High-Performance Hydrogel-Encapsulated Engineered Exosomes for Supporting Endoplasmic Reticulum Homeostasis and Boosting Diabetic Bone Regeneration.

The regeneration of bone defects in diabetic patients still faces challenges, as the intrinsic healing process is impaired by hyperglycemia. Inspired by the discovery that the endoplasmic reticulum (ER) is in a state of excessive stress and dysfunction under hyperglycemia, leading to osteogenic disorder, a novel engineered exosome is proposed to modulate ER homeostasis for restoring the function of mesenchymal stem cells (MSCs). The results indicate that the constructed engineered exosomes efficiently regulate ER homeostasis and dramatically facilitate the function of MSCs in the hyperglycemic niche. Additionally, the underlying therapeutic mechanism of exosomes is elucidated. The results reveal that exosomes can directly provide recipient cells with SHP2 for the activation of mitophagy and elimination of mtROS, which is the immediate cause of ER dysfunction. To maximize the therapeutic effect of engineered exosomes, a high-performance hydrogel with self-healing, bioadhesive, and exosome-conjugating properties is applied to encapsulate the engineered exosomes for in vivo application. In vivo, evaluation in diabetic bone defect repair models demonstrates that the engineered exosomes delivering hydrogel system intensively enhance osteogenesis. These findings provide crucial insight into the design and biological mechanism of ER homeostasis-based tissue-engineering strategies for diabetic bone regeneration.

Shape memory and hemostatic silk-laponite scaffold for alveolar bone regeneration after tooth extraction trauma.

Persistent bleeding and the absence of alveolar bone stress following tooth loss can hinder socket healing, complicating future dental implant procedures, and potentially leading to neighboring tooth instability. Therefore, developing materials that promote alveolar bone regeneration and possess both hemostatic and osteogenic properties is crucial for preserving the extraction sites. This study introduces a silk-based laponite composite scaffold material with proficient hemostatic and osteogenic functions, and excellent shape-memory properties for efficient extraction- site filling. In vitro studies research demonstrated that the scaffold's inherent negative charge of the scaffold significantly enhanced blood coagulation and thrombin generation. Moreover, its porous structure and slightly rough inner surface promoted blood cell adhesion and, improved the hemostatic performance. Furthermore, the scaffold facilitated stem cell osteogenic differentiation by activating the TRPM7 channel through the released of magnesium ions. In vivo tests using rat models confirmed its effectiveness in promoting coagulation and mandibular regeneration. Thus, this study proposes a promising approach for post-extraction alveolar bone regenerative repair. The composite scaffold material, with its hemostatic and osteogenic capabilities and shape-memory features, can potentially enhance dental implant success and overall oral health.

Highly Active Frozen Nanovesicles Microneedles for Senile Wound Healing via Antibacteria, Immunotherapy, and Skin Regeneration.

Senile wound healing risks a variety of health complications and makes both economic and psychological burdens on patients greatly. Poor activity of aged dermal fibroblasts (A-FBs) and local disordered immunoreaction in the deep dermis contribute to delayed wound healing. Therefore, the locally complex microenvironment in deep requires additional processing. Herein, a novel double-layer hyaluronic acid methacrylate (HAMA)/polyvinyl alcohol (PVA) microneedle patch (MNP) coated by young fibroblast-derived exosomes (Y-EXOs) (Y-EXOs@HAMA/PVA MNP) is presented for deep drug delivery, aged wound healing and immunoregulation. A spraying and freeze-drying method is applied for keeping the bioactivity of the nanovesicles. An ideal loading of Y-EXOs and enhanced strength for penetration have realized after circulation for times. The Y-EXOs@HAMA/PVA MNP shows an excellent influence on delayed wound healing of aged skin with active A-FBs, more deposition of collagen and less production of IL-17A compared with application of aged fibroblast-derived exosomes (A-EXOs). Moreover, the content microRNAs in Y-EXOs and A-EXOs are sequenced for further study. This study initiatively demonstrates that Y-EXOs have effective function on both anti-aging and anti-inflammation and Y-EXOs@HAMA/PVA MNP is expected as a novel strategy for deep drug delivery for promoting hard wound healing in aged skin in future clinical application.

