Laminin-Augmented Decellularized Extracellular Matrix Ameliorating Neural Differentiation and Neuroinflammation in Human Mini-Brains.

Non-neural extracellular matrix (ECM) has limited application in humanized physiological neural modeling due to insufficient brain-specificity and safety concerns. Although brain-derived ECM contains enriched neural components, certain essential components are partially lost during the decellularization process, necessitating augmentation. Here, it is demonstrated that the laminin-augmented porcine brain-decellularized ECM (P-BdECM) is xenogeneic factor-depleted as well as favorable for the regulation of human neurons, astrocytes, and microglia. P-BdECM composition is comparable to human BdECM regarding brain-specificity through the matrisome and gene ontology-biological process analysis. As augmenting strategy, laminin 111 supplement promotes neural function by synergic effect with laminin 521 in P-BdECM. Annexin A1(ANXA1) and Peroxiredoxin(PRDX) in P-BdECM stabilized microglial and astrocytic behavior under normal while promoting active neuroinflammation in response to neuropathological factors. Further, supplementation of the brain-specific molecule to non-neural matrix also ameliorated glial cell inflammation as in P-BdECM. In conclusion, P-BdECM-augmentation strategy can be used to recapitulate humanized pathophysiological cerebral environments for neurological study.

The Correlation between Waist Circumference and the Pro-Inflammatory Adipokines in Diabetic Retinopathy of Type 2 Diabetes Patients.

Central obesity is one of the major risk factors for type 2 diabetes mellitus (DM), and the most common complication of DM is diabetic retinopathy. However, the exact relationship between obesity and DR remains unknown. In this study, we evaluate the effect of obesity on DR by comparing the aqueous humor-derived adipokines. For the analysis, 37 DR patients and 29 non-DR-patients participated. To evaluate the obesity of the patients, body mass index (BMI) and waist circumference (WC) were used. By comparing the concentrations of adipokines obtained from the aqueous humor of the two groups, the relationship between DR and adipokines was analyzed. In addition, by analyzing the correlation between obesity and adipokines in patients, the relationship between central obesity and DR was finally confirmed. The WC was significantly higher in patients than in the non-patient group. The concentrations of all adipokines compared in this study were significantly higher in the DR group than in the non-DM group (p < 0.05). Among them, adiponectin, leptin, TNF-α, Factor D (adipsin), lipocalin-2 (NGAL), Serpin E1 (PAI-1), and CXCL8 (IL-8) were confirmed to have a positive correlation with central obesity (defined as WC). These findings suggest that central obesity is strongly associated with the risk of DR.

Decellularized Extracellular Matrix-based Bioinks for Engineering Tissue- and Organ-specific Microenvironments.

Biomaterials-based biofabrication methods have gained much attention in recent years. Among them, 3D cell printing is a pioneering technology to facilitate the recapitulation of unique features of complex human tissues and organs with high process flexibility and versatility. Bioinks, combinations of printable hydrogel and cells, can be utilized to create 3D cell-printed constructs. The bioactive cues of bioinks directly trigger cells to induce tissue morphogenesis. Among the various printable hydrogels, the tissue- and organ-specific decellularized extracellular matrix (dECM) can exert synergistic effects in supporting various cells at any component by facilitating specific physiological properties. In this review, we aim to discuss a new paradigm of dECM-based bioinks able to recapitulate the inherent microenvironmental niche in 3D cell-printed constructs. This review can serve as a toolbox for biomedical engineers who want to understand the beneficial characteristics of the dECM-based bioinks and a basic set of fundamental criteria for printing functional human tissues and organs.

Tissue-Specific Decellularized Extracellular Matrix Bioinks for Musculoskeletal Tissue Regeneration and Modeling Using 3D Bioprinting Technology.

The musculoskeletal system is a vital body system that protects internal organs, supports locomotion, and maintains homeostatic function. Unfortunately, musculoskeletal disorders are the leading cause of disability worldwide. Although implant surgeries using autografts, allografts, and xenografts have been conducted, several adverse effects, including donor site morbidity and immunoreaction, exist. To overcome these limitations, various biomedical engineering approaches have been proposed based on an understanding of the complexity of human musculoskeletal tissue. In this review, the leading edge of musculoskeletal tissue engineering using 3D bioprinting technology and musculoskeletal tissue-derived decellularized extracellular matrix bioink is described. In particular, studies on in vivo regeneration and in vitro modeling of musculoskeletal tissue have been focused on. Lastly, the current breakthroughs, limitations, and future perspectives are described.

