Growth of Intestinal Neomucosa on Pedicled Gastric Wall Flap, a Novel Technique in an Animal Model.

Background: Short bowel syndrome (SBS) remains an unsolved issue in modern medicine. Numerous experimental surgical techniques have been proposed in the attempt to increase the intestinal absorptive capacity.Materials and Methods: Ten female Landrace pigs, divided in two groups of 5 (A and B), were explored through a midline incision. A spindle-shaped vascularized full-thickness gastric wall flap (GWF) consisting of part of the major curvature with the gastroepiploic arch preserved was de-epithelialized and then placed as a "patch" to cover an antimesenteric border defect of either a nonfunctional blind intestinal loop (group A) or a functional intestinal loop of the gastrointestinal tract (group B). A spindle-shaped curved, rigid, low density polyethylene (LDPE) splint was sutured on the external surface of the patch in order to prevent shrinkage of GWF and collapse of the intestinal wall in group A.Results: There was a decrease of both dimensions of the patch. Microscopically a thin layer of columnar epithelial cells covered the center of the patch, evolving in shorter, blunt, poorly developed villi with increasing maturation laterally. The patch surface was covered by nearly 90%. In the three animals that died prematurely the coverage of GWF was negligent or suboptimal directly dependent on the length of survival.Conclusions: The hereby-described patching technique demonstrated the growth of intestinal neomucosa on the GWF. The capability of the stomach to provide large flaps and the advantages of the use of native tissues render this animal model valuable for the future research in the field.

A High-Cholesterol Diet Increases Toll-like Receptors and Other Harmful Factors in the Rabbit Myocardium: The Beneficial Effect of Statins.

BACKGROUND: A high-cholesterol diet (HCD) induces vascular atherosclerosis through vascular inflammatory and immunological processes via TLRs. The aim of this study is to investigate the mRNA expression of TLRs and other noxious biomarkers expressing inflammation, fibrosis, apoptosis, and cardiac dysfunction in the rabbit myocardium during (a) high-cholesterol diet (HCD), (b) normal diet resumption and (c) fluvastatin or rosuvastatin treatment. METHODS: Forty-eight male rabbits were randomly divided into eight groups (n = 6/group). In the first experiment, three groups were fed with HCD for 1, 2 and 3 months. In the second experiment, three groups were fed with HCD for 3 months, followed by normal chow for 1 month and administration of fluvastatin or rosuvastatin for 1 month. Control groups were fed with normal chow for 90 and 120 days. The whole myocardium was removed; total RNA was isolated from acquired samples, and polymerase chain reaction, reverse transcription PCR and quantitative real-time PCR were performed. RESULTS: mRNA of TLRs 2, 3, 4 and 8; interleukin-6; TNF-a; metalloproteinase-2; tissue inhibitor of metalloproteinase-1; tumor protein 53; cysteinyl aspartate specific proteinase-3; and brain natriuretic peptide (BNP) increased in HCD. Statins but not resumption of a normal diet decreased levels of these biomarkers and increased levels of antifibrotic factors. CONCLUSIONS: HCD increases the levels of TLRs; inflammatory, fibrotic and apoptotic factors; and BNP in the rabbit myocardium. Atherogenic diets adversely affect the myocardium at a molecular level and are reversed by statins.

Insights into Biomechanical and Proteomic Characteristics of Small Diameter Vascular Grafts Utilizing the Human Umbilical Artery.

The gold standard vascular substitutes, used in cardiovascular surgery, are the Dacron or expanded polytetrafluoroethylene (ePTFE)-derived grafts. However, major adverse reactions accompany their use. For this purpose, decellularized human umbilical arteries (hUAs) may be proven as a significant source for the development of small diameter conduits. The aim of this study was the evaluation of a decellularization protocol in hUAs. To study the effect of the decellularization to the hUAs, histological analysis was performed. Then, native and decellularized hUAs were biochemically and biomechanically evaluated. Finally, broad proteomic analysis was applied. Histological analysis revealed the successful decellularization of the hUAs. Furthermore, a great amount of DNA was removed from the decellularized hUAs. Biomechanical analysis revealed statistically significant differences in longitudinal direction only in maximum stress (p < 0.013) and strain (p < 0.001). On the contrary, all parameters tested for circumferential direction exhibited significant differences (p < 0.05). Proteomic analysis showed the preservation of the extracellular matrix and cytoskeletal proteins in both groups. Proteomic data are available via ProteomeXchange with identifier PXD020187. The above results indicated that hUAs were efficiently decellularized. The tissue function properties of these conduits were well retained, making them ideal candidates for the development of small diameter vascular grafts.

Cilostazol Mediates Immune Responses and Affects Angiogenesis During the Acute Phase of Hind Limb Ischemia in a Mouse Model.

