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.

Optimising a self-assembling peptide hydrogel as a Matrigel alternative for 3-dimensional mammary epithelial cell culture.

Three-dimensional (3D) organoid models have been instrumental in understanding molecular mechanisms responsible for many cellular processes and diseases. However, established organic biomaterial scaffolds used for 3D hydrogel cultures, such as Matrigel, are biochemically complex and display significant batch variability, limiting reproducibility in experiments. Recently, there has been significant progress in the development of synthetic hydrogels for in vitro cell culture that are reproducible, mechanically tuneable, and biocompatible. Self-assembling peptide hydrogels (SAPHs) are synthetic biomaterials that can be engineered to be compatible with 3D cell culture. Here we investigate the ability of PeptiGel® SAPHs to model the mammary epithelial cell (MEC) microenvironment in vitro. The positively charged PeptiGel®Alpha4 supported MEC viability, but did not promote formation of polarised acini. Modifying the stiffness of PeptiGel® Alpha4 stimulated changes in MEC viability and changes in protein expression associated with altered MEC function, but did not fully recapitulate the morphologies of MECs grown in Matrigel. To supply the appropriate biochemical signals for MEC organoids, we supplemented PeptiGels® with laminin. Laminin was found to require negatively charged PeptiGel® Alpha7 for functionality, but was then able to provide appropriate signals for correct MEC polarisation and expression of characteristic proteins. Thus, optimisation of SAPH composition and mechanics allows tuning to support tissue-specific organoids.

Laminin mimetic angiogenic and collagen peptide hydrogel for enhance dermal wound healing.

Laminins are essential in basement membrane architecture and critical in re-epithelialization and angiogenesis. These processes and collagen deposition are vital in skin wound healing. The role of angiogenic peptides in accelerating the wound-healing process has been known. The bioactive peptides could be a potential approach due to their similar effects as growth factors and inherent biocompatible and biodegradable nature with lower cost. They can also recognize ligand-receptor interaction and mimic the extracellular matrix. Here, we report novel angiogenic DYVRLAI, CDYVRLAI, angiogenic-collagen PGPIKVAV, and Ac-PGPIKVAV peptides conjugated sodium carboxymethyl cellulose hydrogel, which was designed from laminin. The designed peptide exhibits a better binding with the α3ß1, αvß3, and α5ß1 integrins and CXCR2 receptor, indicating their angiogenic and collagen binding efficiency. The peptides were evaluated to stimulate wound healing in full-thickness excision wounds in normal and diabetic mice (type II). They demonstrated their efficacy in terms of angiogenesis (CD31), re-epithelialization through regeneration of the epidermis (H&E), and collagen deposition (MT). The synthesized peptide hydrogel (DYVRLAI and CDYVRLAI) showed enhanced wound contraction up to 10.1 % and 12.3 % on day 7th compared to standard becaplermin gel (49 %) in a normal wound model. The encouraging results were also observed with the diabetic model, where these peptides showed a significant decrease of 5.20 and 5.17 % in wound size on day 10th compared to the commercial gel (9.27 %). These outcomes signify that the modified angiogenic peptide is a cost effective, novel peptide motif to promote dermal wound healing in both models.

LAMC2 mitigates ER stress by enhancing ER-mitochondria interaction via binding to MYH9 and MYH10.

Highly proliferative and metastatic tumors are constantly exposed to both intrinsic and extrinsic factors that induce adaptation to stressful conditions. Chronic adaptation to endoplasmic reticulum (ER) ER stress is common to many different types of cancers, and poses a major challenge for acquired drug resistance. Here we report that LAMC2, an extracellular matrix protein upregulated in many types of cancers, is localized in the ER of lung, breast, and liver cancer cells. Under tunicamycin-induced ER stress, protein level of LAMC2 is upregulated. Transfection of cancer cells with LAMC2 resulted in the attenuation of ER stress phenotype, accompanied by elevation in mitochondrial membrane potential as well as reduction in reactive oxygen species (ROS) levels and apoptosis. In addition, LAMC2 forms protein complexes with MYH9 and MYH10 to promote mitochondrial aggregation and increased ER-mitochondria interaction at the perinuclear region. Moreover, overexpression of LAMC2 counteracts the effects of ER stress and promotes tumor growth in vivo. Taken together, our results revealed that in complex with MYH9 and MYH10, LAMC2 is essential for promoting ER-mitochondria interaction to alleviate ER stress and allow cancer cells to adapt and proliferate under stressful conditions. This study provides new insights and highlights the promising potential of LAMC2 as a therapeutic target for cancer treatment.

