Synthetic biology-based bacterial extracellular vesicles displaying BMP-2 and CXCR4 to ameliorate osteoporosis.

Osteoporosis (OP) is a systematic bone disease characterized by low bone mass and fragile bone microarchitecture. Conventional treatment for OP has limited efficacy and long-term toxicity. Synthetic biology makes bacterial extracellular vesicle (BEVs)-based therapeutic strategies a promising alternative for the treatment of OP. Here, we constructed a recombinant probiotics Escherichia coli Nissle 1917-pET28a-ClyA-BMP-2-CXCR4 (ECN-pClyA-BMP-2-CXCR4), in which BMP-2 and CXCR4 were overexpressed in fusion with BEVs surface protein ClyA. Subsequently, we isolated engineered BEVs-BMP-2-CXCR4 (BEVs-BC) for OP therapy. The engineered BEVs-BC exhibited great bone targeting in vivo. In addition, BEVs-BC had good biocompatibility and remarkable ability to promote osteogenic differentiation of BMSCs. Finally, the synthetic biology-based BEVs-BC significantly prevented the OP in an ovariectomized (OVX) mouse model. In conclusion, we constructed BEVs-BC with both bone-targeting and bone-forming in one-step using synthetic biology, which provides an effective strategy for OP and has great potential for industrialization.

Expert consensus on Prospective Precision Diagnosis and Treatment Strategies for Osteoporotic Fractures.

Osteoporotic fractures are the most severe complications of osteoporosis, characterized by poor bone quality, difficult realignment and fixation, slow fracture healing, and a high risk of recurrence. Clinically managing these fractures is relatively challenging, and in the context of rapid aging, they pose significant social hazards. The rapid advancement of disciplines such as biophysics and biochemistry brings new opportunities for future medical diagnosis and treatment. However, there has been limited attention to precision diagnosis and treatment strategies for osteoporotic fractures both domestically and internationally. In response to this, the Chinese Medical Association Orthopaedic Branch Youth Osteoporosis Group, Chinese Geriatrics Society Geriatric Orthopaedics Committee, Chinese Medical Doctor Association Orthopaedic Physicians Branch Youth Committee Osteoporosis Group, and Shanghai Association of Integrated Traditional Chinese and Western Medicine Osteoporosis Professional Committee have collaborated to develop this consensus. It aims to elucidate emerging technologies that may play a pivotal role in both diagnosis and treatment, advocating for clinicians to embrace interdisciplinary approaches and incorporate these new technologies into their practice. Ultimately, the goal is to improve the prognosis and quality of life for elderly patients with osteoporotic fractures.

Boosting cartilage repair with silk fibroin-DNA hydrogel-based cartilage organoid precursor.

Osteoarthritis (OA), a common degenerative disease, is characterized by high disability and imposes substantial economic impacts on individuals and society. Current clinical treatments remain inadequate for effectively managing OA. Organoids, miniature 3D tissue structures from directed differentiation of stem or progenitor cells, mimic native organ structures and functions. They are useful for drug testing and serve as active grafts for organ repair. However, organoid construction requires extracellular matrix-like 3D scaffolds for cellular growth. Hydrogel microspheres, with tunable physical and chemical properties, show promise in cartilage tissue engineering by replicating the natural microenvironment. Building on prior work on SF-DNA dual-network hydrogels for cartilage regeneration, we developed a novel RGD-SF-DNA hydrogel microsphere (RSD-MS) via a microfluidic system by integrating photopolymerization with self-assembly techniques and then modified with Pep-RGDfKA. The RSD-MSs exhibited uniform size, porous surface, and optimal swelling and degradation properties. In vitro studies demonstrated that RSD-MSs enhanced bone marrow mesenchymal stem cells (BMSCs) proliferation, adhesion, and chondrogenic differentiation. Transcriptomic analysis showed RSD-MSs induced chondrogenesis mainly through integrin-mediated adhesion pathways and glycosaminoglycan biosynthesis. Moreover, in vivo studies showed that seeding BMSCs onto RSD-MSs to create cartilage organoid precursors (COPs) significantly enhanced cartilage regeneration. In conclusion, RSD-MS was an ideal candidate for the construction and long-term cultivation of cartilage organoids, offering an innovative strategy and material choice for cartilage regeneration and tissue engineering.

Extracellular derivatives for bone metabolism.

