Thrombogenicity assessment of perfusable tissue engineered constructs: a systematic review.

Vascular surgery faces a critical demand for novel vascular grafts that are biocompatible and thromboresistant. This urgency particularly applies to bypass operations involving small caliber vessels. In the realm of tissue engineering, the development of fully vascularized organs holds great promise as a solution to organ shortage for transplantation. To achieve this, it is imperative to (re-)construct a biocompatible and non-thrombogenic vascular network within these organs. In this systematic review, we identify, classify and discuss basic principles and methods used to perform in vitro/ex vivo dynamic thrombogenicity testing of perfusable tissue engineered organs and tissues. We conducted a pre-registered systematic review of studies published in the last 23 years according to PRISMA-P Guidelines, comprising a systematic data extraction, in-depth analysis and risk of bias assessment of 116 included studies. We identified shaking (n=28), flow loop (n=17), ex vivo (arterio-venous shunt, n=33) and dynamic in vitro models (n=38) as main approaches for thrombogenicity assessment. This comprehensive review unveils a prevalent lack of standardization and serves as a valuable guide in the design of standardized experimental setups.

Distinctive protein expression in elderly livers in a Sprague-Dawley rat model of normothermic ex vivo liver machine perfusion.

BACKGROUND: Liver grafts are frequently declined due to high donor age or age mismatch with the recipient. To improve the outcome of marginal grafts, we aimed to characterize the performance of elderly vs. young liver grafts in a standardized rat model of normothermic ex vivo liver machine perfusion (NMP). METHODS: Livers from Sprague-Dawley rats aged 3 or 12 months were procured and perfused for 6 h using a rat NMP system or collected as a reference group (n = 6/group). Tissue, bile, and perfusate samples were used for biochemical, and proteomic analyses. RESULTS: All livers cleared lactate during perfusion and continued to produce bile after 6 h of perfusion (614 mg/h). Peak urea levels in 12-month-old animals were higher than in younger animals. Arterial and portal venous pressure, bile production and pH did not differ between groups. Proteomic analysis identified a total of 1477 proteins with oxidoreductase and catalytic activity dominating the gene ontology analysis. Proteins such as aldehyde dehydrogenase 1A1 and 2-Hydroxyacid oxidase 2 were significantly more present in livers of older age. CONCLUSIONS: Young and elderly liver grafts exhibited similar viability during NMP, though proteomic analyses indicated that older grafts are less resilient to oxidative stress. Our study is limited by the elderly animal age, which corresponds to mature but not elderly human age typically seen in marginal human livers. Nevertheless, reducing oxidative stress could be a promising therapeutic target in the future.

Quality Assessment by Bile Composition in Normothermic Machine Perfusion of Rat Livers.

Background: The persistent challenge of organ scarcity in liver transplantation leads to an escalating dependence on organs obtained from extended criteria donors (ECD). Normothermic machine perfusion (NMP) is used for improved preservation. Due to the mimicked in vivo conditions during normothermic machine perfusion, the liver is metabolically active, which allows quality assessment during perfusion. Bile seems to be of rising interest in clinical studies, as it is easily collectible for analysis. As there are currently no data on biliary bile acids during NMP, the primary objective of this study was to use our experimental rodent NMP model to assess changes in bile composition through organ damage during perfusion to inform clinical evaluation of donor organs during NMP. Methods: Thirty livers from male Sprague-Dawley rats in five groups underwent 6 h of NMP using either erythrocyte-supplemented DMEM or Steen solution, with or without 30 min of warm ischemia time (WIT). We conducted regular measurements of AST, ALT, LDH, and urea levels in the perfusate at 3-hour intervals. Bile samples were analyzed for biliary pH, LDH, and gamma glutamyltransferase, as well as biliary bile acids via mass spectrometry and UHPLC. Results: Compared with regular livers, liver injury parameters were significantly higher in our donation after circulatory death (DCD) model. Bile production was significantly reduced in livers exposed to WIT, and the bile showed a significantly more alkaline pH. This correlated with the concentration of total bile acids, which was significantly higher in livers experiencing WIT. However, regular livers produced a higher total amount of biliary bile acids during perfusion. Taurocholic acid and its metabolites were most prominent. Secondary bile acids were significantly reduced during perfusion due to the missing enterohepatic circulation. Conclusions: WIT-induced liver injury affects bile composition within our small-animal NMP model. We hypothesize this phenomenon to be attributed to the energy-driven nature of bile secretion, potentially explaining why DCD livers produce less, yet more concentrated, bile. Our results may inform clinical studies, in which biliary bile acids might have a potential as a quantifiable viability marker in human NMP liver transplantation studies.

