Gastric distension causes changes in heart rate and arterial blood pressure by affecting the crosstalk between vagal and splanchnic systems in anesthetised rats.

Various hindbrain nuclei have been demonstrated to be involved in the control of the cardiovascular reflexes elicited by both non-noxious and noxious gastric distension, through parasympathetic and sympathetic activation. The different role played by the branches of autonomic nervous system in exerting these effects and their crosstalk in relation to low-/high-pressure distension rate has not been examined yet. Therefore, in the present work, monolateral and bilateral vagotomy and splanchnicotomy were performed in anesthetised rats to analyse the involvement of hindbrain nuclei in haemodynamic changes caused by gastric distension at high (80 mmHg) and low (15 mmHg) pressure. The analysis of c-Fos expression in neuronal areas involved in cardiovascular control allowed us to examine their recruitment in response to various patterns of gastric distension and the crosstalk between vagal and splanchnic systems. The results obtained show that the low-pressure (non-noxious) gastric distension increases both heart rate and arterial blood pressure. In addition, the vagus nerve and hindbrain nuclei, such as nucleus ambiguous, ventrolateral medulla and lateral reticular nucleus, appear to be primarily involved in observed responses. In particular, we have found that although vagus nerve plays a central role in exerting those cardiovascular reflex changes at low gastric distension, for its functional expression an intact splanchnic system is mandatory. Hence, the absence of splanchnic input attenuates pressor responses or turns them into depressor responses. Instead at high-pressure (noxious) gastric distension, the splanchnic nerve represents the primary component in regulating the reflex cardiovascular effects.

Effect of retinoic acid and vitamin D3 on osteoblast differentiation and activity in aging.

Several studies have evidenced that in aging, osteoblast functional activity is impaired: osteoblast proliferation is slower and matrix deposition is less efficient. Because peroxisome-proliferator-activated receptor γ2 (PPARγ2) and fatty acids are important inhibitory signals in osteoblast development, we have investigated in human primary osteoblasts obtained from patients of different ages, the influence of retinoic acid and calcitriol on enzymes involved in differentiative (PPARγ2, ß-catenin, and insulin-like growth factor 1) and metabolic (carnitine palmitoyltransferase 1) intracellular pathways, and on transglutaminase 2, as enzyme fundamental for stabilizing the newly deposited extracellular matrix in bone. Retinoic acid and calcitriol influenced, respectively, proliferation and differentiation of osteoblasts, and an increase in PPARγ2 expression was observed following retinoic acid administration, whereas a decrease was observed following calcitriol administration. Aging widely influenced all parameters analyzed (the proliferation, differentiation, and new matrix deposition are significantly reduced in aged osteoblasts), with the exception of PPARγ2, which we found to be constitutively overexpressed and not modulated by retinoic acid or calcitriol administration. Our findings show the impaired ability of aged osteoblasts to perform adequate functional response and draw attention to the therapeutic approaches for bone healing in elderly patients.

A study of the mechanical properties of ePTFE suture used as artificial mitral chordae.

BACKGROUND AND AIM OF THE STUDY: We investigated the dimensional and mechanical properties of polyetetrafluorene (ePTFE) sutures used as artificial chordae during mitral valve repair. METHODS: Mechanical properties of ePTFE synthetic chordae tendineae were tested with a servo hydraulic testing machine. Several different lengths from 2 to 14 cm were studied under both single and multiple mechanical traction. RESULTS: The mechanical behavior of artificial chordae reveals that three centimeters is the length over which we observe a significant increase in stiffness. The chordae stiffness grows further at the length greater than seven centimeters following a low number of traction cycles. CONCLUSION: The increase of the length of artificial ePTFE chordae is accompanied by an increasing stiffness that compromises the long-term resistance of the chordae. ePTFE length can alter the performance of artificial chordae. This suggests that mitral valve repairs which anchor ePTFE neochordae to the ventricular apex may have less durability than when anchored to the tips of the papillary muscles.

Effects and differentiation activity of IGF-I, IGF-II, insulin and preptin on human primary bone cells.

