Bioprinting of Perfusable, Biocompatible Vessel-like Channels with dECM-Based Bioinks and Living Cells.

There is a growing interest in the production of bioinks that on the one hand, are biocompatible and, on the other hand, have mechanical properties that allow for the production of stable constructs that can survive for a long time after transplantation. While the selection of the right material is crucial for bioprinting, there is another equally important issue that is currently being extensively researched-the incorporation of the vascular system into the fabricated scaffolds. Therefore, in the following manuscript, we present the results of research on bioink with unique physico-chemical and biological properties. In this article, two methods of seeding cells were tested using bioink B and seeding after bioprinting the whole model. After 2, 5, 8, or 24 h of incubation, the flow medium was used in the tested systems. At the end of the experimental trial, for each time variant, the canals were stored in formaldehyde, and immunohistochemical staining was performed to examine the presence of cells on the canal walls and roof. Cells adhered to both ways of fiber arrangement; however, a parallel bioprint with the 5 h incubation and the intermediate plating of cells resulted in better adhesion efficiency. For this test variant, the percentage of cells that adhered was at least 20% higher than in the other analyzed variants. In addition, it was for this variant that the lowest percentage of viable cells was found that were washed out of the tested model. Importantly, hematoxylin and eosin staining showed that after 8 days of culture, the cells were evenly distributed throughout the canal roof. Our study clearly shows that neovascularization-promoting cells effectively adhere to ECM-based pancreatic bioink. Summarizing the presented results, it was demonstrated that the proposed bioink compositions can be used for bioprinting bionic organs with a vascular system formed by endothelial cells and fibroblasts.

Bioprinted 3D Bionic Scaffolds with Pancreatic Islets as a New Therapy for Type 1 Diabetes-Analysis of the Results of Preclinical Studies on a Mouse Model.

Recently, tissue engineering, including 3D bioprinting of the pancreas, has acquired clinical significance and has become an outstanding potential method of customized treatment for type 1 diabetes mellitus. The study aimed to evaluate the function of 3D-bioprinted pancreatic petals with pancreatic islets in the murine model. A total of 60 NOD-SCID (Nonobese diabetic/severe combined immunodeficiency) mice were used in the study and divided into three groups: control group; IsletTx (porcine islets transplanted under the renal capsule); and 3D bioprint (3D-bioprinted pancreatic petals with islets transplanted under the skin, on dorsal muscles). Glucose, C-peptide concentrations, and histological analyses were performed. In the obtained results, significantly lower mean fasting glucose levels (mg/dL) were observed both in a 3D-bioprint group and in a group with islets transplanted under the renal capsule when compared with untreated animals. Differences were observed in all control points: 7th, 14th, and 28th days post-transplantation (129, 119, 118 vs. 140, 139, 140; p < 0.001). Glucose levels were lower on the 14th and 28th days in a group with bioprinted petals compared to the group with islets transplanted under the renal capsule. Immunohistochemical staining indicated the presence of secreted insulin-living pancreatic islets and neovascularization within 3D-bioprinted pancreatic petals after transplantation. In conclusion, bioprinted bionic petals significantly lowered plasma glucose concentration in studied model species.

Streptozotocin-Induced Diabetes in a Mouse Model (BALB/c) Is Not an Effective Model for Research on Transplantation Procedures in the Treatment of Type 1 Diabetes.