Depletion of donor dendritic cells ameliorates immunogenicity of both skin and hind limb transplants.

Acute cellular rejection remains a significant obstacle affecting successful outcomes of organ transplantation including vascularized composite tissue allografts (VCA). Donor antigen presenting cells (APCs), particularly dendritic cells (DCs), orchestrate early alloimmune responses by activating recipient effector T cells. Employing a targeted approach, we investigated the impact of donor-derived conventional DCs (cDCs) and APCs on the immunogenicity of skin and skin-containing VCA grafts, using mouse models of skin and hind limb transplantation. By post-transplantation day 6, skin grafts demonstrated severe rejections, characterized by predominance of recipient CD4 T cells. In contrast, hind limb grafts showed moderate rejection, primarily infiltrated by CD8 T cells. Notably, the skin component exhibited heightened immunogenicity when compared to the entire VCA, evidenced by increased frequencies of pan (CD11b-CD11c+), mature (CD11b-CD11c+MHCII+) and active (CD11b-CD11c+CD40+) DCs and cDC2 subset (CD11b+CD11c+ MHCII+) in the lymphoid tissues and the blood of skin transplant recipients. While donor depletion of cDC and APC reduced frequencies, maturation and activation of DCs in all analyzed tissues of skin transplant recipients, reduction in DC activities was only observed in the spleen of hind limb recipients. Donor cDC and APC depletion did not impact all lymphocyte compartments but significantly affected CD8 T cells and activated CD4 T in lymph nodes of skin recipients. Moreover, both donor APC and cDC depletion attenuated the Th17 immune response, evident by significantly reduced Th17 (CD4+IL-17+) cells in the spleen of skin recipients and reduced levels of IL-17E and lymphotoxin-α in the serum samples of both skin and hind limb recipients. In conclusion, our findings underscore the highly immunogenic nature of skin component in VCA. The depletion of donor APCs and cDCs mitigates the immunogenicity of skin grafts while exerting minimal impact on VCA.

On the relationship between viscoelasticity and water diffusion in soft biological tissues.

Magnetic resonance elastography (MRE) and diffusion-weighted imaging (DWI) are complementary imaging techniques that detect disease based on viscoelasticity and water mobility, respectively. However, the relationship between viscoelasticity and water diffusion is still poorly understood, hindering the clinical translation of combined DWI-MRE markers. We used DWI-MRE to study 129 biomaterial samples including native and cross-linked collagen, glycosaminoglycans (GAGs) with different sulfation levels, and decellularized specimens of pancreas and liver, all with different proportions of solid tissue, or solid fractions. We developed a theoretical framework of the relationship between mechanical loss and tissue-water mobility based on two parameters, solid and fluid viscosity. These parameters revealed distinct DWI-MRE property clusters characterizing weak, moderate, and strong water-network interactions. Sparse networks interacting weakly with water, such as collagen or diluted decellularized tissue, resulted in marginal changes in water diffusion over increasing solid viscosity. In contrast, dense networks with larger solid fractions exhibited both free and hindered water diffusion depending on the polarity of the solid components. For example, polar and highly sulfated GAGs as well as native soft tissues hindered water diffusion despite relatively low solid viscosity. Our results suggest that two fundamental properties of tissue networks, solid fraction and network polarity, critically influence solid and fluid viscosity in biological tissues. Since clinical DWI and MRE are sensitive to these viscosity parameters, the framework we present here can be used to detect tissue remodeling and architectural changes in the setting of diagnostic imaging. STATEMENT OF SIGNIFICANCE: The viscoelastic properties of biological tissues provide a wealth of information on the vital state of cells and host matrix. Combined measurement of viscoelasticity and water diffusion by medical imaging is sensitive to tissue microarchitecture. However, the relationship between viscoelasticity and water diffusion is still poorly understood, hindering full exploitation of these properties as a combined clinical biomarker. Therefore, we analyzed the parameter space accessible by diffusion-weighted imaging (DWI) and magnetic resonance elastography (MRE) and developed a theoretical framework for the relationship between water mobility and mechanical parameters in biomaterials. Our theory of solid material properties related to particle motion can be translated to clinical radiology using clinically established MRE and DWI.

