Measuring mechanical cues for modeling the stromal matrix in 3D cell cultures.

A breast-cancer tumor develops within a stroma, a tissue where a complex extracellular matrix surrounds cells, mediating the cancer progression through biomechanical and -chemical cues. Current materials partially mimic the stromal matrix in 3D cell cultures but methods for measuring the mechanical properties of the matrix at cell-relevant-length scales and stromal-stiffness levels are lacking. Here, to address this gap, we developed a characterization approach that employs probe-based microrheometry and Bayesian modeling to quantify length-scale-dependent mechanics and mechanical heterogeneity as in the stromal matrix. We examined the interpenetrating network (IPN) composed of alginate scaffolds (for adjusting mechanics) and type-1 collagen (a stromal-matrix constituent). We analyzed viscoelasticity: absolute-shear moduli (stiffness/elasticity) and phase angles (viscous and elastic characteristics). We determined the relationship between microrheometry and rheometry information. Microrheometry reveals lower stiffness at cell-relevant scales, compared to macroscale rheometry, with dependency on the length scale (10 to 100 µm). These data show increasing IPN stiffness with crosslinking until saturation (≃15 mM of Ca2+). Furthermore, we report that IPN stiffness can be adjusted by modulating collagen concentration and interconnectivity (by polymerization temperature). The IPNs are heterogeneous structurally (in SEM) and mechanically. Interestingly, increased alginate crosslinking changes IPN heterogeneity in stiffness but not in phase angle, until the saturation. In contrast, such changes are undetectable in alginate scaffolds. Our nonlinear viscoelasticity analysis at tumor-cell-exerted strains shows that only the softer IPNs stiffen with strain, like the stromal-collagen constituent. In summary, our approach can quantify the stromal-matrix-related viscoelasticity and is likely applicable to other materials in 3D culture.

Toward morphologically relevant extracellular matrix: nanofiber-hydrogel composites for tumor cell culture.

The natural extracellular matrix (ECM) consists of a continuous integrated fibrin network and a negatively charged proteoglycan-based matrix. In this work, we report a novel three-dimensional nanofiber hydrogel composite that mimics the natural ECM structure, exhibiting both degradability and mechanical characteristics comparable to that of tumor tissue. The embedded nanofiber improves the hydrogel mechanical properties, and varying the fiber density can match the elastic modulus of different tumor tissues (1.51-10.77 kPa). The degradability of the scaffold gives sufficient space for tumor cells to secrete and remodel the ECM. The expression levels of cancer stem cell markers confirmed the development of aggressive and metastatic phenotypes of prostate cancer cells in the 3D scaffold. Similar results were obtained in terms of anticancer resistance of prostate cancer cells in 3D scaffolds showing stem cell-like properties, suggesting that the current bionic 3D scaffold tumor model has broad potential in the development of effective targeted agents.

Comparative Study of Basement-membrane Matrices for Human Stem Cell Maintenance and Intestinal Organoid Generation.

Extracellular matrix (ECM) plays a critical role in cell behavior and development. Organoids generated from human induced pluripotent stem cells (hiPSCs) are in the spotlight of many research areas. However, the lack of physiological cues in classical cell culture materials hinders efficient iPSC differentiation. Incorporating commercially available ECM into stem cell culture provides physical and chemical cues beneficial for cell maintenance. Animal-derived commercially available basement membrane products are composed of ECM proteins and growth factors that support cell maintenance. Since the ECM holds tissue-specific properties that can modulate cell fate, xeno-free matrices are used to stream up translation to clinical studies. While commercially available matrices are widely used in hiPSC and organoid work, the equivalency of these matrices has not been evaluated yet. Here, a comparative study of hiPSC maintenance and human intestinal organoids (hIO) generation in four different matrices: Matrigel (Matrix 1-AB), Geltrex (Matrix 2-AB), Cultrex (Matrix 3-AB), and VitroGel (Matrix 4-XF) was conducted. Although the colonies lacked a perfectly round shape, there was minimal spontaneous differentiation, with over 85% of the cells expressing the stem cell marker SSEA-4. Matrix 4-XF led to the formation of 3D round clumps. Also, increasing the concentration of supplement and growth factors in the media used to make the Matrix 4-XF hydrogel solution improved hiPSC expression of SSEA-4 by 1.3-fold. Differentiation of Matrix 2-AB -maintained hiPSC led to fewer spheroid releases during the mid-/hindgut stage compared to the other animal-derived basement membranes. Compared to others, the xeno-free organoid matrix (Matrix 4-O3) leads to larger and more mature hIO, suggesting that the physical properties of xeno-free hydrogels can be harnessed to optimize organoid generation. Altogether, the results suggest that variations in the composition of different matrices affect stages of IO differentiation. This study raises awareness about the differences in commercially available matrices and provides a guide for matrix optimization during iPSC and IO work.

