Adipose tissue reconstruction facilitated with low immunogenicity decellularized adipose tissue scaffolds.

There is currently an urgent need to develop engineered scaffolds to support new adipose tissue formation and facilitate long-term maintenance of function and defect repair to further generate prospective bioactive filler materials capable of fulfilling surgical needs. Herein, adipose regeneration methods were optimized and decellularized adipose tissue (DAT) scaffolds with good biocompatibility were fabricated. Adipose-like tissues were reconstructed using the DAT and 3T3-L1 preadipocytes, which have certain differentiation potential, and the regenerative effects of the engineered adipose tissuesin vitroandin vivowere explored. The method improved the efficiency of adipose removal from tissues, and significantly shortened the time for degreasing. Thus, the DAT not only provided a suitable space for cell growth but also promoted the proliferation, migration, and differentiation of preadipocytes within it. Following implantation of the constructed adipose tissuesin vivo, the DAT showed gradual degradation and integration with surrounding tissues, accompanied by the generation of new adipose tissue analogs. Overall, the combination of adipose-derived extracellular matrix and preadipocytes for adipose tissue reconstruction will be of benefit in the artificial construction of biomimetic implant structures for adipose tissue reconstruction, providing a practical guideline for the initial integration of adipose tissue engineering into clinical medicine.

Distinct cellular microenvironment with cytotypic effects regulates orderly regeneration of vascular tissues.

Regeneration of the architecturally complex blood vascular system requires precise temporal and spatial control of cell behaviours. Additional components must be integrated into the structure to achieve clinical success for in situ tissue engineering. Consequently, this study proposed a universal method for including any substrate type in vascular cell extracellular matrices (VCEM) via regulating selective adhesion to promote vascular tissue regeneration. The results uncovered that the VCEM worked as cell adhesion substrates, exhibited cell type specificity, and functioned as an address signal for recognition by vascular cells, which resulted in matching with the determined cells. The qPCR and immunofluorescence results revealed that a cell type-specific VCEM could be designed to promote or inhibit cell adhesion, consistenting with the expression patterns of eyes absent 3 (Eya3). In addition, a 3D vascular graft combined with VCEM which could recapitulate the vascular cell-like microenvironment was fabricated. The vascular graft revealed a prospective role for cellular microenvironment in the establishment of vascular cell distribution and tissue architecture, and potentiated the orderly regeneration and functional recovery of vascular tissues in vivo. The findings demonstrate that differential adhesion between cell types due to the cellular microenvironment is sufficient to drive the complex assembly of engineered blood vessel functional units, and underlies hierarchical organization during vascular regeneration.

Construction of engineered 3D islet micro-tissue using porcine decellularized ECM for the treatment of diabetes.

Islet transplantation improves diabetes patients' long-term blood glucose control, but its success and utility are limited by cadaver availability, quality, and considerable islet loss after transplantation due to ischemia and inadequate angiogenesis. This study used adipose, pancreatic, and liver tissue decellularized extracellular matrix (dECM) hydrogels in an effort to recapitulate the islet sites inside the pancreas in vitro, and successfully generated viable and functional heterocellular islet micro-tissues using islet cells, human umbilical vein endothelial cells, and adipose-derived mesenchymal stem cells. The three-dimensional (3D) islet micro-tissues maintained prolonged viability and normal secretory function, and showed high drug sensitivity in drug testing. Meanwhile, the 3D islet micro-tissues significantly enhanced survival and graft function in a mouse model of diabetes. These supportive 3D physiomimetic dECM hydrogels can be used not only for islet micro-tissue culture in vitro, but also have great promise for islet transplantation for the treatment of diabetes.

E3 ligase MAEA-mediated ubiquitination and degradation of PHD3 promotes glioblastoma progression.

