Engineering vascularized organotypic tissues via module assembly

Adequate vascularization is a critical determinant for the successful construction and clinical implementation of complex organotypic tissue models. Currently, low cell and vessel density and insufficient vascular maturation make vascularized organotypic tissue construction difficult,greatly limiting its use in tissue engineering and regenerative medicine. To address these limitations, recent studies have adopted pre-vascularized microtissue assembly for the rapid generation of functional tissue analogs with dense vascular networks and high cell density. In this article, we summarize the development of module assembly-based vascularized organotypic tissue construction and its application in tissue repair and regeneration, organ-scale tissue biomanufacturing, as well as advanced tissue modeling.

In situ forming injectable MSC-loaded GelMA hydrogels combined with PD for vascularized sweat gland regeneration

Dear Editor,Three dimensional(3D)bioprinted extracellular matrix(ECM)can be used to provide both biochemical and biophysical cues to direct mesenchymal stem cells(MSCs)differentiation,and then differentiated cells were isolated for implantation in vivo using surgical procedures.However,the reduced cell activity after cell isolation from 3D constructs and low cell retention in injured sites limit its application[1].Methacrylated gelatin(GelMA)hydrogel has the advantage of fast crosslinking,which could resemble complex architectures of tissue construct in vivo[2].Here,we adopted a noninvasive bioprinting procedure to imitate the regenerative microenvironment that could simultaneously direct the sweat gland(SG)and vascular differentiation from MSCs and ultimately promote the replacement of glandular tissue in situ(Fig.1a).

Animal models of vascularized nerve grafts:a systematic review

The aim of this review is to present and compare the various animal models of vascularized nerve grafts described in the literature as well as to summarize preclinical evidence for superior functional results compared to non-vascularized free nerve grafts. We also will present the state of the art on prefabricated vascularized nerve grafts. A systematic literature review on vascularized nerve graft models was conducted via the retrieval with the Pub Med database on March 30, 2019. Data on the animal, nerve, and vascularization model, the recipient bed, the evaluation time points and methods, and the results of the study results were extracted and analyzed from selected articles. The rat sciatic nerve was the most popular model for vascularized nerve grafts, followed by the rabbit;however, rabbit models allow for longer nerve grafts, which are suitable for translational evaluation, and produced more cautious results on the superiority of vascularized nerve grafts. Compared to free nerve grafts, vascularized nerve grafts have better early but similar long-term results, especially in an avascular bed. There are few studies on avascular receiving beds and prefabricated nerve grafts. The clinical translation potential of available animal models is limited, and current experimental knowledge cannot fully support that the differences between vascularized nerve grafts and free nerve grafts yield a clinical advantage that justifies the complexity of the procedure.

Intelligent Vascularized 3D/4D/5D/6D‑Printed Tissue Scaffolds

Blood vessels are essential for nutrient and oxygen delivery and waste removal.Scaffold-repairing materials with functional vascular networks are widely used in bone tissue engineering.Additive manufacturing is a manufacturing technology that creates three-dimensional solids by stacking substances layer by layer,mainly including but not limited to 3D printing,but also 4D printing,5D printing and 6D printing.It can be effectively combined with vascularization to meet the needs of vascularized tissue scaffolds by precisely tuning the mechanical structure and biological properties of smart vascular scaffolds.Herein,the development of neovascularization to vascularization to bone tissue engineering is systematically discussed in terms of the importance of vascularization to the tissue.Additionally,the research progress and future prospects of vascularized 3D printed scaffold materials are highlighted and presented in four categories:functional vascularized 3D printed scaffolds,cell-based vascularized 3D printed scaffolds,vascularized 3D printed scaffolds loaded with specific carriers and bionic vascularized 3D printed scaffolds.Finally,a brief review of vascularized additive manufacturing-tissue scaffolds in related tissues such as the vascular tissue engineering,cardiovascular system,skeletal muscle,soft tissue and a discussion of the challenges and development efforts leading to significant advances in intelligent vascularized tissue regeneration is presented.

Key psychosocial challenges in vascularized composite allotransplantation

Psychosocial factors are important elements in the assessment and follow-up care for vascularized composite allotransplantation(VCA) and require multidisciplinary evaluation protocols. This review will highlight differences between VCA with solid organ transplantation(SOT), provide information on the psychosocial selection of VCA candidates, ethical issues, psychological outcomes, and on the need for multicenter research. VCA is primarily a life-enhancing procedure to improve recipients' quality of life and psychological well-being and it represents a potential option to provide reproduction in case of penile or uterine transplantation. The risk benefit ratio is distinctly different than SOT with candidates desiring life enhancing outcomes including improved body image, return to occupations, restored touch, and for uterine transplant, pregnancy. The Chauvet Workgroup has been convened with membership from a number of transplant centers to address these issues and to call for multicenter research. A multicenter research network would share similar evaluation approaches so that meaningful research on psychosocial variables could inform the transplant community and patients about factors that increase risk of non-adherence and other adverse psychosocial and medical outcomes.