Recombinant Laminin-511 Fragment (iMatrix-511) Coating Supports Maintenance of Human Nucleus Pulposus Progenitor Cells In Vitro.

The angiopoietin-1 receptor (Tie2) marks specific nucleus pulposus (NP) progenitor cells, shows a rapid decline during aging and intervertebral disc degeneration, and has thus sparked interest in its utilization as a regenerative agent against disc degeneration. However, the challenge of maintaining and expanding these progenitor cells in vitro has been a significant hurdle. In this study, we investigated the potential of laminin-511 to sustain Tie2+ NP progenitor cells in vitro. We isolated cells from human NP tissue (n = 5) and cultured them for 6 days on either standard (Non-coat) or iMatrix-511 (laminin-511 product)-coated (Lami-coat) dishes. We assessed these cells for their proliferative capacity, activation of Erk1/2 and Akt pathways, as well as the expression of cell surface markers such as Tie2, GD2, and CD24. To gauge their regenerative potential, we examined their extracellular matrix (ECM) production capacity (intracellular type II collagen (Col2) and proteoglycans (PG)) and their ability to form spherical colonies within methylcellulose hydrogels. Lami-coat significantly enhanced cell proliferation rates and increased Tie2 expression, resulting in a 7.9-fold increase in Tie2-expressing cell yields. Moreover, the overall proportion of cells positive for Tie2 also increased 2.7-fold. Notably, the Col2 positivity rate was significantly higher on laminin-coated plates (Non-coat: 10.24% (±1.7%) versus Lami-coat: 26.2% (±7.5%), p = 0.010), and the ability to form spherical colonies also showed a significant improvement (Non-coat: 40.7 (±8.8)/1000 cells versus Lami-coat: 70.53 (±18.0)/1000 cells, p = 0.016). These findings demonstrate that Lami-coat enhances the potential of NP cells, as indicated by improved colony formation and proliferative characteristics. This highlights the potential of laminin-coating in maintaining the NP progenitor cell phenotype in culture, thereby supporting their translation into prospective clinical cell-transplantation products.

Fibrin hydrogels fortified with FGF-7/10 and laminin-1 peptides promote regeneration of irradiated salivary glands.

Ionizing radiation, commonly used for head and neck cancer treatment, typically damages the salivary glands, resulting in hyposalivation. The development of treatments to restore this lost function is crucial for improving the quality of life for patients suffering from this condition. To address this clinical need, we have developed an innovative hydrogel by chemically conjugating laminin-1 peptides (A99 and YIGSR) and growth factors, FGF-7 and FGF-10, to fibrin hydrogels. Our results demonstrate that FGF-7/10 and laminin-1 peptides fortified fibrin hydrogel [enhanced laminin-1 peptides fibrin hydrogel (Ep-FH)] promotes salivary gland regeneration and functionality by improving epithelial tissue organization, establishing a healthy network of blood vessels and nerves, while reducing fibrosis in a head and neck irradiated mouse model. These results indicate that fibrin hydrogel-based implantable scaffolds containing pro-regenerative signals promote sustained secretory function of irradiated salivary glands, offering a potential alternative treatment for hyposalivation in head and neck cancer patients undergoing radiation treatment. These unique findings emphasize the potential of fibrin hydrogel-based implantable scaffolds enriched with pro-regenerative signals in sustaining the secretory function of irradiated salivary glands and offer a promising alternative treatment for addressing hyposalivation in head and neck cancer patients undergoing radiation therapy. STATEMENT OF SIGNIFICANCE: Radiation therapies used to treat head and neck cancers often result in damaged salivary gland, leading to severe dryness of the oral cavity. In this study, we engineered FGF-7 and FGF-10 and immobilized them into L1p-FH. The resulting hydrogel, Ep-FH, restored irradiated salivary gland functionality by enhancing epithelial tissue organization, promoting the development of a healthy network of blood vessels and nerves as well as reduction of fibrosis.

