Current Ti-6Al-4V bone implants lack trabecular structure and pro‑angiogenic cues, both essential for regeneration. Herein, a dual biomimetic strategy is devised that integrates a 3D-printed biomimetic trabecular porous Ti-6Al-4V scaffold (BTPS) with exosome-loaded PEGDA/GelMA hydrogel microspheres (PGHExo) designed for sustained release. BTPS is designed using Voronoi algorithms and imaging data, and replicates the geometry and mechanical properties of natural bone. Hypoxia-induced human umbilical vein endothelial cell (HUVEC) derived exosomes (HExo) are encapsulated in PGHExo microspheres via microfluidic technology, enabling controlled release of HExo, and anchored onto BTPS using polydopamine (pDA) modification (BTPS&pDA@PGHExo). BTPS exhibited an elastic modulus of ≈3.2 GPa and a permeability of 11.52 × 10-8 mm2, mimicking natural bone. In vitro assays demonstrated that BTPS&pDA@PGHExo significantly enhanced osteogenesis and angiogenesis. mRNA-Seq analysis suggested that BTPS&pDA@PGHExo regulates osteogenic and angiogenic gene expression through the activation of pathways including MAPK, mTOR, HIF-1, and VEGF. In vivo, BTPS&pDA@PGHExo improved bone volume, density, and neovascularization in a rabbit model. This dual biomimetic strategy offers a promising clinical solution, addressing the limitations of conventional Ti-6Al-4V scaffolds and providing an innovative approach for personalized bone defect repair.

Extracellular vesicles (EVs) have emerged as a promising strategy for treating a wide spectrum of pathologies, as they can deliver their cargo to recipient cells and regulate the signaling pathway of these cells to modulate their fate. Despite the great potential of EVs in clinical applications, their low yield and the challenges of cargo loading remain significant obstacles, hindering their transition from experimental research to clinical practice. Therefore, promoting EV release and enhancing EV cargo-loading are promising fields with substantial research potential and broad application prospects. In this review, we summarize the clinical applications of EVs, the methods and technologies for their large-scale production, engineering, and modification, as well as the challenges that must be addressed during their development. We also discuss the future perspectives of this exciting field of research to facilitate its transformation from bench to clinical reality.

Exosomes have garnered attention for use in bone regeneration, but their low activity, rapid degradation, and inaccurate delivery have been obstacles to their use in clinical applications. As such, there exists a need for an exosome-integrated delivery platform. Calcium silicate (Ca-Si) is considered one of the most promising bioceramics for bone regeneration because of its remarkable ability to promote hydroxyapatite formation, osteoblast proliferation, and differentiation. However, Ca-Si has limitations, such as a high degradation rate leading to high pH values. Here, we propose a bone regeneration platform: three-dimensional-fabricated Ca-Si scaffolds immersed in polycaprolactone (PCL) coated with exosomes. This setup enhanced porosity, mechanical strength, and natural hydroxyapatite formation. Ca-Si incorporation increased the quantity of attached exosomes on the scaffold and enabled more sustainable control of their release compared to bare PCL. The exosome-coated scaffold exhibited excellent cell attachment and osteogenic differentiation, significantly increasing biocompatibility and the in situ recruitment of stem cells when transplanted into the subcutaneous tissue of mice. The bone regenerating efficacy of the exosome-attached scaffold was confirmed using a mouse calvarial bone defect animal model. These findings suggest a potential application of exosome-coated Ca-Si/PCL scaffolds as an osteogenic platform for critical bone defects.

Bone regeneration is a complex biological process that relies on the orchestrated interplay of various cellular and molecular events. Bone tissue engineering is currently the most promising method for treating bone regeneration. However, the immunogenicity, stable and cell quantity of seed cells limited their application. Recently, exosomes, which are small extracellular vesicles released by cells, have been found to effectively address these problems and better induce bone regeneration. Meanwhile, a growing line of research has shown the cargos of exosomes may provide effective therapeutic and biomarker tools for bone repair, including miRNA, lncRNA, and proteins. Moreover, engineered scaffolds loaded with exosomes can offer a cell-free bone repair strategy, addressing immunogenicity concerns and providing a more stable functional performance. Herein, we provide a comprehensive summary of the role played by scaffolds loaded with exosomes in bone regeneration, drawing on a systematic analysis of relevant literature available on PubMed, Scopus, and Google Scholar database.

