Versatile Hypoxic Extracellular Vesicles Laden in an Injectable and Bioactive Hydrogel for Accelerated Bone Regeneration

Extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) have emerged as an appealing alternative to cell therapy in regenerative medicine. Unlike bone marrow MSCs (BMSCs) cultured in vitro with normoxia, bone marrow in vivo is exposed to a hypoxic environment. To date, it remains unclear whether hypoxia preconditioning can improve the function of BMSC-derived EVs and be more conducive to bone repair. Herein, it is found that hypoxia preconditioned BMSCs secrete more biglycan (Bgn)-rich EVs via proteomics analysis, and these hypoxic EVs (Hypo-EVs) significantly promote osteoblast proliferation, migration, differentiation, and mineralization by activating the phosphatidylinositide 3-kinase/protein kinase B pathway. Subsequently, an injectable bioactive hydrogel composed of poly(ethylene glycol)/polypeptide copolymers is developed to improve the stability and retention of Hypo-EVs in vivo. The Hypo-EVs-laden hydrogel shows continuous liberation of Hypo-EVs for 3 weeks and substantially accelerates bone regeneration in 5-mm rat cranial defects. Finally, it is confirmed that Bgn in EVs is a pivotal protein regulating osteoblast differentiation and mineralization and exerts its effects through paracrine mechanisms. Therefore, this study shows that hypoxia stimulation is an effective approach to optimize the therapeutic effects of BMSC-derived EVs and that injectable hydrogel-based EVs delivery is a promising strategy for tissue regeneration.     

Fibrous Viscoelastic Extracellular Matrix Assists Precise Neuronal Connectivity

Synapse formation in complex neuronal network is a pivotal process for normal functioning of nervous system. Although intense research has been conducted, how neurons and axons are guided toward the target remains largely unclear. In traditional opinions, axons are directed through chemotaxis, while recently mechanotaxis has been brought up as a potential complementary mechanism, as it can provide delicately controlled signals in addition to the random diffusive chemical cues. To further explore the path-finding mechanism, a quasi-3D in vitro model for neuronal cells is constructed by integrating hydrogel collagen I as extracellular matrix (ECM), and primary mouse cortical neurons and PC12 cells are tested. It is strikingly found out that axons and neuronal cells can be precisely guided toward target neurites via ECM. By developing a label-free traction force microscopy technique, the force networks among neurons are presented, validating that the fibrous matrix-transmitted paratensile signals can assist the axon pathfinding. This precise axon guidance is related to the activation of mechanosensitive ion channels, calcium signaling, and probably the following F-actin assembly. This mechanism can potentially assist developing clinical applications and designing biomaterials in near future.     

Functionalized Graphene as Extracellular Matrix Mimics: Toward Well‐Defined 2D Nanomaterials for Multivalent Virus Interactions

Polysulfated nanomaterials that mimic the extracellular cell matrix are of great interest for their potential to modulate cellular responses and to bind and neutralize pathogens. However, control over the density of active functional groups on such biomimetics is essential for efficient interactions, and this remains a challenge. In this regard, producing polysulfated graphene derivatives with control over their functionality is an intriguing accomplishment in order to obtain highly effective 2D platforms for pathogen interactions. Here, a facile and efficient method for the controlled attachment of a heparin sulfate mimic on the surface of graphene is reported. Dichlorotriazine groups are conjugated to the surface of graphene by a one‐pot [2+1] nitrene cycloaddition reaction at ambient conditions, providing derivatives with defined functionality. Consecutive step by step conjugation of hyperbranched polyglycerol to the dichlorotriazine groups and eventual conversion to the polyglycerol sulfate result in the graphene based heparin biomimetics. Scanning force microscopy, cryo‐transmission electron microscopy, and in vitro bioassays reveal strong interactions between the functionalized graphene (thoroughly covered by a sulfated polymer) and vesicular stomatitis virus. Infection experiments with highly sulfated versions of graphene drastically promote the infection process, leading to higher viral titers compared to nonsulfated analogues.     

