[Extracellular Matrix Stiffness Induces Mitochondrial Morphological Heterogeneity via AMPK Activation]. / 细胞外基质硬度通过AMPK调控干细胞线粒体的形态异质性.

Objective: To investigate the mechanical responses of mitochondrial morphology to extracellular matrix stiffness in human mesenchymal stem cells (hMSCs) and the role of AMP-activated protein kinase (AMPK) in the regulation of mitochondrial mechanoresponses. Methods: Two polyacrylamide (PAAm) hydrogels, a soft one with a Young's modulus of 1 kPa and a stiff one of 20 kPa, were prepared by changing the monomer concentrations of acrylamide and bis-acrylamide. Then, hMSCs were cultured on the soft and stiff PAAm hydrogels and changes in mitochondrial morphology were observed using a laser confocal microscope. Western blot was performed to determine the expression and activation of AMPK, a protein associated with mitochondrial homeostasis. Furthermore, the activation of AMPK was regulated on the soft and stiff matrixes by AMPK activator A-769662 and the inhibitor Compound C, respectively, to observe the morphological changes of mitochondria. Results: The morphology of the mitochondria in hMSCs showed heterogeneity when there was a change in gel stiffness. On the 1 kPa soft matrix, 74% mitochondria exhibited a dense, elongated filamentous network structure, while on the 20 kPa stiff matrix, up to 63.3% mitochondria were fragmented or punctate and were sparsely distributed. Western blot results revealed that the phosphorylated AMPK (p-AMPK)/AMPK ratio on the stiff matrix was 1.6 times as high as that on the soft one. Immunofluorescence assay results revealed that the expression of p-AMPK was elevated on the hard matrix and showed nuclear localization, which indicated that the activation of intracellular AMPK increased continuously along with the increase in extracellular matrix stiffness. When the hMSCs on the soft matrix were treated with A-769662, an AMPK activator, the mitochondria transitioned from a filamentous network morphology to a fragmented morphology, with the ratio of filamentous network decreasing from 74% to 9.5%. Additionally, AMPK inhibition with Compound C promoted mitochondrial fusion on the stiff matrix and significantly reduced the generation of punctate mitochondria. Conclusion: Extracellular matrix stiffness regulates mitochondrial morphology in hMSCs through the activation of AMPK. Stiff matrix promotes the AMPK activation, resulting in mitochondrial fission and the subsequent fragmentation of mitochondria. The impact of matrix stiffness on mitochondrial morphology can be reversed by altering the level of AMPK phosphorylation.

The study of wound healing activity of Thespesia populnea L. bark, an approach for accelerating healing through nanoparticles and isolation of main active constituents.

The present study provides an evaluation for the wound healing activity of the ethanolic extract of Thespesia populnea L. bark (EBE) and its successive fractions in two doses level (1&2%), designed for determining the most bioactive fraction and the suitable dose. Furthermore, development of the most convenient formulation for these bioactive fractions through either their direct incorporation into hydrogel formulations or incorporation of chitosan-loaded nanoparticles with these bioactive fractions into hydrogel formulations. The highest excision wound healing activity was observed in petroleum ether (Pet-B) followed by ethyl acetate (Etac-B) fractions at the high dose (2%). The most suitable formulation designed for the Etac-B fraction was found to be the chitosan-loaded nanoparticles incorporated in the hydrogel formulation, while the conventional hydrogel formulation was observed to be the highly acceptable formulation for Pet-B fraction. Further phytochemical studies of the bioactive fractions led to the isolation of many compounds of different chemical classes viz; beta-sitosterol and lupeol acetate isolated from the Pet-B, in addition to cyanidin and delphinidin from the Etac-B. Our results revealed that EBE and its bioactive fractions (Pet-B & Etac-B) could be considered as strong wound healers through their anti-oxidant and anti-inflammatory activities, in addition to stimulating collagen synthesis.

