Biomimetic approach for an articular cartilage patch: Combination of decellularized cartilage matrix and silk-elastin-like-protein (SELP) hydrogel.

Articular cartilage degradation due to injury, disease and aging is a common clinical issue as current regenerative therapies are unable to fully replicate the complex microenvironment of the native tissue which, being avascular, is featured by very low ability to self-regenerate. The extracellular matrix (ECM), constituting almost 90% of the entire tissue, plays a critical role in its function and resistance to compressive forces. In this context, the current tissue engineering strategies are only partially effective in restoring the biology and function of the native tissue. A main issue in tissue regeneration is treatment failure due to scarce integration of the engineered construct, often following a gradual detachment of the graft. In this scenario, we aimed to create an adhesive patch able to adequately support cartilage regeneration as a promising tool for the treatment of cartilage injuries and diseases. For this, we produced an engineered construct composed of decellularized ECM (dECM) obtained from horse joint cartilage, to support tissue regeneration, coupled with a Silk-Elastin-Like Proteins (SELP) hydrogel, which acts as a biological glue, to guarantee an adequate adherence to the host tissue. Following the production of the two biomaterials we characterized them by assessing: 1) dECM morphological, chemical, and ultrastructural features along with its capability to support chondrocyte proliferation, specific marker expression and ECM synthesis; 2) SELP microarchitecture, cytocompatibility and mechanical properties. Our results demonstrated that both materials hold unique properties suitable to be exploited to produce a tailored microenvironment to support cell growth and differentiation providing a proof of concept concerning the in vitro biological and mechanical efficacy of the construct. The SELP hydrogel displayed a very interesting physical behavior due to its high degree of resistance to mechanical stress, which is generally associated with physiological mechanical load during locomotion. Intriguingly, the shear-thinning behavior of the hydrogel may also make it suitable to be applied and spread over non-homogeneous surfaces, therefore, we hypothesize that the hybrid biomaterial proposed may be a real asset in the treatment of cartilage defects and injuries.

The myokine irisin: localization and effects in swine late medium and large antral ovarian follicle.

Irisin is mainly synthesized by skeletal muscle tissue, where it is believed to be responsible for the benefits of exercise on metabolism and cardiovascular system. In adipose tissue, its best-known effect is the browning of white adipocytes, resulting in the increase of thermogenesis and energy expenditure. As it has been largely documented that metabolic dysfunctions can frequently be associated with reductions in fertility, the possible involvement of this molecule in the regulation of reproductive processes represents an issue to be addressed. On this basis, the first aim of this work was the evaluation of the presence of irisin in the swine ovary; then, we investigated the expression of the associated molecules FNDC5, PGC-1α, and PPAR-γ. To verify a potential modulatory role both on ovarian function and on redox status, cell growth, steroidogenesis, production of superoxide anion and nitric oxide, the nonenzymatic antioxidant scavengers, were assessed in vitro on granulosa cells treated with increasing concentrations of irisin (50, 100, and 150 ng/mL). The data collected demonstrate the presence of irisin in swine ovarian follicle. Moreover, the highest concentrations tested stimulated metabolic activity and inhibited cell proliferation (P < 0.05); the peptide exerted a biphasic effect on progesterone (P < 0.01) production and, at the highest concentrations, inhibited nitric oxide while stimulated the nonenzymatic antioxidant power (P < 0.05). Superoxide anion and estradiol 17ß were unaffected. The demonstration of the local presence of irisin at the ovarian level and the highlighted effects allow us to qualify this molecule as a potential physiological regulator of follicular function.

Orexin A in swine corpus luteum.

Orexin A (OXA) is a hypothalamic neuropeptide which acts on 2 known G-protein-coupled receptors. It has been demonstrated that OXA is a central molecular link between food intake and reproduction. More recently, its peripheral role has been investigated, and we demonstrated its involvement in regulating ovarian follicle function. The present study was undertaken to explore a potential physiological role of orexin system in swine corpus luteum, a transient ovarian endocrine organ. Our aim was, first, to analyze the localization and eventual colocalization of OXA and its 2 receptors within the different cell types composing the corpus luteum structure. Second, we wanted to explore the effects of OXA on isolated luteal cells, and finally to verify a potential involvement of OXA in angiogenesis, a crucial event in corpus luteum development. Our data demonstrate the local expression of OXA and its receptors in swine corpus luteum. Luteal cell functions were affected by treatment with OXA. In particular, progesterone production was inhibited (P < 0.05) and nonenzymatic scavenging activity was increased (P < 0.05). Moreover, OXA inhibited (P < 0.05) new vessel growth. Our results suggest that OXA could act locally to play a role in corpus luteum demise.

