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.

Modulation of the somatotropic axis, adiponectin and cytokine secretion during highly pathogenic porcine reproductive and respiratory syndrome virus type 1 (HP-PRRSV-1) infection.

Porcine reproductive and respiratory syndrome virus (PRRSV) is known to be clinically responsible for reproductive failure in sows and post-weaning respiratory disease in growing piglets. During the last years, highly pathogenic PRRSV isolates have been discovered. In Italy, a PRRSV-1 subtype 1 strain (namely PR40/2014) characterized by high pathogenicity was isolated and experimental infection was characterized in terms of virological/clinical features and immune modulation (Canelli et al., 2017; Ferrari et al., 2018). The present study was performed in 4-week-old pigs experimentally infected with the highly pathogenic PRRSV1_PR40/2014 (HP-PR40) or with the conventional PRRSV1_PR11/2014 (PR11). The aim was to evaluate the interrelation between plasmatic hormones and cytokines in infected pigs compared to uninfected controls in order to address potential effects on the course of an experimental infection. The time-related changes of growth hormone (GH), insulin-like growth factor-1 (IGF-1), adiponectin, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) levels appear to be modulated by the infection depending on the PRRSV isolate (HP-PR40 vs. PR11). In particular, in HP-PR40 infected animals, the association between high GH levels and viremia may testify the need to block the anabolic action of GH in order to shift available energy towards the immune response. This need appeared to be delayed in PR11 animals, given the lower pathogenicity of the isolate. Adiponectin, IL-6 and TNF-α course supports the hypothesis of GH resistance mechanisms to guarantee homeostasis in HP-PR40 animals and underlines the key role of energy availability in events leading to an effective response to the virus.

Effects of a dietary crude fibre concentrate on growth in weaned piglets.

Many fibre sources can help the adaptation of piglets at weaning, improving the growth. In this study, the effects of a dietary crude fibre concentrate (CFC) on piglet's growth was investigated. From 31 to 51 days of age, 108 weaned piglets (D×(Lw×L)), had access to two isofibrous, isoenergetic and isonitrogenous diets, supplemented with 1% of CFC (CFC group) or not (control (CON) group). From days 52 to 64 all piglets received the same starter diet. During the dietary treatment period the CFC group showed higher average daily gain, average daily feed intake and feed efficiency (P<0.001) than CON group. At 64 days of age, BW was higher in CFC group compared with CON group (P<0.001). Blood samples were collected at days 31, 38, 45 and 52 of age. From days 31 to 52 significant differences in the somatotropic axis between groups were observed. In particular, growth hormone levels were higher only at the end of the 1st week of dietary treatment (P<0.05) in CFC group animals compared with CON group animals. The IGF-I trend was similar between groups even if the IGF-I levels were higher in the CFC group than CON group 1 week after starting treatment (P<0.01). The IGF-binding protein 3 (IGFBP-3) levels were higher in the first 2 weeks of dietary treatment and lower in the 3rd week in CON group compared with CFC group (P<0.01). Specifically, the IGFBP-3 profile was consistent with that of IGF-I in CFC group but not in CON group. At the same time, an increase of leptin in CFC compared with CON group was observed (P<0.05). Piglets fed the CFC diet showed a lower diarrhoea incidence (P<0.05) and a lower number of antibiotic interventions (P<0.05) than CON diet from 31 to 51 days of age. Pig-major acute-phase protein plasma level (P<0.01) and interleukin-6 gene expression (P<0.05) were higher in CON group than CFC group at the end of 1st week of dietary treatment. In conclusion, this study showed that CFC diet influences the hormones related to energy balance enhancing the welfare and growth of piglets. Furthermore, the increase in feed intake during 3 weeks of dietary treatment improved the feed efficiency over the entire post-weaning period.

Anterior pituitary influence on adipokine expression and secretion by porcine adipocytes.

