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The objective of this study was to create injectable photo‐crosslinkable biomaterials, using gelatin methacryloyl (GelMA) hydrogel, combined with a decellularized bone matrix (BMdc) and a deproteinized (BMdp) bovine bone matrix. These were intended to serve as bioactive scaffolds for dentin regeneration. The parameters for GelMA hydrogel fabrication were initially selected, followed by the incorporation of BMdc and BMdp at a 1% (w/v) ratio. Nano‐hydroxyapatite (nHA) was also included as a control. A physicochemical characterization was conducted, with FTIR analysis indicating that the mineral phase was complexed with GelMA, and BMdc was chemically bonded to the amide groups of gelatin. The porous structure was preserved post‐BMdc incorporation, with bone particles incorporated alongside the pores. Conversely, the mineral phase was situated inside the pore opening, affecting the degree of porosity. The mineral phase did not modify the degradability of GelMA, even under conditions of type I collagenase‐mediated enzymatic challenge, allowing hydrogel injection and increased mechanical strength. Subsequently, human dental pulp cells (HDPCs) were seeded onto the hydrogels. The cells remained viable and proliferative, irrespective of the GelMA composition. All mineral phases resulted in a significant increase in alkaline phosphatase activity and mineralized matrix deposition. However, GelMA‐BMdc exhibited higher cell expression values, significantly surpassing those of all other formulations. In conclusion, our results showed that GelMA‐BMdc produced a porous and stable hydrogel, capable of enhancing odontoblastic differentiation and mineral deposition when in contact with HDPCs, thereby showing potential for dentin regeneration. [ABSTRACT FROM AUTHOR]

Cartilage damage presents a significant clinical challenge due to its intrinsic avascular nature which limits self-repair. Addressing this, our study focuses on an alginate-based bioink, integrating human articular cartilage, for cartilage tissue engineering. This novel bioink was formulated by encapsulating C20A4 human articular chondrocytes in sodium alginate, polyvinyl alcohol, gum arabic, and cartilage extracellular matrix powder sourced from allograft femoral condyle shavings. Using a 3D bioprinter, constructs were biofabricated and cross-linked, followed by culture in standard medium. Evaluations were conducted on cellular viability and gene expression at various stages. Results indicated that the printed constructs maintained a porous structure conducive to cell growth. Cellular viability was 87% post printing, which decreased to 76% after seven days, and significantly recovered to 86% by day 14. There was also a notable upregulation of chondrogenic genes, COL2A1 (p = 0.008) and SOX9 (p = 0.021), suggesting an enhancement in cartilage formation. This study concludes that the innovative bioink shows promise for cartilage regeneration, demonstrating substantial viability and gene expression conducive to repair and suggesting its potential for future therapeutic applications in cartilage repair. [ABSTRACT FROM AUTHOR]

Heart failure is a major health problem in which the heart is unable to pump enough blood to meet the body’s needs. It is a progressive disease that becomes more severe over time and can be caused by a variety of factors, including heart attack, cardiomyopathy and heart valve disease. There are various methods to cure this disease, which has many complications and risks. The advancement of knowledge and technology has proposed new methods for many diseases. One of the promising new treatments for heart failure is tissue engineering. Tissue engineering is a field of research that aims to create living tissues and organs to replace damaged or diseased tissue. The goal of tissue engineering in heart failure is to improve cardiac function and reduce the need for heart transplantation. This can be done using the three important principles of cells, biomaterials and signals to improve function or replace heart tissue. The techniques for using cells and biomaterials such as electrospinning, hydrogel synthesis, decellularization, etc. are diverse. Treating heart failure through tissue engineering is still under development and research, but it is hoped that there will be no transplants or invasive surgeries in the near future. In this study, based on the most important research in recent years, we will examine the power of tissue engineering in the treatment of heart failure.

Peripheral nerve injury (PNI) is one of the major clinical treatment challenges following an impact on the body. When PNI manifests as nerve gaps, surgical connections and exogenous grafts are required. Recently, electrically conductive polymers (CPs) based nerve guidance conduits have yielded promising results for treating PNI. Polypyrrole (PPy) has become one of the most commonly used CPs in PNI repair due to its advantages of high conductivity and excellent biocompatibility. In this study, we combined different PPy concentrations with a chitosan (CS) temperature-sensitive hydrogel system containing decellularized nerve matrix (DNM) to construct the electrically conductive nerve conduits. We evaluated the physical and biological properties of four groups of nerve conduits. It was found that the PPy concentrations were proportional to the electrical conductivity of the nerve conduits. The mechanical properties of the nerve conduits increased with higher PPy concentrations but decreased when the PPy concentration was as high as 8%. Meanwhile, the co-blending of PPy and DNM gave the nerve conduit suitable degradation properties. Furthermore, in vitro cytotoxicity assay and live/dead assay demonstrated these conduits could support the adhesion and growth of cells. In summary, the electrically conductive nerve conduits with high conductivity, mechanical properties, biodegradation characteristics, and cytocompatibility had potential applications in the field of peripheral nerve regeneration. [ABSTRACT FROM AUTHOR]

Cell source is the key to decellularized matrix (DM) strategy. This study compared 3 cell types, osteocytes with/without dominant active Wnt/β-catenin signaling (daCO and WTO) and bone marrow stromal cells (BMSCs) for their DMs in bone repair. Decellularization removes all organelles and >95% DNA, and retained >74% collagen and >71% GAG, maintains the integrity of cell basement membrane with dense boundaries showing oval and honeycomb structure in osteocytic DM and smooth but irregular shape in the BMSC-DM. DM produced higher cell survival rate (90%) and higher proliferative activity. In vitro, daCO-DM induces more and longer stress fibers in BMSCs, conducive to cell adhesion, spreading, and osteogenic differentiation. 8-wk after implantation of the critical-sized parietal bone defect model, daCO-DM formed tight structures, composed of a large number of densely-arranged type-I collagen under polarized light microscope, which is similar to and integrated with host bone. BV/TV (>54%) was 1.5, 2.9, and 3.5 times of WTO-DM, BMSC-DM, and none-DM groups, and N.Ob/T.Ar (3.2 × 102/mm2) was 1.7, 2.9, and 3.3 times. At 4-wk, daCO-DM induced osteoclastogenesis, 2.3 times higher than WTO-DM; but BMSC-DM or none-DM didn't. daCO-DM increased the expression of RANKL and MCSF, Vegfa and Angpt1, and Ngf in BMSCs, which contributes to osteoclastogenesis, angiogenesis, and neurogenesis, respectively. daCO-DM promoted H-type vessel formation and nerve markers β3-tubulin and NeuN expression. Conclusion: daCO-DM produces metabolic and neurovascularized organoid bone to accelerate the repair of bone defects. These features are expected to achieve the effect of autologous bone transplantation, suitable for transformation application.

Decellularized extracellular matrices show а great promise as materials for tissue engineering and regenerative medicine. Recently, there has been an increasing interest in the use of cell-derived extracellular matrices (CD-ECMs). The present mini-review focuses on advantages and disadvantages of the CD-ECMs, describes the variety of approaches to modify the CD-ECMs and discusses the CD-ECMs application fields. In particular, CD-ECMs were shown to serve as cell culture substrate, as base for biocompatible scaffold production, as drug for cell-free therapy and as component of disease models. [ABSTRACT FROM AUTHOR]

Annulus fibrosus (AF) repair remains a challenge because of its limited self-healing ability. Endogenous repair strategies combining scaffolds and growth factors show great promise in AF repair. Although the unique and beneficial characteristics of decellularized extracellular matrix (ECM) in tissue repair have been demonstrated, the poor mechanical property of ECM hydrogels largely hinders their applications in tissue regeneration. In the present study, we combined polyethylene glycol diacrylate (PEGDA) and decellularized annulus fibrosus matrix (DAFM) to develop an injectable, photocurable hydrogel for AF repair. We found that the addition of PEGDA markedly improved the mechanical strength of DAFM hydrogels while maintaining their porous structure. Transforming growth factor-β1 (TGF-β1) was further incorporated into PEGDA/DAFM hydrogels, and it could be continuously released from the hydrogel. The in vitro experiments showed that TGF-β1 facilitated the migration of AF cells. Furthermore, PEGDA/DAFM/TGF-β1 hydrogels supported the adhesion, proliferation, and increased ECM production of AF cells. In vivo repair performance of the hydrogels was assessed using a rat AF defect model. The results showed that the implantation of PEGDA/DAFM/TGF-β1 hydrogels effectively sealed the AF defect, prevented nucleus pulposus atrophy, retained disc height, and partially restored the biomechanical properties of disc. In addition, the implanted hydrogel was infiltrated by cells resembling AF cells and well integrated with adjacent AF tissue. In summary, findings from this study indicate that TGF-β1-supplemented DAFM hydrogels hold promise for AF repair.

