In situ crosslinkable multi-functional and cell-responsive alginate 3D matrix via thiol-maleimide click chemistry.

In vivo, cells interact with the extracellular matrix (ECM), which provides a multitude of biophysical and biochemical signals that modulate cellular behavior. Inspired by this, we explored a new methodology to develop a more physiomimetic polysaccharide-based matrix for 3D cell culture. Maleimide-modified alginate (AlgM) derivatives were successfully synthesized using DMTMM to activate carboxylic groups. Thiol-terminated cell-adhesion peptides were tethered to the hydrogel network to promote integrin binding. Rapid and efficient in situ hydrogel formation was promoted by thiol-Michael addition "click" chemistry via maleimide reaction with thiol-flanked protease-sensitive peptides. Alginate derivatives were further ionically crosslinked by divalent ions present in the medium, which led to greater stability and allowed longer cell culture periods. By tailoring alginate's biofunctionality we improved cell-cell and cell-matrix interactions, providing an ECM-like 3D microenvironment. We were able to systematically and independently vary biochemical and biophysical parameters to elicit specific cell responses, creating custom-made 3D matrices. DMTMM-mediated maleimide incorporation is a promising approach to synthesizing AlgM derivatives that can be leveraged to produce ECM-like matrices for a broad range of applications, from in vitro tissue modeling to tissue regeneration.

Liquid nitrogen improves the decellularization effectiveness of whole-ovary.

BACKGROUND: Ovarian tissue cryopreservation for fertility preservation carries a risk of malignant cell re-seeding. Artificial ovary is a promising method to solve such a problem. However, ovary decellularization protocols are limited. Hence, further studies are necessary to get better ovarian decellularization techniques for the construction of artificial ovary scaffolds. OBJECTIVE: To establish an innovative decellularization technique for whole porcine ovaries by integrating liquid nitrogen with chemical agents to reduce the contact time between the scaffolds and chemical reagents. MATERIALS AND METHODS: Porcine ovaries were randomly assigned to three groups: novel decellularized group, conventional decellularized group and fresh group. The ovaries in the novel decellularized group underwent three cycles of freezing by liquid nitrogen and thawing at temperatures around 37 degree C before decellularization. The efficiency of the decellularization procedure was assessed through histological staining and DNA content analysis. The maintenance of ovarian decellularized extracellular matrix(ODECM) constituents was determined by analyzing the content of matrix proteins. Additionally, we evaluated the biocompatibility of the decellularized extracellular matrix(dECM) by observing the growth of granulosa cells on the ODECM scaffold in vitro. RESULTS: Hematoxylin and eosin staining, DAPI staining and DNA quantification techniques collectively confirm the success of the novel decellularization methods in removing cellular and nuclear components from ovarian tissue. Moreover, quantitative assessments of ODECM contents revealed that the novel decellularization technique preserved more collagen and glycosaminoglycan compared to the conventional decellularized group (P<0.05). Additionally, the novel decellularized scaffold exhibited a significantly higher number of granulosa cells than the conventional scaffold during in vitro co-culture (P<0.05). CONCLUSION: The novel decellularized method demonstrated high efficacy in eliminating DNA and cellular structures while effectively preserving the extracellular matrix. As a result, the novel decellularized method holds significant promise as a viable technique for ovarian decellularization in forthcoming studies. Doi.org/10.54680/fr24310110212.

Cryo-storage of porcine hides at the industrial scale for tissue engineering and regenerative medicine application.