LAMC2 regulates proliferation, migration, and invasion mediated by the Pl3K/AKT/mTOR pathway in oral.

Background: Oral squamous cell carcinoma (OSCC) is a common malignant tumor. Recently, Laminin Gamma 2 (LAMC2) has been shown to be abnormally expressed in OSCC; however, how LAMC2 signaling contributes to the occurrence and development of OSCC and the role of autophagy in OSCC has not been fully explored. This study aimed to analyze the role and mechanism of LAMC2 signaling in OSCC and the involvement of autophagy in OSCC. Methods: To explore the mechanism by which LAMC2 is highly expressed in OSCC, we used small interfering RNA (siRNA) to knock down LAMC2 to further observe the changes in the signaling pathway. Furthermore, we used cell proliferation assays, Transwell invasion assays, and wound-healing assays to observe the changes in OSCC proliferation, invasion, and metastasis. RFP-LC3 was used to detect the level of autophagy intensity. A cell line-derived xenograft (CDX) model was used to detect the effect of LAMC2 on tumor growth in vivo. Results: This study found that the level of autophagy was correlated with the biological behavior of OSCC. The downregulation of LAMC2 activated autophagy and inhibited OSCC proliferation, invasion, and metastasis via inhibiting the PI3K/AKT/mTOR pathway. Moreover, autophagy has a dual effect on OSCC, and the synergistic downregulation of LAMC2 and autophagy can inhibit OSCC metastasis, invasion, and proliferation via the PI3K/AKT/mTOR pathway. Conclusions: LAMC2 interacts with autophagy to regulate OSCC metastasis, invasion, and proliferation via the PI3K/AKT/mTOR pathway. LAMC2 down-regulation can synergistically modulate autophagy to inhibit OSCC migration, invasion, and proliferation.

A Novel Three-Dimensional Follicle Culture System Decreases Oxidative Stress and Promotes the Prolonged Culture of Human Granulosa Cells.

Tissue engineering advancements have made it possible to modify biomaterials to reconstruct a similar three-dimensional structure of the extracellular matrix (ECM) for follicle development and to supply the required biological signals. We postulated that an artificial polysaccharide hydrogel modified with an ECM mimetic peptide may produce efficient irritation signals by binding to specific integrins providing a suitable environment for follicular development and influencing the behavior of human granulosa cells (hGCs). Laminin, an important component of the extracellular matrix, can modulate hGCs and oocyte growth. Specifically, follicles of mice were randomly divided into two-dimensional (2D) and three-dimensional (3D) culture systems established by a hydrogel modified with RGD or laminin mimetic peptides (IKVAV and YIGSR) and RGD (IYR). Our results showed that 3D cultured systems significantly improved follicle survival, growth, and viability. IYR peptides enhanced the oocyte meiosis competence. Additionally, we explored the effect of 3D culture on hGCs, which improved hGCs viability, increased the proportion of S- and G2/M-phase cells, and inhibited cell apoptosis of hGCs. On days 1 and 2, the secretion of progesterone was reduced in 3D-cultured hGCs. Notably, 3D-cultured hGCs exhibited delayed senescence, decreased oxidative stress, and elevated mitochondrial membrane potential. Moreover, the expression levels of cumulus expansion-related genes (COX2, HAS2, and PTX3) and integrin α6ß1 were upregulated in 3D-cultured hGCs. In conclusion, a 3D culture utilizing hydrogels modified with Laminin-mimetic peptides can provide a durable physical environment suitable for follicular development. The laminin-mimetic peptides may regulate the biological activity of hGCs by attaching to the integrin α6ß1.

Indirect co-culture of islet cells in 3D biocompatible collagen/laminin scaffold with angiomiRs transfected mesenchymal stem cells.