Nutritional stressors may cause negative effects on animal health and growth and lead to significant economic impact. Adipose tissue is an endocrine organ producing, mediators and hormones, called adipokines. They play a dynamic role in body homeostasis and in the regulation of energy expenditure, interacting with feeding behavior, hormones and growth factors. This in vitro study aimed to investigate how nutritional conditions and growth hormone (GH) can influence nitric oxide (NO) production and the expression and secretion of three important adipokines, that is leptin, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), by swine adipocytes. In our experimental model, mesenchymal stem cells from omental adipose tissue were induced to adipogenic differentiation. After differentiation, adipocytes were incubated for 24 h (T0) with DMEM/Ham's F12 (group A) or DMEM/Ham's F12 salts (group B), a DMEM/Ham's F12 formulation deprived of nutritional components. Primary adipocyte cells were also co-cultured for 4 h (T+4) or 12 h (T+12) with or without anterior pituitary slices. To stimulate GH secretion by pituitary cells, growth hormone releasing hormone at 10-8 M was added at the start of the incubation times (4 or 12 h). At T0, T+4 and T+12, NO production, leptin, IL-6 and TNF-α expression and secretion were measured. NO increased (P<0.05) up to twofold in restricted culture conditions. Deprived medium and coincubation with anterior pituitary positively influenced leptin secretion and expression. TNF-α was expressed and secreted only in deprived culture condition groups (B, B1 and B2). Nutrients availability and pituitary co-culture did not affect IL-6 expression and secretion. Our study shows an endocrine function for porcine adipocytes. In our model, adipocytes readily responded to nutritional inputs by secretion of molecules affecting energy balance. This secretion capacity was modulated by GH. Improving our knowledge of the role of adipocyte in the endocrine system, may lead to a more complete understanding of regulating energy balance in swine.

Monitoring blood plasma leptin and lactogenic hormones in pregnant sows.

The mechanism of action of leptin in pregnant breeding sows, in which hyperphagia is managed through dietary strategies, is yet to be clarified. The aim of this study was to monitor leptin concentrations and their interactions with lactogenic hormones in Large White×Landrace breeding multiparous sows (n=15). All sows showed a normal body condition (mean body condition score: 2.96). Blood samples were collected the day after weaning the litters, at insemination, every 15 days up to day 45 of pregnancy and every 7 days from day 46 to farrowing. At delivery, the placenta was collected for the analysis of leptin and leptin receptor expressions. Plasma leptin levels increased from the end of mid gestation (day 72) and remained high until farrowing (P<0.05). As expected, plasma prolactin (PRL), low during most of pregnancy, increased during the 2 weeks before farrowing (P<0.05), whereas progesterone levels reached plateau at 30 days of gestation and decreased at farrowing (P<0.05). Cortisol levels peaked close to farrowing (P<0.05). Leptin was expressed in the placenta, where the receptor expression analysis showed the presence of the short form but not of the long form. A positive correlation was found between leptin and PRL concentrations during mid (r=0.430; P<0.001) and late (r=0.687; P<0.001) pregnancy, and with progesterone in early pregnancy (r=0.462; P<0.05). During late gestation, a positive correlation was observed between leptin and cortisol (r=0.585; P<0.001). Our results suggested that, in restrictively fed pregnant sows, the leptin levels increased from the end of mid pregnancy to delivery, confirming the presence of leptin resistance. We showed a correlation between leptin and lactogenic hormones during different stages of pregnancy in sows. Lactogenic hormones show pregnancy-specific changes in their secretion and all may become involved in modulating leptin signal.

Effect of urethral infusion of atracurium besylate on manual bladder expression in dogs and cats with spinal cord injuries: a randomised trial.

The aim of this randomised trial was to assess the effect of urethral infusion of atracurium besylate in dogs and cats with signs of urinary retention secondary to lesions affecting spinal cord segments T3-L3. Eighteen dogs and six cats with urinary retention were examined and scored before treatment on the degree of difficulty of inducing bladder emptying by manual bladder compression. Animals were subsequently treated in a blinded fashion by the same operator with urethral infusion of 2-4 ml of either a solution of 0.5 mg/ml of atracurium (treatment group) or placebo (control group) and, after five minutes, a second attempt was made to induce bladder emptying by manual compression and a post-treatment score assigned. Pretreatment scores did not differ between the treatment and control groups (5.6±0.8 v 6.2±0.7, respectively; P=0.22); however, post-treatment scores were significantly lower in the treatment group compared with the control group (2.9±0.4 v 5.9±0.3; P<0.05). Urethral infusion of atracurium facilitates manual bladder expression in dogs and cats with urinary retention secondary to spinal cord injuries. No side effects were recognised.