Enhancement of peri-implant bone osteogenic activity induced by a peptidomimetic functionalization of titanium.

Osteoblast cell adhesion to the extracellular matrix is established through two main pathways: one is mediated by the binding between integrin and a minimal adhesion sequence (RGD) on the extracellular protein, the other is based on the interactions between transmembrane proteoglycans and heparin-binding sequences found in many matrix proteins. The aim of this study is the evaluation in an in vivo endosseous implant model of the early osteogenic response of the peri-implant bone to a biomimetic titanium surface functionalized with the retro-inverso 2DHVP peptide, an analogue of Vitronectin heparin binding site. The experimental plan is based on a bilateral study design of Control and 2DHVP implants inserted respectively in the right and left femur distal metaphysis of adult male Wistar rats (n=16) weighing about 300grams and evaluated after 15days. Fluorochromic bone vital markers were given in a specific time frame, in order to monitor the dynamic of new bone deposition. The effect inducted by the peptidomimetic coating on the surrounding bone were qualitatively and quantitatively evaluated by means of static and dynamic histomorphometric analyses performed within three concentric and subsequent circular Regions of Interest (ROI) of equivalent thickness (220µm), ROI1 adjacent to the interface, ROI2, the middle, and ROI3 the farthest. The data indicated that these functionalized implants stimulated a higher bone apposition rate (p<0,01) and larger and rapid osteoblast activation in terms of mineralizing surface within ROI1 compared to the control (p<0,01). These higher osteoblast recruitment and activation leads to a greater bone-to-implant contact reached for DHVP samples (p<0,5). This represents an initial stimulus of the osteogenic activity that might results in a faster and better osteointegration process.

Orexin system in swine ovarian follicles.

Successful reproduction is strictly linked to metabolic cues. The orexins are a family of hypothalamic neurohormones, well known for their key role in the control of food intake and the involvement in several aspects of the reproductive process. The biological actions of both orexins are carried out through binding to the related Orexin 1 (OX1R) and Orexin 2 (OX2R) G-protein-coupled receptors. The purpose of this study was to investigate the presence of orexin system components in the porcine ovaries, to contribute to expand the knowledge about their pleiotropic role. First, we investigated the localization of orexin A (OXA) and its receptors by immunochemistry in different ovarian districts. Thereafter, we evaluated the expression of the prepro-orexin (PPO) gene and OXA effects on granulosa cell functions. Immunohistochemical study revealed the presence of orexinergic system components in porcine ovarian follicles. Moreover, our data show the expression of PPO messenger RNA in swine ovarian follicles >5 mm. In addition, OXA influences proliferation (P < 0.05), steroidogenic activity (P < 0.05), and redox status of granulosa cells (P < 0.05). Therefore, we hypothesize that OXA could exert a local physiological role in swine ovarian follicles even if further studies are required to deeply define the function of this pleiotropic system.

Effect of the supplementation with a blend containing short and medium chain fatty acid monoglycerides in milk replacer on rumen papillae development in weaning calves.

Feeding of neonates with artificial milk formulas is a popular trend toward early weaning of newborn dairy calves. These milk replacers (MR) should accelerate the rumen development, determining early solid feed intake and leading to better performances in cattle. Previous research demonstrated that sodium butyrate supplementation in MR can affect both small intestine and rumen development. Also acetate and propionate showed similar properties, while only a few studies indicate some potential benefit of monoglycerides on gut functions. The present study is aimed to determine the effect of the supplementation of a blend containing short and medium chain fatty acids monoglycerides (SMCFA) in milk replacer on rumen papillae development and growth performances in weaning calves. Twenty bull calves (about 2 weeks old, weighing around 43kg) were randomly allocated into two groups: control (C) and treated (T). Besides MR and starter diet, the latter offered at libitum, T calves received 0.2% SMCFA in MR. Animals were slaughtered after 56 days from the beginning of the trial. No difference was found between groups either in growth performances or in mean number of papillae/cm(2) of mucosa, total surface of papillae (mm(2))/cm(2) of mucosa or papillary size. In both groups, the morphology of the rumen epithelium was typical of parakeratosis. The cells of the stratum spinosum were directly transformed into swollen, ovoid, still nucleated keratinocytes, particularly at the papillary tip, probably as a result of unphysiological osmolarities caused by high concentrate intake. Degenerated squamous horn cells covered the "balloon like" cells forming several layers, particularly in the places of the rumen mucosa more protected from an abrasive action of solid feed. This was more evident in C animals. The squamous cells covering the papillary tip showed cytoplasmic protrusion, representing remains of the attachment sites of desmosomes, which increased the total absorptive surface and were more numerous and higher in T compared to C animals. It might be hypothesized that SMCFA supplementation in MR could better regulate epithelial cell proliferation and probably have an "emollient effect" leading to an easier "peeling" that might increase efficiency for nutrient transport across the epithelium.

