Transduction profiles in minipig following MRI guided delivery of AAV-5 into thalamic and corona radiata areas.

BACKGROUND: As a step towards clinical use of AAV-mediated gene therapy, brains of large animals are used to settle delivery parameters as most brain connections, and relative sizes in large animals and primates, are reasonably common. Prior to application in the clinic, approaches that have shown to be successful in rodent models are tested in larger animal species, such as dogs, non-human primates, and in this case, minipigs. NEW METHOD: We evaluated alternate delivery routes to target the basal ganglia by injections into the more superficial corona radiata, and, deeper into the brain, the thalamus. Anatomically known connections can be used to predict the expression of the transgene following infusion of AAV5. For optimal control over delivery of the vector with regards to anatomical location in the brain and spread in the tissue, we have used magnetic resonance image-guided convection-enhanced diffusion delivery. RESULTS: While the transduction of the cortex was observed, only partial transduction of the basal ganglia was achieved via the corona radiata. Thalamic administration, on the other hand, resulted in widespread transduction from the midbrain to the frontal cortex COMPARISON WITH EXISTING METHODS: Compared to other methods, such as delivery directly to the striatum, thalamic injection may provide an alternative when for instance, injection into the basal ganglia directly is not feasible. CONCLUSIONS: The study results suggest that thalamic administration of AAV5 has significant potential for indications where the transduction of specific areas of the brain is required.

Semi-spherical Radiofrequency Bipolar Device - A New Technique for Liver Resection: Experimental In Vivo Study on the Porcine Model.

The incidence of colorectal carcinoma is still growing in the Czech Republic and also all around the world. With success of oncological treatment is also growing a number of potential patients with liver metastases, who can profit from surgical therapy. The aim of this study was to confirm on porcine models that this method by using new surgical device is effective and safe for patients who have to undergo liver resection. The primary hypothesis of the study was to evaluate whether this new device is able to consistently produce homogeneous and predictable areas of coagulation necrosis without the Pringle maneuver of vascular inflow occlusion. The secondary hypothesis of the study was to compare the standard linear radiofrequency device and a new semi-spherical bipolar device for liver ablation and resection in a hepatic porcine model. Twelve pigs were randomly divided into two groups. Each pig underwent liver resection from both liver lobes in the marginal, thinner part of liver parenchyma. The pigs in first group were operated with standard using device and in the second group we used new developed semi-spherical device. We followed blood count in 0(th), 14(th) and 30(th) day from operation. 14(th) day from resection pigs underwent diagnostic laparoscopy to evaluate of their state, and 30(th) day after operation were all pigs euthanized and subjected to histopathological examination. Histopathological evaluation of thermal changes at the resection margin showed strong thermal alteration in both groups. Statistical analysis of collected dates did not prove any significant (p < 0.05) differences between standard using device and our new surgical tool. We proved safety of new designed semi-spherical surgical. This device can offer the possibility of shortening the ablation time and operating time, which is benefit for patients undergoing the liver resection.

[Radiofrequency ablation of pancreatic neuroendocrine tumor]. / Radiofrekvencní ablace neuroendokrinního nádoru pankreatu.

BACKGROUNDS: Pancreatic neuroendocrine tumors (PNETs) include a broad range of neoplasms spanning from relatively benign to malignant. Radical resection has been advocated as the only curative method. Debulking (R2) resection can be indicated for locally unresectable PNETs. Debulking surgery improves the quality of life and prolongs overall survival. The disadvantages of this approach include bleeding, pancreatic fistula and tumor spread. No alternative method that would eliminate these complications has been published yet. Considering the encouraging results of the studies describing radiofrequency ablation (RFA) of locally advanced pancreatic cancer, a question arises, whether it might be possible to use RFA as a R2 resection alternative in PNETs. CASE: A 73-year-old gentleman had been admitted due to abdominal pain and hyperglycaemic syndrome. Contrast-enhanced CT showed a tumor of pancreatic head invading portal vein (PV) and superior mesenteric vein (VMS). A surgery was indicated on the basis of EUS-guided FNAB that verified a PNET of uncertain biological behaviour. The surgery confirmed a locally advanced tumor of pancreatic head invading the PV and SMV. Due to the polymorbidity, radical pancreatoduodenectomy with SMV resection was not indicated. Because of the presence of symptoms, RFA of the PNET using ValleyLab generator with cooltip cluster electrode, was performed. Postoperative course was uneventful. Final immunohistochemical examination verified a well-differentiated grade 1 PNET. The patient was regularly monitored during a three-year follow-up. The quality of life was evaluated using standardized EORT QLQ-30 questionnaire. Pain was assessed by a ten-point visual analogue scale (VAS). Ablated area was evaluated annually by contrast-enhanced CT. Postoperatively, abdominal pain ceased (pain decrease from 2 to 0 on VAS). Insulin dose was reduced from 46 IU (international units) to 20 IU of Humulin-R per day. CT verified tumor regression according to RECIST (response evaluation criteria in solid tumors). During the three-year follow-up, no local progression or tumor dissemination was observed. CONCLUSION: We present the first case report of a patient with locally advanced symptomatic pancreatic neuroendocrine tumor successfully treated by intraoperative radiofrequency ablation. More clinical studies are needed to evaluate the clinical relevance of this cytoreductive method in the PNET indication.

