Rise of tissue- and species-specific 3D bioprinting based on decellularized extracellular matrix-derived bioinks and bioresins.

Thanks to its natural complexity and functionality, decellularized extracellular matrix (dECM) serves as an excellent foundation for creating highly cell-compatible bioinks and bioresins. This enables the bioprinted cells to thrive in an environment that closely mimics their native ECM composition and offers customizable biomechanical properties. To formulate dECM bioinks and bioresins, one must first pulverize and/or solubilize the dECM into non-crosslinked fragments, which can then be chemically modified as needed. In bioprinting, the solubilized dECM-derived material is typically deposited and/or crosslinked in a layer-by-layer fashion to build 3D hydrogel structures. Since the introduction of the first liver-derived dECM-based bioinks, a wide variety of decellularized tissue have been employed in bioprinting, including kidney, heart, cartilage, and adipose tissue among others. This review aims to summarize the critical steps involved in tissue-derived dECM bioprinting, starting from the decellularization of the ECM to the standardized formulation of bioinks and bioresins, ultimately leading to the reproducible bioprinting of tissue constructs. Notably, this discussion also covers photocrosslinkable dECM bioresins, which are particularly attractive due to their ability to provide precise spatiotemporal control over the gelation in bioprinting. Both in extrusion printing and vat photopolymerization, there is a need for more standardized protocols to fully harness the unique properties of dECM-derived materials. In addition to mammalian tissues, the most recent bioprinting approaches involve the use of microbial extracellular polymeric substances in bioprinting of bacteria. This presents similar challenges as those encountered in mammalian cell printing and represents a fascinating frontier in bioprinting technology.

Toward a 3D Printed Perfusable Islet Embedding Structure: Technical Notes and Preliminary Results.

To date, islet transplantation to treat type 1 diabetes mellitus remains unsuccessful in long-term follow-up, mainly due to failed engraftment and reconstruction of the islet niche. Alternative approaches, such as islet embedding structures (IESs) based on 3D printing have been developed. However, most of them have been implanted subcutaneously and only a few are intended for direct integration into the vascular system through anastomosis. In this study, we 3D printed a proof-of-concept IES using gelatin methacrylate biocompatible ink. This structure consisted of a branched vascular system surrounding both sides of a central cavity dedicated to islets of Langerhans. Furthermore, we designed a bioreactor optimized for these biological structures. This bioreactor allows seeding and perfusion experiments under sterile and physiological conditions. Preliminary experiments aimed to analyze if the vascular channel could successfully be seeded with mature endothelial cells and the central cavity with rat islets. Subsequently, the structures were used for a humanized model seeding human endothelial progenitor cells (huEPC) within the vascular architecture and human islets co-cultured with huEPC within the central cavity. The constructs were tested for hemocompatibility, suture strength, and anastomosability. The 3D printed IES appeared to be hemocompatible and anastomosable using an alternative cuff anastomosis in a simple ex vivo perfusion model. While rat islets alone could not successfully be embedded within the 3D printed structure for 3 days, human islets co-cultivated with huEPC successfully engrafted within the same time. This result emphasizes the importance of co-culture, nursing cells, and islet niche. In conclusion, we constructed a proof-of-concept 3D printed islet embedding device consisting of a vascular channel that is hemocompatible and perspectively anastomosable to clinical scale blood vessels. However, there are numerous limitations in this model that need to be overcome to transfer this technology to the bedside.

Optimized protocol for quantification of extracellular nicotinamide adenine dinucleotide: evaluating clinical parameters and pre-analytical factors for translational research.