Type 1 diabetes (T1D) is characterized by the destruction of over 90% of the ß-cells. C-peptide is a parameter for evaluating T1D. Streptozotocin (STZ) is a standard method of inducing diabetes in animals. Eight protocols describe the administration of STZ in mice; C-peptide levels are not taken into account. The aim of the study is to determine whether the STZ protocol for the induction of beta-cell mass destruction allows for the development of a stable in vivo mouse model for research into new transplant procedures in the treatment of type 1 diabetes. Materials and methods: Forty BALB/c mice were used. The animals were divided into nine groups according to the STZ dose and a control group. The STZ doses were between 140 and 400 mg/kg of body weight. C-peptide was taken before and 2, 7, 9, 12, 14, and 21 days after STZ. Immunohistochemistry was performed. The area of the islet and insulin-/glucagon-expressing tissues was calculated. Results: Mice who received 140, 160, 2 × 100, 200, and 250 mg of STZ did not show changes in mean fasting C-peptide in comparison to the control group and to day 0. All animals with doses of 300 and 400 mg of STZ died during the experiment. The area of the islets did not show any differences between the control and STZ-treated mice in groups below 300 mg. The reduction of insulin-positive areas in STZ mice did not exceed 50%. Conclusions: Streptozotocin is not an appropriate method of inducing a diabetes model for further research on transplantation treatments of type 1 diabetes, having caused the destruction of more than 90% of the ß-cell mass in BALB/c mice.

Impact of Porcine Pancreas Decellularization Conditions on the Quality of Obtained dECM.

Due to the limited number of organ donors, 3D printing of organs is a promising technique. Tissue engineering is increasingly using xenogeneic material for this purpose. This study was aimed at assessing the safety of decellularized porcine pancreas, together with the analysis of the risk of an undesirable immune response. We tested eight variants of the decellularization process. We determined the following impacts: rinsing agents (PBS/NH3·H2O), temperature conditions (4 °C/24 °C), and the grinding method of native material (ground/cut). To assess the quality of the extracellular matrix after the completed decellularization process, analyses of the following were performed: DNA concentration, fat content, microscopic evaluation, proteolysis, material cytotoxicity, and most importantly, the Triton X-100 content. Our analyses showed that we obtained a product with an extremely low detergent content with negligible residual DNA content. The obtained results confirmed the performed histological and immuno-fluorescence staining. Moreover, the TEM microscopic analysis proved that the correct collagen structure was preserved after the decellularization process. Based on the obtained results, we chose the most favorable variant in terms of quality and biology. The method we chose is an effective and safe method that gives a chance for the development of transplant and regenerative medicine.

Bionic Organs: Shear Forces Reduce Pancreatic Islet and Mammalian Cell Viability during the Process of 3D Bioprinting.

BACKGROUND: 3D bioprinting is the future of constructing functional organs. Creating a bioactive scaffold with pancreatic islets presents many challenges. The aim of this paper is to assess how the 3D bioprinting process affects islet viability. METHODS: The BioX 3D printer (Cellink), 600 µm inner diameter nozzles, and 3% (w/v) alginate cell carrier solution were used with rat, porcine, and human pancreatic islets. Islets were divided into a control group (culture medium) and 6 experimental groups (each subjected to specific pressure between 15 and 100 kPa). FDA/PI staining was performed to assess the viability of islets. Analogous studies were carried out on α-cells, ß-cells, fibroblasts, and endothelial cells. RESULTS: Viability of human pancreatic islets was as follows: 92% for alginate-based control and 94%, 90%, 74%, 48%, 61%, and 59% for 15, 25, 30, 50, 75, and 100 kPa, respectively. Statistically significant differences were observed between control and 50, 75, and 100 kPa, respectively. Similar observations were made for porcine and rat islets. CONCLUSIONS: Optimal pressure during 3D bioprinting with pancreatic islets by the extrusion method should be lower than 30 kPa while using 3% (w/v) alginate as a carrier.

The Influence of the Flow of Detergent and Donor Characteristics on the Extracellular Matrix Composition After Human Pancreas Decellularization.