Glioblastoma (GBM) is the most common malignant glioma, with a high recurrence rate and a poor prognosis. However, the molecular mechanism behind the malignant progression of GBM is still unclear. In the present study, through the tandem mass tag (TMT)-based quantitative proteomic analysis of clinical primary and recurrent glioma samples, we identified that aberrant E3 ligase MAEA was expressed in recurrent samples. The results of bioinformatics analysis showed that the high expression of MAEA was related to the recurrence and poor prognosis of glioma and GBM. Functional studies showed that MAEA could promote proliferation, invasion, stemness and temozolomide (TMZ) resistance. Mechanistically, the data indicated that MAEA targeted prolyl hydroxylase domain 3 (PHD3) K159 to promote its K48-linked polyubiquitination and degradation, thus enhancing the stability of HIF-1α, thereby promoting the stemness and TMZ resistance of GBM cells through upregulating CD133. The in vivo experiments further confirmed that knocking down MAEA could inhibit the growth of GBM xenograft tumors. In summary, MAEA enhances the expression of HIF-1α/CD133 through the degradation of PHD3 and promotes the malignant progression of GBM.

In vitroadipogenesis and long-term adipocyte culture in adipose tissue-derived cell banks.

There is a critical need to developin vitroculture systems appropriate for the expansion of adipose tissue, in order to gain new insights into metabolic diseases and to assist in the restoration of tissue defects. Conventional two- or three-dimensional (2D or 3D)in vitromodels of adipocytes require a combination of supplements to induce adipocyte maturation that greatly increases the cost of large-scale industrial production. In the present study, a microporous, perforated bacterial cellulose (BC)-assisted culture system was developed that promoted the adhesion, proliferation, and adipogenic differentiation of preadipocytes. Additionally, the system maintained the cells as mature unilocular adipocytesex vivoin normal cell culture medium in long-term culture. All cells were derived from isolated adipose tissue without the use of expensive enzymes for tissue digestion. In contrast to culture in hard tissue culture plates, preadipocytes in the soft 3D environments formed multidimensional interlaced cell contacts, undergoing significant spontaneous lipid accumulation and could be cultured for up to threemonths in maintenance medium. More importantly, the cultured adipose tissue-derived cell bank created here was able to produce injury repair activators that promoted the proliferation of fibroblasts with little fibrosis and the functional differentiation of myoblasts, displaying the potential for use in adipose reconstruction. Thus, the present study demonstrates the potential of a mechanically flexible BC scaffold to generate volume tunable adipose constructs and provides a low-cost and user-friendly strategy for large-scale industrial production of adipose tissue.

3D printed in vitro tumor tissue model of colorectal cancer.

Rationale: The tumor microenvironment (TME) determines tumor progression and affects clinical therapy. Its basic components include cancer-associated fibroblasts (CAFs) and tumor-associated endothelial cells (TECs), both of which constitute the tumor matrix and microvascular network. The ability to simulate interactions between cells and extracellular matrix in a TME in vitro can assist the elucidation of cancer growth and evaluate the efficiency of therapies. Methods: In the present study, an in vitro 3D model of tumor tissue that mimicked in vivo cell physiological function was developed using tumor-associated stromal cells. Colorectal cancer cells, CAFs, and TECs were co-cultured on 3D-printed scaffolds so as to constitute an extracellular matrix (ECM) that allowed cell processes such as adhesion, stemness, proliferation, and vascularization to take place. Normal stromal cells were activated and reprogrammed into tumor-related stromal cells to construct a TME of tumor tissues. Results: The activated stromal cells overexpressed a variety of tumor-related markers and remodeled the ECM. Furthermore, the metabolic signals and malignant transformation of the in vitro 3D tumor tissue was substantially similar to that observed in tumors in vivo. Conclusions: The 3D tumor tissue exhibited physiological activity with high drug resistance. The model is suitable for research studies of tumor biology and the development of personalized treatments for cancer.

3D printed intelligent scaffold prevents recurrence and distal metastasis of breast cancer.

Rationale: Tumors are commonly treated by resection, which usually leads to massive hemorrhage and tumor cell residues, thereby increasing the risk of local recurrence and distant metastasis. Methods: Herein, an intelligent 3D-printed poly(lactic-co-glycolic acid), gelatin, and chitosan scaffold loaded with anti-cancer drugs was prepared that showed hemostatic function and good pH sensitivity. Results: Following in situ implantation in wounds, the scaffolds absorbed hemorrhage and cell residues after surgery, and promoted wound healing. In an in vivo environment, the scaffold responded to the slightly acidic environment of the tumor to undergo sustained drug release to significantly inhibit the recurrence and growth of the tumor, and reduced drug toxicity, all without causing damage to healthy tissues and with good biocompatibility. Conclusions: The multifunctional intelligent scaffold represents an excellent treatment modality for breast cancer following resection, and provides great potential for efficient cancer therapy.

