HIF-1α activation impairs dendrites formation and elongation in osteocytogenesis.

Osteocytes are terminally differentiated cells derived from osteoblasts and are deeply embedded within the bone matrix. They play a critical role in bone remodeling by generating a lacuno-canalicular network (LCN) and controlling the transport of nutrients. Due to the absence of blood vessels within the bone matrix, it is widely believed that osteocytes develop in a hypoxic environment. However, the mechanisms of osteocytogenesis and the role of oxygen sensing in this process remain unclear. Hypoxia-inducible factors (HIFs) are major transcriptional factors involved in oxygen sensing. Previous studies have shown that accumulation of HIFs in osteoblasts leads to abnormal bone remodeling, potentially linked with the alterations of the LCN network. Specifically, HIF-1α is hypothesized to play a more significant role in regulating bone remodeling compared to HIF-2α. Therefore, we investigated the functions of HIF-1α in dendrite formation and the establishment of the LCN network during osteocytogenesis. Immunostaining and scanning electron microscopy revealed that the E11 protein aggregates to form a ring structure that defines the site for dendrite initiation. This process is followed by activation of the ERM/RhoA pathway and recruitment of matrix metalloproteinase 14 (MMP14) to facilitate extracellular matrix degradation, enabling dendrite elongation. However, both hypoxic treatment and overexpression of HIF-1α impair ring formation, resulting in reduced ERM/RhoA activity and decreased matrix degradation capability. These findings suggest that abnormal HIF-1α activity in local areas could contribute to impaired LCN network formation and abnormal bone remodeling observed in bone diseases such as osteopenia and aging.

Activation of the osteoblastic HIF-1α pathway partially alleviates the symptoms of STZ-induced type 1 diabetes mellitus via RegIIIγ.

The hypoxia-inducible factor-1α (HIF-1α) pathway coordinates skeletal bone homeostasis and endocrine functions. Activation of the HIF-1α pathway increases glucose uptake by osteoblasts, which reduces blood glucose levels. However, it is unclear whether activating the HIF-1α pathway in osteoblasts can help normalize glucose metabolism under diabetic conditions through its endocrine function. In addition to increasing bone mass and reducing blood glucose levels, activating the HIF-1α pathway by specifically knocking out Von Hippel‒Lindau (Vhl) in osteoblasts partially alleviated the symptoms of streptozotocin (STZ)-induced type 1 diabetes mellitus (T1DM), including increased glucose clearance in the diabetic state, protection of pancreatic ß cell from STZ-induced apoptosis, promotion of pancreatic ß cell proliferation, and stimulation of insulin secretion. Further screening of bone-derived factors revealed that islet regeneration-derived protein III gamma (RegIIIγ) is an osteoblast-derived hypoxia-sensing factor critical for protection against STZ-induced T1DM. In addition, we found that iminodiacetic acid deferoxamine (SF-DFO), a compound that mimics hypoxia and targets bone tissue, can alleviate symptoms of STZ-induced T1DM by activating the HIF-1α-RegIIIγ pathway in the skeleton. These data suggest that the osteoblastic HIF-1α-RegIIIγ pathway is a potential target for treating T1DM.

Positioning regulation of organelle network via Chinese microneedle.

The organelle network is a key factor in the repair and regeneration of lesion. However, effectively intervening in the organelle network which has complex interaction mechanisms is challenging. In this study, on the basis of electromagnetic laws, we constructed a biomaterial-based physical/chemical restraint device. This device was designed to jointly constrain electrical and biological factors in a conductive screw-threaded microneedle (ST-needle) system, identifying dual positioning regulation of the organelle network. The unique physical properties of this system could accurately locate the lesion and restrict the current path to the lesion cells through electromagnetic laws, and dynamic Van der Waals forces were activated to release functionalized hydrogel microspheres. Subsequently, the mitochondria-endoplasmic reticulum (ER) complex was synergistically targeted by increasing mitochondrial ATP supply to the ER via electrical stimulation and by blocking calcium current from the ER to the mitochondria using microspheres, and then the life activity of the lesion cells was effectively restored.

Phillygenin prevents osteoclast differentiation and bone loss by targeting RhoA.

