Hypoxia as a Stimulus for the Maturation of Meniscal Cells: Highway to Novel Tissue Engineering Strategies?

The meniscus possesses low self-healing properties. A perfect regenerative technique for this tissue has not yet been developed. This work aims to evaluate the role of hypoxia in meniscal development in vitro. Menisci from neonatal pigs (day 0) were harvested and cultured under two different atmospheric conditions: hypoxia (1% O2) and normoxia (21% O2) for up to 14 days. Samples were analysed at 0, 7 and 14 days by histochemical (Safranin-O staining), immunofluorescence and RT-PCR (in both methods for SOX-9, HIF-1α, collagen I and II), and biochemical (DNA, GAGs, DNA/GAGs ratio) techniques to record any possible differences in the maturation of meniscal cells. Safranin-O staining showed increments in matrix deposition and round-shape "fibro-chondrocytic" cells in hypoxia-cultured menisci compared with controls under normal atmospheric conditions. The same maturation shifting was observed by immunofluorescence and RT-PCR analysis: SOX-9 and collagen II increased from day zero up to 14 days under a hypoxic environment. An increment of DNA/GAGs ratio typical of mature meniscal tissue (characterized by fewer cells and more GAGs) was observed by biochemical analysis. This study shows that hypoxia can be considered as a booster to achieve meniscal cell maturation, and opens new opportunities in the field of meniscus tissue engineering.

Postnatal morpho-functional development of a dog's meniscus.

This study evaluates the morpho-functional modifications that characterize meniscal development from neonatal to adult dogs. Even if menisci are recognized as essential structures for the knee joint, poor information is available about their morphogenesis, in particular in dog models. Menisci from a group of Dobermann Pinchers aged 0, 10, 30 days, and 4 years (T0, T10, T30, adult, respectively) were analyzed by SEM, histochemistry (Safranin O and Picro Sirius Red Staining analyzed under a polarized light microscope), immunofluorescences (collagen type I and II), biomechanical (compression) and biochemical analyses (glycosaminoglycans, GAGs, and DNA content). SEM analyses revealed that the T0 meniscus is a bulgy structure that during growth tends to flatten, firstly in the inner zone (T10) and then even in the outer zone (T30), until the achievement of the completely smooth adult final shape. These results were further supported by the histochemistry analyses in which the deposition of GAGs started from T30, and the presence of type I birefringent collagen fibers was observed from T0 to T30, while poorly refringent type III collagen fibers were observed in the adult dogs. Double immunofluorescence analyses also evidenced that the neonatal meniscus contains mainly type I collagen fibers, as well as the T10 meniscus, and demonstrated a more evident regionalization and crimping in the T30 and adult meniscus. Young's elastic modulus of the meniscus in T0 and T10 animals was lower than the T30 animals, and this last group was also lower than adult ones (T0-T10 vs T30 vs adult). Biochemical analysis confirmed that cellularity decreases over time from neonatal to adult (p < 0.01). The same decreasing trend was observed in GAGs deposition. These results may suggest that the postnatal development of canine meniscus may be related to the progressive functional locomotory development: after birth, the meniscus acquires its functionality over time, through movement, load, and growth itself.

Hind limb ossification centre hypoplasia and deformities induced by quadriceps contracture: Radiographic and Computed Tomographic study in 13 Doberman Pinscher littermates.

Quadriceps contracture (QC) is reported in dogs mainly as a complication of trauma or parasitic infection. QC causes progressive hind limb deviation, muscular hypotrophy and degenerative joint disease and, in puppies, bone hypoplasia. The aim of this clinical case series is to describe the radiographic and computed tomographic (CT) changes in hind limb ossification centres in thirteen 55 to 57-days-old Doberman Pinscher related littermates induced by QC after repeated intramuscular injections. The presence, size, and shape of ossification centres of affected and unaffected hind limbs were compared. Affected limbs were hyperextended and externally rotated, with genu recurvatum and proximo-medial patellar luxation. QC had no influence on the time of appearance of ossification centres however, it was associated with femoral head flattening, hip subluxation, flattening of the femoral distal epiphysis. The tibial plateau was tilted caudoproximally-craniodistally and wedged into the growth plate. Thirty-two out of fifty-five ossification centres (including diaphyseal and epiphyseal centres, such as femoral head and tibial plateau) were significantly smaller in affected limbs (p < 0.05). Lack of weight-bearing could account for the smaller size of ossification centres in affected tarsi and metatarsi. Progressive limb hyperextension and external rotation might have induced gradual loading withdrawal on the medial aspect of the foot justifying the reduced size observed only for the medial ossification centres of the digits of affected limbs. To the best of our knowledge, this is the first study describing CT findings of hind limb ossification centre changes in puppies with QC contracture.