Substrate stiffness modulates cardiac fibroblast activation, senescence, and proinflammatory secretory phenotype.

In vitro cultures of primary cardiac fibroblasts (CFs), the major extracellular matrix (ECM)-producing cells of the heart, are used to determine molecular mechanisms of cardiac fibrosis. However, the supraphysiologic stiffness of tissue culture polystyrene (TCPS) triggers the conversion of CFs into an activated myofibroblast-like state, and serial passage of the cells results in the induction of replicative senescence. These phenotypic switches confound the interpretation of experimental data obtained with cultured CFs. In an attempt to circumvent TCPS-induced activation and senescence of CFs, we used poly(ethylene glycol) (PEG) hydrogels as cell culture platforms with low and high stiffness formulations to mimic healthy and fibrotic hearts, respectively. Low hydrogel stiffness converted activated CFs into a quiescent state with a reduced abundance of α-smooth muscle actin (α-SMA)-containing stress fibers. Unexpectedly, lower substrate stiffness concomitantly augmented CF senescence, marked by elevated senescence-associated ß-galactosidase (SA-ß-Gal) activity and increased expression of p16 and p21, which are antiproliferative markers of senescence. Using dynamically stiffening hydrogels with phototunable cross-linking capabilities, we demonstrate that premature, substrate-induced CF senescence is partially reversible. RNA-sequencing analysis revealed widespread transcriptional reprogramming of CFs cultured on low-stiffness hydrogels, with a reduction in the expression of profibrotic genes encoding ECM proteins, and an attendant increase in expression of NF-κB-responsive inflammatory genes that typify the senescence-associated secretory phenotype (SASP). Our findings demonstrate that alterations in matrix stiffness profoundly impact CF cell state transitions, and suggest mechanisms by which CFs change phenotype in vivo depending on the stiffness of the myocardial microenvironment in which they reside.NEW & NOTEWORTHY Our findings highlight the advantages and pitfalls associated with culturing cardiac fibroblasts on hydrogels of varying stiffness. The findings also define stiffness-dependent signaling and transcriptional networks in cardiac fibroblasts.

3D in vitro hydrogel models to study the human lung extracellular matrix and fibroblast function.

The pulmonary extracellular matrix (ECM) is a macromolecular structure that provides mechanical support, stability and elastic recoil for different pulmonary cells including the lung fibroblasts. The ECM plays an important role in lung development, remodeling, repair, and the maintenance of tissue homeostasis. Biomechanical and biochemical signals produced by the ECM regulate the phenotype and function of various cells including fibroblasts in the lungs. Fibroblasts are important lung structural cells responsible for the production and repair of different ECM proteins (e.g., collagen and fibronectin). During lung injury and in chronic lung diseases such as asthma, idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD), an abnormal feedback between fibroblasts and the altered ECM disrupts tissue homeostasis and leads to a vicious cycle of fibrotic changes resulting in tissue remodeling. In line with this, using 3D hydrogel culture models with embedded lung fibroblasts have enabled the assessment of the various mechanisms involved in driving defective (fibrotic) fibroblast function in the lung's 3D ECM environment. In this review, we provide a summary of various studies that used these 3D hydrogel models to assess the regulation of the ECM on lung fibroblast phenotype and function in altered lung ECM homeostasis in health and in chronic respiratory disease.

Biomimetic approach for an articular cartilage patch: Combination of decellularized cartilage matrix and silk-elastin-like-protein (SELP) hydrogel.

