Edible mycelium as proliferation and differentiation support for anchorage-dependent animal cells in cultivated meat production.

Cultivated meat production requires bioprocess optimization to achieve cell densities that are multiple orders of magnitude higher compared to conventional cell culture techniques. These processes must maximize resource efficiency and cost-effectiveness by attaining high cell growth productivity per unit of medium. Microcarriers, or carriers, are compatible with large-scale bioreactor use, and offer a large surface-area-to-volume ratio for the adhesion and proliferation of anchorage-dependent animal cells. An ongoing challenge persists in the efficient retrieval of cells from the carriers, with conflicting reports on the effectiveness of trypsinization and the need for additional optimization measures such as carrier sieving. To surmount this issue, edible carriers have been proposed, offering the advantage of integration into the final food product while providing opportunities for texture, flavor, and nutritional incorporation. Recently, a proof of concept (POC) utilizing inactivated mycelium biomass derived from edible filamentous fungus demonstrated its potential as a support structure for myoblasts. However, this POC relied on a model mammalian cell line combination with a single mycelium species, limiting realistic applicability to cultivated meat production. This study aims to advance the POC. We found that the species of fungi composing the carriers impacts C2C12 myoblast cell attachment-with carriers derived from Aspergillus oryzae promoting the best proliferation. C2C12 myoblasts effectively differentiated on mycelium carriers when induced in myogenic differentiation media. Mycelium carriers also supported proliferation and differentiation of bovine satellite cells. These findings demonstrate the potential of edible mycelium carrier technology to be readily adapted in product development within the cultivated meat industry.

Extracellular Vesicles from Highly Metastatic Osteosarcoma Cells Induce Pro-Tumorigenic Macrophage Phenotypes.

Metastasis is the principal factor in poor prognosis for individuals with osteosarcoma (OS). Understanding the events that lead to metastasis is critical to develop better interventions for this disease. Alveolar macrophages are potentially involved in priming the lung microenvironment for OS metastasis, yet the mechanisms involved in this process remain unclear. Since extracellular vesicles (EVs) are a known actor in primary tumor development, their potential role in OS metastagenesis through macrophage modulation is explored here. The interaction of EVs isolated from highly metastatic (K7M2) and less metastatic (K12) osteosarcoma cell lines is compared with a peritoneal macrophage cell line. An EV concentration that reproducibly induced macrophage migration is identified first, then used for later experiments. By confocal microscopy, both EV types associated with M0 or M1 macrophages; however, only K7M2-EVs are associated with M2 macrophages, an interaction that is abrogated by EV pre-treatment with anti-CD47 antibody. Interestingly, all interactions appeared to be surface binding, not internalized. In functional studies, K7M2-EVs polarized fewer macrophages to M1. Together, these data suggest that K7M2-EVs have unique interactions with macrophages that can contribute to the production of a higher proportion of pro-tumor type macrophages, thereby accelerating metastasis.

Mechanoregulation of MSC spheroid immunomodulation.

Mesenchymal stromal cells (MSCs) are widely used in cell-based therapies and tissue regeneration for their potent secretome, which promotes host cell recruitment and modulates inflammation. Compared to monodisperse cells, MSC spheroids exhibit improved viability and increased secretion of immunomodulatory cytokines. While mechanical stimulation of monodisperse cells can increase cytokine production, the influence of mechanical loading on MSC spheroids is unknown. Here, we evaluated the effect of controlled, uniaxial cyclic compression on the secretion of immunomodulatory cytokines by human MSC spheroids and tested the influence of load-induced gene expression on MSC mechanoresponsiveness. We exposed MSC spheroids, entrapped in alginate hydrogels, to three cyclic compressive regimes with varying stress (L) magnitudes (i.e., 5 and 10 kPa) and hold (H) durations (i.e., 30 and 250 s) L5H30, L10H30, and L10H250. We observed changes in cytokine and chemokine expression dependent on the loading regime, where higher stress regimes tended to result in more exaggerated changes. However, only MSC spheroids exposed to L10H30 induced human THP-1 macrophage polarization toward an M2 phenotype compared to static conditions. Static and L10H30 loading facilitated a strong, interlinked F-actin arrangement, while L5H30 and L10H250 disrupted the structure of actin filaments. This was further examined when the actin cytoskeleton was disrupted via Y-27632. We observed downregulation of YAP-related genes, and the levels of secreted inflammatory cytokines were globally decreased. These findings emphasize the essential role of mechanosignaling in mediating the immunomodulatory potential of MSC spheroids.