Meniscus Matrix Remodeling in Response to Compressive Forces in Dogs.

Joint motion and postnatal stress of weight bearing are the principal factors that determine the phenotypical and architectural changes that characterize the maturation process of the meniscus. In this study, the effect of compressive forces on the meniscus will be evaluated in a litter of 12 Dobermann Pinschers, of approximately 2 months of age, euthanized as affected by the quadriceps contracture muscle syndrome of a single limb focusing on extracellular matrix remodeling and cell-extracellular matrix interaction (i.e., meniscal cells maturation, collagen fibers typology and arrangement). The affected limbs were considered as models of continuous compression while the physiologic loaded limbs were considered as controls. The results of this study suggest that a compressive continuous force, applied to the native meniscal cells, triggers an early maturation of the cellular phenotype, at the expense of the proper organization of collagen fibers. Nevertheless, an application of a compressive force could be useful in the engineering process of meniscal tissue in order to induce a faster achievement of the mature cellular phenotype and, consequently, the earlier production of the fundamental extracellular matrix (ECM), in order to improve cellular viability and adhesion of the cells within a hypothetical synthetic scaffold.

Nutritional Regulation of Gut Barrier Integrity in Weaning Piglets.

Weaning is very stressful for piglets and leads to alterations in the intestinal barrier, a reduction in nutrient absorption and a higher susceptibility to intestinal diseases with heavy economic losses. This review describes the structures involved in the intestinal barrier: the epithelial barrier, immune barrier and the enteric nervous system. Here, new insights into the interactions between feed components and the physiology and morphology of the epithelial barrier are highlighted. Dietary strategies focused on improving gut health are also described including amino acids, phytochemicals and organic acids.

Swine Meniscus: Are Femoral-Tibial Surfaces Properly Tuned to Bear the Forces Exerted on the Tissue?

IMPACT STATEMENT: The importance of the present study is linked to how the contact forces act on the knee meniscus in particular, considering the femoral condyles and tibial plateau: this can be useful as a base for the ultimate creation of tissue-engineered biphasic scaffolds, which can mimic the native tissue complex, for meniscal repair or regeneration.

Novel contributions in canine craniometry: Anatomic and radiographic measurements in newborn puppies.

The largest differences in intraspecific head shape among the Carnivora order are to be found in dogs. Based on their skull morphotypes, dog breeds are currently classified as dolichocephalic, mesaticephalic and brachycephalic. Due to the fact that some breeds have not been yet defined, this classification is incomplete; moreover, multi-breed studies on the skull morphology of puppies have never been performed. The aim of this work was to verify (i) whether differences in the skull conformation of purebred puppies are already present within the first week of age; (ii) whether radiographic and anatomic measures could be considered interchangeable, and (iii) to possibly classify puppies from non-categorized breeds thanks to their radiographic cranial measurements using neural nets. One hundred and thirty-seven dead puppies aged 0-7 days were examined considering their anatomic and radiographic measures. All linear measures and anatomic indices significantly differed among brachycephalic and non-brachycephalic puppies. Radiographic indices, with the exception of CI, identified the three skull morphotypes (p<0.05, for all comparisons). Radiographic and anatomic measures proved to be non-interchangeable in newborn puppies. Finally, nineteen puppies belonging to 5 non-categorized breeds could be classified thanks to neural nets in the three skull morphotypes with different probability (P between 0,66 and 0,95).