Articular cartilage degradation due to injury, disease and aging is a common clinical issue as current regenerative therapies are unable to fully replicate the complex microenvironment of the native tissue which, being avascular, is featured by very low ability to self-regenerate. The extracellular matrix (ECM), constituting almost 90% of the entire tissue, plays a critical role in its function and resistance to compressive forces. In this context, the current tissue engineering strategies are only partially effective in restoring the biology and function of the native tissue. A main issue in tissue regeneration is treatment failure due to scarce integration of the engineered construct, often following a gradual detachment of the graft. In this scenario, we aimed to create an adhesive patch able to adequately support cartilage regeneration as a promising tool for the treatment of cartilage injuries and diseases. For this, we produced an engineered construct composed of decellularized ECM (dECM) obtained from horse joint cartilage, to support tissue regeneration, coupled with a Silk-Elastin-Like Proteins (SELP) hydrogel, which acts as a biological glue, to guarantee an adequate adherence to the host tissue. Following the production of the two biomaterials we characterized them by assessing: 1) dECM morphological, chemical, and ultrastructural features along with its capability to support chondrocyte proliferation, specific marker expression and ECM synthesis; 2) SELP microarchitecture, cytocompatibility and mechanical properties. Our results demonstrated that both materials hold unique properties suitable to be exploited to produce a tailored microenvironment to support cell growth and differentiation providing a proof of concept concerning the in vitro biological and mechanical efficacy of the construct. The SELP hydrogel displayed a very interesting physical behavior due to its high degree of resistance to mechanical stress, which is generally associated with physiological mechanical load during locomotion. Intriguingly, the shear-thinning behavior of the hydrogel may also make it suitable to be applied and spread over non-homogeneous surfaces, therefore, we hypothesize that the hybrid biomaterial proposed may be a real asset in the treatment of cartilage defects and injuries.

Engineered biomechanical microenvironment of articular chondrocytes based on heterogeneous GelMA hydrogel composites and dynamic mechanical compression.

Tissue-engineered articular cartilage constructs are currently not able to equal native tissues in terms of mechanical and biological properties. A major cause lies in the deficiency in engineering the biomechanical microenvironment (BMME) of articular chondrocytes. In this work, to engineer the BMME of articular chondrocytes, heterogeneous hydrogel structures of gelatin methacrylated (GelMA) containing differential-stiffness domains were first fabricated, and then periodic dynamic mechanical stimulations were applied to the hydrogel structures. The chondrocyte phenotype of ATDC5 cells was enhanced as the spatial differentiation in stiffness was increased in the hydrogel structures and was further strengthened by dynamic mechanical stimulation. It was speculated that the mechanical signals generated by the engineered BMME were sensed by the cells through the integrin ß1-FAK signaling pathway. This study revealed the key role of the combined effects of differential and dynamic BMME on the chondrocyte phenotype, which could provide theoretical guidance for highly active tissue-engineered articular cartilage.

3D bioprinting of tissue constructs employing dual crosslinking of decellularized extracellular matrix hydrogel.

Bioprinted tissues are currently being utilized for drug and cosmetic screening mostly, but the long-term goal is to achieve human scale functional tissues and organs for transplantation. Hence, recapitulating the multiscale architecture, 3D structures, and complexity of native tissues is the key to produce bioengineered tissues/organs. Decellularized extracellular matrix (dECM)-based biomaterials are widely being used as bioinks for 3D bioprinting for tissue engineering applications. Their potential to provide excellent biocompatibility for the cells drove researchers to use them extensively. However, the decellularization process involves many detergents and enzymes which may contribute to their loss of mechanical properties. Moreover, thermal gelation of dECM-based hydrogels is typically slow which affects the shape fidelity, printability, and physical properties while printing complex structures with 3D printing. But, thermally gelled dECM hydrogels provide excellent cell viability and functionality. To overcome this, a novel dual crosslinking of unmodified dECM has been proposed in this study to render shape fidelity and enhance cell viability and functionality. The dECM-based bioink can be initially polymerized superficially on exposure to light to achieve immediate stability and can attain further stability upon thermal gelation. This dual crosslinking mechanism can maintain the microenvironment of the structure, hence allowing the printing of stable flexible structures. Optimized concentrations of novel photo crosslinkers have been determined and printing of a few complex-shaped anatomical structures has been demonstrated. This approach of fabricating complex scaffolds employing dual crosslinking can be used for the bioprinting of different complex tissue structures with tissue-specific dECM based bioinks.

