True Stress Theory of Matrix in A Composite: A Topical Review.

Whereas mechanics theories for isotropic materials are almost matured, only linear elastic theories for composites were essentially established. This is because only homogenized or approximated stresses are obtainable for a composite. Its mechanical properties must be estimated on a true stress level. According to Eshelby, the true stresses of the fiber are the same as its homogenized counterparts. The true stress theory for the matrix was systematically established by the author, and is reviewed and summarized in the paper. An Excel table-based program for calculating all of the possible true stress components is provided as a supplement for the reader to download. As most composite failures are caused by matrix failures, the true stress theory plays a predominant role in estimating the composite properties outside a linear elastic range. Some challenging composite failures were resolved upon the matrix true stresses, and are highlighted in the paper.

Tensile Strength Prediction of Short Fiber Reinforced Composites.

Essentially, every failure of a short fiber reinforced composite (SFRC) under tension is induced from a matrix failure, the prediction of which is of fundamental importance. This can be achieved only when the homogenized stresses of the matrix are converted into true values in terms of stress concentration factors (SCFs) of the matrix in an SFRC. Such an SCF cannot be determined in the classical way. In this paper, a closed-form formula for the longitudinal tensile SCF in the SFRC is derived from the matrix stresses determined through an elastic approach. The other directional SCFs in an SFRC are the same as those in a continuous fiber composite already available. A bridging model was used to calculate the homogenized stresses explicitly, and a failure prediction of the SFRC with arbitrary fiber aspect ratio and fiber content was made using only the original constituent strength data. Results showed that the volume fraction, the aspect ratio, and the orientation of the fiber all have significant effect on the tensile strength of an SFRC. In a certain range, the tensile strength of an SFRC increases with the increase in fiber aspect ratio and fiber volume content, and the strength of the oriented short fiber is higher than that of the random short fiber arrangement. Good correlations between the predicted and the available measured strengths for a number of SFRCs show the capability of the present method.

Genomic and epigenomic perspectives of T-cell exhaustion in cancer.

In chronic infection and cancer, T cells gradually become exhausted because of the persistent stimulation by antigens. In this process, the overexpression of multiple inhibitory receptors is induced, the production of effective cytokines decreases and the cytotoxicity and proliferation of T cells impairs, all contributing to the failure of T cells in fighting against cancer. Reversing T-cell exhaustion is a promising immunotherapy for cancer that has yielded encouraging results. In this review, we discuss the genomic and epigenomic landscape of T-cell exhaustion in cancer. Also, we introduce the relevant therapeutic interventions for T-cell exhaustion in clinical trials.

CD8+ T-cell exhaustion in cancer: mechanisms and new area for cancer immunotherapy.

Immunotherapies have emerged as the most promising area in cancer treatments in recent years. CD8+ T cells, as one of the primary effector cells of anticancer immunity, however, when infiltrating in cancer tissues, are generally in dysfunctional states termed T-cell exhaustion. Exhausted CD8+ T cells are characterized by impaired activity and proliferative ability, increased apoptotic rate and reduced production of effector cytokines. Such dysfunctional CD8+ T cells serve as a barrier in successful cancer elimination. Investigation on the mechanism of T-cell exhaustion was aiming to sustain or restore the efficiency of CD8+ T cells infiltrating in cancer, which may help to develop novel strategies to overcome cancer. Recent studies have found several vital mechanisms of CD8+ T-cell exhaustion and provided novel avenues through targeting CD8+ T-cell exhaustion to enhance anticancer immunity. Here, we review the recent progress in the study of CD8+ T-cell exhaustion to make a summary and to provide a framework for further researches.

T cell exhaustion in cancer: Mechanisms and clinical implications.

During chronic viral infection or cancer, the immune system usually induces a corresponding immune response against pathogens or cancer cells so as to prevent worsening disease. T cell exhaustion in which reduced and dysfunctional effector T cells lead to immune escape is one of the mechanisms that pathogens or cancer cells get rid of control from the immune system. In this review, we discuss some mechanisms associated with T cell exhaustion and enumerate current methods of reversing T cell exhaustion. We also summarize current targeted treatment strategies and put forward following aspects that required to research.

The protective effect of total phenolics from Oenanthe Javanica on acute liver failure induced by D-galactosamine.

