Evaluation of polymorphisms and expression of PTPN22, NLRP1 and TYR genes in vitiligo patients.

Introduction: Vitiligo is a pigmentary disorder associated with a selective loss of melanocytes in the skin, its appendages and mucous membranes. Aim: The aim of the study was to evaluate the association between the rs2476601 polymorphism of the PTPN22 gene, the rs2670660 and rs6502867 polymorphisms of the NLRP1 gene and the rs1847134 and rs1393350 polymorphisms of the TYR gene and vitiligo. Another aim was to compare the gene expression in lesional and symmetrically non-lesional skin of vitiligo patients and healthy controls. Material and methods: The experimental group consisted of 42 patients and the control group consisted of 38 healthy volunteers. The polymorphisms of the genes were assessed with PCR-RFLP technique and gene expression with qRT-PCR technique. Results: We found that the CT genotype of the PTPN22 rs2476601 polymorphism is more frequent in vitiligo patients, in the case of the NLRP1 rs2670660 polymorphism it was the AG genotype, in the NLRP1 rs6502867 polymorphism they were the CT and CC genotypes and in the TYR rs1393350 polymorphism it was the AG genotype. There was no association between vitiligo and the TYR rs1847134 polymorphism. We found statistically significant differences in gene expression in the lesional and symmetrical non-lesional skin of vitiligo patients compared to the control group. Conclusions: Our analysis showed genotypes predisposing to vitiligo. We found that the gene expression is different not only in lesional but also in non-lesional skin of vitiligo patients, what may change the approach to treatment of the disease.

Resveratrol as a factor preventing skin aging and affecting its regeneration.

Resveratrol, a compound belonging to polyphenols, besides its action on the cardiovascular system, affects also wound healing, regeneration, and photoaging of the skin. By interactions with numerous substances and pathways, e.g. MAPK, MAPKK, FOXO3, TGF or metalloproteinase 1, it protects the skin against the harmful effects of type B ultraviolet radiation, which is the main factor in the skin aging processes. It also enhances collagen synthesis by activating the oestrogen receptor and reduces wrinkles. In damaged tissues, it accelerates skin regeneration and healing by activating, among others, VEGF. Based on the review of the literature, there is no doubt that resveratrol has the potential to be used in cosmetology, dermatology and plastic surgery. It can be used as a compound of anti-aging products or as a topical treatment of scars and wounds. In the future this polyphenol might be applied in pharmacotherapy of many dermatoses.

Biostimulative effect of laser on growth of mesenchymal stem/stromal cells in vitro.

INTRODUCTION: Human adipose tissue-derived mesenchymal stem/stromal cells (hAT-MSCs) are multipotent stromal cells with a high potential application in tissue engineering and regenerative medicine. Laser irradiation of the place where the cells were implanted can stimulate their proliferation, increase the secretion of growth factors and thus increase the therapeutic effect. AIM: To evaluate the influence of two lasers: Er:YAG and diode on the growth of hAT-MSCs in vitro. MATERIAL AND METHODS: hAT-MSCs were isolated from human subcutaneous adipose tissue. Immunophenotype of hAT-MSCs was confirmed by flow cytometry. Multipotency of hAT-MSCs was confirmed by differentiation into adipogenic, osteogenic and chondrogenic lineages. hAT-MSCs were irradiated with Er:YAG laser (wavelength 2940 nm, frequency 5, 10 Hz, doses: 0.1-1.2 J/cm2) for 2 s and 4 s and diode laser (wavelength 635 nm and doses: 1-8 J/cm2) for 5, 10, 20, 30 and 40 s. Cell viability was analysed 24 h after the exposure using MTT assay. RESULTS: Growth stimulation of hAT-MSCs after 5 Hz Er:YAG laser exposure, 0.1 J/cm2 dose for 4 s and 0.3 J/cm2 dose for 4 s was shown in comparison with the control group. Significant growth stimulation of hAT-MSCs after diode laser irradiation in doses of 1-4 J/cm2 was demonstrated compared to the control group. CONCLUSIONS: The presented results indicate that both lasers, Er:YAG and diode can be used to stimulate stem/stromal cell growth in vitro. The biostimulative effect of laser therapy on stromal cells may be used in the future in aesthetic dermatology in combined laser and cell therapy.