Dominoes with interlocking consequences triggered by zinc: involvement of microelement-stimulated MSC-derived exosomes in senile osteogenesis and osteoclast dialogue.

As societal aging intensifies, senile osteoporosis has become a global public health concern. Bone microdamage is mainly caused by processes such as enhancing osteoclast activity or reducing bone formation by osteoblast-lineage cells. Compared with young individuals, extracellular vesicles derived from senescent bone marrow mesenchymal stem cells(BMSCs) increase the transient differentiation of bone marrow monocytes (BMMs) to osteoclasts, ultimately leading to osteoporosis and metal implant failure. To address this daunting problem, an exosome-targeted orthopedic implant composed of a nutrient coating was developed. A high-zinc atmosphere used as a local microenvironmental cue not only could inhibit the bone resorption by inhibiting osteoclasts but also could induce the reprogramming of senile osteogenesis and osteoclast dialogue by exosome modification. Bidirectional regulation of intercellular communication via cargoes, including microRNAs carried by exosomes, was detected. Loss- and gain-of-function experiments demonstrated that the key regulator miR-146b-5p regulates the protein kinase B/mammalian target of rapamycin pathway by targeting the catalytic subunit gene of PI3K-PIK3CB. In vivo evaluation using a naturally-aged osteoporotic rat femoral defect model further confirmed that a nutrient coating substantially augments cancellous bone remodeling and osseointegration by regulating local BMMs differentiation. Altogether, this study not only reveals the close link between senescent stem cell communication and age-related osteoporosis but also provides a novel orthopedic implant for elderly patients with exosome modulation capability.

Orchestration of energy metabolism and osteogenesis by Mg2+ facilitates low-dose BMP-2-driven regeneration.

The clinical application of bone morphogenetic protein-2 (BMP-2) is limited by several factors, including ineffectiveness at low doses and severe adverse effects at high doses. To address these efficacy and safety limitations, we explored whether orchestration of energy metabolism and osteogenesis by magnesium ion (Mg2+) could reduce the dose and thereby improve the safety of BMP-2. Our results demonstrated that rapid metabolic activation triggered by BMP-2 was indispensable for subsequent osteogenesis. Moreover, inadequate metabolic stimulation was shown to be responsible for the ineffectiveness of low-dose BMP-2. Next, we identified that Mg2+, as an ''energy propellant", substantially increased cellular bioenergetic levels to support the osteogenesis via the Akt-glycolysis-Mrs2-mitochondrial axis, and consequently enhanced the osteoinductivity of BMP-2. Based on the mechanistic discovery, microgel composite hydrogels were fabricated as low-dose BMP-2/Mg2+ codelivery system through microfluidic and 3D printing technologies. An in vivo study further confirmed that rapid and robust bone regeneration was induced by the codelivery system. Collectively, these results suggest that this bioenergetic-driven, cost-effective, low-dose BMP-2-based strategy has substantial potential for bone repair.

Marginal sealing around integral bilayer scaffolds for repairing osteochondral defects based on photocurable silk hydrogels.