ETHNOPHARMACOLOGY RELEVANCE: Water dropwort [Oenanthe javanica (O. javanica)] is an aquatic perennial herb cultivated in East Asian countries. It has been popularly used in traditional Chinese medicine which is beneficial for the treatment of many diseases, including jaundice and various types of chronic and acute hepatitis. In the present study, we investigated the hepatoprotective effect of total phenolics from O. javanica (TPOJ) against D-galactosamine (D-GalN) induced liver injury in mice. MATERIAL AND METHODS: The hepatoprotective activity of TPOJ (125, 250 and 500mg/kg) was investigated on D-GalN (800mg/kg)-induced liver damages in mice. Blood and liver were collected for biochemical and microscopic analysis. RT-PCR was used to determine the changes in hepatic nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression. Protein levels of iNOS, COX-2, superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) were determined by western blotting. RESULTS: In the animal studies, TPOJ could improve the survival of acute liver failure model significantly and prevente the D-GalN-induced elevation of the serum enzymatic markers and nonenzymatic markers levels significantly. Meanwhile, TPOJ-treatment decreased the malondialdehyde (MDA) level and elevated the content of glutathione (GSH) in the liver as compared to those in the D-GalN group. Hepatic activities and protein expressions of antioxidative enzymes, including SOD, GPx, and CAT were enhanced dose dependently with TPOJ. At the same time, application of TPOJ effectively suppressed the D-GalN-induced proinflammatory mRNA and protein expression of iNOS and COX-2. Subsequently, the serum levels of proinflammatory mediators, nitric oxide (NO) and prostaglandin E2 (PGE2) were reduced. Additionally, histological analyses also showed that TPOJ reduced the extent of liver lesions induced by D-GalN. CONCLUSION: Our investigation demonstrated the hepatoprotective activity of TPOJ and revealed that TPOJ attributed its significance in the traditional use for treating liver diseases.

Investigations of the total flavonoids extracted from flowers of Abelmoschus manihot (L.) Medic against α-naphthylisothiocyanate-induced cholestatic liver injury in rats.

ETHNOPHARMACOLOGY RELEVANCE: The decoction of the flowers of Abelmoschus manihot (L.) Medic was traditionally used for the treatment of jaundice and various types of chronic and acute hepatitis in Anhui and Jiangsu Provinces of China for hundreds of years. Phytochemical studies have indicated that total flavonoids extracted from flowers of A. manihot (L.) Medic (TFA) were the major constituents of the flowers. Our previous studies have investigated the hepatoprotective effects of the TFA against carbon tetrachloride (CCl4) induced hepatocyte damage in vitro and liver injury in vivo. This study aimed to investigate the protective effects and mechanisms of TFA on α-naphthylisothiocyanate (ANIT)-induced cholestatic liver injury in rats. MATERIAL AND METHODS: The hepatoprotective activities of TFA (125, 250 and 500mg/kg) were investigated on ANIT-induced cholestatic liver injury in rats. The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) were used as indices of hepatic cell damage and measured. Meanwhile, the serum levels of alkaline phosphatase (ALP), gamma-glutamyltransferase (GGT), total bilirubin (TBIL), direct bilirubin (DBIL), and total bile acid (TBA) were used as indices of biliary cell damage and cholestasis and evaluated. Hepatic malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), glutathione transferase (GST), tumor necrosis factor-α (TNF-α) and nitric oxide (NO) were measured in the liver homogenates. The bile flow in 4h was estimated and the histopathology of the liver tissue was evaluated. Furthermore, the expression of transporters, bile salt export pump (BSEP), multidrug resistance-associated protein 2 (MRP2), and Na(+)-taurocholate cotransporting polypeptide (NTCP) were studied by western blot and reverse transcription-quantitative real-time polymerase chain reaction (RT-PCR) to elucidate the protective mechanisms of TFA against ANIT-induced cholestasis. RESULTS: The oral administration of TFA to ANIT-treated rats could reduce the increases in serum levels of ALT, AST, LDH, ALP, GGT, TBIL, DBIL and TBA. Decreased bile flow by ANIT was restored with TFA treatment. Concurrent administration of TFA reduced the severity of polymorphonuclear neutrophil infiltration and other histological damages, which were consistent with the serological tests. Hepatic MDA and GSH contents in liver tissue were reduced, while SOD and GST activities, which had been suppressed by ANIT, were elevated in the groups pretreated with TFA. With TFA intervention, levels of TNF-α and NO in liver were decreased. Additionally, TFA was found to increase the expression of liver BSEP, MRP2, and NTCP in both protein and mRNA levels in ANIT-induced liver injury with cholestasis. CONCLUSION: TFA exerted protective effects against ANIT-induced liver injury. The possible mechanisms could be related to anti-oxidative damage, anti-inflammation and regulating the expression of hepatic transporters. It layed the foundation for the further research on the mechanisms of cholestasis as well as the therapeutic effects of A. manihot (L.) Medic for the treatment of jaundice.