Osteogenic response and osteoprotective effects in vivo of a nanostructured titanium surface with antibacterial properties.

In implantology, as an alternative approach to the use of antibiotics, direct surface modifications of the implant addressed to inhibit bacterial adhesion and to limit bacterial proliferation are a promising tactic. The present study evaluates in an in vivo normal model the osteogenic response and the osteointegration of an anodic spark deposition nanostructured titanium surface doped with gallium (ASD + Ga) in comparison with two other surface treatments of titanium: an anodic spark deposition treatment without gallium (ASD) and an acid etching treatment (CTR). Moreover the study assesses the osteoprotective potential and the antibacterial effect of the previously mentioned surface treatments in an experimentally-induced peri-implantitis model. The obtained data points out a more rapid primary fixation in ASD and ASD + Ga implants, compared with CTR surface. Regarding the antibacterial properties, the ASD + Ga surface shows osteoprotective action on bone peri-implant tissue in vivo as well as an antibacterial effect within the first considered time point.

Localization of peripheral autonomic neurons innervating the boar urinary bladder trigone and neurochemical features of the sympathetic component.

The urinary bladder trigone (UBT) is a limited area through which the majority of vessels and nerve fibers penetrate into the urinary bladder and where nerve fibers and intramural neurons are more concentrated. We localized the extramural post-ganglionic autonomic neurons supplying the porcine UBT by means of retrograde tracing (Fast Blue, FB). Moreover, we investigated the phenotype of sympathetic trunk ganglion (STG) and caudal mesenteric ganglion (CMG) neurons positive to FB (FB+) by coupling retrograde tracing and double-labeling immunofluorescence methods. A mean number of 1845.1±259.3 FB+ neurons were localized bilaterally in the L1-S3 STG, which appeared as small pericarya (465.6±82.7 µm2) mainly localized along an edge of the ganglion. A large number (4287.5±1450.6) of small (476.1±103.9 µm2) FB+ neurons were localized mainly along a border of both CMG. The largest number (4793.3±1990.8) of FB+ neurons was observed in the pelvic plexus (PP), where labeled neurons were often clustered within different microganglia and had smaller soma cross-sectional area (374.9±85.4 µm2). STG and CMG FB+ neurons were immunoreactive (IR) for tyrosine hydroxylase (TH) (66±10.1% and 52.7±8.2%, respectively), dopamine beta-hydroxylase (DßH) (62±6.2% and 52±6.2%, respectively), neuropeptide Y (NPY) (59±8.2% and 65.8±7.3%, respectively), calcitonin-gene-related peptide (CGRP) (24.1±3.3% and 22.1±3.3%, respectively), substance P (SP) (21.6±2.4% and 37.7±7.5%, respectively), vasoactive intestinal polypeptide (VIP) (18.9±2.3% and 35.4±4.4%, respectively), neuronal nitric oxide synthase (nNOS) (15.3±2% and 32.9±7.7%, respectively), vesicular acetylcholine transporter (VAChT) (15±2% and 34.7±4.5%, respectively), leu-enkephalin (LENK) (14.3±7.1% and 25.9±8.9%, respectively), and somatostatin (SOM) (12.4±3% and 31.8±7.3%, respectively). UBT-projecting neurons were also surrounded by VAChT-, CGRP-, LENK-, and nNOS-IR fibers. The possible role of these neurons and fibers in the neural pathways of the UBT is discussed.

Neurochemical features of boar lumbosacral dorsal root ganglion neurons and characterization of sensory neurons innervating the urinary bladder trigone.