Osteochondral repair remains a major challenge in current clinical practice despite significant advances in tissue engineering. In particular, the lateral integration of neocartilage into surrounding native cartilage is a difficult and inadequately addressed problem that determines the success of tissue repair. Here, a novel design of an integral bilayer scaffold combined with a photocurable silk sealant for osteochondral repair is reported. First, we fabricated a bilayer silk scaffold with a cartilage layer resembling native cartilage in surface morphology and mechanical strength and a BMP-2-loaded porous subchondral bone layer that facilitated the osteogenic differentiation of BMSCs. Second, a TGF-ß3-loaded methacrylated silk fibroin sealant (Sil-MA) exhibiting biocompatibility and good adhesive properties was developed and confirmed to promote chondrocyte migration and differentiation. Importantly, this TGF-ß3-loaded Sil-MA hydrogel provided a bridge between the cartilage layer of the scaffold and the surrounding cartilage and then guided new cartilage to grow towards and replace the degraded cartilage layer from the surrounding native cartilage in the early stage of knee repair. Thus, osteochondral regeneration and superior lateral integration were achieved in vivo by using this composite. These results demonstrate that the new approach of marginal sealing around the cartilage layer of bilayer scaffolds with Sil-MA hydrogel has tremendous potential for clinical use in osteochondral regeneration.

Rapid construction and enhanced vascularization of microtissue using a magnetic control method.

Stem cells play critical roles in tissue repair and regeneration. The construction of stem cell-derived microtissue is a promising strategy for transplanting cells into defects to improve tissue regeneration efficiency. However, rapidly constructing larger microtissues and promoting vascularization to ensure the cellular nutrient supply remain major challenges. Here, we have developed a magnetic device to rapidly construct and regulate millimeter-scale microtissues derived from magnetic nanoparticle-labeled cells. When the microtissue was cultured under a specific magnetic field, the shape of the microtissue could be changed. Importantly, cell proliferation was maintained, and angiogenesis was activated in the process of microtissue deformation. We developed a magnetic control method to treat microtissue, and the implanted microtissue showed excellent vascularizationin vivo. In brief, this magnetic control technology provides a promising strategy for vascularized regenerative medicine.

A rapidly magnetically assembled stem cell microtissue with "hamburger" architecture and enhanced vascularization capacity.

With the development of magnetic manipulation technology based on magnetic nanoparticles (MNPs), scaffold-free microtissues can be constructed utilizing the magnetic attraction of MNP-labeled cells. The rapid in vitro construction and in vivo vascularization of microtissues with complex hierarchical architectures are of great importance to the viability and function of stem cell microtissues. Endothelial cells are indispensable for the formation of blood vessels and can be used in the prevascularization of engineered tissue constructs. Herein, safe and rapid magnetic labeling of cells was achieved by incubation with MNPs for 1 h, and ultrathick scaffold-free microtissues with different sophisticated architectures were rapidly assembled, layer by layer, in 5 min intervals. The in vivo transplantation results showed that in a stem cell microtissue with trisection architecture, the two separated human umbilical vein endothelial cell (HUVEC) layers would spontaneously extend to the stem cell layers and connect with each other to form a spatial network of functional blood vessels, which anastomosed with the host vasculature. The "hamburger" architecture of stem cell microtissues with separated HUVEC layers could promote vascularization and stem cell survival. This study will contribute to the construction and application of structural and functional tissues or organs in the future.

Magnesium Ammonium Phosphate Composite Cell-Laden Hydrogel Promotes Osteogenesis and Angiogenesis In Vitro.

Injectable hydrogels provide an effective strategy for minimally invasive treatment on irregular bony defects in the maxillofacial region. To improve the osteoinduction of gelatin methacrylate (GelMA), we fabricated a three-dimensional (3D) culture system based on the incorporation of magnesium ammonium phosphate hexahydrate (struvite) into GelMA. The optimal concentration of struvite was investigated using the struvite extracts, and 500 µg mL-1 was found to be the most suitable concentration for the osteogenesis of dental pulp stem cells (DPSCs) and angiogenesis of human umbilical vein endothelial cells (HUVECs). We prepared the GelMA composite (MgP) with 500 µg mL-1 struvite. Struvite did not affect the cross-linking of GelMA and released Mg2+ during degradation. The cell delivery system using MgP improved the laden-cell viability, upregulated the expression of osteogenic and angiogenic-differentiation-related genes, and promoted cell migration. Overall, the modifications made to the GelMA in this study improved osteoinduction and demonstrated great potential for application in vascularized bone tissue regeneration.