Development of 3D Printed Bruch's Membrane-Mimetic Substance for the Maturation of Retinal Pigment Epithelial Cells.

Retinal pigment epithelium (RPE) is a monolayer of the pigmented cells that lies on the thin extracellular matrix called Bruch's membrane. This monolayer is the main component of the outer blood-retinal barrier (BRB), which plays a multifunctional role. Due to their crucial roles, the damage of this epithelium causes a wide range of diseases related to retinal degeneration including age-related macular degeneration, retinitis pigmentosa, and Stargardt disease. Unfortunately, there is presently no cure for these diseases. Clinically implantable RPE for humans is under development, and there is no practical examination platform for drug development. Here, we developed porcine Bruch's membrane-derived bioink (BM-ECM). Compared to conventional laminin, the RPE cells on BM-ECM showed enhanced functionality of RPE. Furthermore, we developed the Bruch's membrane-mimetic substrate (BMS) via the integration of BM-ECM and 3D printing technology, which revealed structure and extracellular matrix components similar to those of natural Bruch's membrane. The developed BMS facilitated the appropriate functions of RPE, including barrier and clearance functions, the secretion of anti-angiogenic growth factors, and enzyme formation for phototransduction. Moreover, it could be used as a basement frame for RPE transplantation. We established BMS using 3D printing technology to grow RPE cells with functions that could be used for an in vitro model and RPE transplantation.

Scenarios of energy reduction potential of zero energy building promotion in the Asia-Pacific region to year 2050.

Building energy consumption in the Asia-Pacific region continues to rise. It is important to understand the energy use and future trends of 21 members of Asia-Pacific Economic Cooperation (APEC) and to find more effective ways to achieve APEC's dual goals of reducing energy intensity by 45% of 2005 levels by 2035 and doubling the share of renewable energy in the energy mix between 2010 and 2030. Recently, promoting building toward ultra-low energy, nearly zero energy and zero energy is becoming a consensus trend. This paper aims to explore how zero energy building promotion could influence the total energy demand in the mid to long term. An EUPP (Economic, Urbanization, Population and Purchasing power parity) model was established to show the relationship between building energy consumption and its influencing factors, and the potential development path of building energy consumption in APEC was predicted by using the model. The results show that in the Business As Usual (BAU) model, building energy demand will increase from 1387.4 Mtoe in 2016 to 2456.8 Mtoe in 2050 while in the CAP model, building energy demand will be constrained to under 2000 Mtoe before 2050. In the ZEB promotion model, 897.8 to 1945.3 Mtoe could be saved separately. The share of end demand supplied by onsite renewable energy production could reach 11%-54%. The building sector has the potential to become the largest contributor to achieve the APEC energy goal and thus to the climate change goal.

3D Cell Printing of Tissue/Organ-Mimicking Constructs for Therapeutic and Drug Testing Applications.

The development of artificial tissue/organs with the functional maturity of their native equivalents is one of the long-awaited panaceas for the medical and pharmaceutical industries. Advanced 3D cell-printing technology and various functional bioinks are promising technologies in the field of tissue engineering that have enabled the fabrication of complex 3D living tissue/organs. Various requirements for these tissues, including a complex and large-volume structure, tissue-specific microenvironments, and functional vasculatures, have been addressed to develop engineered tissue/organs with native relevance. Functional tissue/organ constructs have been developed that satisfy such criteria and may facilitate both in vivo replenishment of damaged tissue and the development of reliable in vitro testing platforms for drug development. This review describes key developments in technologies and materials for engineering 3D cell-printed constructs for therapeutic and drug testing applications.

Effect of an oxygen-generating scaffold on the viability and insulin secretion function of porcine neonatal pancreatic cell clusters.