A new bicornuate model of rat uterus transplantation.

INTRODUCTION: Uterus transplantation has revolutionized reproductive medicine for women with absolute uterine factor infertility, resulting in more than 40 reported successful live births worldwide to date. Small animal models are pivotal to refine this surgical and immunological challenging procedure aiming to enhance safety for both the mother and the child. MATERIAL AND METHODS: We established a syngeneic bicornuate uterus transplantation model in young female Lewis rats. All surgical procedures were conducted by an experienced and skilled microsurgeon who organized the learning process into multiple structured steps. Animals underwent meticulous preoperative preparation and postoperative care. Transplant success was monitored by sequential biopsies, monitoring graft viability and documenting histological changes long-term. RESULTS: Bicornuate uterus transplantation were successfully established achieving an over 70% graft survival rate with the passage of time. The bicornuate model demonstrated safety and feasibility, yielding outcomes comparable to the unicornuate model in terms of ischemia times and complications. Longitudinal biopsies were well-tolerated, enabling comprehensive monitoring throughout the study. CONCLUSIONS: Our novel bicornuate rat uterus transplantation model provides a distinctive opportunity for sequential biopsies at various intervals after transplantation and, therefore, comprehensive monitoring of graft health, viability, and identification of potential signs of rejection. Furthermore, this model allows for different interventions in each horn for comparative studies without interobserver differences contrary to the established unicornuate model. By closely replicating the clinical setting, this model stands as a valuable tool for ongoing research in the field of uterus transplantation, promoting further innovation and deeper insights into the intricacies of the uterus transplant procedure.

Chemistry matters: A side-by-side comparison of two chemically distinct methacryloylated dECM bioresins for vat photopolymerization.

Decellularized extracellular matrix (dECM) is an excellent natural source for 3D bioprinting materials due to its inherent cell compatibility. In vat photopolymerization, the use of dECM-based bioresins is just emerging, and extensive research is needed to fully exploit their potential. In this study, two distinct methacryloyl-functionalized, photocrosslinkable dECM-based bioresins were prepared from digested porcine liver dECM through functionalization with glycidyl methacrylate (GMA) or conventional methacrylic anhydride (MA) under mild conditions for systematic comparison. Although the chemical modifications did not significantly affect the structural integrity of the dECM proteins, mammalian cells encapsulated in the respective hydrogels performed differently in long-term culture. In either case, photocrosslinking during 3D (bio)printing resulted in transparent, highly swollen, and soft hydrogels with good shape fidelity, excellent biomimetic properties and tunable mechanical properties (~ 0.2-2.5 kPa). Interestingly, at a similar degree of functionalization (DOF ~ 81.5-83.5 %), the dECM-GMA resin showed faster photocrosslinking kinetics in photorheology resulting in lower final stiffness and faster enzymatic biodegradation compared to the dECM-MA gels, yet comparable network homogeneity as assessed via Brillouin imaging. While human hepatic HepaRG cells exhibited comparable cell viability directly after 3D bioprinting within both materials, cell proliferation and spreading were clearly enhanced in the softer dECM-GMA hydrogels at a comparable degree of crosslinking. These differences were attributed to the additional hydrophilicity introduced to dECM via methacryloylation through GMA compared to MA. Due to its excellent printability and cytocompatibility, the functional porcine liver dECM-GMA biomaterial enables the advanced biofabrication of soft 3D tissue analogs using vat photopolymerization-based bioprinting.

From morbidity reduction to cost-effectiveness: Enhanced recovery after surgery (ERAS) society recommendations in minimal invasive liver surgery.

