Prevascularized Tissue Research Articles

Hierarchically vascularized and suturable tissue constructs created through angiogenesis from tissue-engineered vascular grafts

A major roadblock in implementing engineered tissues clinically lies in their limited vascularization. After implantation, such tissues do not integrate with the hostʼs circulation as quickly as needed, commonly resulting in loss of viability and functionality. This study presents a solution to the vascularization problem that could enable the survival and function of large, transplantable, and vascularized engineered tissues. The technique allows vascularization of a cell laden hydrogel through angiogenesis from a suturable tissue-engineered vascular graft (TEVG) constructed from electrospun polycaprolactone with macropores. The graft is surrounded by a layer of cell-laden gelatin-methacryloyl hydrogel. The constructs are suturable and possess mechanical properties like native vessels. Angiogenesis occurs through the pores in the graft, resulting in a hydrogel containing an extensive vascular network that is connected to an implantable TEVG. The size of the engineered tissue and the degree of vascularization can be increased by adding multiple TEVGs into a single construct. The engineered tissue has the potential to be immediately perfused by the patient's blood upon surgical anastomosis to host vessels, enabling survival of implanted cells. These findings provide a meaningful step to address the longstanding problem of fabricating suturable pre-vascularized tissues which could survive upon implantation in vivo. Statement of significanceCreating vascularized engineered tissues that can be transplanted and rapidly perfused by the host blood supply is a major challenge which has limited the clinical impact of tissue engineering. In this study we demonstrate a technique to fabricate vascularized tissue constructs via angiogenesis from a suturable tissue-engineered vascular graft. The macroporous graft is surrounded with hydrogel, allowing endothelial cells to migrate from the lumen and vascularize the hydrogel layer with capillary-like structures connected to the macrovessel. The graft has comparable mechanical properties to native blood vessels and larger constructs can be fabricated by incorporating multiple grafts. These constructs could potentially be connected surgically to the circulation at an implantation site to support their immediate perfusion and survival.

Engineering pre-vascularized 3D tissue and rapid vascular integration with host blood vessels via co-cultured spheroids-laden hydrogel

Recent advances in regenerative medicine and tissue engineering have enabled the biofabrication of three-dimensional (3D) tissue analogues with the potential for use in transplants and disease modeling. However, the practical use of these biomimetic tissues has been hindered by the challenge posed by reconstructing anatomical-scale micro-vasculature tissues. In this study, we suggest that co-cultured spheroids within hydrogels hold promise for regenerating highly vascularized and innervated tissues, both in vitro and in vivo. Human adipose-derived stem cells (hADSCs) and human umbilical vein cells (HUVECs) were prepared as spheroids, which were encapsulated in gelatin methacryloyl hydrogels to fabricate a 3D pre-vascularized tissue. The vasculogenic responses, extracellular matrix production, and remodeling depending on parameters like co-culture ratio, hydrogel strength, and pre-vascularization time for in vivo integration with native vessels were then delicately characterized. The co-cultured spheroids with 3:1 ratio (hADSCs/HUVECs) within the hydrogel and with a pliable storage modulus showed the greatest vasculogenic potential, and ultimately formed in vitro arteriole-scale vasculature with a longitudinal lumen structure and a complex vascular network after long-term culturing. Importantly, the pre-vascularized tissue also showed anastomotic vascular integration with host blood vessels after transplantation, and successful vascularization that was positive for both CD31 and alpha-smooth muscle actin covering 18.6 ± 3.6 μm2 of the luminal area. The described co-cultured spheroids-laden hydrogel can therefore serve as effective platform for engineering 3D vascularized complex tissues.

Perfusability and immunogenicity of implantable pre-vascularized tissues recapitulating features of native capillary network.

Vascularization is a key pre-requisite to engineered anatomical scale three dimensional (3-D) constructs to ensure their nutrient and oxygen supply upon implantation. Presently, engineered pre-vascularized 3-D tissues are limited to only micro-scale hydrogels, which meet neither the anatomical scale needs nor the complexity of natural extracellular matrix (ECM) environments. Anatomical scale perfusable constructs are critically needed for translational applications. To overcome this challenge, we previously developed pre-vascularized ECM sheets with long and oriented dense microvascular networks. The present study further evaluated the patency, perfusability and innate immune response toward these pre-vascularized constructs. Macrophage-co-cultured pre-vascularized constructs were evaluated in vitro to confirm micro-vessel patency and perturbations in macrophage metabolism. Subcutaneously implanted pre-vascularized constructs remained viable and formed a functional anastomosis with host vasculature within 3 days of implantation. This completely biological pre-vascularized construct holds great potential as a building block to engineer perfusable anatomical scale tissues.