Diabetes is an autoimmune disease in which the pancreatic islets produce insufficient insulin. One of the treatment strategies is islet isolation, which may damage these cells as they lack vasculature. Biocompatible scaffolds are one of the efficient techniques for dealing with this issue. The current study is aimed to determine the effect of transfected BM-MSCS with angiomiR-126 and -210 on the survival and functionality of islets loaded into a 3D scaffold via laminin (LMN). AngiomiRs/Poly Ethylenimine polyplexes were transfected into bone marrow-mesenchymal stem cells (BM-MSCs), followed by 3-day indirect co-culturing with islets laden in collagen (Col)-based hydrogel scaffolds containing LMN. Islet proliferation and viability were significantly increased in LMN-containing scaffolds, particularly in the miRNA-126 treated group. Insulin gene expression was superior in Col scaffolds, especially, in the BM-MSCs/miRNA-126 treated group. VEGF was upregulated in the LMN-containing scaffolds in both miRNA-treated groups, specifically in the miRNA-210, leading to VEGF secretion. MiRNAs' target genes showed no downregulation in LMN-free scaffolds; while a drastic downregulation was seen in the LMN-containing scaffolds. The highest insulin secretion was recorded in the Oxidized dextran (Odex)/ColLMN+ group with miRNA-126. LMN-containing biocompatible scaffolds, once combined with angiomiRs and their downstream effectors, promote islets survival and restore function, leading to enhanced angiogenesis and glycemic status.

Comparison of human recombinant protein coatings and fibroblast-ECM to Matrigel for induced pluripotent stem cell culture and renal podocyte differentiation.

Human induced pluripotent stem cells (hiPSCs) offer great opportunities within the 3R framework. In the field of toxicology, they may contribute greatly to the reduction and eventually replacement of animal models. However, culturing hiPSCs as well as differentiation of hiPSCs into target cells that are used for toxicity testing depend on the presence of extracellular matrix (ECM) coating the growth surface. The most widely used ECM is MatrigelR, an animal product that is derived from mouse sarcoma. Drawbacks of Matrigel are widely recognized and include batch-to batch variations, use of animal rather than human material, and ethical concerns about its production. While alternative coatings exist, higher cost and limited characterizations may hinder their broader uptake by the scientific community. Here, we report an extensive comparison of three commercially available human ECM coatings, vitronectin, laminin-511, and laminin-521, to Matrigel in three different hiPSC lines in long-term culture (≥ 9 passages). Characterization included expression of pluripotent markers in a genome-wide transcriptomics study (TempO-Seq), capacity to differentiate into embryoid bodies, and karyotype stability assessed by analyzing copy number variations by shallow DNA sequencing. Furthermore, a low-cost, decellularized ECM produced by human neonatal dermal fibroblasts was tested. In addition, all alternative coatings were tested for hiPSC differentiation into renal podocyte-like cells in a genome-wide transcriptomics screen. Our results show that all tested coatings were highly comparable to animal-derived Matrigel for both hiPSC maintenance and differentiation into renal podocyte-like cells. Furthermore, decellularized fibroblast-ECM could be a novel, attractive low-cost coating material.

Glioblastoma spheroid growth and chemotherapeutic responses in single and dual-stiffness hydrogels.