BACKGROUND: The industrial scale cryo-storage of raw tissue materials requires a robust, low-cost and easy-to-operate method that can facilitate the down-stream process. OBJECTIVE: The study was aimed to develop the multifunctional protective solutions (MPS) for transportation at ambient conditions and also subsequent cryo-storage below -20 degree C of raw porcine hides for tissue engineering and regenerative medicine. MATERIALS AND METHODS: Protective solutions with antimicrobial activity and proteinase-inhibiting activity were developed and tested for its efficacy in preserving the extracellular matrix of porcine dermis from microbial spoilage, proteolytic degradation, freeze damage and excessive dehydration during shipping and cryo-storage. The MPSs contained phosphate-buffered saline with ethylene diamine tetra acetic acid (EDTA) added as chelator and proteinase inhibitor, as well as glycerol or maltodextrin (M180) as cryoprotectants. RESULTS: MPSs prepared with EDTA and glycerol or M180 had significant antimicrobial activity and proteinase-inhibiting activity during the period of shipping and handling. Glycerol and M180 prevented eutectic salt precipitation and excessive freeze dehydration upon cryo-storage of porcine hides. Without glycerol or M180, hides could be freeze-dehydrated to the low hydration at ~0.4 g/g dw, and formed irreversible plications after freezing. A critical hydration (0.8~0.9 g/g dw) was observed for the extracellular matrix of porcine dermis, and dehydration to a lower level could impose enormous stress and potential damage. The soaking of porcine hides in MPSs decreased water content as glycerol and M180 entered into dermis. Upon equilibration, the glycerol content in the tissue was about 94% of the incubating glycerol solution, but the M180 content in the tissue was only about 50% of the incubating M180 solution, indicating that M180 did not get into the entire aqueous domain within dermis. MPSs reduced ice formation and increased the unfrozen water content of porcine raw hides upon cryo-storage. CONCLUSION: MPSs prepared with EDTA and glycerol or M180 have antimicrobial activity and proteinase-inhibiting activity, which can be used for transportation and cryo-storage of raw hides at the industrial scale. Glycerol at 7.5% w/v and M180 at 20% w/v were sufficient to prevent freeze damage and excessive freeze dehydration. Doi.org/10.54680/fr24310110312.

Schiff base crosslinked hyaluronic acid hydrogels with tunable and cell instructive time-dependent mechanical properties.

The dynamic interplay between cells and their native extracellular matrix (ECM) influences cellular behavior, imposing a challenge in biomaterial design. Dynamic covalent hydrogels are viscoelastic and show self-healing ability, making them a potential scaffold for recapitulating native ECM properties. We aimed to implement kinetically and thermodynamically distinct crosslinkers to prepare self-healing dynamic hydrogels to explore the arising properties and their effects on cellular behavior. To do so, aldehyde-substituted hyaluronic acid (HA) was synthesized to generate imine, hydrazone, and oxime crosslinked dynamic covalent hydrogels. Differences in equilibrium constants of these bonds yielded distinct properties including stiffness, stress relaxation, and self-healing ability. The effects of degree of substitution (DS), polymer concentration, crosslinker to aldehyde ratio, and crosslinker functionality on hydrogel properties were evaluated. The self-healing ability of hydrogels was investigated on samples of the same and different crosslinkers and DS to obtain hydrogels with gradient properties. Subsequently, human dermal fibroblasts were cultured in 2D and 3D to assess the cellular response considering the dynamic properties of the hydrogels. Moreover, assessing cell spreading and morphology on hydrogels having similar modulus but different stress relaxation rates showed the effects of matrix viscoelasticity with higher cell spreading in slower relaxing hydrogels.

Fabrication and Characterization of Colorectal Cancer Organoids from SW1222 Cell Line in Ultrashort Self-Assembling Peptide Matrix.

Ultrashort self-assembling peptides (SAPs) can spontaneously form nanofibers that resemble the extracellular matrix. These fibers allow the formation of hydrogels that are biocompatible, biodegradable, and non-immunogenic. We have previously proven that SAPs, when biofunctionalized with protein-derived motifs, can mimic the extracellular matrix characteristics that support colorectal organoid formation. These biofunctional peptide hydrogels retain the original parent peptide's mechanical properties, tunability, and printability while incorporating cues that allow cell-matrix interactions to increase cell adhesion. This paper presents the protocols needed to evaluate and characterize the effects of various biofunctional peptide hydrogels on cell adhesion and lumen formation using an adenocarcinoma cancer cell line able to form colorectal cancer organoids cost-effectively. These protocols will help evaluate biofunctional peptide hydrogel effects on cell adhesion and luminal formation using immunostaining and fluorescence image analysis. The cell line used in this study has been previously utilized for generating organoids in animal-derived matrices.

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.

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.

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.