The different expression of key markers on urothelial holoclonal, meroclonal, and paraclonal cells in in vitro culture.

Urothelial cell populations which differ in morphology and proliferation capacities can be isolated from the urinary bladder. The goal of this study was to analyze a clonal, proliferative, and self-renewing potential of porcine urothelial cells and to compare expression of selected adhesion and tight junction molecules, urothelial and stem cell markers for the urothelial clone types. Urothelial cells were isolated from 10 porcine urinary bladders. Three different clone types: holoclone-, meroclone-and paraclone-like colonies were identified based on their morphology. To characterize and compare the urothelial clones the immunofluorescent stains were performed. Expression of pancytokeratin (PanCK), Ki-67 and p63 was higher for holoclone- like cells compared to meroclone-and paraclone-like cells (P < 0.05). Meroclone-like cells expressed higher levels of p63 compared to paraclone- like cells (P < 0.05). The level of Ki-67 and PanCK for meroclone- and paraclone- like cells was comparable (P > 0.05). ß1 and ß4 integrins were not expressed. Expression of zonula occludens-1 (ZO-1) in cell-cell junctions for paraclone-, meroclone-and holoclone-like cells was 17.6 ± 0.6, 14.7 ± 0.5, and 16.1 ± 0.4, respectively. The results of actin filaments (F-actin) expression were 253,634 ± 6,920 for meroclone-like cells, 198,512 ± 7,977 for paraclone-like cells and 133,544 ± 3,169 for holoclone-like cells. Three urothelial cell types with differing features can be isolated from the bladder. Holoclone-like cells are the richest in stem cells and should be used in further studies for construction of neo-bladder or neo-conduit using tissue engineering methods.

Stem cells and differentiated cells differ in their sensitivity to urine in vitro.

Urinary tract regeneration using tissue engineering is one of the most challenging issues in the field of reconstructive urology. Cells seeded on scaffold are exposed to urine immediately after the implantation. The outcome of urinary bladder regeneration is depended on the ability of these cells to survive, proliferate, and regenerate. The aim of this study was to compare a sensitivity of three different cell lines to urine in vitro. Three different cell lines were isolated from porcine bladder (urothelial cells, UCs and smooth muscle cells, SMCs) and adipose tissue (adipose-derived stem cells, ADSCs). Cell viability (MTT assay), proliferation (real-time cell analysis using xCELLigence system) and apoptosis/necrosis (flow cytometry) were analyzed after exposition to urine. ADSCs were the most sensitive to urine compared to two other tested cell lines. Among the bladder cell lines the UCs were more resistant to urine than SMCs. Twenty four hour incubation of UCs, SMCs, and ADSCs with urine lead to ∼40%, ∼70%, and ∼90% reduction of their viability, respectively. The mechanism of urine mediated cytotoxicity differed depending on the tested cell type. Urothelial and SMCs seems to be more suitable for urinary bladder regeneration compared to mesenchymal stem cells, however, these cells have limited application especially in the case of urinary bladder cancer.

Vascularization Potential of Electrospun Poly(L-Lactide-co-Caprolactone) Scaffold: The Impact for Tissue Engineering.

BACKGROUND Electrospun nanofibers have widespread putative applications in the field of regenerative medicine and tissue engineering. When compared to naturally occurring collagen matrices, electrospun nanofiber scaffolds have two distinct advantages: they do not induce a foreign body reaction and they are not at risk for biological contamination. However, the exact substrate, structure, and production methods have yet to be defined. MATERIAL AND METHODS In the current study, tubular-shaped poly(L-lactide-co-caprolactone) (PLCL) constructs produced using electrospinning technology were evaluated for their potential application in the field of tissue regeneration in two separate anatomic locations: the skin and the abdomen. The constructs were designed to have an internal diameter of 3 mm and thickness of 200 µm. Using a rodent model, 20 PLCL tubular constructs were surgically implanted in the abdominal cavity and subcutaneously. The constructs were then evaluated histologically using electron microscopy at 6 weeks post-implantation. RESULTS Histological evaluation and analysis using scanning electron microscopy showed that pure scaffolds by themselves were able to induce angiogenesis after implantation in the rat model. Vascularization was observed in both tested groups; however, better results were obtained after intraperitoneal implantation. Formation of more and larger vessels that migrated inside the scaffold was observed after implantation into the peritoneum. In this group no evidence of inflammation and better integration of scaffold with host tissue were noticed. Subcutaneous implantation resulted in more fibrotic reaction, and differences in cell morphology were also observed between the two tested groups. CONCLUSIONS This study provides a standardized evaluation of a PLCL conduit structure in two different anatomic locations, demonstrating the excellent ability of the structure to achieve vascularization. Functional, histological, and mechanical data clearly indicate prospective clinical utilization of PLCL in critical size defect regeneration.