INTRODUCTION: The extracellular matrix (ECM) consists, among others, of polysaccharides, glycosaminoglycans, and proteins. It is being increasingly used in tissue bioengineering. Obtaining ECM of the highest quality through decellularization is a big challenge because of some differences in organ structure. To deprive organs of the cellular part, chemical, enzymatic, or mechanical methods are used. After decellularization, we get a scaffold made of a variety of proteins, and it is the role of these proteins that can significantly affect the maintenance of the spatial structure and be a suitable environment for cells to rebuild a specific organ. AIM: Estimation of the detergent (Triton X-100) flow parameters and anthropometric donors' decellularization process accuracy on the final ECM composition. MATERIALS: Five human pancreata, rejected from transplantation, were used for decellularization. All organs were harvested from brain-dead donors age 13 to 60 years. METHODS: Decellularization was carried out using the flow method with Triton X-100 as an active agent. The experiment compared 5 different flow values. After decellularization, an assessment of the final DNA concentration and the protein composition was performed. Results were compared to anthropometric data of donors. In addition, a microscopic analysis was also carried out. RESULTS: The best results were obtained using a flow of 120 mL/minute. A higher detergent flow was associated with a lower concentration of residual DNA in scaffold. Analysis of the protein profile with anthropometric data has shown that LAM A2 was increasing with age and LAMA5 was decreasing. Being overweight was associated with a higher proportion of COL1 and 4 and a smaller proportion of COL6.

Type 1 diabetes: genes associated with disease development.

Type 1 diabetes (T1D) is the third most common autoimmune disease which develops due to genetic and environmental risk factors. Based on the World Health Organization (WHO) report from 2014 the number of people suffering from all types of diabetes ascended to 422 million, compared to 108 million in 1980. It was calculated that this number will double by the end of 2030. In 2015 American Diabetes Association (ADA) announced that 30.3 million Americans (that is 9.4% of the overall population) had diabetes of which only approximately 1.25 million had T1D. Nowadays, T1D represents roughly 10% of adult diabetes cases total. Multiple genetic abnormalities at different loci have been found to contribute to type 1 diabetes development. The analysis of genome-wide association studies (GWAS) of T1D has identified over 50 susceptible regions (and genes within these regions). Many of these regions are defined by single nucleotide polymorphisms (SNPs) but molecular mechanisms through which they increase or lower the risk of diabetes remain unknown. Genetic factors (in existence since birth) can be detected long before the emergence of immunological or clinical markers. Therefore, a comprehensive understanding of the multiple genetic factors underlying T1D is extremely important for further clinical trials and development of personalized medicine for diabetic patients. We present an overview of current studies and information about regions in the human genome associated with T1D. Moreover, we also put forward information about epigenetic modifications, non-coding RNAs and environmental factors involved in T1D development and onset.

Reoperation in Early Kidney Post-transplant Period as a Strong Risk Factor of Surgical Site Infection Occurrence.

BACKGROUND: One of the most common infective complications after kidney transplant (KTx) is surgical site infection (SSI). Providing indications of improvement of perioperative antibiotic prophylaxis (PAP) and allowing the characterization of risk factors are critical to reduce SSI. The purpose of this study was to evaluate the SSI risk factors and impact of reoperation in the early post-transplant period on SSI occurrence and assess if standard PAP in those cases is a best consideration. METHODS: Between April 2014 and October 2015, a total of 236 KTxs were performed in our center. Deceased donor data, recipient data, and data related to surgical procedures were collected. RESULTS: Surgical site infections were reported in 5.6% (12/214) of patients. Seven patients were diagnosed as having superficial SSI (7/12; 58.3%), 2 with deep SSI (2/12; 16.6%), and 4 with organ-specific SSI (4/12; 33.3%). Extended criteria donor-related transplant, cold ischemia time > 22 hours, dialysis period > 30 months, recipient age older than 45 years, recipient body mass index > 27, induction therapy prior to transplant, diabetes prior to transplant, and ≥ 1 reoperation during 30 days of observation were independent risk factors of SSI occurrence. A total of 19 reoperations were performed in 17 patients. In 8 of all 12 patients with SSI diagnosis, the reoperation was performed (66.7%). In 202 patients of non-SSI patients, only 9 reoperations were performed (4.5%). CONCLUSIONS: Early reoperation after Ktx is a strong risk factor of SSI occurrence. There is a probability that > 4 SSI risk factors and reoperation in the early post-transplant period could require different and more aggressive proceeding, as standard PAP in those cases is insufficient.