Biomimetic Thermo-Sensitive Hydrogel Encapsulating Hemangiomas Stem Cell Derived Extracellular Vesicles Promotes Microcirculation Reconstruction in Diabetic Wounds

Tissue repair and regeneration in ischemic areas require effective strategies for angiogenesis and microcirculation reconstruction. In this research, extracellular vesicles (EVs) derived from hemangioma stem cells (HemSC) are used as bio-functional materials for angiogenesis. In addition, a novel thermo-sensitive hydrogel based on chitosan (CS) and modified with hyaluronic oligosaccharides (oHA), is specially developed as an ideal carrier of HemSC-EVs. The oHA/CS^gel provides a sustained-release delivery system for EVs, enhances the angiogenic function of HemSC-EVs, and exerts other bio-functions to comprehensively accelerate tissue repair. The physical properties of EVs@oHA/CS^gel proved to be soft, highly elastic deformable, biocompatible, and can maintain the shape and structure of EVs. In vitro co-culture assay verifies the angiogenic effect of EVs@oHA/CS^gel, and this effect is also evaluated in a diabetic wound model. Compared to untreated group, the EVs@oHA/CS^gel group has only 26.9% wound area on day 14, and 226.8% blood flux mark on day 17. Pharmacological mechanisms of HemSC-EVs are predicted by RNA-seq, and cluster analysis of miRNAs predicted targets presents several up-regulated biological processes involving in angiogenesis and wound healing. In conclusion, it is suggested that EVs@oHA/CS^gel as a satisfying therapeutic system for microcirculation reconstruction in tissue repair.     

Biomaterials Functionalized with MSC Secreted Extracellular Vesicles and Soluble Factors for Tissue Regeneration

The therapeutic benefits of mesenchymal stromal cell (MSC) transplantation are attributed to their secreted factors, including extracellular vesicles (EVs) and soluble factors. The potential of employing the MSC secretome as an alternative acellular approach to cell therapy is being investigated in various tissue injury indications, but EVs administered via bolus injections are rapidly sequestered and cleared. However, biomaterials offer delivery platforms to enhance EV retention rates and healing efficacy. This review highlights the mechanisms underpinning the therapeutic effects of MSC‐EVs and soluble factors as effectors of immunomodulation and tissue regeneration, conferred primarily via their nucleic acid and protein contents. Discussed is how manipulating the cell culture microenvironment or genetic modification of MSCs can further augment the potency of their secretions. The most recent advances in the development of EV‐functionalized biomaterials that mediate enhanced angiogenesis and cell survival, while attenuating inflammation and fibrosis, are presented. Finally, some technical challenges to be considered for the clinical translation of biomaterials carrying MSC‐secreted bioactive cargo are discussed.     

Recellularization and Integration of Dense Extracellular Matrix by Percolation of Tissue Microparticles

Cells embedded in the extracellular matrix of tissues play a critical role in maintaining homeostasis while promoting integration and regeneration following damage or disease. Emerging engineered biomaterials utilize decellularized extracellular matrix as a tissue-specific support structure; however, many dense, structured biomaterials unfortunately demonstrate limited formability, fail to promote cell migration, and result in limited tissue repair. Here, a reinforced composite material of densely packed acellular extracellular matrix microparticles in a hydrogel, termed tissue clay, that can be molded and crosslinked to mimic native tissue architecture is developed. Hyaluronic acid-based hydrogels are utilized, amorphously packed with acellular cartilage tissue particulated to ≈125–250 microns in diameter and defined a percolation threshold of 0.57 (v/v) beyond which the compressive modulus exceeded 300 kPa. Remarkably, primary chondrocytes recellularize particles within 48 h, a process driven by chemotaxis, exhibit distributed cellularity in large engineered composites, and express genes consistent with native cartilage repair. In addition, broad utility of tissue clays through recellularization and persistence of muscle, skin, and cartilage composites in an in vivo mouse model is demonstrated. The findings suggest optimal material architectures to balance concurrent demands for large-scale mechanical properties while also supporting recellularization and integration of dense musculoskeletal and connective tissues.     