Compliant substrates mitigate the senescence associated phenotype of stress induced mesenchymal stromal cells.

Mesenchymal stromal cells (MSCs) are a promising cell population for musculoskeletal cell-based therapies due to their multipotent differentiation capacity and complex secretome. Cells from younger donors are mechanosensitive, evidenced by changes in cell morphology, adhesivity, and differentiation as a function of substrate stiffness in both two- and three-dimensional culture. However, MSCs from older individuals exhibit reduced differentiation potential and increased senescence, limiting their potential for autologous use. While substrate stiffness is known to modulate cell phenotype, the influence of the mechanical environment on senescent MSCs is poorly described. To address this question, we cultured irradiation induced premature senescent MSCs on polyacrylamide hydrogels and assessed expression of senescent markers, cell morphology, and secretion of inflammatory cytokines. Compared to cells on tissue culture plastic, senescent MSCs exhibited decreased markers of the senescence associated secretory phenotype (SASP) when cultured on 50 kPa gels, yet common markers of senescence (e.g., p21, CDKN2A, CDKN1A) were unaffected. These effects were muted in a physiologically relevant heterotypic mix of healthy and senescent MSCs. Conditioned media from senescent MSCs on compliant substrates increased osteoblast mineralization compared to conditioned media from cells on TCP. Mixed populations of senescent and healthy cells induced similar levels of osteoblast mineralization compared to healthy MSCs, further indicating an attenuation of the senescent phenotype in heterotypic populations. These data indicate that senescent MSCs exhibit a decrease in senescent phenotype when cultured on compliant substrates, which may be leveraged to improve autologous cell therapies for older donors.

Skeletal Muscle Spheroids as Building Blocks for Engineered Muscle Tissue.

Spheroids exhibit enhanced cell-cell interactions that facilitate improved survival and mimic the physiological cellular environment in vivo. Cell spheroids have been successfully used as building blocks for engineered tissues, yet the viability of this approach with skeletal muscle spheroids is poorly understood, particularly when incorporated into three-dimensional (3D) constructs. Bioprinting is a promising strategy to recapitulate the hierarchical organization of native tissue that is fundamental to its function. However, the influence of bioprinting on skeletal muscle cell spheroids and their function are yet to be interrogated. Using C2C12 mouse myoblasts and primary bovine muscle stem cells (MuSCs), we characterized spheroid formation as a function of duration and cell seeding density. We then investigated the potential of skeletal muscle spheroids entrapped in alginate bioink as tissue building blocks for bioprinting myogenic tissue. Both C2C12 and primary bovine MuSCs formed spheroids of similar sizes and remained viable after bioprinting. Spheroids of both cell types fused into larger tissue clusters over time within alginate and exhibited tissue formation comparable to monodisperse cells. Compared to monodisperse cells in alginate gels, C2C12 spheroids exhibited greater MyHC expression after 2 weeks, while cells within bovine MuSC spheroids displayed increased cell spreading. Both monodisperse and MuSC spheroids exhibited increased expression of genes denoting mid- and late-stage myogenic differentiation. Together, these data suggest that skeletal muscle spheroids have the potential for generating myogenic tissue via 3D bioprinting and reveal areas of research that could enhance myogenesis and myogenic differentiation in future studies.

Conductive Microgel Annealed Scaffolds Enhance Myogenic Potential of Myoblastic Cells.

Conductive biomaterials may capture native or exogenous bioelectric signaling, but incorporation of conductive moieties is limited by cytotoxicity, poor injectability, or insufficient stimulation. Microgel annealed scaffolds are promising as hydrogel-based materials due to their inherent void space that facilitates cell migration and proliferation better than nanoporous bulk hydrogels. Conductive microgels are generated from poly(ethylene) glycol (PEG and poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT: PSS) to explore the interplay of void volume and conductivity on myogenic differentiation. PEDOT: PSS increases microgel conductivity two-fold while maintaining stiffness, annealing strength, and viability of associated myoblastic cells. C2C12 myoblasts exhibit increases in the late-stage differentiation marker myosin heavy chain as a function of both porosity and conductivity. Myogenin, an earlier marker, is influenced only by porosity. Human skeletal muscle-derived cells exhibit increased Myod1, insulin like growth factor-1, and insulin-like growth factor binding protein 2 at earlier time points on conductive microgel scaffolds compared to non-conductive scaffolds. They also secrete more vascular endothelial growth factor at early time points and express factors that led to macrophage polarization patterns observe during muscle repair. These data indicate that conductivity aids myogenic differentiation of myogenic cell lines and primary cells, motivating the need for future translational studies to promote muscle repair.