Do We Need Insulin Independence After Islet Transplantation?

INTRODUCTION: Most life-threatening diabetes-related complications involve the kidneys, eyes, cardiovascular system, and autonomic nervous system. Clinical islet transplantation (CITx) may be a therapeutic option for some patients. In this study, we analyzed the progression of diabetic complications after CITx and in patients waiting for islet transplantation. METHODS: From 2008 to 2015, 67 patients were listed for pancreatic or islet transplantation. We compared beta scores, islet scores, and secondary complications between patients who underwent islet allotransplantation (CITx group, n = 6) and the patients awaiting islet transplantation (wait group, n = 19) at baseline and during the 1-year follow-up. RESULTS: In the CITx group, good islet function was observed in 80% of patients 1 month post-transplantation and 40% of patients 1 year post-transplantation; however, no patient achieved insulin independence. One patient who underwent simultaneous islet-kidney transplantation died on day 8 because of severe bleeding in the retroperitoneal space. In 1 case, islet primary nonfunction was observed. Mean islet score in the CITx group 1 year post-transplantation was significantly higher than the pretransplant score and wait group scores at enrollment and 1 year later (P < .01). Increased albuminuria was observed in 3 of 11 (27%) patients in the wait group and 0 patients in the CITx group (P = .08). One patient (9%) in the wait group developed chronic renal failure requiring hemodialysis. Ophthalmologic procedures were required by 47% of patients in the wait group and 0 patients in the CITx group in the first year after transplantation (P < .01). CONCLUSION: Successful islet transplantation slows the progression of diabetic complications.

Time of Cold Storage Prior to Start of Hypothermic Machine Perfusion and Its Influence on Graft Survival.

BACKGROUND: Hypothermic machine perfusion (HMP) has become a standard method of preservation for kidneys procured from expanded-criteria donors and donors after cardiac death. There are different systems and approaches to the HMP preservation period, with cold storage prior to HMP sometimes taking several hours. This study evaluated whether the time at which kidneys receive HMP had any influence on the outcomes of kidney transplantation. METHODS: In this analysis, patient and graft survival were evaluated over a 1-year post-transplantation period. Patients who received HMP kidneys (n = 379) were divided into 2 groups: those who received kidneys with a cold ischemia time (CIT) prior to HMP <295 minutes (group G1; n = 254) and those who received kidneys with CIT prior to HMP >295 minutes (group G2; n = 125). RESULTS: Delayed graft function was observed in 31.8% (81/254) of patients in group G1 vs 46.4% (58/125) of patients in group G2 (P = .007). One-year graft survival was statistically higher in the group G1 (93.2%; 233/254) vs group G2 (86.5%; 105/125, P = .029). Mean 1-year estimated glomerular filtration rate was significantly better in the group G1. CONCLUSIONS: In conclusion, introduction of HMP up to 295 minutes from procurement led to better early and 1-year graft results. Kidneys should receive HMP as soon as possible after retrieval, preferably during procurement.

Islet Autotransplantation in Diabetic Patients.

INTRODUCTION: Painful chronic pancreatitis (CP) is the main indication for analgesic pancreatectomy with simultaneous islet autotransplantation to prevent postoperative diabetes mellitus (DM). However, advanced CP may lead to insulin secretion disorders and DM. There are doubts as to whether islet autotransplantation in such cases is an appropriate procedure. The aim of this study was to analyze the results of islet autotransplantation in patients with CP with already diagnosed with DM. METHOD: Between 2008 and 2015, at the Department of General and Transplantation Surgery, patients with CP and unsatisfying pain treatment with positive fasting C-peptide ( > 0.3 ng/mL) level were qualified for simultaneous pancreatectomy and islet autotransplantation. Eight procedures were performed. In 5 cases patients had DM diagnosed prior to the procedure (DM group n = 5). Three patients without DM diagnosed prior to surgery were the control group (n = 3). RESULT: There were no cases of procedure-related deaths in either group. Pain relief without analgesics was reported by all patients. Good islet function was observed in 80% (4/5) of the DM group vs 100% (3/3) in the control group (P = ns). Brittle diabetes was diagnosed in 1 patient in the DM group as a result of islet primary non-function. CONCLUSION: Patients with CP-related severe pain and DM patients with positive C-peptides should be considered for pancreatectomy and islet autotransplantation.