Directly coaxial bioprinting of 3D vascularized tissue using novel bioink based on decellularized human amniotic membrane

Despite several progressions in the biofabrication of large-scale engineered tissues, direct biopri nting of perfusable three-dimensional (3D) vasculature remained unaddressed. Developing a feasible method to generate cell-laden thick tissue with an effective vasculature network to deliver oxygen and nutrient is crucial for preventing the formation of necrotic spots and tissue death. In this study, we developed a novel technique to directly bioprint 3D cell-laden prevascularized construct. We developed a novel bioink by mixing decellularized human amniotic membrane (dHAM) and alginate (Alg) in various ratios. The bioink with encapsulated human vein endothelial cells (HUVECs) and a crosslinker, CaCl2, were extruded via sheath and core nozzle respectively to directly bioprint a perfusable 3D vasculature construct. The various concentration of bioink was assessed from several aspects like biocompatibility, porosity, swelling, degradation, and mechanical characteristics, and accordingly, optimized concentration was selected (Alg 4 %w/v – dHAM 0.6 %w/v). Then, the crosslinked bioink without microchannel and the 3D bioprinted construct with various microchannel distances (0, 1.5 mm, 3 mm) were compared. The 3D bioprinted construct with a 1.5 mm microchannels distance demonstrated superiority owing to its 492 ± 18.8 % cell viability within 14 days, excellent tubulogenesis, remarkable expression of VEGFR-2 which play a crucial role in endothelial cell proliferation, migration, and more importantly angiogenesis, and neovascularization. This perfusable bioprinted construct also possess appropriate mechanical stability (32.35 ± 5 kPa Young's modulus) for soft tissue. Taking these advantages into the account, our new bioprinting method possesses a prominent potential for the fabrication of large-scale prevascularized tissue to serve for regenerative medicine applications like implantation, drug-screening platform, and the study of mutation disease.

New Insights and Advanced Strategies for In Vitro Construction of Vascularized Tissue Engineering.

Inadequate vascularization is a significant barrier to clinical application of large-volume tissue engineered grafts. In contrast to in vivo vascularization, in vitro prevascularization shortens the time required for host vessels to grow into the graft core and minimizes necrosis in the core region of the graft. However, the challenge of prevascularization is to construct hierarchical perfusable vascular networks, increase graft volume, and form a vascular tip that can anastomose with host vessels. Understanding advances in in vitro prevascularization techniques and new insights into angiogenesis could overcome these obstacles. In the present review, we discuss new perspectives on angiogenesis, the differences between in vivo and in vitro tissue vascularization, the four elements of prevascularized constructs, recent advances in perfusion-based in vitro prevascularized tissue fabrication, and prospects for large-volume prevascularized tissue engineering.

Reconstructing vascular networks promotes the repair of skeletal muscle following volumetric muscle loss by pre-vascularized tissue constructs

Current treatment for complex and large-scale volumetric muscle loss (VML) injuries remains a limited success and have substantial disadvantages, due to the irreversible loss of muscle mass, slow muscle regeneration, and rapid formation of non-functional fibrosis scars. These VML injuries are accompanied by denervation and the destruction of native vasculature which increases difficulties in the functional restoration of muscle. Here, reconstruction of the vascular network at the injury site was offered as a possible solution for improving the repair of muscle defects through the timely supply of nutrients and oxygen to surrounding cells. A hydrogel-based tissue construct containing various densities of the vascular network was successfully created in the subcutaneous space of mice by manipulating hydrogel properties, and then implanted into the VML injury site. One month after implantation, the mouse treated with the highly vascularized tissue had extensive muscle repair at the injury site and only spent a shorter time completing the inclined plane tests. These findings suggest that the reconstruction of the functional vascular network at the VML injury site accelerated muscle fiber repair through a timely supply of sufficient blood and avoided invasion by host fibroblasts.