The importance of the complex interrelated regulatory pathways involving IGF factors and pancreatic hormones can be observed in several metabolic diseases, where the deregulation of these factors has a wide impact on bone health. These findings have stimulated us to compare the effect of IGF-I, IGF-II, insulin and preptin on human bone cells. The effect on cell differentiation and cell activity of osteoblasts and osteoclasts has been analysed. We have observed a significant effect by IGF-I, a modest effect by IGF-II and preptin and no effect after insulin administration on human primary osteoblast-like cells. All studied factors have shown an induction on human primary osteoclast differentiation and bone resorption activity, with IGF-I being the most potent factor. We hypothesize that these findings may be on the basis of decreased bone mass density observed in several diseases.

In vitro toxicity of photodynamic antimicrobial chemotherapy on human keratinocytes proliferation.

This in vitro experimental study has been designed to assess the effects of photodynamic antimicrobial chemotherapy (PACT) on human keratinocytes proliferation. Human keratinocytes (HaCaT) monolayers (∼0.5 cm(2)) have been irradiated with 635 nm red laser light with a fluence of 82.5 or 112.5 J/cm(2) in the absence or presence of toluidine (TB). Cell proliferation, monolayer area coverage, cytokeratin 5 (K5) and filaggrin (Fil) expression, and metalloproteinase (MMP)-2 and MMP-9 activity were measured after 72 h from laser irradiation. HaCaT proliferation was reduced by TB staining. Cell exposure to both low- and high-fluence laser irradiation in both presence and absence of TB staining reduced their proliferation and monolayer area extension. Moreover both laser treatments were able to reduce K5 and Fil expression and MMP-9 production in keratinocytes not treated with TB. These data indicate that PACT could exert toxic effects on normal proliferating keratinocytes present around parodontal pockets. The observed reduced cell proliferation along with a reduced production of enzymes involved in wound healing could alter the clinical outcome of the patients treated with PACT.

Gelatin-based anionic hydrogel as biocompatible substrate for human keratinocyte growth.

Ionic hydrogels are biocompatible candidates for skin tissue engineering. Two hydrogels synthesized by crosslinking gelatin with polylysine (positively charged HG1) or polyglutamic acid (negatively charged HG2) were tested using spontaneously immortalized human keratinocytes (HaCaT). HaCaT cells displayed higher adhesion and proliferation onto HG2, forming a continuous and stratified epithelium after 7 days. Moreover HaCaT cells grown onto HG2 showed a decreased Epilysin and Filaggrin expression, while transglutaminase-1 expression was increased. Those data indicate that human keratinocyte can form a stratified epithelium onto HG2 that could therefore be an useful tool for skin tissue engineering.

Low concentration amino-bisphosphonates stimulate human keratinocyte proliferation and in vitro wound healing.

Amino-bisphosphonates (N-BPs) are widely used to treat a great variety of clinical conditions involving altered calcium metabolism, as well as to prevent bone metastases. The use of N-BPs, however, display well-known side effects, including cellular toxicity, mainly at soft tissue and mucosal level, that arise from N-BPs ability to induce cell apoptosis when administered at clinically relevant concentrations. The aim of this study was to evaluate, in an in vitro wound healing model, the effect of N-BPs low concentration (10 nM-10 µM) stimulation on keratinocyte cellular behaviour. Human keratinocytes were treated with neridronate and zoledronate, two N-BPs with different chemical structure and clinical potency, but sharing a common pharmacological target: farnesyl pyrophosphate (FPP) synthase. Surprisingly, at the tested concentrations, both drugs stimulated keratinocytes proliferation, upregulating cytokeratin 5 while downregulating filaggrin expression, and wound healing ability, without any significant effect on matrix metalloproteinase (MMP)-9 activity. The lack of N-BPs effect on MMP-9 activity indicates that wound closure, in our experimental model, is mainly due to an increase in cell proliferation rather than to an increase in cell migration. Therefore, it can be hypothesised that the observed wound healing results could be ascribed to an N-BPs mediated reduction of FPP endogenous levels, thus suggesting new possible clinical applications for these compounds.

Nicotine modulates gelatinase B (MMP-9) and epilysin (MMP-28) expression in reconstituted human oral epithelium.