Biomaterials are the key to unlock spheroid function and therapeutic potential.

Spheroids are three-dimensional cell aggregates that mimic fundamental aspects of the native tissue microenvironment better than single cells, making them a promising platform for the study of tissue development and therapeutics. Spheroids have been investigated for decades as models in cancer research, yet we have only just scratched the surface of their potential clinical utility in cell-based therapies. Like many cells, spheroids commonly exhibit a loss of key attributes upon implantation, motivating the need for strategies to regulate their function in situ. Biomaterials offer numerous opportunities to preserve spheroid function and guide spheroid behavior by tailoring the local microenvironment.

Engineered bone marrow as a clinically relevant ex vivo model for primary bone cancer research and drug screening.

Osteosarcoma (OS) is the most common primary malignant bone cancer in children and adolescents. While numerous other cancers now have promising therapeutic advances, treatment options for OS have remained unchanged since the advent of standard chemotherapeutics and offer less than a 25% 5-y survival rate for those with metastatic disease. This dearth of clinical progress underscores a lack of understanding of OS progression and necessitates the study of this disease in an innovative system. Here, we adapt a previously described engineered bone marrow (eBM) construct for use as a three-dimensional platform to study how microenvironmental and immune factors affect OS tumor progression. We form eBM by implanting acellular bone-forming materials in mice and explanting the cellularized constructs after 8 wk for study. We interrogate the influence of the anatomical implantation site on eBM tissue quality, test ex vivo stability under normoxic (5% O2) and standard (21% O2) culture conditions, culture OS cells within these constructs, and compare them to human OS samples. We show that eBM stably recapitulates the composition of native bone marrow. OS cells exhibit differential behavior dependent on metastatic potential when cultured in eBM, thus mimicking in vivo conditions. Furthermore, we highlight the clinical applicability of eBM as a drug-screening platform through doxorubicin treatment and show that eBM confers a protective effect on OS cells that parallel clinical responses. Combined, this work presents eBM as a cellular construct that mimics the complex bone marrow environment that is useful for mechanistic bone cancer research and drug screening.

Conductive microgel annealed scaffolds enhance myogenic potential of myoblastic cells.

Bioelectricity is an understudied phenomenon to guide tissue homeostasis and regeneration. Conductive biomaterials may capture native or exogenous bioelectric signaling, but incorporation of conductive moieties is limited by cytotoxicity, poor injectability, or insufficient stimulation. Microgel annealed scaffolds are promising as hydrogel-based materials due to their inherent void space that facilitates cell migration and proliferation better than nanoporous bulk hydrogels. We generated conductive microgels from poly(ethylene) glycol and poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) to explore the interplay of void volume and conductivity on myogenic differentiation. PEDOT:PSS increased microgel conductivity over 2-fold while maintaining stiffness, annealing strength, and viability of associated myoblastic cells. C2C12 myoblasts exhibited increases in the late-stage differentiation marker myosin heavy chain as a function of both porosity and conductivity. Myogenin, an earlier marker, was influenced only by porosity. Human skeletal muscle derived cells exhibited increased Myod1 , IGF-1, and IGFBP-2 at earlier timepoints on conductive microgel scaffolds compared to non-conductive scaffolds. They also secreted higher levels of VEGF at early timepoints and expressed factors that led to macrophage polarization patterns observed during muscle repair. These data indicate that conductivity aids myogenic differentiation of myogenic cell lines and primary cells, motivating the need for future translational studies to promote muscle repair.

Hydrogel degradation promotes angiogenic and regenerative potential of cell spheroids for wound healing.