Glioblastoma (GBM) is the deadliest brain tumor for which there is no cure. Bioengineered GBM models, such as hydrogel-encapsulated spheroids, that capture both cell-cell and cell-matrix interactions could facilitate testing of much needed therapies. Elucidation of specific microenvironment properties on spheroid responsiveness to therapeutics would enhance the usefulness of GBM models as predictive drug screening platforms. Here, GBM spheroids consisting of U87 or patient-derived GBM cells were encapsulated in soft (∼1 kPa), stiff (∼7 kPa), and dual-stiffness polyethylene glycol-based hydrogels, with GBM spheroids seeded at the stiffness interface. Spheroids were cultured for 7 days and examined for viability, size, invasion, laminin expression, hypoxia, proliferation, and response to the chemotherapeutic temozolomide (TMZ). We noted excellent cell viability in all hydrogels, and higher infiltration in soft compared to stiff hydrogels for U87 spheroids. In dual gels spheroids mostly infiltrated away from the stiffness interface with minimal crossing over it and some individual cell migration along the interface. U87 spheroids were equally responsive to TMZ in the soft and stiff hydrogels, but cell viability in the spheroid periphery was higher than the core for stiff hydrogels whereas the opposite was true for soft hydrogels. HIF1A expression was higher in the core of spheroids in the stiff hydrogels, while there was no difference in cell proliferation between spheroids in the stiff vs soft hydrogels. Patient-derived GBM spheroids did not show stiffness-dependent drug responses. U87 cells showed similar laminin expression in soft and stiff hydrogels with higher expression in the spheroid periphery compared to the core. Our results indicate that microenvironment stiffness needs to be considered in bioengineered GBM models including those designed for use in drug screening applications. STATEMENT OF SIGNIFICANCE: Recent work on tumor models engineered for use in drug screening has highlighted the potential of hydrogel-encapsulated spheroids as a simple, yet effective platform that show drug responses similar to native tumors. It has also been shown that substrate stiffness, in vivo and in vitro, affects cancer cell responses to drugs. This is particularly important for glioblastoma (GBM), the deadliest brain cancer, as GBM cells invade by following the stiffer brain structures such as white matter tracks and the perivascular niche. Invading cells have also been associated with higher resistance to chemotherapy. Here we developed GBM spheroid models using soft, stiff and dual-stiffness hydrogels to explore the connection between substrate stiffness, spheroid invasion and drug responsiveness in a controlled environment.

Epithelial folding of alveolar cells derived from human induced pluripotent stem cells on artificial basement membrane.

Epithelial folding depends on mechanical properties of both epithelial cells and underlying basement membrane (BM). While folding is essential for tissue morphogenesis and functions, it is difficult to recapitulate features of a growing epithelial monolayer for in vitro modeling due to lack of in vivo like BM. Herein, we report a method to overcome this difficulty by culturing on an artificial basement membrane (ABM) the primordial lung progenitors (PLPs) from human induced pluripotent stem cells (hiPSCs). The ABM was achieved by self-assembling collagen IV and laminin, the two principal natural BM proteins, in the pores of a monolayer of crosslinked gelatin nanofibers deposited on a honeycomb micro-frame. The hiPSC-PLPs were seeded on the ABM for alveolar differentiation under submerged and air-liquid interface culture conditions. As results, the forces generated by the growing epithelial monolayer led to a geometry-dependent folding. Analysis of strain distribution in a clamped membrane provided instrumental insights into some of the observed phenomena. Moreover, the forces generated by the growing epithelial layer led to a high-level expression of surfactant protein C and a high percentage of aquaporin 5 positive cells compared with the results obtained with a nanofiber-covered bulk substrate. Thus, this work demonstrated the importance of recapitulating natural BM for advanced epithelial modeling. STATEMENT OF SIGNIFICANCE: The effort to develop in vitro epithelial models has not been entirely successful to date, due to lack of in vivo like basement membrane (BM). This lack has been overcome by using a microfabricated dense thin and pliable sheet like structure made of natural BM proteins. With such an artificial BM, alveolar epithelial deformation and folding could be studied and date could be correlated to numerical analyses of a plate theory. This method is simple and effective, enabling further developments in epithelial tissue modeling.

Bovine and human endometrium-derived hydrogels support organoid culture from healthy and cancerous tissues.