Cornea-Specific Human Adipose Stem Cell-Derived Extracellular Matrix for Corneal Stroma Tissue Engineering.

Utilizing tissue-specific extracellular matrices (ECMs) is vital for replicating the composition of native tissues and developing biologically relevant biomaterials. Human- or animal-derived donor tissues and organs are the current gold standard for the source of these ECMs. To overcome the several limitations related to these ECM sources, including the highly limited availability of donor tissues, cell-derived ECM offers an alternative approach for engineering tissue-specific biomaterials, such as bioinks for three-dimensional (3D) bioprinting. 3D bioprinting is a state-of-the-art biofabrication technology that addresses the global need for donor tissues and organs. In fact, there is a vast global demand for human donor corneas that are used for treating corneal blindness, often resulting from damage in the corneal stromal microstructure. Human adipose tissue is one of the most abundant tissues and easy to access, and adipose tissue-derived stem cells (hASCs) are a highly advantageous cell type for tissue engineering. Furthermore, hASCs have already been studied in clinical trials for treating corneal stromal pathologies. In this study, a corneal stroma-specific ECM was engineered without the need for donor corneas by differentiating hASCs toward corneal stromal keratocytes (hASC-CSKs). Furthermore, this ECM was utilized as a component for corneal stroma-specific bioink where hASC-CSKs were printed to produce corneal stroma structures. This cost-effective approach combined with a clinically relevant cell type provides valuable information on developing more sustainable tissue-specific solutions and advances the field of corneal tissue engineering.

Magnetic biocomposite scaffold based on decellularized tendon ECM and MNP-deposited halloysite nanotubes: physicochemical, thermal, rheological, mechanical andin vitrobiological evaluations.

The development of new three-dimensional biomaterials with advanced versatile properties is critical to the success of tissue engineering (TE) applications. Here, (a) bioactive decellularized tendon extracellular matrix (dECM) with a sol-gel transition feature at physiological temperature, (b) halloysite nanotubes (HNT) with known mechanical properties and bioactivity, and (c) magnetic nanoparticles (MNP) with superparamagnetic and osteogenic properties were combined to develop a new scaffold that could be used in prospective bone TE applications. Deposition of MNPs on HNTs resulted in magnetic nanostructures without agglomeration of MNPs. A completely cell-free, collagen- and glycosaminoglycan- rich dECM was obtained and characterized. dECM-based scaffolds incorporated with 1%, 2% and 4% MNP-HNT were analysed for their physical, chemical, andin vitrobiological properties. Fourier-transform infrared spectroscopy, x-ray powder diffractometry and vibrating sample magnetometry analyses confirmed the presence of dECM, HNT and MNP in all scaffold types. The capacity to form apatite layer upon incubation in simulated body fluid revealed that dECM-MNP-HNT is a bioactive material. Combining dECM with MNP-HNT improved the thermal stability and compressive strength of the macroporous scaffolds upto 2% MNP-HNT.In vitrocytotoxicity and hemolysis experiments showed that the scaffolds were essentially biocompatible. Human bone marrow mesenchymal stem cells adhered and proliferated well on the macroporous constructs containing 1% and 2% MNP-HNT; and remained metabolically active for at least 21 din vitro. Collectively, the findings support the idea that magnetic nanocomposite dECM scaffolds containing MNP-HNT could be a potential template for TE applications.

