Human ECM Research Articles

Combining human liver ECM with topographically featured electrospun scaffolds for engineering hepatic microenvironment

Liver disease cases are rapidly expanding worldwide, and transplantation remains the only effective cure for end-stage disease. There is an increasing demand for developing potential drug treatments, and regenerative therapies using in-vitro culture platforms. Human decellularized extracellular matrix (dECM) is an appealing alternative to conventional animal tissues as it contains human-specific proteins and can serve as scaffolding materials. Herein we exploit this with human donor tissue from discarded liver which was not suitable for transplant using a synergistic approach to combining biological and topographical cues in electrospun materials as an in-vitro culture platform. To realise this, we developed a methodology for incorporating human liver dECM into electrospun polycaprolactone (PCL) fibres with surface nanotopographies (230–580 nm). The hybrid scaffolds were fabricated using varying concentrations of dECM; their morphology, mechanical properties, hydrophilicity and stability were analysed. The scaffolds were validated using HepG2 and primary mouse hepatocytes, with subsequent results indicating that the modified scaffolds-maintained cell growth and influenced cell attachment, proliferation and hepatic-related gene expression. This work demonstrates a novel approach to harvesting the potential from decellularized human tissues in the form of innovative in-vitro culture platforms for liver.

The Development of a 3D PET Fibrous Scaffold Modified with an Umbilical Cord dECM for Liver Tissue Engineering.

Organ and tissue dysfunction represents a clinically significant condition. By integrating cell biology with materials science, tissue engineering enables the reconstruction and restoration of damaged tissues or organs, offering a noninvasive repair approach. In our study, we replicated the cellular growth environment by utilizing a human umbilical cord-derived decellularized extracellular matrix (dECM) as a modifying agent for the polyethylene terephthalate (PET) polymeric fiber scaffold. This allowed us to create a dECM-coated polyester fiber-based scaffold, PET-dECM, tailored for liver tissue engineering purposes. We effectively produced a decellularized human umbilical cord-derived ECM through a combined decellularization process involving trypsin/EDTA, TritonX-100, and sodium deoxycholate. The application of the dECM coating onto the PET material was accomplished through several steps, such as ester hydrolysis, EDC/NHS-activated crosslinking, and dECM conjugation. The biological performance of the PET-dECM was validated using RG cell culture assays. Notably, the dECM coating significantly improved PET's hydrophilicity and biocompatibility, thereby aiding cell adhesion, proliferation, and functional differentiation (p < 0.05). It was further found that the hepatocyte function of HepaRG was significantly enhanced on the PET-dECM, which may be attributed to the dECM's ability to facilitate the restoration of cell polarity. The PET-dECM holds promise as an effective hepatocyte culture carrier and could potentially find application in liver tissue engineering.

Assessment of inflammatory suppression and fibroblast infiltration in tissue remodelling by supercritical CO2 acellular dermal matrix (scADM) utilizing Sprague Dawley models.

Human skin-derived ECM aids cell functions but can trigger immune reactions; therefore it is addressed through decellularization. Acellular dermal matrices (ADMs), known for their regenerative properties, are used in tissue and organ regeneration. ADMs now play a key role in plastic and reconstructive surgery, enhancing aesthetics and reducing capsular contracture risk. Innovative decellularization with supercritical carbon dioxide preserves ECM quality for clinical use. The study investigated the cytotoxicity, biocompatibility, and anti-inflammatory properties of supercritical CO2 acellular dermal matrix (scADM) in vivo based on Sprague Dawley rat models. Initial experiments in vitro with fibroblast cells confirmed the non-toxic nature of scADM and demonstrated cell infiltration into scADMs after incubation. Subsequent tests in vitro revealed the ability of scADM to suppress inflammation induced by lipopolysaccharides (LPS) presenting by the reduction of pro-inflammatory cytokines TNF-α, IL-6, IL-1β, and MCP-1. In the in vivo model, histological assessment of implanted scADMs in 6months revealed a decrease in inflammatory cells, confirmed further by the biomarkers of inflammation in immunofluorescence staining. Besides, an increase in fibroblast infiltration and collagen formation was observed in histological staining, which was supported by various biomarkers of fibroblasts. Moreover, the study demonstrated vascularization and macrophage polarization, depicting increased endothelial cell formation. Alteration of matrix metalloproteinases (MMPs) was analyzed by RT-PCR, indicating the reduction of MMP2, MMP3, and MMP9 levels over time. Simultaneously, an increase in collagen deposition of collagen I and collagen III was observed, verified in immunofluorescent staining, RT-PCR, and western blotting. Overall, the findings suggested that scADMs offer significant benefits in improving outcomes in implant-based procedures as well as soft tissue substitution.