Chronic nonhealing wounds are debilitating and diminish one's quality of life, necessitating the development of improved strategies for effective treatment. Biomaterial- and cell-based therapies offer an alternative treatment compared to conventional wound care for regenerating damaged tissues. Cell-based approaches frequently utilize endothelial cells (ECs) to promote vascularization and mesenchymal stromal cells (MSCs) for their potent secretome that promotes host cell recruitment. Spheroids have improved therapeutic potential over monodisperse cells, while degradable scaffolds can influence cellular processes conducive to long-term tissue regeneration. However, the role of biomaterial degradation on the therapeutic potential of heterotypic EC-MSC spheroids for wound healing is largely unknown. We formed poly(ethylene) glycol (PEG) hydrogels with varying ratios of matrix metalloproteinase (MMP)-degradable and non-degradable crosslinkers to develop three distinct constructs - fully degradable, partially degradable, and non-degradable - and interrogate the influence of degradation rate on engineered cell carriers for wound healing. We found that the vulnerability to degradation was critical for cellular proliferation, while inhibition of degradation impaired spheroid metabolic activity. Higher concentrations of degradable crosslinker promoted robust cell spreading, outgrowth, and secretion of proangiogenic cytokines (i.e., VEGF, HGF) that are critical in wound healing. The degree of degradation dictated the unique secretory profile of spheroids. When applied to a clinically relevant full-thickness ex vivo skin model, degradable spheroid-loaded hydrogels restored stratification of the epidermal layer, confirming the efficacy of scaffolds to promote wound healing. These results highlight the importance of matrix remodeling and its essential role in the therapeutic potential of heterotypic spheroids.

Prostate cancer and bone: clinical presentation and molecular mechanisms.

Prostate cancer (PCa) is an increasingly prevalent health problem in the developed world. Effective treatment options exist for localized PCa, but metastatic PCa has fewer treatment options and shorter patient survival. PCa and bone health are strongly entwined, as PCa commonly metastasizes to the skeleton. Since androgen receptor signaling drives PCa growth, androgen-deprivation therapy whose sequelae reduce bone strength constitutes the foundation of advanced PCa treatment. The homeostatic process of bone remodeling - produced by concerted actions of bone-building osteoblasts, bone-resorbing osteoclasts, and regulatory osteocytes - may also be subverted by PCa to promote metastatic growth. Mechanisms driving skeletal development and homeostasis, such as regional hypoxia or matrix-embedded growth factors, may be subjugated by bone metastatic PCa. In this way, the biology that sustains bone is integrated into adaptive mechanisms for the growth and survival of PCa in bone. Skeletally metastatic PCa is difficult to investigate due to the entwined nature of bone biology and cancer biology. Herein, we survey PCa from origin, presentation, and clinical treatment to bone composition and structure and molecular mediators of PCa metastasis to bone. Our intent is to quickly yet effectively reduce barriers to team science across multiple disciplines that focuses on PCa and metastatic bone disease. We also introduce concepts of tissue engineering as a novel perspective to model, capture, and study complex cancer-microenvironment interactions.

The Year 2022 in biomaterials research: A perspective from the editors of six leading journals.

The field of biomaterials science is highly active, with a steadily increasing number of publications and new journals being founded. This article brings together contributions from the editors of six leading journals in the area of biomaterials science and engineering. Each contributor highlights specific advances, topics, and trends that have emerged through the publications in their respective journal in the calendar year 2022. It presents a global perspective on a wide range of material types, functionalities, and applications. The highlighted topics include a diversity of biomaterials; from proteins, polysaccharides, and lipids to ceramics, metals, advanced composites, and a variety of new forms of these materials. Important advances in dynamically functional materials are presented, including a range of fabrication techniques such as bioassembly, 3D bioprinting and microgel formation. Similarly, several applications are highlighted in drug and gene delivery, biological sensing, cell guidance, immunoengineering, electroconductivity, wound healing, infection resistance, tissue engineering, and treatment of cancer. The goal of this paper is to provide the reader with both a broad view of recent biomaterials research, as well as expert commentary on some of the key advances that will shape the future of biomaterials science and engineering.

Macromolecular crowding and decellularization method increase the growth factor binding potential of cell-secreted extracellular matrices.

Recombinant growth factors are used in tissue engineering to stimulate cell proliferation, migration, and differentiation. Conventional methods of growth factor delivery for therapeutic applications employ large amounts of these bioactive cues. Effective, localized growth factor release is essential to reduce the required dose and potential deleterious effects. The endogenous extracellular matrix (ECM) sequesters native growth factors through its negatively charged sulfated glycosaminoglycans. Mesenchymal stromal cells secrete an instructive extracellular matrix that can be tuned by varying culture and decellularization methods. In this study, mesenchymal stromal cell-secreted extracellular matrix was modified using λ-carrageenan as a macromolecular crowding (MMC) agent and decellularized with DNase as an alternative to previous decellularized extracellular matrices (dECM) to improve growth factor retention. Macromolecular crowding decellularized extracellular matrix contained 7.7-fold more sulfated glycosaminoglycans and 11.7-fold more total protein than decellularized extracellular matrix, with no significant difference in residual DNA. Endogenous BMP-2 was retained in macromolecular crowding decellularized extracellular matrix, whereas BMP-2 was not detected in other extracellular matrices. When implanted in a murine muscle pouch, we observed increased mineralized tissue formation with BMP-2-adsorbed macromolecular crowding decellularized extracellular matrix in vivo compared to conventional decellularized extracellular matrix. This study demonstrates the importance of decellularization method to retain endogenous sulfated glycosaminoglycans in decellularized extracellular matrix and highlights the utility of macromolecular crowding to upregulate sulfated glycosaminoglycan content. This platform has the potential to aid in the delivery of lower doses of BMP-2 or other heparin-binding growth factors in a tunable manner.