Decellularized Extracellular Matrix for Modeling Cardiac Extracellular Microenvironment.

The extracellular matrix (ECM) forms most of the tissue microenvironment and is in a constant and dynamic equilibrium with cells. The decellularization process employs physical or chemical methods, or a combination of them, to remove the cellular components of tissues and organs while preserving the architecture and composition of the ECM. Depending on the methodology used, the decellularized ECM (dECM) is then suitable for research or clinical applications. Here, we describe an optimized protocol for the efficient decellularization of the human myocardium to generate 3D scaffolds of well-preserved cardiac extracellular matrix that can be used for in vitro or in vivo studies.

SimuCell3D: three-dimensional simulation of tissue mechanics with cell polarization.

The three-dimensional (3D) organization of cells determines tissue function and integrity, and changes markedly in development and disease. Cell-based simulations have long been used to define the underlying mechanical principles. However, high computational costs have so far limited simulations to either simplified cell geometries or small tissue patches. Here, we present SimuCell3D, an efficient open-source program to simulate large tissues in three dimensions with subcellular resolution, growth, proliferation, extracellular matrix, fluid cavities, nuclei and non-uniform mechanical properties, as found in polarized epithelia. Spheroids, vesicles, sheets, tubes and other tissue geometries can readily be imported from microscopy images and simulated to infer biomechanical parameters. Doing so, we show that 3D cell shapes in layered and pseudostratified epithelia are largely governed by a competition between surface tension and intercellular adhesion. SimuCell3D enables the large-scale in silico study of 3D tissue organization in development and disease at a great level of detail.

Engineering fibronectin-templated multi-component fibrillar extracellular matrices to modulate tissue-specific cell response.

Cells assemble fibronectin, the major extracellular matrix (ECM) protein, into fibrillar matrices, which serve as 3D architectural scaffolds to provide, together with other ECM proteins tissue-specific environments. Although recent approaches enable to bioengineer 3D fibrillar fibronectin matrices in vitro, it remains elusive how fibronectin can be co-assembled with other ECM proteins into complex 3D fibrillar matrices that recapitulate tissue-specific compositions and cellular responses. Here, we introduce the engineering of fibrillar fibronectin-templated 3D matrices that can be complemented with other ECM proteins, including vitronectin, collagen, and laminin to resemble ECM architectures observed in vivo. For the co-assembly of different ECM proteins, we employed their innate fibrillogenic mechanisms including shear forces, pH-dependent electrostatic interactions, or specific binding domains. Through recapitulating various tissue-specific ECM compositions and morphologies, the large scale multi-composite 3D fibrillar ECM matrices can guide fibroblast adhesion, 3D fibroblast tissue formation, or tissue morphogenesis of epithelial cells. In other examples, we customize multi-composite 3D fibrillar matrices to support the growth of signal propagating neuronal networks and of human brain organoids. We envision that these 3D fibrillar ECM matrices can be tailored in scale and composition to modulate tissue-specific responses across various biological length scales and systems, and thus to advance manyfold studies of cell biological systems.

Construction of dental pulp decellularized matrix by cyclic lavation combined with mechanical stirring and its proteomic analysis.

The decellularized matrix has a great potential for tissue remodeling and regeneration; however, decellularization could induce host immune rejection due to incomplete cell removal or detergent residues, thereby posing significant challenges for its clinical application. Therefore, the selection of an appropriate detergent concentration, further optimization of tissue decellularization technique, increased of biosafety in decellularized tissues, and reduction of tissue damage during the decellularization procedures are pivotal issues that need to be investigated. In this study, we tested several conditions and determined that 0.1% Sodium dodecyl sulfate and three decellularization cycles were the optimal conditions for decellularization of pulp tissue. Decellularization efficiency was calculated and the preparation protocol for dental pulp decellularization matrix (DPDM) was further optimized. To characterize the optimized DPDM, the microstructure, odontogenesis-related protein and fiber content were evaluated. Our results showed that the properties of optimized DPDM were superior to those of the non-optimized matrix. We also performed the 4D-Label-free quantitative proteomic analysis of DPDM and demonstrated the preservation of proteins from the natural pulp. This study provides a optimized protocol for the potential application of DPDM in pulp regeneration.