BACKGROUND: Islet encapsulation techniques have shown limited success in maintaining islet survival and function because encapsulation decreases oxygen supply. In this study, an oxygen-generating scaffold was fabricated to prevent hypoxic cell damage and improve the viability and insulin secretion of islets. METHODS: We fabricated an oxygen-generating scaffold by mixing calcium peroxide (CaO2 ) with polydimethylsiloxane (PDMS). We evaluated the effects of the oxygen-generating PDMS + CaO2 scaffold on viability, caspase-3 and caspase-7 activity, oxygen consumption rate (OCR), glucose-stimulated insulin secretion (GSIS), hypoxic cell marker expression, and reactive oxygen species (ROS) levels in porcine neonatal pancreatic cell clusters (NPCCs). We also fabricated a microfluidic device that allowed measuring the effects of the oxygen-generating scaffold on viability. RESULTS: Oxygen generation by the PDMS + CaO2 scaffold was sustained for more than 24 hours in vitro. NPCCs encapsulated in PDMS + CaO2 showed higher viability than NPCCs in PDMS scaffolds and control NPCCs grown without a scaffold. PDMS + CaO2 -encapsulated NPCCs showed lower caspase-3 and caspase-7 activity, hypoxic cell expression, and ROS levels, and higher OCR and GSIS than those in PDMS or control cells. Using the microfluidic device, we observed that the viability of PDMS + CaO2 -encapsulated NPCCs was higher than that of PDMS-encapsulated NPCCs. CONCLUSIONS: NPCCs in PDMS + CaO2 scaffolds show higher viability and insulin secretion than do NPCCs in PDMS scaffolds and control cells. Therefore, this oxygen-generating scaffold has potential for application in future islet transplantation studies.

Comparison of tracheal reconstruction with allograft, fresh xenograft and artificial trachea scaffold in a rabbit model.

This study evaluated the possibility of tracheal reconstruction with allograft, pig-to-rabbit fresh xenograft or use of a tissue-engineered trachea, and compared acute rejection of three different transplanted tracheal segments in rabbits. Eighteen healthy New Zealand White rabbits weighing 2.5-3.1 kg were transplanted with three different types of trachea substitutes. Two rabbits and two alpha 1, 3-galactosyltransferase gene-knockout pigs weighing 5 kg were used as donors. The rabbits were divided into three groups: an allograft control group consisting of rabbit-to-rabbit allotransplantation animals (n = 6), a fresh xenograft group consisting of pig-to-rabbit xenotransplantation animals (n = 6), and an artificial trachea scaffold group (n = 6). All animals were monitored for 4 weeks for anastomotic complications or infection. The recipients were sacrificed at 28 days after surgery and the grafts were evaluated. On bronchoscopy, all of the fresh xenograft group animals showed ischemic and necrotic changes at 28 days after trachea replacement. The allograft rabbits and the tissue-engineered rabbits showed mild mucosal granulation. The levels of interleukin-2 and interferon-γ in the fresh xenograft group were higher than in other groups. Histopathologic examination of the graft in the fresh xenograft rabbits showed ischemic and necrotic changes, including a loss of epithelium, mucosal granulation, and necrosis of cartilaginous rings. The pig-to-rabbit xenografts showed more severe acute rejection within a month than the rabbits with allograft or artificial trachea-mimetic graft. In addition, the artificial tracheal scaffold used in the present experiment is superior to fresh xenograft and may facilitate tracheal reconstruction in the clinical setting.

Three-Dimensional Bio-Printed Scaffold Sleeves With Mesenchymal Stem Cells for Enhancement of Tendon-to-Bone Healing in Anterior Cruciate Ligament Reconstruction Using Soft-Tissue Tendon Graft.