BACKGROUND: Bone metabolism can maintain the normal homeostasis and function of bone tissue. Once the bone metabolism balance is broken, it will cause osteoporosis, osteoarthritis, bone defects, bone tumors, or other bone diseases. However, such orthopedic diseases still have many limitations in clinical treatment, such as drug restrictions, drug tolerance, drug side effects, and implant rejection. AIM OF REVIEW: In complex bone therapy and bone regeneration, extracellular derivatives have become a promising research focus to solve the problems of bone metabolic diseases. These derivatives, which include components such as extracellular matrix, growth factors, and extracellular vesicles, have significant therapeutic potential. It has the advantages of good biocompatibility, low immune response, and dynamic demand for bone tissue. The purpose of this review is to provide a comprehensive perspective on extracellular derivatives for bone metabolism and elucidate the intrinsic properties and versatility of extracellular derivatives. Further discussion of them as innovative advanced orthopedic materials for improving the effectiveness of bone therapy and regeneration processes. KEY SCIENTIFIC CONCEPTS OF REVIEW: In this review, we first listed the types and functions of three extracellular derivatives. Then, we discussed the effects of extracellular derivatives of different cell sources on bone metabolism. Subsequently, we collected applications of extracellular derivatives in the treatment of bone metabolic diseases and summarized the advantages and challenges of extracellular derivatives in clinical applications. Finally, we prospected the extracellular derivatives in novel orthopedic materials and clinical applications. We hope that the comprehensive understanding of extracellular derivatives in bone metabolism will provide new solutions to bone diseases.

Microenvironment-targeted strategy steers advanced bone regeneration.

Treatment of large bone defects represents a great challenge in orthopedic and craniomaxillofacial surgery. Traditional strategies in bone tissue engineering have focused primarily on mimicking the extracellular matrix (ECM) of bone in terms of structure and composition. However, the synergistic effects of other cues from the microenvironment during bone regeneration are often neglected. The bone microenvironment is a sophisticated system that includes physiological (e.g., neighboring cells such as macrophages), chemical (e.g., oxygen, pH), and physical factors (e.g., mechanics, acoustics) that dynamically interact with each other. Microenvironment-targeted strategies are increasingly recognized as crucial for successful bone regeneration and offer promising solutions for advancing bone tissue engineering. This review provides a comprehensive overview of current microenvironment-targeted strategies and challenges for bone regeneration and further outlines prospective directions of the approaches in construction of bone organoids.

M2 macrophage-derived exosomes promote diabetic fracture healing by acting as an immunomodulator.

Diabetes mellitus is a chronically inflamed disease that predisposes to delayed fracture healing. Macrophages play a key role in the process of fracture healing by undergoing polarization into either M1 or M2 subtypes, which respectively exhibit pro-inflammatory or anti-inflammatory functions. Therefore, modulation of macrophage polarization to the M2 subtype is beneficial for fracture healing. Exosomes perform an important role in improving the osteoimmune microenvironment due to their extremely low immunogenicity and high bioactivity. In this study, we extracted the M2-exosomes and used them to intervene the bone repair in diabetic fractures. The results showed that M2-exosomes significantly modulate the osteoimmune microenvironment by decreasing the proportion of M1 macrophages, thereby accelerating diabetic fracture healing. We further confirmed that M2-exosomes induced the conversion of M1 macrophages into M2 macrophages by stimulating the PI3K/AKT pathway. Our study offers a fresh perspective and a potential therapeutic approach for M2-exosomes to improve diabetic fracture healing.

Fluorescence lifetime tracking and imaging of single moving particles assisted by a low-photon-count analysis algorithm.

Fluorescence lifetime imaging microscopy (FLIM) has been widely used in the field of biological research because of its high specificity, sensitivity, and quantitative ability in the sensing cellular microenvironment. The most commonly used FLIM technology is based on time-correlated single photon counting (TCSPC). Although the TCSPC method has the highest temporal resolution, the data acquisition time is usually long, and the imaging speed is slow. In this work, we proposed a fast FLIM technology for fluorescence lifetime tracking and imaging of single moving particles, named single particle tracking FLIM (SPT-FLIM). We used feedback-controlled addressing scanning and Mosaic FLIM mode imaging to reduce the number of scanned pixels and the data readout time, respectively. Moreover, we developed a compressed sensing analysis algorithm based on alternating descent conditional gradient (ADCG) for low-photon-count data. We applied the ADCG-FLIM algorithm on both simulated and experimental datasets to evaluate its performance. The results showed that ADCG-FLIM could achieve reliable lifetime estimation with high accuracy and precision in the case of a photon count less than 100. By reducing the photon count requirement for each pixel from, typically, 1000 to 100, the acquisition time for a single frame lifetime image could be significantly shortened, and the imaging speed could be improved to a great extent. On this basis, we obtained lifetime trajectories of moving fluorescent beads using the SPT-FLIM technique. Overall, our work offers a powerful tool for fluorescence lifetime tracking and imaging of single moving particles, which will promote the application of TCSPC-FLIM in biological research.