Abstract One of the severe complications occurring because of the patient’s intubation is tracheal stenosis. Its incidence has significantly risen because of the COVID-19 pandemic and tends only to increase. Here, we propose an alternative to the donor trachea and synthetic prostheses—the tracheal equivalent. To form it, we applied the donor trachea samples, which were decellularized, cross-linked, and treated with laser to make wells on their surface, and inoculated them with human gingiva-derived mesenchymal stromal cells. The fabricated construct was assessed in vivo using nude (immunodeficient), immunosuppressed, and normal mice and rabbits. In comparison with the matrix ones, the tracheal equivalent samples demonstrated the thinning of the capsule, the significant vessel ingrowth into surrounding tissues, and the increase in the submucosa resorption. The developed construct was shown to be highly biocompatible and efficient in trachea restoration. These results can facilitate its clinical translation and be a base to design clinical trials. Graphical Abstract

A healthy lymphatic system is required to return excess interstitial fluid back to the venous circulation. However, up to 49% of breast cancer survivors eventually develop breast cancer-related lymphedema due to lymphatic injuries from lymph node dissections or biopsies performed to treat cancer. While early-stage lymphedema can be ameliorated by manual lymph drainage, no cure exists for late-stage lymphedema when lymph vessels become completely dysfunctional. A viable late-stage treatment is the autotransplantation of functional lymphatic vessels. Here we report on a novel engineered lymphatic flap that may eventually replace the skin flaps used in vascularized lymph vessel transfers. The engineered flap mimics the lymphatic and dermal compartments of the skin by guiding multi-layered tissue organization of mesenchymal stem cells and lymphatic endothelial cells with an aligned decellularized fibroblast matrix. The construct was tested in a novel bilayered wound healing model and implanted into athymic nude rats. The in vitro model demonstrated capillary invasion into the wound gaps and deposition of extracellular matrix fibers, which may guide anastomosis and vascular integration of the graft during wound healing. The construct successfully anastomosed in vivo, forming chimeric vessels of human and rat cells. Overall, our flap replacement has high potential for treating lymphedema. [ABSTRACT FROM AUTHOR]

The sufficient imitation of tissue structures and components represents an effective and promising approach for tissue engineering and regenerative medicine applications. Dental pulp disease is one of the most common oral diseases, although functional pulp regeneration remains challenging. Herein, we propose a strategy that employs hydrogel microspheres incorporated with decellularized dental pulp matrix-derived bioactive factors to simulate a pulp-specific three-dimensional (3D) microenvironment. The dental pulp microenvironment-specific microspheres constructed by this regenerative strategy exhibited favorable plasticity, biocompatibility, and biological performances. Human dental pulp stem cells (hDPSCs) cultured on the constructed microspheres exhibited enhanced pulp-formation ability in vitro. Furthermore, the hDPSCs-microcarriers achieved the regeneration of pulp-like tissue and new dentin in a semi-orthotopic model in vivo. Mechanistically, the decellularized pulp matrix-derived bioactive factors mediated the multi-directional differentiation of hDPSCs to regenerate the pulp tissue by eliciting the secretion of crucial bioactive cues. Our findings demonstrated that a 3D dental pulp-specific microenvironment facilitated by hydrogel microspheres and dental pulp-specific bioactive factors regenerated the pulp-dentin complex and could be served as a promising treatment option for dental pulp disease. Injectable bioscaffolds are increasingly used for regenerative endodontic treatment. Despite their success related to their ability to load stem cells, bioactive factors, and injectability, conventional bulk bioscaffolds have drawbacks such as ischemic necrosis in the central region. Various studies have shown that ischemic necrosis in the central region can be corrected by injectable hydrogel microspheres. Unfortunately, pristine microspheres or microspheres without dental pulp-specific bioactive factor would oftentimes fail to regulate stem cells fates in dental pulp multi-directional differentiation. Our present study reported the biofabrication of dental pulp-derived decellularized matrix functionalized gelatin microspheres, which contained dental pulp-specific bioactive factors and have the potential application in endodontic regeneration. [Display omitted] [ABSTRACT FROM AUTHOR]

The limited bioactivity of scaffold materials is an important factor that restricts the development of bone tissue engineering. Wnt3a activates the classicWnt/β-catenin signaling pathway which effects bone growth and development by the accumulation of β-catenin in the nucleus. In this study, we fabricated 3D printed PCL scaffold with Wnt3a-induced murine bone marrow-derived stromal cell line ST2 decellularized matrix (Wnt3a-ST2-dCM-PCL) and ST2 decellularized matrix (ST2-dCM-PCL) by freeze-thaw cycle and DNase decellularization treatment which efficiently decellularized >90% DNA while preserved most protein. Compared to ST2-dCM-PCL, Wnt3a-ST2-dCM-PCL significantly enhanced newly-seeded ST2 proliferation, osteogenic differentiation and upregulated osteogenic marker genes alkaline phosphatase (Alp), Runx2, type I collagen (Col 1) and osteocalcin (Ocn) mRNA expression. After 14 days of osteogenic induction, Wnt3a-ST2-dCM-PCL promoted ST2 mineralization. These results demonstrated that Wnt3a-induced ST2 decellularized matrix improve scaffold materials' osteoinductivity and osteoconductivity. [ABSTRACT FROM AUTHOR]

Abstract Background Colorectal cancer is a commonly diagnosed cancer worldwide. Unfortunately, many patients do not respond to standard chemotherapy treatments and develop disease relapse and metastases. Besides cancer cell specific genetic changes, heterogeneity in the tumor microenvironment contribute to the clinical presentation of the disease and can potentially also influence drug resistance. By using a recently developed patient-derived scaffold method monitoring how a standardized reporter cancer cell line adapts to various microenvironments treated with chemotherapy, we wanted to clarify how individual patient specific microenvironments influence the chemotherapy response in colorectal cancer. Methods Surgically resected colorectal cancer specimens from 89 patients were decellularized to produce patient-derived scaffold, which were seeded with HT29 cells, cultured for 3 weeks, and treated with 5-fluorouracil. Gene expression changes of adapted and treated HT29 cells were monitored by qPCR and compared with clinical parameters including disease-free survival. Results The effects of 5-fluorouracil treatment varied between different patient-derived scaffold, but generally induced a reduced expression of proliferation genes and increased expression of pluripotency and epithelial-to-mesenchymal transition genes. Interestingly, patient-derived scaffold cultures obtained from patients with disease recurrences showed a significantly less pronounced anti-proliferative effect of 5-fluorouracil and more pronounced increase of pluripotency, with MKI67 and POU5F1 being among the most significant genes linked to disease relapse in colorectal cancer. Conclusions Colorectal patient-derived scaffold can decode clinically relevant tumor microenvironmental influence of 5-fluorouracil treatment effects opening up for optimized precision medicine in colorectal cancer treatment.

Intervertebral disc degeneration (IDD)-induced low back pain significantly influences the quality of life, placing a burden on public health systems worldwide. Currently available therapeutic strategies, such as conservative or operative treatment, cannot effectively restore intervertebral disc (IVD) function. Decellularized matrix (DCM) is a tissue-engineered biomaterial fabricated using physical, chemical, and enzymatic technologies to eliminate cells and antigens. By contrast, the extracellular matrix (ECM), including collagen and glycosaminoglycans, which are well retained, have been extensively studied in IVD regeneration. DCM inherits the native architecture and specific-differentiation induction ability of IVD and has demonstrated effectiveness in IVD regeneration in vitro and in vivo. Moreover, significant improvements have been achieved in the preparation process, mechanistic insights, and application of DCM for IDD repair. Herein, we comprehensively summarize and provide an overview of the roles and applications of DCM for IDD repair based on the existing evidence to shed a novel light on the clinical treatment of IDD.

