Age-related modulation of angiogenesis-regulating factors in the swine meniscus.

An in-depth knowledge of the native meniscus morphology and biomechanics in its different areas is essential to develop an engineered tissue. Meniscus is characterized by a great regional variation in extracellular matrix components and in vascularization. Then, the aim of this work was to characterize the expression of factors involved in angiogenesis in different areas during meniscus maturation in pigs. The menisci were removed from the knee joints of neonatal, young and adult pigs, and they were divided into the inner, intermediate and outer areas. Vascular characterization and meniscal maturation were evaluated by immunohistochemistry and Western blot analysis. In particular, expression of the angiogenic factor Vascular Endothelial Growth Factor (VEGF) and the anti-angiogenic marker Endostatin (ENDO) was analysed, as well as the vascular endothelial cadherin (Ve-CAD). In addition, expression of Collagen II (COLL II) and SOX9 was examined, as markers of the fibro-cartilaginous differentiation. Expression of VEGF and Ve-CAD had a similar pattern in all animals, with a significant increase from the inner to the outer part of the meniscus. Pooling the zones, expression of both proteins was significantly higher in the neonatal meniscus than in young and adult menisci. Conversely, the young meniscus revealed a significantly higher expression of ENDO compared to the neonatal and adult ones. Analysis of tissue maturation markers showed an increase in COLL II and a decrease in SOX9 expression with age. These preliminary data highlight some of the changes that occur in the swine meniscus during growth, in particular the ensemble of regulatory factors involved in angiogenesis.

Meniscus maturation in the swine model: changes occurring along with anterior to posterior and medial to lateral aspect during growth.

The meniscus plays important roles in knee function and mechanics and is characterized by a heterogeneous matrix composition. The changes in meniscus vascularization observed during growth suggest that the tissue-specific composition may be the result of a maturation process. This study has the aim to characterize the structural and biochemical variations that occur in the swine meniscus with age. To this purpose, menisci were collected from young and adult pigs and divided into different zones. In study 1, both lateral and medial menisci were divided into the anterior horn, the body and the posterior horn for the evaluation of glycosaminoglycans (GAGs), collagen 1 and 2 content. In study 2, the menisci were sectioned into the inner, the intermediate and the outer zones to determine the variations in the cell phenotype along with the inner-outer direction, through gene expression analysis. According to the results, the swine meniscus is characterized by an increasing enrichment in the cartilaginous component with age, with an increasing deposition in the anterior horn (GAGs and collagen 2; P < 0.01 both); moreover, this cartilaginous matrix strongly increases in the inner avascular and intermediate zone, as a consequence of a specific differentiation of meniscal cells towards a cartilaginous phenotype (collagen 2, P < 0.01). The obtained data add new information on the changes that accompany meniscus maturation, suggesting a specific response of meniscal cells to the regional mechanical stimuli in the knee joint.

Necdin mediates skeletal muscle regeneration by promoting myoblast survival and differentiation.

Regeneration of muscle fibers that are lost during pathological muscle degeneration or after injuries is sustained by the production of new myofibers. An important cell type involved in muscle regeneration is the satellite cell. Necdin is a protein expressed in satellite cell-derived myogenic precursors during perinatal growth. However, its function in myogenesis is not known. We compare transgenic mice that overexpress necdin in skeletal muscle with both wild-type and necdin null mice. After muscle injury the necdin null mice show a considerable defect in muscle healing, whereas mice that overexpress necdin show a substantial increase in myofiber regeneration. We also find that in muscle, necdin increases myogenin expression, accelerates differentiation, and counteracts myoblast apoptosis. Collectively, these data clarify the function and mechanism of necdin in skeletal muscle and show the importance of necdin in muscle regeneration.

Follistatin induction by nitric oxide through cyclic GMP: a tightly regulated signaling pathway that controls myoblast fusion.

The mechanism of skeletal myoblast fusion is not well understood. We show that endogenous nitric oxide (NO) generation is required for myoblast fusion both in embryonic myoblasts and in satellite cells. The effect of NO is concentration and time dependent, being evident only at the onset of differentiation, and direct on the fusion process itself. The action of NO is mediated through a tightly regulated activation of guanylate cyclase and generation of cyclic guanosine monophosphate (cGMP), so much so that deregulation of cGMP signaling leads to a fusion-induced hypertrophy of satellite-derived myotubes and embryonic muscles, and to the acquisition of fusion competence by myogenic precursors in the presomitic mesoderm. NO and cGMP induce expression of follistatin, and this secreted protein mediates their action in myogenesis. These results establish a hitherto unappreciated role of NO and cGMP in regulating myoblast fusion and elucidate their mechanism of action, providing a direct link with follistatin, which is a key player in myogenesis.

Development and biological validation of a cyclic stretch culture system for the ex vivo engineering of tendons.

