Photochemical Crosslinking of Tarsal Collagen as a Treatment for Eyelid Laxity: Evaluation in Ex Vivo Human Tissue.

PURPOSE: Experimental investigation in human eyelids to confirm that exposing excised tarsal plates to ultraviolet-A radiation can induce a stiffening effect through the riboflavin-photosensitized crosslinking of tarsal collagen. METHODS: Thirteen tarsal plates excised from nonfrozen human cadavers were irradiated with ultraviolet-A rays (365 nm wavelength) at an irradiance of 75 mW/cm2 for 3 minutes, equivalent to a radiation fluence of 13.5 J/cm2, in the presence of a riboflavin derivative as a photosensitizer. The tensile stress (strength) and Young's modulus (stiffness) of both nonirradiated and irradiated specimens were measured with the BioTester 5000 in the uniaxial mode at a strain of 10% and analyzed statistically. Individual specimens excised from 2 cadavers were also examined by routine histopathologic protocols to assess the effect of radiation on the Meibomian glands and collagen organization. RESULTS: The irradiation enhanced both stiffness and strength of the human tarsal specimens, the difference between the test samples and controls being statistically significant (p < 0.0002 for n = 13). Histology indicated no damage to tarsal connective tissue or to Meibomian glands, and revealed a more compact packing of the collagen network located around the glands, which may be beneficial. The existence of collagen compaction was also supported by the reduction of samples' thickness after irradiation (p = 0.0645). CONCLUSIONS: The irradiation of tarsal tissue with ultraviolet-A light of tarsus appears to be a safe and effective method for reducing eyelid laxity in human patients.

Cardio Protective Effects of Lipid Emulsion against Ropivacaine-Induced Local Anesthetic Systemic Toxicity-An Experimental Study.

Inadvertent intravascular injection of local anesthetics (LA) during regional anesthesia causes Local Anesthetic Systemic Toxicity (LAST). Theories of lipid rescue in the case of LAST proved that the administration of lipids in LAST has beneficial effects. One possible mechanism of action is based on the lipophilic properties of LA which allow plasma-free LA to be bound by the molecules of Lipid Emulsion (LE). The association LA-LE is shuttled towards organs such as liver and the kidneys, and the half-life of LA is shortened. The main objective of this experimental study was to assess the possible cardio-prophylactic effect of LE administration before the induction of LAST by intravenous administration of Ropivacaine. This was an experimental, interventional, prospective, and non-randomized study. The subjects were divided into groups and received, under general anesthesia, LE 20% first 0.3-0.4 mL, followed by 0.1 mL Ropivacaine 2 mg/mL, or Ropivacaine alone. At the end of the experiment, the subjects were sacrificed, and tissue samples of kidney, heart and liver were harvested for histopathological examination. LE, when administered as prophylaxis in Ropivacaine-induced LAST, had protective cardiac effects in rats. The LE known side effects were not produced if the substance was administered in the low doses used for LAST prophylaxis.

Preclinical Testing of Living Tissue-Engineered Heart Valves for Pediatric Patients, Challenges and Opportunities.

Introduction: Pediatric patients with cardiac congenital diseases require heart valve implants that can grow with their natural somatic increase in size. Current artificial valves perform poorly in children and cannot grow; thus, living-tissue-engineered valves capable of sustaining matrix homeostasis could overcome the current drawbacks of artificial prostheses and minimize the need for repeat surgeries. Materials and Methods: To prepare living-tissue-engineered valves, we produced completely acellular ovine pulmonary valves by perfusion. We then collected autologous adipose tissue, isolated stem cells, and differentiated them into fibroblasts and separately into endothelial cells. We seeded the fibroblasts in the cusp interstitium and onto the root adventitia and the endothelial cells inside the lumen, conditioned the living valves in dedicated pulmonary heart valve bioreactors, and pursued orthotopic implantation of autologous cell-seeded valves with 6 months follow-up. Unseeded valves served as controls. Results: Perfusion decellularization yielded acellular pulmonary valves that were stable, no degradable in vivo, cell friendly and biocompatible, had excellent hemodynamics, were not immunogenic or inflammatory, non thrombogenic, did not calcify in juvenile sheep, and served as substrates for cell repopulation. Autologous adipose-derived stem cells were easy to isolate and differentiate into fibroblasts and endothelial-like cells. Cell-seeded valves exhibited preserved viability after progressive bioreactor conditioning and functioned well in vivo for 6 months. At explantation, the implants and anastomoses were intact, and the valve root was well integrated into host tissues; valve leaflets were unchanged in size, non fibrotic, supple, and functional. Numerous cells positive for a-smooth muscle cell actin were found mostly in the sinus, base, and the fibrosa of the leaflets, and most surfaces were covered by endothelial cells, indicating a strong potential for repopulation of the scaffold. Conclusions: Tissue-engineered living valves can be generated in vitro using the approach described here. The technology is not trivial and can provide numerous challenges and opportunities, which are discussed in detail in this paper. Overall, we concluded that cell seeding did not negatively affect tissue-engineered heart valve (TEHV) performance as they exhibited as good hemodynamic performance as acellular valves in this model. Further understanding of cell fate after implantation and the timeline of repopulation of acellular scaffolds will help us evaluate the translational potential of this technology.