Mesenchymal stem cell-derived exosomes (MSC-Exos) have emerged as promising candidates for cell-free therapy in tissue regeneration. However, the native osteogenic and angiogenic capacities of MSC-Exos are often insufficient to repair critical-sized bone defects, and the underlying immune mechanisms remain elusive. Furthermore, achieving sustained delivery and stable activity of MSC-Exos at the defect site is essential for optimal therapeutic outcomes. Here, we extracted exosomes from osteogenically pre-differentiated human bone marrow mesenchymal stem cells (hBMSCs) by ultracentrifugation and encapsulated them in gelatin methacryloyl (GelMA) hydrogel to construct a composite scaffold. The resulting exosome-encapsulated hydrogel exhibited excellent mechanical properties and biocompatibility, facilitating sustained delivery of MSC-Exos. Osteogenic pre-differentiation significantly enhanced the osteogenic and angiogenic properties of MSC-Exos, promoting osteogenic differentiation of hBMSCs and angiogenesis of human umbilical vein endothelial cells (HUVECs). Furthermore, MSC-Exos induced polarization of Raw264.7 cells from a pro-inflammatory phenotype to an anti-inflammatory phenotype under simulated inflammatory conditions, thereby creating an immune microenvironment conducive to osteogenesis. RNA sequencing and bioinformatics analysis revealed that MSC-Exos activate the p53 pathway through targeted delivery of internal microRNAs and regulate macrophage polarization by reducing DNA oxidative damage. Our study highlights the potential of osteogenic exosome-encapsulated composite hydrogels for the development of cell-free scaffolds in bone tissue engineering.

Due to the spatiotemporal complexity of the composition, structure, and cell population of the tendon-bone interface (TBI), it is difficult to achieve true healing. Recent research is increasingly focusing on engineered exosomes, which are a promising strategy for TBI regeneration.

This review discusses the physiological and pathological characteristics of TBI and the application and limitations of natural exosomes in the field of tendon-bone healing. The definition, loading strategies, and spatiotemporal properties of engineered exosomes were elaborated. We also summarize the application and future research directions of engineered exosomes in the field of tendon-bone healing.

Engineered exosomes can spatially deliver cargo to targeted sites and temporally realize the sustained release of therapeutic molecules in TBI. This review expounds on the multidifferentiation of engineered exosomes for tendon-bone healing, which effectively improves the biological and biomechanical properties of TBI. Engineered exosomes could be a promising strategy for tendon-bone healing.

Modulating the inflammatory microenvironment to reconstruct the fibrocartilaginous layer while promoting tendon repair is crucial for enhancing tendon-to-bone healing in rotator cuff repair (RCR), a persistent challenge in orthopedics. Small extracellular vesicles (sEVs) hold significant potential to modulate inflammation, yet the efficient production of highly bioactive sEVs remains a substantial barrier to their clinical application. Moreover, achieving minimally invasive local delivery of sEVs to the tendon-to-bone interface presents significant technical difficulties. Herein, the circadian rhythm of adipose-derived stem cells is modulated to increase the yield and enhance the inflammatory regulatory capacity of sEVs. Circadian rhythm-regulated sEVs (CR-sEVs) enhance the cyclic adenosine monophosphate signaling pathway in macrophage (Mφ) via platelet factor 4 delivery, thereby inhibiting Mφ M1 polarization. Subsequently, a triphasic microneedle (MN) scaffold with a tip, stem, and base is designed for the local delivery of CR-sEVs (CR-sEVs/MN) at the tendon-to-bone junction, incorporating tendon-derived decellularized extracellular matrix in the base to facilitate tendon repair. CR-sEVs/MN mitigates inflammation, promotes fibrocartilage regeneration, and enhances tendon healing, thereby improving biomechanical strength and shoulder joint function in a rat RCR model. Combining CR-sEVs with this triphasic microneedle delivery system presents a promising strategy for enhancing tendon-to-bone healing in clinical settings.