Bioactivated gellan gum hydrogels affect cellular rearrangement and cell response in vascular co-culture and subcutaneous implant models

Hydrogels are suitable soft tissue mimics and capable of creating pre-vascularized tissues, that are useful for in vitro tissue engineering and in vivo regenerative medicine. The polysaccharide gellan gum (GG) offers an intriguing matrix material but requires bioactivation in order to support cell attachment and transfer of biomechanical cues. Here, four versatile modifications were investigated: Purified NaGG; avidin-modified NaGG combined with biotinylated fibronectin (NaGG-avd); oxidized GG (GGox) covalently modified with carbohydrazide-modified gelatin (gelaCDH) or adipic hydrazide-modified gelatin (gelaADH). All materials were subjected to rheological analysis to assess their viscoelastic properties, using a time sweep for gelation analysis, and subsequent amplitude sweep of the formed hydrogels. The sweeps show that NaGG and NaGG-avd are rather brittle, while gelatin-based hydrogels are more elastic. The degradation of preformed hydrogels in cell culture medium was analyzed with an amplitude sweep and show that gelatin-containing hydrogels degrade more dramatically. A co-culture of GFP-tagged HUVEC and hASC was performed to induce vascular network formation in 3D for up to 14 days. Immunofluorescence staining of the αSMA+ network showed increased cell response to gelatin-GG networks, while the NaGG-based hydrogels did not allow for the elongation of cells. Preformed, 3D hydrogels disks were implanted to subcutaneous rat skin pockets to evaluate biological in vivo response. As visible from the hematoxylin and eosin-stained tissue slices, all materials are biocompatible, however gelatin-GG hydrogels produced a stronger host response. This work indicates, that besides the biochemical cues added to the GG hydrogels, also their viscoelasticity greatly influences the biological response.

Establishment and biological effect evaluation of prevascularized porous β-tricalcium phosphate tissue engineered bone

To evaluate the biological effect on vascularization during bone repair of prevascularized porous β-tricalcium phosphate (β-TCP) tissue engineered bone (hereinafter referred to as prevascularized tissue engineered bone), which was established by co-culture of endothelial progenitor cells (EPCs) and bone marrow mesenchymal stem cells (BMSCs) based on tissue engineering technology. EPCs and BMSCs were isolated from iliac bone marrow of New Zealand white rabbits by density gradient centrifugation and differential adhesion method. The cells were identified by immunophenotypic detection, BMSCs-induced differentiation, and EPCs phagocytosis. After identification, the third-generation cells were selected for subsequent experiments. First, in vitro tubule formation in EPCs/BMSCs direct contact co-culture (EPCs/BMSCs group) was detected by Matrigel tubule formation assay and single EPCs (EPCs group) as control. Then, the prevascularized tissue engineered bone were established by co-culture of EPCs/BMSCs in porous β-TCP scaffolds for 7 days (EPCs/BMSCs group), taking EPCs in porous β-TCP scaffolds as a control (EPCs group). Scanning electron microscopy and laser scanning confocal microscopy were used to observe the adhesion, proliferation, and tube formation of cells. Femoral condyle defect models of 12 New Zealand white rabbits were used for implantation of prevascularized tissue engineered bone as the experimental group ( n=6) and porous β-TCP scaffold as the control group ( n=6). The process of vascularization of β-TCP scaffolds were observed. The numbers, diameter, and area fraction of neovascularization were quantitatively evaluated by Microfill perfusion, Micro-CT scanning, and vascular imaging under fluorescence at 4 and 8 weeks. The isolated cells were BMSCs and EPCs through identification. EPCs/BMSCs co-culture gradually formed tubular structure. The number of tubules and branches, and the total length of tubules formed in the EPCs/BMSCs group were significantly more than those in the EPCs group on Matrigel ( P<0.05) after 6 hours. After implanting and culturing in porous β-TCP scaffold for 7 days, EPCs formed cell membrane structure and attached to the material in EPCs group, and the cells attached more tightly, cell layers were thicker, the number of cells and the formation of tubular structures were significantly more in the EPCs/BMSCs group than in the EPCs group. At 4 weeks after implantation, neovascularization was observed in both groups. At 8 weeks, remodeling of neovascularization occurred in both groups. The number, diameter, and area fraction of neovascularization in the experimental group were higher than those in the control group ( P<0.05), except for area fraction at 4 weeks after implantation ( P>0.05). The prevascularized tissue engineered bone based on direct contact co-culture of BMSCs and EPCs can significantly promote the early vascularization process during bone defects repair.

A Biofabrication Strategy for a Custom-Shaped, Non-Synthetic Bone Graft Precursor with a Prevascularized Tissue Shell.