Melatonin Promotes Differentiation of Human Spermatogonial Stem Cells Cultured on Three-Dimensional Decellularized Human Testis Matrix.

The use of 3D (3-Dimensional) culture systems supported cell-to-cell and cell-to-extracellular matrix (ECM) interactions, proliferation, and differentiation of SSCs (Spermatogonial stem cells). The potential advantages of ECM-based scaffolds for in vitro spermatogenesis have been indicated in human and animal experiments. Furthermore, the strong antioxidant and anti-inflammatory activities of melatonin have improved in vitro manipulation of human SSCs in culture conditions. SSCs were isolated from the testis of three dead-brain patients and then propagated for four weeks. The characterization of SSC colonies was done using real-time PCR (Polymerase chain reaction), ICC (Immunocytochemistry), and xenotransplantation to mice model. Decellularization of the human testis was performed using 0.3% sodium dodecyl sulfate (SDS) solution and 1% Triton X-100. Also, various characterizations of DTM (Decellularized testicular matrix ) were carried out using histological staining and DNA content analysis. The optimum dose of melatonin was selected by MTT (Methyl thiazol tetrazolium). SSCs were cultured in 4 groups: control, melatonin, ECM, and ECM-melatonin in a differentiation medium for four weeks. The expression of differentiation genes was evaluated by real-time polymerase chain reaction. In addition, the viability of cultured cells was assessed by MTT assay. The results of ICC and real-time PCR showed the expression of undifferentiated SSC markers (PLZF and GRFA1) in SSC colonies following the 2D culture of isolated SSCs. The presence of testicular ECM components after different staining methods; and the reduction of DNA content confirmed the proper decellularization process. Germ cell apoptosis significantly decreased in melatonin and ECM groups, and the higher viability of SSCs was seen in the ECM-melatonin group. The relative expression of GFRA1 and PRM2 decreased and increased in ECM and ECM-melatonin groups, respectively. Our study showed that the addition of melatonin to the human naturally-derived ECM scaffold could provide a suitable platform for inducing the differentiation and preserving the viability of SSCs.

Puerarin modulates proliferation, inflammation and ECM metabolism in human nucleus pulposus mesenchymal stem cells via the lncRNA LINC01535