PURPOSE: To investigate the efficacy of the insertion of 3-dimensional (3D) bio-printed scaffold sleeves seeded with mesenchymal stem cells (MSCs) to enhance osteointegration between the tendon and tunnel bone in anterior cruciate ligament (ACL) reconstruction in a rabbit model. METHODS: Scaffold sleeves were fabricated by 3D bio-printing. Before ACL reconstruction, MSCs were seeded into the scaffold sleeves. ACL reconstruction with hamstring tendon was performed on both legs of 15 adult rabbits (aged 12 weeks). We implanted 15 bone tunnels with scaffold sleeves with MSCs and implanted another 15 bone tunnels with scaffold sleeves without MSCs before passing the graft. The specimens were harvested at 4, 8, and 12 weeks. H&E staining, immunohistochemical staining of type II collagen, and micro-computed tomography of the tunnel cross-sectional area were evaluated. Histologic assessment was conducted with a histologic scoring system. RESULTS: In the histologic assessment, a smooth bone-to-tendon transition through broad fibrocartilage formation was identified in the treatment group, and the interface zone showed abundant type II collagen production on immunohistochemical staining. Bone-tendon healing histologic scores were significantly higher in the treatment group than in the control group at all time points. Micro-computed tomography at 12 weeks showed smaller tibial (control, 9.4 ± 0.9 mm2; treatment, 5.8 ± 2.9 mm2; P = .044) and femoral (control, 9.6 ± 2.9 mm2; treatment, 6.0 ± 1.0 mm2; P = .03) bone-tunnel areas in the treated group than in the control group. CONCLUSIONS: The 3D bio-printed scaffold sleeve with MSCs exhibited excellent results in osteointegration enhancement between the tendon and tunnel bone in ACL reconstruction in a rabbit model. CLINICAL RELEVANCE: If secure biological healing between the tendon graft and tunnel bone can be induced in the early postoperative period, earlier, more successful rehabilitation may be facilitated. Three-dimensional bio-printed scaffold sleeves with MSCs have the potential to accelerate bone-tendon healing in ACL reconstruction.

Smart Microbubble Eluting Theranostic Stent for Noninvasive Ultrasound Imaging and Prevention of Restenosis.

A pH-responsive microbubble-eluting theranostic stent is developed for real-time ultrasound imaging of stent implanted blood vessels and dissolution of fat-rich plaques to prevent the blocking of blood vessels in rats. This smart theranostic stent can be effectively applied to facilitate noninvasive monitoring and prevent restenosis after stent implantation.

Development of Liver Decellularized Extracellular Matrix Bioink for Three-Dimensional Cell Printing-Based Liver Tissue Engineering.

The liver is an important organ and plays major roles in the human body. Because of the lack of liver donors after liver failure and drug-induced liver injury, much research has focused on developing liver alternatives and liver in vitro models for transplantation and drug screening. Although numerous studies have been conducted, these systems cannot faithfully mimic the complexity of the liver. Recently, three-dimensional (3D) cell printing technology has emerged as one of a number of innovative technologies that may help to overcome this limitation. However, a great deal of work in developing biomaterials optimized for 3D cell printing-based liver tissue engineering remains. Therefore, in this work, we developed a liver decellularized extracellular matrix (dECM) bioink for 3D cell printing applications and evaluated its characteristics. The liver dECM bioink retained the major ECM components of the liver while cellular components were effectively removed and further exhibited suitable and adjustable properties for 3D cell printing. We further studied printing parameters with the liver dECM bioink to verify the versatility and fidelity of the printing process. Stem cell differentiation and HepG2 cell functions in the liver dECM bioink in comparison to those of commercial collagen bioink were also evaluated, and the liver dECM bioink was found to induce stem cell differentiation and enhance HepG2 cell function. Consequently, the results demonstrate that the proposed liver dECM bioink is a promising bioink candidate for 3D cell printing-based liver tissue engineering.

Effects of 3D-Printed Polycaprolactone/ß-Tricalcium Phosphate Membranes on Guided Bone Regeneration.

This study was conducted to compare 3D-printed polycaprolactone (PCL) and polycaprolactone/ß-tricalcium phosphate (PCL/ß-TCP) membranes with a conventional commercial collagen membrane in terms of their abilities to facilitate guided bone regeneration (GBR). Fabricated membranes were tested for dry and wet mechanical properties. Fibroblasts and preosteoblasts were seeded into the membranes and rates and patterns of proliferation were analyzed using a kit-8 assay and by scanning electron microscopy. Osteogenic differentiation was verified by alizarin red S and alkaline phosphatase (ALP) staining. An in vivo experiment was performed using an alveolar bone defect beagle model, in which defects in three dogs were covered with different membranes. CT and histological analyses at eight weeks after surgery revealed that 3D-printed PCL/ß-TCP membranes were more effective than 3D-printed PCL, and substantially better than conventional collagen membranes in terms of biocompatibility and bone regeneration and, thus, at facilitating GBR.

Fabrication of a nanofibrous mat with a human skin pattern.