Evaluation of osteogenic potential of demineralized dentin matrix hydrogel for bone formation.

OBJECTIVES: Dentin, the bulk material of the tooth, resemble the bone's chemical composition and is considered a valuable bone substitute. In the current study, we assessed the cytotoxicity and osteogenic potential of demineralized dentin matrix (DDM) in comparison to HA nanoparticles (n-HA) on bone marrow mesenchymal stem cells (BMMSCs) using a hydrogel formulation. MATERIALS AND METHODS: Human extracted teeth were minced into particles and treated via chemical demineralization using ethylene diamine tetra-acetic acid solution (EDTA) to produce DDM particles. DDM and n-HA particles were added to the sodium alginate then, the combination was dripped into a 5% (w/v) calcium chloride solution to obtain DDM hydrogel (DDMH) or nano-hydroxyapatite hydrogel (NHH). The particles were evaluated by dynamic light scattering (DLS) and the hydrogels were evaluated via scanning electron microscope (SEM). BMMSCs were treated with different hydrogel concentrations (25%, 50%, 75% and neat/100%) and cell viability was evaluated using MTT assay after 72 h of culture. Collagen-I (COL-I) gene expression was studied with real-time quantitative polymerase chain reaction (RT-qPCR) after 3 weeks of culture and alkaline phosphatase (ALP) activity was assessed using enzyme-linked immune sorbent assay (ELISA) over 7th, 10th, 14th and 21st days of culture. BMMSCs seeded in a complete culture medium were used as controls. One-way ANOVA was utilized to measure the significant differences in the tested groups. RESULTS: DLS measurements revealed that DDM and n-HA particles had negative values of zeta potential. SEM micrographs showed a porous microstructure of the tested hydrogels. The viability results revealed that 100% concentrations of either DDMH or NHH were cytotoxic to BMMSCs after 72 h of culture. However, the cytotoxicity of 25% and 50% concentrations of DDMH were not statistically significant compared to the control group. RT-qPCR showed that COL-I gene expression was significantly upregulated in BMMSCs cultured with 50% DDMH compared to all other treated or control groups (P < 0.01). ELISA analysis revealed that ALP level was significantly increased in the groups treated with 50% DDMH compared to 50% NHH after 21 days in culture (P < 0.001). CONCLUSION: The injectable hydrogel containing demineralized dentin matrix was successfully formulated. DDMH has a porous structure and has been shown to provide a supporting matrix for the viability and differentiation of BMMSCs. A 50% concentration of DDMH was revealed to be not cytotoxic to BMMSCs and may have a great potential to promote bone formation ability.

5-Azacytidine incorporated skeletal muscle-derived hydrogel promotes rat skeletal muscle regeneration.

Decellularized skeletal muscle is a promising biomaterial for muscle regeneration due to the mimicking of the natural microenvironment. Previously, it has been reported that 5-Azacytidine (5-Aza), a DNA methyltransferase inhibitor, induces myogenesis in different types of stem cells. In the current study, we investigated the effect of 5-Aza incorporated muscle-derived hydrogel on the viability and proliferation of muscle-derived stem cells (MDSCs) in vitro and muscle regeneration in vivo. Wistar rat skeletal muscles were decellularized using a physico-chemical protocol. The decellularized tissue was analyzed using SEM, histological staining and evaluation of DNA content. Then, muscle-derived hydrogel was made from Pepsin-digested decellularized muscle tissues. 5-Aza was physically adsorbed in prepared hydrogels. Then, MDSCs were cultured on hydrogels with/without 5-Aza, and their proliferation and cell viability were determined using LIVE/DEAD and DAPI staining. Moreover, myectomy lesions were done in rat femoris muscles, muscle-derived hydroges with/without 5-Aza were injected to the myectomy sites, and histological evaluation was performed after three weeks. The analysis of decellularized muscle tissues showed that they maintained extracellular matrix components of native muscles, while they lacked DNA. LIVE/DEAD and DAPI staining showed that the hydrogel containing 5-Aza supported MDSCs viability. Histological analysis of myectomy sites showed an improvement in muscle regeneration after administration of 5-Aza incorporated hydrogel. These findings suggest that the combination of 5-Aza with skeletal muscle hydrogel may serve as an alternative treatment option to improve the regeneration of injured muscle tissue.