BackgroundIntervertebral disc degeneration (IVDD) is a highly prevalent musculoskeletal disorder characterized by progressive destruction of the intervertebral disc, leading to chronic low back pain and disability. Emerging evidence suggests that dysregulation of ferroptosis, a recently discovered form of regulated cell death, participates in IVDD pathogenesis. Puerarin, a natural flavonoid compound from Pueraria lobata, has shown promise in modulating ferroptosis in various diseases. MethodsHuman nucleus pulposus-derived mesenchymal stem cells (NPMSCs) were isolated and identified by flow cytometry. We investigated the effects of puerarin on human NPMSCs and examined the underlying molecular mechanisms. ResultsPuerarin significantly promoted human NPMSC proliferation, as evidenced by the increased cell viability and colony formation ability. Furthermore, puerarin suppressed the expression of cyclooxygenase-2 and the proinflammatory cytokine interleukin-6 in NPMSCs, demonstrating the anti-inflammatory properties of the compound. Notably, puerarin attenuated ECM breakdown by downregulating the ECM-degrading enzymes MMP3, MMP13 and ADAMTS5, and it increased ECM component synthesis, including collagen type II and aggrecan, by NPMSCs. Moreover, puerarin inhibited ferroptosis in NPMSCs by modulating the expression of key ferroptosis-related genes, including ACSL4, PTGS2 and GPX4. Depletion of LINC01535 abolished the effects of puerarin on proliferation, inflammation and ECM metabolism, suggesting a key role of this lncRNA in mediating the effects of puerarin. ConclusionOur findings show that puerarin promotes the proliferation of human NPMSCs and ECM synthesis by these cells. Furthermore, puerarin inhibits inflammation and ECM degradation by suppressing ferroptosis via LINC01535. These results provide insights into the molecular mechanisms underlying the therapeutic effects of puerarin in IVDD. Targeting ferroptosis and its regulatory factors, such as LINC01535, may have therapeutic potential for the treatment of IDD and other degenerative disorders of the intervertebral disc. Further studies are needed to uncover the translational potential of puerarin and its downstream targets in preclinical and clinical applications.

Effect of human heart valve-derived ECM and NP/PCL electrospun nanofibrous sheet on mice bone marrow mononuclear cells and cardiac repair

BackgroundBiomaterials can improve cardiac repair combined with transplantation of bone marrow mononuclear cells (BMMNCs). In this study, we compared the phenotype and cardiac repair between human heart valve-derived scaffold (hHVS) and natural protein/polycaprolactone (NP/PCL) anchored BMNNCs. Methods and resultsBMMNCs were obtained from mice five days following myocardial infarction. Subsequently, BMMNCs were separately cultured on hHVS and PCL. Proliferation and cardiomyogenic differentiation were detected in vitro. Cardiac function was measured after transplantation of cell-seeded cardiac patch on MI mice. After that, the BMMNCs were collected for mRNA sequencing after culturing on the scaffolds. Upon anchoring onto hHVS or PCL, BMMNCs exhibited an increased capacity for proliferation in vitro, however, the cells on hHVS exhibited superior cardiomyogenic differentiation ability. Moreover, both BMMNCs-seeded biomaterials effectively improved cardiac function after 4 weeks of transplantation, with reduced infarction area and restricted LV remodeling. Cell-seeded hHVS was superior to cell-seeded PCL. ConclusionBMMNCs on hHVS showed better capacity in both cell cardiac repairing and improvement for cardiac function than on PCL. Compared with seeded onto PCL, BMMNCs on hHVS had 253 genes up regulated and 189 genes down regulated. The reason for hHVS’ better performance than PCL as a scaffold for BMMNCs might be due to the fact that optimized method of decellularization let more cytokines in ECM retained.

Human hepatocytes response to pathological shifts in liver fibrosis

Protracted liver assault results in hepatic fibrosis, frequently advancing into cirrhosis, hepatic failure, portal hypertension and hepatocellular carcinoma or degeneration. Hepatic fibrosis emanates from the wound-healing response of the liver to persistent assault. Following acute liver insult, such as viral hepatitis, parenchymal cells regenerate and substitute the necrotic or apoptotic cells. A salient proportion of the affected population of NAFLD advances to nonalcoholic steatohepatitis, NASH, and is characterised by inflammation, hepatocellular swelling, with ensuing eruption of deteriorating fibrosis. An untreated or persistent NASH can progressively culminate in liver cirrhosis and hepatocellular carcinoma. Chronic liver derangement results in pathologic ECM protein or liver fibrosis aggregation. In essence, the encompassing characterisation of human ECM molecular composition provides for excursions into mechanisms of liver disease or hepatocellular degeneration. Hepatocytes are characterized by elevated mitochondrial concentrations. The difference between NASH and fibrosis is evident that the progression of NASH to cirrhosis commences as inflamed liver tissue evolves into scar tissue or fibrosis that is capable of obliterating the liver from optimum functionality. A third or more of persons presenting with NASH develop cirrhosis , and also elevates and accelerates the risk in the development of liver cancer. The hepatic stellate cells are responsible for liver fibrosis as they are substantially involved in the initiation, progression, and regression of liver fibrosis via the secretion of fibrogenic factors which promote portal fibrocytes, fibroblasts, and bone marrow-derived myofibroblasts in the formation of collagen, and consequential propagation of fibrosis.