Leptin regulates GH gene expression and secretion and nitric oxide production in pig pituitary cells.

The aim of this study was to investigate the direct effect of leptin on GH gene expression and secretion and the role of nitric oxide as a possible mediator in pig anterior pituitary cells. Pituitary cells from adult sows were treated for 4 or 24 h with rhleptin (from 0.1 nM to 1 microM) alone or in association with GHRH (10 nM) or hexarelin (10 nM). At the end of incubation, medium was collected for GH and nitric oxide determination by ELISA and Griess test, respectively. Total RNA was collected from cells, and GH gene expression was measured by RT-PCR. Leptin significantly (P < 0.001) stimulated GH secretion in both incubation periods. The maximum response was induced by 10 nM leptin; furthermore, a significant interaction (P < 0.002) between leptin and GHRH (P < 0.03) and between leptin and hexarelin was observed when the molecules were used in association. GH gene expression was significantly increased (at least P < 0.05) by hexarelin, GHRH, and leptin (1000 and 100 nM) after 24 h of treatment. Leptin (10 nM and 1 microM) significantly (P < 0.05) increased nitric oxide production, whereas S-nitroso-N-acetyl-penicillamine (from 0.01-1000 nM) significantly (P < 0.05) stimulated GH secretion. These data demonstrate that leptin directly influences GH regulation at the pituitary level, and nitric oxide may be involved in this function.

Systemic and local immune response in pigs intradermally and intramuscularly injected with inactivated Mycoplasma hyopneumoniae vaccines.

The systemic and respiratory local immune response induced by the intradermal administration of a commercial inactivated Mycoplasma hyopneumoniae whole-cell vaccine (Porcilis(®) MHYO ID ONCE - MSD AH) in comparison with two commercial vaccines administered via the intramuscular route and a negative control (adjuvant only) was investigated. Forty conventional M. hyopneumoniae-free pigs were randomly assigned to four groups (ten animals each): Group A=intradermal administration of the test vaccine by using the needle-less IDAL(®) vaccinator at a dose of 0.2 ml; Group B=intramuscular administration of a commercially available vaccine (vaccine B); Group C=intramuscular administration of the adjuvant only (2 ml of X-solve adjuvant); Group D=intramuscular administration of a commercially available vaccine (vaccine D). Pigs were vaccinated at 28 days of age. Blood and bronchoalveolar lavage (BAL) fluid samples were collected at vaccination (blood only), 4 and 8 weeks post-vaccination. Serum and BAL fluid were tested for the presence of antibodies by ELISA test. Peripheral blood monomorphonuclear cells (PBMC) were isolated to quantify the number of IFN-γ secreting cells by ELISpot. Moreover, cytokine gene expression from the BAL fluid was performed. Total antibodies against M. hyopneumoniae and specific IgG were detected in serum of intradermally and intramuscularly (vaccine B only) vaccinated pigs at 4 and 8 weeks post-vaccination. M. hyopneumoniae specific IgA were detected in BAL fluid from vaccinated animals (Groups A and B) but not from controls and animals vaccinated with the bacterin D (p<0.05). Significantly higher gene expression of IL-10 was observed in the BAL fluid at week 8 post-vaccination in the intradermally vaccinated pigs (p<0.05). The results support that the intradermal administration of an adjuvanted bacterin induces both systemic and mucosal immune responses. Moreover, the intramuscularly administered commercial vaccines each had a different ability to stimulate the immune response both systemically and locally.

Prostaglandin F2α and control of reproduction in female swine: a review.

