Precision spinal gene delivery-induced functional switch in nociceptive neurons reverses neuropathic pain.

Second-order spinal cord excitatory neurons play a key role in spinal processing and transmission of pain signals to the brain. Exogenously induced change in developmentally imprinted excitatory neurotransmitter phenotypes of these neurons to inhibitory has not yet been achieved. Here, we use a subpial dorsal horn-targeted delivery of AAV (adeno-associated virus) vector(s) encoding GABA (gamma-aminobutyric acid) synthesizing-releasing inhibitory machinery in mice with neuropathic pain. Treated animals showed a progressive and complete reversal of neuropathic pain (tactile and brush-evoked pain behavior) that persisted for a minimum of 2.5 months post-treatment. The mechanism of this treatment effect results from the switch of excitatory to preferential inhibitory neurotransmitter phenotype in dorsal horn nociceptive neurons and a resulting increase in inhibitory activity in regional spinal circuitry after peripheral nociceptive stimulation. No detectable side effects (e.g., sedation, motor weakness, loss of normal sensation) were seen between 2 and 13 months post-treatment in naive adult mice, pigs, and non-human primates. The use of this treatment approach may represent a potent and safe treatment modality in patients suffering from spinal cord or peripheral nerve injury-induced neuropathic pain.

Huntingtin Co-Isolates with Small Extracellular Vesicles from Blood Plasma of TgHD and KI-HD Pig Models of Huntington's Disease and Human Blood Plasma.

(1) Background: Huntington's disease (HD) is rare incurable hereditary neurodegenerative disorder caused by CAG repeat expansion in the gene coding for the protein huntingtin (HTT). Mutated huntingtin (mHTT) undergoes fragmentation and accumulation, affecting cellular functions and leading to neuronal cell death. Porcine models of HD are used in preclinical testing of currently emerging disease modifying therapies. Such therapies are aimed at reducing mHTT expression, postpone the disease onset, slow down the progression, and point out the need of biomarkers to monitor disease development and therapy efficacy. Recently, extracellular vesicles (EVs), particularly exosomes, gained attention as possible carriers of disease biomarkers. We aimed to characterize HTT and mHTT forms/fragments in blood plasma derived EVs in transgenic (TgHD) and knock-in (KI-HD) porcine models, as well as in HD patients' plasma. (2) Methods: Small EVs were isolated by ultracentrifugation and HTT forms were visualized by western blotting. (3) Results: The full length 360 kDa HTT co-isolated with EVs from both the pig model and HD patient plasma. In addition, a ~70 kDa mutant HTT fragment was specific for TgHD pigs. Elevated total huntingtin levels in EVs from plasma of HD groups compared to controls were observed in both pig models and HD patients, however only in TgHD were they significant (p = 0.02). (4) Conclusions: Our study represents a valuable initial step towards the characterization of EV content in the search for HD biomarkers.

Novel porcine model of Crohn's disease anastomotic stricture suitable for evaluation and training of advanced endoscopic techniques.

BACKGROUND AND AIMS: Currently, treatment options in postsurgical recurrence of stricturing Crohn's disease (CD) are limited. However, development of new invasive endoscopic techniques in clinical practice has safety constraints. The aim of this study was to create a large animal model of anastomotic stricture with CD properties to enable development of new techniques and training. METHODS: A side-to-side ileocolonic anastomosis was created in a modified Roux-en-Y manner with bowel continuity preserved. Two weeks after surgery, we began endoscopic submucosal injections of phenol/trinitrobenzenesulfonic acid solution. This solution was injected every 2 weeks in each quadrant of the anastomosis until development of a stricture. The anastomosis site was assessed endoscopically 2 weeks after the last application (baseline) and then every 2 months until month 6. Endoscopically nonpassable strictures were treated with balloon dilation, endoscopic stricturotomy, and stent placement to confirm the feasibility of such interventions. RESULTS: Nineteen minipigs were included with no postoperative adverse events. After a mean of 4.4 ± .7 injection sessions with 10.5 ± 3.0 mL of the solution, anastomotic strictures were created in 16 pigs (84.2%). Mean diameter of the strictures at baseline was 11.6 ± 2.2 mm. The strictures were inflamed, and the endoscope could not pass. Follow-up was successfully completed in 15 animals (79.0%) with the mean deviation from the initial diameter in every measurement of -.02 ± 2.26 mm (P = .963) and a mean final diameter of 11.7 ± 3.4 mm. The histopathologic evaluation revealed the presence of submucosal fibrosis, chronic inflammation, and microgranulomas. All strictures were amenable to endoscopic therapeutic interventions. CONCLUSIONS: We developed a novel, reproducible porcine model of anastomotic stricture with histologically verified changes mimicking CD and stable diameter for more than 6 months. It is suitable for further endoscopic interventions.