Fabrication and evaluation of some characteristics of the skin's dermis gel direction to application in regenerative medicine

Objective:The tight connection between the cell-extracellular matrix (ECM - Extracellular matrix) is the foundation of tissue development and repair. The extracellular framework simulates the biological properties of organism-based ECM. The ECM of the skin’s dermis is a natural material, containing ingredients ideal for creating products that help tissue heal. This study focuses on the production of gel products from human skin ECM. The skin ECM is gelled by dissolving in a pepsin enzyme solution.Method: 3D gel formation process using neutral enzyme solution and incubation under physiological conditions. Gel samples were evaluated for speed and formability. Additionally, the dermal gel preparation was evaluated for its effects on the survival and proliferation of human fibroblasts in vitro by toxicity testing and proliferation testing, for 10 days. Results: Direct and extract toxicity assessments showed that the ECM gel regimen was safe for fibroblast survival. At the same time, fibroblasts had proliferation properties after 10 days in culture conditions with gel extract.Conclusion: These results showed that the ECM gel product regimen has the ability to positively impact the biological properties of cells and has the potential for application in the field of regenerative medicine.

The Effect of MultilayeredElectrospun PLLA Nanofibers Coated with Human Amnion or Bladder ECM Proteins on Epithelialization and Smooth Muscle Regeneration in the Rabbit Bladder.

Nanofibrous scaffolds have attracted much attention in bladder reconstruction approaches due to their excellent mechanical properties. In addition, their biological properties can be improved by combination with biological materials. Taking into account the advantages of nanofibrous scaffolds and decellularized extracellular matrix (dECM) in tissue engineering, scaffolds of poly-L-lactic acid (PLLA) coated with decellularized human amnion membrane (hAM) or sheep bladder (SB)-derived ECM proteins are developed (amECM-coated PLLA and sbECM-coated PLLA, respectively). The bladder regenerative potential of modified electrospun PLLA scaffolds is investigated in rabbits. The presence of ECM proteins is confirmed on the nanofibers' surface. Coating the surface of the PLLA nanofibers improves cell adhesion and proliferation. Histological and immunohistochemical evaluations show that rabbits subjected to cystoplasty with a multilayered PLLA scaffold show de novo formation and maturation of the multilayered urothelial layer. However, smooth muscle bundles (myosin heavy chain [MHC] and α-smooth muscle actin [α-SMA] positive) are detected only in ECM-coated PLLA groups. All groups show no evidence of a diverticulumor fistula in the urinary bladder. These results suggest that the biofunctionalization of electrospun PLLA nanofibers with ECM proteins can be a promising option for bladder tissue engineering. Furthermore, hAM can also replace animal-sourced ECM proteins in bladder tissue regeneration approaches.

Human adipose ECM alleviates radiation-induced skin fibrosis via endothelial cell-mediated M2 macrophage polarization

Radiation therapy can lead to late radiation-induced skin fibrosis (RISF), causing movement restriction, pain, and organ dysfunction. This study evaluated adipose-derived extracellular matrix (Ad-ECM) as a mitigator of RISF. Female C57BL/6J mice that were irradiated developed fibrosis, which was mitigated by a single local Ad-ECM injection, improving limb movement and reducing epithelium thickness and collagen deposition. Ad-ECM treatment resulted in decreased expression of pro-inflammatory and fibrotic genes, and upregulation of anti-inflammatory cytokines, promoting M2 macrophage polarization. Co-culture of irradiated human fibroblasts with Ad-ECM down-modulated fibrotic gene expression and enhanced bone marrow cell migration. Ad-ECM treatment also increased interleukin (IL)-4, IL-5, and IL-15 expression in endothelial cells, stimulating M2 macrophage polarization and alleviating RISF. Prophylactic use of Ad-ECM showed effectiveness in mitigation. This study suggests Ad-ECM's potential in treating chronic-stage fibrosis.