Oral epithelial keratinocytes express nicotinic cholinergic receptors which activation modulates keratinocytes differentiation and migration through different metabolic pathways. Matrix metalloproteinases (MMPs) are Zn-dependent enzyme involved in cell migration. Among them, gelatinase B (MMP-9) and epilysin (MMP-28) are two MMPs expressed by human keratinocytes during both wound healing and proliferation. Their expression has been investigated in a reconstituted human oral epithelium (HOE) exposed to nicotine (Nic, 1-50 µM) for 72 h both in the absence and presence of the nicotinic antagonist mecamylamine (Mec), H7, a PKC inhibitor and PD98059, a MAPK inhibitor (PD). At the end of treatment, MMP-28 expression has been analyzed in epithelium sections using an anti-MMP-28 antibody, whereas MMP-9 presence and activity has been measured in cell-conditioned medium analyzed by gelatine zymography. The expression of MMP-9 was reduced by Nic in a dose-dependent fashion and this effect was antagonized by Mec, H7 and PD. On the other hand, Nic increased the expression of MMP-28, and this effect was blocked both by H7 and PD, whereas Mec even enforced it. Nic effects on MMP-9 and MMP-28 expression by oral keratinocytes were not previously reported and these data suggest MMPs expression mediated by PKC and MAPK as a possible target for Nic toxicity in oral epithelium.

Processing Adipose Tissue to Make it More Stable When Used for Refilling: A Morphologic and Immunohistochemistry Evaluation.

Breast reconstruction has gained from lipofilling the possibility to recover the aesthetic outcome of anatomical profile in a more natural appearance. However, until today, the long-term graft survival remains unpredictable, and sometimes it does not guarantee a well-adequate aesthetic result. In the present work, the morphological changes, occurring in fat mass used for refilling, harvested by the Coleman's procedure or through the washing/fragmenting procedure were analysed. Adipocyte size; immunohistochemistry against CD8, CD31, CD68 and M2-type macrophages and catalase enzyme, were analysed in vitro on fat mass cultured for 4 weeks. Our observation reveals an increase of connective tissue around the mass and a high number of immune cells occurrence in fat mass harvested by the Coleman's procedure. Instead, the washing/fragmented procedure would reduce the number of immune cells within the fat mass, increase the size of adipocytes, and cause a wider presence of active vessels profile and greater catalase expression. We hypothesize that the fat mass processed by the Coleman's procedure would remain more reactive due to a higher number of immune and macrophages cells, prone to develop cystic formation, leading to a suboptimal integration in the recipient site. On the other hand, the conditions more prone to realize an optimal integration would occur in the fat mass processed by the washing/fragmenting procedure: a reduced number of immune cells, low amount of connective tissue, presence of larger adipocytes. Follow-up monitoring did support our conclusion, as we observed a reduction of re-intervention for refilling procedure in patients treated with the washing/fragmenting procedure.

The Italian law on body donation: A position paper of the Italian College of Anatomists.

In Italy, recent legislation (Law No. 10/2020) has tuned regulations concerning the donation of one's postmortem body and tissues for study, training, and scientific research purposes. This study discusses several specific issues to optimise the applicability and effectiveness of such an important, novel regulatory setting. Critical issues arise concerning the learners, the type of training and teaching activities that can be planned, the position of academic anatomy institutes, the role of family members in the donation process, the time frame of the donation process, the eligibility of partial donation, or the simultaneous donation of organs and tissues to patients awaiting transplantation. In particular, a universal time limit for donations (i.e., one year) makes it impossible to plan the long-term use of specific body parts, which could be effectively preserved for the advanced teaching and training of medical students and surgeons. The abovementioned conditions lead to the limited use of corpses, thus resulting in the inefficiency of the whole system of body donation. Overall, the donors' scope for the donation of their body could be best honoured by a more flexible and tuneable approach that can be used on a case-by-case basis. Furthermore, it is deemed necessary to closely monitor the events scheduled for corpses in public nonacademic institutions or private enterprises. This paper presents useful insights from Italian anatomists with the hope of providing inspiration for drafting the regulations. In conclusion, this paper focuses on the critical issues derived from the recently introduced Italian law on the donation and use of the body after death and provides suggestions to lawmakers for future implementations.

Regulation of osteoblast and osteoclast functions by FGF-6.