Organoid technology has provided unique insights into human organ development, function, and diseases. Patient-derived organoids are increasingly used for drug screening, modeling rare disorders, designing regenerative therapies, and understanding disease pathogenesis. However, the use of Matrigel to grow organoids represents a major challenge in the clinical translation of organoid technology. Matrigel is a poorly defined mixture of extracellular matrix proteins and growth factors extracted from the Engelbreth-Holm-Swarm mouse tumor. The extracellular matrix is a major driver of multiple cellular processes and differs significantly between tissues as well as in healthy and disease states of the same tissue. Therefore, we envisioned that the extracellular matrix derived from a native healthy tissue would be able to support organoid growth akin to organogenesis in vivo. Here, we have developed hydrogels from decellularized human and bovine endometrium. These hydrogels supported the growth of mouse and human endometrial organoids, which was comparable to Matrigel. Organoids grown in endometrial hydrogels were proteomically more similar to the native tissue than those cultured in Matrigel. Proteomic and Raman microspectroscopy analyses showed that the method of decellularization affects the biochemical composition of hydrogels and, subsequently, their ability to support organoid growth. The amount of laminin in hydrogels correlated with the number and shape of organoids. We also demonstrated the utility of endometrial hydrogels in developing solid scaffolds for supporting high-throughput, cell culture-based applications. In summary, endometrial hydrogels overcome a major limitation of organoid technology and greatly expand the applicability of organoids to understand endometrial biology and associated pathologies.

Protease-Sensitive, VEGF-Mimetic Peptide, and IKVAV Laminin-Derived Peptide Sequences within Elastin-Like Recombinamer Scaffolds Provide Spatiotemporally Synchronized Guidance of Angiogenesis and Neurogenesis.

Spatiotemporal control of vascularization and innervation is a desired hallmark in advanced tissue regeneration. For this purpose, we design a 3D model scaffold, based on elastin-like recombinamer (ELR) hydrogels. This contains two interior and well-defined areas, small cylinders, with differentiated bioactivities with respect to the bulk. Both are constructed on a protease sensitive ELR with a fast-proteolyzed domain, but one bears a VEGF-mimetic peptide (QK) and the other a laminin-derived pentapeptide (IKVAV), to promote angiogenesis and neurogenesis, respectively. The outer bulk is based on a slow proteolytic sequence and RGD cell adhesion domains. In vitro studies show the effect of QK and IKVAV peptides on the promotion of endothelial cell and axon spreading, respectively. The subcutaneous implantation of the final 3D scaffold demonstrates the ability to spatiotemporally control angiogenesis and neurogenesis in vivo. Specifically, the inner small cylinder containing the QK peptide promotes fast endothelialization, whereas the one with IKVAV peptide promotes fast neurogenesis. Both, vascularization and innervation take place in advance of the bulk scaffold infiltration. This scaffold shows that it is possible to induce vascularization and innervation in predetermined areas of the scaffold well ahead to the bulk infiltration. That significantly increases the efficiency of the regenerative activity.

Effect of Sulfonation Group on Polyaniline Copolymer Scaffolds for Tissue Engineering with Laminin Treatment under Electrical Stimulation.

Sulfonated copolyanilines (SPANs), SPAN-40 and SPAN-75, were prepared and applied in this tissue engineering study. SPAN scaffolds (SPANs) and control group polyaniline (PANI) were synthesized by performing oxidative polymerization. To further research the effects of neuron regeneration, PC12 cells were cultured on as-prepared PANI and SPANs with laminin (La) treatment under electrical stimulation. The effects on PC12 cell differentiation were investigated by controlling the amount of sulfonated groups (-SO3H) in the SPAN chain, the electrical stimulation voltage, and the presence or absence of La coating. The adhesion and proliferation of cells increased with the degree of sulfonation; La and electrical stimulation further promoted neuronal cell differentiation as increased neurite length was demonstrated in the micrograph analyses. In summary, the sulfonated copolyaniline coated with La had the best effect on neuronal differentiation under electrical stimulation, suggesting its potential as a substrate for nerve tissue engineering.

3D bioprinting of gellan gum-based hydrogels tethered with laminin-derived peptides for improved cellular behavior.