Patient-derived extracellular matrix demonstrates role of COL3A1 in blood vessel mechanics.

Vascular Ehlers-Danlos Syndrome (vEDS) is a rare autosomal dominant disease caused by mutations in the COL3A1 gene, which renders patients susceptible to aneurysm and arterial dissection and rupture. To determine the role of COL3A1 variants in the biochemical and biophysical properties of human arterial ECM, we developed a method for synthesizing ECM directly from vEDS donor fibroblasts. We found that the protein content of the ECM generated from vEDS donor fibroblasts differed significantly from ECM from healthy donors, including upregulation of collagen subtypes and other proteins related to ECM structural integrity. We further found that ECM generated from a donor with a glycine substitution mutation was characterized by increased glycosaminoglycan content and unique viscoelastic mechanical properties, including increased time constant for stress relaxation, resulting in a decrease in migratory speed of human aortic endothelial cells when seeded on the ECM. Collectively, these results demonstrate that vEDS patient-derived fibroblasts harboring COL3A1 mutations synthesize ECM that differs in composition, structure, and mechanical properties from healthy donors. These results further suggest that ECM mechanical properties could serve as a prognostic indicator for patients with vEDS, and the insights provided by the approach demonstrate the broader utility of cell-derived ECM in disease modeling. STATEMENT OF SIGNIFICANCE: The role of collagen III ECM mechanics remains unclear, despite reported roles in diseases including fibrosis and cancer. Here, we generate fibrous, collagen-rich ECM from primary donor cells from patients with vascular Ehlers-Danlos syndrome (vEDS), a disease caused by mutations in the gene that encodes collagen III. We observe that ECM grown from vEDS patients is characterized by unique mechanical signatures, including altered viscoelastic properties. By quantifying the structural, biochemical, and mechanical properties of patient-derived ECM, we identify potential drug targets for vEDS, while defining a role for collagen III in ECM mechanics more broadly. Furthermore, the structure/function relationships of collagen III in ECM assembly and mechanics will inform the design of substrates for tissue engineering and regenerative medicine.

Potentiation of Sphingolipids and TGF-β in the human corneal stroma reveals intricate signaling pathway crosstalks

Corneal haze brought on by fibrosis due to insult can lead to partial or complete vision loss. Currently, corneal transplantation is the gold standard for treating severe corneal fibrosis, which comes with the risk of rejection and the issue of donor tissue shortages. Sphingolipids (SPLs) are known to be associated with fibrosis in various tissues and organs, including the cornea. We previously reported that SPLs are tightly related to Transforming Growth Factor β (TGF-β) signaling and corneal fibrogenesis. This study aimed to elucidate the interplay of SPLs, specifically sphingosine-1-phosphate (S1P) signaling, and its’ interactions with TGF-β signaling through detailed analyses of the corresponding downstream signaling targets in the context of corneal fibrosis, in vitro. Healthy human corneal fibroblasts (HCFs) were isolated, plated on polycarbonate membranes, and stimulated with a stable Vitamin C derivative. The 3D constructs were treated with either 5 μM sphingosine-1-phosphate (S1P), 5 μM SPHK I2 (I2; inhibitor of sphingosine kinase 1, one of the two enzymes responsible for generating S1P in mammalian cells), 0.1 ng/mL TGF-β1, or 0.1 ng/mL TGF-β3. Cultures with control medium-only served as controls. All 3D constructs were examined for protein expression of fibrotic markers, SPLs, TGF-βs, and relevant downstream signaling pathways. This data revealed no significant changes in any LTBP (latent TGF-β binding proteins) expression when stimulated with S1P or I2. However, LTBP1 was significantly upregulated via stimulation of TGF-β1 and TGF-β3, whereas LTBP2 was significantly upregulated only with TGF-β3 stimulation. Significant downregulation of TGF-β receptor II (TGF-βRII) following S1P stimulation but significant upregulation following I2 stimulation was observed. Following TGF-β1, S1P, and I2 stimulation, phospho-SMAD2 (pSMAD2) was significantly downregulated. Furthermore, I2 stimulation led to significant downregulation of SMAD4. Adhesion/proliferation/transcription regulation targets, SRC, FAK, and pERK 1/2 were all significantly downregulated by exogenous S1P, whereas I2 only significantly downregulated FAK. Exogenous TGF-β3 caused significant upregulation of AKT. Interestingly, both I2 and TGF-β3 caused significant downregulation of JNK expression. Lastly, TGF-β1 led to significant upregulation of sphingosine kinase 1 (SphK1) and sphingosine-1-phosphate receptor 3 (S1PR3), whereas TGF-β3 caused significant upregulation of only SphK1. Together with previously published work from our group and others, S1P inhibition exhibits great potential as an efficacious anti-fibrotic modality in human corneal stromal ECM. The current findings shed further light on a very complex and rather incompletely investigated mechanism, and cement the intricate crosstalk between SPLs and TGF-β in corneal fibrogenesis. Future studies will dictate the potential of utilizing SPLs/TGF-β signaling modulators as novel therapeutics in corneal fibrosis.