The role of a drug-loaded poly (lactic co-glycolic acid) (PLGA) copolymer stent in the treatment of ovarian cancer.

Objectives: Cisplatin (CDDP) is a widely used and effective basic chemotherapeutic drug for the treatment of a variety of tumors, including ovarian cancer. However, adverse side effects and acquired drug resistance are observed in the clinical application of CDDP. Identifying a mode of administration that can alleviate side effects and reduce drug resistance has become a promising strategy to solve this problem. Methods: In this study, 3D printing technology was used to prepare a CDDP-poly (lactic-co-glycolic acid) (CDDP-PLGA) polymer compound stent, and its physicochemical properties and cytotoxicity were evaluated both in vitro and in vivo. Results: The CDDP-PLGA stent had a significant effect on cell proliferation and apoptosis and clearly decreased the size of subcutaneous tumors in nude mice, whereas the systemic side effects were mild compared with those of intraperitoneal CDDP injection. Compared with the control group, CDDP-PLGA significantly increased the mRNA and protein levels of p-glycoprotein (P < 0.01; P < 0.01) and decreased vascular endothelial growth factor mRNA (P < 0.05) and protein levels (P < 0.01), however, CDDP-PLGA significantly decreased the mRNA and protein levels of p-glycoprotein (P < 0.01; P < 0.01) and vascular endothelial growth factor (P < 0.01; P < 0.01), which are associated with chemoresistance, in subcutaneous tumor tissue. Immunohistochemistry assay results revealed that, in the CDDP-PLGA group, the staining of the proliferation-related genes Ki67 and PCNA were lightly, and the apoptosis-related gene caspase-3 stained deeply. Conclusions: PLGA biomaterials loaded with CDDP, as compared with the same amount of free CDDP, showed good efficacy in terms of cytotoxicity, as evidenced by changes in apoptosis. Continuous local CDDP release can decrease the systemic side effects of this drug and the occurrence of drug resistance and angiogenesis, and improve the therapeutic effect. This new approach may be an effective strategy for the local treatment of epithelial ovarian cancer.

E-jet 3D printed drug delivery implants to inhibit growth and metastasis of orthotopic breast cancer.

Drug-loaded implants have attracted considerable attention in cancer treatment due to their precise delivery of drugs into cancer tissues. Contrary to injected drug delivery, the application of drug-loaded implants remains underutilized given the requirement for a surgical operation. Nevertheless, drug-loaded implants have several advantages, including a reduction in frequency of drug administration, minimal systemic toxicity, and increased delivery efficacy. Herein, we developed a new, precise, drug delivery device for orthotopic breast cancer therapy able to suppress breast tumor growth and reduce pulmonary metastasis using combination chemotherapy. Poly-lactic-co-glycolic acid scaffolds were fabricated by 3D printing to immobilize 5-fluorouracil and NVP-BEZ235. The implantable scaffolds significantly reduced the required drug dosages and ensured curative drug levels near tumor sites for prolonged period, while drug exposure to normal tissues was minimized. Moreover, long-term drug release was achieved, potentially allowing one-off implantation and, thus, a major reduction in the frequency of drug administration. This drug-loaded scaffold has great potential in anti-tumor treatment, possibly paving the way for precise, effective, and harmless cancer therapy.

E-Jet 3D-Printed Scaffolds as Sustained Multi-Drug Delivery Vehicles in Breast Cancer Therapy.

PURPOSE: Combination chemotherapy is gradually receiving more attention because of its potential synergistic effect and reduced drug doses in clinical application. However, how to precisely control drug release dose and time using vehicles remains a challenge. This work developed an efficient drug delivery system to combat breast cancer, which can enhance drug effects despite reducing its concentration. METHODS: Controlled-release poly-lactic-co-glycolic acid (PLGA) scaffolds were fabricated by E-jet 3D printing to deliver doxorubicin (DOX) and cisplatin (CDDP) simultaneously. RESULTS: This drug delivery system allowed the use of a reduced drug dosage resulting in a better effect on the human breast cancer cell apoptosis and inhibiting tumor growth, compared with the effect of each drug and the two drugs administrated without PLGA scaffolds. Our study suggested that DOX-CDDP-PLGA scaffolds could efficiently destroy MDA-MB-231 cells and restrain tumor growth. CONCLUSIONS: The 3D printed PLGA scaffolds with their time-programmed drug release might be useful as a new multi-drug delivery vehicle in cancer therapy, which has a potential advantage in a long term tumor cure and prevention of tumor recurrence.