The treatment of skeletal muscle defects is still a topic of noteworthy concern since surgical intervention is not capable of recovering muscle function. Herein, we propose myoblasts laden in laminin-inspired biofunctionalized gellan gum hydrogels as promising tissue-engineered skeletal muscle surrogates. Gellan gum-based hydrogels were developed by combining native gellan gum (GG) and GG tethered with laminin-derived peptides (CIKVAVS (V), KNRLTIELEVRTC (T) or RKRLQVQLSIRTC (Q)), using different polymer content (0.75%-1.875%). Hydrogels were characterized in terms of compressive modulus, molecules trafficking, and C2C12 adhesion. Hydrogels with higher polymeric content (1.125%-1.875%) showed higher stiffness whereas hydrogels with lower polymer content (0.75%-1.125%) showed higher fluorescein isothiocyanate-dextran molecules diffusion. Cell spreading was achieved regardless of the laminin-derived peptide but preferred in hydrogels with higher polymer content (1.125%-1.875%). Taken together, hydrogels with 1.125% of polymer content were selected for printability analysis. GG-based inks showed a non-newtonian, shear-thinning, and thixotropic behavior suitable for printing. Accordingly, all inks were printable, but inks tethered with T and Q peptides presented some signs of clogging. Cell viability was affected after printing but increased after 7 days of culture. After 7 days, cells were spreading but not showing significant signs of cell-cell communications. Therefore, cell density was increased, thus, myocytes loaded in V-tethered GG-based inks showed higher cell-cell communication, spreading morphology, and alignment 7, 14 days post-printing. Overall, myoblasts laden in laminin-inspired biofunctionalized GG-based hydrogels are a promising skeletal muscle surrogate with the potential to be used as in vitro model or explored for further in vivo applications.

Injectable laminin-biofunctionalized gellan gum hydrogels loaded with myoblasts for skeletal muscle regeneration.

Moderate muscular injuries that exceed muscular tissue's auto-healing capacity are still a topic of noteworthy concern. Tissue engineering appeared as a promising therapeutic strategy capable of overcoming this unmet clinical need. To attain such goal, herein we propose an in situ-crosslinking gellan gum (GG)-based hydrogel tethered with a skeletal muscle-inspired laminin-derived peptide RKRLQVQLSIRTC(Q) and encapsulated with skeletal muscle cells (SMCs). Pre-hydrogel solutions presented decreasing shear viscosity with increasing shear rate and shear stress, and required low forces for extrusion, validating their injectability. The GGDVS hydrogel was functionalized with Q-peptide with 30% of efficiency. C2C12 were able to adhere to the developed hydrogel, remained living and spreading 7 days post-encapsulation. Q-peptide release studies indicated that 25% of the unbound peptide can be released from the hydrogels up to 7 days, dependent on the hydrogel formulation. Treatment of a chemically-induced muscular lesion in mice with an injection of C2C12-laden hydrogels improved myogenesis, primarily promoted by the C2C12. In accordance, a high density of myoblasts (α-SA+ and MYH7+) were localized in tissues treated with the C2C12 (alone or encapsulated in the hydrogel). α-SA protein levels were significantly increased 8 weeks post-treatment with C2C12-laden hydrogels and MHC protein levels were increased in all experimental groups 4 weeks post-treatment, in relation to the SHAM. Neovascularization and neoinnervation was also detected in the defects. Altogether, this study indicates that C2C12-laden hydrogels hold great potential for skeletal muscle regeneration. STATEMENT OF SIGNIFICANCE: We developed an injectable gellan gum-based hydrogel for delivering C2C12 into localized myopathic model. The gellan gum was biofunctinalized with laminin-derived peptide to mimic the native muscular ECM. In addition, hydrogel was physically tuned to mimic the mechanical properties of native tissue. To the best of our knowledge, this formula was used for the first time under the context of skeletal muscle tissue regeneration. The injectability of the developed hydrogel provided non-invasive administration method, combined with a reliable microenvironment that can host C2C12 with nominal inflammation, indicated by the survival and adhesion of encapsulated cells post-injection. The treatment of skeletal muscle defect with the cell-laden hydrogel approach significantly enhanced the regeneration of localized muscular trauma.

Decidual macrophage derived MMP3 contributes to extracellular matrix breakdown in spiral artery remodeling in early human pregnancy.