The Translation from In Vitro Bioactive Ion Concentration Screening to In Vivo Application for Preventing Peri-implantitis.

Peri-implantitis is a typical pathological condition characterized by the destructive inflammation in the soft tissue and the progressive loss of supporting bones. As the current effective treatments and preventive measures are inconsistent and unpredictable, the use of biomaterials as carriers of bioactive ion coatings is a promising approach. However, the translation from lab to large-scale production and clinical applications is difficult due to a technology barrier. Determining the effective dosage of each ion to achieve an in vivo application of the in vitro screening is challenging. Here, we selected zinc and strontium ions to provide multiple effects on antibacterial activity and osteogenesis. The optimal coating with effective release concentrations of the two ions was obtained after the two-step screening from in vitro testing. The results showed that this type of in vivo bioactive ion usage leads to an enhanced osseointegration during the immediate implantation in a periodontitis-affected environment and prevents soft tissue inflammation and bone resorption in an inflammatory environment. The new biologically active ion screening method could verify the effectiveness of this clinical translation and its potential for large-scale production and could determine the effective dosage of each ion for a specific application.

Preservation of alveolar ridge height through mechanical memory: A novel dental implant design.

Irreversible marginal bone loss can hinder recovery around dental implants. Insufficient alveolar osteogenesis and stress concentration during chewing contribute to marginal bone resorption and can result in implant failure. A biomaterial with a micropore-channel structure was developed using 3D printing technology. This design facilitated bony ingrowth and provided similar mechanical stimulation at the implant neck during mastication to a natural tooth. The micropore channels provided a guiding structure for bone mesenchymal stem cell proliferation and differentiation without the need for growth factors. Specifically, this was achieved through mechanical transduction by F-actin remodeling and the activation of Yes-associated protein (YAP). The implants were verified in a canine dental implant surgery model, which demonstrated the promising use of biomaterial-based dental implants in future clinical applications.

In situ gas foaming based on magnesium particle degradation: A novel approach to fabricate injectable macroporous hydrogels.

Injectable hydrogels are attractive biomaterials for cell delivery in tissue engineering. However, the in vivo viability of transplanted cells remains limited. Typically, macroporous structures constructed in hydrogels are utilized to enhance oxygen and nutrients diffusion for cell survival and to promote integration between the material and host tissue. A new gas-foaming method to generate pores was proposed by directly adding Mg particles into cell-laden hydrogel solutions, taking advantage of the H2 gas formed during the degradation of Mg. The optimization design of the size and amount of Mg particles added into the hydrogels was investigated. Improved cell viability and proliferation were demonstrated in the group with Mg particles. Additionally, Mg2+ ions generated during Mg degradation facilitated the osteogenic differentiation of stem cells encapsulated in hydrogels. Extensive vascularized bone regeneration in the femoral defects of rats revealed that the use of Mg particles as the foaming agent is feasible, endowing injectable hydrogels with optimized porosity and enhanced bioactivity, and providing a new strategy for future designs of porous hydrogels in tissue engineering.

Enhanced corrosion resistance and bioactivity of Mg alloy modified by Zn-doped nanowhisker hydroxyapatite coatings.

In this work, Zn is doped into a hydroxyapatite coating on the surface of ZK60 magnesium alloys using a one-pot hydrothermal method to obtain a corrosion-resistant implant with abilities of osteogenic differentiation and bacterial inhibition. With the addition of Zn, the morphology changes with a nanowhisker structure appearing on the coating. Electrochemical measurements show that the nanowhisker hydroxyapatite coating provides a high corrosion resistance. Compared with hydroxyapatite coating, the nanowhisker coating not only effectively inhibits bacteria, but also promotes the adhesion and differentiation of rat bone marrow mesenchymal stem cells at appropriate Zn concentrations. In conclusion, a novel nanowhisker structure prepared by a single variable Zn doping can significantly improve the corrosion resistance and biological activity of hydroxyapatite coatings.

