Clinical anatomy of the meniscus in animal models: pros and cons.

Nowadays, despite the possibility to use in vitro or computer models in research, animal models are still essential. Different animal models are available for meniscal repair investigation. Although a unique perfect model for the structure of the human's knee does not exist, the choice of the proper animal model is crucial for a correct research. The principal animal models in the meniscal repair are sheep, goats, pigs and dogs. Each of these has pros and cons for their utilization. Analysing each pro and con is essential for optimizing the choice of the animal model, which depends on the experimental question, avoiding unnecessary waste of resources and minimizing the animal suffering, according to the Russell and Burch's three "Rs" principles (Reduce, Refine and Recycle). In this concise review, we resume the meniscus anatomical features of the main large animals, to help choose the most suitable animal model for subsequent studies on meniscal repair.

Histological assessment of new bone formation with biomimetic scaffold in the presence of bone loss in trauma surgery.

Open reduction and internal fixation (ORIF) surgery may require the use of bone grafts (usually allogeneic). In the context of traumatology surgeries, the use of autologous grafts is almost never used and allogeneic grafts are not always available. In recent years, bone substitutes have been introduced in clinical practice to overcome these limitations. The purpose of this paper is to report two cases in which the use of a bone substitute was used to overcome the bone loss during surgeries of ORIF. Two patients, one with a tibial plateau fracture (Schatzker 6) and one with a proximal humerus fracture (Neer 4), underwent ORIF surgery. In both cases, due to a loss of bone stock, a synthetic bone substitute (OrthOss®) was used. One year after surgery, the complete osseointegration of the synthetic bone substitute was seen, both radiologically and histologically. This bone substitute may represent a safe and effective alternative to autologous bone grafts, avoiding adverse events related to donor-site morbidity.

Decorin age-related variations in the distribution of pig extracellular matrix meniscus.

Menisci act like shock absorbers and transmit load across the tibiofemoral joint by increasing congruency during movements or body weight load. This leads to decreasing the resultant stress on the articular cartilages. The meniscus has a dense extracellular matrix (ECM) composed of water, different types of collagens, and proteoglycans, such as decorin, aggrecan and biglycan. Decorin (DCN) regulates collagen fibrillogenesis acting on collagen fibrils diameter and fibrils orientation to achieve the proper assembly of its network. This work investigates the spatial disposition of this fundamental protein in pig meniscus' matrix by immunohistochemistry and western blot analysis. DCN shows an increasing trend, moving from neonatal to adult pig menisci. Adult meniscus, in porcine species, is the only one that could be considered fully mature and functional, and, even if an increasing trend is seen, no precise phenotypical switch points are seen in the age stages considered in this study.

Age assessment in puppies: Coming to terms with forensic requests.

Age estimation in growing dogs is crucial not only in clinical practice but increasingly so in forensic practice as well. In the last few years, it has assumed great importance for correctly identifying the age of puppies illegally imported to Italy as well as to other European countries. Puppies are, in fact, transported when they are too young to be moved, which can cause both animal/public health and animal welfare issues. Therefore, the movement of animals within the European Community is governed by strict rules, and veterinarians are often required to evaluate the age of the imported puppies in a forensic scenario as accurately as possible. To date, X-ray evaluation of limb bones ossification centers (OCs) is generally accepted as a valid tool to assess the age of puppies. A wealth of information exists on this topic but it is not always easily available. This work is a historical review of the existing literature and proposes two tables illustrating the timelines of limb OCs appearance and closure, coming to terms with forensic requests to evaluate the age of a puppy. The timelines reported indicate the need to improve methodology to enhance the accuracy and to reduce the error in age estimation.

Small-sized newborn dogs skeletal development: radiologic, morphometric, and histological findings obtained from spontaneously dead animals.

BACKGROUND: Very little is known about neonatal skeletal development in small-sized purebred dogs. In order to improve this knowledge, 27 spontaneously dead puppies belonging to small-sized breeds were enrolled in this study for radiologic, histological and morphometric investigations. RESULTS: The appearance of the limb secondary ossification centers and the onset of their formation were clearly observed by x rays and confirmed by histological evidences. Radiographic and anatomic measurements of limb bones length and skull length and width were positively correlated with body weight and age of the subjects and the body weight was positively correlated with radius bone mineral density, as demonstrated by dual-energy x-rays absorptiometry. CONCLUSIONS: These data provided original information on the growth of newborn small-sized breed dogs, and suggest that cadavers may be useful to study skeletal development.

Cartilage canals in newborn dogs: histochemical and immunohistochemical findings.

Cartilage canals (CCs) are microscopic structures involved in secondary ossification centers (SOCs) development. The features of CCs were investigated in the humeral and femoral proximal epiphyses of small-sized newborn dogs (from premature to 28 days after birth) with histochemical and immunohistochemical approaches. Masson's Trichrome revealed a ring-shaped area around CCs, which changes in colour from green (immature collagen) to red (mature collagen) as ossification progresses; perichondrium staining always matched the ring colour. Safranin-O was always negative. Immunohistochemical analysis revealed immunopositivity for both collagen type I and V around the CCs; collagen type II was negative. CCs count showed a tendency to be higher in the humerus than in the femur. This work enlightened for the first time changes in composition of CCs surrounding matrix during SOCs development in dogs, paving the way to further investigations.