Tuneable hydrogel patterns in pillarless microfluidic devices.

Organ-on-chip (OOC) technology has recently emerged as a powerful tool to mimic physiological or pathophysiological conditions through cell culture in microfluidic devices. One of its main goals is bypassing animal testing and encouraging more personalized medicine. The recent incorporation of hydrogels as 3D scaffolds into microfluidic devices has changed biomedical research since they provide a biomimetic extracellular matrix to recreate tissue architectures. However, this technology presents some drawbacks such as the necessity for physical structures as pillars to confine these hydrogels, as well as the difficulty in reaching different shapes and patterns to create convoluted gradients or more realistic biological structures. In addition, pillars can also interfere with the fluid flow, altering the local shear forces and, therefore, modifying the mechanical environment in the OOC model. In this work, we present a methodology based on a plasma surface treatment that allows building cell culture chambers with abutment-free patterns capable of producing precise shear stress distributions. Therefore, pillarless devices with arbitrary geometries are needed to obtain more versatile, reliable, and biomimetic experimental models. Through computational simulation studies, these shear stress changes are demonstrated in different designed and fabricated geometries. To prove the versatility of this new technique, a blood-brain barrier model has been recreated, achieving an uninterrupted endothelial barrier that emulates part of the neurovascular network of the brain. Finally, we developed a new technology that could avoid the limitations mentioned above, allowing the development of biomimetic OOC models with complex and adaptable geometries, with cell-to-cell contact if required, and where fluid flow and shear stress conditions could be controlled.

Development of a novel bioartificial cornea using 3D bioprinting based on electrospun micro-nanofibrous decellularized extracellular matrix.

Corneal damage contributes to blindness in millions of people. Simulating natural corneas with artificial corneas is challenging due to material and manufacturing limitations, including poor mechanical properties, complex manufacturing processes, and ocular histocompatibility. In this study, electrospun micro-nanofibrous decellularized extracellular matrix (dECM) is combined with digital light processing 3D bioprinting and validated as a bioartificial cornea for the first time. Electrospinning gives the material a controllable shape, and the electrospun micro-nanofibrous dECM, with preserved inherent biochemical components, can better mimic the natural ECM native microenvironment. An efficient platform can be developed for creating novel structural materials, when combined with intelligent manufacturing. Artificial biological corneas developed using this method showed five-fold improvements in mechanical properties (248.5 ± 35.67 kPa vs. 56.91 ± 3.68 kPa,p< 0.001), superior guidance for cell organization and adhesion, and better maintenance of the cellular phenotype of keratocytes. In animal studies,in vivotransplantation of this artificial cornea showed better regeneration, which accelerated corneal epithelialization and maintained corneal transparency. This method has potential for biomedical applications, and bioartificial corneas manufactured by this method have ideal properties as an alternative to lamellar keratoplasty, with promise for clinical transformation.

Fiber Flexibility Reconciles Matrix Recruitment and the Fiber Modulus to Promote Cell Mechanosensing.

The mechanical interaction between cells and the extracellular matrix is pervasive in biological systems. On fibrous substrates, cells possess the ability to recruit neighboring fibers, thereby augmenting their own adhesion and facilitating the generation of mechanical cues. However, the matrices with high moduli impede fiber recruitment, restricting the cell mechanoresponse. Herein, by harnessing the inherent swelling properties of gelatin, the flexible gelatin methacryloyl network empowers cells to recruit fibers spanning a broad spectrum of physiological moduli during adhesion. The high flexibility concurrently facilitates the optimization of fiber distribution, deformability, and modulus, contributing to the promotion of cell mechanosensing. Consequently, the randomly distributed flexible fibers with high moduli maximize the cell adhesive forces. This study uncovers the impact of fiber recruitment on cell mechanosensing and introduces fiber flexibility as a previously unexplored property, offering an innovative perspective for the design and development of novel biomaterials.

Distinct spectral signatures unfold ECM stiffness-triggered biochemical changes in breast cancer cells.