It is common for maladies and trauma to cause significant bone deterioration in the craniofacial bone, which can cause patients to experience complications with their appearance and their ability to function. Regarding grafting procedures' complications and disadvantages, the newly emerging field of tissue regeneration has shown promise. Tissue -engineered technologies and their applications in the craniofacial region are increasingly gaining prominence with limited postoperative risk and cost. MSCs-derived exosomes are widely applied in bone tissue engineering to provide cell-free therapies since they not only do not cause immunological rejection in the same way that cells do, but they can also perform a cell-like role. Additionally, the hydrogel system is a family of multipurpose platforms made of cross-linked polymers with considerable water content, outstanding biocompatibility, and tunable physiochemical properties for the efficient delivery of commodities. Therefore, the promising exosome-loaded hydrogels can be designed for craniofacial bone regeneration. This review lists the packaging techniques for exosomes and hydrogel and discusses the development of a biocompatible hydrogel system and its potential for exosome continuous delivery for craniofacial bone healing.

The utilization of extracellular vesicles (EVs) in wound healing has been well-documented. However, the direct administration of free EVs via subcutaneous injection at wound sites may result in the rapid dissipation of bioactive components and diminished therapeutic efficacy. Functionalized hydrogels provide effective protection, as well as ensure the sustained release and bioactivity of EVs during the wound healing process, making them an ideal candidate material for delivering EVs. In this review, we introduce the mechanisms by which EVs accelerate wound healing, and then elaborate on the construction strategies for engineered EVs. Subsequently, we discuss the synthesis strategies and application of hydrogels as delivery systems for the sustained release of EVs to enhance complicated wound healing. Furthermore, in the face of complicated wounds, functionalized hydrogels with specific wound microenvironment regulation capabilities, such as antimicrobial, anti-inflammatory, and immune regulation, used for loading engineered EVs, provide potential approaches to addressing these healing challenges. Ultimately, we deliberate on potential future trajectories and outlooks, offering a fresh viewpoint on the advancement of artificial intelligence (AI)-energized materials and 3D bio-printed multifunctional hydrogel-based engineered EVs delivery dressings for biomedical applications.

The increasing prevalence of bone replacements and complications associated with bone replacement procedures underscores the need for innovative tissue restoration approaches. Existing synthetic grafts cannot fully replicate bone vascularization and mechanical characteristics. This study introduces a novel strategy utilizing pectin, chitosan, and polyvinyl alcohol to create interpenetrating polymeric network (IPN) scaffolds incorporated with extracellular vesicles (EVs) isolated from human mesenchymal stem cells (hMSCs). We assess the osteointegration and osteoconduction abilities of these modelsin vitrousing hMSCs and MG-63 osteosarcoma cells. Additionally, we confirm exosome properties through Transmission Electron Microscopy (TEM), immunoblotting, and Dynamic Light Scattering (DLS).In vivo, chick allantoic membrane assay investigates vascularization characteristics. The study did not includein vivoanimal experiments. Our results demonstrate that the IPN scaffold is highly porous and interconnected, potentially suitable for bone implants. EVs, approximately 100 nm in size, enhance cell survival, proliferation, alkaline phosphatase activity, and the expression of osteogenic genes. EVs-mediated IPN scaffolds demonstrate promise as precise drug carriers, enabling customized treatments for bone-related conditions and regeneration efforts. Therefore, the EVs-mediated IPN scaffolds demonstrate promise as precise carriers for the transport of drugs, allowing for customized treatments for conditions connected to bone and efforts in regeneration.

Exosomes, nanoparticles secreted by various cells, composed of a bilayer lipid membrane, and containing bioactive substances such as proteins, nucleic acids, metabolites, etc., have been intensively investigated in tissue engineering owing to their high biocompatibility and versatile biofunction. However, there is still a lack of a high-quality review on bone defect regeneration potentiated by exosomes. In this review, the biogenesis and isolation methods of exosomes are first introduced. More importantly, the engineered exosomes of the current state of knowledge are discussed intensively in this review. Afterward, the biomaterial carriers of exosomes and the mechanisms of bone repair elucidated by compelling evidence are presented. Thus, future perspectives and concerns are revealed to help devise advanced modalities based on exosomes to overcome the challenges of bone regeneration. It is totally believed this review will attract special attention from clinicians and provide promising ideas for their future works.