A Magnesium-Enriched 3D Culture System that Mimics the Bone Development Microenvironment for Vascularized Bone Regeneration.

The redevelopment/regeneration pattern of amputated limbs from a blastema in salamander suggests that enhanced regeneration might be achieved by mimicking the developmental microenvironment. Inspired by the discovery that the expression of magnesium transporter-1 (MagT1), a selective magnesium (Mg) transporter, is significantly upregulated in the endochondral ossification region of mouse embryos, a Mg-enriched 3D culture system is proposed to provide an embryonic-like environment for stem cells. First, the optimum concentration of Mg ions (Mg2+) for creating the osteogenic microenvironment is screened by evaluating MagT1 expression levels, which correspond to the osteogenic differentiation capacity of stem cells. The results reveal that Mg2+ selectively activates the mitogen-activated protein kinase/extracellular regulated kinase (MAPK/ERK) pathway to stimulate osteogenic differentiation, and Mg2+ influx via MagT1 is profoundly involved in this process. Then, Mg-enriched microspheres are fabricated at the appropriate size to ensure the viability of the encapsulated cells. A series of experiments show that the Mg-enriched microenvironment not only stimulates the osteogenic differentiation of stem cells but also promotes neovascularization. Obvious vascularized bone regeneration is achieved in vivo using these Mg-enriched cell delivery vehicles. The findings suggest that biomaterials mimicking the developmental microenvironment might be promising tools to enhance tissue regeneration.

Recent advances in cell sheet technology for bone and cartilage regeneration: from preparation to application.

Bone defects caused by trauma, tumour resection, infection and congenital deformities, together with articular cartilage defects and cartilage-subchondral bone complex defects caused by trauma and degenerative diseases, remain great challenges for clinicians. Novel strategies utilising cell sheet technology to enhance bone and cartilage regeneration are being developed. The cell sheet technology has shown great clinical potential in regenerative medicine due to its effective preservation of cell-cell connections and extracellular matrix and its scaffold-free nature. This review will first introduce several widely used cell sheet preparation systems, including traditional approaches and recent improvements, as well as their advantages and shortcomings. Recent advances in utilising cell sheet technology to regenerate bone or cartilage defects and bone-cartilage complex defects will be reviewed. The key challenges and future research directions for the application of cell sheet technology in bone and cartilage regeneration will also be discussed.

Early effects of parathyroid hormone on vascularized bone regeneration and implant osseointegration in aged rats.

The decreased bone mass and impaired osteogenesis capacities that occur with aging may influence the outcome of dental implants. Parathyroid hormone (PTH) (1-34) is an anabolic agent for the treatment of osteoporosis. However, little is known about its effects and mechanisms on vascularized bone regeneration and implant osseointegration in aging. In current study, we adopted both in vivo and in vitro approaches to explore the mechanisms of early actions of PTH (1-34) on the angiogenic and osteogenic microenvironment to enhance implant osseointegration in aged rats. Daily subcutaneous injections of 30 µg/kg PTH (1-34) were given to female rats aged 20 months beginning on next day of implantation and lasting for 5 weeks. Radiological and histological analysis confirmed that PTH (1-34) improved new bone formation, angiogenesis and implant osseointegration in aged rats in the early stage. The osteogenic potential of aged bone mesenchymal stem cells (BMSCs) was enhanced, while their adipogenesis capacity was attenuated. Furthermore, PTH (1-34) was shown to promote angiogenesis directly via endothelial cell migration and blood vessel formation in vitro. Meanwhile, PTH (1-34) stimulated more osteoclasts participation in bone remodeling by secreting angiogenic and osteogenic growth factors to induce early vascularization and stimulate the migration or differentiation of BMSCs indirectly. Together, these results demonstrate mechanistic insight into how PTH (1-34) regulates the angiogenic and osteogenic microenvironment to result in more active bone remodeling and new bone formation, making it an excellent potential therapeutic agent for rapid vascularized bone regeneration and implant osseointegration in the aged population.