Biochemical and immunomodulatory insights of extracellular matrix from decellularized human whole cervix: recellularization andin vivoECM remodeling interplay.

Extracellular matrix (ECM) rich whole organ bio-scaffolds, preserving structural integrity and essential growth factors, has potential towards regeneration and reconstruction. Women with cervical anomalies or trauma can benefit from clinical cervicovaginal repair using constructs rich in site specific ECM. In this study, complete human cervix decellularization was achieved using a modified perfusion-based stir bench top decellularization method. This was followed by physico-chemical processes including perfusion of ionic agents, enzymatic treatment and washing using detergent solutions for a duration of 10-12 d. Histopathological analysis, as well as DNA quantification confirmed the efficacy of the decellularization process. Tissue ultrastructure integrity was preserved and the same was validated via scanning electron microscopy and transmission electron microscopy studies. Biochemical analysis and structural characterizations like Fourier transform infrared, Raman spectroscopy of decellularized tissues demonstrated preservation of important proteins, crucial growth factors, collagen, and glycosaminoglycans.In vitrostudies, using THP-1 and human umbilical vein endothelial cell (HUVEC) cells, demonstrated macrophage polarization from M1 to M2 and vascular functional genes enhancement, respectively, when treated with decellularized human cervical matrix (DHCp). Crosslinked DHC scaffolds were recellularized with site specific human cervical epithelial cells and HUVEC, showing non-cytotoxic cell viability and enhanced proliferation. Furthermore, DHC scaffolds showed immunomodulatory effectsin vivoon small rodent model via upregulation of M2 macrophage genes as compared to decellularized rat cervix matrix scaffolds (DRC). DHC scaffolds underwent neo-vascularization followed by ECM remodeling with enhanced tissue integration.

Decellularized kidney extracellular matrix-based hydrogels for renal tissue engineering.

Kidney regeneration is hindered by the limited pool of intrinsic reparative cells. Advanced therapies targeting renal regeneration have the potential to alleviate the clinical and financial burdens associated with kidney disease. Delivery systems for cells, extracellular vesicles, or growth factors aimed at enhancing regeneration can benefit from vehicles enabling targeted delivery and controlled release. Hydrogels, optimized to carry biological cargo while promoting regeneration, have emerged as promising candidates for this purpose. This study aims to develop a hydrogel from decellularized kidney extracellular matrix (DKECM) and explore its biocompatibility as a biomaterial for renal regeneration. The resulting hydrogel crosslinks with temperature and exhibits a high concentration of extracellular matrix. The decellularization process efficiently removes detergent residues, yielding a pathogen-free biomaterial that is non-hemolytic and devoid of α-gal epitope. Upon interaction with macrophages, the hydrogel induces differentiation into both pro-inflammatory and anti-inflammatory phenotypes, suggesting an adequate balance to promote biomaterial functionality in vivo. Renal progenitor cells encapsulated in the DKECM hydrogel demonstrate higher viability and proliferation than in commercial collagen-I hydrogels, while also expressing tubular cells and podocyte markers in long-term culture. Overall, the injectable biomaterial derived from porcine DKECM is anticipated to elicit minimal host reaction while fostering progenitor cell bioactivity, offering a potential avenue for enhancing renal regeneration in clinical settings. STATEMENT OF SIGNIFICANCE: The quest to improve treatments for kidney disease is crucial, given the challenges faced by patients on dialysis or waiting for transplants. Exciting new therapies combining biomaterials with cells can revolutionize kidney repair. In this study, researchers created a hydrogel from pig kidney. This gel could be used to deliver cells and other substances that help in kidney regeneration. Despite coming from pigs, it's safe for use in humans, with no harmful substances and reduced risk of immune reactions. Importantly, it promotes a balanced healing response in the body. This research not only advances our knowledge of kidney repair but also offers hope for more effective treatments for kidney diseases.

Chemistry matters: A side-by-side comparison of two chemically distinct methacryloylated dECM bioresins for vat photopolymerization.