A controlled release system of titanocene dichloride by electrospun fiber and its antitumor activity in vitro.

In order to improve both safety and efficacy of cancer chemotherapy of titanocene dichloride and overcome the shortcomings such as instability and short half-life in the human body, we report a controlled release system of titanocene dichloride by electrospun fiber and its in vitro antitumor activity against human lung tumor spca-1 cells. The system was developed by electrospinning. The release profiles of titanocene dichloride in PBS were researched by UV-Vis spectrophotometer. In vitro antitumor activities of the fibers were examined by MTT method. Titanocene dichloride was well incorporated in biodegradable poly(L-lactic acid) fibers. XRD results suggest that titanocene dichloride exists in the amorphous form in the fibers. The controlled release of titanocene dichloride can be gained for long time. MTT showed actual titanocene dichloride content 40, 80, 160 and 240 mg/L from the fibers mat, cell growth inhibition rates of 11.2%, 22.1%, 44.2% and 68.2% were achieved, respectively. The titanocene dichloride released has obvious inhibition effect against lung tumor cells. The system has an effect of controlled release of titanocene dichloride and may be used as an implantable anticancer drug in clinical applications in the future.

Development of braided drug-loaded nanofiber sutures.

The objectives of this work are twofold. Firstly, while most work on electrospinning is limited to the development of only functional materials, a structural application of electrospun nanofibers is explored. Secondly, a drug-loaded tissue suture is fabricated and its various properties are characterized. Braided drug-loaded nanofiber sutures are obtained by combining an electrospinning process with a braiding technique followed by a coating procedure. Two different electrospinning techniques, i.e. blend and coaxial electrospinning, to incorporate a model drug cefotaxime sodium (CFX-Na) into poly(L-lactic acid) (PLLA) nanofibers have been applied and compared with each other. Properties of the braided drug-loaded sutures are characterized through a variety of methods including SEM, TEM and tensile testing. The results show that the nanofibers had a preferable micromorphology. The drug was incorporated into the polymer nanofibers homogeneously, with no cross-linking. The nanofibers maintained their fibrous structures. An in vitro release study indicates that the drug-loaded nanofibers fabricated by blend electrospinning and coaxial electrospinning had a different drug release behavior. An inhibition zone experiment shows that both sutures obtained from the nanofibers of the different electrospinning techniques had favorable antibacterial properties. The drug-loaded sutures had preferable histological compatibility performance compared with commercial silk sutures in an in vivo comparative study.

[Protective effect of extract of Apocynum venetum on kidneys of streptozotocin-induced diabetic rats].

The aim of this study is to investigate the effects and mechanism of extract of Apocynum venetum (AV) on kidneys of streptozotocin-induced diabetic rats. Diabetes mellitus (DM) was induced in rats by intraperitoneal injection of streptozotocin (STZ). The indexes of the blood glucose, renal function and oxidative stress were observed. The DM rats were administrated with the AV for 8 weeks, the above-mentioned indexes were detected. The blood glucose level, BUN, 24 h urine protein excretion, urine volume, renal index, renal cortex's MDA level in model groups all increased significantly. Renal cortex's SOD and GSH activities decreased significantly compared with the normal control group (P < 0.05). The above-mentioned indexes were significantly improved by the AV treatment (P < 0.05). AV have protective effects on renal function of kidneys of streptozotocin-induced diabetic rats, and maybe via inhibition of the renal oxidative stress.

