Vascular, cardiac, and renal lesions attributed to primary systemic hypertension in western pygmy marmosets (Cebuella pygmaea).

In a retrospective study of a western pygmy marmoset (Cebuella pygmaea) colony, postmortem examination of 1/8 juvenile and 29/47 adult animals identified vascular, cardiac, and renal lesions consistent with systemic hypertension. This included frequent renal arteriolar hypertrophy, hyaline and proliferative arteriolosclerosis, fibrinoid necrosis of arterioles, glomerulosclerosis, and nephrosclerosis. Affected animals ranged from 0.6 to 12 years of age (mean 6 years) and had an observed male predominance. Genealogical relatedness was evident in several breeding pairs and spanned multiple generations. Concurrent cardiac and renal disease was commonly identified, although frequently subclinical, and both were important causes of morbidity and mortality in affected animals. Cardiomegaly and hypertrophy were typical features and were accompanied by left atrial thrombosis in 10 animals. Signs of heart failure included chronic pulmonary edema in 20 cases and body cavity effusions in 17. In the kidneys, 19 cases had glomerular disease and hypertensive vasculopathy, and 26 cases had nephrosclerosis or glomerulosclerosis. Common extrarenal secondary causes of hypertension were excluded by necropsy examination. The pathogenesis is suggested to involve primary hypertension leading to renal and cardiac disease. Elevated sympathetic activity might be an underlying factor in the frequent development of primary systemic hypertension in the pygmy marmoset, as for the owl monkey.

Effects of antigen removal on a porcine osteochondral xenograft for articular cartilage repair.

Given the limited availability of fresh osteochondral allografts and uncertainty regarding performance of decellularized allografts, this study was undertaken as part of an effort to develop an osteochondral xenograft for articular cartilage repair. The purpose was to evaluate a simple antigen removal procedure based mainly on treatment with SDS and nucleases. Histology demonstrated a preservation of collagenous structure and removal of most nuclei. Immunohistochemistry revealed the apparent retention of α-Gal within osteocyte lacunae unless the tissue underwent an additional α-galactosidase processing step. Cytoplasmic protein was completely removed as shown by Western blot. Quantitatively, the antigen removal protocol was found to extract approximately 90% of DNA from cartilage and bone, and it extracted over 80% of glycosaminoglycan from cartilage. Collagen content was not affected. Mechanical testing of cartilage and bone were performed separately, in addition to testing the cartilage-bone interface, and the main effect of antigen removal was an increase in cartilage hydraulic permeability. In vivo immunogenicity was assessed by subcutaneous implantation into DBA/1 J mice, and the response was typical of a foreign body rather than immune reaction. Thus, an osteochondral xenograft produced as described has the potential for further development into a treatment for osteochondral lesions in the human knee. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2251-2260, 2018.

Comparison of natural crosslinking agents for the stabilization of xenogenic articular cartilage.

Osteochondral xenografts are potentially inexpensive, widely available alternatives to fresh allografts. However, antigen removal from xenogenic cartilage may damage the extracellular matrix and reduce compressive stiffness. Non-crosslinked xenogenic cartilage may also undergo rapid enzymatic degradation in vivo. We hypothesized that natural crosslinking agents could be used in place of glutaraldehyde to improve the mechanical properties and enzymatic resistance of decellularized cartilage. This study compared the effects of genipin (GNP), proanthocyanidin (PA), and epigallocatechin gallate (EGCG), on the physical and mechanical properties of decellularized porcine cartilage. Glutaraldehyde (GA) served as a positive control. Porcine articular cartilage discs were decellularized in 2% sodium dodecyl sulfate and DNase I followed by fixation in 0.25% GNP, 0.25% PA, 0.25% EGCG, or 2.5% GA. Decellularization decreased DNA by 15% and GAG by 35%. For natural crosslinkers, the average degree of crosslinking ranged from approximately 50% (EGCG) to 78% (GNP), as compared to 83% for the GA control. Among the natural crosslinkers, only GNP significantly affected the disc diameter, and shrinkage was under 2%. GA fixation had no significant effect on disc diameter. Decellularization decreased aggregate modulus; GA and GNP, but not EGCG and PA, were able to restore it to its original level. GNP, PA, and GA conferred a similar, almost complete resistance to collagenase degradation. EGCG also conferred substantial resistance but to a lesser degree. Overall, the data support our hypothesis and suggest that natural crosslinkers may be suitable alternatives to glutaraldehyde for stabilization of decellularized cartilage. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1037-1046, 2016.