Effect of vitrification of feline ovarian cortex on follicular and oocyte quality and competence.

Cryopreservation of ovarian cortex has important implications in the preservation of fertility and biodiversity in animal species. Slow freezing of cat ovarian tissue resulted in the preservation of follicular morphology and in the follicular development after xenografting. Vitrification has been recently applied to ovarian tissues of different species, but no information is available on the effect of this method on feline ovarian cortex. Moreover, meiotic competence of fully grown oocytes isolated from cryopreserved tissue has not been reported. The aim of this study was to evaluate the effect of vitrification of feline ovarian cortex on follicular morphology and oocyte integrity, as well as meiotic competence. A total of 352 fragments (1.5-2 mm(3) ) were obtained from ovarian cortical tissues: 176 were vitrified and 176 were used fresh as control. Histological evaluation of fresh and vitrified fragments showed intact follicles after cryopreservation procedures with no statistically significant destructive effect from primordial to antral follicles. After IVM, oocytes collected from vitrified ovarian fragment showed a higher proportion of gametes arrested at germinal vesicle (GV) stage compared to those isolated from fresh control tissue (33.8% vs 2.9%; p < 0.001). However, oocytes isolated from vitrified tissues were able to resume meiosis, albeit at lower rate than those collected from fresh tissues (39.8% vs 85.9%; p < 0.00001). Vitrification induced changes in the organization of cytoskeletal elements (actin microfilaments and microtubules) of oocytes, but significantly only for actin network (p < 0.001). Finally, chromatin configuration within the GV was not affected by the cryopreservation procedure. Our study demonstrated that vitrification preserves the integrity of ovarian follicles and that oocytes retrieved from cryopreserved tissue maintain the capability of resuming meiosis. To our knowledge, this has not previously been reported in the cat.

The endothelial nitric oxide synthase/nitric oxide system is involved in the defective quality of bovine oocytes from low mid-antral follicle count ovaries.

In a previous survey concerning cows of reproductive age, we demonstrated that oocytes isolated from ovaries with <10 medium antral follicles of 2 to 6 mm in diameter (low ovaries; Lo) show less developmental competence than oocytes collected from ovaries with >10 medium antral follicles (high ovaries; Hi). The aim of the present study was to evaluate whether a defective endothelial nitric oxide synthase/nitric oxide (eNOS/NO) system and vasculature in healthy medium antral follicles is likely to reduce oocyte competence from Lo ovaries. Thus, experiments were conducted to 1) immunolocalize eNOS protein during folliculogenesis; 2) quantify eNOS protein/vasculature in the follicle wall; and 3) verify if NO donor, S-nitroso acetyl penicillamine (SNAP) administration during in vitro maturation affects developmental competence of oocytes isolated from Lo ovaries. Endothelial nitric oxide synthase protein was detected in granulosa and theca cells, as well as in blood vessels from primordial to antral follicles. Quantitative analysis indicated that in medium antral follicles from Lo ovaries, eNOS protein expression and vasculature were reduced (P < 0.05). The addition of SNAP improved blastocyst and hatching rates of oocytes from Lo ovaries, promoting a percentage similar to oocytes from Hi ovaries, and reduced the percentage of apoptotic nuclei in in vitro-produced blastocysts (P < 0.05). Results from our study suggest that in bovine ovaries with small mid antral follicle number, a defective eNOS/NO system is related to a reduced follicle vasculature and may affect oocyte quality, thus inducing a premature decline of fertility.

Localization of DNA methyltransferase-1 during oocyte differentiation, in vitro maturation and early embryonic development in cow.

DNA methyltransferase-1 (Dnmt1) is involved in the maintenance of DNA methylation patterns and is crucial for normal mammalian development. The aim of the present study was to assess the localization of Dnmt1 in cow, during the latest phases of oocyte differentiation and during the early stages of segmentation. Dnmt1 expression and localization were assessed in oocytes according to the chromatin configuration, which in turn provides an important epigenetic mechanism for the control of global gene expression and represents a morphological marker of oocyte differentiation. We found that the initial chromatin condensation was accompanied by a slight increase in the level of global DNA methylation, as assessed by 5-methyl-cytosine immunostaining followed by laser scanning confocal microscopy analysis (LSCM). RT-PCR confirmed the presence of Dnmt1 transcripts throughout this phase of oocyte differentiation. Analogously, Dnmt1 immunodetection and LSCM indicated that the protein was always present and localized in the cytoplasm, regardless the chromatin configuration and the level of global DNA methylation. Moreover, our data indicate that while Dnmt1 is retained in the cytoplasm in metaphase II stage oocytes and zygotes, it enters the nuclei of 8-16 cell stage embryos. As suggested in mouse, the functional meaning of the presence of Dnmt1 in the bovine embryo nuclei could be the maintainement of the methylation pattern of imprinted genes. In conclusion, the present work provides useful elements for the study of Dnmt1 function during the late stage of oocyte differentiation, maturation and early embryonic development in mammals.