Forsythia suspensa tea is a popular traditional Chinese medicine decoction for its healthy and therapeutic benefits. However, its effects in bone metabolism were not clear. In recent study, we uncovered anti-osteoclastogenesis property of Phillygenin (Phi), a compound abundant in Forsythia suspensa leaves, and aimed to investigate the effect and mechanism of Phi on bone metabolism in vivo and in vitro. Lipopolysaccharides-induced murine calvaria osteolysis and ovariectomy-induced bone loss animal models were used to identify the bone-protective effect of Phi in vivo and micro-CT, pQCT, and TRAP staining were applied. We used CCK8, TUNEL, BrdU, and TRAP staining to evaluate the efficacy of Phi on the proliferation and formation of OCs in primary mBMMs. RNA sequence, activity-based protein profiling, molecular docking, G-LISA, and WB were used to inspect the target and underlying mechanism of Phi's actions in mBMMs. We found Phi significantly inhibited bone resorption in vivo and inhibited mBMMs osteoclastogenesis in vitro. Ras homolog gene family member A (RhoA) was identified as the direct target of Phi. It counteracted the effects of RhoA activator and acted as a RhoA inhibitor. By targeting RhoA, Phi modulated Rho-associated coiled-coil containing protein kinase 1 (ROCK1) activity and regulated its downstream NF-κB/NFATc1/c-fos pathway. Furthermore, Phi depressed the disassembling of F-actin ring through cofilin and myosin1a. Our findings provided Phi as a potential option for treating bone loss diseases by targeting RhoA and highlighted the importance of F. suspensa as a preventive approach in bone disorders.

Injectable Nano-Micro Composites with Anti-bacterial and Osteogenic Capabilities for Minimally Invasive Treatment of Osteomyelitis.

The effective management of osteomyelitis remains extremely challenging due to the difficulty associated with treating bone defects, the high probability of recurrence, the requirement of secondary surgery or multiple surgeries, and the difficulty in eradicating infections caused by methicillin-resistant Staphylococcus aureus (MRSA). Hence, smart biodegradable biomaterials that provide effective and precise local anti-infection effects and can promote the repair of bone defects are actively being developed. Here, a novel nano-micro composite is fabricated by combining calcium phosphate (CaP) nanosheets with drug-loaded GelMA microspheres via microfluidic technology. The microspheres are covalently linked with vancomycin (Van) through an oligonucleotide (oligo) linker using an EDC/NHS carboxyl activator. Accordingly, a smart nano-micro composite called "CaP@MS-Oligo-Van" is synthesized. The porous CaP@MS-Oligo-Van composites can target and capture bacteria. They can also release Van in response to the presence of bacterial micrococcal nuclease and Ca2+, exerting additional antibacterial effects and inhibiting the inflammatory response. Finally, the released CaP nanosheets can promote bone tissue repair. Overall, the findings show that a rapid, targeted drug release system based on CaP@MS-Oligo-Van can effectively target bone tissue infections. Hence, this agent holds potential in the clinical treatment of osteomyelitis caused by MRSA.

Hydrogen Ion Capturing Hydrogel Microspheres for Reversing Inflammaging.

Inflammaging is deeply involved in aging-related diseases and can be destructive during aging. The maintenance of pH balance in the extracellular microenvironment can alleviate inflammaging and repair aging-related tissue damage. In this study, the hydrogen ion capturing hydrogel microsphere (GMNP) composed of mineralized transforming growth factor-ß (TGF-ß) and catalase (CAT) nanoparticles is developed via biomimetic mineralization and microfluidic technology for blocking the NLRP3 cascade axis in inflammaging. This GMNP can neutralize the acidic microenvironment by capturing excess hydrogen ions through the calcium carbonate mineralization layer. Then, the subsequent release of encapsulated TGF-ß and CAT can eliminate both endogenous and exogenous stimulus of NLRP3, thus suppressing the excessive activation of inflammaging. In vitro, GMNP can suppress the excessive activation of the TXNIP/NLRP3/IL-1ß cascade axis and enhance extracellular matrix (ECM) synthesis in nucleus pulposus cells. In vivo, GMNP becomes a sustainable and stable niche with microspheres as the core to inhibit inflammaging and promote the regeneration of degenerated intervertebral discs. Therefore, this hydrogen ion-capturing hydrogel microsphere effectively reverses inflammaging by interfering with the excessive activation of NLRP3 in the degenerated tissues.

Regulating Chondro-Bone Metabolism for Treatment of Osteoarthritis via High-Permeability Micro/Nano Hydrogel Microspheres.