Surface Microdialysis for Detection of Colorectal Anastomosis Ischemia-An Experimental Study.

BACKGROUND: Inadequate blood supply is one of the major risk factors for anastomotic leak after low anterior rectal resection. Early detection of ischemia might predict complicated healing and enable anastomotic salvage, which is associated with better outcomes. A microdialysis offers a real-time evaluation of adequate bowel perfusion through monitoring of tissue metabolism. In this experimental study, we assessed the role of microdialysis in detecting early ischemia after colorectal anastomosis. MATERIALS AND METHODS: Colorectal anastomosis was performed in six miniature pigs. A microdialysis catheter was placed on the bowel serosa 5 mm proximal to the anastomosis. Metabolic changes were monitored hourly before and after initiating ischemia, which was induced by ligation of the inferior mesenteric artery and skeletonization of the mesocolon. RESULTS: Significant increase in tissue levels of lactate was detected after 60 min of ischemia (13.6 [10.4-16.1] versus 6.75 [1.8-9.6] mmol/L at baseline; P < 0.005). The lactate/pyruvate ratio increased accordingly. The concentration of glycerol increased significantly after 2 hours-from a baseline value of 29.5 (3-84) to 125 (79-201) mmol/L (P < 0.005). The decrease in glucose levels was also significant after 60 minutes-0 (0-0.2) versus 4.7 (3.3-6.8) mmol/L at baseline. However, its values started to decline before ischemia. CONCLUSIONS: Surface microdialysis can detect ischemic changes early and may be a promising method in postoperative monitoring of colorectal anastomosis.

A novel postgraduate endoscopic course using a large animal model of secondary Crohn's disease stricture.

BACKGROUND: The increasing complexity of advanced endoscopic techniques places a high demand on the endoscopist's expertise. Thus, live porcine models have been more frequently used for training. We briefly describe a hands-on postgraduate endoscopic course regarding a novel method of treatment of anastomotic strictures in a porcine model. METHODS: The porcine model of Crohn's disease anastomotic stricture with two artificial side-to-side ileo-colonic anastomoses was used. Participants performed endoscopic stricturotomy under supervision at one of two equipped endoscopic stations. Available animals were endoscopically re-examined 3 months after the course. RESULTS: Twelve anastomoses were prepared for the course. Eleven circumferential stricturotomies together with horizontal cut and clip placement were conducted. All anastomoses were passable for the scope after the procedure, and no case of perforation or bleeding occurred. All anastomoses available for re-examination remained passable for the endoscope after 3 months. CONCLUSION: We successfully organised the first endoscopic hands-on course for the training of endoscopic stricturotomy on a large animal model.

Positive impact of dynamic seeding of mesenchymal stem cells on bone-like biodegradable scaffolds with increased content of calcium phosphate nanoparticles.

One of the main aims of bone tissue engineering, regenerative medicine and cell therapy is development of an optimal artificial environment (scaffold) that can trigger a favorable response within the host tissue, it is well colonized by resident cells of organism and ideally, it can be in vitro pre-colonized by cells of interest to intensify the process of tissue regeneration. The aim of this study was to develop an effective tool for regenerative medicine, which combines the optimal bone-like scaffold and colonization technique suitable for cell application. Accordingly, this study includes material (physical, chemical and structural) and in vitro biological evaluation of scaffolds prior to in vivo study. Thus, porosity, permeability or elasticity of two types of bone-like scaffolds differing in the ratio of collagen type I and natural calcium phosphate nanoparticles (bCaP) were determined, then analyzes of scaffold interaction with mesenchymal stem cells (MSCs) were performed. Simultaneously, dynamic seeding using a perfusion bioreactor followed by static cultivation was compared with standard static cultivation for the whole period of cultivation. In summary, cell colonization ability was estimated by determination of cell distribution within the scaffold (number, depth and homogeneity), matrix metalloproteinase activity and gene expression analysis of signaling molecules and differentiation markers. Results showed, the used dynamic colonization technique together with the newly-developed collagen-based scaffold with high content of bCaP to be an effective combined tool for producing bone grafts for bone implantology and regenerative medicine.