In female swine, PGF2α is an important regulator of corpora luteal (CL) function, uterine contractility, ovulation, and embryo attachment. High affinity PGF2α receptors are present in the CL at all stages of the estrous cycle and they are functional. Therefore, a lack of luteolytic capacity of PGF2α is related to other factors that have not been well identified. In female pigs, a single exogenous PGF2α injection produces a short lasting decrease in plasma progesterone levels but does not induce luteolysis before day 12 of the estrous cycle. However, multiple injections of PGF2α can induce luteolysis before day 12 of the estrous cycle and may be utilized in the development of protocols for ovulation synchronization and timed AI. Most commonly, PGF2α is used for the induction of farrowing and so facilitation of cross fostering. Further, since PGF2α is a smooth muscle stimulant, treatment to stimulate myometrial contractions and uterine evacuation of residual products from parturition or infectious debris, may have beneficial effects on post-weaning fertility. Administration of PGF2α at the moment of insemination has been shown to improve reproductive performances when fertility is otherwise low, such as in sow under summer heat stress.

Effects of dietary nucleotide supplementation on growth performance and hormonal and immune responses of piglets.

The effects of dietary nucleotide supplementation from 9 days of age until the end of post-weaning on piglets hormonal and immune responses and on growth performance were investigated. During lactation (days 9 to 21) and post-weaning (days 22 to 55) 10 [HBI Fomeva11 × (Large White × Landrace)] litters (n = 108 piglets) had ad libitum access to two standard diets, both supplemented with 0% (T0 group) or 0.1% (T1 group) of yeast extract nucleotides. BW of piglets at days 21 (P < 0.10), 35 and 55 (P < 0.05) was greater in T1 compared with T0. Feed intake was not different between groups (P > 0.05). Cortisol content was lower in T1 than in T0 at days 28 and 35 (P < 0.05), whereas growth hormone was lower at day 35 (P < 0.05). Levels of IGF-1 were similar across groups (P > 0.05). Nucleotide-supplemented diets increased lymphocyte subpopulation CD4-CD8+high at days 21 and 35 (P < 0.05), whereas CD4+CD8- cells were higher in T1 than in T0 at day 21 (P < 0.05). Peripheral blood mononuclear cells cytokine expression was influenced by dietary nucleotide supplementation. At weaning, interleukin (IL)-6 and IL-1ß expression was lower (P < 0.05) in T1 compared with T0, whereas the expression of interferon (IFN)-γ and IL-10 was higher (P < 0.05). At day 28, piglets in T1 showed higher values of tumor necrosis factor (TNF)-α expression than T0 and lower values of IL-10 expression (P < 0.05). Dietary nucleotide supplementation had a suppressive effect on IL-6 and IL-10 expression (P < 0.05) at day 35. On the contrary, the expression of IFN-γ, TNF-α and IL-1ß was enhanced (P < 0.05). In conclusion, these results suggest that starting a dietary nucleotide supplementation before weaning can improve the adaptive capabilities of weaned piglets to the stressors, enhancing the growth performance.

Peripheral cytokine expression in Standardbred mares at different adiposity during the periparturient period.

The aim of this study was to evaluate whether the peripheral cytokine expression could be affected by differences in fat thickness in healthy mares during the periparturient period (last weeks of pregnancy and first weeks of lactation). At the beginning of the 11th month of gestation, 18 mares with normal body condition score (BCS; 5) were divided into two groups with low (L; ≤12 mm; n = 12) and high (H; >12 mm; n = 6) fat thickness. Blood samples were collected every 10 days from the 11th month of gestation to 20 days post partum. Peripheral blood mononuclear cells were separated and tumour necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-1α (IL-1α), -1ß (ILß), -2 (IL-2) and -6 (IL-6) gene expressions were performed by using reverse transcriptase PCR. TNF-α and IFN-γ expressions were affected by fatness group, as the sampling period influenced the expression of all cytokines with the exception of IFN-γ. The interaction between fatness group and sampling period tended to be significant (P < 0.10) only for IL-1ß, TNF-α and IL-2. In H group, compared with L group, IL-1ß and IL-2 significantly (P < 0.05) increased at parturition. TNF-α peripheral expression increased from parturition to reach the peak at +10 days (P < 0.05) only in H group. In conclusion, in normal BCS periparturient mares, small body fat thickness variations influence the peripheral cytokine expression, showing a possible role for cytokines in parturition and lactation, events strongly linked to adipose tissue availability.