Isolation, expansion and characterization of porcine urinary bladder smooth muscle cells for tissue engineering.

BACKGROUND: A key requirements for therapy utilizing the tissue engineering methodologies is use of techniques which have the capability to yield a high number of cells, from small tissue biopsy in a relatively short time. Up to date there was no optimal methods of isolation and expansion of urinary bladder smooth muscle cells (UB-SMCs). The aim of this study was to compare isolation and expansion techniques of UB-SMCs to select the most repeatable and efficient one. METHOD: Five protocols of porcine UB- SMCs isolation including enzymatic and explant techniques and three expansion techniques were compared. Isolation effectiveness was evaluated using trypan blue assay. Cell phenotype was confirmed by immunofluorescence staining. Proliferation rate was analyzed using MTT and X- Celligence system. Cellular senescence was assessed measuring ß-galactosidase activity. RESULTS: Enzymatic methods using collagenase with dispase (method I) or collagenase only (method III) allowed to isolate much larger number of cells than the methods using trypsin with collagenase (method II) and collagenase after digestion with trypsin (method IV). The success rate of establishment of primary culture was the highest when the isolated cells were cultured in the Smooth muscle Growth Medium-2 (SmGM-2). Expression of the smooth muscle markers- alpha smooth muscle actin and smoothelin was the highest for cells isolated by enzymatic method I and cultured in SmGM-2. There was no significant signs of cell senescence until the 8th passage. CONCLUSION: The most efficient method of establishment of porcine UB-SMCs culture is enzymatic digestion of urinary bladder tissue with collagenase and dispase and culture of isolated cells in SmGM-2. This method was up to 10 times more efficient than other methods used for isolation and culture of UB-SMCs. This is an easy and consistent method for obtaining high numbers of urinary bladder smooth muscle cells.

Optimization of porcine urothelial cell cultures: Best practices, recommendations, and threats.

Many experimental approaches have been conducted in order to isolate urothelial cells from bladder tissue biopsies, but each method described has utilized different protocols and sources of bladder tissue. In this study, we compared the different methods of urothelial cell isolation available in literature together with standardized methods in order to obtain more unified results. Five methods for primary porcine urothelial culture establishment were compared: tissue explants and four enzymatic methods utilizing collagenase II, dispase II, combination of dispase II and trypsin, and trypsin alone. The average number of isolated cells, cell morphology, success of established culture, average number of cells from the first passage, expression of p63 and pancytokeratin and the characterization of urothelial cell growth, and aging were analyzed during the in vitro culture. The method utilizing dispase II was the most efficient and reproducible method for the isolation and culture of porcine urothelial cells when compared to the other tested methods. Urothelial cells obtained by this method grew considerably well and the cultures were established with high efficiency, which enabled us in obtaining a large quantity of cells with normal morphology. Contamination with fibroblasts in this method was the lowest. The utilization of a proper method for urothelial cell isolation is a critical step in the urinary tract regeneration when using tissue engineering techniques. In summary, this study demonstrated that by utilizing the described method with dispase II, a suitable number of cells was achieved, proving the method useful for tissue regeneration.

Is regenerative medicine a new hope for kidney replacement?