Tuning the Microenvironment to Create Functionally Distinct Mesenchymal Stromal Cell Spheroids.

Mesenchymal stromal cells (MSCs) are under investigation for wound healing and tissue regeneration due to their potent secretome. Compared to monodisperse cells, MSC spheroids exhibit increased cell survival and enhanced secretion of endogenous factors such as vascular endothelial growth factor (VEGF) and prostaglandin E2 (PGE2), two key factors in wound repair. We previously upregulated the proangiogenic potential of homotypic MSC spheroids by manipulating microenvironmental culture conditions. However, this approach depends on the responsiveness of host endothelial cells (ECs)-a limitation when attempting to restore large tissue deficits and for patients with chronic wounds in which ECs are dysfunctional and unresponsive. To address this challenge, we used a Design of Experiments (DOE) approach to engineer functionally distinct MSC spheroids that maximize VEGF production (VEGFMAX) or PGE2 production (PGE2,MAX) while incorporating ECs that could serve as the basic building blocks for vessel formation. VEGFMAX produced 22.7-fold more VEGF with enhanced endothelial cell migration compared to PGE2,MAX, while PGE2,MAX produced 16.7-fold more PGE2 with accelerated keratinocyte migration compared to VEGFMAX. When encapsulated together in engineered protease-degradable hydrogels as a model of cell delivery, VEGFMAX and PGE2,MAX spheroids exhibited robust spreading into the biomaterial and enhanced metabolic activity. The distinct bioactivities of these MSC spheroids demonstrate the highly tunable nature of spheroids and provide a new approach to leverage the therapeutic potential of cell-based therapies.

Electroconductive agarose hydrogels modulate mesenchymal stromal cell adhesion and spreading through protein adsorption.

Electrically conductive biomaterials direct cell behavior by capitalizing on the effect of bioelectricity in tissue homeostasis and healing. Many studies have leveraged conductive biomaterials to influence cells and improve tissue healing, even in the absence of external stimulation. However, most studies using electroactive materials neglect characterizing how the inclusion of conductive additives affects the material's mechanical properties, and the interplay between substrate electrical and mechanical properties on cell behavior is poorly understood. Furthermore, mechanisms dictating how electrically conductive materials affect cell behavior in the absence of external stimulation are not explicit. In this study, we developed a mechanically and electrically tunable conductive hydrogel using agarose and the conductive polymer PEDOT:PSS. Under certain conditions, we observed that the hydrogel physical and electrical properties were decoupled. We then seeded human mesenchymal stromal cells (MSCs) onto the hydrogels and observed enhanced adhesion and spreading of MSCs on conductive substrates, regardless of the hydrogel mechanical properties, and despite the gels having no cell-binding sites. To explain this observation, we measured protein interaction with the gels and found that charged proteins adsorbed significantly more to conductive hydrogels. These data demonstrate that conductivity promotes cell adhesion, likely by facilitating increased adsorption of proteins associated with cell binding, providing a better understanding of the mechanism of action of electrically conductive materials.

Multisized Photoannealable Microgels Regulate Cell Spreading, Aggregation, and Macrophage Phenotype through Microporous Void Space.

Microgels are an emerging platform for in vitro models and guiding cell fate due to their inherent porosity and tunability. This work describes a light-based technique for rapidly annealing microgels across a range of diameters. Utilizing 8-arm poly(ethylene) glycol-vinyl sulfone, the number of arms available for crosslinking, functionalization, and annealing is stoichiometrically controlled. Small and large microgels are fabricated to explore how microgel diameter impacts void space and the role of porosity on cell spreading, cell aggregation, and macrophage polarization. Mesenchymal stromal cells spread rapidly in both formulations, yet the smaller microgels permit a higher cell density. When seeded with macrophages, the smaller microgels promote an M1 phenotype, while larger microgels promote an M2 phenotype. As another application, the inherent porosity of annealed microgels is leveraged to induce cell aggregation. Finally, the microgels are implanted to examine how different size microgels influence endogenous cell invasion and macrophage polarization. The use of ultraviolet light allows for microgels to be noninvasively injected into a desired mold or wound defect before annealing, and microgels of different properties combined to create a heterogeneous scaffold. This approach is clinically relevant given its tunability and fast annealing time.