Challenges in Perioperative Animal Care for Orthotopic Implantation of Tissue-Engineered Pulmonary Valves in the Ovine Model.

BACKGROUND: Development of valvular substitutes meeting the performance criteria for surgical correction of congenital heart malformations is a major research challenge. The sheep is probably the most widely used animal model in heart valves regenerative medicine. Although the standard cardiopulmonary bypass (CPB) technique and various anesthetic and surgical protocols are reported to be feasible and safe, they are associated with significant morbidity and mortality rates. The premise of this paper is that the surgical technique itself, especially the perioperative animal care and management protocol, is essential for successful outcomes and survival. METHODS: Ten juvenile and adult female sheep aged 7.8-37.5 months and weighing 32.0-58.0 kg underwent orthotopic implantation of tissue-engineered pulmonary valve conduits on beating heart under normothermic CPB. The animals were followed-up for 6 months before scheduled euthanasia. RESULTS: Based on our observations, we established a guide for perioperative care, follow-up, and treatment containing information regarding the appropriate clinical, biological, and ultrasound examinations and recommendations for feasible and safe anesthetic, surgical, and euthanasia protocols. Specific recommendations were also included for perioperative care of juvenile versus adult sheep. CONCLUSION: The described surgical technique was feasible, with a low mortality rate and minimal surgical complications. The proposed anesthetic protocol was safe and effective, ensuring both adequate sedation and analgesia as well as rapid recovery from anesthesia without significant complications. The established guide for postoperative care, follow-up and treatment in sheep after open-heart surgery may help other research teams working in the field of heart valves tissue regeneration.

Intestinal fatty acid-binding protein, as a marker of anastomotic leakage after colonic resection in rats.

AIM: The aim of this experimental study was to determine if the type of termino-terminal anastomosis of the colon affect the process of healing of the intestinal mucosa and if the severity of the anastomotic leakage (AL) can be predicted based on the circulating level of intestinal fatty acid-binding protein (I-FABP). MATERIALS AND METHODS: In 18 healthy Wistar rats, two types of open colon resection with termino-terminal anastomosis were performed: group A (n=9) - without inversing the vascularization and group B (n=9) - with inversing the vascularization. To assess the intestinal barrier function, circulating level of I-FABP was measured pre- and post-operatively. I-FABP tissue expression was immunohistochemically assessed in the anastomotic and perianastomotic colonic mucosa. The rats were sacrificed at three, five, and seven days after surgery. RESULTS: In both groups, the post-operative serum level of I-FABP increased 3-4 times at 3-5 days and seven times in the seventh post-operative day. In the six cases that showed AL, the increased level was significantly higher: seven times at three days (n=2) or five days (n=2) and 30 times at seven days (n=2). The I-FABP epithelial expression was lost in all cases from group B (as result of prolonged ischemia) and in cases with AL from group A. The I-FABP was translocated in the intraglandular mucus. CONCLUSIONS: The post-operative level of I-FABP can be appreciated based on the pre-operative value only. A 7-8 increased value in first five days might indicate a risk of AL. After seven days, a seven times increased value is an indicator of a proper healing process but an increasing amount higher that 30 times might predict risk for AL, fistula, peritonitis and septic shock. The risk of AL does not depend on the anastomotic method, although the level of ischemia is higher in anastomoses that involve vascular damage.

A mitochondrial DNA phylogeny of the endangered vipers of the Vipera ursinii complex.

The last two populations of the Hungarian meadow viper Vipera ursinii rakosiensis were thought to persist in the steppe fragments of Hungary until meadow vipers were discovered in central Romania (Transylvania), suggesting a possible existence of remnant populations elsewhere. We assessed the phylogenetic position of the Transylvanian vipers using 2030 bp of mitochondrial DNA sequence. We showed that they were closely related to the Hungarian vipers, while those from northeastern Romania (Moldavia) and Danube Delta belonged to the subspecies Vipera ursinii moldavica. Montane subspecies from Europe (Vipera ursinii ursinii and Vipera ursinii macrops) formed a sister clade to the two lowland subspecies. Vipera renardi formed a sister clade to V. ursinii, with populations from the Greater Caucasus (Vipera renardi lotievi) and Tien Shan (Vipera renardi tienshanica) as the sister group to Vipera renardi renardi, and Vipera renardi eriwanensis from the Lesser Caucasus as the most basal taxon in the species. Our results illustrate that the divergence between the lowland and montane populations occurred separately in each species and several times in V. renardi. We demonstrated that the recently discovered Transylvanian population is the third surviving population of V. u. rakosiensis and the only known population outside of Hungary.