Osteochondral defects involving cartilage and subchondral bone are difficult to repair. Recently, scientists have attempted to develop composite scaffolds for repair of osteochondral injuries. However, the available composite scaffolds do not fully recapitulate the functions of the cartilage, subchondral bone, and cancellous bone. In this study, we developed a bioactive multiphase scaffold by decellularizing an intact osteochondral graft for osteochondral repair. The porosity and mechanical properties of the scaffolds were optimized using laser drilling and collagen digestion. The scaffold promoted the recellularization of the cartilage layer. The experimental results showed that the multiphasic scaffolds had excellent mechanical properties and structural stability similar to that of the normal rabbit osteochondral tissue. The in vitro analysis showed that the scaffold promoted zone-specific gene expression. Approximately 12 weeks after in vivo implantation, the multiphasic scaffold significantly facilitated the concurrent regeneration of cartilage and subchondral bone in a rabbit model (detected using gross and micro-computed tomography images, histological staining, immunohistochemistry, and visualization of the collagen network). Overall, this study provides ideas for the development of new multiphasic scaffolds for osteochondral defect repair.

Conjunctival defects severely impair ocular surface homeostasis and vision. However, the existing conjunctival reconstruction methods are still unable to achieve satisfactory functional replacement of conjunctiva. Scaffold materials that provide a more stem-cell-friendly microenvironment are in urgent need of improvement. Herein we reported a functional conjunctival reconstruction scaffold constructed from decellularized matrix derived from rabbit subconjunctival fibroblasts (DM-SCF) loaded with conjunctival epithelial stem cells (CjESCs) for ocular surface repair. The DM-SCF had a thickness and mechanical strength close to that of normal conjunctiva and showed excellent biocompatibility. CjESCs inoculated on DM-SCF maintained good stem cell properties, which showed long-term maintenance of low differentiation and good proliferation activity. DM-SCF transplantation with CjESCs showed satisfactory results in rabbit models of a large conjunctival defect. The conjunctiva was reconstructed with abundant goblet cell expression and regular fiber arrangement. Protein mass spectrometry revealed the active extracellular matrix components of DM-SCF, regulating the activation of Wnt/β-catenin and Notch pathways for stem cell maintenance. Overall, our study developed a promising new strategy for ocular surface reconstruction by optimizing bioactive scaffold and providing functional stem cell niche, so as to achieve long-standing and superior functional repair.

Background: Owing to the disappointing regenerative ability of osteochondral tissue, without treatment an osteochondral defect would progress to osteoarthritis. This situation motivates the need for new strategies to enhance the regeneration of osteochondral defects. Purpose: To develop a tissue-engineering scaffold by tethering bone morphogenetic protein 2 (BMP2) and transforming growth factor beta 3 (TGFβ3) in a layer-specific manner on a slotted decellularized osteochondral matrix (SDOM) and to evaluate the efficacy of this scaffold for osteochondral regeneration. Study Design: Controlled laboratory study. Methods: Normal osteochondral tissue from the rabbit patellofemoral groove was sectioned into a slot shape and decellularized for fabricating an SDOM. The collagen-binding domain (CBD) was fused into the N-terminus of BMP2 or TGFβ3 to synthesize 2 recombinant growth factors (GFs) (CBD-BMP2 or CBD-TGFβ3), which were tethered to the bone layer and cartilage layer, respectively, of the SDOM to prepare a tissue-engineering scaffold (namely, CBD-GFs/SDOM). After examining the influence of the CBD-GFs/SDOM on the viability and layer-specific differentiation of bone marrow mesenchymal stem cells in vitro, we determined the regeneration potential of the CBD-GFs/SDOM on osteochondral regeneration in a rabbit model. A total of 72 New Zealand White rabbits with a cylindrical osteochondral defect in the patellofemoral groove were randomly assigned to 3 groups: defect only (control [CTL] group), defect patched with an SDOM (SDOM group), and defect patched with the CBD-GFs/SDOM (CBD-GFs/SDOM group). At 6 or 12 weeks postoperatively, the rabbits were euthanized to harvest the knee joint, which was then evaluated via gross observation, micro–computed tomography, histological staining, and mechanical testing. Results: In vitro, the CBD-GFs/SDOM was noncytotoxic, showed high biomimetics with normal osteochondral tissue, was suitable for cell adhesion and growth, and had good layer-specific ability in inducing stem cell differentiation. Macroscopic images showed that the CBD-GFs/SDOM group had significantly better osteochondral regeneration than the CTL and SDOM groups had. Micro–computed tomography demonstrated that much more bony tissue was formed at the defect sites in the CBD-GFs/SDOM group compared with the defect sites in the CTL or SDOM group. Histological analysis showed that the CBD-GFs/SDOM group had a significant enhancement in osteochondral regeneration at 6 and 12 weeks postoperatively in comparison with the CTL or SDOM group. At 12 weeks postoperatively, the mechanical properties of reparative tissue were significantly better in the CBD-GFs/SDOM group than in the other groups. Conclusion: The CBD-GFs/SDOM is a promising scaffold for osteochondral regeneration. Clinical Relevance: The findings of this study indicated that the CBD-GFs/SDOM is an excellent candidate for reconstructing osteochondral defects, which may be translated for clinical use in the future. [ABSTRACT FROM AUTHOR]

Nasal septum defects can currently only be reconstructed using autologous cartilage grafts. In this study, we examine the reconstruction of septal cartilage defects in a rabbit model using porcine decellularized nasal septal cartilage (DNSC) functionalized with recombinant platelet-derived growth factor-BB (PDFG-BB). The supportive function of the transplanted DNSC was estimated by the degree of septum deviation and shrinkage using magnetic resonance imaging (MRI). The biocompatibility of the transplanted scaffolds was evaluated by histology according to international standards. A study group with an autologous septal transplant was used as a reference. In situ regeneration of cartilage defects was assessed by histological evaluation 4 and 16 weeks following DNSC transplantation. A study group with non-functionalized DNSC was introduced for estimation of the effects of PDFG-BB functionalization. DNSC scaffolds provided sufficient structural support to the nasal septum, with no significant shrinkage or septal deviations as evaluated by the MRI. Biocompatibility analysis after 4 weeks revealed an increased inflammatory reaction of the surrounding tissue in response to DNSC as compared to the autologous transplants. The inflammatory reaction was, however, significantly attenuated after 16 weeks in the PDGF-BB group whereas only a slight improvement of the biocompatibility score was observed in the untreated group. In situ regeneration of septal cartilage, as evidenced by the degradation of the DNSC matrix and production of neocartilage, was observed in both experimental groups after 16 weeks but was more pronounced in the PDFG-BB group. Overall, DNSC provided structural support to the nasal septum and stimulated in situ regeneration of the cartilage tissue. Furthermore, PDFG-BB augmented the regenerative potential of DNSC and enhanced the healing process, as demonstrated by reduced inflammation after 16 weeks. [ABSTRACT FROM AUTHOR]

Sterilization is the process of killing all microorganisms, while disinfection is the process of killing or removing all kinds of pathogenic microorganisms except bacterial spores. Biomaterials involved in cell experiments, animal experiments, and clinical applications need to be in the aseptic state, but their physical and chemical properties as well as biological activities can be affected by sterilization or disinfection. Decellularized matrix (dECM) is the low immunogenicity material obtained by removing cells from tissues, which retains many inherent components in tissues such as proteins and proteoglycans. But there are few studies concerning the effects of sterilization or disinfection on dECM, and the systematic introduction of sterilization or disinfection for dECM is even less. Therefore, this review systematically introduces and analyzes the mechanism, advantages, disadvantages, and applications of various sterilization and disinfection methods, discusses the factors influencing the selection of sterilization and disinfection methods, summarizes the sterilization and disinfection methods for various common dECM, and finally proposes a graphical route for selecting an appropriate sterilization or disinfection method for dECM and a technical route for validating the selected method, so as to provide the reference and basis for choosing more appropriate sterilization or disinfection methods of various dECM.