Mesenchymal stromal cell spheroids in sulfated alginate enhance muscle regeneration.

The therapeutic efficacy of mesenchymal stromal cells (MSCs) for tissue regeneration is critically linked to the potency of the complex mixture of growth factors, cytokines, exosomes, and other biological cues that they secrete. The duration of cell-based approaches is limited by rapid loss of cells upon implantation, motivating the need to prolong cell viability and extend the therapeutic influence of the secretome. We and others demonstrated that the secretome is upregulated when MSCs are formed into spheroids. Although the efficacy of the MSC secretome has been characterized in the literature, no studies have reported the therapeutic benefit of in situ sequestration of the secretome within a wound site using engineered biomaterials. We previously demonstrated the capacity of sulfated alginate hydrogels to sequester components of the MSC secretome for prolonged presentation in vitro, yet the efficacy of this platform has not been evaluated in vivo. In this study, we used sulfated alginate hydrogels loaded with MSC spheroids to aid in the regeneration of a rat muscle crush injury. We hypothesized that the use of sulfated alginate to bind therapeutically relevant growth factors from the MSC spheroid secretome would enhance muscle regeneration by recruiting host cells into the tissue site. The combination of sulfated alginate and MSC spheroids resulted in decreased collagen deposition, improved myogenic marker expression, and increased neuromuscular junctions 2 weeks after injury. These data indicate that MSC spheroids delivered in sulfated alginate represent a promising approach for decreased fibrosis and increased functional regeneration of muscle. STATEMENT OF SIGNIFICANCE: The therapeutic efficacy of mesenchymal stromal cells (MSCs) for tissue regeneration is attributed to the complex diversity of the secretome. Cell-based approaches are limited by rapid cell death, motivating the need to extend the availability of the secretome. We previously demonstrated that sulfated alginate hydrogels sequester components of the MSC secretome for prolonged presentation in vitro, yet no studies have reported the in situ sequestration of the secretome. Herein, we transplanted MSC spheroids in sulfated alginate hydrogels to promote muscle regeneration. MSC spheroids in sulfated alginate decreased collagen deposition, improved myogenic marker expression, and increased neuromuscular junctions. These data indicate that MSC spheroids delivered in sulfated alginate represent a promising approach for decreasing fibrosis and increasing functional muscle regeneration.

A bioactive material with dual integrin-targeting ligands regulates specific endogenous cell adhesion and promotes vascularized bone regeneration in adult and fetal bone defects.

Significant progress has been made in designing bone materials capable of directing endogenous cells to promote vascularized bone regeneration. However, current strategies lack regulation of the specific endogenous cell populations for vascularized bone regeneration, thus leading to adverse tissue formation and decreased regenerative efficiency. Here, we engineered a biomaterial to regulate endogenous cell adhesion and promote vascularized bone regeneration. The biomaterial works by presenting two synthetic ligands, LLP2A and LXW7, explicitly targeting integrins α4ß1 and αvß3, respectively, expressed on the surfaces of the cells related to bone formation and vascularization, such as mesenchymal stem cells (MSCs), osteoblasts, endothelial progenitor cells (EPCs), and endothelial cells (ECs). In vitro, the LLP2A/LXW7 modified biomaterial improved the adhesion of MSCs, osteoblasts, EPCs, and ECs via integrin α4ß1 and αvß3, respectively. In an adult rat calvarial bone defect model, the LLP2A/LXW7 modified biomaterial enhanced bone formation and vascularization by synergistically regulating endogenous cells with osteogenic and angiogenic potentials, such as DLX5+ cells, osteocalcin+ cells, CD34+/CD45- cells and CD31+ cells. In a fetal sheep spinal bone defect model, the LLP2A/LXW7 modified biomaterial augmented bone formation and vascularization without any adverse effects. This innovative biomaterial offers an off-the-shelf, easy-to-use, and biologically safe product suitable for vascularized bone regeneration in both fetal and adult disease environments.