Cancer progression often accompanies the stiffening of extracellular matrix (ECM) in and around the tumor, owing to extra deposition and cross-linking of collagen. Stiff ECM has been linked with poor prognosis and is known to fuel invasion and metastasis, notably in breast cancer. However, the underlying biochemical or metabolic changes and the cognate molecular signatures remain elusive. Here, we explored Raman spectroscopy to unveil the spectral fingerprints of breast cancer cells in response to extracellular mechanical cues. Using stiffness-tuneable hydrogels, we showed that cells grown on stiff ECM displayed morphological changes with high proliferation. We further demonstrated that Raman Spectroscopy, a label-free and non-invasive technique, could provide comprehensive information about the biochemical environment of breast cancer cells in response to varying ECM stiffness. Raman spectroscopic analysis classified the cells into distinct clusters based on principal component-based linear discriminant analysis (PC-LDA). Multivariate curve resolution-alternating least squares (MCR-ALS) analysis indicated that cells cultured on stiff ECM exhibited elevated nucleic acid content and lesser lipids. Interestingly, increased intensity of Raman bands corresponding to cytochrome-c was also observed in stiff ECM conditions, suggesting mitochondrial modulation. The key findings harboured by spectral profiles were also corroborated by transmission electron microscopy, confirming altered metabolic status as reflected by increased mitochondria number and decreased lipid droplets in response to ECM stiffening. Collectively, these findings not only give the spectral signatures for mechanoresponse but also provide the landscape of biochemical changes in response to ECM stiffening.

Plasma-treated collagen functionalized with chondroitin sulfate as bioactive and nanostructured extracellular matrix mimics.

Aim: Cell microenvironment contains a plethora of information that influences cell modulation. Indeed, the extracellular matrix plays a central role in tissue development. Reproducing the cell-extracellular matrix crosstalk able to recapitulate both physical and biochemical signals is crucial to obtain functional tissue models or regenerative strategies. Materials & methods: Here, a combined method is proposed to easily functionalize collagen surface films, tailoring morphological properties. Oxygen nonthermal plasma treatment and glyco-conjugation with chondroitin sulfate are used to modify surface properties. Results: It results in higher adhesion, proliferation and morphological organization of U87 glioblastoma cells. Conclusion: Our finding suggests new promising strategies for the development of collagen-based biomaterials, which can be employed for advanced in vitro models.

Multimodal characterization of the collagen hydrogel structure and properties in response to physiologically relevant pH fluctuations.

pH fluctuations within the extracellular matrix (ECM) and its principal constituent collagen, particularly in solid tumors and chronic wounds, may influence its structure and function. Whereas previous research examined the impact of pH on collagen fibrillogenesis, this study focuses on determining how pH fluctuations affect collagen hydrogels that mimic the physiological ECM. Utilizing a type I collagen hydrogel, we examined the influence of pH fluctuations on its structure, properties, and function while keeping the collagen hydrated. We show that collagen's secondary structure remains unaltered during pathologically relevant microenvironmental pH changes. By employing cryo scanning electron microscopy and artificial intelligence-assisted image analysis, we show that at physiological pH, collagen hydrogel presents densely packed, aligned, and elongated fibrils, which upon a decrease to pH 6.5, are transformed into shorter, sparser, and disoriented fibrils. The collagen possesses a higher storage modulus yet a lower permeability at pH 7 and 7.8 compared with pH 6.5 and 7.4. Exposing acidified collagen to a basic buffer reinstates its native structure and viscoelastic properties. Our study offers an innovative approach to analyze and characterize perturbations in hydrated collagen-based systems with potential implications for better understanding and combating disease progression. STATEMENT OF SIGNIFICANCE: As the main component of the extracellular matrix, collagen undergoes conformational changes associated with pH changes during disease. We analyze the impact of pH on pre-formed collagen fibers mimicking healthy tissues subjected to disease, and do not focus on the more studied fibrillogenesis process. Using cryogenic SEM, which allowed imaging close to the native state, we show that even minor fluctuations in the pH affect the collagen thickness, length, fiber alignment, and rheological properties. Following exposure to acidic pH, the collagen had short fibers, lacked orientation, and had low mechanical strength. This acidic collagen restored its original properties after returning to a neutral pH. These findings can help determine how pH changes can be modulated to restore healthy collagen properties.