Early debridement and delayed primary vascularized cover in forearm electrical burns: A prospective study

AIM To look into the management options of early debridement of the wound, followed by vascularized cover to bring in fresh blood supply to remaining tissue in electrical burns. METHODS A total of 16 consecutive patients sustaining full thickness forearm burns over a period of one year were included in the study group. Debridement was undertaken within 48 h in 13 patients. Three patients were taken for debridement after 48 h. Debridement was repeated within 2-4 d after daily wound assessment and need for further debridement. RESULTS On an average two debridements(range 1-4) was required in our patients for the wound to be ready for definitive cover. Interval between each debridement ranged from 2-18 d. Fourteen patients were provided vascularized cover after final debridement(6 free flaps, 8 pedicled flaps). Functional assessment of gross hand function done at 6 wk, 2 mo, 3 mo and 6 mo follow-up. CONCLUSION High-tension electrical burns lead to significant morbi-dity. These injuries are best managed by early decompression followed by multiple serial debridements. The ideal timing of free flap coverage needs further investigation.

Donor defects after lymph vessel transplantation and free vascularized lymph node transfer:A comparison and evaluation of complications

BACKGROUND Secondary lymphedema after surgical interventions is a progressive,chronic disease that is still not completely curable.Over the past years,a multitude of surgical therapy options have been described.AIM To summarize the single-center complications in lymph vessel(LVTx)and free vascularized lymph node transfer(VLNT).METHODS In total,the patient collective consisted of 87 patients who were undergoing treatment for secondary leg lymphedema during the study period from March 2010 to April 2020.The data collection was performed preoperatively during consultations,as well as three weeks,six months and twelve months after surgical treatment.In the event of complications,more detailed follow-up checks were carried out.In total n=18 robot-assisted omental lymph node transplantations,n=33 supraclavicular lymph node transplantations and n=36 Lymph vessel transplantations were analyzed.An exemplary drawing is shown in Figure 1.A graphical representation of patient selection is shown in Figure 2.Robotic harvest was performed with the Da Vinci Xi Robot Systems(Intuitive Surgical,CA,United States).RESULTS In total,11 male and 76 female patients were operated on.The mean age of the patients at study entry was:omental VLNT:57.45±8.02 years;supraclavicular VLNT:49.76±4.16 years and LVTx:49.75±4.95 years.The average observation time postoperative was:omental VLNT:18±3.48 mo;supraclavicular VLNT:14.15±4.9 and LVTx:14.84±4.46 mo.In our omental VLNT,three patients showed a slight abdominal sensation of tension within the first 12 postoperative days.No other donor side morbidities occurred.No intraoperative conversion to open technique was needed.Our supraclavicular VLNT collective showed 10 lift defect morbidities with one necessary surgical intervention.In our LVTx collective,12 cases of donor side morbidity were registered.In one case,surgical intervention was necessary.CONCLUSION Concerning donor side morbidity,robot-assisted omental VLNT is clearly superior to supraclavicular lymph node transplantation and LVTx.

A hierarchical vascularized engineered bone inspired by intramembranous ossification for mandibular regeneration

Mandibular defects caused by injuries,tumors,and infections are common and can severely affect mandibular function and the patient's appearance.However,mandible reconstruction with a mandibular bionic structure remains challenging.Inspired by the process of intramembranous ossification in mandibular development,a hierarchical vascularized engineered bone consisting of angiogenesis and osteogenesis modules has been produced.Moreover,the hierarchical vascular network and bone structure generated by these hierarchical vascularized engineered bone modules match the particular anatomical structure of the mandible.The ultra-tough polyion complex has been used as the basic scaffold for hierarchical vascularized engineered bone for ensuring better reconstruction of mandible function.According to the results of in vivo experiments,the bone regenerated using hierarchical vascularized engineered bone is similar to the natural mandibular bone in terms of morphology and genomics.The sonic hedgehog signaling pathway is specifically activated in hierarchical vascularized engineered bone,indicating that the new bone in hierarchical vascularized engineered bone underwent a process of intramembranous ossification identical to that of mandible development.Thus,hierarchical vascularized engineered bone has a high potential for clinical application in mandibular defect reconstruction.Moreover,the concept based on developmental processes and bionic structures provides an effective strategy for tissue regeneration.