In terms of method of production, collagen carriers are subdivided into materials obtained on the basis of extracellular matrix (ECM) components, particularly collagen-containing hydrogels and decellularized tissue.Objective: to compare in vitro the ability of biopolymer microheterogeneous collagen-containing hydrogel (BMCH) and tissue-specific matrix from decellularized porcine articular cartilage (DPAC) to support adhesion, proliferation and chondrogenic differentiation of human adipose-derived mesenchymal stem cells (hAMSCs).Materials and methods. For cartilage decellularization, we carried out treatment with surfactants (sodium dodecyl sulfate, Triton X-100) followed by exposure in DNAase. The metabolic activity of hAMSCs was assessed by PrestoBlue™ (Invitrogen, USA) staining. The morphological study of cell-engineered constructs (CECs) formed by culturing hAMSCs in the presence of matrices was performed using histological staining and scanning electron microscopy (SEM) with lanthanide contrasting.Results. The number of cells on the surface of both BMCH and DPAC increased within 14 days. Mitochondrial activity of the cells was 1.7, 1.7, and 1.3 times higher on days 3, 10, and 14 when cultured on DPAC compared to BMCH, respectively. On day 14 of cultivation in the chondrogenic culture medium, hAMSCs formed cell layers on the DPAC surface and on the BMCH surface. Cytoplasm of the cells included numerous granules, which, when stained, resembled the matrix itself. On the DPAC matrix surface, cells were more evenly distributed, whereas in the case of BMCH, cell adhesion and proliferation were observed only in certain areas. The ECM produced by the cells contained collagen and glycosaminoglycans (GAGs).Conclusion. The ability of DPAC obtained according to the developed protocol to form CECs with hAMSCs with uniform distribution of cells and their production of specific collagen- and GAG-containing ECM suggests that DPAC is effective in regeneration of damaged cartilage. Chondrogenic differentiation of hAMSCs was observed both when cultured with BMCH and with DPAC. When creating a tissue equivalent of cartilage in vitro, the advantage of using tissue-specific matrix over BMCH should be considered.

One of the severe complications occurring because of the patient's intubation is tracheal stenosis. Its incidence has significantly risen because of the COVID-19 pandemic and tends only to increase. Here, we propose an alternative to the donor trachea and synthetic prostheses—the tracheal equivalent. To form it, we applied the donor trachea samples, which were decellularized, cross-linked, and treated with laser to make wells on their surface, and inoculated them with human gingiva-derived mesenchymal stromal cells. The fabricated construct was assessed in vivo using nude (immunodeficient), immunosuppressed, and normal mice and rabbits. In comparison with the matrix ones, the tracheal equivalent samples demonstrated the thinning of the capsule, the significant vessel ingrowth into surrounding tissues, and the increase in the submucosa resorption. The developed construct was shown to be highly biocompatible and efficient in trachea restoration. These results can facilitate its clinical translation and be a base to design clinical trials. [ABSTRACT FROM AUTHOR]

Background: Colorectal cancer is a commonly diagnosed cancer worldwide. Unfortunately, many patients do not respond to standard chemotherapy treatments and develop disease relapse and metastases. Besides cancer cell specific genetic changes, heterogeneity in the tumor microenvironment contribute to the clinical presentation of the disease and can potentially also influence drug resistance. By using a recently developed patient-derived scaffold method monitoring how a standardized reporter cancer cell line adapts to various microenvironments treated with chemotherapy, we wanted to clarify how individual patient specific microenvironments influence the chemotherapy response in colorectal cancer.Methods: Surgically resected colorectal cancer specimens from 89 patients were decellularized to produce patient-derived scaffold, which were seeded with HT29 cells, cultured for 3 weeks, and treated with 5-fluorouracil. Gene expression changes of adapted and treated HT29 cells were monitored by qPCR and compared with clinical parameters including disease-free survival.Results: The effects of 5-fluorouracil treatment varied between different patient-derived scaffold, but generally induced a reduced expression of proliferation genes and increased expression of pluripotency and epithelial-to-mesenchymal transition genes. Interestingly, patient-derived scaffold cultures obtained from patients with disease recurrences showed a significantly less pronounced anti-proliferative effect of 5-fluorouracil and more pronounced increase of pluripotency, with MKI67 and POU5F1 being among the most significant genes linked to disease relapse in colorectal cancer.Conclusions: Colorectal patient-derived scaffold can decode clinically relevant tumor microenvironmental influence of 5-fluorouracil treatment effects opening up for optimized precision medicine in colorectal cancer treatment. [ABSTRACT FROM AUTHOR]

Decellularized extracellular matrices (DECM) are among the most common types of materials used in tissue engineering due to their cell instructive properties, biodegradability, and accessibility. Particularly in cartilage, a natural collagen type II matrix can be a promising means to provide the necessary cues and support for chondrogenic stem and progenitor cells (CSPCs). However, efficient remodeling of the transplanted DECM is largely dependent on the host immune response, with macrophages playing the central role in orchestrating both inflammatory and regenerative processes. Here we assessed the reaction of human primary macrophages to the cartilage DECM. Our findings show that the xenogeneic collagen matrix can elicit a mixed response in human macrophages, whereby the inflammatory response (M1) and the activation of remodeling (M2) type of macrophages are both present. Additionally, we demonstrate the inhibitory effect of macrophage response on the migratory capacity of human CSPCs. We further show that the inflammatory reaction of macrophages to the cartilage DECM, as well as the resulting inhibitory effects on CSPC migration, can be attenuated by interleukin‐4 (IL‐4). Finally, we demonstrate that IL‐4 can effectively bind the matrix, thereby modulating macrophage response by reducing the inflammatory reaction and inducing the M2 phenotype. [ABSTRACT FROM AUTHOR]

Skin models are used for many applications such as research and development or grafting. Unfortunately, most lack a proper microenvironment producing poor mechanical properties and inaccurate extra-cellular matrix composition and organization. In this report we focused on mechanical properties, extra-cellular matrix organization and cell interactions in human skin samples reconstructed with pure collagen or dermal decellularized extra-cellular matrices (S-dECM) and compared them to native human skin. We found that Full-thickness S-dECM samples presented stiffness two times higher than collagen gel and similar to ex vivo human skin, and proved for the first time that keratinocytes also impact dermal mechanical properties. This was correlated with larger fibers in S-dECM matrices compared to collagen samples and with a differential expression of F-actin, vinculin and tenascin C between S-dECM and collagen samples. This is clear proof of the microenvironment's impact on cell behaviors and mechanical properties. In vitro skin models have been used for a long time for clinical applications or in vitro knowledge and evaluation studies. However, most lack a proper microenvironment producing a poor combination of mechanical properties and appropriate biological outcomes, partly due to inaccurate extra-cellular matrix (ECM) composition and organization. This can lead to limited predictivity and weakness of skin substitutes after grafting. This study shows, for the first time, the importance of a complex and rich microenvironment on cell behaviors, matrix macro- and micro-organization and mechanical properties. The increased composition and organization complexity of dermal skin decellularized extra-cellular matrix populated with differentiated cells produces in vitro skin models closer to native human skin physiology. [Display omitted] [ABSTRACT FROM AUTHOR]

Abstract Background Colorectal cancer is the second most common cause of cancer‐related death worldwide and standardized therapies often fail to treat the more aggressive and progressive types of colorectal cancer. Tumor cell heterogeneity and influence from the surrounding tumor microenvironment (TME) contribute to the complexity of the disease and large variability in clinical outcomes. Methods To model the heterogeneous nature of colorectal cancer, we used patient‐derived scaffolds (PDS), which were obtained via decellularization of surgically resected tumor material, as a growth substrate for standardized cell lines. Results After confirmation of native cell absence and validation of the structural and compositional integrity of the matrix, 89 colorectal PDS were repopulated with colon cancer cell line HT29. After 3 weeks of PDS culture, HT29 cells varied their gene and protein expression profiles considerably compared to 2D‐grown HT29 cells. Markers associated with proliferation were consistently decreased, while markers associated with pluripotency were increased in PDS‐grown cells compared to their 2D counterparts. When comparing the PDS‐induced changes in HT29 cells with clinically relevant tumor information from individual patients, we observed significant associations between stemness/pluripotency markers and tumor location, and between epithelial‐to‐mesenchymal transition (EMT) markers and cancer mortality. Kaplan–Meier analysis revealed that low PDS‐induced EMT correlated with worse cancer‐specific survival. Conclusions The colorectal PDS model can be used as a simplified personalized tool that can potentially reveal important diagnostic and pathophysiological information related to the TME.