[Treatment of a bone bridge by transplantation of mesenchymal stem cells and chondrocytes in a composite scaffold in pigs: experimental study]. / Lécba kostního mustku transplantací mezenchymových kmenových bunek a chondrocytu v kompozitním nosici u prasat - experimentální studie.

PURPOSE OF THE STUDY: The presented experimental study describes the results of using a combination of allogeneic mesenchymal cells (MSCs) with chondrocytes (CHCs) and a novel scaffold based on type I collagen and chitosan fibres. This biocomposite was transplanted into a defect produced by excision of a bone bridge to induce new cartilaginous tissue formation. The left femur was treated by transplantation into a defect of distal epiphysis; the right femur with implantation of the scaffold only served as control. A better therapeutic result was therefore expected in the left femur - the reduction of growth and angular deformities, and the histological finding of a tissue similar to the cartilage excised from the left femur.. MATERIAL AND METHODS: The miniature pig was selected as an experimental model and 10 pigs were used. Mesenchymal stem cells derived from femoral bone marrow and chondrocytes derived from a sample harvested from the non-weight-bearing articular surface of the distal end of the femur were cultured in medium. The novel scaffold was based on collagen containing chitosan nanofibres. To make manipulation during implantation easier, the cilindrical scaffolds after lyophilisation were again placed in 96-well plates for seeding. The scaffolds before implantation were seeded with 2x106 allogeneic MSCs and 1x106 allogeneic CHCs. The outcomes of treatment were assessed by measuring the length of bone and the degree of distal femoral valgus deformity, and by the histological findings obtained (properties and maturity of the newly-formed tissue, detection of type II collagen, PAS reaction). RESULTS: The right and left legs were examined for longitudinal bone growth and the valgus angle and compared. The treated left leg showed a higher average value for longitudinal growth than the untreated right leg (p = 0.004). The average degree of angular deformity was lower in the left leg than in the right leg (p = 0.008). The microscopic findings showed that a tissue similar to hyaline cartilage was more frequently present in the femoral bone defect of the left leg, as compared with that of the right leg. Type II collagen was detected more frequently and at higher amounts on the left than the right side (p = 0.033). The PAS reaction was positive in all left limbs, with a high degree of positivity in 80 % of them, while this was not achieved in any of the right limbs (p = 0.001). DISCUSSION: The use of stem cells in the indication reported here has only been the matter of time since the information on encouraging results in neurology and cardiology was published. First studies with positive results have soon been reported. The initial hydrogel scaffolds were based on tissue adhesives. However, they were not stable enough and were difficult to handle during surgery. In further studies, therefore, the use was made of a three-dimensional scaffold with a self-supporting structure of collagen fibres. This structure also facilitated its hydrodynamic seeding with MSCs and CHCs, which is an effective and sparing procedure for the transplanted cells. Studies concerned with MSCs and/or CHCs transplantation for re - pair of a physeal defect following bone bridge excision, i.e. for bone bridge treatment, in a broader experimental design, however, are still missing. CONCLUSION: Transplantation of a composite scaffold seeded with mesenchymal stem cells and chondrocytes into a physeal defect following bone bridge excision prevented growth disturbance and angular deformity development in the distal femoral epi - physis. In comparison with the control group, it resulted in a more frequent production of a tissue similar to hyaline cartilage, with a cell formation reminiscent of a typical columnar arrangement of the growth plate. Key words: mesenchymal stem cells, growth plate, bone bridge, scaffold.

Quality of newly formed cartilaginous tissue in defects of articular surface after transplantation of mesenchymal stem cells in a composite scaffold based on collagen I with chitosan micro- and nanofibres.

The aim of this study was to evaluate macroscopically, histologically and immunohistochemically the quality of newly formed tissue in iatrogenic defects of articular cartilage of the femur condyle in miniature pigs treated with the clinically used method of microfractures in comparison with the transplantation of a combination of a composite scaffold with allogeneic mesenchymal stem cells (MSCs) or the composite scaffold alone. The newly formed cartilaginous tissue filling the defects of articular cartilage after transplantation of the scaffold with MSCs (Group A) had in 60 % of cases a macroscopically smooth surface. In all lesions after the transplantation of the scaffold alone (Group B) or after the method of microfractures (Group C), erosions/fissures or osteophytes were found on the surface. The results of histological and immunohistochemical examination using the modified scoring system according to O'Driscoll were as follows: 14.7+/-3.82 points after transplantations of the scaffold with MSCs (Group A); 5.3+/-2.88 points after transplantations of the scaffold alone (Group B); and 5.2+/-0.64 points after treatment with microfractures (Group C). The O'Driscoll score in animals of Group A was significantly higher than in animals of Group B or Group C (p<0.0005 both). No significant difference was found in the O'Driscoll score between Groups B and C. The treatment of iatrogenic lesions of the articular cartilage surface on the condyles of femur in miniature pigs using transplantation of MSCs in the composite scaffold led to the filling of defects by a tissue of the appearance of hyaline cartilage. Lesions treated by implantation of the scaffold alone or by the method of microfractures were filled with fibrous cartilage with worse macroscopic, histological and immunohistochemical indicators.