Fabrication and characterization of chitosan coated braided PLLA wire using aligned electrospun fibers.

The development of functionalized braided wires coated with chitosan that can be used for tissue suturing and tissue regeneration is the subject of this work. Poly(L: -lactic acid) (PLLA) braided wires were successfully fabricated by combining an electrospinning technique and alignment collection with a mini-type braiding method. The resulting PLLA wires with and without chitosan coating were characterized through a variety of methods including scanning electron microscopy (SEM), X-ray photoelectronic spectra (XPS) and tensile mechanical testing. Hemolytic property, kinetic hemostasis behavior, platelet adhesion, erythrocyte adhesion, and water uptake ability of the wires were explored. The results showed that a nearly comparable mechanical behavior of the braided wires with some commercial suture could be obtained with well-aligned fibers, and no significant difference in tensile performances were recognized with and without the introduction of chitosan. The PLLA wires coated with chitosan were found to have better prohemostatic activity than those without a chitosan coating.

Fabrication of drug-loaded electrospun aligned fibrous threads for suture applications.

In this work, drug-loaded fibers and threads were successfully fabricated by combining electrospinning with aligned fibers collection. Two different electrospinning processes, that is, blend and coaxial electrospinning, to incorporate a model drug tetracycline hydrochloride (TCH) into poly(L-lactic acid) (PLLA) fibers have been used and compared with each other. The resulting composite ultrafine fibers and threads were characterized through scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, X-ray diffraction, differential scanning calorimetry, and tensile testing. It has been shown that average diameters of the fibers made from the same polymer concentration depended on the processing method. The blend TCH/PLLA fibers showed the smallest fiber diameter, whereas neat PLLA fibers and core-shell TCH-PLLA fibers showed a larger proximal average diameter. Higher rotating speed of a wheel collector is helpful for obtaining better-aligned fibers. Both the polymer and the drug in the electrospun fibers have poor crystalline property. In vitro release study indicated that threads made from the core-shell fibers could suppress the initial burst release and provide a sustained drug release useful for the release of growth factor or other therapeutic drugs. On the other hand, the threads from the blend fibers produced a large initial burst release that may be used to prevent bacteria infection. A combination of these results suggests that electrospinning technique provides a novel way to fabricate medical agents-loaded fibrous threads for tissue suturing and tissue regeneration applications.

[Effects of hyperin on the cccDNA of duck hepatitis B virus and its immunological regulation].

The aim of this study is to investigate the effect of hyperin on the cccDNA of duck hepatitis B virus and its immunological regulation. Duck hepatitis B virus (DHBV) infection model and normal mouse spleen lymphocyte were used to evaluate the anti-HBV and immunoregulation effects. The DHBV-DNA of serum was detected at different time points by using serum DOT-BLOT hybridization. Polymerase chain reaction (PCR) was used for the determination of nuclear covalent closed circular DNA (cccDNA). Cytokine secretion was determined by ELISA method. DHBV-DNA were inhibited by hyperin (25 or 50 mg x kg(-1)), while cccDNA of liver could be eliminated efficiently by hyperin (25 or 50 mg x kg(-1), P < 0.05, P < 0.01). The T helper 1 effector cytokine was markedly enhanced by hyperin (25 or 50 microg x mL(-1), P < 0.01). In conclusion, hyperin has anti-HBV activity via multiple targets and pathways, and cccDNA may be one of the important targets.

In vivo and in vitro anti-hepatitis B virus activity of total phenolics from Oenanthe javanica.