Critical-sized defects of irregular bones requiring bone grafting, such as in craniofacial reconstruction, are particularly challenging to repair. With bone-grafting procedures growing in number annually, there is a reciprocal growing interest in bone graft substitutes to meet the demand. Autogenous osteo(myo)cutaneous grafts harvested from a secondary surgical site are the gold standard for reconstruction but are associated with donor-site morbidity and are in limited supply. We developed a bone graft strategy for irregular bone-involved reconstruction that is customizable to defect geometry and patient anatomy, is free of synthetic materials, is cellularized, and has an outer pre-vascularized tissue layer to enhance engraftment and promote osteogenesis. The graft, comprised of bioprinted human-derived demineralized bone matrix blended with native matrix proteins containing human mesenchymal stromal cells and encased in a simple tissue shell containing isolated, human adipose microvessels, ossifies when implanted in rats. Ossification follows robust vascularization within and around the graft, including the formation of a vascular leash, and develops mechanical strength. These results demonstrate an early feasibility animal study of a biofabrication strategy to manufacture a 3D printed patient-matched, osteoconductive, tissue-banked, bone graft without synthetic materials for use in craniofacial reconstruction. The bone fabrication workflow is designed to be performed within the hospital near the Point of Care.

Microvascular Fragments in Microcirculation Research and Regenerative Medicine.

Adipose tissue-derived microvascular fragments (MVF) are functional vessel segments, which rapidly reassemble into new microvasculatures under experimental in vitro and in vivo conditions. Accordingly, they have been used for many years in microcirculation research to study basic mechanisms of endothelial cell function, angiogenesis, and microvascular network formation in two- and three-dimensional environments. Moreover, they serve as vascularization units for musculoskeletal regeneration and implanted biomaterials as well as for the treatment of myocardial infarction and the generation of prevascularized tissue organoids. Besides, multiple factors determining the vascularization capacity of MVF have been identified, including their tissue origin and cellular composition, the conditions for their short- and long-term storage, as well as their implantation site and the general health status and medication of the recipient. The next challenging step is now the successful translation of all these promising experimental findings into clinical practice. If this succeeds, a multitude of future therapeutic applications may significantly benefit from the remarkable properties of MVF. Impact Statement The present review provides a complete overview of the broad application spectrum of adipose tissue-derived microvascular fragments (MVF) in angiogenesis research and regenerative medicine. Moreover, it systematically describes factors determining their vascularization capacity. These factors may be used to further improve the remarkable properties of MVF in personalized medicine and to promote their rapid introduction as vascularization units into clinical practice.

Regulation of vascular branch formation in 3D bioprinted tissues using confining force

The quality of the vascular network is key to the success of skin transplants. For skin grafts prepared using a tissue engineering approach, vascularization is a critical step determining tissue survival. Prevascularization of bioengineered tissues is the basis for the effective establishment of high-quality vascular networks. Previously, we established a 3D bioprinted model to simulate the mechanical stimulation of vascular tissue development. Based on this, here, we create a prevascularized tissue using 3D bioprinting and show that the vascular branches of the tissue can be controlled by the confining forces created by changing the size of the polycaprolactone (PCL) framework. In addition, it was found that the Yes-associated protein (YAP) participates in the regulation of vascular branch formation in the tissue. A close relationship was found between the width of the cell-fibrin strip, the magnitude of the force and the number of vascular branches in the bioprinted tissue exists. Together, our findings indicate that vascularization of engineered skin tissue is a complex yet controllable process, and precise mechanical control can lead to effective vascular network generation.

Cell-assembled extracellular matrix (CAM): a human biopaper for the biofabrication of pre-vascularized tissues able to connect to the host circulation in vivo

When considering regenerative approaches, the efficient creation of a functional vasculature, that can support the metabolic needs of bioengineered tissues, is essential for their survival after implantation. However, it is widely recognized that the post-implantation microenvironment of the engineered tissues is often hypoxic due to insufficient vascularization, resulting in ischemia injury and necrosis. This is one of the main limitations of current tissue engineering applications aiming at replacing significant tissue volumes. Here, we have explored the use of a new biomaterial, the cell-assembled extracellular matrix (CAM), as a biopaper to biofabricate a vascular system. CAM sheets are a unique, fully biological and fully human material that has already shown stable long-term implantation in humans. We demonstrated, for the first time, the use of this unprocessed human ECM as a microperforated biopaper. Using microvalve dispensing bioprinting, concentrated human endothelial cells (30 millions ml−1) were deposited in a controlled geometry in CAM sheets and cocultured with HSFs. Following multilayer assembly, thick ECM-based constructs fused and supported the survival and maturation of capillary-like structures for up to 26 d of culture. Following 3 weeks of subcutaneous implantation in a mice model, constructs showed limited degradative response and the pre-formed vasculature successfully connected with the host circulatory system to establish active perfusion.This mechanically resilient tissue equivalent has great potential for the creation of more complex implantable tissues, where rapid anastomosis is sine qua non for cell survival and efficient tissue integration.