Injectable Decellularized Extracellular Matrix Hydrogel Containing Stromal Cell-Derived Factor 1 Promotes Transplanted Cardiomyocyte Engraftment and Functional Regeneration after Myocardial Infarction

Transplantation of exogenous cardiomyocytes (CMs) is a hopeful method to treat myocardial infarction (MI). However, its clinical application still remains challenging due to low retention and survival rates of the transplanted cells. Herein, a stromal cell-derived factor 1 (SDF-1)-loaded injectable hydrogel based on a decellularized porcine extracellular matrix (dECM) is developed to encapsulate and deliver CMs locally to the infarct area of the heart. The soluble porcine cardiac dECM is composed of similar components such as the human cardiac ECM, which could be self-assembled into a nanofibrous hydrogel at physiological temperature to improve the retention of transplanted CMs. Furthermore, the chemokine SDF-1 could recruit endogenous cells to promote angiogenesis, mitigating the ischemic microenvironment and improving the survival of CMs. The results in vitro show that this composite hydrogel exhibits good biocompatibility, anti-apoptosis property, and chemotactic effects for mesenchymal stromal cells and endothelial cells through SDF-1-CXCR4 axis. Moreover, intramyocardial injection of this composite hydrogel to the infarcted area leads to the promotion of angiogenesis and inhibition of fibrosis, reducing the infarction size and improving the cardiac function. The combination of natural biomaterials, exogenous cells, and bioactive factors shows potential for MI treatment in the clinical application.

Use and Efficacy of Porcine Urinary Bladder Matrix for Tissue Regeneration: A Review

Porcine-derived UBM, a type of acellular ECM, has demonstrated clinical utility for tissue repair and regeneration across various body systems. UBM acts as a full-thickness, exogenic skin substitute and scaffolding for soft tissue reconstruction while mimicking the function and properties of human ECM. This review presents an overview of the current literature evaluating UBM’s clinical and preclinical utility across a broad range of applications. A compilation of studies of human and animal patients with a multitude of tissue defects resulting from various pathologic or injurious processes were systematically reviewed. The types of reconstructions included were categorized by the following surgical domains: abdominal wall; cardiothoracic and pulmonary; gastrointestinal; neurosurgery; oral and maxillofacial; otolaryngology or head and neck; ophthalmology; orthopedic or plastic or orthoplastic surgery; burn and wound care; and urology and gynecology. This systematic review illustrates that UBM may perform as well as or better than other ECM mimetics across various parameters, including reduced time to definitive wound closure, recurrence of wound, infection and/or complication rates, and immunogenic transplant rejection; reduction in overall cost burden to the patient, improved patient satisfaction, and ease of use and maintenance for providers; increased cellular recruitment, invasion, differentiation, and proliferation; and increased repair and regeneration of tissue. This tissue regeneration tends to be more functionally, mechanically, and histologically similar to native tissue through tissue-specific functional remodeling and maturation. This clinical outcome can be seen in various tissue types, levels of injury, and/or defect severity. UBM also proves valuable because of its ability to be used off-the-shelf in surgical, nonsurgical, or office and in-the-field treatment settings.