The topography of fibrous scaffolds modulates the paracrine function of Ad-MSCs in the regeneration of skin tissues.

Injuries to the skin are common in daily life, and a certain type or size of defect is not easily restored using conventional dressings or naturally. The repair of these defects requires restoration of function in regenerated tissues. In this study, a tissue engineered skin was designed and fabricated using a bio-3D printing system. Polycaprolactone and bacterial cellulose comprised the scaffold, due to their excellent biocompatibility and multifunctionality. Adipose-derived mesenchymal stem cells (Ad-MSCs) were seeded onto the scaffold to functionalize it as an artificial skin. The finished artificial skin had mechanical properties similar to that of natural skin, and its fibrous structure providing a unique micro-environment that could regulate the paracrine function of the Ad-MSCs. This effect could be greatly increased by changes in the characteristics of the biomaterials. The artificial skin exhibited high biological activity, strong induction of cell recruitment, migration, growth and up-regulation of gene expression of relevant factors, resulting in excellent wound healing characteristics. This study clarified novel design aspects of cell-material interactions in which the topographical characteristics of materials can be further developed to establish cell signaling or communication networks that take advantage of the paracrine actions of Ad-MSCs to promote specific tissue regeneration or repair characteristics.

Transportan-derived cell-penetrating peptide delivers siRNA to inhibit replication of influenza virus in vivo.

INTRODUCTION: In this study, we report on the development of an effective delivery system for siRNAs; a novel cell-penetrating peptide (CPP), T9(dR), obtained from transportan (TP), was used for in vivo and in vitro testing. METHODS: In this study, toxicity of T9(dR) and TP and efficient delivery of siRNA were tested in 293T, MDCK, RAW, and A549 cells. Furthermore, T9(dR)- and TP-delivered siRNAs against nucleoprotein (NP) gene segment of influenza virus (siNP) were studied in both cell lines and mice. RESULTS: Gel retardation showed that T9(dR) effectively condensed siRNA into nanoparticles sized between 350 and 550 nm when the mole ratio of T9(dR) to siRNA was ≥4:1. In vitro studies demonstrated that T9(dR) successfully delivered siRNA with low cellular toxicity into several cell lines. It was also observed that T9(dR)-delivered siRNAs inhibited replication of influenza virus more efficiently as compared to that delivered by TP into the MDCK and A549 cells. It was also noticed that when given a combined tail vein injection of siNP and T9(dR) or TP, all mice in the 50 nmol siNP group infected with PR8 influenza virus survived and showed weight recovery at 2 weeks post-infection. CONCLUSION: This study indicates that T9(dR) is a promising siRNA delivery tool with potential application for nucleotide drug delivery.

Enhanced growth and differentiation of myoblast cells grown on E-jet 3D printed platforms.

BACKGROUND: Skeletal muscle tissue engineering often involves the prefabrication of muscle tissues in vitro by differentiation and maturation of muscle precursor cells on a platform which provides an environment that facilitates the myogenic differentiation of the seeded cells. METHODS: Poly lactic-co-glycolic acid (PLGA) 3D printed scaffolds, which simulate the highly complex structure of extracellular matrix (ECM), were fabricated by E-jet 3D printing in this study. The scaffolds were used as platforms, providing environment that aids in growth, differentiation and other properties of C2C12 myoblast cells. RESULTS: The C2C12 myoblast cells grown on the PLGA 3D printed platforms had enhanced cell adhesion and proliferation. Moreover, the platforms were able to induce myogenic differentiation of the myoblast cells by promoting the formation of myotubes and up-regulating the expressions of myogenic genes (MyHC and MyOG). CONCLUSION: The fabricated 3D printed platforms have excellent biocompatibility, thereby can potentially be used as functional cell culture platforms in skeletal tissue engineering and regeneration.