Engineering Tissue‐Specific, Multiscale Microvasculature with a Capillary Network for Prevascularized Tissue (Small Methods 10/2021)

Back Cover In article number 2100632, Kang and co-workers provide a novel printing technology for the fabrication of multiscale microvasculature to mimic the hierarchical architecture of the human microvascular system. The millimeter-scale vessels were produced with direct printing of sacrificial bio-ink and capillary-scale vessels were generated within the user-designed bridge pattern and induced by the angiogenic factors from the fibroblast pattern.

Periosteal Flaps Enhance Prefabricated Engineered Bone Reparative Potential

The clinical translation of bone tissue engineering for reconstructing large bone defects has not advanced without hurdles. The in vivo bioreactor (IVB) concept may therefore bridge between bone tissue engineering and reconstructive surgery by employing the patient body for prefabricating new prevascularized tissues. Ideally, IVB should minimize the need for exogenous growth factors/cells. Periosteal tissues are promising for IVB approaches to prefabricate tissue-engineered bone (TEB) flaps. However, the significance of preserving the periosteal vascular supply has not been adequately investigated. This study assessed muscle IVB with and without periosteal/pericranial grafts and flaps for prefabricating TEB flaps to reconstruct mandibular defects in sheep. The sheep (n = 14) were allocated into 4 groups: muscle IVB (M group; nM = 3), muscle + periosteal graft (MP group; nMP = 4), muscle + periosteal flap (MVP group; nMVP = 4), and control group (nControl = 3). In the first surgery, alloplastic bone blocks were implanted in the brachiocephalic muscle (M) with a periosteal graft (MP) or with a vascularized periosteal flap (MVP). After 9 wk, the prefabricated TEB flaps were transplanted to reconstruct a mandibular angle defect. In the control group, the defects were reconstructed by non-prevascularized bone blocks. Computed tomography (CT) scans were performed after 13 wk and after 23 wk at termination, followed by micro-CT (µCT) and histological analyses. Both CT and µCT analysis revealed enhanced new bone formation and decreased residual biomaterial volume in the MVP group compared with control and MP groups, while the M group showed less new bone formation and more residual biomaterial. The histological analysis showed that most of the newly formed bone emerged from defect edges, but larger areas of new bone islands were found in MP and MVP groups. The MVP group showed enhanced vascularization and higher biomaterial remodeling rates. The periosteal flaps boosted the reconstructive potential of the prefabricated TEB flaps. The regenerative potential of the periosteum was manifested after the transplantation into the mechanically stimulated bony defect microenvironment.

Engineering Tissue-Specific, Multiscale Microvasculature with a Capillary Network for Prevascularized Tissue.

Although there are various pre-existing technologies for engineering vasculatures, multiscale modeling of the architecture of human vasculature at a capillary scale remains a challenge. In this study, a novel technology is developed for the production of a functional, multiscale microvasculature comprising of endothelialized channels and tissue-specific capillary networks. Perfusable, endothelialized channels are bioprinted, after which angiogenic sprouts are grown into user-designed capillary networks. The induction of branched and liver-lobule-like capillary networks confirm that the technology can produce various types of tissue-specific multiscale microvasculatures. Further, the channels and capillaries are deemed to be functional when evaluated in vitro. An ex vivo assay demonstrates that the microvasculature can induce neovessel ingrowth, integrate with host vessels, and facilitate blood flow. Remarkably, blood flows through the implanted capillary network without any change in its morphology. Finally, the technology is applied to produce a vascularized liver tissue; it significantly improves its hepatic function. It is believed that this new technology will create new possibilities in the development of highly vascularized and functional tissues/organs on a clinically relevant scale.