Nicotinamide adenine dinucleotide (NAD+), a coenzyme for more than 500 enzymes, plays a central role in energy production, metabolism, cellular signaling, and DNA repair. Until recently, NAD+ was primarily considered to be an intracellular molecule (iNAD+), however, its extracellular species (eNAD+) has recently been discovered and has since been associated with a multitude of pathological conditions. Therefore, accurate quantification of eNAD+ in bodily fluids such as plasma is paramount to answer important research questions. In order to create a clinically meaningful and reliable quantitation method, we analyzed the relationship of cell lysis, routine clinical laboratory parameters, blood collection techniques, and pre-analytical processing steps with measured plasma eNAD+ concentrations. Initially, NAD+ levels were assessed both intracellularly and extracellularly. Intriguingly, the concentration of eNAD+ in plasma was found to be approximately 500 times lower than iNAD+ in peripheral blood mononuclear cells (0.253 ± 0.02 µM vs. 131.8 ± 27.4 µM, p = 0.007, respectively). This stark contrast suggests that cellular damage or cell lysis could potentially affect the levels of eNAD+ in plasma. However, systemic lactate dehydrogenase in patient plasma, a marker of cell damage, did not significantly correlate with eNAD+ (n = 33; r = -0.397; p = 0.102). Furthermore, eNAD+ was negatively correlated with increasing c-reactive protein (CRP, n = 33; r = -0.451; p = 0.020), while eNAD+ was positively correlated with increasing hemoglobin (n = 33; r = 0.482; p = 0.005). Next, variations in blood drawing, sample handling and pre-analytical processes were examined. Sample storage durations at 4°C (0-120 min), temperature (0° to 25°C), cannula sizes for blood collection and tourniquet times (0 - 120 s) had no statistically significant effect on eNAD+ (p > 0.05). On the other hand, prolonged centrifugation (> 5 min) and a faster braking mode of the centrifuge rotor (< 4 min) resulted in a significant decrease in eNAD+ levels (p < 0.05). Taken together, CRP and hemoglobin appeared to be mildly correlated with eNAD+ levels whereas cell damage was not correlated significantly to eNAD+ levels. The blood drawing trial did not show any influence on eNAD+, in contrast, the preanalytical steps need to be standardized for accurate eNAD+ measurement. This work paves the way towards robust eNAD+ measurements, for use in future clinical and translational research, and provides an optimized hands-on protocol for reliable eNAD+ quantification in plasma.

Solid fraction determines stiffness and viscosity in decellularized pancreatic tissues.

The role of extracellular matrix (ECM) composition and turnover in mechano-signaling and the metamorphic fate of cells seeded into decellularized tissue can be elucidated by recent developments in non-invasive imaging and biotechnological analysis methods. Because these methods allow accurate quantification of the composition and structural integrity of the ECM, they can be critical in establishing standardized decellularization protocols. This study proposes quantification of the solid fraction, the single-component fraction and the viscoelasticity of decellularized pancreatic tissues using compact multifrequency magnetic resonance elastography (MRE) to assess the efficiency and quality of decellularization protocols. MRE of native and decellularized pancreatic tissues showed that viscoelasticity parameters depend according to a power law on the solid fraction of the decellularized matrix. The parameters can thus be used as highly sensitive markers of the mechanical integrity of soft tissues. Compact MRE allows consistent and noninvasive quantification of the viscoelastic properties of decellularized tissue. Such a method is urgently needed for the standardized monitoring of decellularization processes, evaluation of mechanical ECM properties, and quantification of the integrity of solid structural elements remaining in the decellularized tissue matrix.

Surface modification of decellularized bovine carotid arteries with human vascular cells significantly reduces their thrombogenicity.

BACKGROUND: Since autologous veins are unavailable when needed in more than 20% of cases in vascular surgery, the production of personalized biological vascular grafts for implantation has become crucial. Surface modification of decellularized xenogeneic grafts with vascular cells to achieve physiological luminal coverage and eventually thromboresistance is an important prerequisite for implantation. However, ex vivo thrombogenicity testing remains a neglected area in the field of tissue engineering of vascular grafts due to a multifold of reasons. METHODS: After seeding decellularized bovine carotid arteries with human endothelial progenitor cells and umbilical cord-derived mesenchymal stem cells, luminal endothelial cell coverage (LECC) was correlated with glucose and lactate levels on the cell supernatant. Then a closed loop whole blood perfusion system was designed. Recellularized grafts with a LECC > 50% and decellularized vascular grafts were perfused with human whole blood for 2 h. Hemolysis and complete blood count evaluation was performed on an hourly basis, followed by histological and immunohistochemical analysis. RESULTS: While whole blood perfusion of decellularized grafts significantly reduced platelet counts, platelet depletion from blood resulting from binding to re-endothelialized grafts was insignificant (p = 0.7284). Moreover, macroscopic evaluation revealed thrombus formation only in the lumen of unseeded grafts and histological characterization revealed lack of CD41 positive platelets in recellularized grafts, thus confirming their thromboresistance. CONCLUSION: In the present study we were able to demonstrate the effect of surface modification of vascular grafts in their thromboresistance in an ex vivo whole blood perfusion system. To our knowledge, this is the first study to expose engineered vascular grafts to human whole blood, recirculating at high flow rates, immediately after seeding.