Bone/cartilage organoid on-chip: Construction strategy and application.

The necessity of disease models for bone/cartilage related disorders is well-recognized, but the barrier between ex-vivo cell culture, animal models and the real human body has been pending for decades. The organoid-on-a-chip technique showed opportunity to revolutionize basic research and drug screening for diseases like osteoporosis and arthritis. The bone/cartilage organoid on-chip (BCoC) system is a novel platform of multi-tissue which faithfully emulate the essential elements, biologic functions and pathophysiological response under real circumstances. In this review, we propose the concept of BCoC platform, summarize the basic modules and current efforts to orchestrate them on a single microfluidic system. Current disease models, unsolved problems and future challenging are also discussed, the aim should be a deeper understanding of diseases, and ultimate realization of generic ex-vivo tools for further therapeutic strategies of pathological conditions.

Maintaining hypoxia environment of subchondral bone alleviates osteoarthritis progression.

Abnormal subchondral bone remodeling featured by overactivated osteoclastogenesis leads to articular cartilage degeneration and osteoarthritis (OA) progression, but the mechanism is unclear. We used lymphocyte cytosolic protein 1 (Lcp1) knockout mice to suppress subchondral osteoclasts in a mice OA model with anterior cruciate ligament transection (ACLT), and Lcp1-/- mice showed decreased bone remodeling in subchondral bone and retarded cartilage degeneration. For mechanisms, the activated osteoclasts in subchondral bone induced type-H vessels and elevated oxygen concentration, which ubiquitylated hypoxia-inducible factor 1 alpha subunit (HIF-1α) in chondrocytes and led to cartilage degeneration. Lcp1 knockout impeded angiogenesis, which maintained hypoxia environment in joints and delayed the OA progression. Stabilization of HIF-1α delayed cartilage degeneration, and knockdown of Hif1a abolished the protective effects of Lcp1 knockout. Last, we showed that Oroxylin A, an Lcp1-encoded protein l-plastin (LPL) inhibitor, could alleviate OA progression. In conclusion, maintaining hypoxic environment is an attractive strategy for OA treatment.

Selection and identification of a novel ssDNA aptamer targeting human skeletal muscle.

Skeletal muscle disorders have posed great threats to health. Selective delivery of drugs and oligonucleotides to skeletal muscle is challenging. Aptamers can improve targeting efficacy. In this study, for the first time, the human skeletal muscle-specific ssDNA aptamers (HSM01, etc.) were selected and identified with Systematic Evolution of Ligands by Exponential Enrichment (SELEX). The HSM01 ssDNA aptamer preferentially interacted with human skeletal muscle cells in vitro. The in vivo study using tree shrews showed that the HSM01 ssDNA aptamer specifically targeted human skeletal muscle cells. Furthermore, the ability of HSM01 ssDNA aptamer to target skeletal muscle cells was not affected by the formation of a disulfide bond with nanoliposomes in vitro or in vivo, suggesting a potential new approach for targeted drug delivery to skeletal muscles via liposomes. Therefore, this newly identified ssDNA aptamer and nanoliposome modification could be used for the treatment of human skeletal muscle diseases.

Revealing the structure and organization of intercellular tunneling nanotubes (TNTs) by STORM imaging.

Tunneling nanotubes (TNTs) are nanoscale, actin-rich, transient intercellular tubes for cell-to-cell communication, which transport various cargoes between distant cells. The structural complexity and spatial organization of the involved components of TNTs remain unknown. In this work, the STORM super-resolution imaging technique was applied to elucidate the structural organization of microfilaments and microtubules in intercellular TNTs at the nanometer scale. Our results reveal different distributions of microfilaments and intertwined structures of microtubules in TNTs, which promote the knowledge of TNT communications.

Loss of Bcl-3 delays bone fracture healing through activating NF-κB signaling in mesenchymal stem cells.