Serious orthopedic disorders resulting from myriad diseases and impairments continue to pose a considerable challenge to contemporary clinical care. Owing to its limited regenerative capacity, achieving complete bone tissue regeneration and complete functional restoration has proven challenging with existing treatments. By virtue of cellular regenerative and paracrine pathways, stem cells are extensively utilized in the restoration and regeneration of bone tissue; however, low survival and retention after transplantation severely limit their therapeutic effect. Meanwhile, biomolecule materials provide a delivery platform that improves stem cell survival, increases retention, and enhances therapeutic efficacy. In this review, we present the basic concepts of stem cells and extracellular vesicles from different sources, emphasizing the importance of using appropriate expansion methods and modification strategies. We then review different types of biomolecule materials, focusing on their design strategies. Moreover, we summarize several forms of biomaterial preparation and application strategies as well as current research on biomacromolecule materials loaded with stem cells and extracellular vesicles. Finally, we present the challenges currently impeding their clinical application for the treatment of orthopedic diseases. The article aims to provide researchers with new insights for subsequent investigations.

Canine mesenchymal stromal cells (MSCs) possess the capacity to differentiate into a variety of cell types and secrete a wide range of bioactive molecules in the form of soluble and membrane-bound exosomes. Extracellular vesicles/exosomes are nano-sized vesicles that carry proteins, lipids, and nucleic acids and can modulate recipient cell response in various ways. The process of exosome formation is a physiological interaction between cells. With a significant increase in basic research over the last two decades, there has been a tremendous expansion in research in MSC exosomes and their potential applications in canine disease models. The characterization of exosomes has demonstrated considerable variations in terms of source, culture conditions of MSCs, and the inclusion of fetal bovine serum or platelet lysate in the cell cultures. Furthermore, the amalgamation of exosomes with various nano-materials has become a novel approach to the fabrication of nano-exosomes. The fabrication of exosomes necessitates the elimination of extrinsic proteins, thus enhancing their potential therapeutic uses in a variety of disease models, including spinal cord injury, osteoarthritis, and inflammatory bowel disease. This review summarizes current knowledge on the characteristics, biological functions, and clinical relevance of canine MSC exosomes and their potential use in human and canine research. As discussed, exosomes have the ability to control lethal vertebrate diseases by administration directly at the injury site or through specific drug delivery mechanisms.

The development of static hydrogels as an optimal choice for bone tissue engineering (BTE) remains a difficult challenge primarily due to the intricate nature of bone healing processes, continuous physiological functions, and pathological changes. Hence, there is an urgent need to exploit smart hydrogels with programmable properties that can effectively enhance bone regeneration. Increasing evidence suggests that photoresponsive hydrogels are promising bioscaffolds for BTE due to their advantages such as controlled drug release, cell fate modulation, and the photothermal effect. Here, we review the current advances in photoresponsive hydrogels. The mechanism of photoresponsiveness and its advanced applications in bone repair are also elucidated. Future research would focus on the development of more efficient, safer, and smarter photoresponsive hydrogels for BTE. This review is aimed at offering comprehensive guidance on the trends of photoresponsive hydrogels and shedding light on their potential clinical application in BTE.

Extracellular vesicles (EVs) can deliver various bioactive molecules among cells, making them promising diagnostic and therapeutic alternatives in diseases. Mesenchymal stem cell-derived EVs (MSC-EVs) have shown therapeutic potential similar to MSCs but with drawbacks such as lower yield, reduced biological activities, off-target effects, and shorter half-lives. Improving strategies utilizing biotechniques to pretreat MSCs and enhance the properties of released EVs, as well as modifying MSC-EVs to enhance targeting abilities and achieve controlled release, shows potential for overcoming application limitations and enhancing therapeutic effects in treating bone-related diseases. This review focuses on recent advances in functionalizing MSC-EVs to treat bone-related diseases. Firstly, we underscore the significance of MSC-EVs in facilitating crosstalk between cells within the skeletal environment. Secondly, we highlight strategies of functional-modified EVs for treating bone-related diseases. We explore the pretreatment of stem cells using various biotechniques to enhance the properties of resulting EVs, as well as diverse approaches to modify MSC-EVs for targeted delivery and controlled release. Finally, we address the challenges and opportunities for further research on MSC-EVs in bone-related diseases.

Mesenchymal stem cells (MSCs) modulated by various exogenous signals have been applied extensively in regenerative medicine research. Notably, nanosecond pulsed electric fields (nsPEFs), characterized by short duration and high strength, significantly influence cell phenotypes and regulate MSCs differentiation via multiple pathways. Consequently, we used transcriptomics to study changes in messenger RNA (mRNA), long noncoding RNA (lncRNA), microRNA (miRNA), and circular RNA expression during nsPEFs application.

To explore gene expression profiles and potential transcriptional regulatory mechanisms in MSCs pretreated with nsPEFs.