Objective: Decellularized adipose-derived matrix (DAM) can promote adipogenic differentiation and adipose tissue remodeling, but the biological impact of tissue origin on DAM remains unknown. The present study aimed to investigate the effects of tissue origins on the adipogenic capacity of the decellularized matrix by comparing the cellular and tissue responses of DAM versus acellular dermal matrix (ADM).Methods: The in vitro response of adipose-derived stem/stromal cells (ADSCs) to DAM and ADM was characterized by proliferation and differentiation. The in vivo remodeling response was evaluated in the subcutaneous injection model of immunocompromised mice, using histology, protein expression, and transcriptome analysis.Results: Both DAM and ADM exhibited excellent decellularization effects and cytocompatibility. In the absence of exogenous stimuli, DAM could induce adipogenic differentiation of ADSCs compared with ADM. In the animal model, the levels of PDGF, VEGF, and ACRP30 were higher in the DAM groups than in the ADM group, and more neovascularization and extensive adipose tissue remodeling were observed. The mRNA-seq analysis indicated that the DAM implant regulated tissue remodeling by modulating Lat1/2 expression along with Hippo Signaling pathway in the early stage.Conclusion: Tissue origin can influence the biological response of the decellularized matrix. DAM can retain favorable tissue-specific characteristics after the decellularization process and have unique adipogenic effects in vitro and vivo, which can be fully utilized for soft tissue repair and regeneration.

Nasal septum defects can currently only be reconstructed using autologous cartilage grafts. In this study, we examine the reconstruction of septal cartilage defects in a rabbit model using porcine decellularized nasal septal cartilage (DNSC) functionalized with recombinant platelet-derived growth factor-BB (PDFG-BB). The supportive function of the transplanted DNSC was estimated by the degree of septum deviation and shrinkage using magnetic resonance imaging (MRI). The biocompatibility of the transplanted scaffolds was evaluated by histology according to international standards. A study group with an autologous septal transplant was used as a reference. In situ regeneration of cartilage defects was assessed by histological evaluation 4 and 16 weeks following DNSC transplantation. A study group with non-functionalized DNSC was introduced for estimation of the effects of PDFG-BB functionalization. DNSC scaffolds provided sufficient structural support to the nasal septum, with no significant shrinkage or septal deviations as evaluated by the MRI. Biocompatibility analysis after 4 weeks revealed an increased inflammatory reaction of the surrounding tissue in response to DNSC as compared to the autologous transplants. The inflammatory reaction was, however, significantly attenuated after 16 weeks in the PDGF-BB group whereas only a slight improvement of the biocompatibility score was observed in the untreated group. In situ regeneration of septal cartilage, as evidenced by the degradation of the DNSC matrix and production of neocartilage, was observed in both experimental groups after 16 weeks but was more pronounced in the PDFG-BB group. Overall, DNSC provided structural support to the nasal septum and stimulated in situ regeneration of the cartilage tissue. Furthermore, PDFG-BB augmented the regenerative potential of DNSC and enhanced the healing process, as demonstrated by reduced inflammation after 16 weeks. [ABSTRACT FROM AUTHOR]

End-stage liver disease is one of the leading causes of death around the world. Since insufficient sources of transplantable liver and possible immune rejection severely hinder the wide application of conventional liver transplantation therapy, artificial three-dimensional (3D) liver culture and assembly from stem cells have become a new hope for patients with end-stage liver diseases, such as cirrhosis and liver cancer. However, the induced differentiation of single-layer or 3D-structured hepatocytes from stem cells cannot physiologically support essential liver functions due to the lack of formation of blood vessels, immune regulation, storage of vitamins, and other vital hepatic activities. Thus, there is emerging evidence showing that 3D organogenesis of artificial vascularized liver tissue from combined hepatic cell types derived from differentiated stem cells is practical for the treatment of end-stage liver diseases. The optimization of novel biomaterials, such as decellularized matrices and natural macromolecules, also strongly supports the organogenesis of 3D tissue with the desired complex structure. This review summarizes new research updates on novel differentiation protocols of stem cell-derived major hepatic cell types and the application of new supportive biomaterials. Future biological and clinical challenges of this concept are also discussed.

Decellularized extracellular matrix (dECM) has been considered as a promising scaffold in xenotransplantation, yet natural tissue dECM is often mechanically weak and rapidly degraded, compromising the outcomes. How to restore the mechanical strength and optimise the in vivo degradation, but maintain the microstructure and maximumly suppress the immune rejection, remains challenging. For this aim, we prepared and characterised various crosslinked decellularized rabbit uterus matrix (dUECM) and evaluated in vivo performance after uterus xenotransplantation from rabbit to rat. Naturally derived genipin (GP) and procyanidins (PC) were chosen to crosslink the dUECM, producing significant mechanical enhanced crosslinked-dUECM along with prolonged enzymatic degradation rate. Xenogeneic subcutaneous graft studies revealed that PC- and GP-crosslinked dUECM experienced significant cell infiltration and caused low immune reactions, indicating the desired biocompatibility. In vivo transplantation of GP- and PC-crosslinked dUECM to a uterus circular excised rat yielded excellent recellularization ability and promoted uterus regeneration after 90 days. While the reconstruction efficacy of crosslinked dUECM is highly depended on the crosslinking degree, crosslinking condition must be carefully evaluated to balance the role of crosslinked dECM in mechanical and biological support for tissue regeneration promotion.

A healthy lymphatic system is required to return excess interstitial fluid back to the venous circulation. However, up to 49% of breast cancer survivors eventually develop breast cancer-related lymphedema due to lymphatic injuries from lymph node dissections or biopsies performed to treat cancer. While early-stage lymphedema can be ameliorated by manual lymph drainage, no cure exists for late-stage lymphedema when lymph vessels become completely dysfunctional. A viable late-stage treatment is the autotransplantation of functional lymphatic vessels. Here we report on a novel engineered lymphatic flap that may eventually replace the skin flaps used in vascularized lymph vessel transfers. The engineered flap mimics the lymphatic and dermal compartments of the skin by guiding multi-layered tissue organization of mesenchymal stem cells and lymphatic endothelial cells with an aligned decellularized fibroblast matrix. The construct was tested in a novel bilayered wound healing model and implanted into athymic nude rats. The in vitro model demonstrated capillary invasion into the wound gaps and deposition of extracellular matrix fibers, which may guide anastomosis and vascular integration of the graft during wound healing. The construct successfully anastomosed in vivo, forming chimeric vessels of human and rat cells. Overall, our flap replacement has high potential for treating lymphedema.

Heart failure is a major health problem in which the heart is unable to pump enough blood to meet the body's needs. It is a progressive disease that becomes more severe over time and can be caused by a variety of factors, including heart attack, cardiomyopathy and heart valve disease. There are various methods to cure this disease, which has many complications and risks. The advancement of knowledge and technology has proposed new methods for many diseases. One of the promising new treatments for heart failure is tissue engineering. Tissue engineering is a field of research that aims to create living tissues and organs to replace damaged or diseased tissue. The goal of tissue engineering in heart failure is to improve cardiac function and reduce the need for heart transplantation. This can be done using the three important principles of cells, biomaterials and signals to improve function or replace heart tissue. The techniques for using cells and biomaterials such as electrospinning, hydrogel synthesis, decellularization, etc. are diverse. Treating heart failure through tissue engineering is still under development and research, but it is hoped that there will be no transplants or invasive surgeries in the near future. In this study, based on the most important research in recent years, we will examine the power of tissue engineering in the treatment of heart failure., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Rayat Pisheh, Nojabaei, Darvishi, Rayat Pisheh and Sani.)