Decellularized extracellular matrix (dECM) is an excellent natural source for 3D bioprinting materials due to its inherent cell compatibility. In vat photopolymerization, the use of dECM-based bioresins is just emerging, and extensive research is needed to fully exploit their potential. In this study, two distinct methacryloyl-functionalized, photocrosslinkable dECM-based bioresins were prepared from digested porcine liver dECM through functionalization with glycidyl methacrylate (GMA) or conventional methacrylic anhydride (MA) under mild conditions for systematic comparison. Although the chemical modifications did not significantly affect the structural integrity of the dECM proteins, mammalian cells encapsulated in the respective hydrogels performed differently in long-term culture. In either case, photocrosslinking during 3D (bio)printing resulted in transparent, highly swollen, and soft hydrogels with good shape fidelity, excellent biomimetic properties and tunable mechanical properties (~ 0.2-2.5 kPa). Interestingly, at a similar degree of functionalization (DOF ~ 81.5-83.5 %), the dECM-GMA resin showed faster photocrosslinking kinetics in photorheology resulting in lower final stiffness and faster enzymatic biodegradation compared to the dECM-MA gels, yet comparable network homogeneity as assessed via Brillouin imaging. While human hepatic HepaRG cells exhibited comparable cell viability directly after 3D bioprinting within both materials, cell proliferation and spreading were clearly enhanced in the softer dECM-GMA hydrogels at a comparable degree of crosslinking. These differences were attributed to the additional hydrophilicity introduced to dECM via methacryloylation through GMA compared to MA. Due to its excellent printability and cytocompatibility, the functional porcine liver dECM-GMA biomaterial enables the advanced biofabrication of soft 3D tissue analogs using vat photopolymerization-based bioprinting.

Bone morphogenetic protein signaling inhibitor improves differentiation and function of 3D muscle construct fabricated using C2C12.

Functional tissue-engineered artificial skeletal muscle tissue has great potential for pharmacological and academic applications. This study demonstrates an in vitro tissue engineering system to construct functional artificial skeletal muscle tissues using self-organization and signal inhibitors. To induce efficient self-organization, we optimized the substrate stiffness and extracellular matrix (ECM) coatings. We modified the tissue morphology to be ring-shaped under optimized self-organization conditions. A bone morphogenetic protein (BMP) inhibitor was added to improve overall myogenic differentiation. This supplementation enhanced the myogenic differentiation ratio and myotube hypertrophy in two-dimensional cell cultures. Finally, we found that myotube hypertrophy was enhanced by a combination of self-organization with ring-shaped tissue and a BMP inhibitor. BMP inhibitor treatment significantly improved myogenic marker expression and contractile force generation in the self-organized tissue. These observations indicated that this procedure may provide a novel and functional artificial skeletal muscle for pharmacological studies.

Decellularized Brain Extracellular Matrix Hydrogel Aids the Formation of Human Spinal-Cord Organoids Recapitulating the Complex Three-Dimensional Organization.

The intricate electrophysiological functions and anatomical structures of spinal cord tissue render the establishment of in vitro models for spinal cord-related diseases highly challenging. Currently, both in vivo and in vitro models for spinal cord-related diseases are still underdeveloped, complicating the exploration and development of effective therapeutic drugs or strategies. Organoids cultured from human induced pluripotent stem cells (hiPSCs) hold promise as suitable in vitro models for spinal cord-related diseases. However, the cultivation of spinal cord organoids predominantly relies on Matrigel, a matrix derived from murine sarcoma tissue. Tissue-specific extracellular matrices are key drivers of complex organ development, thus underscoring the urgent need to research safer and more physiologically relevant organoid culture materials. Herein, we have prepared a rat decellularized brain extracellular matrix hydrogel (DBECMH), which supports the formation of hiPSC-derived spinal cord organoids. Compared with Matrigel, organoids cultured in DBECMH exhibited higher expression levels of markers from multiple compartments of the natural spinal cord, facilitating the development and maturation of spinal cord organoid tissues. Our study suggests that DBECMH holds potential to replace Matrigel as the standard culture medium for human spinal cord organoids, thereby advancing the development of spinal cord organoid culture protocols and their application in in vitro modeling of spinal cord-related diseases.