Highly Efficient Cardiac Differentiation and Maintenance by Thrombin-Coagulated Fibrin Hydrogels Enriched with Decellularized Porcine Heart Extracellular Matrix.

Biochemical and biophysical properties instruct cardiac tissue morphogenesis. Here, we are reporting on a blend of cardiac decellularized extracellular matrix (dECM) from porcine ventricular tissue and fibrinogen that is suitable for investigations employing an in vitro 3D cardiac cell culture model. Rapid and specific coagulation with thrombin facilitates the gentle inclusion of cells while avoiding sedimentation during formation of the dECM-fibrin composite. Our investigations revealed enhanced cardiogenic differentiation in the H9c2 myoblast cells when using the system in a co-culture with Nor-10 fibroblasts. Further enhancement of differentiation efficiency was achieved by 3D embedding of rat neonatal cardiomyocytes in the 3D system. Calcium imaging and analysis of beating motion both indicate that the dECM-fibrin composite significantly enhances recovery, frequency, synchrony, and the maintenance of spontaneous beating, as compared to various controls including Matrigel, pure fibrin and collagen I as well as a fibrin-collagen I blend.

Anisotropic dense collagen hydrogels with two ranges of porosity to mimic the skeletal muscle extracellular matrix.

Despite the crucial role of the extracellular matrix (ECM) in the organotypic organization and function of skeletal muscles, most 3D models do not mimic its specific characteristics, namely its biochemical composition, stiffness, anisotropy, and porosity. Here, a novel 3D in vitro model of muscle ECM was developed reproducing these four crucial characteristics of the native ECM. An anisotropic hydrogel mimicking the muscle fascia was obtained thanks to unidirectional 3D printing of dense collagen with aligned collagen fibrils. The space between the different layers was tuned to generate an intrinsic network of pores (100 µm) suitable for nutrient and oxygen diffusion. By modulating the gelling conditions, the mechanical properties of the construct reached those measured in the physiological muscle ECM. This artificial matrix was thus evaluated for myoblast differentiation. The addition of large channels (600 µm) by molding permitted to create a second range of porosity suitable for cell colonization without altering the physical properties of the hydrogel. Skeletal myoblasts embedded in Matrigel®, seeded within the channels, organized in 3D, and differentiated into multinucleated myotubes. These results show that porous and anisotropic dense collagen hydrogels are promising biomaterials to model skeletal muscle ECM.

Locust bean gum hydrogels are bioadhesive and improve indole-3-carbinol cutaneous permeation: influence of the polysaccharide concentration

Abstract The locust bean gum (LBG) is a polysaccharide with thickening, stabilizing and gelling properties and it has been used in the preparation of pharmaceutical formulations. Hydrogels (HGs) are obtained from natural or synthetic materials that present interesting properties for skin application. This study aimed to develop HGs from LBG using indole-3-carbinol (I3C) as an asset model for cutaneous application. HGs were prepared by dispersing LBG (2%, 3% and 4% w/v) directly in cold water. The formulations showed content close to 0.5 mg/g (HPLC) and pH ranging from 7.25 to 7.41 (potentiometry). The spreadability factor (parallel plate method) was inversely proportional to LBG concentration. The rheological evaluation (rotational viscometer) demonstrated a non-Newtonian pseudoplastic flow behavior (Ostwald De Weale model), which is interesting for cutaneous application. The HET-CAM evaluation showed the non-irritating characteristic of the formulations. The bioadhesive potential demonstrated bioadhesion in a concentration-dependent manner. Permeation in human skin using Franz cells showed that the highest LBG concentration improved the skin distribution profile with greater I3C amounts in the viable skin layers. The present study demonstrated the feasibility of preparing HGs with LBG and the formulation with the highest polymer concentration was the most promising to transport active ingredients through the skin.