Objective: To investigate the construction of a novel tissue engineered meniscus scaffold based on low temperature deposition three-dimenisonal (3D) printing technology and evaluate its biocompatibility., Methods: The fresh pig meniscus was decellularized by improved physicochemical method to obtain decellularized meniscus matrix homogenate. Gross observation, HE staining, and DAPI staining were used to observe the decellularization effect. Toluidine blue staining, safranin O staining, and sirius red staining were used to evaluate the retention of mucopolysaccharide and collagen. Then, the decellularized meniscus matrix bioink was prepared, and the new tissue engineered meniscus scaffold was prepared by low temperature deposition 3D printing technology. Scanning electron microscopy was used to observe the microstructure. After co-culture with adipose-derived stem cells, the cell compatibility of the scaffolds was observed by cell counting kit 8 (CCK-8), and the cell activity and morphology were observed by dead/live cell staining and cytoskeleton staining. The inflammatory cell infiltration and degradation of the scaffolds were evaluated by subcutaneous experiment in rats., Results: The decellularized meniscus matrix homogenate appeared as a transparent gel. DAPI and histological staining showed that the immunogenic nucleic acids were effectively removed and the active components of mucopolysaccharide and collagen were remained. The new tissue engineered meniscus scaffolds was constructed by low temperature deposition 3D printing technology and it had macroporous-microporous microstructures under scanning electron microscopy. CCK-8 test showed that the scaffolds had good cell compatibility. Dead/live cell staining showed that the scaffold could effectively maintain cell viability (>90%). Cytoskeleton staining showed that the scaffolds were benefit for cell adhesion and spreading. After 1 week of subcutaneous implantation of the scaffolds in rats, there was a mild inflammatory response, but no significant inflammatory response was observed after 3 weeks, and the scaffolds gradually degraded., Conclusion: The novel tissue engineered meniscus scaffold constructed by low temperature deposition 3D printing technology has a graded macroporous-microporous microstructure and good cytocompatibility, which is conducive to cell adhesion and growth, laying the foundation for the in vivo research of tissue engineered meniscus scaffolds in the next step.

Background: Colorectal cancer is the second most common cause of cancer‐related death worldwide and standardized therapies often fail to treat the more aggressive and progressive types of colorectal cancer. Tumor cell heterogeneity and influence from the surrounding tumor microenvironment (TME) contribute to the complexity of the disease and large variability in clinical outcomes. Methods: To model the heterogeneous nature of colorectal cancer, we used patient‐derived scaffolds (PDS), which were obtained via decellularization of surgically resected tumor material, as a growth substrate for standardized cell lines. Results: After confirmation of native cell absence and validation of the structural and compositional integrity of the matrix, 89 colorectal PDS were repopulated with colon cancer cell line HT29. After 3 weeks of PDS culture, HT29 cells varied their gene and protein expression profiles considerably compared to 2D‐grown HT29 cells. Markers associated with proliferation were consistently decreased, while markers associated with pluripotency were increased in PDS‐grown cells compared to their 2D counterparts. When comparing the PDS‐induced changes in HT29 cells with clinically relevant tumor information from individual patients, we observed significant associations between stemness/pluripotency markers and tumor location, and between epithelial‐to‐mesenchymal transition (EMT) markers and cancer mortality. Kaplan–Meier analysis revealed that low PDS‐induced EMT correlated with worse cancer‐specific survival. Conclusions: The colorectal PDS model can be used as a simplified personalized tool that can potentially reveal important diagnostic and pathophysiological information related to the TME. [ABSTRACT FROM AUTHOR]

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., (Creative Commons Attribution license.)

The arteriovenous loop (AVL) model allows the in vivo engineering of axially vascularized flaps, the so-called AVL flaps. Although AVL flaps can be transplanted microsurgically to cover tissue defects, they lack an epithelial layer on the surface. Therefore, the objective of this study was to engineer axially vascularized AVL flaps with an accompanying epithelial layer for local defect reconstruction. In this study, AVLs were established in 20 male Lewis rats. Minimally invasive injection of keratinocytes onto the surface of the AVL flaps was performed on postoperative day (POD) 21. AVL flaps were explanted from 12 rats on POD 24 or POD 30, then the epithelium formed by the keratinocytes on the surface of the flaps was evaluated using immunofluorescence staining. In six other rats, the AVL flap was locally transposed to cover a critical defect in the rats' leg on POD 30 and explanted for analysis on POD 40. In two control rats, sodium chloride was applied instead of keratinocytes. These control flaps were also transplanted on POD 30 and explanted on POD 40. Our results revealed that 3 days after keratinocyte application, a loose single-layered epithelium was observed histologically on the AVL flaps surface, whereas after 9 days, a multilayered and structured epithelium had grown. The epithelium on the transplanted AVL flaps showed its physiological differentiation when being exposed to an air-liquid interface. Histologically, a layered epithelium identical to the rats' regular skin was formed. In the sodium chloride control group, no epithelium had been grown. This study clearly demonstrates that axially vascularized AVL flaps can be processed in the subcutaneous chamber by minimally invasive injection of keratinocytes. Thus, AVL flaps with an intact epithelial layer were engineered and could be successfully transplanted for local defect coverage in a small animal model.

Decellularized extracellular matrix (dECM) has been considered as a promising scaffold in xenotransplantation, yet natural tissue dECM is often mechanically weak and rapidly degraded, compromising the outcomes. How to restore the mechanical strength and optimise the in vivo degradation, but maintain the microstructure and maximumly suppress the immune rejection, remains challenging. For this aim, we prepared and characterised various crosslinked decellularized rabbit uterus matrix (dUECM) and evaluated in vivo performance after uterus xenotransplantation from rabbit to rat. Naturally derived genipin (GP) and procyanidins (PC) were chosen to crosslink the dUECM, producing significant mechanical enhanced crosslinked-dUECM along with prolonged enzymatic degradation rate. Xenogeneic subcutaneous graft studies revealed that PC- and GP-crosslinked dUECM experienced significant cell infiltration and caused low immune reactions, indicating the desired biocompatibility. In vivo transplantation of GP- and PC-crosslinked dUECM to a uterus circular excised rat yielded excellent recellularization ability and promoted uterus regeneration after 90 days. While the reconstruction efficacy of crosslinked dUECM is highly depended on the crosslinking degree, crosslinking condition must be carefully evaluated to balance the role of crosslinked dECM in mechanical and biological support for tissue regeneration promotion. [ABSTRACT FROM AUTHOR]

Tarsal plate regeneration has always been a challenge in the treatment of eyelid defects. The commonly used clinical treatments such as hard palate mucosa grafts cannot achieve satisfactory repair effects. Tissue engineering has been considered as a promising technology. However, tarsal plate tissue engineering is difficult to achieve due to its complex structure and lipid secretion function. Three-dimensional (3D) printing technology has played a revolutionary role in tissue engineering because it can fabricate complex scaffolds through computer aided design (CAD). In this study, it was novel in applying 3D printing technology to the fabrication of tarsal plate scaffolds using poly-caprolactone (PCL). The decellularized matrix of adipose-derived mesenchymal stromal cells (DMA) was coated on the surface of the scaffold, and its biofunction was further studied. Immortalized human SZ95 sebocytes were seeded on the scaffolds so that neutral lipids were secreted for replacing meibocytes. In vitro experiments revealed excellent biocompatibility of DMA-PCL scaffolds with sebocytes. In vivo experiments revealed excellent sebocytes proliferation on the DMA–PCL scaffolds. Meanwhile, sebocytes seeded on the scaffolds secreted abundant neutral lipid in vitro and in vivo. In conclusion, a 3D-printed PCL scaffold modified with DMA was found to be a promising substitute for tarsal plate tissue engineering.

Regenerative therapies for intervertebral disc (IVD) injuries are currently a major challenge that is addressed in different ways by scientists working in this field. Extracellular matrix (ECM) deriving from decellularized non-autologous tissues has been established as a biomaterial with remarkable regenerative capacity and its potential as a therapeutic agent is rising. In the present study, we investigated the potential of decellularized Wharton’s jelly matrix (DWJM) from human umbilical cord to act as an ECM-based scaffold for IVD cell culturing. An efficient detergent-enzymatic treatment (DET) was used to produce DWJM maintaining its native microarchitecture. Afterward, immunofluorescence, biochemical assays and electron microscopy analysis showed that DWJM was able to produce sizeable 3D cell aggregates, when combined with human mesenchymal stromal cells isolated from WJ (MSCs) and IVD cells. These latter cells are characterized by the loss of their chondrocyte-like phenotype since they have been isolated from degenerated IVD and in vitro expanded to further de-differentiate. While the effect exerted by DWJM on MSCs was essentially the induction of proliferation, conversely, on IVD cells the DWJM promoted cell differentiation toward a discogenic phenotype. Notably, for the first time, the ability of DWJM to improve the degenerated phenotype of human IVD cells was demonstrated, showing that the mere presence of the matrix maintained the viability of the cells, and positively affected the expression of critical regulators of IVD homeostasis, such as SOX2, SOX9, and TRPS1 transcription factors at specific culture time. Our data are in line with the hypothesis that the strengthening of cell properties in terms of viability and expression of specific proteins at precise times represents an important condition in the perspective of guiding the recovery of cellular functionality and triggering regenerative potential. Currently, there are no definitive surgical or pharmacological treatments for IVD degeneration (IDD) able to restore the disc structure and function. Therefore, the potential of DWJM to revert degenerated IVD cells could be exploited in the next future an ECM-based intradiscal injectable therapeutic.