Comparative analysis of supercritical fluid-based and chemical-based decellularization techniques for nerve tissue regeneration.

Axon regeneration and Schwann cell proliferation are critical processes in the repair and functional recovery of damaged neural tissues. Biomaterials can play a crucial role in facilitating cell proliferative processes that can significantly impact the target tissue repair. Chemical decellularization and supercritical fluid-based decellularization methods are similar approaches that eliminate DNA from native tissues for tissue-mimetic biomaterial production by using different solvents and procedures to achieve the final products. In this study, we conducted a comparative analysis of these two methods in the context of nerve regeneration and neuron cell differentiation efficiency. We evaluated the efficacy of each method in terms of biomaterial quality, preservation of extracellular matrix components, promotion of neuronal cell differentiation and nerve tissue repair ability in vivo. Our results indicate that while both methods produce high-quality biomaterials, supercritical fluid-based methods have several advantages over conventional chemical decellularization, including better preservation of extracellular matrix components and mechanical properties and superior promotion of cellular responses. We conclude that supercritical fluid-based methods show great promise for biomaterial production for nerve regeneration and neuron cell differentiation applications.

[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.

Injectable extracellular matrix-mimetic hydrogel based on electrospun Janus fibers.

To date, the reported injectable hydrogels have failed to mimic the fibrous architecture of the extracellular matrix (ECM), limiting their biological effects on cell growth and phenotype. Additionally, they lack the micro-sized pores present within the ECM, which is unfavorable for the facile transport of nutrients and waste. Herein, an injectable ECM-mimetic hydrogel (IEMH) was fabricated by shortening and dispersing Janus fibers capable of self-curling at body temperature into pH 7.4 phosphate buffer solution. The IEMH could be massively prepared through a side-by-side electrospinning process combined with ultraviolet irradiation. The IEMHs with only 5 wt% fibers could undergo sol-gel transition at body temperature to become solid gels with desirable stability, sturdiness, and elasticity and self-healing ability. In addition, they possessed notable pseudoplasticity, which is beneficial to injection at room temperature. The results obtained from characterization analysis via scanning electron microscopy, total internal reflection fluorescence microscopy, nuclear magnetic resonance spectroscopy, and Fourier-transform infrared spectroscopy indicate that their sol-gel transition under physiological conditions stems from the synergistic action of the tight entanglements between thermally-induced self-curling fibers and the hydrophobic interaction between the fibers. An MTT assay using C2C12 myoblast cells was performed to examine the in vitro cytotoxicity of IEMHs for biomedical applications, and the cell viability was found to be more than 95%.

It's more than the amount that counts: implications of collagen organization on passive muscle tissue properties revealed with micromechanical models and experiments.

Collagen accumulation is often used to characterize skeletal muscle fibrosis, but the role of collagen in passive muscle mechanics remains debated. Here we combined finite-element models and experiments to examine how collagen organization contributes to macroscopic muscle tissue properties. Tissue microstructure and mechanical properties were measured from in vitro biaxial experiments and imaging in dystrophin knockout (mdx) and wild-type (WT) diaphragm muscle. Micromechanical models of intramuscular and epimuscular extracellular matrix (ECM) regions were developed to account for complex microstructure and predict bulk properties, and directly calibrated and validated with the experiments. The models predicted that intramuscular collagen fibres align primarily in the cross-muscle fibre direction, with greater cross-muscle fibre alignment in mdx models compared with WT. Higher cross-muscle fibre stiffness was predicted in mdx models compared with WT models and differences between ECM and muscle properties were seen during cross-muscle fibre loading. Analysis of the models revealed that variation in collagen fibre distribution had a much more substantial impact on tissue stiffness than ECM area fraction. Taken together, we conclude that collagen organization explains anisotropic tissue properties observed in the diaphragm muscle and provides an explanation for the lack of correlation between collagen amount and tissue stiffness across experimental studies.