A number of studies on skin tissue regeneration and wound healing have been conducted. Electrospun nanofibers have numerous advantages for use in wound healing dressings. Here, we present an electrospinning method for alteration of the surface morphological properties of electrospun mats because most previous studies focused on the materials used or the introduction of bioactive healing agents. In this study, a micromachined human skin pattern mold was used as a collector in an electrospinning setup to replicate the pattern onto the surface of the electrospun mat. We demonstrated the successful fabrication of a nanofibrous mat with a human skin pattern. To verify its suitability for wound healing, a 14-day in vitro cell culture was carried out. The results indicated that the fabricated mat not only induces equivalent cell viability to the conventional electrospun mat, but also exhibits guidance of cells along the skin pattern without significant deterioration of pattern geometry.

Morphometric analysis of the proximal ulna using three-dimensional computed tomography and computer-aided design: varus, dorsal, and torsion angulation.

PURPOSE: The proximal ulna, particularly the course of the posterior border, has a complex three-dimensional (3D) morphology which has been highlighted recently due to its clinical relevance in relation to surgical treatments. 3D computed tomography (CT) reconstruction and computer-aided design (CAD) based software can help to visualize the complex anatomy and thus aid the investigation of the more detailed morphology of the proximal ulna. METHODS: In our current study, 3D CT reconstruction images of 20 cadavers were imported into the 3D CAD program. Three morphologic angle parameters of the proximal ulna were measured including the dorsal, varus and torsion angulation. The torsion angulation was measured using the flat spot of olecranon dorsal aspect. We measured the total length of the ulna and the distance between the olecranon tip and the apex of dorsal and varus angulation. Furthermore, the thickness of olecranon was also measured for all the specimens. RESULTS: The results showed that the mean dorsal, varus, and torsion angulation was 4.3° (range 2.6°-5.9°), 12.1° (range 7.9°-17.6°), and 22.5° (range 16.6°-30.5°), respectively. The average length ratio of the dorsal and varus angulation apex to the total ulnar length was 26.4 % (range 19.8-30.7 %) and 32.7 % (range 27.5-37.5 %), respectively. The average of olecranon thickness at the proximal tip, mid-olecranon fossa, and at coronoid tip level was 17.8 mm (range 14.1-22.8 mm), 19.7 mm (range 15.8-23.1 mm), and 35.1 mm (range 27.9-41.8 mm), respectively. CONCLUSION: In conclusion, variations in the proximal ulna have to be considered when anatomically contoured dorsal plates are applied. Knowledge of the 3D morphologic anatomy of the proximal ulna would provide important information on fracture reductions, and the design of a precontoured dorsal plate or a prosthetic ulnar stem.

Degradation-controlled tissue extracellular sponge for rapid hemostasis and wound repair after kidney injury.

Patients diagnosed with T1a cancer undergo partial nephrectomy to remove the tumors. In the process of removing the tumors, loss of kidney volume is inevitable, and current surgical methods focus solely on hemostasis and wound closure. Here, we developed an implantable form of decellularized extracellular matrix sponge to target both hemostasis and wound healing at the lesion site. A porous form of kidney decellularized matrix was achieved by fabricating a chemically cross-linked cryogel followed by lyophilization. The prepared kidney decellularized extracellular matrix sponge (kdES) was then characterized for features relevant to a hemostasis as well as a biocompatible and degradable biomaterial. Finally, histological evaluations were made after implantation in rat kidney incision model. Both gelatin sponge and kdES displayed excellent hemocompatibility and biocompatibility. However, after a 4-week observation period, kdES exhibited more favorable wound healing results at the lesion site. This suggests a promising potential for kdES as a supportive material in facilitating wound closure during partial nephrectomy surgery. KdES not only achieved rapid hemostasis for managing renal hemorrhage that is comparable to commercial hemostatic sponges, but also demonstrated superior wound healing outcomes.

A new method of fabricating robust freeform 3D ceramic scaffolds for bone tissue regeneration.