In Vitro Study of Colon Cancer Cell Migration Using E-Jet 3D Printed Cell Culture Platforms.

The development of accurate and predictive in vitro experimental models of human tumors consistent with in vivo tumor microenvironments has garnered great attention in modern cancer research. 3D scaffolds are fabricated in this study by E-jet 3D printing with the aim of replicating the functionalities of tumor microenvironments in vitro which could be applicable as screening platforms for novel therapeutic strategies. Tumor protein 53 (p53) plays an important role in penetration and migration in 2D cell culture. However, whether or not p53 has the same function in 3D cell culture and the underlying mechanisms are poorly understood. Results show that p53 deletion significantly decreases the speed of migration and proliferation of cancer cells within 3D environments. This study unveils aspects of cancer cell motility and migration and the steps involved in subsequent cancer metastases, which provides a new perspective and platform for the research of tumor metastasis therapy.

Triple-Layer Vascular Grafts Fabricated by Combined E-Jet 3D Printing and Electrospinning.

Small-diameter tissue-engineered vascular grafts are urgently needed for clinic arterial substitute. To simulate the structures and functions of natural blood vessels, we designed a novel triple-layer poly(ε-caprolactone) (PCL) fibrous vascular graft by combining E-jet 3D printing and electrospinning techniques. The resultant vascular graft consisted of an interior layer comprising 3D-printed highly aligned strong fibers, a middle layer made by electrospun densely fibers, and an exterior structure composed of mixed fibers fabricated by co-electrospraying. The biocompatible triple-layer graft was used for in vivo implantation, and results demonstrated that the longitudinally-aligned fibers within the lumen of the graft could enhance the proliferation and migration of endothelial cells, while maintained good mechanical properties. The exterior layer provided a pathway that encouraged cells to migrate into the scaffold after implantation. This experimental graft overcame the limitations of conventionally electrospun vascular grafts of inadequate porosity and lowly cell penetration. The unique structure of the triple-layer vascular graft promoted cell growth and infiltration in vivo, thus provided an encouraging substitute for in situ tissue engineering.

An effective thermal therapy against cancer using an E-jet 3D-printing method to prepare implantable magnetocaloric mats.

Magnetic hyperthermia has been rapidly developed as a potential cancer treatment in recent years. Artificially induced hyperthermia close to a tumor can raise the temperature to 45°C causing tumor cell death. Herein, we introduce a novel method for rapid preparation of anti-cancer magnetocaloric PCL/Fe3 O4 mats capable of high-performance hyperthermia using E-jet 3D printing technology. Our 3D printed mats not only maintained the heating efficiency of traditional techniques for magnetic hyperthermia but also prolonged the effective therapy in vivo. When Fe3 O4 nanoparticles (NPs) were used in mats at a concentration of 6 mmol/L, 0.07 g PCL/Fe3 O4 mats were able to increase the temperature peripherally to 45°C under an alternating magnetic field (AMF) within 45 min. Moreover, the reproducibility experiment indicated that the maximum temperature was achieved following repeated heating and cooling cycles. Cell toxicity tests showed a high cell death rate during one treatment cycle. In vivo experiments indicated clear signs of tumor growth inhibitory and prolonged survival time of tumor-bearing mice after 4 weeks of treatment. The present magnetic mats may be a potential candidate for a novel heat-generating substrate for localized hyperthermia cancer therapy. Furthermore, the main advantage of such implantable magnetic mats is the local and precise delivery of Fe3 O4 NPs, ideal for the hyperthermia treatment of easily accessible tumors. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1827-1841, 2018.

Control of cell growth on 3D-printed cell culture platforms for tissue engineering.