Development and clinical application of hydrogel formulations containing papain and urea for wound healing

Abstract Hydrogels are used for wound treatment, as they may contain one or more active components and protect the wound bed. Papain is one of the active substances that have been used with this purpose, alongside urea. In this paper, carboxypolymethylene hydrogels containing papain (2% and 10% concentrations) and urea (5% concentration) were produced. Physical-chemical stability was performed at 0, 7, 15 and 30 days at 2-8ºC, 25ºC and 40ºC, as well as the rheological aspects and proteolytic activity of papain by gel electrophoresis. Clinical efficacy of the formulations in patients with lower limb ulcers was also evaluated in a prospective, single-center, randomized, double-blind and comparative clinical trial. The results showed 7-day stability for the formulations under 25ºC, in addition to approximately 100% and 15% of protein activity for 10% and 2% papain hydrogel, respectively. The rheological profile was non-Newtonian for the 10% papain hydrogel tested. There were no significant differences regarding the mean time for healing of the lesions, although 10% papain presented a better approach to be used in all types of tissue present in the wound bed.

A modified hydrogel production protocol to decrease cellular content.

PURPOSE: To analyze the cytotoxicity and cell in porcine-derived decellularized skin matrix. METHODS: We analyzed the effect of multiple decellularization processes by histological analysis, DNA quantification, and flow cytometry. Subsequently, we analyzed the most appropriate hydrogel concentration to minimize cytotoxicity on fibroblast culture and to maximize cell proliferation. RESULTS: After the fourth decellularization, the DNA quantification showed the lowest DNA concentration (< 50 ng/mg). Histological analysis showed no cell components in the hydrogel. Moreover, hematoxylin and eosin showed a heterogeneous structure of collagen fibers. The best hydrogel concentration ranged from 3 to 25%, and there was no significant difference between the 24 hours and seven days. CONCLUSIONS: The process of hydrogel production was effective for removing cells and DNA elements. The best hydrogel concentration ranged from 3 to 25%.

Tunable PEG Hydrogels for Discerning Differential Tumor Cell Response to Biomechanical Cues.

Increased extracellular matrix (ECM) density in the tumor microenvironment has been shown to influence aspects of tumor progression such as proliferation and invasion. Increased matrix density means cells experience not only increased mechanical properties, but also a higher density of bioactive sites. Traditional in vitro ECM models like Matrigel and collagen do not allow these properties to be investigated independently. In this work, a poly(ethylene glycol)-based scaffold is used which modifies with integrin-binding sites for cell attachment and matrix metalloproteinase 2 and 9 sensitive sites for enzyme-mediated degradation. The polymer backbone density and binding site concentration are independently tuned and the effect each of these properties and their interaction have on the proliferation, invasion, and focal complex formation of two different tumor cell lines is evaluated. It is seen that the cell line of epithelial origin (Hs 578T, triple negative breast cancer) proliferates more, invades less, and forms more mature focal complexes in response to an increase in matrix adhesion sites. Conversely, the cell line of mesenchymal origin (HT1080, fibrosarcoma) proliferates more in 2D culture but less in 3D culture, invades less, and forms more mature focal complexes in response to an increase in matrix stiffness.

Biophysical properties of hydrogels for mimicking tumor extracellular matrix.