The impact of nsPEFs on the MSCs transcriptome was investigated through whole transcriptome sequencing. MSCs were pretreated with 5-pulse nsPEFs (100 ns at 10 kV/cm, 1 Hz), followed by total RNA isolation. Each transcript was normalized by fragments per kilobase per million. Fold change and difference significance were applied to screen the differentially expressed genes (DEGs). Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were performed to elucidate gene functions, complemented by quantitative polymerase chain reaction verification.

In total, 263 DEGs were discovered, with 92 upregulated and 171 downregulated. DEGs were predominantly enriched in epithelial cell proliferation, osteoblast differentiation, mesenchymal cell differentiation, nuclear division, and wound healing. Regarding cellular components, DEGs are primarily involved in condensed chromosome, chromosomal region, actin cytoskeleton, and kinetochore. From aspect of molecular functions, DEGs are mainly involved in glycosaminoglycan binding, integrin binding, nuclear steroid receptor activity, cytoskeletal motor activity, and steroid binding. Quantitative real-time polymerase chain reaction confirmed targeted transcript regulation.

Our systematic investigation of the wide-ranging transcriptional pattern modulated by nsPEFs revealed the differential expression of 263 mRNAs, 2 miRNAs, and 65 lncRNAs. Our study demonstrates that nsPEFs may affect stem cells through several signaling pathways, which are involved in vesicular transport, calcium ion transport, cytoskeleton, and cell differentiation.

Bone defects are often difficult to treat due to their complexity and specificity, and therefore pose a serious threat to human life and health. Currently, the clinical treatment of bone defects is mainly surgical. However, this treatment is often more harmful to patients and there is a potential risk of rejection and infection. Hydrogels have a unique three-dimensional structure that can accommodate a variety of materials, including particles, polymers and small molecules, making them ideal for treating bone defects. Therefore, emerging composite hydrogels are considered one of the most promising candidates for the treatment of bone defects. This review describes the use of different types of composite hydrogel in the treatment of bone defects. We present the basic concepts of hydrogels, different preparation techniques (including chemical and physical crosslinking), and the clinical requirements for hydrogels used to treat bone defects. In addition, a review of numerous promising designs of different types of hydrogel doped with different materials (e.g., nanoparticles, polymers, carbon materials, drugs, and active factors) is also highlighted. Finally, the current challenges and prospects of composite hydrogels for the treatment of bone defects are presented. This review will stimulate research efforts in this field and promote the application of new methods and innovative ideas in the clinical field of composite hydrogels.

The management of critical-sized bone defects caused by nonunion, trauma, infection, malignancy, pseudoarthrosis, and osteolysis poses complex reconstruction challenges for orthopedic surgeons. Current treatment modalities, including autograft, allograft, and distraction osteogenesis, are insufficient for the diverse range of pathology encountered in clinical practice, with significant complications associated with each. Therefore, there is significant interest in the development of delivery vehicles for growth factors to aid in bone repair in these settings. This article reviews innovative strategies for the management of critical-sized bone loss, including novel scaffolds designed for controlled release of rhBMP, bioengineered extracellular vesicles for delivery of intracellular signaling molecules, and advances in regional gene therapy for sustained signaling strategies. Improvement in the delivery of growth factors to areas of significant bone loss has the potential to revolutionize current treatment for this complex clinical challenge.

Bone regeneration therapy is clinically important, and targeted regulation of endoplasmic reticulum (ER) stress is important in regenerative medicine. The processing of proteins in the ER controls cell fate. The accumulation of misfolded and unfolded proteins occurs in pathological states, triggering ER stress. ER stress restores homeostasis through three main mechanisms, including protein kinase-R-like ER kinase (PERK), inositol-requiring enzyme 1ɑ (IRE1ɑ) and activating transcription factor 6 (ATF6), collectively known as the unfolded protein response (UPR). However, the UPR has both adaptive and apoptotic effects. Modulation of ER stress has therapeutic potential for numerous diseases. Repair of bone defects involves both angiogenesis and bone regeneration. Here, we review the effects of ER stress on osteogenesis and angiogenesis, with emphasis on ER stress under high glucose (HG) and inflammatory conditions, and the use of ER stress inducers or inhibitors to regulate osteogenesis and angiogenesis. In addition, we highlight the ability for exosomes to regulate ER stress. Recent advances in the regulation of ER stress mediated osteogenesis and angiogenesis suggest novel therapeutic options for bone defects.