Treatment of Scaphoid Nonunion: Pedicled Vascularized Bone Graft vs. Traditional Bone Graft

The clinical results of the application of pedicled vascularized bone graft （VBG） from Lister＇s tubercle vs. traditional bone graft （TBG） were evaluated and compared. Thirteen cases of symptomatic scaphoid nonunion were treated between January 2011 and December 2012, including 7 cases subject to VBG and the rest 6 cases to TBG, respectively. Outcomes were assessed by modified Mayo wrist score system. All cases were followed up for an average period of 3.5 months after opera- tion. The results showed that total scores in VBG group were 86.4i9.4 after operation with excellent result in 4 cases, good in 2 and acceptable in one, and those in TBG group were 71.7±9.3 after operation with good result in 2 cases, acceptable in 3 and disappointing in one. Total score of wrist function was significantly improved in VBG group as compared with TBG group （P〈0.05）. Our study suggests that VBG method is more effective for treating scaphoid nonunion than TBG method.

Reconstruction Using a Free Vascularized Fibular Graft after Frozen Autograft Reconstruction for Osteosarcoma of the Distal Tibia: A Case Report

Recently we have been performing biological reconstruction for malignant bone tumors of the extremities using frozen autografts. Here we present a case treated with free vascularized fibular graft (FVFG) after this method. A 23-year-old man developed osteosarcoma in his left distal tibia. There was nonunion after frozen autograft reconstruction, which we treated with FVFG. Twenty-four months later, bridging between the host bone and the frozen autograft was achieved. Our department has achieved bone union in almost all cases, but we sometimes encounter cases of nonunion after this method because of delayed blood supply. In these instances, reconstruction using FVFG may represent an attractive choice for salvage treatment.

Scalp and Dura Matter Complex Reconstruction Using Free Anterolateral Thigh Flap with Vascularized Fascia

The reconstruction of large scalp and dural defects is difficult. Anterolateral thigh (ALT) flap is now widely used because of its reliable blood supply to the skin paddle. Additionally, ALT can be harvested with a large skin paddle and large, well-vascularized fascia. We have successfully treated eight scalp and dural composite defect cases (five male and three female) using ALT with vascularized fascia. The patients’ mean age was 59.1 ± 20.4 years ranging from 31 to 83 years. The mean dural defect size was 73 ± 21 cm2, ranging from 50 to 120 cm2. There were no postoperative infections, bleeding, cerebrospinal fluid leakage, or meningitis. Further discussion about the usefulness of vascularized fascia may be required and we believe that plastic surgeons, head and neck surgeons, and neurosurgeons should report on the results of dural reconstruction.

Prevascularized Micro‑/Nano‑Sized Spheroid/Bead Aggregates for Vascular Tissue Engineering

Efficient strategies to promote microvascularization in vascular tissue engineering,a central priority in regenerative medicine,are still scarce;nano-and micro-sized aggregates and spheres or beads harboring primitive microvascular beds are promising methods in vascular tissue engineering.Capillaries are the smallest type and in numerous blood vessels,which are distributed densely in cardiovascular system.To mimic this microvascular network,specific cell components and proangiogenic factors are required.Herein,advanced biofabrication methods in microvascular engineering,including extrusion-based and droplet-based bioprinting,Kenzan,and biogripper approaches,are deliberated with emphasis on the newest works in prevascular nano-and micro-sized aggregates and microspheres/microbeads.

A novel strategy for engineering vascularized grafts in vitro

Tissue engineering is an interdisciplinary field promising new therapeutic means for replacing lost or severely damaged tissues or organs. However, the fabrication of complex engineered tissues has been hampered due to the lack of vascularization to provide sufficient blood supply after implantation. In this article, we propose using rapid prototyping technology to prefabricate a scaffold with an inside hollowed vascular system including an arterial end, a venous end and capillary networks between them. The scaffold will be ''printed'' layer by layer. When printing every layer, a ''low-melting point'' material will be used to form a blood vessel network and a tissue-specific material will be used outside it. Hereafter the ‘low-melting point’ material will be evacuated by vaporization to ensure a hollowed vessel network. Then the inside hollowed capillary network can be endothelialized by using autologous endothelial cells in a cycling bioreactor while the outside material can be embedded with tissue-special cells. In the end, the new vascularized autologous grafts could be transferred to the defect site by using microsurgical techniques to connect the grafts with the host artery and vein. The strategy would facilitate construction of complex tissue engineering if the hypothesis proved to be practical.