PURPOSE: Minimal-invasive liver surgery (MILS) reduces surgical trauma and is associated with fewer postoperative complications. To amplify these benefits, perioperative multimodal concepts like Enhanced Recovery after Surgery (ERAS), can play a crucial role. We aimed to evaluate the cost-effectiveness for MILS in an ERAS program, considering the necessary additional workforce and associated expenses. METHODS: A prospective observational study comparing surgical approach in patients within an ERAS program compared to standard care from 2018-2022 at the Charité - Universitätsmedizin Berlin. Cost data were provided by the medical controlling office. ERAS items were applied according to the ERAS society recommendations. RESULTS: 537 patients underwent liver surgery (46% laparoscopic, 26% robotic assisted, 28% open surgery) and 487 were managed by the ERAS protocol. Implementation of ERAS reduced overall postoperative complications in the MILS group (18% vs. 32%, p = 0.048). Complications greater than Clavien-Dindo grade II incurred the highest costs (€ 31,093) compared to minor (€ 17,510) and no complications (€13,893; p < 0.001). In the event of major complications, profit margins were reduced by a median of € 6,640. CONCLUSIONS: Embracing the ERAS society recommendations in liver surgery leads to a significant reduction of complications. This outcome justifies the higher cost associated with a well-structured ERAS protocol, as it effectively offsets the expenses of complications.

Optimizing environmental enrichment for Sprague Dawley rats: Exemplary insights into the liver proteome.

BACKGROUND: Considering the expected increase in the elderly population and the growing emphasis on aging-related biomedical research, the demand for aged laboratory animals has surged, challenging established husbandry practices. Our objective was to establish a cost-effective method for environmental enrichment, utilizing the liver as a representative organ to assess potential metabolic changes in response to differing enrichment levels. METHODS: We conducted a six-month study involving 24 male Sprague Dawley rats, randomly assigned to four environmental enrichment groups. Two groups were housed in standard cages, while the others were placed in modified rabbit cages. Half of the groups received weekly playtime in an activity focused rat housing unit. We evaluated hormone levels, playtime behavior, and subjective handling experience. Additionally, liver tissue proteomic analysis was performed. RESULTS: Initial corticosterone levels and those after 3 and 6 months showed no significant differences. Yet, testosterone levels were lower in the control group by the end of the study (p = 0.007). We observed 1871 distinct proteins in liver tissue, with 77% being common across groups. In gene ontology analysis, no specific pathways were overexpressed. In semiquantitative analysis, we observed differences in proteins associated in lipid metabolism such as Apolipoprotein A-I and Acyl-CoA 6-desaturase, which were lower in the control group (p = 0.024 and p = 0.009). Rats in the intervention groups with weekly playtime displayed the least amount of reported distress during inspection or upon room entry and were less prone to accepting treats. Removing animals from their enclosure was most effortless for those in the large cage group. Over time, there was a decrease in conflicts among rats that interacted only twice weekly during playpen time. DISCUSSION: In summary, refining husbandry practices for aging rats is both simple and budget-friendly, with no apparent adverse effects on stress levels, animal development, or relevant metabolic changes in the liver.

Improved outcomes after hypothermic oxygenated machine perfusion in liver transplantation-Long-term follow-up of a multicenter randomized controlled trial.

BACKGROUND: While 4 randomized controlled clinical trials confirmed the early benefits of hypothermic oxygenated machine perfusion (HOPE), high-level evidence regarding long-term clinical outcomes is lacking. The aim of this follow-up study from the HOPE-ECD-DBD trial was to compare long-term outcomes in patients who underwent liver transplantation using extended criteria donor allografts from donation after brain death (ECD-DBD), randomized to either HOPE or static cold storage (SCS). METHODS: Between September 2017 and September 2020, recipients of liver transplantation from 4 European centers receiving extended criteria donor-donation after brain death allografts were randomly assigned to HOPE or SCS (1:1). Follow-up data were available for all patients. Analyzed endpoints included the incidence of late-onset complications (occurring later than 6 months and graded according to the Clavien-Dindo Classification and the Comprehensive Complication Index) and long-term graft survival and patient survival. RESULTS: A total of 46 patients were randomized, 23 in both arms. The median follow-up was 48 months (95% CI: 41-55). After excluding early perioperative morbidity, a significant reduction in late-onset morbidity was observed in the HOPE group (median reduction of 23 Comprehensive Complication Index-points [p=0.003] and lower incidence of major complications [Clavien-Dindo ≥3, 43% vs. 85%, p=0.009]). Primary graft loss occurred in 13 patients (HOPE n=3 vs. SCS n=10), resulting in a significantly lower overall graft survival (p=0.029) and adverse 1-, 3-, and 5-year survival probabilities in the SCS group, which did not reach the level of significance (HOPE 0.913, 0.869, 0.869 vs. SCS 0.783, 0.606, 0.519, respectively). CONCLUSIONS: Our exploratory findings indicate that HOPE reduces late-onset morbidity and improves long-term graft survival providing clinical evidence to further support the broad implementation of HOPE in human liver transplantation.

