolated mesenchymal cells from other species. After classified the UC-WJ cells as mesenchymal stromal phenotype the in vitro 3D cultures was performed using the AlgiMatrix® protocol. Based on the size of spheroids (283,07 μm ± 43,10 μm) we found that three weeks of culture was the best period to growth the UC-WJ cells on 3D dimension. The initial cell density was measured and the best value was 1.5 × 106 cells/well.ConclusionsWe described for the first time the isolation and characterization of UC-WJ cells in a serum-free condition and maintenance of primitive mesenchymal phenotype. The culture was stable under 60 consecutive passages with no genetic abnormalities and proliferating ratios. Taken together all results, it was possible to demonstrate an easy way to isolate and culture of bovine-derived UC-WJ cells under 2D and 3D serum-free condition, from fetal adnexa with a great potential in cell therapy and biotechnology.

The inability of the avascular region of the meniscus to regenerate has led to the use of tissue engineering to treat meniscal injuries. The aim of this study was to evaluate the ability of fibrochondrocytes preseeded on PLDLA/PCL-T [poly(L-co-D,L-lactic acid)/poly(caprolactone-triol)] scaffolds to stimulate regeneration of the whole meniscus. Porous PLDLA/PCL-T (90/10) scaffolds were obtained by solvent casting and particulate leaching. Compressive modulus of 9.5±1.0 MPa and maximum stress of 4.7±0.9 MPa were evaluated. Fibrochondrocytes from rabbit menisci were isolated, seeded directly on the scaffolds, and cultured for 21 days. New Zealand rabbits underwent total meniscectomy, after which implants consisting of cell-free scaffolds or cell-seeded scaffolds were introduced into the medial knee meniscus; the negative control group consisted of rabbits that received no implant. Macroscopic and histological evaluations of the neomeniscus were performed 12 and 24 weeks after implantation. The polymer scaffold implants adapted well to surrounding tissues, without apparent rejection, infection, or chronic inflammatory response. Fibrocartilaginous tissue with mature collagen fibers was observed predominantly in implants with seeded scaffolds compared to cell-free implants after 24 weeks. Similar results were not observed in the control group. Articular cartilage was preserved in the polymeric implants and showed higher chondrocyte cell number than the control group. These findings show that the PLDLA/PCL-T 90/10 scaffold has potential for orthopedic applications since this material allowed the formation of fibrocartilaginous tissue, a structure of crucial importance for repairing injuries to joints, including replacement of the meniscus and the protection of articular cartilage from degeneration.

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BackgroundRecent studies clearly characterize the anatomical parameters of the knee anterolateral ligament (ALL). The potential clinical importance of this ligament is exemplified by some patients with possible combined Anterior Cruciate Ligament (ACL) and ALL rupture who do not progress satisfactorily following isolated ACL reconstruction. Previous biomechanical studies have assessed the resistance parameters of the ALL in order to address potential reconstruction strategies; however, these have reported conflicting results. Thus, this study aimed to evaluate the linear resistance of the ALL by means of a biomechanical study in cadaveric knees.MethodsFourteen cadaveric knees were used. The ALL was dissected, and all structures that connect the femur and the tibia, except for the ALL, were sectioned. The ALL was subjected to a tensile test with the knee around 30 to 40 degrees, in a way that the ALL was aligned with the machine. The strength at the maximum resistance limit, deformation and stiffness of the ALL were evaluated.ResultsThe mean maximum strength of the ALL was 204.8 +/- 114.9 N. The stiffness was 41.9 +/- 25.7 N/mm and the deformation 10.3 +/- 3.5 mm.ConclusionThe ALL has a mean ultimate tensile strength of 204.8 N. This suggests that simple bands of all autologous or homologous grafts commonly used in clinical practice for ligament reconstruction around the knee possess the required biomechanical resistance characteristics for ALL reconstruction.

A sensitive reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the rapid visual detection of turkey coronavirus (TCoV) infection. The reaction is performed in one step in a single tube at 65 °C for 45 min, with hydroxynaphthol blue (HNB) dye added prior to amplification. The detection limit of the RT-LAMP assay was approximately 10(2) EID(50/50 μl) TCoV genome, and no cross-reaction with other avian viruses was observed. The assay was evaluated further in tissue suspensions prepared from the ileum and ileum-caecal junctions of infected turkey embryos; 100% of these samples were positive in the RT-LAMP assay. All individual feces samples collected in the field were considered positive by both conventional RT-PCR and RT-LAMP. In conclusion, RT-LAMP with HNB dye was shown to be a sensitive, simple assay for the rapid diagnosis of TCoV infection, either directly from feces or in association with virus isolation methods.

Background:No consensus exists regarding the anatomic characteristics of the knee anterolateral ligament (ALL). A critical analysis of the dissections described in previous studies allows the division of the ALL into 2 groups with similar characteristics. The presence of considerable variability suggests that the authors may not be referring to the same structure.Purpose/Hypothesis:To perform a lateral anatomic dissection, by layers, seeking to characterize the 2 variants described for the ALL on the same knee. We hypothesized that we would identify the 2 variants described for the ALL and that these variants would have distinct characteristics.Study Design:Descriptive laboratory study.Methods:Thirteen unpaired cadaveric knees were used in this study. The dissection protocol followed the parameters described in previous studies. Immediately below the iliotibial tract, we isolated a structure designated as the superficial ALL, whereas between this structure and the articular capsule, we isolated a structure designated as the deep ALL. The 2 structures were measured for length at full extension and at 90° of flexion and for distance from the tibial insertion relative to the Gerdy tubercle. Potential contact with the lateral meniscus was also evaluated. After measurements were obtained, the 2 dissected structures underwent histologic analysis.Results:The superficial ALL presented a posterior and proximal origin to the center of the lateral epicondyle, its length increased on knee extension, and it exhibited no contact with the lateral meniscus. The deep ALL was located in the center of the lateral epicondyle, its length increased on knee flexion, and it presented a meniscal insertion. Both structures had a similar tibial insertion site; however, the insertion site of the deep ALL was located more posteriorly. The analysis of the histological sections for both structures indicated the presence of dense and well-organized collagen fibers.Conclusion:This anatomic study clearly identified 2 structures, described as the superficial and deep ALL, which were consistent with previous but conflicting descriptions of the ALL.Clinical Relevance:This study clarifies numerous controversies encountered in anatomic studies of the ALL. Knowledge regarding the existence of 2 distinct structures in the anterolateral knee will allow more accurate evaluation of their functions and characteristics.

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