Changes in Gene Expression of Selected Genes in Patients with Type 1 Diabetes and Pancreas Transplant in Peripheral Blood.

BACKGROUND: Diabetes is an autoimmunologic disease that may have a different background. The aim of our study was to show that type 1 diabetes is accompanied by changes in gene expression in peripheral blood mononuclear cells. We analyzed the genes characteristic of pancreatic islet cells and genes playing a big part in autoimmune diseases and cancer. DESIGN: The study included 21 patients and was performed to examine the expression of 9 genes. The patients were divided into 3 research groups: people with type 1 diabetes, people with diabetes after pancreas transplant, and a control group of healthy patients. To assess the level of expression, RNA material was obtained from peripheral blood collected from individuals qualified for the study. RESULTS: The results of the study showed many interesting changes in the expression level of the analyzed genes. It was demonstrated that CASR gene expression was significantly higher in transplant patients than in diabetic patients. Differences in the level of activity are also noted in genes that take part in autoimmune diseases. PROPOSAL: Profiling gene expression in peripheral blood samples may be a useful and noninvasive diagnostic tool that allows early detection of changes leading to the onset or resumption of diabetes.

[Islet transplantation as a treatment for complications of type I diabetes]. / Przeszczepienie wysp trzustkowych jako metoda leczenia powiklan cukrzycy typu I.

Reduced physical activity and high calories up-take along with carbohydrates based diet are considered to be a leading cause of diabetes mellitus rise in western countries. Together with rise in DM morbidity, increase of complicated diabetes is also observed. Pancreas transplantation occurred to be a milestone in diabetic patient management. Guine pig pancreatic islets isolation performed for the first time by Moskalewski in 1965 and updates of his method have given an opportunity to introduce allogenic isolated islets transplantation to clinical usage. For the first time in Poland clinical allotransplantation of isolated pancreatic islets took place in Department of General Surgery and Transplantology of Medical University of Warsaw in 12's June 2008. Unfortunately, unsatisfying results of islet transplantation, specially short period of insulin independence after successful transplantation related with multifactor islet function lost, reduce clinical indications. In this publication we have analyzed known and potential factors of islet lost and we have tried to find way to prevent them, with a long period insulin-independence after transplantation as a main goal.

One-year results of a prospective, randomized trial comparing two machine perfusion devices used for kidney preservation.

Studies have shown beneficial effects of machine perfusion (MP) on early kidney function and long-term graft survival. The aim of this study was to investigate whether the type of perfusion device could affect outcome of transplantation of deceased donor kidneys. A total of 50 kidneys retrieved from 25 donors were randomized to machine perfusion using a flow-driven (FD) device (RM3; Waters Medical Inc) or a pressure-driven (PD) device (LifePort; Organ Recovery Systems), 24 of these kidneys (n = 12 pairs; 48%) were procured from expanded criteria donors (ECD). The primary endpoints were kidney function after transplantation defined using the incidence of delayed graft function (DGF), the number of hemodialysis sessions required, graft function at 12 months, and analyses of biopsy. DGF was similar in both groups (32%; 8/25). Patients with DGF in the FD group required a mean of 4.66 hemodialysis sessions versus 2.65 in the PD group (P = 0.005). Overall, 1-year graft survival was 80% (20/25) vs. 96% (24/25) in the FD and PD groups. One-year graft survival of ECD kidneys was 66% (8/12) in the FD group versus 92% (11/12) in the PD group. Interstitial fibrosis and tubular atrophy were significantly more common in the FD group - 45% (5/11) vs. 0% (0/9) (P = 0.03) in PD group. There were no differences in creatinine levels between the groups. Machine perfusion using a pressure-driven device generating lower pulse stress is superior to a flow-driven device with higher pulse stress for preserving kidney function.