The availability of kidney and other organs from matching donors is not enough for many patients on demand for organ transplant. Unfortunately, this situation is not better despite the many of new interesting projects of promoting family, cross or domino transplants. These inexorable global statistics forced medical researchers to find a new potential therapeutic option that would guarantee safety and efficacy for the treatment of ESRD comparable to kidney transplantation. The aim of our review is to summarize the scientific literature that relating to the modern as well as innovative experimental methods and possibilities of kidney regeneration and, in addition, to find whether the regenerative medicine field will be a new hope for curing the patient with renal disease complications. The most important achievements in the field of regenerative medicine of kidney, which were mentioned and described here, are currently cumulated in 4 areas of interest: stem cell-based therapies, neo-kidneys with specially designed scaffolds or cell-seeded matrices, bioartificial kidneys and innovative nanotechnologically bioengineered solutions. Nowadays, we can add some remarks that the regenerative medicine is still insufficient to completely replace current therapy methods used in patients with chronic kidney disease especially with the end-stage renal disease where in many cases kidney transplantation is the only one chance. But we think that development of regenerative medicine especially in the last 20 years brings us more and more closer to solve many of today's problems at the frontier of nephrology and transplantology.

Filling effects, persistence, and safety of dermal fillers formulated with stem cells in an animal model.

BACKGROUND: Research is scarce regarding the effectiveness of dermal fillers containing autologous stem cells. OBJECTIVES: The authors sought to determine the local and systemic effects of adipose-derived stem cells (ADSCs) as a component of dermal fillers in an animal model. METHODS: Wistar rats were injected with 1 of the following dermal fillers: ADSCs combined with hyaluronic acid (ADSC-HA), ADSCs combined with fish collagen (ADSC-COL), HA alone (CONTROL-HA), or COL alone (CONTROL-COL). Fillers were injected into the glabella, dorsum, and chest of each animal. The ADSCs were labeled with PKH26 to assess cell migration. Filling effects (FEs) were measured immediately after injection and at 1.5 months and 3 months after injection. Skin specimens were stained with hematoxylin and eosin to assess localization and persistence of ADSCs. RESULTS: Mean FEs in animals implanted with ADSCs were greater and persisted longer than those of controls. No inflammatory responses were observed in any group. Three months after injection, PKH26-positive cells comprised nearly 70% of cells at the injection site in animals treated with ADSC-HA. PKH26 fluorescence also was detected in the spleen but not in the brain, kidney, or lung. CONCLUSIONS: Stem cells have the potential to improve the aesthetic effects and longevity of dermal fillers.

A comparison of five methods to maximize RNA and DNA isolation yield from adipose tissue.

Adipose tissue in the human body occurs in various forms with different functions. It is an energy store, a complex endocrine organ, and a source of cells used in medicine. Many molecular analyses require the isolation of nucleic acids, which can cause some difficulties connected with the large amount of lipids in adipocytes. Ribonucleic acid isolation is particularly challenging due to its low stability and easy degradation by ribonucleases. The study aimed to compare and evaluate five RNA and DNA isolation methods from adipose tissue. The tested material was subcutaneous porcine adipose tissue subjected to different homogenization methods and RNA or DNA purification. A mortar and liquid nitrogen or ceramic beads were used for homogenization. The organic extraction (TriPure Reagent), spin columns with silica-membrane (RNeasy Mini Kit or High Pure PCR Template Preparation Kit), and the automatic MagNA Pure system were used for the purification. Five combinations were compared for RNA and DNA isolation. Obtained samples were evaluated for quantity and quality. The methods were compared in terms of yield (according to tissue mass), purity (A260/280 and A260/230), and nucleic acid degradation (RNA Integrity Number, RIN; DNA Integrity Number, DIN). The results were analyzed statistically. The average RNA yield was highest in method I, which used homogenization with ceramic beads and organic extraction. Low RNA concentration didn't allow us to measure degradation for all samples in method III (homogenization with ceramic beads and spin-column purification). The highest RNA quality was achieved with method IV using homogenization in liquid nitrogen and spin column purification, which makes it the most effective for RNA isolation from adipose tissue. Required values of DNA yield, purity, and integrity were achieved only with spin column-based methods (III and IV). The most effective method for DNA isolation from adipose tissue is method III, using spin-columns without additional homogenization.

[Tissue engineering - experimental method of urinary bladder regeneration]. / Inzynieria tkankowa - eksperymentalna metoda regeneracji pecherza moczowego.