Porcine lumbosacral dorsal root ganglion (DRG) neurons were neurochemically characterized by using six neuronal markers: calcitonin gene-related peptide (CGRP), substance P (SP), neuronal nitric oxide synthase (nNOS), neurofilament 200kDa (NF200), transient receptor potential vanilloid 1 (TRPV1), and isolectin B4 (IB4) from Griffonia simplicifolia. In addition, the phenotype and cross-sectional area of DRG neurons innervating the urinary bladder trigone (UBT) were evaluated by coupling retrograde tracer technique and immunohistochemistry. Lumbar and sacral DRG neuronal subpopulations were immunoreactive (IR) for CGRP (30 ± 3% and 29 ± 3%, respectively), SP (26 ± 8% and 27 ± 12%, respectively), nNOS (21 ± 4% and 26 ± 7%, respectively), NF200 (75 ± 14% and 81 ± 7%, respectively), and TRPV1 (48 ± 13% and 43 ± 6%, respectively), and labeled for IB4 (56 ± 6% and 43 ± 10%, respectively). UBT sensory neurons, which were distributed from L2 to Ca1 DRG, had a segmental localization, showing their highest density in L4-L5 and S2-S4 DRG. Lumbar and sacral UBT sensory neurons expressed similar percentages of NF200 immunoreactivity (64 ± 33% and 58 ± 12%, respectively) but showed a significantly different immunoreactivity for CGRP, SP, nNOS, and TRPV1 (56 ± 9%, 39 ± 15%, 17 ± 13%, 62 ± 10% vs. 16 ± 6%, 16 ± 11%, 6 ± 1%, 45 ± 24%, respectively). Lumbar and sacral UBT sensory neurons also showed different IB4 labeling (67 ± 19% and 48 ± 16, respectively). Taken together, these data indicate that the lumbar and sacral pathways probably play different roles in sensory transmission from the UBT. The findings related to cell size also reinforced this hypothesis, because lumbar UBT sensory neurons were significantly larger than sacral ones (1,112 ± 624 µm(2) vs. 716 ± 421 µm(2) ).

Experimental study on the location of neurons associated with the first sacral sympathetic trunk ganglion of the pig.

The neurons associated with the left first sacral sympathetic trunk ganglion (STG S1), an autonomic ganglion particularly concerned in the innervation of the smooth and striated musculature associated with pelvic organs, were identified in the pig, using the non-trans-synaptic fluorescent retrograde neuronal tracer Fast Blue. The labelled neurons were located mostly ipsilaterally, in the intermediolateral nucleus of the spinal cord segments T10-L5, in the sympathetic trunk ganglia L3-Co1, in the caudal mesenteric ganglia, in the pelvic ganglia, and in the spinal ganglia T13-S4. Our results could indicate the existence of visceral neuronal circuits concerning the ganglia of the sympathetic trunk and the caudal mesenteric, pelvic and spinal ganglia with or without the intervention of the central nervous system, whose identification and preservation during surgical treatments could be helpful in reducing the risk of subsequent urinary and sexual disfunctions.

Double labelling immunohistochemical characterization of autonomic sympathetic neurons innervating the sow retractor clitoridis muscle.

Retrograde neuronal tracing and immunohistochemical methods were used to define the neurochemical content of sympathetic neurons projecting to the sow retractor clitoridis muscle (RCM). Differently from the other smooth muscles of genital organs, the RCM is an isolated muscle that is tonically contracted in the rest phase and relaxed in the active phase. This peculiarity makes it an interesting experimental model. The fluorescent tracer fast blue was injected into the RCM of three 50 kg subjects. After a one-week survival period, the ipsilateral paravertebral ganglion S1, that in a preliminary study showed the greatest number of cells projecting to the muscle, was collected from each animal. The co-existence of tyrosine hydroxylase with choline acetyltransferase, neuronal nitric oxide synthase, calcitonin gene-related peptide, leu-enkephalin, neuropeptide Y, substance P and vasoactive intestinal polypeptide was studied under a fluorescent microscope on cryostat sections. Tyrosine hydroxylase was present in about 58% of the neurons projecting to the muscle and was found to be co-localized with each of the other tested substances. Within fast blue-labelled cells negative to the adrenergic marker, small populations of neurons singularly containing each of the other enzymatic markers or peptides were also observed. The present study documents the complexity of the neurochemical interactions that regulate the activity of the smooth myocytes of the RCM and their vascular components.

Peripheral ganglia supplying the genital smooth musculature in the female pig: an experimental study.

The aim of the present study was to locate the sensory and autonomic ganglia innervating the female genital musculature in pigs. The retrograde neuronal tracers horseradish peroxidase (HRP) or fast blue (FB) were injected into the left retractor clitoridis muscle (RCM), which was treated as a typical model of the genital smooth musculature. Labelled cells were found in ipsilateral dorsal root ganglia S1-S4, in bilateral sympathetic paravertebral ganglia from L5-L6 or L6-L7 to S3 and in the left and right caudal mesenteric ganglion. In two of the five animals treated, presumably preganglionic parasympathetic cells were labelled in the ipsilateral intermediate grey substance of the segments S1-S2.