Fabrication of three-dimensional (3D) scaffolds with appropriate mechanical properties and desired architecture for promoting cell growth and new tissue formation is one of the most important efforts in tissue engineering field. Scaffolds fabricated from bioactive ceramic materials such as hydroxyapatite and tricalcium phosphate show promise because of their biological ability to support bone tissue regeneration. However, the use of ceramics as scaffold materials is limited because of their inherent brittleness and difficult processability. The aim of this study was to create robust ceramic scaffolds, which have a desired architecture. Such scaffolds were successfully fabricated by projection-based microstereolithography, and dilatometric analysis was conducted to study the sintering behavior of the ceramic materials. The mechanical properties of the scaffolds were improved by infiltrating them with a polycaprolactone solution. The toughness and compressive strength of these ceramic/polymer scaffolds were about twice those of ceramic scaffolds. Furthermore, the osteogenic gene expression on ceramic/polymer scaffolds was better than that on ceramic scaffolds. Through this study, we overcame the limitations of previous research on fabricating ceramic scaffolds and these new robust ceramic scaffolds may provide a much improved 3D substrate for bone tissue regeneration.

Design and assessment of a microfluidic network system for oxygen transport in engineered tissue.

Oxygen and nutrients cannot be delivered to cells residing in the interior of large-volume scaffolds via diffusion alone. Several efforts have been made to meet the metabolic needs of cells in a scaffold by constructing mass transport channels, particularly in the form of bifurcated networks. In contrast to progress in fabrication technologies, however, an approach to designing an optimal network based on experimental evaluation has not been actively reported. The main objective of this study was to establish a procedure for designing an effective microfluidic network system for a cell-seeded scaffold and to develop an experimental model to evaluate the design. We proposed a process to design a microfluidic network by combining an oxygen transport simulation with biomimetic principles governing biological vascular trees. The simulation was performed with the effective diffusion coefficient (D(e,s)), which was experimentally measured in our previous study. Porous scaffolds containing an embedded microfluidic network were fabricated using the lost mold shape-forming process and salt leaching method. The reliability of the procedure was demonstrated by experiments using the scaffolds. This approach established a practical basis for designing an effective microfluidic network in a cell-seeded scaffold.

Evaluation of the effective diffusivity of a freeform fabricated scaffold using computational simulation.

In scaffold-based tissue engineering, sufficient oxygen and nutrient supply into cells within a scaffold is essential to increase cell viability and the proliferation rate. Generally, oxygen and nutrients reach the cells through the media by diffusion in vitro or in vivo, assuming there is no convection flow through a scaffold with small-sized pores. The scaffold diffusion rate depends mainly on the scaffold pore architecture. Thus, understanding the effect of scaffold pore architecture on the diffusion mechanism is necessary to design an efficient scaffold model. This study proposes a computational method to estimate diffusivity using the finite element analysis (FEA). This method can be applied to evaluate and analyze the effective diffusivity of a freeform fabricated 3D scaffold. The diffusion application module of commercial FEA software was used to calculate the spatial oxygen concentration gradient in a scaffold model medium. The effective diffusivities of each scaffold could be calculated from the oxygen concentration data, which revealed that the scaffold pore architecture influences its effective diffusivity. The proposed method has been verified experimentally and can be applied to design pore architectures with efficient diffusion by increasing our understanding of how the diffusion rate within a scaffold is affected by its pore architecture.

Flexure-based device for cyclic strain-mediated osteogenic differentiation.

Application of low-magnitude strains to cells on small-thickness scaffolds, such as those for rodent calvarial defect models, is problematic, because general translation systems have limitations in terms of generating low-magnitude smooth signals. To overcome this limitation, we developed a cyclic strain generator using a customized, flexure-based, translational nanoactuator that enabled generation of low-magnitude smooth strains at the subnano- to micrometer scale to cells on small-thickness scaffolds. The cyclic strain generator we developed showed predictable operational characteristics by generating a sinusoidal signal of a few micrometers (4.5 µm) without any distortion. Three-dimensional scaffolds fitting the critical-size rat calvarial defect model were fabricated using poly(caprolactone), poly(lactic-co-glycolic acid), and tricalcium phosphate. Stimulation of human adipose-derived stem cells (ASCs) on these fabricated scaffolds using the cyclic strain generator we developed resulted in upregulated osteogenic marker expression compared to the nonstimulated group. These preliminary in vitro results suggest that the cyclic strain generator successfully provided mechanical stimulation to cells on small-thickness scaffolds, which influenced the osteogenic differentiation of ASCs.