Drug-loading ZIF-8 for modification of microporous bone scaffold to promote vascularized bone regeneration

Surface modification of microporous bone scaffolds using nanoparticles has been broadly studied in bone tissue engineering.Aiming at improving vascularized bone regeneration(VBR),zeolitic imidazolate framework-8(ZIF-8)was encapsulated with dimethyloxallyl glycine(DMOG)and the drug-carrying nanoparticles(D@Z)could be uniformly coated onto the surface of the bone scaffold.The osteogenic and angiogenic actions of D@Z are closely correlated with the amount of slowly released DMOG,and in general,exhibited a favorable association.Then,the D7.5@Z group,which showed the greatest capacity to induce in vitro osteogenesis-angiogenesis coupling,was utilized for surface modification of the bone scaffold.Biological processes including phosphate-containing compound metabolic process,cell differentiation,cell proliferation and cell motility might contribute to enhanced ability to induce VBR by the coated scaffold and signaling pathways such as Rap1,Ras,phosphatidylinositol 3-kinase/protein kinase B(PI3K-AKT)and vascular endothelial growth factor(VEGF)signaling pathways participated in these processes.Finally,as depicted by in vitro real time-polymerase chain reaction(RT-PCR),Western blot(WB)and in vivo cranial bone defect model,the microporous scaffold coated with nano-D7.5@Z greatly promoted VBR.To conclude,nano-D@Z has significant promise for practical application in modification of microporous bone scaffolds to enhance VBR,and DMOG loading quantity has a beneficial influence on D@Z to improve osteogenesis-angiogenesis coupling.

Construction of millimeter-scale vascularized engineered myocardial tissue using a mixed gel

Engineering myocardium has shown great clinal potential for repairing permanent myocardial injury.However,the lack of perfusing blood vessels and difficulties in preparing a thick-engineered myocardium result in its limited clinical use.We prepared a mixed gel containing fibrin(5 mg/ml)and collagen I(0.2 mg/ml)and verified that human umbilical vein endothelial cells(HUVECs)and human-induced pluripotent stem cell-derived cardiomyocytes(hiPSC-CMs)could fom microvascular lumens and myocardial cell clusters by harnessing the low-hardness and hyperelastic characteristics of fibrin.hiPSC-CMs and HUVECs in the mixed gel formed self-organized cell clusters,which were then cultured in different media using a three-phase approach.The successfully constructed vascularized engineered myocardial tissue had a spherical structure and final diameter of 1-2mm.The tissue exhibited autonomous beats that occured at a frequency similar to a normal human heart rate.The internal microvascular lumen could be maintained for 6 weeks and showed good results during preliminary surface re-vascularization in vitro and vascular remodeling in vivo.In summary,we propose a simple method for constructing vascularized engineered myocardial tissue,through phased cultivation that does not rely on high-end manufacturing equipment and cutting-edge preparation techniques.The constructed tissue has potential value for clinical use after preliminary evaluation.

Vascularized lymph node transfer using axilla as recipient site restores lymphatic flow in upper limb lymphedema:Evidence from magnetic resonance lymphangiography

Background:Lymphedema is a debilitating condition that frequently occurs after breast cancer treatment.Vas-cularized lymph node transfer(VLNT)is a promising approach to reduce lymphedema.This study used magnetic resonance lymphangiography(MRL)to assess lymphatic reconnections post-VLNT in patients with breast cancer-related lymphedema(BCRL).Methods:The clinical records of six female patients with unilateral upper limb BCRL who underwent VLNT(4 cases)or VLNT combined with breast reconstruction(2 cases)were retrospectively reviewed.All patients were examined using MRL preoperatively and at the 1-year follow-up.The morphological characteristics of the lymphatic network,dermal backflow patterns,and architecture of the lymph nodes were evaluated.Clinical outcomes,patient satisfaction,and complications were assessed.Results:At the 1-year follow-up,reduction in tissue edema and limb circumference was achieved in all six patients.In MRL,the implanted lymph nodes in the axillary region of the affected upper arm were enhanced and visualized in all six patients.Reconnected lymphatic vessels in the subcutaneous tissue associated with the implanted lymph nodes were observed in four patients.Decreased dermal backflow and lymphatic vessel dilation of the affected limbs were observed in all six patients.No disruption of the lymph flow in the donor area was detected.Conclusion:This is the first study to provide direct imaging evidence for the reconnection of afferent lymphatic channels between implanted lymph nodes and the recipient lymphatic system in patients with BCRL.Overall,our study demonstrates the mechanism and efficacy of VLNT in reducing lymphedema.