Fibroblast growth factor-6 (FGF-6) is known to be the key ligand for fibroblast growth factor receptor 4 (FGFR4) during muscle regeneration but its role in bone has yet to be verified. FGFR signaling is known to be important in the initiation and regulation of osteogenesis, so in this study the actions of FGF-6 on human osteoblasts and osteoclasts were investigated. Human primary osteoblasts (hOB) were used to study the effect of FGF-6 on proliferation (by ATP quantification), signal transduction (by ERK and AKT phosphorylation), differentiation (by alkaline phosphatase activity, APA), and mineralization (by calcein staining). To study FGF-6 activity on osteoclast differentiation, human bone marrow cells were used and tartrate-resistant acid phosphatase (TRAP) multinucleated cells together with actin filaments arrangements were quantified. Human primary mature osteoclasts were used to evaluate the effect of FGF-6 on osteoclast reabsorbing activity by reabsorbed pit measurements. FGF-6 >10(-9) M as FGF-2 10(-7) M induced hOB proliferation mediated by pERK together with a reduction in APA and reduced mineralization of the treated cells. Moreover FGF-6 increased the formation of TRAP-positive multinucleated cells in a dose-dependent manner (maximal effect at 10(-8) M). FGF-6-treated cells showed also a greater percentage of cells that formed typical osteoclast sealing zones. Mature osteoclasts cultured on dentine slice increased the area of reabsorption with a maximal effect of FGF-6 at 10(-12) M. FGF-6 may be considered a regulator of bone metabolism as shown by its activity on both osteoblasts and osteoclasts.

Controlled delivery of the heparan sulfate/FGF-2 complex by a polyelectrolyte scaffold promotes maximal hMSC proliferation and differentiation.

Growth factors and other regulatory molecules are required to direct differentiation of bone marrow-derived human mesenchymal stem cells (hMSC) along specific lineages. However, the therapeutic use of growth factors is limited by their susceptibility to degradation, and the need to maintain prolonged local release of growth factor at levels sufficient to stimulate hMSC. The aim of this study was to investigate whether a device containing heparan sulfate (HS), which is a co-factor in growth factor-mediated cell proliferation and differentiation, could potentiate and prolong the delivery of fibroblast growth factor-2 (FGF-2) and thus enhance hMSC stimulation. To this aim, we synthesized cationic polyelectrolyte polymers covalently and non-covalently anchored to HS and evaluated their effect on hMSC proliferation. Polymers non-covalently bound to HS resulted in the release of an HS/FGF-2 complex rather than FGF-2 alone. The release of this complex significantly restored hMSC proliferation, which was abolished in serum-free medium and only partially restored by the release of FGF-2 alone as occurred with polymer covalently bound to HS. We also demonstrate that exposure to HS/FGF-2 during early growth but not during post-confluence is essential for hMSC differentiation down the fibroblast lineage, which suggests that both factors are required to establish the correct stem cell commitment that is necessary to support subsequent differentiation. In conclusion, the delivery platform described here is a step towards the development of a new class of biomaterial that enables the prolonged, non-covalent binding and controlled delivery of growth factors and cofactors without altering their potency.

Pleiotrophin: Analysis of the endothelialisation potential.

PURPOSE: Endothelialisation of vascular substitutes, in fact, remains one of the most unsolved problems in cardiovascular diseases treatment. Stromal Derived Factor 1 (SDF-1) has been largely investigated as an endothelialisation promoter and Pleiotrophin is a promising alternative. Although it has been known to exert beneficial effects on different cell types, its potential as an inducer of proliferation and migration of endothelial cells was not investigated. Therefore, this work is aimed to compare the effects of Pleiotrophin on proliferation and migration of endothelial cells with respect to SDF-1. MATERIALS/METHODS: Endothelial cell line EA.hy926 was treated with Pleiotrophin (50 ng/ml) or SDF-1 (50 ng/ml). Cell viability was evaluated by MTT assay and migration assays were performed in Transwell chambers. Wound healing potential was evaluated by scratch wound assay. CXCR4, RPTP ß/ζ, PCNA and Rac1 expression was detected by Western Blot. RESULTS: Interestingly, Pleiotrophin significantly increased the viability of the treated endothelial cells with respects to SDF-1. The migratory ability of the endothelial cells was also improved in the presence of Pleiotrophin with reference to the SDF-1 treatment. Moreover, Western Blot analysis showed how the treatment with Pleiotrophin can induce an increase in the expression of RPTP ß/ζ, PCNA and Rac1 compared to SDF-1. CONCLUSION: Due to the significant effects exerted on viability, migration and repair ability of endothelial cells compared to SDF-1, Pleiotrophin can be considered as an interesting molecule to promote re-endothelialisation.