Advanced Collagen‐Based Biomaterials for Regenerative Biomedicine

In recent decades, collagen is one of the most versatile biomaterials used in biomedical applications, mostly due to its biomimetic and structural composition in the extracellular matrix (ECM). Several attempts are proposed for designing innovative collagen‐based biomaterials and applying them in tissue regeneration. The regeneration of different tissues is prompted by different types and diverse physical forms of collagen‐based biomaterials prepared by various methods. Based on such concepts, the source, structure, and classification of collagen are briefly introduced in this review. Here, the commonly used physical forms and modification methods of collagen‐based biomaterials are reviewed, including hydrogels, scaffolds, and microspheres, followed by their applications in the regeneration of tissues and organs. Important proof‐of‐concept examples are described to demonstrate the outcomes on material characteristics, cellular reactions, and tissue regeneration. A concise assessment of the limitations that still exist and the developing trends in the future of collagen‐based biomaterials are put forward.     

Using Cartilage Extracellular Matrix (CECM) Membrane to Enhance the Reparability of the Bone Marrow Stimulation Technique for Articular Cartilage Defect in Canine Model

Bone marrow stimulation techniques (BSTs) are widely used in clinics to treat cartilage defects, but yet have a critical limitation from the loss of blood clots. In this work, a novel cartilage extracellular matrix (CECM) membrane is developed to protect blood clots after BSTs. The CECM membrane was made of ECM fabricated naturally by cultured porcine chondrocytes, and then decellularized and multi‐layered to confer optimal mechanical strength. Highly compatible with cells, the CECM membrane did not show any cytotoxicity or immune responses in vivo. The CECM membrane was very thin (30–60 μm thick) and bendable, but had good tensile strength (85.64 N), suitable for protecting blood clots from leakage in rabbit cartilage defect. Moreover, the CECM membrane showed low but enough diffusion coefficient to allow delivery of small proteins in synovial fluid into the repaired tissue. In a beagle model, covering the cartilage defect with the CECM membrane after BST generated more hyaline cartilage‐like tissues than the BST alone in histology and chemical analyses at 18 weeks. Its ICRS score was approximately 2.5 times higher than that of the BST alone. Therefore, the CECM membrane is proposed as a useful tool that can improve the outcome of BSTs to treat cartilage defects.     

Free‐Standing Cell Sheet Assembled with Ultrathin Extracellular Matrix as an Innovative Approach for Biomimetic Tissues

Current artificial tissue‐substitutes have limited clinical applications due to unmatched complex combination of cells and extracellular matrix (ECM) as seen in native tissues. From a developmental perspective, the construction of effective biomimetic tissues is from the bottom (one‐dimensional nanoparticles or two‐dimensional membranes) up (three‐dimensional scaffolds or more complex composite). In a hierarchical architecture, each sub‐structure can be assembled in a flexible way with specific regulators and cells, which overcomes the deficiency of one‐for‐all scaffold. Here, a cell‐compatible cell‐lined layered nano‐membrane is developed. Bioactive molecules are mounted on a nano‐membrane and later released to its lined cell sheet. The cell‐lined membrane is in a free‐standing form to regulate cellular functions. The major advantage of this methodology is to provide a versatile approach to construct biomimetic tissues for clinical applications.     

Tissue Beads: Tissue‐Specific Extracellular Matrix Microbeads to Potentiate Reprogrammed Cell‐Based Therapy

Microbeads have been utilized as efficient cell culture carriers and injectable scaffolds for cell transplantation. However, various polymers currently used to generate microbeads have limited applicability due to loss of biological functions and tissue‐specific effects. Here, a tissue bead platform is reported that can provide a tissue‐specific microenvironment to facilitate cell culture and potentiate cell therapy. Using a flow‐focusing microfluidic device, uniform‐sized tissue microbeads are fabricated with extracellular matrix (ECM) from various decellularized tissues. The tissue microbeads are tested for tissue‐specific encapsulation of induced hepatic (iHep), induced cardiac (iCar), and induced myogenic (iMyo) cells, which are directly reprogrammed from mouse primary fibroblasts. Tissue‐specific microbeads significantly enhanced the viability, lineage‐specific maturation, and functionality of each type of reprogrammed cell, as compared to functionality when using conventional microbeads from a single ECM component (collagen). Finally, tissue microbeads are confirmed to mediate the successful in vivo engraftment of reprogrammed cells (iHep and iMyo) after transplantation, potentiating cell therapy and promoting functional tissue regeneration in tissue defective animal models. The study suggests that the use of a decellularized tissue matrix combined with a microfluidic technique can be employed to produce tissue‐specific ECM microbeads with increased versatility and efficacy for reprogrammed cell‐based therapy.     