Effects of different culture conditions and leptin on GH mRNA expression and GH secretion by pig pituitary cells.

Growth hormone (GH) is enhanced in malnutrition; physiological increments in GH secretion seem to play an important role in regulating metabolism during fasting. Leptin has also been shown to play a role, amongst others, in modulating the somatotropic axis. In this study, we investigated how the composition of culture media could influence basal and leptin-stimulated GH secretion and expression in pig pituitary cells. Pituitary cells from 8-month-old sows were incubated for 48 h in presence and absence of 10% fetal calf serum, either in DMEM/Ham's F12, in arginine-free DMEM/Ham's F-12, or in DMEM/Ham's F12 Salts. Cells were then treated for 24 h with GHRH or recombinant human leptin (rhLep) individually or in association with GHRH; cell proliferation, nitric oxide (NO) production and GH expression and secretion were determined. The absence of nutritional factors induced a decrease in cell proliferation, but stimulated both GH secretion and expression. Furthermore, rhLep significantly increased GH expression and secretion irrespective of culture conditions. NO production was only significantly enhanced by leptin under DMEM/Ham's F12 culture conditions. These observations lead us to hypothesize that the adaptive capabilities of pituitary cells may overcome the negative effects of undernutrition; in this context, leptin does not seem to depend on NO pathways in modulating GH secretion.

Modulation by galanin of growth hormone and gonadotropin secretion from perifused pituitary and median eminence of prepubertal male calves.

Galanin is widely distributed in the peripheral and central nervous system and has been indicated as a putative hypothalamic-hypophysiotropic hormone. This study was performed to investigate the effects of galanin on both growth (GH) and luteinizing hormones (LH) from pituitaries of young male calves. Pituitary slices (P, 500 microm in thickness) were perfused alone or coincubated with median eminence terminals (ME) in DMEM-F12 plus BSA 0.1% and antibiotics. The perifusion chambers were kept in equilibrium for 150 min, and medium samples were collected every 10 min for 240 min and stored at -20 degrees C until the measurement of LH and GH levels. Basal GH release increased up to 60% after galanin infusion (p < 0.01 vs. baseline levels) for 60 min in P alone; in P + ME coincubation, galanin-stimulated GH secretion was further increased by up to 200%. Basal LH release in chambers with P was significantly increased (up to 25%; p < 0.05) for 70 min after galanin infusion; P + ME coincubation showed a galanin-mediated increase in LH release of up to 50%. GH and LH responsiveness to exogenous GH-releasing hormone and gonadotropin-releasing hormone was not significantly modulated by galanin in our experimental model. In conclusion, galanin is demonstrated to have a significant stimulatory role in the secretion of GH and LH, with a combined action at both the hypothalamic and pituitary levels. ......................

IGF-I, IGFBP-2 and -3 but not GH concentrations are different in normal and poor growing piglets.

In this study we investigated the somatotropic axis in piglets with evident growth delay. Female Suffolk crossbred piglets (30 days old; N = 12) were divided into normal weight (10 +/- 0.9 kg) and poor growing subjects (7 +/- 0.5 kg) and bled for growth hormone (GH), Insulin-like growth factor-I (IGF-I), Insulin-like growth factor binding protein 2 and 3 (IGFBP-2 and -3) determination. Basal and induced-GH levels were not different in the groups. Plasma IGF-I concentrations were significantly different (p < 0.001): 101.8 +/- 9.8 ng x mL(-1) (normal weight group) and 39.5 +/- 4.0 ng x mL(-1) (poor growing group). IGFBP-2 and -3 concentrations were significantly (p < 0.001) lower in poor growing than in normal piglets. Piglet weight was positively correlated (r = 0.98, p < 0.001) with IGF-I and IGFBP-2 or -3 concentrations. Our data indicate that growth rate was not correlated to basal or secretagogue-induced GH secretion.