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

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

Cell-assembled extracellular matrix (CAM): a human biopaper for the biofabrication of pre-vascularized tissues able to connect to the host circulation in vivo

When considering regenerative approaches, the efficient creation of a functional vasculature, that can support the metabolic needs of bioengineered tissues, is essential for their survival after implantation. However, it is widely recognized that the post-implantation microenvironment of the engineered tissues is often hypoxic due to insufficient vascularization, resulting in ischemia injury and necrosis. This is one of the main limitations of current tissue engineering applications aiming at replacing significant tissue volumes. Here, we have explored the use of a new biomaterial, the cell-assembled extracellular matrix (CAM), as a biopaper to biofabricate a vascular system. CAM sheets are a unique, fully biological and fully human material that has already shown stable long-term implantation in humans. We demonstrated, for the first time, the use of this unprocessed human ECM as a microperforated biopaper. Using microvalve dispensing bioprinting, concentrated human endothelial cells (30 millions ml−1) were deposited in a controlled geometry in CAM sheets and cocultured with HSFs. Following multilayer assembly, thick ECM-based constructs fused and supported the survival and maturation of capillary-like structures for up to 26 d of culture. Following 3 weeks of subcutaneous implantation in a mice model, constructs showed limited degradative response and the pre-formed vasculature successfully connected with the host circulatory system to establish active perfusion.This mechanically resilient tissue equivalent has great potential for the creation of more complex implantable tissues, where rapid anastomosis is sine qua non for cell survival and efficient tissue integration.

Tunable Human Myocardium Derived Decellularized Extracellular Matrix for 3D Bioprinting and Cardiac Tissue Engineering.

The generation of 3D tissue constructs with multiple cell types and matching mechanical properties remains a challenge in cardiac tissue engineering. Recently, 3D bioprinting has become a powerful tool to achieve these goals. Decellularized extracellular matrix (dECM) is a common scaffold material due to providing a native biochemical environment. Unfortunately, dECM’s low mechanical stability prevents usage for bioprinting applications alone. In this study, we developed bioinks composed of decellularized human heart ECM (dhECM) with either gelatin methacryloyl (GelMA) or GelMA-methacrylated hyaluronic acid (MeHA) hydrogels dual crosslinked with UV light and microbial transglutaminase (mTGase). We characterized the bioinks’ mechanical, rheological, swelling, printability, and biocompatibility properties. Composite GelMA–MeHA–dhECM (GME) hydrogels demonstrated improved mechanical properties by an order of magnitude compared to the GelMA–dhECM (GE) hydrogels. All hydrogels were extrudable and compatible with human induced pluripotent stem cell derived cardiomyocytes (iCMs) and human cardiac fibroblasts (hCFs). Tissue-like beating of the printed constructs with striated sarcomeric alpha-actinin and connexin 43 expression was observed. The order of magnitude difference between the elastic modulus of these hydrogel composites offers applications in in vitro modeling of the myocardial infarct boundary. Here, as a proof of concept, we created an infarct boundary region with control over the mechanical properties along with the cellular and macromolecular content through printing iCMs with GE bioink and hCFs with GME bioink.

Restoration of Autophagic Flux Rescues Oxidative Damage and Mitochondrial Dysfunction to Protect against Intervertebral Disc Degeneration.