BACKGROUND: Cilostazol is a drug of choice for the treatment of intermittent claudication that also affects innate and adaptive immune cells. The purpose of our study was the evaluation of cilostazol's impact on the immune and angiogenic response in murine models of hind limb ischemia. METHODS: We used 108 immunodeficient NOD.CB17-Prkdcscid/J mice and 108 wild-type CB17 mice. At day 0 (D0), all animals underwent hind limb ischemia. Half of them in both groups received daily cilostazol starting at D0 and for the next 7 postoperative days, while the rest of them served as controls, receiving vehicle. Interleukin (IL) 2, IL-4, IL-6, IL-10, IL-17A, tumor necrosis factor α (TNF-α), and interferon γ (IFN-γ) serum concentrations were measured by flow cytometry on postsurgery days D1, D3, D5, and D7. On D7, both groups underwent positron emission tomography scan with 68Ga-RGD. Mice were euthanatized and gastrocnemius muscles were obtained for histological evaluation. RESULTS: There was a statistically significant augmentation (P < .05) in IL-4, IL-10, IL-6, and IFN-γ concentrations in treated CB17 animals, while IL-2 was significantly suppressed. Significant difference was detected between the CiBisch and Bisch groups on D1 and D7 (P < .05) in CD31 staining. In treated NOD.CB17 animals, TNF-α, IL-6, and IFN-γ presented significant augmentation, while 68Ga-NODAGA-RGDfK uptake and CD31 expression were found significantly lower for both legs in comparison to the control. CONCLUSION: Cilostazol seems to significantly increase angiogenesis in wild-type animals during the first postoperational week. It also influences immune cells, altering the type of immune response by promoting anti-inflammatory cytokine production in wild-type animals, while it helps toward inflammation regression in immunodeficient animals.

Advanced Therapy Medicinal Products Challenges and Perspectives in Regenerative Medicine.

Recently, the design and development of a modern health policy in the field of regenerative medicine leads to the formation of a new and integrated cognitive field, which requires systematic research and study in order to produce innovative answers and best practices. Advanced therapy medicinal products (ATMPs) is a new product category, which is at the heart of concern since it has to deal with diseases in which traditional medicine has proven to be ineffective so far. The aim of this review is to provide evidence for the state of the art ATMPs and their modern applications in the field of regenerative medicine. The ATMPs are characterized by a great heterogeneity and variation in methods of isolation, which cover the entire spectrum from a single intravenous injection to a surgical placement. Clinical development of ATMP encounters specific challenges due to the nature of the product and the limited availability of non-clinical data. The gold standard of a controlled, randomized, clinical trial may not be feasible or ethically justified for all indications, particularly in life-threatening diseases, where there is no satisfactory standard of care. Therefore, the European Commission (EC) took initiatives in order to set standards and operating rules concerning authorization and supervision of ATMPs and on pharmacovigilance in relation to them. The European Union (EU) Regulation 1394/2007 provides the possibility of exceptions. In particular, the "hospital exemption" allows for the administration of an ATMP without a license on certain conditions. Although the Regulation 1394/2007 has led to the commercial exploitation of ATMPs, the reality today, 11 years after its first implementation, is completely different. While the Committee for Advanced Therapies (CAT) has already registered 285 products as ATMPs, only 10 licenses were granted which only remained six (the rest related to products withdrawn). The key players in the development and delivery of ATMPs still remain the academic/research centers and small and medium-sized enterprises; while the involvement of pharmaceutical companies is focusing on recent developments in the treatment of oncological incidents with in vitro modified cytotoxic T lymphocytes, and chimeric antigen receptor (CAR)-T cells.

Toll-Like Receptors -2, -3, -4 and -7 Expression Patterns in the Liver of a CLP-Induced Sepsis Mouse Model.

Objective: To investigate the expression of toll-like receptors (TLRs) in the liver of septic mouse model. Materials and methods: For this study seventy-two C57BL/6J mice were utilized. Sepsis was induced by cecal ligation and puncture (CLP) in the mice of the three septic (S) groups (euthanized at 24 hours, 48 hours and 72 hours). Sham (laparotomy)- operated mice constituted the control (C) groups (euthanized at 24, 48 and 72 hours). Blood samples were drawn and liver tissues were extracted and examined histologically. The expression of TLRs 2, 3, 4 and 7 was assessed via immunohistochemistry (IHC) and qrt-PCR (quantitative- Polymerase Chain Reaction). Results: Liver function tests were elevated in all S-groups in contrast to their time-equivalent control groups (S24 versus C24, S48 versus C48 and S72 versus C72) (p < 0.05). Liver histology displayed progressive deterioration in the septic groups. IHC and qrt-PCR both showed an increased expression of all TLRs in the septic mice in comparison to their analogous control ones (p < 0.05). Analysis of livers and intestines of the septic animals proved that all TLRs were significantly expressed in higher levels in the intestinal tissues at 24h and 48h (p < 0.05) except for TLR 3 in S48 (p > 0.05); whereas at 72 hours only TLR 4 levels were significantly elevated in the intestine (p < 0.05). Conclusion: TLRs seem to be expressed in significant levels in the livers of septic rodents, indicating that they have a possible role in the pathophysiology of liver damage in septic conditions.