The most common cause of bladder reconstruction is radical cystectomy for invasive bladder cancer. Currently, bowel segments remains, the most widely used in reconstruction of urinary tract. Using of bowel as a "material" for bladder reconstruction is associated with numerous complications. Tissue engineering methods provide opportunities to construct bladder tissue in vitro from autologous cells obtained from non urinary tract system. So far, the most useful cell and matrix type for bladder reconstruction have not been defined. In this work actual knowledge about tissue engineering in bladder regeneration was presented.

[Modern biomaterials as hemostatic dressings in kidney nephron sparing surgery (NSS)--murine model. A preliminary report]. / Nowoczesne biomaterialy jako opatrunki hemostatyczne w chirurgii oszczedzajacej miazsz nerki--model zwierzecy. Doniesienie wstepne.

INTRODUCTION: Kidney cancer is now days, one of the main problems in oncological urology. More frequent cases detection of this type of cancer and the implementation of modern methods of treatment, involves the public and good diagnostic radiological imaging methods. Approximately 40% of renal tumors are detected clinically as a changes in T1N0M0 stage. This means that in these patients, surgery can be performed using the method of nephron sparing surgery (NSS), far from consisting the implementation of radical nephrectomy. Unfortunately, despite the saving nature of this type of treatment, NSS methods are associated with local recurrence of tumor formation. Another problem is intra operative bleeding, that's why in order to stop this negative process surgeons currently use hemostatic dressings. Potentially and clinically significant solution could be a combination of this two main problematics points of concern, through the use of modern biomaterials coated on oncostatic substances as a haemostatic dressings, to the prevention of tumor recurrence. AIM OF THE WORK: The aim of this work, was to present preliminary report of the use of advanced biomaterials, as haemostatic dressings in an experimental technique of nephron sparing surgery on an murine model. MATERIAL AND METHODS: In the experiment we use two types of biomaterials and the standard haemostatic dressing used in the nephron sparing surgery (NSS) as a control. We use a polycaprolactone biomaterial obtained by electrospinning. As a second type of biomaterial, we use a homogeneous material with a structure similar to wool, also obtained from medical polycaprolactone by electrospinning. As an murine (in vivo) model in the study, we use 10 C57BL/J mice (with the local ethical committee permission). 8 mice were used in the present study, 2 mice were constituted as a separate control for obtaining the bleeding data. Kidney melanoma cells were implanted under the C57B1/J B16 mouse kidney fibrous capsule, one week before NSS. After 3 weeks the animals were sacrificed for comparison of hemostatic dressings function. RESULTS: Used biomaterials fulfilled their role as a hameostatic dresings. The material (Type I) was convenient and good for suturing. Haemostatic action times were as follows: (Type I) - 30 seconds. (Type III) - 50 seconds. In the control group were also observed, a proper hemostatic function after 30 seconds. In sectional observation was also found in 3 kidneys section preparation samples, a local tumor recurrence and metastasis to the other tissues of the abdomen. CONCLUSION: The tested biomaterials fulfill their hemostatic effect on kidney after NSS, without any significant difference acording to a standard hemostatic dressing used clinically. This data may be a potential factor for use in further studies to determine their continued relevance in the prevention of local tumor recurrence after nephron sparing surgery.

Urinary bladder augmentation with acellular biologic scaffold-A preclinical study in a large animal model.

Current strategies in urinary bladder augmentation include use of gastrointestinal segments, however, the technique is associated with inevitable complications. An acellular biologic scaffold seems to be a promising option for urinary bladder augmentation. The aim of this study was to evaluate the utility of bladder acellular matrix (BAM) for reconstruction of clinically significant large urinary bladder wall defects in a long-term porcine model. Urinary bladders were harvested from 10 pig donors. Biological scaffolds were prepared by chemically removing all cellular components from urinary bladder tissue. A total of 10 female pigs underwent hemicystectomy and subsequent bladder reconstruction with BAM. The follow-up study was 6 months. Reconstructed bladders were subjected to radiological, macroscopic, histological, immunohistochemical, and molecular evaluations. Six out of ten animals survived the 6-month follow-up period. Four pigs died during observation due to mechanical failure of the scaffold, anastomotic dehiscence between the scaffold and native bladder tissue, or occluded catheter. Tissue engineered bladder function was normal without any signs of postvoid residual urine in the bladder or upper urinary tracts. Macroscopically, graft shrinkage was observed. Urothelium completely covered the luminal surface of the graft. Smooth muscle regeneration was observed mainly in the peripheral graft region and gradually decreased toward the center of the graft. Expression of urothelial, smooth muscle, blood vessel, and nerve markers were lower in the reconstructed bladder wall compared to the native bladder. BAM seems to be a promising biomaterial for reconstruction of large urinary bladder wall defects. Further research on cell-seeded BAM to enhance urinary bladder regeneration is required.