AAV5-miHTT Gene Therapy Demonstrates Broad Distribution and Strong Human Mutant Huntingtin Lowering in a Huntington's Disease Minipig Model.

Huntington's disease (HD) is a fatal neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the huntingtin gene. Previously, we showed strong huntingtin reduction and prevention of neuronal dysfunction in HD rodents using an engineered microRNA targeting human huntingtin, delivered via adeno-associated virus (AAV) serotype 5 vector with a transgene encoding an engineered miRNA against HTT mRNA (AAV5-miHTT). One of the challenges of rodents as a model of neurodegenerative diseases is their relatively small brain, making successful translation to the HD patient difficult. This is particularly relevant for gene therapy approaches, where distribution achieved upon local administration into the parenchyma is likely dependent on brain size and structure. Here, we aimed to demonstrate the translation of huntingtin-lowering gene therapy to a large-animal brain. We investigated the feasibility, efficacy, and tolerability of one-time intracranial administration of AAV5-miHTT in the transgenic HD (tgHD) minipig model. We detected widespread dose-dependent distribution of AAV5-miHTT throughout the tgHD minipig brain that correlated with the engineered microRNA expression. Both human mutant huntingtin mRNA and protein were significantly reduced in all brain regions transduced by AAV5-miHTT. The combination of widespread vector distribution and extensive huntingtin lowering observed with AAV5-miHTT supports the translation of a huntingtin-lowering gene therapy for HD from preclinical studies into the clinic.

Impact factors on fetal descent rates in the active phase of labor: a retrospective cohort study.

AIM: To assess fetal descent rates of nulliparous and multiparous women in the active phase of labor and to evaluate significant impact factors. METHODS: In a retrospective cohort study at the University Hospital of Zurich, Switzerland, we evaluated 6045 spontaneous vaginal deliveries with a singleton in vertex presentation between January 2007 and July 2014 at 34 0/7 to 42 0/7 gestational weeks. Median fetal descent rates and their 10th and 90th percentiles were assessed in the active phase of labor and different impact factors were evaluated. RESULTS: Fetal descent rates are exponentially increasing. Nulliparous women have slower fetal descent than multiparous women (P<0.001), ranging from 0 to 5.81 cm/h and from 0 to 15 cm/h, respectively. The total duration of fetal descent in labor is 5.42 h for nulliparous and 2.71 h for multiparous women. Accelerating impact factors are a lower fetal station, multiparity, increasing maternal weight and fetal occipitoanterior position, whereas epidural anesthesia decelerates fetal descent (P<0.001). CONCLUSIONS: Fetal descent is a hyperbolic increasing process with faster descent in multiparous women compared to nulliparous women, is highly inter individual and is associated with different impact factors. The diagnosis of labor arrest or prolonged labor should therefore be based on such rates as well as on individual evaluation of every parturient.

Impact factors on cervical dilation rates in the first stage of labor.

AIMS: To assess cervical dilation rates of nulliparous and multiparous women in the active first stage of labor and to evaluate significant impact factors. METHODS: In a retrospective cohort study between January 2007 and July 2014 at the University Hospital of Zurich in Switzerland, we analyzed 8378 women with singleton pregnancies in vertex presentation with a vaginal delivery at 34+0 to 42+5 gestational weeks. Median cervical dilation rates were calculated and different impact factors evaluated. RESULTS: Cervical dilation rates increase during labor progress with faster rates in multiparous compared with nulliparous women (P<0.001). Dilation rates exceed 1 cm/h at a dilatation of 6-7 cm, but are very individual. Accelerating impact factors are multiparity, a greater amount of cervical dilation and fetal occipitoanterior position, whereas the use of epidural anesthesia, a higher fetal weight and head circumference decelerate dilation (P<0.001). CONCLUSION: Cervical dilation is a hyperbolic increasing process, with faster dilation rates in multiparous compared to nulliparous women and a reversal point of labor around 6-7 cm, respectively. Besides, cervical dilation is highly individual and affected by several impact factors. The diagnosis of labor arrest or prolonged labor should therefore be based on such rates and on the individual evaluation of every woman.

Gradual Phenotype Development in Huntington Disease Transgenic Minipig Model at 24 Months of Age.