Oxidative stress-induced mitochondrial dysfunction and nucleus pulposus (NP) cell apoptosis play crucial roles in the development of intervertebral disc degeneration (IDD). Increasing studies have shown that interventions targeting impaired autophagic flux can maintain cellular homeostasis by relieving oxidative damage. Here, we investigated the effect of curcumin (CUR), a known autophagy activator, on IDD in vitro and in vivo. CUR suppressed tert-butyl hydroperoxide- (TBHP-) induced oxidative stress and mitochondrial dysfunction and thereby inhibited human NP cell apoptosis, senescence, and ECM degradation. CUR treatment induced autophagy and enhanced autophagic flux in an AMPK/mTOR/ULK1-dependent manner. Notably, CUR alleviated TBHP-induced interruption of autophagosome-lysosome fusion and impairment of lysosomal function and thus contributed to the restoration of blocked autophagic clearance. These protective effects of CUR in TBHP-stimulated human NP cells resembled the effects produced by the autophagy inducer rapamycin, but the effects were partially eliminated by 3-methyladenine- and compound C-mediated inhibition of autophagy initiation or chloroquine-mediated obstruction of autophagic flux. Lastly, CUR also exerted a protective effect against puncture-induced IDD progression in vivo. Our results showed that suppression of excessive ROS production and mitochondrial dysfunction through enhancement of autophagy coupled with restoration of autophagic flux ameliorated TBHP-induced human NP cell apoptosis, senescence, and ECM degradation. Thus, maintenance of the proper functioning of autophagy represents a promising therapeutic strategy for IDD, and CUR might serve as an effective therapeutic agent for IDD.

Evaluation of NV556, a Novel Cyclophilin Inhibitor, as a Potential Antifibrotic Compound for Liver Fibrosis.

Hepatic fibrosis can result as a pathological response to nonalcoholic steatohepatitis (NASH). Cirrhosis, the late stage of fibrosis, has been linked to poor survival and an increased risk of developing hepatocellular carcinoma, with limited treatment options available. Therefore, there is an unmet need for novel effective antifibrotic compounds. Cyclophilins are peptidyl-prolyl cis-trans isomerases that facilitate protein folding and conformational changes affecting the function of the targeted proteins. Due to their activity, cyclophilins have been presented as key factors in several stages of the fibrotic process. In this study, we investigated the antifibrotic effects of NV556, a novel potent sanglifehrin-based cyclophilin inhibitor, in vitro and in vivo. NV556 potential antifibrotic effect was evaluated in two well-established animal models of NASH, STAM, and methionine-choline-deficient (MCD) mice, as well as in an in vitro 3D human liver ECM culture of LX2 cells, a human hepatic stellate cell line. We demonstrate that NV556 decreased liver fibrosis in both STAM and MCD in vivo models and decreased collagen production in TGFβ1-activated hepatic stellate cells in vitro. Taken together, these results present NV556 as a potential candidate for the treatment of liver fibrosis.

Adipose tissue-derived ECM hydrogels and their use as 3D culture scaffold

Adipose tissue has the therapeutic capacity in the form of a fat graft, for example, for treatment of irradiation-induced scars and difficult to heal dermal wounds. For large-scale clinical application, an off-the-shelf product is warranted. In recent years, ECM-derived hydrogels are postulated to harbour therapeutic capacity and might even replicate the beneficial effects of adipose tissue. In normal homeostasis, the natural ECM acts as a deposit of growth factors, that releases them over time. In the healing of lesions, this might promote cell accumulation and proliferation which in turn stimulates angiogenesis and repair. The decellularization of tissue and the generation of hydrogels may leave cytotoxic traces. Therefore, our research assessed the cytotoxic effect of human adipose tissue-derived ECM hydrogels on connective tissue cells i.e. fibroblasts. The results showed no cytotoxicity, meaning the hydrogels caused no cell death. Cell migration and survival were observed when cultured in ECM hydrogels and followed for 7 days. Cell survival in the hydrogel was confirmed with CFDA staining and also cells showed the ability to penetrate and migrate throughout the gel. We conclude that ECM hydrogels are promising to use as innovative therapy for wound healing.