Exogenous FGF-2 prolongs endothelial connection in multilayered human skeletal muscle cell sheet

Angiogenesis is a pressing issue in tissue engineering associated with restoration of blood supply to ischemic tissues and promotion of rapid vascularization of tissue-engineered grafts. Fibroblast growth factor-2 (FGF-2) plays a vital role in processes such as angiogenesis and is an attractive candidate for tissue engineering. While skeletal muscle tissue engineering is established, the role of FGF-2 in endothelial function to promote angiogenesis after transplantation is unclear. Here, a culture system comprising a five-layered sheet of human skeletal muscle cells co-incubated on green fluorescent protein-expressing human umbilical vein endothelial cells (GFP-HUVECs) mimicking invivo angiogenesis was used to investigate the role of FGF-2 in vascularization of engineered tissues. The basal level of FGF-2 in cultured media of skeletal muscle cell sheets was undetectable. Therefore, cell sheets co-incubated with GFP-HUVECs were exogenously treated with 10ng/mL FGF-2, and endothelial network formation was evaluated. After prolonged culture, the endothelial network length and connectivity increased following treatment with FGF-2 as compared with control treatment. The numbers of medium and long endothelial networks significantly increased inside the sheet longer than 0.2 and 0.4cm, respectively, after FGF-2 treatment. Time-lapse microscopy monitoring dynamic endothelial behavior revealed that FGF-2-mediated maintenance of endothelial connection and retardation of endothelial network disconnection after 72h. The present study suggests the precise role of FGF-2 in maintaining endothelial connection and the extent of the endothelial network in skeletal muscle cell sheets. This understanding can be applied to design invitro pre-vascularized tissue and graft integration prospects.

Endothelialised Tissue Engineered Substitutes Produced by the Self-Assembly Approach for Implantation and Research: Past, Present and Future

Recent innovations in medicine include tissue engineering to produce tissues or organs for replacement/repair surgeries but also to serve as relevant tridimensional research models. For both applications, presence of microvascular networks inside the reconstructed tissues is necessary to obtain structures that are both complex and more complete. Indeed, rapid inosculation of graft to host is essential for positive clinical outcomes but pre-vascularized tissues are also a need to obtain thick tissue where diffusion of nutrients and oxygen cannot be only passive. In the last two decades, the self-assembly approach was developed at LOEX and has allowed breakthroughs in many organ/tissue reconstructions. This unique technique relies on the production of a stroma scaffold by the mesenchymal cells themselves without the need of exogenous materials. Endothelialisation of such tissues shows a great impact not only on graft reperfusion but also in the improvement of research models such as cancer and psoriatic models. The presence of a vascular or lymphatic network now opens the door to the development of complex and configurable models which may be available soon.

Injectable pre-cultured tissue modules catalyze the formation of extensive functional microvasculature in vivo

Revascularization of ischemic tissues is a major barrier to restoring tissue function in many pathologies. Delivery of pro-angiogenic factors has shown some benefit, but it is difficult to recapitulate the complex set of factors required to form stable vasculature. Cell-based therapies and pre-vascularized tissues have shown promise, but the former require time for vascular assembly in situ while the latter require invasive surgery to implant vascularized scaffolds. Here, we developed cell-laden fibrin microbeads that can be pre-cultured to form primitive vascular networks within the modular structures. These microbeads can be delivered in a minimally invasive manner and form functional microvasculature in vivo. Microbeads containing endothelial cells and stromal fibroblasts were pre-cultured for 3 days in vitro and then injected within a fibrin matrix into subcutaneous pockets on the dorsal flanks of SCID mice. Vessels deployed from these pre-cultured microbeads formed functional connections to host vasculature within 3 days and exhibited extensive, mature vessel coverage after 7 days in vivo. Cellular microbeads showed vascularization potential comparable to bulk cellular hydrogels in this pilot study. Furthermore, our findings highlight some potentially advantageous characteristics of pre-cultured microbeads, such as volume preservation and vascular network distribution, which may be beneficial for treating ischemic diseases.

A NOVEL PRE-VASCULARIZED TISSUE ENGINEERING CHAMBER SERVES AS A BIO-ARTIFICIAL PANCREAS FOR AN ATTRACTIVE SITE OF ALLOGENIC AND XENOGENEIC ISLET TRANSPLANTATION