BACKGROUND: Huntington disease (HD) is an incurable neurodegenerative disease caused by the expansion of a polyglutamine sequence in a gene encoding the huntingtin (Htt) protein, which is expressed in almost all cells of the body. In addition to small animal models, new therapeutic approaches (including gene therapy) require large animal models as their large brains are a more realistic model for translational research. OBJECTIVE: In this study, we describe phenotype development in transgenic minipigs (TgHD) expressing the N-terminal part of mutated human Htt at the age of 24 months. METHODS: TgHD and wild-type littermates were compared. Western blot analysis and subcellular fractionation of different tissues was used to determine the fragmentation of Htt. Immunohistochemistry and optical analysis of coronal sections measuring aggregates, Htt expression, neuroinflammation, and myelination was applied. Furthermore, the expression of Golgi protein acyl-CoA binding domain containing 3 (ACBD3) was analyzed. RESULTS: We found age-correlated Htt fragmentation in the brain. Among various tissues studied, the testes displayed the highest fragmentation, with Htt fragments detectable even in cell nuclei. Also, Golgi protein ACBD3 was upregulated in testes, which is in agreement with previously reported testicular degeneration in TgHD minipigs. Nevertheless, the TgHD-specific mutated Htt fragments were also present in the cytoplasm of striatum and cortex cells. Moreover, microglial cells were activated and myelination was slightly decreased, suggesting the development of a premanifest stage of neurodegeneration in TgHD minipigs. CONCLUSIONS: The gradual development of a neurodegenerative phenotype, ac-companied with testicular degeneration, is observed in 24- month-old TgHD minipigs.

A new experimental model of calculous cholecystitis suitable for the evaluation and training of minimally invasive approaches to cholecystectomy.

BACKGROUND: Novel, less invasive approaches such as single-incision laparoscopic cholecystectomy or natural orifice transluminal endoscopic surgery require preclinical evaluation and training. Therefore, there is a need for an experimental model closely mimicking the clinical situation. The aim of our study was to create an experimental model of calculous cholecystitis in a large laboratory animal and test its feasibility for the evaluation of different techniques of cholecystectomy. METHODS: In 11 laboratory pigs, gallstones were placed inside the gallbladder laparoscopically. Levels of inflammatory markers-leucocytes (WBC), C-reactive protein (CRP) and interleukin 6 (IL-6)-were monitored on the postoperative days (POD) 1, 2, 3, 7 and 30. Abdominal ultrasound was performed 2 and 4 weeks after the operation. Four weeks after the lithiasis induction, laparoscopic cholecystectomy was performed. The control group consisted of ten healthy animals in which a cholecystectomy was performed. The pigs were monitored for 30 days after surgery. All removed gallbladders were assessed histologically. RESULTS: The induction of lithiasis took 42 (35-52) min with no morbidity and mortality. The values of WBC, CRP and IL-6 increased significantly (vs. baseline) on POD 1, 2 and 3 (p < 0.05) and then normalised. Ultrasonography confirmed the presence of chronic calculous cholecystitis in all cases after 4 weeks. Laparoscopic cholecystectomy was significantly longer in animals with lithiasis, 63 (42-91) versus 46 (31-62) min (p = 0.018). Perioperative gallbladder wall perforation was significantly more frequent in the model group (8/11 vs. 1/10; p = 0.04). In contrast to healthy animals, all gallbladders with stones showed histological signs of chronic inflammation. CONCLUSIONS: A new animal model of calculous cholecystitis was created. Laparoscopic cholecystectomy was more technically difficult compared to operating on a healthy gallbladder. This model may be a suitable tool for effective preclinical training and also for the evaluation of different techniques of cholecystectomy.

Mutated Huntingtin Causes Testicular Pathology in Transgenic Minipig Boars.