Human Placenta Decellularized Extracellular Matrix Hydrogel Promotes the Generation of Human Spinal Cord Organoids with Dorsoventral Organization from Human Induced Pluripotent Stem Cells.

Spinal cord organoids are of significant value in the research of spinal cord-related diseases by simulating disease states, thereby facilitating the development of novel therapies. However, the complexity of spinal cord structure and physiological functions, along with the lack of human-derived inducing components, presents challenges in the in vitro construction of human spinal cord organoids. Here, we introduce a novel human decellularized placenta-derived extracellular matrix hydrogel (DPECMH) and, combined with a new induction protocol, successfully construct human spinal cord organoids. The human placenta-sourced decellularized extracellular matrix (dECM), verified through hematoxylin and eosin staining, DNA quantification, and immunofluorescence staining, retained essential ECM components such as elastin, fibronectin, type I collagen, laminin, and so forth. The temperature-sensitive hydrogel made from human placenta dECM demonstrated good biocompatibility and promoted the differentiation of human induced pluripotent stem cell (hiPSCs)-derived spinal cord organoids into neurons. It displayed enhanced expression of laminar markers in comparison to Matrigel and showed higher expression of laminar markers compared to Matrigel, accelerating the maturation process of spinal cord organoids and demonstrating its potential as an organoid culture substrate. DPECMH has the potential to replace Matrigel as the standard additive for human spinal cord organoids, thus advancing the development of spinal cord organoid culture protocols and their application in the in vitro modeling of spinal cord-related diseases.

Tissue requirements for the application of aortic valve neocuspidization - appropriate pericardium properties and homogeneity?

OBJECTIVE: Aortic valve neocuspidization (AVNeo) using autologous pericardium is a promising technique. Expected advantages are reduced immune response, appropriate biomechanics and lower treatment expenses. Nevertheless, autologous pericardium can be affected by patient's age and comorbidities. Usually, glutaraldehyde (GA) - fixed bovine pericardium is the basic material for aortic valve prostheses, easy available and carefully pre-examined in a standardized fabrication process. Aim of the study is the verification of autologous pericardial tissue homogeneity by analysing tissue thickness, biomechanics and extracellular matrix (ECM) composition. METHODS: Segments of human GA-fixed pericardium selected by the surgeon based on visual criteria for cusp pre-cut and remaining after surgical AV replacement were investigated in comparison to bovine standard tissue treated equivalently. Pericardium sampling was performed at up to three positions of each sutured cusp for histological or biomechanical analysis, according to tissue availability. RESULTS AND CONCLUSIONS: Human pericardia exhibited a higher heterogeneity in collagen content, density of vessel structures and elastic moduli. Thickness, vessel density and collagen and elastin content differed significantly between the species. In contrast, significant interindividual differences were detected in most properties investigated for human pericardial samples but only for tissue thickness in bovine tissues. Higher heterogeneity of human pericardium, differing vessel and collagen content compared to bovine state-of-the-art material might be detrimental for long term AV functionality or deterioration and have to be intensely investigated in patients follow up after autologous cusp replacement.

An injectable active hydrogel based on BMSC-derived extracellular matrix for cartilage regeneration enhancement.

Articular cartilage injury impairs joint function and necessitates orthopedic intervention to restore the structure and function of the cartilage. Extracellular matrix (ECM) scaffolds derived from bone marrow mesenchymal stem cells (BMSCs) can effectively promote cell adhesion, proliferation, and chondrogenesis. However, pre-shaped ECM scaffolds have limited applicability due to their poor fit with the irregular surface of most articular cartilage defects. In this study, we fabricated an injectable active ECM hydrogel from autologous BMSCs-derived ECM by freeze-drying, liquid nitrogen milling, and enzymatic digestion. Moreover, our in vitro and in vivo results demonstrated that the prepared hydrogel enhanced chondrocyte adhesion and proliferation, chondrogenesis, cartilage regeneration, and integration with host tissue, respectively. These findings indicate that active ECM components can provide trophic support for cell proliferation and differentiation, restoring the structure and function of damaged cartilage.