Background: Bone fracture healing is a postnatal regenerative process in which fibrocartilaginous callus formation and bony callus formation are important. Bony callus formation requires osteoblastic differentiation of MSCs. Materials and methods: The formation of callus was assessed by µCT, Safranin-O, H&E and Masson trichrome staining. Osteogenesis of MSCs was analyzed by ALP staining, ARS staining, qRT-PCR and WB. And we also used IF and TOP/FOP Flash luciferase reporter to assess the nuclear translocation of PP65. Results: In this study, we found Bcl-3 showed a significant correlation with bone fracture healing. Results of µCT showed that loss of Bcl-3 delays bone fracture healing. Safranin-O, H&E and Masson trichrome staining confirmed that loss of Bcl-3 impacted the formation of cartilage and woven bone in callus. Further experiments in vitro manifested that Bcl-3-knockdown could inhibit MSCs osteoblastic differentiation through releasing the inhibition on NF-κB signaling by Co-IP, IF staining and luciferase reporter assay. Conclusions: We unveiled that loss of Bcl-3 could lead to inhibited osteogenic differentiation of MSCs via promoting PP65 nuclear translocation. The translational potential of this article: Our data demonstrated that overexpression of Bcl-3 accelerates bone fracture healing, which serves as a promising therapeutic target for bone fracture treatment.

Bacterial extracellular vesicles as bioactive nanocarriers for drug delivery: Advances and perspectives.

Nanosized extracellular vesicles derived from bacteria contain diverse cargo and transfer intercellular bioactive molecules to cells. Due to their favorable intercellular interactions, cell membrane-derived bacterial extracellular vesicles (BEVs) have great potential to become novel drug delivery platforms. In this review, we summarize the biogenesis mechanism and compositions of various BEVs. In addition, an overview of effective isolation and purification techniques of BEVs is provided. In particular, we focus on the application of BEVs as bioactive nanocarriers for drug delivery. Finally, we summarize the advances and challenges of BEVs after providing a comprehensive discussion in each section. We believe that a deeper understanding of BEVs will open new avenues for their exploitation in drug delivery applications.

LPS/Bcl3/YAP1 signaling promotes Sox9+HNF4α+ hepatocyte-mediated liver regeneration after hepatectomy.

Recent reports have demonstrated that Sox9+HNF4α+ hepatocytes are involved in liver regeneration after chronic liver injury; however, little is known about the origin of Sox9+HNF4α+ hepatocytes and the regulatory mechanism. Employing a combination of chimeric lineage tracing, immunofluorescence, and immunohistochemistry, we demonstrate that Sox9+HNF4α+ hepatocytes, generated by transition from mature hepatocytes, play an important role in the initial phase after partial hepatectomy (PHx). Additionally, knocking down the expression of Sox9 suppresses hepatocyte proliferation and blocks the recovery of lost hepatic tissue. In vitro and in vivo assays demonstrated that Bcl3, activated by LPS, promotes hepatocyte conversion and liver regeneration. Mechanistically, Bcl3 forms a complex with and deubiquitinates YAP1 and further induces YAP1 to translocate into the nucleus, resulting in Sox9 upregulation and mature hepatocyte conversion. We demonstrate that Bcl3 promotes Sox9+HNF4α+ hepatocytes to participate in liver regeneration, and might therefore be a potential target for enhancing regeneration after liver injury.

Lipophilic Red-Emitting Carbon Dots for Detecting and Tracking Lipid Droplets in Live Cells.

Lipid droplets (LDs), a dynamic organelle, are of vital importance in regulating the storage of neutral lipids and energy homeostasis. The aberrant expression of LDs is found to be highly associated with diverse metabolic diseases. Thus, detecting and monitoring LDs are essential to study the pathological and physiological processes of LDs in living bodies. However, it remains challenging to obtain suitable imaging probes to track LDs in vivo. Fortunately, the emergence of carbon dots (CDs), which are fluorescent nanomaterials with good biocompatibility and high stability, has provided us an unprecedented choice. In this work, CDs were synthesized via a solvothermal treatment of commercial reagents, 3-dimethylaminophenol. Interestingly, the prepared CDs show an intense red emission in non-hydrogen-bonding solution and have strong LD-targeting ability without any postmodification of ligands. Moreover, due to their low phototoxicity and excellent photostability, CDs were successfully applied to track the dynamics of LDs in live cells and image LDs in different cell lines and lipid-rich tissues. Overall, this work here proposed an LD-specific red-emitting CD probe, which will be of great value for learning more about LD-associated behaviors and diseases.

Optimizing the strontium content to achieve an ideal osseointegration through balancing apatite-forming ability and osteogenic activity.