The Positive Impact of Donor Bone Marrow Cells Transplantation into Immunoprivileged Compartments on the Survival of Vascularized Skin Allografts.

Using the vascularized skin allograft (VSA) model, we compared the tolerogenic effects of different allogeneic bone marrow transplantation (BMT) delivery routes into immunoprivileged compartments under a 7-day protocol immunosuppressive therapy. Twenty-eight fully MHC mismatched VSA transplants were performed between ACI (RT1a) donors and Lewis (RT11) recipients in four groups of seven animals each, under a 7-day protocol of alfa/beta TCRmAb/CsA (alpha/beta-TCR monoclonal antibodies/Cyclosporine A therapy). Donor bone marrow cells (BMC) (100 × 106 cells) were injected into three different immunoprivileged compartments: Group 1: Control, without cellular supportive therapy, Group 2: Intracapsular BMT, Group 3: Intragonadal BMT, Group 4: Intrathecal BMT. In Group 2, BMC were transplanted under the kidney capsule. In Group 3, BMC were transplanted into the right testis between tunica albuginea and seminiferous tubules, and in Group 4, cells were injected intrathecally. The assessment included: skin evaluation for signs and grade of rejection and immunohistochemistry for donor cells engraftment into host lymphoid compartments. Donor-specific chimerism for MHC class I (RT1a) antigens and the presence of CD4+/CD25+ T cells were assessed in the peripheral blood of recipients. The most extended allograft survival, 50-78 days, was observed in Group 4 after intrathecal BMT. The T cells CD4+/CD25+ in the peripheral blood were higher after intrathecal BMC injection than other experimental groups at each post-transplant time point. Transplantation of BMC into immunoprivileged compartments delayed rejection of fully mismatched VSA and induction of robust, donor-specific chimerism.

Donor Recipient Chimeric Cells Induce Chimerism and Extend Survival of Vascularized Composite Allografts.

This study evaluated the efficacy of donor recipient chimeric cell (DRCC) therapy created by fusion of donor and recipient derived bone marrow cells (BMC) in chimerism and tolerance induction in a rat vascularized composite allograft (VCA) model. Twenty-four VCA (groin flaps) from MHC-mismatched ACI (RT1a) donors were transplanted to Lewis (RT1l) recipients. Rats were randomly divided into (n = 6/group): Group 1-untreated controls, Groups 2-7-day immunosuppression controls, Group 3-DRCC, and Group 4-DRCC with 7-day anti-αßTCR monoclonal antibody and cyclosporine A protocol. DRCC created by polyethylene glycol-mediated fusion of ACI and Lewis BMC were cultured and transplanted (2-4 × 106) to VCA recipients via intraosseous delivery route. Flow cytometry assessed peripheral blood chimerism while fluorescent microscopy and PCR tested the presence of DRCC in the recipient's blood, bone marrow (BM), and lymphoid organs at the study endpoint (VCA rejection). No complications were observed after DRCC intraosseous delivery. Group 4 presented the longest average VCA survival (79.3 ± 30.9 days) followed by Group 2 (53.3 ± 13.6 days), Group 3 (18 ± 7.5 days), and Group 1 (8.5 ± 1 days). The highest chimerism level was detected in Group 4 (57.9 ± 6.2%) at day 7 post-transplant. The chimerism declined at day 21 post-transplant and remained at 10% level during the entire follow-up period. Single dose of DRCC therapy induced long-term multilineage chimerism and extended VCA survival. DRCC introduces a novel concept of customized donor-recipient cell-based therapy supporting solid organ and VCA transplants.