Allogeneic transplantation of isolated islet cells in clinical practice.

The only clinically acceptable radical treatment for patients with insulin-dependent diabetes mellitus is a whole pancreas transplantation, or alternatively an infusion of isolated islet cells into the hepatic portal venous system. Allogeneic transplantation of isolated islet cells is a procedure used only in a highly specific group of recipients, whereas intensive insulin treatment still remains the best therapy to achieve glycemia control in most patients with type 1 diabetes. Two groups of allograft recipients should be taken into consideration when scheduled for islet cell transplantation. The first group comprises allogeneic kidney recipients with a stabilized graft function for >6 months who receive chronic immunosuppression and require transplantation for end-stage renal disease caused by diabetic nephropathy. The second group consists of patients with unsatisfactory glycemic control despite insulin therapy, life-threatening hypoglycemic episodes and a rapid progression of long-term complications. Despite increasingly beneficial outcomes, islet cell transplantation has several limitations. Maintaining normoglycemia without exogenous insulin administration and appropriate selection of immunosuppressive agents to prolong graft survival are the major challenges. The aim of related studies has been to optimize all phases of islet cell transplantation in order to achieve total insulin independence and prolong graft survival.

TransEndoscopic Gastric SubMucosa Islet Transplantation (eGSM-ITx) in pigs with streptozotocine induced diabetes - technical aspects of the procedure - preliminary report.

BACKGROUND: Islets and pancreas transplantation have become standard treatments of patients with diabetic complications. However pancreas transplantation is associated with high incidence of complications and the long-term results of islet transplantation are still unsatisfactory. Loss of pancreatic islets grafts is caused not only by immunological reactions but also due to the site of grafting and IBMIR. Gastric submucosal space could be an alternative site for transplantation. The aim of this study was to assess the possibility of endoscopic islets transplantation into the gastric submucosa-its efficacy and potential complications.
MATERIAL/METHOD: 20 Landrace pigs weighing 19-24 kg were obtained for the study. Seven animals were controls (C-group) and 13 formed the transplantation group (TX group). In both groups diabetes was induced by streptozotocine (stz) infusion at a dose of 200 mg/kg. At 7 days post stz infusion pigs of both groups underwent endoscopy-in group C to assess the feasibility of gastroscopic examination under general anaesthesia in pigs with diabetes and to study the influence of basiliximab infusion on pigs, in the Tx-group to perform endoscopic submucosal islet transplantation (eGSM-ITx). Immunosuppression consisted of tacrolimus 0.2 mg/kg and sirolimus 6 mg/m(2). At 7 days post transplantation, control gastroscopy was performed to assess the gastric mucosa and to obtain biopsies for histopathology. 10 to 30 days after eGSM-ITx, magnetic resonance (MRI) scan was performed. Stomach and pancreas were obtained at autopsy for histopathology. Glycemia was assessed twice daily during the experiment. For 10 days after diabetes induction (up to three days after eGSM-ITx) in both groups, insulin was given to reach glycemia between 150-200 mg/dl, after that period insulin was given only when glycemia exceeded 600 mg/dl.
RESULTS: There were no differences in insulin requirement and glycemia up to the day of eGSM-ITx between the groups. Tx-group animals received a mean of 6000+/-3170 IEQ/kg. Tx-group animals had a significantly lower insulin requirement and significantly lower mean glycemia since the first day post transplantation. C-group animals all required insulin once daily to keep glycemia below 600 mg/dl. There were no signs of perforation, ulceration or bleeding after eGSM-ITx on gastroscopy and histopathological examination. MRI scans revealed unspecific thickening of gastric wall at sites of islet deposition.
CONCLUSIONS: Transendoscopic islets transplantation into gastric submucosa is feasible and a safe procedure in an experimental animal setting. Its potential for clinical application in human subjects needs further studies.