Implant failure caused by unsatisfying osseointegration is still a noteworthy clinical problem. Strontium (Sr) has been confirmed to be a bioactive element that facilitates bone growth. In this study, Sr was surface incorporated in titanium (Ti) implant with different contents. The XRD results demonstrated that Sr existed mainly in the form of SrTiO3. All Sr-contained implants showed sustainable Sr2+ release behavior. Meanwhile, the Sr2+ release rate was proportional to the Sr content. The in vitro immersing test showed that the apatite-forming ability on the implant surface was decreased with the increase of Sr content. Conversely, the cell experiments manifested that implants with high content of Sr were more favorable to cell spreading, proliferation, osteogenic differentiation, and extracellular matrix mineralization. The in vivo implant experiment revealed that Sr-incorporation could improve osseointegration, new bone formation and mineralization, and bone-implant bonding strength. In addition, Ti5Sr, which possessed a combined good osteogenic activity and apatite-forming ability, exhibited the best in vivo overall performance. In summary, we first put forward the competitive effect of osteogenic activity and apatite-forming ability on bone-implant osseointegration, which would provide a new strategy for implant design.

Diterbutyl phthalate attenuates osteoarthritis in ACLT mice via suppressing ERK/c-fos/NFATc1 pathway, and subsequently inhibiting subchondral osteoclast fusion.

Osteoarthritis (OA) is the most common arthritis with a rapidly increasing prevalence. Disease progression is irreversible, and there is no curative therapy available. During OA onset, abnormal mechanical loading leads to excessive osteoclastogenesis and bone resorption in subchondral bone, causing a rapid subchondral bone turnover, cyst formation, sclerosis, and finally, articular cartilage degeneration. Moreover, osteoclast-mediated angiogenesis and sensory innervation in subchondral bone result in abnormal vascularization and OA pain. The traditional Chinese medicine Panax notoginseng (PN; Sanqi) has long been used in treatment of bone diseases including osteoporosis, bone fracture, and OA. In this study we established two-dimensional/bone marrow mononuclear cell/cell membrane chromatography/time of flight mass spectrometry (2D/BMMC/CMC/TOFMS) technique and discovered that diterbutyl phthalate (DP) was the active constituent in PN inhibiting osteoclastogenesis. Then we explored the therapeutic effect of DP in an OA mouse model with anterior cruciate ligament transaction (ACLT). After ACLT was conducted, the mice received DP (5 mg·kg-1·d-1, ip) for 8 weeks. Whole knee joint tissues of the right limb were harvested at weeks 2, 4, and 8 for analysis. We showed that DP administration impeded overactivated osteoclastogenesis in subchondral bone and ameliorated articular cartilage deterioration. DP administration blunted aberrant H-type vessel formation in subchondral bone marrow and alleviated OA pain assessed in Von Frey test and thermal plantar test. In RANKL-induced RAW264.7 cells in vitro, DP (20 µM) retarded osteoclastogenesis by suppressing osteoclast fusion through inhibition of the ERK/c-fos/NFATc1 pathway. DP treatment also downregulated the expression of dendritic cell-specific transmembrane protein (DC-STAMP) and d2 isoform of the vacuolar (H+) ATPase V0 domain (Atp6v0d2) in the cells. In conclusion, we demonstrate that DP prevents OA progression by inhibiting abnormal osteoclastogenesis and associated angiogenesis and neurogenesis in subchondral bone.

Corrigendum: Super-Resolution Microscopy: Shedding New Light on In Vivo Imaging.

[This corrects the article DOI: 10.3389/fchem.2021.746900.].

Super-Resolution Microscopy: Shedding New Light on In Vivo Imaging.

Over the past two decades, super-resolution microscopy (SRM), which offered a significant improvement in resolution over conventional light microscopy, has become a powerful tool to visualize biological activities in both fixed and living cells. However, completely understanding biological processes requires studying cells in a physiological context at high spatiotemporal resolution. Recently, SRM has showcased its ability to observe the detailed structures and dynamics in living species. Here we summarized recent technical advancements in SRM that have been successfully applied to in vivo imaging. Then, improvements in the labeling strategies are discussed together with the spectroscopic and chemical demands of the fluorophores. Finally, we broadly reviewed the current applications for super-resolution techniques in living species and highlighted some inherent challenges faced in this emerging field. We hope that this review could serve as an ideal reference for researchers as well as beginners in the relevant field of in vivo super resolution imaging.