Biomimetic Mineralized Organic–Inorganic Hybrid Macrofiber with Spider Silk‐Like Supertoughness

Spider silk fibers (SSF) have a hierarchical structure composed of proteins with highly repetitive sequences and biomineralization is sophisticated in hierarchical organic–inorganic constructions. By using inorganic hydroxyapatite (HAP) and organic polyvinyl alcohol (PVA) to simulate the rigid crystalline and flexible amorphous protein blocks of SSF, respectively, biomimetic mineralization is herein attempted for the large‐scale preparation of SSF‐like macrofibers with a hierarchical ordered structure, a superhigh tensile strength of 949 ± 38 MPa, a specific toughness of 296 ± 12 J g^?1, and a stretch ability of 80.6%. The hybrid macrofibers consist of microfibers, and their outstanding performance (e.g., extreme tolerance to temperatures ranging from ?196 to 80 °C and superior ability to inhibit the transverse growth of cracks) is attributed to the hierarchical arrangement as well as the organic–inorganic integrated structure within the oriented mineralized polymer chains. The biomineralization‐inspired technique provides a promising tactic that can be used to synthesize functional organic–inorganic fibers that are structurally complex and, furthermore, industrially manufacture SSF‐like artificial fibers with a supertoughness.     

A Bioprinting Process Supplemented with In Situ Electrical Stimulation Directly Induces Significant Myotube Formation and Myogenesis

Electric field stimulation has supported biophysical and biological cues for tissue regeneration approaches to affect cell morphology, alignment, and even cellular phenotypes types. Here, an innovative bioprinting approach supported by in situ electrical stiumlation (E-printing) is used to fabricate a bioengineered skeletal muscle construct composed of human adipose stem cells and methacrylated decellularized extracellular matrix (dECM-Ma) derived from porcine muscle. To obtain highly ordered myofiber-like structures, various parameters of the printing process are optimized. The E-printed structure exhibits higher cell viability and fully aligned cytoskeleton than the conventionally printed cell-bearing structures, due to activation of voltage-gated ion channels that affect various signaling pathways. When using the E-printed structure, expression of myogenesis-related genes is upregulated by 1.9–2.5-fold higher than when using a dECM-Ma structure produced without electrical stimulation. Furthermore, when implanted into a rat model of volumetric muscle loss, the structure yields outstanding myogenesis relative to the conventionally bioprinted structure.     

Preparation of Protamine–Titania Microcapsules Through Synergy Between Layer‐by‐Layer Assembly and Biomimetic Mineralization

A novel approach combining layer‐by‐layer (LbL) assembly with biomimetic mineralization is proposed to prepare protamine–titiania hybrid microcapsules. More specifically, these microcapsules are fabricated by alternative deposition of positively charged protamine layers and negatively charged titania layers on the surface of CaCO₃ microparticles, followed by dissolution of the CaCO₃ microparticles using EDTA. During the deposition process, the protamine layer induces the hydrolysis and condensation of a titania precursor, to form the titania layer. Thereafter, the negatively charged titania layer allows a new cycle of deposition step of the protamine layer, which ensures a continuous LbL process. The morphology, structure, and chemical composition of the microcapsules are characterized by scanning electron microscopy, transmission electron microscopy, Fourier transform infrared, and X‐ray photoelectron spectroscopy. Moreover, these protamine–titania hybrid microcapsules are first employed as the carrier for the immobilization of yeast alcohol dehydrogenase (YADH), and the encapsulated YADH displays enhanced recycling stability. This approach may open a facile, general, and efficient way to prepare organic–inorganic hybrid materials with different compositions and shapes.     