Tingfang Sun,1,* Man Liu,2,* Sheng Yao,1 Yanhui Ji,1 Lei Shi,3 Kai Tang,1 Zekang Xiong,1 Fan Yang,1 Kaifang Chen,1 Xiaodong Guo1 1Department of Orthopedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China; 2Department of Gastroenterology and Hepatology, Taikang Tongji Hospital, Wuhan 430050, China; 3State Key Laboratory of Materials Processing and Die & Mould Technology, School of Materials Science and Engineering, Huazhong University of Science and Technology, Wuhan 430074, China *These authors contributed equally to this work Introduction: At present, the treatment of osteoporotic defects poses a great challenge to clinicians, owing to the lower regeneration capacity of the osteoporotic bone as compared with the normal bone. The guided bone regeneration (GBR) technology provides a promising strategy to cure osteoporotic defects using bioactive membranes. The decellularized matrix from the small intestinal submucosa (SIS) has gained popularity for its natural microenvironment, which induces cell response. Materials and methods: In this study, we developed heparinized mineralized SIS loaded with bone morphogenetic protein 2 (BMP2)-related peptide P28 (mSIS/P28) as a novel GBR membrane for guided osteoporotic bone regeneration. These mSIS/P28 membranes were obtained through the mineralization of SIS (mSIS), followed by P28 loading onto heparinized mSIS. The heparinized mSIS membrane was designed to improve the immobilization efficacy and facilitate controlled release of P28. P28 release from mSIS-heparin-P28 and its effects on the proliferation, viability, and osteogenic differentiation of bone marrow stromal stem cells from ovariectomized rats (rBMSCs-OVX) were investigated in vitro. Furthermore, a critical-sized OVX calvarial defect model was used to assess the bone regeneration capability of mSIS-heparin-P28 in vivo. Results: In vitro results showed that P28 release from mSIS-heparin-P28 occurred in a controlled manner, with a long-term release time of 40 days. Moreover, mSIS-heparin-P28 promoted cell proliferation and viability, alkaline phosphatase activity, and mRNA expression of osteogenesis-related genes in rBMSCs-OVX without the addition of extra osteogenic components. In vivo experiments revealed that mSIS-heparin-P28 dramatically stimulated osteoporotic bone regeneration. Conclusion: The heparinized mSIS loaded with P28 may serve as a potential GBR membrane for repairing osteoporotic defects. Keywords: osteoporotic defect, guided bone regeneration, decellularized matrix, heparin, BMP2-related peptide, control release

Repairing large segmental bone defects above a critical size remains challenging with high risk of delayed union or even non-union. From the perspective of bone development and clinical experience, periosteum plays an indispensable role in bone repair and reconstruction. In this study, we explored the feasibility of using preosteoblast-derived matrix (pODM) as a biomimetic periosteum. By culturing MC3T3-E1 cell sheet on poly(dimethylsiloxane) and performing decellularization, an integral cell-free sheet of pODM could be readily harvested. Bone marrow mesenchymal stem cells (BMSCs) adhered and proliferated well on pODM. In addition, pODM exhibited a chemotactic effect on BMSCs in a concentration-dependent manner and also promoted osteogenic differentiation of BMSCs. Following that, pODM was wrapped around a gelatin methacryloyl (GelMA) hydrogel to construct an engineered periosteum-bone substitute. A rabbit radius segmental bone defect model was used to examine the bone repair efficacy of pODM/GelMA. Upon implantation of pODM/GelMA construct for 12 weeks, the critical-sized bone defects completely healed with remarkable full reconstruction of medullary cavity at the radial diaphysis. Together, this work proposes a high potency of using precursor cell-derived matrix as a biomimetic periosteum, which preserves the beneficial biological factors while avoids the limitations of using exogenous cells for bone regeneration. Combining precursor cell-derived matrix with hydrogel may provide a promising periosteum-bone biomimetic substitute for bone repair. Repairing large segmental bone defects above a critical size remains challenging. As the periosteum plays an essential role in bone repair, this study aimed to explore the use of preosteoblast-derived matrix (pODM), harvested from decellularized MC3T3-E1 cell sheet, as a biomimetic periosteum to facilitate bone repair. We found that in vitro , pODM exhibited considerable chemotactic effect and osteogenic induction capability to bone marrow mesenchymal stem cells (BMSCs). In vivo , implantation of pODM/gelatin methacryloyl (GelMA) constructs as engineered periosteum-bone substitutes effectively repaired the critical-sized segmental bone defects at rabbit radius. Surprisingly, remarkable full reconstruction of medullary cavity at the diaphysis was achieved. Therefore, combining pODM with hydrogel may provide a promising biomimetic substitute for bone repair. Image, graphical abstract [ABSTRACT FROM AUTHOR]

This study aims to evaluate the CD146+ stem cells obtained from the human umbilical cord and their extracellular matrix proteins on in vitro Pseudomonas aeruginosa and Staphylococcus aureus biofilms to understand their possible antimicrobial activity. CD146+ stem cells were determined according to cell surface markers and differentiation capacity. Characterization of the decellularized matrix was done with DAPI, Masson's Trichrome staining and proteome analysis. Cell viability/proliferation of cells in co-cultures was evaluated by WST-1 and crystal-violet staining. The effects of cells and decellularized matrix proteins on biofilms were investigated on a drip flow biofilm reactor and their effects on gene expression were determined by RT-qPCR. We observed that CD146/105+ stem cells could differentiate adipogenically and decellularized matrix showed negative DAPI and positive collagen staining with Masson' s Trichrome. Proteome analysis of the decellularized matrix revealed some matrix components and growth factors. Although the decellularized matrix significantly reduced the cell counts of P. aeruginosa, no significant difference was observed for S. aureus cells in both groups. Supporting data was obtained from the gene expression results of P. aeruginosa with the significant down-regulation of rhlR and lasR. For S. aureus, icaADBC genes were significantly up-regulated when grown on the decellularized matrix. [ABSTRACT FROM AUTHOR]

Tissue engineering (TE) has become a primary research topic for the treatment of diseased or damaged tendon/ligament (T/L) tissue. T/L injuries pose a severe clinical burden worldwide, necessitating the development of effective strategies for T/L repair and tissue regeneration. TE has emerged as a promising strategy for restoring T/L function using decellularized extracellular matrix (dECM)-based scaffolds. dECM scaffolds have gained significant prominence because of their native structure, relatively high bioactivity, low immunogenicity, and ability to function as scaffolds for cell attachment, proliferation, and differentiation, which are difficult to imitate using synthetic materials. Here, we review the recent advances and possible future prospects for the advancement of dECM scaffolds for T/L tissue regeneration. We focus on crucial scaffold properties and functions, as well as various engineering strategies employed for biomaterial design in T/L regeneration. dECM provides both the physical and mechanical microenvironments required by cells to survive and proliferate. Various decellularization methods and sources of allogeneic and xenogeneic dECM in T/L repair and regeneration are critically discussed. Additionally, dECM hydrogels, bio-inks in 3D bioprinting, and nanofibers are briefly explored. Understanding the opportunities and challenges associated with dECM-based scaffold development is crucial for advancing T/L repairs in tissue engineering and regenerative medicine. Schematic illustration of the application of dECM in tendon/ligament repair, including sources, decellularization methods and dECM post-processing methods. [Display omitted] • dECM scaffolds offer a natural environment for tissue development and show remarkable potential for tendon/ligament repair. • Comprehensive overview of the opportunities and challenges using dECM as a scaffold for tendon/ligament repair and regeneration. • Post-processing strategies enhance the dECM application scenarios and show great promise for tendon/ligament repair. [ABSTRACT FROM AUTHOR]