BACKGROUND: Huntington's disease is induced by CAG expansion in a single gene coding the huntingtin protein. The mutated huntingtin (mtHtt) primarily causes degeneration of neurons in the brain, but it also affects peripheral tissues, including testes. OBJECTIVE: We studied sperm and testes of transgenic boars expressing the N-terminal region of human mtHtt. METHODS: In this study, measures of reproductive parameters and electron microscopy (EM) images of spermatozoa and testes of transgenic (TgHD) and wild-type (WT) boars of F1 (24-48 months old) and F2 (12-36 months old) generations were compared. In addition, immunofluorescence, immunohistochemistry, Western blot, hormonal analysis and whole-genome sequencing were done in order to elucidate the effects of mtHtt. RESULTS: Evidence for fertility failure of both TgHD generations was observed at the age of 13 months. Reproductive parameters declined and progressively worsened with age. EM revealed numerous pathological features in sperm tails and in testicular epithelium from 24- and 36-month-old TgHD boars. Moreover, immunohistochemistry confirmed significantly lower proliferation activity of spermatogonia in transgenic testes. mtHtt was highly expressed in spermatozoa and testes of TgHD boars and localized in all cells of seminiferous tubules. Levels of fertility-related hormones did not differ in TgHD and WT siblings. Genome analysis confirmed that insertion of the lentiviral construct did not interrupt any coding sequence in the pig genome. CONCLUSIONS: The sperm and testicular degeneration of TgHD boars is caused by gain-of-function of the highly expressed mtHtt.

Nanofiber Wound Dressing Materials-A Comparative Study of Wound Healing on a Porcine Model.

BACKGROUND: Nanofiber wound dressings remain the domain of in vitro studies. The purpose of our study was to verify the benefits of chitosan (CTS) and polylactide (PLA)-based nanofiber wound dressings on a porcine model of a naturally contaminated standardized wound and compare them with the conventional dressings, i.e., gauze and Inadine. MATERIAL AND METHODS: The study group included 32 pigs randomized into four homogeneous groups according to the wound dressing type. Standardized wounds were created on their backs, and wound dressings were regularly changed. We evaluated difficulty of handling individual dressing materials and macroscopic appearance of the wounds. Wound swabs were taken for bacteriological examination. Blood samples were obtained to determine blood count values and serum levels of acute phase proteins (serum amyloid A, C-reactive protein, and haptoglobin). The crucial point of the study was histological analysis. Microscopic evaluation was focused on the defect depth and tissue reactions, including formation of the fibrin exudate with neutrophil granulocytes, the layer of granulation and cellular connective tissue, and the reepithelialization. Statistical analysis was performed by using SPSS software. The analysis was based on the Kruskal-Wallis H test and Mann-Whitney U test followed by Bonferroni correction. Significance was set at P < .05. RESULTS: Macroscopic examination did not show any difference in wound healing among the groups. However, evaluation of histological findings demonstrated that PLA-based nanofiber dressing accelerated the proliferative (P = .025) and reepithelialization (P < .001) healing phases, while chitosan-based nanofiber dressing potentiated and accelerated the inflammatory phase (P = .006). No statistically significant changes were observed in the blood count or acute inflammatory phase proteins during the trial. Different dynamics were noted in serum amyloid A values in the group treated with PLA-based nanofiber dressing (P = .006). CONCLUSION: Based on the microscopic examination, we have documented a positive effect of nanofiber wound dressings on acceleration of individual phases of the healing process. Nanofiber wound dressings have a potential to become in future part of the common wound care practice.

Derivation of Sendai-Virus-Reprogrammed Human iPSCs-Neuronal Precursors: In Vitro and In Vivo Post-grafting Safety Characterization.

The critical requirements in developing clinical-grade human-induced pluripotent stem cells-derived neural precursors (hiPSCs-NPCs) are defined by expandability, genetic stability, predictable in vivo post-grafting differentiation, and acceptable safety profile. Here, we report on the use of manual-selection protocol for generating expandable and stable human NPCs from induced pluripotent stem cells. The hiPSCs were generated by the reprogramming of peripheral blood mononuclear cells with Sendai-virus (SeV) vector encoding Yamanaka factors. After induction of neural rosettes, morphologically defined NPC colonies were manually harvested, re-plated, and expanded for up to 20 passages. Established NPCs showed normal karyotype, expression of typical NPCs markers at the proliferative stage, and ability to generate functional, calcium oscillating GABAergic or glutamatergic neurons after in vitro differentiation. Grafted NPCs into the striatum or spinal cord of immunodeficient rats showed progressive maturation and expression of early and late human-specific neuronal and glial markers at 2 or 6 months post-grafting. No tumor formation was seen in NPCs-grafted brain or spinal cord samples. These data demonstrate the effective use of in vitro manual-selection protocol to generate safe and expandable NPCs from hiPSCs cells. This protocol has the potential to be used to generate GMP (Good Manufacturing Practice)-grade NPCs from hiPSCs for future clinical use.