Remodeling of the uterine spiral arteries is required for a successful pregnancy. This process requires the co-ordinated activity of a number of different cell types including uterine natural killer cells, decidual macrophages, extravillous trophoblast cells, vascular smooth muscle cells and endothelial cells. We have previously demonstrated that decidual macrophages facilitate breakdown of fibronectin and laminin in a model of spiral artery remodeling. The aim of the current study was to determine which matrix metalloproteinases (MMPs) decidual macrophages express and play roles in extracellular matrix (ECM) breakdown in vascular remodeling. Decidual macrophages were isolated from first trimester decidua and cultured for 24 h to obtain conditioned medium. MMP secretion was assessed by a membrane based array and immunohistochemistry of decidual sections. In addition, the chorionic plate artery (CPA) model was used with decidual macrophage conditioned medium, with and without a MMP3 inhibitor and ECM protein expression assessed using quickscore. The decidual macrophages secreted a wide range of MMPs, with MMP3 being the most predominant. Co-localization of MMP3 to decidual macrophages was confirmed by immunohistochemistry. Decidual macrophage conditioned medium facilitated breakdown of laminin and fibronectin in the CPA model, an effect that was abrogated by the MMP3 inhibitor. These data further support the role of decidual macrophages in tissue remodeling in the first trimester of pregnancy. An alteration in their numbers or phenotype would impact spiral artery remodeling and contribute to the etiology of a number of complications of pregnancy.

Bioorthogonally Cross-Linked Hyaluronan-Laminin Hydrogels for 3D Neuronal Cell Culture and Biofabrication.

Laminins (LNs) are key components in the extracellular matrix of neuronal tissues in the developing brain and neural stem cell niches. LN-presenting hydrogels can provide a biologically relevant matrix for the 3D culture of neurons toward development of advanced tissue models and cell-based therapies for the treatment of neurological disorders. Biologically derived hydrogels are rich in fragmented LN and are poorly defined concerning composition, which hampers clinical translation. Engineered hydrogels require elaborate and often cytotoxic chemistries for cross-linking and LN conjugation and provide limited possibilities to tailor the properties of the materials. Here a modular hydrogel system for neural 3D cell cultures, based on hyaluronan and poly(ethylene glycol), that is cross-linked and functionalized with human recombinant LN-521 using bioorthogonal copper-free click chemistry, is shown. Encapsulated human neuroblastoma cells demonstrate high viability and grow into spheroids. Long-term neuroepithelial stem cells (lt-NES) cultured in the hydrogels can undergo spontaneous differentiation to neural fate and demonstrate significantly higher viability than cells cultured without LN. The hydrogels further support the structural integrity of 3D bioprinted structures and maintain high viability of bioprinted and syringe extruded lt-NES, which can facilitate biofabrication and development of cell-based therapies.

Bisphenol S Impairs Invasion and Proliferation of Extravillous Trophoblasts Cells by Interfering with Epidermal Growth Factor Receptor Signaling.

The placenta supports fetal growth and is vulnerable to exogenous chemical exposures. We have previously demonstrated that exposure to the emerging chemical bisphenol S (BPS) can alter placental endocrine function. Mechanistically, we have demonstrated that BPS interferes with epidermal growth factor receptor (EGFR) signaling, reducing placenta cell fusion. Extravillous trophoblasts (EVTs), a placenta cell type that aids with vascular remodeling, require EGF to invade into the maternal endometrium. We hypothesized that BPS would impair EGF-mediated invasion and proliferation in EVTs. Using human EVTs (HTR-8/SVneo cells), we tested whether BPS could inhibit the EGF response by blocking EGFR activation. We also evaluated functional endpoints of EGFR signaling, including EGF endocytosis, cell invasion and proliferation, and endovascular differentiation. We demonstrated that BPS blocked EGF-induced phosphorylation of EGFR by acting as a competitive antagonist to EGFR. Transwell assay and a three-dimensional microfluidic chip invasion assay revealed that BPS exposure can block EGF-mediated cell invasion. BPS also blocked EGF-mediated proliferation and endovascular differentiation. In conclusion, BPS can prevent EGF-mediated EVT proliferation and invasion through EGFR antagonism. Given the role of EGFR in trophoblast proliferation and differentiation during placental development, our findings suggest that maternal exposure to BPS may contribute to placental dysfunction via EGFR-mediated mechanisms.