Proteomic analysis of decellularized mice liver and kidney extracellular matrices.

BACKGROUND: The extracellular matrix (ECM) is a three-dimensional network of proteins that encases and supports cells within a tissue and promotes physiological and pathological cellular differentiation and functionality. Understanding the complex composition of the ECM is essential to decrypt physiological processes as well as pathogenesis. In this context, the method of decellularization is a useful technique to eliminate cellular components from tissues while preserving the majority of the structural and functional integrity of the ECM. RESULTS: In this study, we employed a bottom-up proteomic approach to elucidate the intricate network of proteins in the decellularized extracellular matrices of murine liver and kidney tissues. This approach involved the use of a novel, perfusion-based decellularization protocol to generate acellular whole organ scaffolds. Proteomic analysis of decellularized mice liver and kidney ECM scaffolds revealed tissue-specific differences in matrisome composition, while we found a predominantly stable composition of the core matrisome, consisting of collagens, glycoproteins, and proteoglycans. Liver matrisome analysis revealed unique proteins such as collagen type VI alpha-6, fibrillin-2 or biglycan. In the kidney, specific ECM-regulators such as cathepsin z were detected. CONCLUSION: The identification of distinct proteomic signatures provides insights into how different matrisome compositions might influence the biological properties of distinct tissues. This experimental workflow will help to further elucidate the proteomic landscape of decellularized extracellular matrix scaffolds of mice in order to decipher complex cell-matrix interactions and their contribution to a tissue-specific microenvironment.

miRNA as potential biomarkers after liver transplantation: A systematic review.

BACKGROUND: Liver transplantation is a life-saving therapy for end-stage liver disease patients, but acute cellular rejection (ACR) and graft complications remain significant postoperative challenges. Early and accurate diagnosis is crucial for timely intervention and improved patient outcomes, but their diagnosis rely currently on invasive biopsy sampling, thus prompting the search for non-invasive Biomarkers. MicroRNA (miRNA) have emerged as promising biomarkers in various pathological conditions, and their potential utility in diagnosing acute cellular rejection after liver transplantation has gained significant interest. METHODS: This systematic review of PubMed, Web of Science, and the ClinicalTrials.gov registry analyzes studies exploring miRNA as biomarkers for ACR and graft dysfunction in liver transplantation (PROSPERO ID CRD42023465278). The Cochrane Collaboration tool for assessing risk of bias was employed. Population data, identified miRNA and their dynamic regulation, as well as event prediction were compared. Data extraction and quality assessment were performed independently by two reviewers. RESULTS: Thirteen studies were included in this systematic review. Various investigated miRNAs were upregulated in association with acute cellular rejection, like miR-122, miR-155, miR-181, miR-483-3p, and miR-885-5p, demonstrating great biomarker potential. Additionally, several studies conducted target gene analysis, revealing insights into cellular mechanisms linked to ACR. Moreover, various miRNA were also capable of predicting different organ complications following transplantation, expanding their versatility. Remaining challenges include the standardization of miRNA profiling, the need for functional validation, and the necessity for long-term studies. CONCLUSION: The results highlight the potential of miRNA as specific, non-invasive biomarkers for ACR and graft dysfunction following liver transplantation. However, further research is needed to validate these findings and establish standardized diagnostic panels to incorporate them into clinical practice and explore miRNA-based therapies in the future.

Moderate LMWH Anticoagulation Improves Success Rate of Hind Limb Allotransplantation in Mice.