Electrospun Extracellular Matrix: Paving the Way to Tailor‐Made Natural Scaffolds for Cardiac Tissue Regeneration

Biomimetic scaffolds generally aim at structurally and compositionally imitating native tissue, thus providing a supportive microenvironment to the transplanted or recruited cells in the tissue. Native decellularized porcine extracellular matrix (ECM) is becoming the ultimate bioactive material for the regeneration of different organs. Particularly for cardiac regeneration, ECM is studied as a patch and injectable scaffolds, which improve cardiac function, yet lack reproducibility and are difficult to control or fine‐tune for the desired properties, like most natural materials. Seeking to harness the natural advantages of ECM in a reproducible, scalable, and controllable scaffold, for the first time, a matrix that is produced from whole decellularized porcine cardiac ECM using electrospinning technology, is developed. This unique electrospun cardiac ECM mat preserves the composition of ECM, self‐assembles into the same microstructure of cardiac ECM ,and ,above all, preserves key cardiac mechanical properties. It supports cell growth and function, and demonstrates biocompatibility in vitro and in vivo. Importantly, this work reveals the great potential of electrospun ECM‐based platforms for a wide span of biomedical applications, thus offering the possibility to produce complex natural materials as tailor‐made, well‐defined structures.     

A Robustly Supported Extracellular Matrix Improves the Intravascular Delivery Efficacy of Endothelial Progenitor Cells

A major barrier in the multi-purpose use of implantable materials is an incomplete integration with surrounding tissues, causing foreign body reactions, such as fibrous encapsulation and chronic inflammation. From its viewpoint, a universal method is suggested for cloaking any kind of substrate in specific cell-made extracellular matrices (ECMs) via robust linkages for delivery of therapeutic cells. In addition, the feasibility of ECM-coated substrates as endothelial progenitor cells (EPCs)-laden coronary stent platform for endovascular implantation is investigated. Characteristics and stability of cell-secreted ECMs anchored on a substrate are evaluated, and structural and componential features are revealed similar to those of native ECMs, with enough strength to enable practical uses. The in vitro experiments demonstrated that the ECM coating effectively supports the adhesion, proliferation, and viability of EPCs and has selectively opposite effects on smooth muscle cells. The in vivo experiments using a porcine coronary model supports that ECM-coated stents enable endothelial regeneration and inhibit neointimal growth, and the cell-laden form is more effective than other stents. These results will allow the preparation of tissue-integrating materials containing cellular microenvironments and safely coat surfaces with cells to enable long-term implantation and the continuous managing of chronic diseases.     

Processed Tissue–Derived Extracellular Matrices: Tailored Platforms Empowering Diverse Therapeutic Applications

Tissue‐derived decellularized extracellular matrices (dECM) have gradually become the gold standard of scaffolds for tissue engineering, owing to their close mirroring of the intricate composition, architecture, and topology of the native extracellular matrix (ECM). Intriguingly, further manipulation of these acellular tissues through various processing techniques has been demonstrated to be an effective strategy to control their characteristics and impart them with ample valuable new traits, thereby expanding their applicability to a significantly wider spectrum of research and translational applications. Herein, state‐of‐the‐art processed dECM platforms and their potential applications are focused on. The ECM characteristics that make it so appealing for tissue engineering are presented, followed by a concise discussion on the main considerations for choosing a dECM source for such applications. The key methodologies for dECM processing, including hydrogel production, bioprinting, electrospinning, and production of porous scaffolds, microcarriers, and microcapsules, as well as their inherent advantages and challenges, are introduced. To demonstrate the use of processed dECM platforms for tissue engineering, selected in vivo and in vitro applications recently developed utilizing these platforms are highlighted. Finally, concluding remarks and a prospective outlook for future developments and improvements in the field of processed dECM‐based devices are given.     

3D‐Printed Biomimetic Scaffold Simulating Microfibril Muscle Structure

In the human body, microfibril structures can be found in several types of tissue, such as muscles, nerves, and even tendons. However, most micropatterned fabrication methods have focused on 2D surface patterned configurations, which imitate the alignment and fusion of cardiac and skeletal muscle cells. Despite the development of these 2D methods, it has continued to be a challenge to fabricate realistic 3D microfibril structures. The goal of this study is to develop a micropatterned polycaprolactone (PCL) microfiber strut. This process uses a microfibrillation/leaching process of poly(vinyl alcohol) (PVA) from a PVA/PCL mixture to imitate skeletal muscle cell alignment and fusion in vitro. To attain the optimal processing conditions, a variety of parameters—including a mixture ratio, processing temperature, and pneumatic pressure—are considered. To increase biocompatibility of a microfibrous PCL bundle, the fabricated structure is supplemented with type‐I collagen. The myoblasts (C2C12 cells) are used to determine the cellular responses of the fabricated structure. By analyzing cell proliferation and myogenic differentiation, it can be confirmed that the hybrid microfibrillated structure can be an important potential platform to obtain efficient regeneration of muscle cells.     