Evaluation of a Decellularization Protocol for the Development of a Decellularized Tracheal Scaffold.

BACKGROUND/AIM: Currently, there are no effective solutions for the treatment of advanced disorders of the airways. The aim of this study was to assess the efficacy of a decellularization protocol of the trachea in order to produce a functional scaffold for serious clinical respiratory disorders. MATERIALS AND METHODS: Rat tracheas were decellularized using a protocol which included constituents with chemical action. Histological analysis was performed in order to evaluate the efficacy of decellularization. Genetic material was assayed and the toxicity of the decellularization protocol was assessed. RESULTS: Histological analysis confirmed the removal of the nuclear and cellular components of the decellularized tissue, as well as maintenance of the extracellular matrix. DNA quantification showed removal of the genetic material. Furthermore, the decellularization protocol did not induce any cytotoxicity on tracheaI tissue. CONCLUSION: The decellularization protocol was effective for tracheal decellularization. The final aim, in the future, would be to create a tissue-engineered airway which will be able to function normally.

The complementary effects of atorvastatin and exercise treatment on the composition and stability of the atherosclerotic plaques in ApoE knockout mice.

AIM: This study aimed to investigate the effects of combined atorvastatin and exercise treatment on the composition and stability of the atherosclerotic plaques in apolipoproteinE (apoE) knockout mice. METHODS: Forty male, apoE-/- mice were fed a high-fat diet for 16 weeks. Thereafter, while maintained on high-fat diet, they were randomized into four (n = 10) groups for 8 additional weeks: Group CO: Control. Group AT: Atorvastatin treatment (10 mg/Kg/day). Group EX: Exercise-training on treadmill. Group AT+EX: Atorvastatin and simultaneous exercise training. At the study's end, plasma cholesterol levels, lipids and triglycerides were measured, along with the circulating concentrations of matrix-metalloproteinases (MMP-2,3,8,9) and their inhibitors (TIMP-1,2,3). Plaque area and the relative concentrations of collagen, elastin, macrophages, smooth muscle cells, MMP-2,3,8,9 and TIMP-1,2,3 within plaques were determined. Lastly, MMP activity was assessed in the aortic arch. RESULTS: All intervention groups showed a lower degree of lumen stenosis, with atheromatous plaques containing more collagen and elastin. AT+EX group had less stenosis and more elastin compared to single intervention groups. MMP-3,-8 -9 and macrophage intra-plaque levels were reduced in all intervention groups. EX group had increased TIMP-1 levels within the lesions, while TIMP-2 was decreased in all intervention groups. The blood levels of the above molecules increased during atherosclerosis development, but they did not change after the therapeutic interventions in accordance to their intra-plaque levels. CONCLUSION: The two therapeutic strategies act with synergy regarding the extent of the lesions and lumen stenosis. They stabilize the plaque, increasing its content in elastin and collagen, by influencing the MMP/TIMP equilibrium, which is mainly associated with the macrophage amount. While the increased MMP-2,-3,-8 -9, as well as TIMP-1 and TIMP-2 circulating levels are markers of atherosclerosis, they are not correlated with their corresponding concentrations within the lesions after the therapeutic interventions, and cannot serve as markers for the disease development/amelioration.

Minimal invasive technique for gene delivery in porcine liver lobe segment.

INTRODUCTION: The feasibility and outcome of large volume injection of gene solution in a segment of a liver lobe, without backflow, were studied in a porcine model, using a custom-designed balloon catheter. METHOD: Eight anesthetized pigs underwent successful injection of 200 ml of gene solution at a rate of 20 ml/s via a minimally invasive technique without backflow. A custom-made balloon catheter was introduced under fluoroscopy guidance into the right lateral liver lobe via the right external jugular vein. The vein of the liver lobe was occluded with the balloon catheter and contrast material was injected to check if total occlusion was achieved. Since there was no backflow an angiographic pump injected the solution. The catheter was left in place for 10 min. Then contrast material was injected to check whether the vein remained occluded. RESULTS: All animals tolerated the procedure without obvious adverse effects. Ultrasound scan showed no gross changes within liver three days following the infusion. A transient rise in platelet count was observed which returned to normal after 13 days and remained stable; all other biochemistry values were normal. CONCLUSIONS: Injecting large volume of gene solution in a liver lobe segment using this minimally invasive technique in a porcine model is possible, making the development of a successful gene transfer protocol in humans feasible.