Background: Although the sites for clinical or experimental islet transplantation have been well established, the pancreatic islet survival and function in homing locations still remain unsatisfied[1]. One of possible reasons that account for this is local hypoxia due to the shortage of blood supply[2]. Here we modified a pre-vascularized tissue engineering chamber (TEC) in vivo which facilitated the vitality and function of seeded islets by providing a microvascular network prior to transplantation. Method: A TEC filled with MatrigelTM and bFGF-2 was created and embedded in the groin of streptozotocin-induced diabetic mice. The micro-vascularization in the TEC was analyzed by histology, real-time PCR and western blotting. Three hundreds of syngeneic islets were seeded into the chamber on day 0, 14, and 28 post-chamber implantation to research the degree of pre-vascularized in the TEC. In addition, different numbers of syngenic islets (300, 200, and 100) were seeded into the chamber on day 28 post implantation to study the TEC function influenced by islets quantities. Furthermore, we explore the feasibility of TEC as a transplantation site for islet allograft and xenograft which was treated by anti-CD45RB and/or anti-CD40L (MR-1). Results and Discussion: A highly developed micro-vascularized network was established in the TEC on day 28 post implantation. The normalization of blood glucose levels of diabetic mice was negatively correlated with the time required for pre-vascularization and the number of seeded syngenic islets. The combined treatment of anti-CD45RB and MR-1 guaranteed the grafts long-term survival after allotransplantation (5/5, 100%) and xenotransplantation (16/20, 80%). Flow cytometry showed the frequency of CD4+Foxp3 Tregs and CD4+IL-4+Th2 cells significantly increased in tolegenic xenograft transplantation, compared with the decrease of CD4+IFN-γTh1 cells. Conclusion: These findings highlight that the highly developed micro-vascularized construct facilitates islet transplantation in the TEC, implicating the possibility of application as an artificial pancreas in the future.Fig. 1. (A) HE images (1st row; scale bars: 10 μm) and histology analysis (2nd and 3rd rows; scale bars: 40 μm) of pre-vascularization TECs on day 7, 14, 28, and 35. Blood vessels were marked by black arrows in HE (1st row; scale bars: 10 μm). The micro-vascularize were stained by anti-VEGF and anti-CD31, and marked by brown and black arrows seperately (2nd and 3rd rows; scale bars: 40 μm). (B)The level of VEGF mRNA was quantified. The extent of pre-vascularization confirmed a significant increase in the 28d group compared with VEGF levels in the 7d and 14d groups (p**<0.001, p***<0.001), and no significant difference between the 28d group and the 35day group (P > 0.05). Sham of inguinal adipose tissue set as control. Data was shown as the mean ± SD and representative of three separate experiments. (C) Western blot analysis showed the protein levels of VEGF at different time points in TEC.Fig. 2. The time required for the blood glucose returning to normal level (11.1mol/dl) in diabetic mice was negatively correlated with the pre-vascularization time (A) and the seeding quantity of syngeneic islets (B). (C) Non-fasting blood glucose maintained normoglycemia after syngeneic islets transplanted in the TEC of diabetic mice, and became hyperglycaemia when the TEC removed (black arrow). (D) Insulin stained donor islet in recipient TEC at 90d posttransplant (shown brown), the islet was pointed by the red arrow (Scale bars:40 μm).Txpl: Transplantation post-implantation.Fig. 3. (A) Islet allografts (Balb/c to C57BL/6) were rejected in the untreated group within 22±2.2 d. Islet allografts treated with anti-CD45RB or anti-CD40L alone were also rejected in recipients within 24±3.12 d, 26±1.74 d. Islet allografts with the dual antibody treatment of anti-CD45RB and anti-CD40L presented long-term graft survival in all recipients. The Kaplan–Meier curves were compared with the log-rank test. (B) Non-fasting blood glucose maintained normoglycemia (5/5, 80%) in the diabetic mice after allogeneic islets transplanted, and became hyperglycaemia when the TEC removed (black arrow) (C) Islet xenograft (SD rat to C57BL/6) rejected in the untreated group, treated-CD45RB group and treated-CD40L group within 13±3.1d, 16±3.12d, 18±1.74d, respectively. Islet xenografts with the dual antibody treatment of anti-CD45RB and anti-CD40L resulted in long-term graft survival in all recipients. The Kaplan–Meier curves were compared with the log-rank test. (D)Non-fasting blood glucose maintained normoglycemia (16/20, 80%) after xenogeneic islets transplanted in the diabetic mice, and showed hyperglycaemia when the TEC removed (black arrow).(E) Another 300 xenogeneic islets were re-transplanted into diabetic mice again, which were failures for the acceptance of the xenograft. Three of four diabetic mice showing that hyperglycemia was ameliorated until the TEC was removed (black arrow). (F)Insulin stained donor allograft and xenograft in recipients TEC at 90d of posttransplant (shown brown), the islets were pointed by the red arrow (Scale bars:40 μm). National Natural Science Foundation of China (nos. 81172832 and 81771723). Special Program for Sichuan Youth Science and Technology Innovation (no. 2014TD0010). Sichuan Youth Science and Technology Foundation (no. 2013JQ0020). The Health and Family Planning Commission of Sichuan Province (no. 110190).