Granulomatous lymphadenitis caused by Talaromyces helicus in a Labrador Retriever.

A 3-year-old spayed female Labrador Retriever was presented for right prescapular lymphadenomegaly. Examination of fine-needle aspirates and impression smears of the node revealed many short hyphal structures found within macrophages and extracellularly. Hyphae were approximately 3 µm in diameter, were irregularly septate with nonparallel walls, and had a small clear halo surrounding a partially stained basophilic internal structure. Hyphae were tapered on one end and had oval to pyriform swellings of 7-10 µm on the other, resulting in a bulbous appearance. Fungal elements stained positively with Gomori methenamine silver and Periodic acid-Schiff stains. The dog was euthanized at the owner's request, and necropsy revealed marked peripheral and visceral lymphadenomegaly. Histopathologic examination of lymph nodes confirmed granulomatous lymphadenitis with many fungal hyphae. Fungal culture yielded pure cultures of organisms that failed to produce ascospores or conidia precluding morphologic identification. PCR was performed using pan-fungal primers, ITS-1 and ITS-2, to amplify the intergenic spacer regions of ribosomal RNA; the PCR product was sequenced and a BLAST search of the GenBank databases at NCBI revealed 100% identity of the organism with Talaromyces helicus, the sexual form of Penicillium helicum. Talaromyces helicus has not previously been reported to cause disease in people or animals.

Effect of a mechanical stimulation bioreactor on tissue engineered, scaffold-free cartilage.

Achieving sufficient functional properties prior to implantation remains a significant challenge for the development of tissue engineered cartilage. Many studies have shown chondrocytes respond well to various mechanical stimuli, resulting in the development of bioreactors capable of transmitting forces to articular cartilage in vitro. In this study, we describe the production of sizeable, tissue engineered cartilage using a novel scaffold-free approach, and determine the effect of perfusion and mechanical stimulation from a C9-x Cartigen bioreactor on the properties of the tissue engineered cartilage. We created sizable tissue engineered cartilage from porcine chondrocytes using a scaffold-free approach by centrifuging a high-density chondrocyte cell-suspension onto an agarose layer in a 50 mL tube. The gross and histological appearances, biochemical content, and mechanical properties of constructs cultured in the bioreactor for 4 weeks were compared to constructs cultured statically. Mechanical properties were determined from unconfined uniaxial compression tests. Constructs cultured in the bioreactor exhibited an increase in total GAG content, equilibrium compressive modulus, and dynamic modulus versus static constructs. Our study demonstrates the C9-x CartiGen bioreactor is able to enhance the biomechanical and biochemical properties of scaffold-free tissue engineered cartilage; however, no additional enhancement was seen between loaded and perfused groups.

Production of hyaline-like cartilage by bone marrow mesenchymal stem cells in a self-assembly model.

A scaffoldless or self-assembly approach to cartilage tissue engineering has been used to produce hyaline cartilage from bone marrow-derived mesenchymal stem cells (bMSCs), but the mechanical properties of such engineered cartilage and the effects the transforming growth factor (TGF) isoform have not been fully explored. This study employs a cell culture insert model to produce tissue-engineered cartilage using bMSCs. Neonatal pig bMSCs were isolated by plastic adherence and expanded in monolayer before being seeded into porous transwell inserts and cultured for 4 or 8 weeks in defined chondrogenic media containing either TGF-beta1 or TGF-beta3. Following biomechanical evaluation in confined compression, colorimetric dimethyl methylene blue and Sircol dye-binding assays were used to analyze glycosaminoglycan (GAG) and collagen contents, respectively. Histological sections were stained with toluidine blue for proteoglycans and with picrosirius red to reveal collagen orientation, and immunostained for detection of collagen types I and II. Neocartilage increased in thickness, collagen, and GAG content between 4 and 8 weeks. Proteoglycan concentration increased with depth from the top surface. The tissue contained much more collagen type II than type I, and there was a consistent pattern of collagen alignment. TGF-beta1-treated and TGF-beta3-treated constructs were similar at 4 weeks, but 8-week TGF-beta1 constructs had a higher aggregate modulus and GAG content compared to TGF-beta3. These results demonstrate that bMSCs can generate functional hyaline-like cartilage through a self-assembling process.