Relevance of inflammation and matrix remodeling in abdominal aortic aneurysm (AAA) and popliteal artery aneurysm (PAA) progression.

Aneurysm is a multifactorial degenerative disease characterized by focal dilatation of blood vessels. Although abdominal aortic (AAA) and popliteal aneurysms (PAA) are the most common dilatative vascular diseases and share some features, a comparison between the different anatomical sites and the relative pathophysiological differences has not been established. In order to gain deeper insights to AAA and PAA, we have characterized the role of matrix remodelling, vascular cells phenotype depletion and the inflammatory process in both diseases. Results show a more extensive presence of T-cell, B-cell and monocyte-macrophage infiltration in AAA with respect to PAA. Concurring with this aspect, IL-6, IL-8 and MCP-1 are 10-fold increased in AAA. Moreover, MMP-9, and metalloproteinase inhibitor 3 (TIMP3) resulted up-regulated in AAA tissues. Regarding the catalytic activity, which is tightly related to the oxidative stress, we found an up-regulation of superoxide dismutase [Mn] mitochondrial (SODM), glutathione peroxidase 3 (GPX3) and peroxiredoxin-1 (PRDX1). Histological analyses clearly showed a massive elastin fragmentation in AAA. This may enhance the inflammatory response, which has a prevalent role in AAA, while PAA is mainly guided by a loss of the contractile phenotype. These findings suggest insight in these potentially devastating diseases in term of their progression, aiming to identify potential specific markers respectively for AAA and PAA treatment.

Cellular behavior of neointima-like cells onto vitamin E-enriched poly(D,L)lactic acid.

In-stent restenosis is a process that occurs in 10-50% of cases currently treated with stent and it is caused by an abnormal smooth muscle cell (SMC) proliferation and migration in the vascular lumen. One of the most promising strategy to reduce restenosis is stent coating with biodegradable polymers to deliver in situ anti-proliferative drugs. Poly(D,L)lactic acid (P(D,L)LA), one of the most interesting candidate for stent coating, has been observed to induce inflammation and neointimal proliferation. In our laboratory, we developed P(D,L)LA enriched with Vitamin E (Vit.E), an anti-oxidative and anti-inflammatory agent that reduces also SMC proliferation. In order to evaluate the in vitro cellular behaviour of neointima cells onto Vitamin E-enriched P(D,L)LA, cell adhesion and proliferation along with the expression of two SMC migration markers (MMP-9 and hyaluronic acid receptor CD44) were measured in rat vascular SMC A10 cells seeded onto control P(D,L)LA (PLA) and P(D,L)LA films containing 10-30% (w/w) Vit.E (PLA10-30). Cell adhesion, proliferation and MMP-9 production were unaffected by the Vit.E presence in the PLA films after 24 h, while proliferation was slowed or blocked after 48 and 72 h onto PLA10, 20 and 30. MMP-9 production was almost blocked and CD44 density decreased significantly after 72 h for cells grew onto PLA30 compare to cells seeded onto PLA. These data indicate that Vit.E-enriched P(D,L)LA could be an interesting polymer for stent coating.

Effect of different growth factors on human osteoblasts activities: a possible application in bone regeneration for tissue engineering.

Cultured human primary osteoblasts reproduce the phenotypic differentiation and maturation of cells in vivo. We have investigated the influence of three isoforms of transforming growth factor beta (TGF-beta1, TGF-beta2 and TGF-beta3), three fibroblast growth factors (FGF-2, FGF-4 and FGF-6) and the active metabolite of Vitamin D [1,25-(OH)(2)D3] on proliferation, alkaline phosphatase activity and mineralization of human osteoblasts during a period of 24 days of culture. TGF-beta isoforms and three FGFs examined have been proved to be inducers of osteoblasts proliferation (higher extent for TGF-beta and FGF-2) and inhibitors of alkaline phosphatase activity and osteoblasts mineralization. Combination of these growth factors with the active form of Vitamin D induced osteodifferentiation. In fact Vitamin D showed an additive effect on alkaline phosphatase activity and calcium content, induced by FGF-2 and TGF-beta in human osteoblast. These results highlight the potential of proliferating cytokines' combination with mineralizing agents for in vitro bone growth induction in bone tissue engineering.