Black phosphorus nanosheets-enabled DNA hydrogel integrating 3D-printed scaffold for promoting vascularized bone regeneration

The classical 3D-printed scaffolds have attracted enormous interests in bone regeneration due to the customized structural and mechanical adaptability to bone defects.However,the pristine scaffolds still suffer from the absence of dynamic and bioactive microenvironment that is analogous to natural extracellular matrix(ECM)to regulate cell behaviour and promote tissue regeneration.To address this challenge,we develop a black phosphorus nanosheets-enabled dynamic DNA hydrogel to integrate with 3D-printed scaffold to build a bioactive gel-scaffold construct to achieve enhanced angiogenesis and bone regeneration.The black phosphorus nanosheets reinforce the mechanical strength of dynamic self-healable hydrogel and endow the gel-scaffold construct with preserved protein binding to achieve sustainable delivery of growth factor.We further explore the effects of this activated construct on both human umbilical vein endothelial cells(HUVECs)and mesenchymal stem cells(MSCs)as well as in a critical-sized rat cranial defect model.The results confirm that the gel-scaffold construct is able to promote the growth of mature blood vessels as well as induce osteogenesis to promote new bone formation,indicating that the strategy of nano-enabled dynamic hydrogel integrated with 3D-printed scaffold holds great promise for bone tissue engineering.

Functionalize d 3D-printe d porous titanium scaffold induces in situ vascularized bone regeneration by orchestrating bone microenvironment

Titanium(Ti)and its alloys have been extensively explored for treating load-bearing bone defects.How-ever,high-stress shielding,weak osteogenic activity,and insufficient vascularization remain key chal-lenges for the long-term clinical outcomes of Ti-based implants.Herein,inspired by structural and func-tional cues of bone regeneration,a silicon-doped nano-hydroxyapatite(nSiHA)/titanium dioxide(TiO_(2))composite coating with a hierarchical micro/nano-network structure is constructed on the surface of a 3D-printed porous Ti scaffold via a combined strategy of acid-alkali(AA)treatment and electrochemi-cal deposition technique,which not only endows the scaffold with excellent osteoinduction ability but can also effectively immobilize and release vascular endothelial growth factor(VEGF).The results of the in vitro cell experiments show that the functionalized Ti scaffold significantly promotes osteogenesis in bone marrow mesenchymal stem cells(BMSCs)and angiogenesis in human umbilical vein endothelial cells(HUVECs)by activating the extracellular signal-regulated protein kinase(ERK)and HIF-1αsignaling pathways.After being implanted into a rat femoral condyle defect model,the functionalized Ti scaffold can induce in situ vascularized bone regeneration by orchestrating the two coupled processes of angio-genesis and osteogenesis.These findings indicate that the functionalized Ti scaffold has great potential in bone tissue regeneration and is a promising candidate for load-bearing bone defect repair.

A bioactive material with dual integrin-targeting ligands regulates specific endogenous cell adhesion and promotes vascularized bone regeneration in adult and fetal bone defects

Significant progress has been made in designing bone materials capable of directing endogenous cells to promote vascularized bone regeneration.However,current strategies lack regulation of the specific endogenous cell populations for vascularized bone regeneration,thus leading to adverse tissue formation and decreased regenerative efficiency.Here,we engineered a biomaterial to regulate endogenous cell adhesion and promote vascularized bone regeneration.The biomaterial works by presenting two synthetic ligands,LLP2A and LXW7,explicitly targeting integrinsα4β1 andαvβ3,respectively,expressed on the surfaces of the cells related to bone formation and vascularization,such as mesenchymal stem cells(MSCs),osteoblasts,endothelial progenitor cells(EPCs),and endothelial cells(ECs).In vitro,the LLP2A/LXW7 modified biomaterial improved the adhesion of MSCs,osteoblasts,EPCs,and ECs via integrinα4β1 andαvβ3,respectively.In an adult rat calvarial bone defect model,the LLP2A/LXW7 modified biomaterial enhanced bone formation and vascularization by synergistically regulating endogenous cells with osteogenic and angiogenic potentials,such as DLX5^(+)cells,osteocalcin^(+)cells,CD34^(+)/CD45-cells and CD31^(+)cells.In a fetal sheep spinal bone defect model,the LLP2A/LXW7 modified biomaterial augmented bone formation and vascularization without any adverse effects.This innovative biomaterial offers an off-the-shelf,easy-to-use,and biologically safe product suitable for vascularized bone regeneration in both fetal and adult disease environments.