The traditional Chinese medicine Oenanthe javanica (OJ) has been used for many years, mainly for the treatment of inflammatory conditions including hepatitis. In this study, human hepatoma Hep G2.2.15 cells culture system and duck hepatitis B virus (DHBV) infection model were used as in vivo and in vitro models to evaluate the anti-HBV effects of total phenolics from Oenanthe javanica (OJTP). The HBeAg and HBsAg concentrations in cell culture medium were determined by using the enzyme immunoassay kit after Hep G2.2.15 cells were treated with OJTP for 9 d. DHBV-DNA in duck serum was analyzed by dot blot hybridization assay. In the cell model, OJTP could dose-dependently inhibit the production of the HBeAg and HBsAg, and the inhibition rates of OJTP on HBeAg and HBsAg in the Hep G2.2.15 cells were 70.12% and 72.61% on day 9, respectively. In the DHBV infection model, OJTP also reduced HBV DNA level in a dose-dependent manner. The DHBV-DNA levels decreased significantly after the treatment with 0.10 g kg(-1)d(-1) and 0.20 g kg(-1)d(-1) OJTP. The inhibition of the peak of viremia was at the maximum at the dose of 0.20 g kg(-1)d(-1) and reached 64.10% on day 5 and 66.48% on day 10, respectively. Histopathological evaluation of the liver revealed significant improvement by OJTP. In conclusion, our results demonstrate that OJTP can efficiently inhibit HBV replication in Hep G2.2.15 cells line in vitro and inhibit DHBV replication in ducks in vivo. OJTP therefore warrants further investigation as a potential therapeutic agent for HBV infections.

In vivo and in vitro antiviral activity of hyperoside extracted from Abelmoschus manihot (L) medik.

AIM: To assess the anti-hepatitis B virus (HBV) effect of hyperoside extracted from Abelmoschus manihot (L) medik. METHODS: The human hepatoma Hep G2.2.15 cell culture system and duck hepatitis B virus (DHBV) infection model were used as in vivo and in vitro models to evaluate the anti-HBV effects. RESULTS: In the cell model, the 50% toxic concentration of hyperoside was 0.115 g/L; the maximum nontoxic concentration was 0.05 g/L. On the maximum nontoxic concentrations, the inhibition rates of hyperoside on HBeAg and HBsAg in the 2.2.15 cells were 86.41% and 82.27% on d 8, respectively. In the DHBV infection model, the DHBV-DNA levels decreased significantly in the treatment of 0.05 g x kg(-1 ) x d(-1 ) and 0.10 g x kg(-1) x d(-1) dosage groups of hyperoside (P<0.01). The inhibition of the peak of viremia was at the maximum at the dose of 0.10 g x kg(-1 ) x d(-1) and reached 60.79% on d 10 and 69.78% on d 13, respectively. CONCLUSION: These results suggested that hyperoside is a strong inhibitor of HBsAg and HBeAg secretion in 2.2.15 cells and DHBV-DNA levels in the HBV-infected duck model.

Ultravilolet-radiation-induced graft polymerization of acrylamide onto the melt-blown polypropylene filter element by dynamic method.

By dynamic method under UV irradiation, commercial melt-blown polypropylene (PPMB) filter element was modified with acrylamide (AAm) using benzophenone (BP) as initiator. Attenuated total reflection-Fourier transform infrared spectroscopy and scanning electron microscope verified that polyacrylamide chain was grafted on the fiber surface of PPMB filter element. Elemental content analysis with energy dispersive X-ray of fibers revealed that the polymerization content in the inner part of filter element was relatively higher than that in the outer. Degree of grafting changed with initiator concentration, monomer concentration, reaction temperature and reached 2.6% at the reaction condition: CBP=0.06 mol/L, CAAm=2.0 mol/L, irradiation time: 80 min, temperature: 600 degrees C. Relative water flux altered with the hydrophilicity and pore size of filter element. In the antifouling test, the modified filter gave greater flux recovery (approximately 70%) after filtration of the water extract of Liuweidihuang, suggesting that the fouling layer was more easily reversible due to the hydrophilic nature of the modified filter.

Encapsulating drugs in biodegradable ultrafine fibers through co-axial electrospinning.

This article describes an electrospinning process to fabricate double-layered ultrafine fibers. A bioabsorbable polymer, Polycaprolactone (PCL), was used as the outer layer or the shell and two medically pure drugs, Resveratrol (RT, a kind of antioxidant) and Gentamycin Sulfate (GS, an antibiotic), were used as the inner layers or the cores. Morphology and microstructure of the ultrafine fibers were characterized by scanning electron microscope (SEM) and transmission electron microscopy (TEM), whereas mechanical performance of them was understood through tensile test. In vitro degradation rates of the nanofibrous membranes were determined by measuring their weight loss when immersed in pH 7.4 phosphate-buffered saline (PBS) mixed with certain amount of Pseudomonas lipase for a maximum of 7 days. The drug release behaviors of the RT and GS were measured using a high performance liquid chromatography (HPLC) and ultraviolet-visible (UV-vis) spectroscopy, respectively. It has been found that the drug solutions without any fiber-forming additive could be encapsulated in the PCL ultrafine fibers, although they alone cannot be made into a fiber form. Beads on the fiber surface influenced the tensile behavior of the ultrafine fibers remarkably. When the core solvent was miscible with the shell solvent, higher drug concentration decreased the bead formation and thus favored the mechanical performance. The situation, however, became different if the two solvents were immiscible with each other. The degradation rate was closely related to hydrophilicity of the drugs in the cores. Higher hydrophilicity apparently led to faster degradation. The release profiles of the RT and GS exhibited a sustained release characteristic, with no burst release phenomenon.