Enhancing Corrosion Resistance, Osteoinduction, and Antibacterial Properties by Zn/Sr Additional Surface Modification of Magnesium Alloy.

To control the degradation of magnesium alloy and enhance its osteoinduction activity and antibacterial properties, we proposed the addition of Zn and Sr ions in the process of surface modification of the magnesium alloy (ZK60) by a one-pot hydrothermal process. We found that, after surface modification, the surface of the materials formed a cluster crystal structure coating layer, and the successful incorporation of Zn and Sr ions in the surface coating did not affect the morphology of the microstructure. The corrosion resistance of the surface of the modified magnesium alloy was significantly improved, and cells grew well on the modified material surfaces. Zn and Sr ions released from the coating layer promote cell osteogenic differentiation, and Zn ions also provide a good antibacterial effect. Thus, the combined use of Zn and Sr offers antibacterial effects and promotes osteogenic differentiation of cells. To summarize, we have developed a controllable and degradable magnesium alloy material that offers both osteoinduction and antibacterial effects. The development of this material provides ideas about the preparation of a novel biodegradable magnesium alloy with better bioactivity for clinical application.

[Effect of overexpression of transcription factor Runx2 and Osterix on osteogenic differentiation of endothelial cells].

PURPOSE: To explore the effect of overexpression of Runx2 and Osterix (OSX) genes on osteogenic differentiation of human umbilical vein endothelial cells (HUVECs). METHODS: Overexpressed Runx2 and OSX lentiviral vectors were transfected into HUVECs respectively. The osteogenic potential of transfected cells was identified by alkaline phosphatase (ALP) staining and ALP activity. Furthermore, real time-PCR, Western blot and immunofluorescence staining were performed to detect the expression of osteogenic genes and proteins in HUVECs. GraphPad Prism 6.01 software was used for statistical analysis. RESULTS: Overexpression of Runx2 gene was beneficial for osteogenic differentiation of HUVECs, while overexpression of osterix gene did not show osteogenic differential potential. Moreover, overexpression of Runx2 gene in HUVECs up-regulated the gene expression level of Runx2, OSX, ALP, bone sialoprotein (BSP), osteopontin (OPN), and osteocalcin (OCN), and up-regulated protein level of OPN and OCN. CONCLUTIONS: Overexpression of Runx2 could promote osteogenic differentiation of HUVECs.

Magnetically Controlled Growth-Factor-Immobilized Multilayer Cell Sheets for Complex Tissue Regeneration.

The scaffold-free cell-sheet technique plays a significant role in stem-cell-based regeneration. Furthermore, growth factors are known to direct stem cell differentiation and enhance tissue regeneration. However, the absence of an effective means to incorporate growth factors into the cell sheets hinders further optimization of the regeneration efficiency. Here, a novel design of magnetically controlled "growth-factor-immobilized cell sheets" is reported. A new Fe3 O4 magnetic nanoparticle (MNP) coated with nanoscale graphene oxide (nGO@Fe3 O4 ) is developed to label stem cells and deliver growth factors. First, the nGO@Fe3 O4 MNPs can be easily swallowed by dental-pulp stem cells (DPSCs) and have no influence on cell viability. Thus, the MNP-labeled cells can be organized via magnetic force to form multilayered cell sheets in different patterns. Second, compared to traditional Fe3 O4 nanoparticles, the graphene oxide coating provides plenty of carboxyl groups to bind and deliver growth factors. Therefore, with these nGO@Fe3 O4 MNPs, bone-morphogenetic-protein-2 (BMP2) is successfully incorporated into the DPSCs sheets to induce more bone formation. Furthermore, an integrated osteochondral complex is also constructed using a combination of DPSCs/TGFß3 and DPSCs/BMP2. All these results demonstrate that the new cell-sheet tissue-engineering approach exhibits promising potential for future use in regenerative medicine.