Expandable Sendai-Virus-Reprogrammed Human iPSC-Neuronal Precursors: In Vivo Post-Grafting Safety Characterization in Rats and Adult Pig.

One of the challenges in clinical translation of cell-replacement therapies is the definition of optimal cell generation and storage/recovery protocols which would permit a rapid preparation of cell-treatment products for patient administration. Besides, the availability of injection devices that are simple to use is critical for potential future dissemination of any spinally targeted cell-replacement therapy into general medical practice. Here, we compared the engraftment properties of established human-induced pluripotent stem cells (hiPSCs)-derived neural precursor cell (NPCs) line once cells were harvested fresh from the cell culture or previously frozen and then grafted into striata or spinal cord of the immunodeficient rat. A newly developed human spinal injection device equipped with a spinal cord pulsation-cancelation magnetic needle was also tested for its safety in an adult immunosuppressed pig. Previously frozen NPCs showed similar post-grafting survival and differentiation profile as was seen for freshly harvested cells. Testing of human injection device showed acceptable safety with no detectable surgical procedure or spinal NPCs injection-related side effects.

Modelling Duchenne muscular dystrophy in vitro with newly generated, blood cell-derived induced pluripotent stem cell line ORIONi003-A.

Here, we present newly derived in vitro model for modeling Duchenne muscular dystrophy. Our new cell line was derived by reprogramming of peripheral blood mononuclear cells (isolated from blood from pediatric patient) with Sendai virus encoding Yamanaka factors. Derived iPS cells are capable to differentiate in vitro into three germ layers as verified by immunocytochemistry. When differentiated in special medium, our iPSc formed spontaneously beating cardiomyocytes. As cardiomyopathy is the main clinical complication in patients with Duchenne muscular dystrophy, the cell line bearing the dystrophin gene mutation might be of interest to the research community.

Subretinal Implantation of Human Primary RPE Cells Cultured on Nanofibrous Membranes in Minipigs.

PURPOSE: The development of primary human retinal pigmented epithelium (hRPE) for clinical transplantation purposes on biodegradable scaffolds is indispensable. We hereby report the results of the subretinal implantation of hRPE cells on nanofibrous membranes in minipigs. METHODS: The hRPEs were collected from human cadaver donor eyes and cultivated on ultrathin nanofibrous carriers prepared via the electrospinning of poly(L-lactide-co-DL-lactide) (PDLLA). "Libechov" minipigs (12-36 months old) were used in the study, supported by preoperative tacrolimus immunosuppressive therapy. The subretinal implantation of the hRPE-nanofibrous carrier was conducted using general anesthesia via a custom-made injector during standard three-port 23-gauge vitrectomy, followed by silicone oil endotamponade. The observational period lasted 1, 2, 6 and 8 weeks, and included in vivo optical coherence tomography (OCT) of the retina, as well as post mortem immunohistochemistry using the following antibodies: HNAA and STEM121 (human cell markers); Bestrophin and CRALBP (hRPE cell markers); peanut agglutining (PNA) (cone photoreceptor marker); PKCα (rod bipolar marker); Vimentin, GFAP (macroglial markers); and Iba1 (microglial marker). RESULTS: The hRPEs assumed cobblestone morphology, persistent pigmentation and measurable trans-epithelial electrical resistance on the nanofibrous PDLLA carrier. The surgical delivery of the implants in the subretinal space of the immunosuppressed minipigs was successfully achieved and monitored by fundus imaging and OCT. The implanted hRPEs were positive for HNAA and STEM121 and were located between the minipig's neuroretina and RPE layers at week 2 post-implantation, which was gradually attenuated until week 8. The neuroretina over the implants showed rosette or hypertrophic reaction at week 6. The implanted cells expressed the typical RPE marker bestrophin throughout the whole observation period, and a gradual diminishing of the CRALBP expression in the area of implantation at week 8 post-implantation was observed. The transplanted hRPEs appeared not to form a confluent layer and were less capable of keeping the inner and outer retinal segments intact. The cone photoreceptors adjacent to the implant scaffold were unchanged initially, but underwent a gradual change in structure after hRPE implantation; the retina above and below the implant appeared relatively healthy. The glial reaction of the transplanted and host retina showed Vimentin and GFAP positivity from week 1 onward. Microglial activation appeared in the retinal area of the transplant early after the surgery, which seemed to move into the transplant area over time. CONCLUSIONS: The differentiated hRPEs can serve as an alternative cell source for RPE replacement in animal studies. These cells can be cultivated on nanofibrous PDLLA and implanted subretinally into minipigs using standard 23-gauge vitrectomy and implantation injector. The hRPE-laden scaffolds demonstrated relatively good incorporation into the host retina over an eight-week observation period, with some indication of a gliotic scar formation, and a likely neuroinflammatory response in the transplanted area despite the use of immunosuppression.