The extracellular matrix (ECM) is an essential component of the tumor microenvironment. It plays a critical role in regulating cell-cell and cell-matrix interactions. However, there is lack of systematic and comparative studies on different widely-used ECM mimicking hydrogels and their properties, making the selection of suitable hydrogels for mimicking different in vivo conditions quite random. This study systematically evaluates the biophysical attributes of three widely used natural hydrogels (Matrigel, collagen gel and agarose gel) including complex modulus, loss tangent, diffusive permeability and pore size. A new and facile method was developed combining Critical Point Drying, Scanning Electron Microscopy imaging and a MATLAB image processing program (CSM method) for the characterization of hydrogel microstructures. This CSM method allows accurate measurement of the hydrogel pore size down to nanometer resolution. Furthermore, a microfluidic device was implemented to measure the hydrogel permeability (Pd) as a function of particle size and gel concentration. Among the three gels, collagen gel has the lowest complex modulus, medium pore size, and the highest loss tangent. Agarose gel exhibits the highest complex modulus, the lowest loss tangent and the smallest pore size. Collagen gel and Matrigel produced complex moduli close to that estimated for cancer ECM. The Pd of these hydrogels decreases significantly with the increase of particle size. By assessing different hydrogels' biophysical characteristics, this study provides valuable insights for tailoring their properties for various three-dimensional cancer models.

Engineered models for placental toxicology: Emerging approaches based on tissue decellularization.

Recent increases in prescriptions and illegal drug use as well as exposure to environmental contaminants during pregnancy have highlighted the critical importance of placental toxicology in understanding and identifying risks to both mother and fetus. Although advantageous for basic science, current in vitro models often fail to capture the complexity of placental response, likely due to their inability to recreate and monitor aspects of the microenvironment including physical properties, mechanical forces and stiffness, protein composition, cell-cell interactions, soluble and physicochemical factors, and other exogenous cues. Tissue engineering holds great promise in addressing these challenges and provides an avenue to better understand basic biology, effects of toxic compounds and potential therapeutics. The key to success lies in effectively recreating the microenvironment. One strategy to do this would be to recreate individual components and then combine them. However, this becomes challenging due to variables present according to conditions such as tissue location, age, health status and lifestyle. The extracellular matrix (ECM) is known to influence cellular fate by working as a storage of factors. Decellularized ECM (dECM) is a recent tool that allows usage of the original ECM in a refurbished form, providing a relatively reliable representation of the microenvironment. This review focuses on using dECM in modified forms such as whole organs, scaffold sheets, electrospun nanofibers, hydrogels, 3D printing, and combinations as building blocks to recreate aspects of the microenvironment to address general tissue engineering and toxicology challenges, thus illustrating their potential as tools for future placental toxicology studies.

Bioinspired in vitro intestinal mucus model for 3D-dynamic culture of bacteria.

The intestinal mucus is a biological barrier that supports the intestinal microbiota growth and filters molecules. To perform these functions, mucus possesses optimized microstructure and viscoelastic properties and it is steadily replenished thus flowing along the gut. The available in vitro intestinal mucus models are useful tools in investigating the microbiota-human cells interaction, and are used as matrices for bacterial culture or as static component of microfluidic devices like gut-on-chips. The aim of this work is to engineer an in vitro mucus models (I-Bac3Gel) addressing in a single system physiological viscoelastic properties (i.e., 2-200 Pa), 3D structure and suitability for dynamic bacterial culture. Homogeneously crosslinked alginate hydrogels are optimized in composition to obtain target viscoelastic and microstructural properties. Then, rheological tests are exploited to assess a priori the hydrogels capability to withstand the flow dynamic condition. We experimentally assess the suitability of I-Bac3Gels in the evolving field of microfluidics by applying a dynamic flow to a bacterial-loaded mucus model and by monitoring E. coli growth and survival. The engineered models represent a step forward in the modelling of the mucus, since they can answer to different urgent needs such as a 3D structure, bioinspired properties and compatibility with dynamic system.

Designing highly customizable human based platforms for cell culture using proteins from the amniotic membrane.