Articular cartilage has a limited capacity to heal itself and the defects often result in the development of osteoarthritis. Current cartilage tissue engineering strategies seek to regenerate injured tissue by creating scaffolds that aim to mimic the unique structure and composition of native articular cartilage. The purpose of this study was to develop a three dimensional acellular cartilage matrix as a powder ACMP for cartilage substitution. Additionally, the prepared scaffolds obtained from lyophilization was evaluated in vitro, by histological, SEM and XRD analysis, SEM showed that the ACMP had a sponge-like structure of high porosity. XRD showed, the broad peaks with relatively low intensity at 2θ = 7.9°, 20.3° and 37.7° is related to presence of semi-crystalline or amorphous organic materials, which are chondroitin sulfate(CS), Collagen Type 1(Col1) and type 2(Col2), hyaluronic Acid(HA) and proteoglycan (PG). This study provides a technique to produce acellular ECM derived from articular bovine cartilage which may serve as a xenogenic scaffold for cartilage tissue regeneration. [ABSTRACT FROM AUTHOR]

Abstract Rapid development of tissue engineering technology provides new methods for tracheal cartilage regeneration. However, the current lack of an ideal scaffold makes engineering of trachea cartilage tissue into a three-dimensional (3-D) tubular structure a great challenge. Although a decellularized trachea matrix (DTM) has become a recognized scaffold for trachea cartilage regeneration, it is difficult for cells to detach from or penetrate the matrix because of its non-porous structure. To tackle these problems, a laser micropore technique (LMT) was applied in the current study to enhance trachea sample porosity, and facilitate decellularizing treatment and cell ingrowth. Furthermore, after optimizing LMT and decellularizing treatment parameters, LMT-treated DTM (LDTM) retained its natural tubular structure with only minor extracellular matrix damage. Moreover, compared with DTM, the current study showed that LDTM significantly improved the adherence rate of cells with perfect cell biocompatibility. Moreover, the optimal implantation cell density for chondrogenesis with LDTM was determined to be 1 × 108 cells/ml. Collectively, the results suggest that the novel LDTM is an ideal scaffold for trachea tissue engineering. Graphical abstract fx1 [ABSTRACT FROM AUTHOR]

Introduction: Different diseases may affect the trachea and, therefore, the quality of life. Tissue engineering may be a therapeutic alternative for patients with such diseases, using decellularized trachea matrixes seeded with cells from the recipient, which do not generate immune response and may even prevent rejection of zoo-transplants. Objective: To evaluate a decellularization method to obtain tracheal extracellular matrixes in the porcine model. Materials and methods: Two study groups, treated and control, were obtained from 5 porcine tracheas. A chemical-enzymatic method for decellularization was used. Histological analyses were performed with hematoxylin-eosin, Masson’s trichromic stain and safranin O. Biomechanical properties of both groups were evaluated by determining the Young modulus, maximum strength and deformation rate. Results: Compared to the controls, there was a 66 % decrease in the cell content in the treated specimens. Collagen was preserved and a reduction of glycosaminoglycans was detected. Biomechanical tests revealed a significant difference in the percentage of deformation, with no alteration of the remaining parameters. Conclusions: The evaluated decellularization method proved to be efficient to reduce the cellular content of porcine tracheas, with a considerable decrease in cost and production time. These advantages could make it a good option for the socio-economic Colombian conditions.

Hydrogels are three-dimensional (3D) materials able to absorb and retain water in large amounts while maintaining their structural stability. Due to their considerable biocompatibility and similarity with the body’s tissues, hydrogels are one of the most promising groups of biomaterials. The main application of these hydrogels is in regenerative medicine, in which they allow the formation of an environment suitable for cell differentiation and growth. Deriving from these hydrogels, it is, therefore, possible to obtain bioactive materials that can regenerate tissues. Because vessels guarantee the right amount of oxygen and nutrients but also assure the elimination of waste products, angiogenesis is one of the processes at the base of the regeneration of a tissue. On the other hand, it is a very complex mechanism and the parameters to consider are several. Indeed, the factors and the cells involved in this process are numerous and, for this reason, it has been a challenge to recreate a biomaterial able to adequately sustain the angiogenic process. However, in this review the focal point is the application of natural hydrogels in angiogenesis enhancing and their potential to guide this process.

Graphical abstract Abstract Stem cell-based tissue engineering is a promising treatment for intervertebral disc (IVD) degeneration. A bio-scaffold that can maintain the function of transplanted cells and possesses favorable mechanical properties is needed in tissue engineering. Decellularized nucleus pulposus (dNP) has the potential to be a suitable bio-scaffold because it mimics the native nucleus pulposus (NP) composition. However, matrix loss during decellularization and difficulty in transplantation limit the clinical application of dNP scaffolds. In this study, we fabricated an injectable dNP-based cell delivery system (NPCS) and evaluated its properties by assessing the microstructure, biochemical composition, water content, biosafety, biostability, and mechanical properties. We also investigated the stimulatory effects of the bio-scaffold on the NP-like differentiation of adipose-derived stem cells (ADSCs) in vitro and the regenerative effects of the NPCS on degenerated NP in an in vivo animal model. The results showed that approximately 68% and 43% of the collagen and sGAG, respectively, remained in the NPCS after 30 days. The NPCS also showed mechanical properties similar to those of fresh NP. In addition, the NPCS was biocompatible and able to induce NP-like differentiation and extracellular matrix (ECM) synthesis in ADSCs. The disc height index (almost 81%) and the MRI index (349.05 ± 38.48) of the NPCS-treated NP were significantly higher than those of the degenerated NP after 16 weeks. The NPCS also partly restored the ECM content and the structure of degenerated NP in vivo. Our NPCS has good biological and mechanical properties and has the ability to promote the regeneration of degenerated NP. Statement of Significance Nucleus pulposus (NP) degeneration is usually the origin of intervertebral disc degeneration. Stem cell-based tissue engineering is a promising treatment for NP regeneration. Bio-scaffolds which have favorable biological and mechanical properties are needed in tissue engineering. Decellularized NP (dNP) scaffold is a potential choice for tissue engineering, but the difficulty in balancing complete decellularization and retaining ECM limits its usage. Instead of choosing different decellularization protocols, we complementing the sGAG lost during decellularization by cross-linking via genipin and fabricating an injectable dNP-based cell delivery system (NPCS) which has similar components as the native NP. We also investigated the biological and mechanical properties of the NPCS in vitro and verified its regenerative effects on degenerated IVDs in an animal model. [ABSTRACT FROM AUTHOR]

Abstract Background The development and application of biomaterials to promote stem cell proliferation and differentiation has undergone major expansion over the last few years. Decellularized stem cell matrix (DSCMs) represent bioactive and biocompatible materials which achieve similar characteristics of native extracellular matrix. DSCMs have given promising outcomes in generating novel cell culture substrates mimicking specific niche microenvironments in tissue engineering. Aims This research aims at producing two different DSCMs obtained from adipose derived mesenchymal stem cells and bone marrow mesenchymal stem cells, characterize them and evaluate the DSCMs bioactivity on mesenchymal stem cells. Methods DSCMs were produced using ascorbic or chondrogenic medium, which were then used as a scaffold for adipose derived mesenchymal stem cells and bone marrow mesenchymal stem cells, respectively. The biological characteristics of both types of DSCMs, including cell attachment, morphology, proliferation, viability, and chondrogenic and osteogenic differentiation were evaluated and compared. Results Differences between ascorbic derived-DSCMs and chondrogenic derived DSCMs were found. Chondrogenic derived-DSCMs remained compact and stronger during extraction and this made their handling easier. Ascorbic derived-DSCMs showed a different protein composition to chondrogenic-DSCMs. Bioactive characteristics analyzed were different depending on the cellular origin of DSCM and the method used to produce them. Conclusions The DSCMs obtained in this work constitutes favorable structure- and growth factors providing a microenvironment which is very similar to that of native ECM, which results in enhanced biological potential of the MSCs and responsiveness to the induction of differentiation. We found differences between ascorbic derived-DSCMs and chondrogenic derived DSCMs. Our results suggest that the cell source used to produce DSCMs is highly related to the bioactive characteristics of DSCMs. [ABSTRACT FROM AUTHOR]