“Tissue Papers” from Organ‐Specific Decellularized Extracellular Matrices

Using an innovative, tissue‐independent approach to decellularized tissue processing and biomaterial fabrication, the development of a series of “tissue papers” derived from native porcine tissues/organs (heart, kidney, liver, muscle), native bovine tissue/organ (ovary and uterus), and purified bovine Achilles tendon collagen as a control from decellularized extracellular matrix particle ink suspensions cast into molds is described. Each tissue paper type has distinct microstructural characteristics as well as physical and mechanical properties, is capable of absorbing up to 300% of its own weight in liquid, and remains mechanically robust (E = 1–18 MPa) when hydrated; permitting it to be cut, rolled, folded, and sutured, as needed. In vitro characterization with human mesenchymal stem cells reveals that all tissue paper types support cell adhesion, viability, and proliferation over four weeks. Ovarian tissue papers support mouse ovarian follicle adhesion, viability, and health in vitro, as well as support, and maintain the viability and hormonal function of nonhuman primate and human follicle‐containing, live ovarian cortical tissues ex vivo for eight weeks postmortem. “Tissue papers” can be further augmented with additional synthetic and natural biomaterials, as well as integrated with recently developed, advanced 3D‐printable biomaterials, providing a versatile platform for future multi‐biomaterial construct manufacturing.     

Tumor Microenvironment‐Tailored Weakly Cell‐Interacted Extracellular Delivery Platform Enables Precise Antibody Release and Function

Precise delivery of extracellularly functional protein drugs is limited by the drawback in that the protective carrier often causes undesirable cellular uptake of these therapeutic agents. Here, the design of a weakly cell‐interacted, nanosized, environment‐responsive vehicle (WINNER) with rational phosphorylcholine (PC) surface filling ratios capable of precise extracellular delivery of therapeutic agents for enhanced tumor suppression is reported. Highly hydrophilic zwitterionic PC and enzyme‐responsive peptides are engineered into the functional shell of WINNER which reasonably covers the inner protein. It is demonstrated that rationally controlled PC surface filling ratios (50.5–58.3%) are necessary for weakening interactions between the cell and WINNER whilst providing enough sites on WINNER for enzyme recognition. Consequently, WINNER (50.5–58.3%) can protect inner cargos from cellular uptake and undergo enzymatic degradation, resulting in precise extracellular release of inner protein, such as therapeutic monoclonal antibody (mAb). After intravenous administration, therapeutic mAb nimotuzumab‐loaded WINNER (51.2%) shows highest in vivo antitumor activity compared with free nimotuzumab or nimotuzumab‐loaded PC‐free nanocarrier in a lung adenocarcinoma xenograft tumor animal model. This work presents a simple and flexible approach to design precise extracellular delivery platform which can uncage the therapeutic power of extracellular targeting therapeutic agents.     

Biomimetic,Hypoxia‐Responsive Nanoparticles Overcome Residual Chemoresistant Leukemic Cells with Co‐Targeting of Therapy‐Induced Bone Marrow Niches

Chemoresistance conferred by leukemia propagating cells (LPCs) in a therapy‐induced niche (TI‐niche) within the bone marrow is one of the main obstacles in leukemia treatment. Effective approaches to circumvent the TI‐niche protection and to eliminate the resident LPCs remain to be exploited. Here, developed is a niche‐targeted nanosystem using leukemic cell membrane‐coated mesoporous silica nanoparticles (DA_azo@CMSN) for co‐delivering daunorubicin for leukemia cell chemotherapy and a TGFβRII neutralizing antibody (aTGFβRII) to block niche signaling. DA_azo@CMSN effectively targets the TI‐niche. Through an azobenzene‐based hypoxia‐responsive linker, sequential delivery of the two active molecules overcomes niche‐mediated chemoresistance, attenuates systemic burden, and prolongs survival in a mouse model of leukemia. This work demonstrates a proof‐of‐principle for biomimetic and microenvironment‐activated multiplexed nanoparticulate drug delivery strategies for overcoming therapy‐induced chemoresistance in leukemia.