Dual-Sensitive Fluorescent Nanoprobes for Simultaneously Monitoring In Situ Changes in pH and Matrix Metalloproteinase Expression in Stiffness-Tunable Three-Dimensional In Vitro Scaffolds.

A tumor microenvironment often presents altered physicochemical characteristics of the extracellular matrix (ECM) including changes in matrix composition, stiffness, protein expression, pH, temperature, or the presence of certain stromal and immune cells. Of these, overexpression of matrix metalloproteinases (MMPs) and extracellular acidosis are the two major hallmarks of cancer that can be exploited for tumor detection. The change in matrix stiffness and the release of certain cytokines (TNF-α) in the tumor microenvironment play major roles in inducing MMP-9 expression in cancerous cells. This study highlights the role of mechanical cues in upregulating MMP-9 expression in cancerous cells using stiffness-tunable matrix compositions and dual-sensitive fluorescent nanoprobes. Ionically cross-linked 3D alginate/gelatin (AG) scaffolds with three stiffnesses were chosen to reflect the ECM stiffnesses corresponding to healthy and pathological tissues. Moreover, a dual-sensitive nanoprobe, an MMP-sensitive peptide conjugated to carbon nanoparticles with intrinsic pH fluorescence properties, was utilized for in situ monitoring of the two cancer hallmarks in the 3D scaffolds. This platform was further utilized for designing a 3D core-shell platform for spatially mapping tumor margins and for visualizing TNF-α-induced MMP-9 expression in cancerous cells.

Organic-inorganic composite hydrogels: compositions, properties, and applications in regenerative medicine.

Hydrogels, formed from crosslinked hydrophilic macromolecules, provide a three-dimensional microenvironment that mimics the extracellular matrix. They served as scaffold materials in regenerative medicine with an ever-growing demand. However, hydrogels composed of only organic components may not fully meet the performance and functionalization requirements for various tissue defects. Composite hydrogels, containing inorganic components, have attracted tremendous attention due to their unique compositions and properties. Rigid inorganic particles, rods, fibers, etc., can form organic-inorganic composite hydrogels through physical interaction and chemical bonding with polymer chains, which can not only adjust strength and modulus, but also act as carriers of bioactive components, enhancing the properties and biological functions of the composite hydrogels. Notably, incorporating environmental or stimulus-responsive inorganic particles imparts smartness to hydrogels, hence providing a flexible diagnostic platform for in vitro cell culture and in vivo tissue regeneration. In this review, we discuss and compare a set of materials currently used for developing organic-inorganic composite hydrogels, including the modification strategies for organic and inorganic components and their unique contributions to regenerative medicine. Specific emphasis is placed on the interactions between the organic or inorganic components and the biological functions introduced by the inorganic components. The advantages of these composite hydrogels indicate their potential to offer adaptable and intelligent therapeutic solutions for diverse tissue repair demands within the realm of regenerative medicine.

Three-dimensional printing and decellularized-extracellular-matrix based methods for advances in artificial blood vessel fabrication: A review.

Blood vessels are the tubes through which blood flows and are divided into three types: millimeter-scale arteries, veins, and capillaries as well as micrometer-scale capillaries. Arteries and veins are the conduits that carry blood, while capillaries are where blood exchanges substances with tissues. Blood vessels are mainly composed of collagen fibers, elastic fibers, glycosaminoglycans and other macromolecular substances. There are about 19 feet of blood vessels per square inch of skin in the human body, which shows how important blood vessels are to the human body. Because cardiovascular disease and vascular trauma are common in the population, a great number of researches have been carried out in recent years by simulating the structures and functions of the person's own blood vessels to create different levels of tissue-engineered blood vessels that can replace damaged blood vessels in the human body. However, due to the lack of effective oxygen and nutrient delivery mechanisms, these tissue-engineered vessels have not been used clinically. Therefore, in order to achieve better vascularization of engineered vascular tissue, researchers have widely explored the design methods of vascular systems of various sizes. In the near future, these carefully designed and constructed tissue engineered blood vessels are expected to have practical clinical applications. Exploring how to form multi-scale vascular networks and improve their compatibility with the host vascular system will be very beneficial in achieving this goal. Among them, 3D printing has the advantages of high precision and design flexibility, and the decellularized matrix retains active ingredients such as collagen, elastin, and glycosaminoglycan, while removing the immunogenic substance DNA. In this review, technologies and advances in 3D printing and decellularization-based artificial blood vessel manufacturing methods are systematically discussed. Recent examples of vascular systems designed are introduced in details, the main problems and challenges in the clinical application of vascular tissue restriction are discussed and pointed out, and the future development trends in the field of tissue engineered blood vessels are also prospected.