Recent development of polymer nanofibers for biomedical and biotechnological applications.

Research in polymer nanofibers has undergone significant progress in the last one decade. One of the main driving forces for this progress is the increasing use of these polymer nanofibers for biomedical and biotechnological applications. This article presents a review on the latest research advancement made in the use of polymer nanofibers for applications such as tissue engineering, controlled drug release, wound dressings, medical implants, nanocomposites for dental restoration, molecular separation, biosensors, and preservation of bioactive agents.

Effect of Oenanthe javanica flavone on human and duck hepatitis B virus infection.

AIM: To study the antiviral effect of Oenanthe javanica flavones (OjF) on human hepatoma HepG2.2.15 culture system and duck hepatitis B virus (DHBV) infection. METHODS: (1) After incubation for 24 h, the 2.2.15 cells were treated with different concentrations of OjF for 12 d. The cell alteration was observed by microscope. The presence of HBsAg and HBeAg were measured using the enzyme immunoassay kit after 2.2.15 cells were treated with OjF for 9 d. (2) Ducklings infected with DHBV intravenously were divided into 5 groups and treated with OjF, acyclovir (ACV), and normal saline respectively for 10 d. All the ducklings were bled before, during, and after treatments at different times, and serum levels of DHBV-DNA were detected by a dot-blot hybridization assay. RESULTS: (1) The 50% toxic concentration (TC50) of OjF was 2.28 g/L. The maximum nontoxic concentration (TC0) was 1.00 g/L. In nontoxic concentrations, OjF significantly inhibited HBsAg and HBeAg in 2.2.15 cells after 9 d of treatment (P<0.05, P<0.01). (2) The DHBV-DNA levels decreased significantly after the treatment with 0.50 and 1.00 g/kg of OjF (P<0.01). The inhibition of the peak of viremia was maximum at a dose of 1.00 g/kg and reached 54.3% on d 5 and 64.5% on d 10, respectively. CONCLUSION: The results demonstrate that OjF is a strong inhibitor of HBsAg and HBeAg secretion in 2.2.15 cells and DHBV-DNA levels in the HBV-infected duck model.

Electrospinning of gelatin fibers and gelatin/PCL composite fibrous scaffolds.

In this article, ultrafine gelatin (Gt) fibers were successfully produced with the use of the electrical spinning or electrospinning technique. A fluorinated alcohol of 2,2,2-trifluoroethanol (TFE) was used as the dissolving solvent. The morphology of the electrospun gelatin fibers was found to be dependent on the alteration of gelatin concentration ranging from 2.5% w/v to 12.5% w/v at 2.5% increment intervals. Based on the electrospun gelatin fibers obtained, 10% w/v gelatin/TFE solution was selected and mixed with 10% w/v poly(epsilon-caprolactone) (PCL) in TFE at a ratio of 50:50 and co-electrospun to produce gelatin/PCL composite membranes. Contact-angle measurement and tensile tests indicated that the gelatin/PCL complex fibrous membrane exhibited improved mechanical properties as well as more favorable wettability than that obtained from either gelatin or PCL alone. The gelatin/PCL fibrous membranes were further investigated as a promising scaffold for bone-marrow stromal cell (BMSC) culture. Scanning electron microscopy (SEM) and laser confocal microscopy observations showed that the cells could not only favorably attach and grow well on the surface of these scaffolds, but were also able to migrate inside the scaffold up to 114 microm within 1 week of culture. These results suggest the potential of using composite gelatin/PCL fibrous scaffolds for engineering three-dimensional tissues.