In vitro modeling of amyotrophic lateral sclerosis with induced pluripotent stem cell technology-derived cell line ORIONi002-A.

We present here a new iPS cell line for modeling sporadic form of ALS. Cell line was generated by reprogramming skin fibroblasts isolated with explant culture technology from skin biopsy, donated by ALS patient. For reprogramming, polycistronic self-replicating RNA vector was used and derived iPS cells were characterized by immunocytochemistry and FACS (pluripotent factors expression), karyotyping, STR fingerprinting analysis and in vitro differentiation assay. New cell line showed normal (46, XY) karyotype and differentiated in vitro into cells from three germ layers. STR analysis proved the origin and originality of the cell line.

Subretinal Implantation of RPE on a Carrier in Minipigs: Guidelines for Preoperative Preparations, Surgical Techniques, and Postoperative Care.

Degenerative disorders of the retina (including age-related macular degeneration), which originate primarily at or within the retinal pigmented epithelial (RPE) layer, lead to a progressive disorganization of the retinal anatomy and the deterioration of visual function. The substitution of damaged RPE cells (RPEs) with in vitro cultured RPE cells using a subretinal cell carrier has shown potential for re-establishing the anatomical structure of the outer retinal layers and is, therefore, being further studied. Here, we present the principles of a surgical technique that allows for the effective subretinal transplantation of a cell carrier with cultivated RPEs into minipigs. The surgeries were performed under general anesthesia and included a standard lens-sparing three-port pars plana vitrectomy (PPV), subretinal application of a balanced salt solution (BSS), a 2.7 mm retinotomy, implantation of a nanofibrous cell carrier into the subretinal space through an additional 3.0 mm sclerotomy, fluid-air exchange (FAX), silicone oil tamponade, and closure of all the sclerotomies. This surgical approach was used in 29 surgeries (18 animals) over the past 8 years with a success rate of 93.1%. Anatomic verification of the surgical placement was carried out using in vivo fundus imaging (fundus photography and optical coherence tomography). The recommended surgical steps for the subretinal implantation of RPEs on a carrier in minipig eyes can be used in future preclinical studies using large-eye animal models.

Vancomycin-Loaded Collagen/Hydroxyapatite Layers Electrospun on 3D Printed Titanium Implants Prevent Bone Destruction Associated with S. epidermidis Infection and Enhance Osseointegration.

The aim of the study was to develop an orthopedic implant coating in the form of vancomycin-loaded collagen/hydroxyapatite layers (COLHA+V) that combine the ability to prevent bone infection with the ability to promote enhanced osseointegration. The ability to prevent bone infection was investigated employing a rat model that simulated the clinically relevant implant-related introduction of bacterial contamination to the bone during a surgical procedure using a clinical isolate of Staphylococcus epidermidis. The ability to enhance osseointegration was investigated employing a model of a minipig with terminated growth. Six weeks following implantation, the infected rat femurs treated with the implants without vancomycin (COLHA+S. epidermidis) exhibited the obvious destruction of cortical bone as evinced via a cortical bone porosity of up to 20% greater than that of the infected rat femurs treated with the implants containing vancomycin (COLHA+V+S. epidermidis) (3%) and the non-infected rat femurs (COLHA+V) (2%). The alteration of the bone structure of the infected COLHA+S. epidermidis group was further demonstrated by a 3% decrease in the average Ca/P molar ratio of the bone mineral. Finally, the determination of the concentration of vancomycin released into the blood stream indicated a negligible systemic load. Six months following implantation in the pigs, the quantified ratio of new bone indicated an improvement in osseointegration, with a two-fold bone ingrowth on the COLHA (47%) and COLHA+V (52%) compared to the control implants without a COLHA layer (27%). Therefore, it can be concluded that COLHA+V layers are able to significantly prevent the destruction of bone structure related to bacterial infection with a minimal systemic load and, simultaneously, enhance the rate of osseointegration.