Morphological characteristics and distribution of the autonomic and sensitive innervation of the prostate in some animal species.

The sensitive and autonomic innervation of the prostate was studied in some mammals (rat, rabbit, horse, donkey and bull) using gold chloride impregnation. In all the species considered the gland was supplied with conspicuous innervation located in the thickness of the capsule and in trabeculae. The autonomic innervation was constituted by ganglion cells of different sizes (small, medium and large), isolated or in groups of large ganglia also, always located along the course of nerve bundles. The sensitive nervous component, placed more frequently in the most superficial layer of the capsule, consisted of delicate networks originating by more fibres and, above all, of capsulated receptors such as Pacini and Golgi-Mazzoni corpuscles. A characteristic finding, but only present in the horse, donkey and bull, was the helicoidal disposition of thin collagen fibres around thin nervous bundles that ended up to the point where the nerve trunk divided.

On the bulky appliances and artero-venous anastomoses in the vascular district of the base of the brain.

It has been studied the vascular territory of the base of the brain and it has been pointed out the presence of characteristical structural arrangements of the vasal wall and artero-venous anastomoses. The just mentioned characteristical structures are placed in points where a vessel divides itself or creates a collateral branch and their functional engagement is target-oriented to control the blood flux.

Further observations on the presence of ganglion cells in the oculomotor nerves of mammals and fish: number, origin and probable functions.

The oculomotor nerves (3rd, 4th and 6th) of some species of fish and mammals have been studied to establish the presence, number, true topography and probable functional role of the ganglion cells located along the trunk. The finding of typical pseudo-unipolar ganglion cells is always unpredictable and extremely variable, from an inter- and intra-specific point of view, in members of the two zoological classes studied.

On the presence of ganglion cells in the intracranial portion of the accessory nerve (XI cranial nerve) in some mammals.

The intracranial tract of the accessory nerve (XI cranial nerve) was studied in some mammals (equines, domestic and wild ruminants, pig, carnivores, rabbit, nutria, guinea pig, hamster, hedgehog). The specimens were embedded in paraffin or paraplast, the sections were stained with cresyl violet, haematoxylin and eosin, or submitted to argentic impregnation. Pseudounipolar ganglion cells were found in all the mammals examined, with the exception of the cat. The number of cells and their variability in the different species and subjects were related. The topography and morphology of the cells were described. This comparative study has demonstrated that the accessory nerve is not a entirely motor nerve, but it is a mixed, motor and sensitive, nerve. Nevertheless, we think further studies are necessary in order to establish the peripheral distribution, the central pathway and the functional role of the pseudounipolar neurons found in the intracranial tract of the accessory nerve.

On the vegetative and sensitive innervation of the Retractor clitoridis muscle in some domestic animals.

The retractor clitoridis muscle originates from the coccygeal vertebrae in the cow, ewe, goat and mare, and from the anal musculature in the sow. It terminates at the base of the clitoris. In all the species considered, a vegetative innervation was found. This was represented by isolated or grouped ganglion cells. Nervous sensitive supply was also present. This was represented by Pacinian, Pacinian-like and Golgi-Mazzoni's corpuscles, and by Krauses's end bulbs. A notable difference was found in the amount and type of these receptors. They were numerous in the sow, ewe and goat, and rare in the cow and mare. Additionally, in the sow, ewe and goat, all the above mentioned receptors were found, while, in the cow and mare, only Pacinian and Pacinian-like corpuscles occurred. The morphology of these receptors was described and hypotheses were made concerning their probable functional role.

Comparative study of sensitive and vegetative innervation of external and internal anal sphincter muscles in different mammals.

Proprioceptive innervation of the external anal sphincter muscle and the organization of the vegetative and sensitive nerve components of the internal and sphincter muscle have been studied in different mammals. The findings of typical muscle spindles in the external anal sphincter muscle were constant in the pig, frequent in the goat and cow, rare in the sheep and horse and absent in the roe and rabbit. In the pig, muscle spindles were observed in the entire extension of the muscle, while in the sheep, goat, cow and horse, the receptors were found only in the cranial portion of the muscle. In all the species studied, the internal anal sphincter muscle had numerous ganglion cells, isolated or grouped, and rare Pacinian, Pacinian-like, and Golgi-Mazzoni corpuscles. Their functional role has been hypothesized.