In the past few years researchers have witnessed a paradigm shift in the development of biomaterials for drug discovery, tissue engineering, and regenerative medicine. After the great advances resulting from the transition of the 2D to the 3D, the new focus has been to increase the clinical relevance of such systems, as well as avoid the use of animals, by developing platforms that better replicate the human physiology in vitro. In this sense, we envisage the use of human matrices extracted from ethically sourced and readily available tissues as an optimal and promising alternative to currently used approaches. Hereupon, we report for the first time the chemical modification of human ECM proteins from the amniotic membrane (AM) with photoresponsive groups to produce bioinks and hydrogel precursors to engineer customizable platforms that are representative of native tissues and capable of supporting long-term cell culture. Our results demonstrated an efficient decellularization, liquefaction and functionalization of AM-derived ECM with methacryloyl domains (AMMA), with production of stable and versatile hydrogels. Mechanical characterization evidenced an increased compression strength as a function of methacrylation degree and decellularized ECM concentration. Three-dimensional (3D) stem cell culture in the AMMA hydrogels resulted in viable and proliferative cells up to 7 days; moreover, the mouldable character of the hydrogel precursors permits the processing of patterned hydrogel constructs allowing the control over cellular alignment and elongation, or microgels with highly tunable shape.

Construction of in vitro 3-D model for lung cancer-cell metastasis study.

BACKGROUND: Cancer metastasis is the main cause of mortality in cancer patients. However, the drugs targeting metastasis processes are still lacking, which is partially due to the short of effective in vitro model for cell invasion studies. The traditional 2-D culture method cannot reveal the interaction between cells and the surrounding extracellular matrix during invasion process, while the animal models usually are too complex to explain mechanisms in detail. Therefore, a precise and efficient 3-D in vitro model is highly desirable for cell invasion studies and drug screening tests. METHODS: Precise micro-fabrication techniques are developed and integrated with soft hydrogels for constructing of 3-D lung-cancer micro-environment, mimicking the pulmonary gland or alveoli as in vivo. RESULTS: A 3-D in vitro model for cancer cell culture and metastasis studies is developed with advanced micro-fabrication technique, combining microfluidic system with soft hydrogel. The constructed microfluidic platform can provide nutrition and bio-chemical factors in a continuous transportation mode and has the potential to form stable chemical gradient for cancer invasion research. Hundreds of micro-chamber arrays are constructed within the collagen gel, ensuring that all surrounding substrates for tumor cells are composed of natural collagen hydrogel, like the in vivo micro-environment. The 3-D in vitro model can also provide a fully transparent platform for the visual observation of the cell morphology, proliferation, invasion, cell-assembly, and even the protein expression by immune-fluorescent tests if needed. The lung-cancer cells A549 and normal lung epithelial cells (HPAEpiCs) have been seeded into the 3-D system. It is found out that cells can normally proliferate in the microwells for a long period. Moreover, although the cancer cells A549 and alveolar epithelial cells HPAEpiCs have the similar morphology on 2-D solid substrate, in the 3-D system the cancer cells A549 distributed sparsely as single round cells on the extracellular matrix (ECM) when they attached to the substrate, while the normal lung epithelial cells can form cell aggregates, like the structure of normal tissue. Importantly, cancer cells cultured in the 3-D in vitro model can exhibit the interaction between cells and extracellular matrix. As shown in the confocal microscope images, the A549 cells present round and isolated morphology without much invasion into ECM, while starting from around Day 5, cells changed their shape to be spindle-like, as in mesenchymal morphology, and then started to destroy the surrounding ECM and invade out of the micro-chambers. CONCLUSIONS: A 3-D in vitro model is constructed for cancer cell invasion studies, combining the microfluidic system and micro-chamber structures within hydrogel. To show the invasion process of lung cancer cells, the cell morphology, proliferation, and invasion process are all analyzed. The results confirmed that the micro-environment in the 3-D model is vital for revealing the lung cancer cell invasion as in vivo.