Politetrafluorene suture used as artificial mitral chord: mechanical properties and surgical implications.

BACKGROUND: Novel surgical approach to repair degenerative mitral regurgitation such as transapical chordae tendineae replacement and "loop in loop" in loop techniques, need of artificial chordae longer than that used in the older techniques of chordae tendineae replacement. This difference in length has been reported as potential critical point for durability of artificial chordae. In the present paper we have investigated the elastic behavior of different diameter and length politetrafluorene (PTFE) suture threads as substitute of native chordae, to identify their reliability to use as long artificial chordae. METHODS: PTFE suture threads with different diameters were investigated in their mechanical properties at different length from 2 to 14 cm, by a servo hydraulic testing machine, to test the elastic properties of the sample in their use as mitral chordae substitutes. RESULTS: Our study shows that the chordae length is an important parameter that can change the performance of chordae itself. The analysis of elastic/properties of suture threads specimen, reveals that long PTFE chords have an optimal mechanical behavior in which elongation is accompanied by a safe elastic properties that make them well resistance during multiple tractions. CONCLUSIONS: In conclusion the use of PTFE as an artificial chordae may represent a valid choice in case of insertion of artificial chordae with extra anatomic length.

* Injectable Graft Substitute Active on Bone Tissue Regeneration.

With the aim to obtain an injectable bioactive scaffold that can accelerate bone formation in sinus lift augmentation, in bony void and fracture repair, we have developed a three-dimensional (3D) jelly collagen containing lysophosphatidic acid (LPA) and 1α,25-dihydroxyvitamin D3 (1,25D3). Using an in vitro 3D culture model of bone fracture, we show that the contraction of the collagen gel is mediated by Rho-kinase activation in osteoblasts. The gel contraction showed dependence on cell concentration and was increased by LPA, which favored apposition and fastening of bone fragments approach. LPA was shown to act through actin cytoskeleton reorganization and myosin light chain phosphorylation of human primary osteoblasts (hOB). Moreover, LPA conferred osteoconductive properties as evidenced by the induction of proliferation, differentiation, and migration of hOB. The addition of 1,25D3 did not enhance cell-mediated gel contraction, but stimulated the maturation of hOB in vitro through the production of extracellular matrix of higher quality. On the basis of these observations, the collagen gel enriched with LPA and 1,25D3 described herein can be considered an injectable natural scaffold that allows the migration of cells from the side of bone defect and a promising candidate to accelerate bone growth and fracture healing.

Decellularized biological matrices: an interesting approach for cardiovascular tissue repair and regeneration.

The repair and replacement of blood vessels is one of the most challenging topics for biomedical research. Autologous vessels are preferred as graft materials, but they still have many issues to overcome: for instance, they need multiple surgical procedures and often patients may not have healthy and surgically valuable arteries useful as an autograft. A tissue-engineering approach is widely desirable to generate biological vascular prostheses. Recently, decellularization of native tissue has gained significant attention in the biomedical research field. This method is used to obtain biological scaffolds that are expected to maintain the complex three-dimensional structure of the extracellular matrix, preserving the biomechanical properties of the native tissues. The decellularizing methods and the biomechanical characteristics of these products are presented in this review. Decellularization of biological matrices induces the loss of major histocompatibility complex (MHC), which is expected to promote an immunological response by the host. All the studies showed that decellularized biomaterials possess adequate properties for xenografting. Concerning their mechanical properties, several studies have demonstrated that, although chemical decellularization methods do not affect the scaffolds' mechanical properties, these materials can be modified through different treatments in order to provide the desired mechanical characteristics, depending on the specific application. A short overview of legislative issues concerning the use of decellularized substitutes and future perspectives in surgical applications is also presented. Copyright © 2015 John Wiley & Sons, Ltd.