Biocompatible tissue growth has excellent prospects for tissue engineering. These tissues are built over scaffolds, which can influence aspects such as cell adhesion, proliferation rate, morphology, and differentiation. However, the ideal 3D biological structure has not been developed yet. Here, we applied the electro-hydrodynamic jet (E-jet) 3D printing technology using poly-(lactic-co-glycolic acid, PLGA) solution to print varied culture platforms for engineered tissue structures. The effects of different parameters (electrical voltage, plotting speed, and needle sizes) on the outcome were investigated. We compared the biological compatibility of the 3D printed culture platforms with that of random fibers. Finally, we used the 3D-printed PLGA platforms to culture fibroblasts, the main cellular components of loose connective tissue. The results show that the E-jet printed platforms could guide and improve cell growth. These highly aligned fibers were able to support cellular alignment and proliferation. Cell angle was consistent with the direction of the fibers, and cells cultured on these fibers showed a much faster migration, potentially enhancing wound healing performance. Thus, the potential of this technology for 3D biological printing is large. This process can be used to grow biological scaffolds for the engineering of tissues. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3281-3292, 2017.

Electrospun vein grafts with high cell infiltration for vascular tissue engineering.

Demand is increasing for functional small-diameter vascular grafts (diameter<6mm) for clinical arterial replacement. In the present study, we develop a bilayer poly(ε-caprolactone, PCL) fibrous vascular graft consisting of a thin internal layer made of longitudinally aligned fibers and a relatively thick highly porous external layer. The internal layer provides a scaffold with the necessary mechanical strength and enhances the growth of endothelial cells, whereas the external layer enhances cell motility through the scaffold bulk. The biocompatibility and biological performance of bilayer fibrous scaffolds are evaluated by in vivo experiments, molecular biology, and histology studies. Our bilayer scaffolds demonstrate much better fiber alignment and higher porosity than do normal electrospun vascular grafts with randomly distributed fibers. The results suggest that the proposed grafts can overcome limitations owing to the inadequate porosity, small pores, and poor cell infiltration of scaffolds fabricated by conventional electrospinning. The unique structure of bilayer scaffolds is satisfactory and promotes cell proliferation, collagen-fiber deposition, and ingrowth of smooth muscle cells and endothelial cells in vivo. The results of this study illustrate the strong potential of such bilayer fibrous scaffolds for vascular tissue engineering and regeneration.

Control of cell proliferation in E-jet 3D-printed scaffolds for tissue engineering applications: the influence of the cell alignment angle.

The ideal 3D scaffold for biological applications has not yet been designed. Our aim is to better match the scaffold performance through fine control of the fabrication process. Here, we applied electro-hydrodynamic jet (E-jet) 3D printing technology using poly-(lactic-co-glycolic acid) (PLGA) solution to construct scaffolds for tissue engineering applications. We fabricated different scaffolds of 1 : 1, 1 : 2 and 1 : 3 aspect ratios and tested the biological compatibility of the 2D and 3D printed scaffolds with fibroblasts. The scaffolds were used to culture fibroblasts, the main cellular components of loose connective tissue. The results show that the E-jet printed scaffolds could guide and improve cell growth. These scaffolds were able to support cellular alignment and proliferation. The cell angle was consistent with the longitudinal direction of the scaffolds, potentially enhancing the wound healing performance. Thus, the potential of this flexible technology for the 3D printing of scaffolds for the engineering of tissues is extensive.

Pazopanib, a novel multi-kinase inhibitor, shows potent antitumor activity in colon cancer through PUMA-mediated apoptosis.

Colon cancer is still the third most common cancer which has a high mortality but low five-year survival rate. Novel tyrosine kinase inhibitors (TKI) such as pazopanib become effective antineoplastic agents that show promising clinical activity in a variety of carcinoma, including colon cancer. However, the precise underlying mechanism against tumor is unclear. Here, we demonstrated that pazopanib promoted colon cancer cell apoptosis through inducing PUMA expression. Pazopanib induced p53-independent PUMA activation by inhibiting PI3K/Akt signaling pathway, thereby activating Foxo3a, which subsequently bound to the promoter of PUMA to activate its transcription. After induction, PUMA activated Bax and triggered the intrinsic mitochondrial apoptosis pathway. Furthermore, administration of pazopanib highly suppressed tumor growth in a xenograft model. PUMA deletion in cells and tumors led to resistance of pazopanib, indicating PUMA-mediated pro-apoptotic and anti-tumor effects in vitro and in vivo. Combing pazopanib with some conventional or novel drugs, produced heightened and synergistic antitumor effects that were associated with potentiated PUMA induction via different pathways. Taken together, these results establish a critical role of PUMA in mediating the anticancer effects of pazopanib in colon cancer cells and provide the rationale for clinical evaluation.