Background: The mouse hind limb model represents a powerful research tool in vascularized composite tissue allotransplantation, but its applicability is limited due to poor graft survival (62%-83%). Vascular thrombosis and massive hemorrhage are the major causes for these drop-outs. We hypothesize that because of better anticoagulation effect and lower risk of thrombocytopenia, application of low molecular weight heparin (LMWH) will minimize vascular complications and enhance graft and animal survival. Methods: Fifty allogeneic hind limb transplantations were performed (C57BL/6 to DBA/2 mice) using five different anticoagulation protocols. Bleeding and thromboembolic events were recorded macroscopically by postoperative hemorrhage and livid discoloration of the graft, respectively. Graft perfusion and survival were monitored daily by capillary-refill-time of graft toes within 2-3 seconds. Vascular congestion and tissue necrosis were examined by histological evaluation of hematoxylin-eosin-stained tissue sections. Results: All transplantations were technically successful. Increase in thromboembolic events and a concomitant decrease in bleeding events were observed with the decreasing concentration of heparin in the perfusion solution. Although treatment of donor and recipient with low dose of LMWH could not reduce thromboembolic events, moderate dose effectively reduced these events. Compared with the poor outcome of graft perfusion with heparin alone, additional treatment of donor and recipient with low dose of LMWH improved graft and animal survival by 18%. Interestingly, animals treated with moderate dose of LMWH demonstrated 100% graft and animal survival. Conclusions: Treatment of donor and recipient mice with a moderate dose of LMWH prevents vascular complications and improves the outcome of murine hind limb transplants.

Minimal Invasive Versus Open Surgery for Colorectal Liver Metastases: A Multicenter German StuDoQ|Liver Registry-Based Cohort Analysis in Germany.

Objective: To compare the outcome of minimally invasive liver surgery (MILS) to open liver surgery (OLS) for resection of colorectal liver metastases (CRLM) on a nationwide level. Background: Colorectal cancer is the third most common malignancy worldwide. Up to 50% of all patients with colorectal cancer develop CRLM. MILS represents an attractive alternative to OLS for treatment of CRLM. Methods: Retrospective cohort study using the prospectively recorded German Quality management registry for liver surgery. Propensity-score matching was performed to account for variance in the extent of resection and patient demographics. Results: In total, 1037 patients underwent liver resection for CRLM from 2019 to 2021. MILS was performed in 31%. Operative time was significantly longer in MILS (234 vs 222 minutes, P = 0.02) compared with OLS. After MILS, median length of hospital stay (LOS) was significantly shorter (7 vs 10 days; P < 0.001). Despite 76% of major resections being OLS, postoperative complications and 90-day morbidity and mortality did not differ. The Pringle maneuver was more frequently used in MILS (48% vs 40%, P = 0.048). After propensity-score matching for age, body mass index, Eastern Cooperative Oncology Group, and extent of resection, LOS remained shorter in the MILS cohort (6 vs 10 days, P < 0.001) and operative time did not differ significantly (P = 0.2). Conclusion: MILS is not the standard for resection of CRLM in Germany. Drawbacks, such as a longer operative time remain. However, if technically possible, MILS is a reasonable alternative to OLS for resection of CRLM, with comparable postoperative complications, reduced LOS, and equal oncological radicality.

Correction: Felsenstein M. et al. Perineural Invasion in Pancreatic Ductal Adenocarcinoma (PDAC): A Saboteur of Curative Intended Therapies? J. Clin. Med. 2022, 11, 2367.

In the original publication [...].

Papain-Based Solubilization of Decellularized Extracellular Matrix for the Preparation of Bioactive, Thermosensitive Pregels.

Solubilized, gel-forming decellularized extracellular matrix (dECM) is used in a wide range of basic and translational research and due to its inherent bioactivity can promote structural and functional tissue remodeling. The animal-derived protease pepsin has become the standard proteolytic enzyme for the solubilization of almost all types of collagen-based dECM. In this study, pepsin was compared with papain, α-amylase, and collagenase for their potential to solubilize porcine liver dECM. Maximum preservation of bioactive components and native dECM properties was used as a decisive criterion for further application of the enzymes, with emphasis on minimal destruction of the protein structure and maintained capacity for physical thermogelation at neutral pH. The solubilized dECM digests, and/or their physically gelled hydrogels were characterized for their rheological properties, gelation kinetics, GAG content, proteomic composition, and growth factor profile. This study highlights papain as a plant-derived enzyme that can serve as a cost-effective alternative to animal-derived pepsin for the efficient solubilization of dECM. The resulting homogeneous papain-digested dECM preserved its thermally triggered gelation properties similar to pepsin digests, and the corresponding dECM hydrogels demonstrated their enhanced bioadhesiveness in single-cell force spectroscopy experiments with fibroblasts. The viability and proliferation of human HepaRG cells on dECM gels were similar to those on pure rat tail collagen type I gels. Papain is not only highly effective and economically attractive for dECM solubilization but also particularly interesting when digesting human-tissue-derived dECM for regenerative applications, where animal-derived materials are to be avoided.