Hyperspectral Mapping of Human Primary and Stem Cells at Cell-Matrix Interfaces.

Extracellular matrices interface with cells to promote cell growth and tissue development. Given this critical role, matrix mimetics are introduced to enable biomedical materials ranging from tissue engineering scaffolds and tumor models to organoids for drug screening and implant surface coatings. Traditional microscopy methods are used to evaluate such materials in their ability to support exploitable cell responses, which are expressed in changes in cell proliferation rates and morphology. However, the physical imaging methods do not capture the chemistry of cells at cell-matrix interfaces. Herein, we report hyperspectral imaging to map the chemistry of human primary and embryonic stem cells grown on matrix materials, both native and artificial. We provide the statistical analysis of changes in lipid and protein content of the cells obtained from infrared spectral maps to conclude matrix morphologies as a major determinant of biochemical cell responses. The study demonstrates an effective methodology for evaluating bespoke matrix materials directly at cell-matrix interfaces.

Construction of Integral Decellularized Cartilage Using a Novel Hydrostatic Pressure Bioreactor.

The decellularized extracellular matrix (ECM) of cartilage is a widely used natural bioscaffold for constructing tissue-engineered cartilage due to its good biocompatibility and regeneration properties. However, current decellularization methods for accessing decellularized cartilaginous tissues require multiple steps and a relatively long duration to produce decellularized cartilage. In addition, most decellularization strategies lead to damage of the microstructure and loss of functional components of the cartilaginous matrix. In this study, a novel decellularization strategy based on a hydrostatic pressure (HP) bioreactor was introduced, which aimed to improve the efficiency of producing integral decellularized cartilage pieces by combining physical and chemical decellularization methods in a perfusing manner. Two types of cartilaginous tissues, auricular cartilage (AC) and nucleus pulposus (NP) fibrocartilage, were selected for comparison of the effects of ordinary, positive, and negative HP-based decellularization according to the cell clearance ratio, microstructural changes, ECM components, and mechanical properties. The results indicated that applying positive HP improved the efficiency of producing decellularized AC, but no significant differences in decellularization efficiency were found between the ordinary and negative HP-treated groups. However, compared with the ordinary HP treatment, the application of the positive or negative HP did not affect the efficiency of decellularized NP productions. Moreover, neither positive nor negative HP influenced the preservation of the microstructure and components of the AC matrix. However, applying negative HP disarranged the fibril distribution of the NP matrix and reduced glycosaminoglycans and collagen type II contents, two essential ECM components. In addition, the positive HP was beneficial for maintaining the mechanical properties of decellularized cartilage. The recellularization experiments also verified the good biocompatibility of the decellularized cartilage produced by the present bioreactor-based decellularization method under positive HP. Overall, applying positive HP-based decellularization resulted in a superior effect on the production of close-to-natural scaffolds for cartilage tissue engineering. Impact statement In this study, we successfully constructed a novel hydrostatic pressure (HP) bioreactor and used this equipment to produce decellularized cartilage by combining physical and chemical decellularization methods in a perfusing manner. We found that positive HP-based decellularization could improve the production efficiency of integral decellularized cartilage pieces and promote the maintenance of matrix components and mechanical properties. This new decellularization strategy exhibited a superior effect in the production of close-to-natural scaffolds and positively impacts cartilage tissue engineering.