In vitro recellularization of decellularized bovine carotid arteries using human endothelial colony forming cells.

BACKGROUND: Many patients suffering from peripheral arterial disease (PAD) are dependent on bypass surgery. However, in some patients no suitable replacements (i.e. autologous or prosthetic bypass grafts) are available. Advances have been made to develop autologous tissue engineered vascular grafts (TEVG) using endothelial colony forming cells (ECFC) obtained by peripheral blood draw in large animal trials. Clinical translation of this technique, however, still requires additional data for usability of isolated ECFC from high cardiovascular risk patients. Bovine carotid arteries (BCA) were decellularized using a combined SDS (sodium dodecyl sulfate) -free mechanical-osmotic-enzymatic-detergent approach to show the feasibility of xenogenous vessel decellularization. Decellularized BCA chips were seeded with human ECFC, isolated from a high cardiovascular risk patient group, suffering from diabetes, hypertension and/or chronic renal failure. ECFC were cultured alone or in coculture with rat or human mesenchymal stromal cells (rMSC/hMSC). Decellularized BCA chips were evaluated for biochemical, histological and mechanical properties. Successful isolation of ECFC and recellularization capabilities were analyzed by histology. RESULTS: Decellularized BCA showed retained extracellular matrix (ECM) composition and mechanical properties upon cell removal. Isolation of ECFC from the intended target group was successfully performed (80% isolation efficiency). Isolated cells showed a typical ECFC-phenotype. Upon recellularization, co-seeding of patient-isolated ECFC with rMSC/hMSC and further incubation was successful for 14 (n = 9) and 23 (n = 5) days. Reendothelialization (rMSC) and partial reendothelialization (hMSC) was achieved. Seeded cells were CD31 and vWF positive, however, human cells were detectable for up to 14 days in xenogenic cell-culture only. Seeding of ECFC without rMSC was not successful. CONCLUSION: Using our refined decellularization process we generated easily obtainable TEVG with retained ECM- and mechanical quality, serving as a platform to develop small-diameter (< 6 mm) TEVG. ECFC isolation from the cardiovascular risk target group is possible and sufficient. Survival of diabetic ECFC appears to be highly dependent on perivascular support by rMSC/hMSC under static conditions. ECFC survival was limited to 14 days post seeding.

Long-term Outcome of Orthotopic Liver Transplantation in Patients With Hemochromatosis: A Summary of a 30-year Transplant Program.

Hemochromatosis (HC) is an autosomal recessive disease characterized by impaired iron metabolism and a rare indication for orthotopic liver transplantation (LT). Data about iron reaccumulation and remodeling of the liver graft after LT are limited. Therefore, we performed an evaluation of the histopathologic changes during long-term follow-up in patients with HC. METHODS: A retrospective analysis of patients undergoing LT at our center between 1990 and 2016 identified 29 patients with HC. End points were the evaluation of post-LT iron reaccumulation and the stage of fibrosis as well as the degree of inflammation of the liver graft. Secondary end points were patient survival and postoperative complications. RESULTS: The median age was 52.7 y, and there were more male (82.8%) than female patients (17.2%). Post-LT serum ferritin values (>1000 µg/L) were only temporarily elevated in 2 patients. The median estimated survival after LT was 45.5 mo (0.1-285.9 mo). Twenty patients (69%) died during follow-up of 10 y. The survival of patients with HC was significantly worse (P = 0.001) when compared with the overall cohort of patients undergoing LT because of to other causes. CONCLUSIONS: There was no significant iron overload detected in patients with HC after LT, and only minimal iron deposits were described in liver biopsies. Nevertheless, patients suffering from HC show a lower post-LT survival when compared with patients without iron storage disease but mostly because of extrahepatic causes.

Engineering an endothelialized, endocrine Neo-Pancreas: Evaluation of islet functionality in an ex vivo model.