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.

Body composition is associated with disease aetiology and prognosis in patients undergoing resection of intrahepatic cholangiocarcinoma.

BACKGROUND: Body composition alterations are frequent in patients with cancer or chronic liver disease, but their prognostic value remains unclear in many cancer entities. OBJECTIVE: We investigated the impact of disease aetiology and body composition after surgery for intrahepatic cholangiocarcinoma (iCCA), a rare and understudied cancer entity in European and North American cohorts. METHODS: Computer tomography-based assessment of body composition at the level of the third lumbar vertebra was performed in 173 patients undergoing curative-intent liver resection for iCCA at the Department of Surgery, Charité - Universitätsmedizin Berlin. Muscle mass and -composition as well as subcutaneous and visceral adipose tissue quantity were determined semi-automatically. (Secondary) sarcopenia, sarcopenic obesity, myosteatosis, visceral and subcutaneous obesity were correlated to clinicopathological data. RESULTS: Sarcopenia was associated with post-operative morbidity (intraoperative transfusions [p = 0.027], Clavien-Dindo ≥ IIIb complications [p = 0.030], post-operative comprehensive complication index, CCI [p < 0.001]). Inferior overall survival was noted in patients with myosteatosis (33 vs. 23 months, p = 0.020). Fifty-eight patients (34%) had metabolic (dysfunction)-associated fatty liver disease (MAFLD) and had a significantly higher incidence of sarcopenic (p = 0.006), visceral (p < 0.001) and subcutaneous obesity (p < 0.001). Patients with MAFLD had longer time-to-recurrence (median: 38 vs. 12 months, p = 0.025, log-rank test). Multivariable cox regression analysis confirmed only clinical, and not body, composition parameters (age > 65, fresh frozen plasma transfusions) as independently prognostic for overall survival. CONCLUSION: This study evidenced a high prevalence of MAFLD in iCCA, suggesting its potential contribution to disease aetiology. Alterations of muscle mass and adipose tissue were more frequent in patients with MAFLD.

Development and systematic evaluation of decellularization protocols in different application models for diaphragmatic tissue engineering.

BACKGROUND: Tissue engineered bioscaffolds based on decellularized composites have gained increasing interest for treatment of various diaphragmatic impairments, including muscular atrophies and diaphragmatic hernias. Detergent-enzymatic treatment (DET) constitutes a standard strategy for diaphragmatic decellularization. However, there is scarce data on comparing DET protocols with different substances in distinct application models in their ability to maximize cellular removal while minimizing extracellular matrix (ECM) damage. METHODS: We decellularized diaphragms of male Sprague Dawley rats with 1 % or 0.1 % sodium dodecyl sulfate (SDS) and 4 % sodium deoxycholate (SDC) by orbital shaking (OS) or retrograde perfusion (RP) through the vena cava. We evaluated decellularized diaphragmatic samples by (1) quantitative analysis including DNA quantification and biomechanical testing, (2) qualitative and semiquantitative analysis by proteomics, as well as (3) qualitative assessment with macroscopic and microscopic evaluation by histological staining, immunohistochemistry and scanning electron microscopy. RESULTS: All protocols produced decellularized matrices with micro- and ultramorphologically intact architecture and adequate biomechanical performance with gradual differences. The proteomic profile of decellularized matrices contained a broad range of primal core and ECM-associated proteins similar to native muscle. While no outstanding preference for one singular protocol was determinable, SDS-treated samples showed slightly beneficial properties in comparison to SDC-processed counterparts. Both application modalities proved suitable for DET. CONCLUSION: DET with SDS or SDC via orbital shaking or retrograde perfusion constitute suitable methods to produce adequately decellularized matrices with characteristically preserved proteomic composition. Exposing compositional and functional specifics of variously treated grafts may enable establishing an ideal processing strategy to sustain valuable tissue characteristics and optimize consecutive recellularization. This aims to design an optimal bioscaffold for future transplantation in quantitative and qualitative diaphragmatic defects.