INTRODUCTION: An innovative approach to the treatment of tendon injury or degeneration is given by engineered grafts, made available through the development of bioreactors that generate tendon tissue in vitro, by replicating in vivo conditions. This work aims at the design of a bioreactor capable of applying a stimulation of cyclic strain on cell constructs to promote the production of bioartificial tissue with mechanical and biochemical properties resembling those of the native tissue. METHODS: The system was actuated by an electromagnet and design specifications were imposed as follows. The stimulation protocol provides to scaffolds a 3% preload, a 10% deformation, and a stimulation frequency rate set at 0.5, 1, and 2 Hz, which alternates stimulation/resting phases. Porcine tenocytes were seeded on collagen scaffolds and cultured in static or dynamic conditions for 7 and 14 days. RESULTS: The culture medium temperature did not exceed 37°C during prolonged culture experiments. The applied force oscillates between 1.5 and 4.5 N. The cyclic stimulation of the engineered constructs let both the cells and the scaffold fibers align along the strain direction in response to the mechanical stimulus. CONCLUSION: We designed a pulsatile strain bioreactor for tendon tissue engineering. The in vitro characterization shows a preferential cell alignment at short time points. Prolonged culture time, however, seems to influence negatively on the survival of the cells indicating the need of further optimization concerning the culture conditions and the mechanical stimulation.

Animal models for meniscus repair and regeneration.

The meniscus plays an important role in knee function and mechanics. Meniscal lesions, however, are common phenomena and this tissue is not able to achieve spontaneous successful repair, particularly in the inner avascular zone. Several animal models have been studied and proposed for testing different reparative approaches, as well as for studying regenerative methods aiming to restore the original shape and function of this structure. This review summarizes the gross anatomy, function, ultrastructure and biochemical composition of the knee meniscus in several animal models in comparison with the human meniscus. The relevance of the models is discussed from the point of view of basic research as well as of clinical translation for meniscal repair, substitution and regeneration. Finally, the advantages and disadvantages of each model for various research directions are critically discussed.

Osteochondral repair by a novel interconnecting collagen-hydroxyapatite substitute: a large-animal study.

A novel three-dimensional bicomponent substitute made of collagen type I and hydroxyapatite was tested for the repair of osteochondral lesions in a swine model. This scaffold was assembled by a newly developed method that guarantees the strict integration between the organic and the inorganic parts, mimicking the biological tissue between the chondral and the osseous phase. Thirty-six osteochondral lesions were created in the trochlea of six pigs; in each pig, two lesions were treated with scaffolds seeded with autologous chondrocytes (cell+group), two lesions were treated with unseeded scaffolds (cell- group), and the two remaining lesions were left untreated (untreated group). After 3 months, the animals were sacrificed and the newly formed tissue was analyzed to evaluate the degree of maturation. The International Cartilage Repair Society (ICRS) macroscopic assessment showed significantly higher scores in the cell- and untreated groups when compared with the cell+ group. Histological evaluation showed the presence of repaired tissue, with fibroblast-like and hyaline-like areas in all groups; however, with respect to the other groups, the cell- group showed significantly higher values in the ICRS II histological scores for "cell morphology" and for the "surface/superficial assessment." While the scaffold seeded with autologous chondrocytes promoted the formation of a reparative tissue with high cellularity but low glycosaminoglycans (GAG) production, on the contrary, the reparative tissue observed with the unseeded scaffold presented lower cellularity but higher and uniform GAG distribution. Finally, in the lesions treated with scaffolds, the immunohistochemical analysis showed the presence of collagen type II in the peripheral part of the defect, indicating tissue maturation due to the migration of local cells from the surroundings. This study showed that the novel osteochondral scaffold was easy to handle for surgical implantation and was stable within the site of lesion; at the end of the experimental time, all implants were well integrated with the surrounding tissue and no signs of synovitis were observed. The quality of the reparative tissue seemed to be superior for the lesions treated with the unseeded scaffolds, indicating the promising potential of this novel biomaterial for use in a one-stage procedure for osteochondral repair.

Collagen scaffold for cartilage tissue engineering: the benefit of fibrin glue and the proper culture time in an infant cartilage model.

This study (i) developed a scaffold made of collagen I designed for hosting the autologous chondrocytes, (ii) focused on the optimization of chondrocytes seeding by the addition of the fibrin glue, and (iii) investigated the culture time for the ideal scaffold maturation in vitro. In the first part of the study, fresh chondrocytes were isolated from infant swine articular cartilage, and immediately seeded onto the collagen sponges either in medium or in fibrinogen in order to show the contribute of fibrin glue in cell seeding and survival into the scaffold. In the second part of the study, chondrocytes were first expanded in vitro and then resuspended in fibrinogen, seeded in collagen sponges, and cultured for 1, 3, and 5 weeks in order to identify the optimal time for the rescue of cell phenotype and for the scaffold maturation into a tissue with chondral properties. The histological and immunohistochemical data from the first part of the study (study with primary chondrocytes) demonstrated that the presence of fibrin glue ameliorated cell distribution and survival into the chondral composites. The second part of this work (study with dedifferentiated chondrocytes) showed that the prolongation of the culture to 3 weeks promoted a significant restoration of the cell phenotype, resulting in a composite with proper morphological features, biochemical composition, and mechanical integrity. In conclusion, this study developed a collagenic-fibrin glue scaffold that was able to support chondrocyte survival and synthetic activity in a static culture; in particular, this model was able to turn the engineered samples into a tissue with chondral-like properties when cultured in vitro for at least 3 weeks.