Islet-based recellularization of decellularized, repurposed rat livers may form a transplantable Neo-Pancreas. The aim of this study is the establishment of the necessary protocols, the evaluation of the organ structure and the analysis of the islet functionality ex vivo. After perfusion-based decellularization of rat livers, matrices were repopulated with endothelial cells and mesenchymal stromal cells, incubated for 8 days in a perfusion chamber, and finally repopulated on day 9 with intact rodent islets. Integrity and quality of re-endothelialization was assessed by histology and FITC-dextran perfusion assay. Functionality of the islets of Langerhans was determined on day 10 and day 12 via glucose stimulated insulin secretion. Blood gas analysis variables confirmed the stability of the perfusion cultivation. Histological staining showed that cells formed a monolayer inside the intact vascular structure. These findings were confirmed by electron microscopy. Islets infused via the bile duct could histologically be found in the parenchymal space. Adequate insulin secretion after glucose stimulation after 1-day and 3-day cultivation verified islet viability and functionality after the repopulation process. We provide the first proof-of-concept for the functionality of islets of Langerhans engrafted in a decellularized rat liver. Furthermore, a re-endothelialization step was implemented to provide implantability. This technique can serve as a bioengineered platform to generate implantable and functional endocrine Neo-Pancreases.

Hepatitis B Immunoglobulin discontinuation in long-term liver transplant patients.

BACKGROUND: Hepatitis B immunoglobulin (HBIG)-as a monotherapy or combined with nucleos(t)ide analogs (NUCs)-has effectively lowered Hepatitis B virus (HBV) reinfection after liver transplantation. However, it is associated with high costs and viral resistance. HBIG-free prophylaxis with novel NUCs (tenofovir, entecavir) composes a viable alternative. We evaluated reinfection rate, histological changes, and outcome associated with HBIG discontinuation. METHODS: A retrospective analysis was performed of patients undergoing liver transplantation due to HBV-induced liver disease at our center since 1988. A controlled HBIG discontinuation was conducted between 2015 and 2017 in 65 patients. Recurrent infection was determined by HbsAg values. Fibrosis and inflammation were evaluated by routine biopsy. The survival of patients after HBIG discontinuation was compared to a control population on HBIG for prophylaxis. RESULTS: From 1988 to 2013, 352 patients underwent liver transplantation due to HBV-induced liver disease. 169 patients could be included for analysis. 104 (51.5%) patients continued a prophylaxis containing HBIG. HBIG was discontinued in 65 (38.5%) patients in a controlled manner, maintaining an oral NUC. None of those patients showed HBV reinfection or graft dysfunction. No significant changes of inflammation grades (P = .067) or fibrosis stages (P = .051) were detected. The survival of patients after HBIG discontinuation was comparable to the control (P = .95). CONCLUSION: HBIG withdrawal under continuation of oral NUC therapy is safe and not related to graft dysfunction, based on blood tests and histology. HBIG-free prophylaxis is not associated with a worse outcome and displays a financial relief as well as a logistic simplification during long-term follow-up.

Development of GelMA/PCL and dECM/PCL resins for 3D printing of acellular in vitro tissue scaffolds by stereolithography.

Gelatin methacryloyl (GelMA) is a chemically modified extracellular matrix (ECM)-derived biopolymer that is widely used for 3D fabrication of tissue engineering scaffolds. However, its tendency for physical gelation limits its use in aqueous 3D printing resins to low concentrations, yielding a poor printing resolution in stereolithography (SLA). To obtain a GelMA-based resin that can be printed into high-resolution tissue scaffolds, we formulated resins of fish and porcine-derived GelMA in formamide using GelMA alone or mixed with star-shaped poly(ε-caprolactone) methacrylate (PCL-MA). We identified GelMA resins and GelMA/PCL-MA hybrid resins with a ratio of 70/30 wt-% to yield a suitable viscosity for SLA at 32 °C and demonstrated the resolution of the new resins in SLA by 3D printing acellular human small intestine-mimicking tissue scaffolds. The presence of PCL-MA in the hybrid resins improved the 3D printing fidelity compared to the neat GelMA resins, while GelMA provided the hybrid materials with enhanced swelling and proliferation of seeded cells. We further demonstrated the transferability of our resin formulation to native organ-derived materials by successfully replacing GelMA in the hybrid resin with solubilized, methacryloyl-functionalized decellularized liver ECM (dECM-MA) and by 3D printing multi-layer dECM/PCL-MA hydrogels.