Research Highlights

Hepatic CEACAM1 Expression Indicates Donor Liver Quality and Prevents Early Transplantation Injury Nakamura K, Kageyama S, Kaldas FM, et al. J Clin Invest. 2020;130:2689–2704. Orthotopic liver transplantation (OLT) is accepted as the gold-standard treatment for patients with liver failure, yet it remains limited by significant donor shortage. A particular challenge is the quality of livers available for transplantation, with a large proportion of deceased donor livers not deemed to be appropriate for transplantation. It is recognized that ischemia-reperfusion injury (IRI) at the time of transplantation contributes to the development of early allograft dysfunction; however, the mechanisms underlying this process are incompletely understood.6 Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), also known as CD66a or biliary glycoprotein-1, is a transmembrane protein expressed in a number of immune and epithelial cells in which it acts as an adhesion molecule but also has a role in hepatic metabolic homeostasis. To investigate the role of CEACAM1 in IRI, Nakamura et al use a mouse model of OLT in which grafts from either wild-type or CC1−/− mice (which possess a global null mutation in CEACAM1) are subjected to extended cold storage (18 h) and subsequently transplanted into wild-type recipients.7 Mice in the CC1−/− group displayed increased evidence of IRI and higher levels of HMGB, a danger-associated molecular pattern, in addition to an enhanced infiltration with macrophages and neutrophils. The CC1−/− group also exhibited signs of injury resulting from cold storage, mediated by the ASK1/p-p38 pathway. Furthermore, preincubation of CC1−/− livers in cold storage with an ASK1 inhibitor improved outcomes. Having established a regulatory role for CEACAM1, the authors next validated the clinical relevance in human OLT. Liver graft biopsies were obtained from 60 patients at the time of liver transplantation. These were screened for CEACAM1 as well as ASK1 and p-p38, identifying a negative correlation between CEACAM1 levels and ASK1. To test the hypothesis that pretransplantation CEACAM1 levels impact clinical outcomes, samples were clustered into high or low CEACAM1 groups based on a median of CEACAM1 levels. Samples with low levels of CEACAM1 correlated with increased liver enzymes postoperatively as well as an augmented frequency of early allograft dysfunction and poorer long-term graft function, although the latter finding did not reach statistical significance. CEACAM1 may therefore represent a useful biomarker of liver quality, as well as a point for intervention, particularly through targeting the ASK1/p-p38 pathway. Transforming the Spleen Into a Liver-like Organ In Vivo Wang L, Wang C, Wang Z, et al. Sci Adv. 2020;6:eaaz9974. Regenerative medicine aims to solve several key challenges in organ transplantation—from the shortage of donor organs to the need for optimized immunosuppression. Significant progress has been made in recent years, but the field is still limited by the inability to generate the necessary vasculature to sustain the engineered tissues1. In the study from Wang et al, this limitation is addressed through a novel concept of transforming an existing organ, the spleen, to perform the function of the liver. The spleen is an ideal subject for this as it is large, has an excellent blood supply, and its absence can be tolerated well. The authors use a mouse model to transform the spleen by remodeling the tissue and subsequently transplanting liver cells into the organ. To facilitate the necessary transformation, the spleen was initially translocated surgically to a subcutaneous location. Thereafter, the spleen was injected with soluble tumor homogenate (STH, from a sarcoma cell line) on days 7, 10, and 14 to suppress immune function and enhance the extracellular matrix. Mice injected with this homogenate demonstrated an increase in spleen size, extracellular matrix composition, and angiogenesis (probably mediated through enhanced vascular endothelial growth factor expression). Interestingly, the transformed spleens provided an immunosuppressive microenvironment, with reduced T and B cells as well as an increase in regulatory T cells. The second step involved the transfer of liver cells into remodeled spleens (“hepatization”). Using luciferase-labeling, the authors were able to detect cells posttransplantation and demonstrated successful graft take of both allografted and xenografted human liver cells, although regular injection of STH was required to achieve this. Cessation of STH injection resulted in grafts being lost 2 wk after the last injection. Transformed spleens were able to produce albumin and metabolize drugs. Furthermore, in mice wherein 90% of the liver was removed, or wherein fulminant liver failure was induced, transformed spleens were able to take over functionality, and the mice survived. The strategy presented offers the exciting possibility of organ repurposing thus allowing organs to be engineered from a baseline prevascularized tissue in situ, providing an advantage over more traditional decellularization-recellularization techniques.