Changes in nitrosative stress biomarkers in swine intestine following dietary intervention with verbascoside.

In farm animals, oxidative stress can be involved in several intestinal pathological disorders, and many antioxidant molecules, especially those of plant origin, can counteract free radicals, thus stabilizing the gut environment and enhancing health. The aim of the study was to investigate whether the use of verbascoside (VB), a polyphenol plant compound, in pig feeding could modulate oxidative and/or nitrosative stress in the gut. Eighteen male piglets (Dalland) were assigned to two groups, which were fed with either a control diet (CON) or a diet supplemented with 5 mg/kg of verbascoside (VB) for 166 days. At slaughter, duodenum and jejunum specimens were collected. Immunohistochemistry and Western blot analyses were performed on the samples to evaluate free radical adducts, including acrolein (ACR), 8-hydroxydeoxyguanosine (8-OHdg) and nitrotyrosine (NT). A KRL test was also used to assess the total blood antioxidant activity, and no difference was observed. Immunohistochemistry and Western blot showed that dietary treatment decreased the levels of nitrotyrosine in enteroendocrine cell populations(P<0.05). Characterization of the enteroendocrine cell typology was then performed, and serotonin-immunoreactive cells were revealed to be directly involved in decreasing the nitrosative stress status. This preliminary study demonstrates the important role of dietary VB in decreasing stress biomarkers in swine gut, thus highlighting a possible intervention aimed at building a large prospective for antioxidant dietary supplementation in food animal species.

Distribution of ghrelin-producing cells in the gastrointestinal tract of pigs at different ages.

Ghrelin is involved in many biological processes, ranging from appetite regulation and the release of growth hormone to the regulation of gastrointestinal motility and secretion processes. Ghrelin expression is not homogenously distributed throughout the gastrointestinal tract; expression is species-specific and can also depend on the animal age. This study was performed to investigate ghrelin immunolocalization in the gastrointestinal tract of pigs at different ages: 1 day (birth), 28 days (weaning), 2 months, 4 months, and 7 months (pre-puberty). Tissue samples were collected along the entire gastrointestinal tract and were examined by immunohistochemistry and double-immunofluorescence. Histometry was performed by counting the number of endocrine ghrelin immunopositive cells in the gastrointestinal mucosa. Ghrelin was found to be present along the swine alimentary canal from the stomach to the caecum. In all regions of the alimentary canal of the animals studied, ghrelin-immunoreactive (IR) cells co-localized with chromogranin-A and were therefore identified as endocrine cells. In the gastric fundus, ghrelin-immunoreactivity was partially detected in co-localization with H-K-adenosine triphosphatase and pepsinogen. Ghrelin-IR endocrine cells were abundant in the oxyntic mucosa but less present in the small intestine and rare in the large intestine. The cell density of the ghrelin-IR endocrine cells was lowest in the oxyntic mucosa of 1-day-old pigs. We can conclude that gastric ghrelin expression is not related merely to age but could also potentially be influenced by food intake.

Fibrin-based model for cartilage regeneration: tissue maturation from in vitro to in vivo.

One of the crucial points for a successful tissue-engineering approach for cartilage repair is represented by the level of in vitro maturation of the engineered tissue before implantation. The purpose of this work was to evaluate the effect of the level of in vitro maturation of engineered cartilaginous samples on the tissue quality after in vivo implantation. Samples were obtained from isolated swine articular chondrocytes embedded in fibrin glue. The cell-fibrin composites were either cultured in vitro or directly implanted in vivo for 1, 5, and 9 weeks. Other experimental samples were precultured for either 1 or 5 weeks in vitro and then implanted in vivo for 4 additional weeks. All the samples were analyzed by histology, immunohistochemistry, biochemistry, and gene expression. The results strongly suggest that the in vivo culture in this model promoted a better tissue maturation than that obtained in the in vitro condition, and that 1 week in vitro preculture resulted in the primary structuring of the engineered composites and their subsequent maturation in vivo, without affecting the cell viability and activity, while a prolonged in vitro preculture caused a cell and matrix degeneration that could not be rescued in vivo.

The low-affinity receptor for neurotrophins p75NTR plays a key role for satellite cell function in muscle repair acting via RhoA.

Regeneration of muscle fibers, lost during pathological muscle degeneration or after injuries, is mediated by the production of new myofibres. This process, sustained by the resident stem cells of the muscle, the satellite cells, is finely regulated by local cues, in particular by cytokines and growth factors. Evidence in the literature suggests that nerve growth factor (NGF) is involved in muscle fiber regeneration; however, its role and mechanism of action were unclear. We have investigated this issue in in vivo mouse models of muscle regeneration and in primary myogenic cells. Our results demonstrate that NGF acts through its low-affinity receptor p75(NTR) in a developmentally regulated signaling pathway necessary to myogenic differentiation and muscle repair in vivo. We also demonstrate that this action of NGF is mediated by the down-regulation of RhoA-GTP signaling in myogenic cells.