Strategies based on organ decellularization and recellularization.

Transplantation is the only curative treatment option available for patients suffering from end-stage organ failure, improving their quality of life and long-term survival. However, because of organ scarcity, only a small number of these patients actually benefit from transplantation. Alternative treatment options are needed to address this problem. The technique of whole-organ decellularization and recellularization has attracted increasing attention in the last decade. Decellularization includes the removal of all cellular components from an organ, while simultaneously preserving the micro and macro anatomy of the extracellular matrix. These bioscaffolds are subsequently repopulated with patient-derived cells, thus constructing a personalized neo-organ and ideally eliminating the need for immunosuppression. However, crucial problems have not yet been satisfyingly addressed and remain to be resolved, such as organ and cell sources. In this review, we focus on the actual state of organ de- and recellularization, as well as the problems and future challenges.

Diagnostic Biomarkers to Diagnose Acute Allograft Rejection After Liver Transplantation: Systematic Review and Meta-Analysis of Diagnostic Accuracy Studies.

Objective: A systematic review and meta-analysis of diagnostic biomarkers for noninvasive diagnosis of acute allograft rejection following liver transplantation. Background: Noninvasive blood and urine markers have been widely explored in recent decades for diagnosing acute rejection after liver transplantation. However, none have been translated into routine clinical use so far due to uncertain diagnostic accuracy, and liver biopsy remains the gold standard. Methods: Systematic literature searches of Medline, Cochrane and Embase were conducted up to February 2019 to identify studies evaluating the use of noninvasive markers in diagnosing allograft rejection following liver transplantation. Meta-analysis was performed using a random effects model with DerSimonian-Laird weighting and the hierarchical summary receiver operating curve. Results: Of 560 identified studies, 15 studies (1,445 patients) met the inclusion criteria. The following markers were tested: acid labile nitroso-compounds (NOx), serum amyloid A protein, procalcitonin, peripheral blood eosinophil count, peripheral blood T-cell activation and interleukin 2 (IL-2) receptor, guanylate-binding protein-2 mRNA, graft-derived cell-free DNA, pi-glutathione S-transferase, alpha-glutathione S-transferase and serum HLA class I soluble antigens. Only eosinophil count was tested in multiple studies, and they demonstrated high heterogeneity (I2 = 72% [95% CI: 0.5-0.99]). IL-2 receptor demonstrated the highest sensitivity (89% [95% CI: 0.78-0.96]) and specificity (81% [95% CI: 0.69-0.89]). Conclusion: IL-2 receptor expression demonstrated the highest diagnostic accuracy, while the peripheral eosinophil count was the only marker tested in more than one study. Presently, liver biopsy remains superior to noninvasive diagnostic biomarkers as most studies exhibited inferior designs, hindering possible translation into clinical application.

The nanomolar sensing of nicotinamide adenine dinucleotide in human plasma using a cycling assay in albumin modified simulated body fluids.

Nicotinamide adenine dinucleotide (NAD), a prominent member of the pyridine nucleotide family, plays a pivotal role in cell-oxidation protection, DNA repair, cell signalling and central metabolic pathways, such as beta oxidation, glycolysis and the citric acid cycle. In particular, extracellular NAD+ has recently been demonstrated to moderate pathogenesis of multiple systemic diseases as well as aging. Herein we present an assaying method, that serves to quantify extracellular NAD+ in human heparinised plasma and exhibits a sensitivity ranging from the low micromolar into the low nanomolar domain. The assay achieves the quantification of extracellular NAD+ by means of a two-step enzymatic cycling reaction, based on alcohol dehydrogenase. An albumin modified revised simulated body fluid was employed as standard matrix in order to optimise enzymatic activity and enhance the linear behaviour and sensitivity of the method. In addition, we evaluated assay linearity, reproducibility and confirmed long-term storage stability of extracellular NAD+ in frozen human heparinised plasma. In summary, our findings pose a novel standardised method suitable for high throughput screenings of extracellular NAD+ levels in human heparinised plasma, paving the way for new clinical discovery studies.