Destruction of cartilage due to the abnormal remodeling of subchondral bone (SB) leads to osteoarthritis (OA), and restoring chondro-bone metabolic homeostasis is the key to the treatment of OA. However, traditional intra-articular injections for the treatment of OA cannot directly break through the cartilage barrier to reach SB. In this study, the hydrothermal method is used to synthesize ultra-small size (≈5 nm) selenium-doped carbon quantum dots (Se-CQDs, SC), which conjugated with triphenylphosphine (TPP) to create TPP-Se-CQDs (SCT). Further, SCT is dynamically complexed with hyaluronic acid modified with aldehyde and methacrylic anhydride (AHAMA) to construct highly permeable micro/nano hydrogel microspheres (SCT@AHAMA) for restoring chondro-bone metabolic homeostasis. In vitro experiments confirmed that the selenium atoms scavenged reactive oxygen species (ROS) from the mitochondria of mononuclear macrophages, inhibited osteoclast differentiation and function, and suppressed early chondrocyte apoptosis to maintain a balance between cartilage matrix synthesis and catabolism. In vivo experiments further demonstrated that the delivery system inhibited osteoclastogenesis and H-vessel invasion, thereby regulating the initiation and process of abnormal bone remodeling and inhibiting cartilage degeneration in SB. In conclusion, the micro/nano hydrogel microspheres based on ultra-small quantum dots facilitate the efficient penetration of articular SB and regulate chondro-bone metabolism for OA treatment.

Black Phosphorus Accelerates Bone Regeneration Based on Immunoregulation.

A fundamental understanding of inflammation and tissue healing suggests that the precise regulation of the inflammatory phase, both in terms of location and timing, is crucial for bone regeneration. However, achieving the activation of early inflammation without causing chronic inflammation while facilitating quick inflammation regression to promote bone regeneration continues to pose challenges. This study reveals that black phosphorus (BP) accelerates bone regeneration by building an osteogenic immunological microenvironment. BP amplifies the acute pro-inflammatory response and promotes the secretion of anti-inflammatory factors to accelerate inflammation regression and tissue regeneration. Mechanistically, BP creates an osteoimmune-friendly microenvironment by stimulating macrophages to express interleukin 33 (IL-33), amplifying the inflammatory response at an early stage, and promoting the regression of inflammation. In addition, BP-mediated IL-33 expression directly promotes osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), which further facilitates bone repair. To the knowledge, this is the first study to reveal the immunomodulatory potential of BP in bone regeneration through the regulation of both early-stage inflammatory responses and later-stage inflammation resolution, along with the associated molecular mechanisms. This discovery serves as a foundation for the clinical use of BP and is an efficient approach for managing the immune microenvironment during bone regeneration.

Intestine-Targeted Explosive Hydrogel Microsphere Promotes Uric Acid Excretion for Gout Therapy.

Uric acid metabolism disorder triggers metabolic diseases, especially gout. However, increasing uric acid excretion remains a challenge. Here, an accelerative uric acid excretion pathway via an oral intestine-explosive hydrogel microsphere merely containing uricase and dopamine is reported. After oral administration, uricase is exposed and immobilized on intestinal mucosa along with an in situ dopamine polymerization via a cascade reaction triggered by the intestinal specific environment. By this means, trace amount of uricase is required to in situ up-regulate uric acid transporter proteins of intestinal epithelial cells, causing accelerated intestinal uric acid excretion. From in vitro data, the uric acid in fecal samples from gout patients could be significantly reduced by up to 37% by the mimic mucosa-immobilized uricase on the isolated porcine tissues. Both hyperuricemia and acute gouty arthritis in vivo mouse models confirm the uric acid excretion efficacy of intestine-explosive hydrogel microspheres. Fecal uric acid excretion is increased around 30% and blood uric acid is reduced more than 70%. In addition, 16S ribosomal RNA sequencing showed that the microspheres optimized intestinal flora composition as well. In conclusion, a unique pathway via the intestine in situ regulation to realize an efficient uric acid intestinal excretion for gout therapy is developed.

Targeted Protein Fate Modulating Functional Microunits Promotes Intervertebral Fusion.

Stable regulation of protein fate is a prerequisite for successful bone tissue repair. As a ubiquitin-specific protease (USP), USP26 can stabilize the protein fate of ß-catenin to promote the osteogenic activity of mesenchymal cells (BMSCs) and significantly increased bone regeneration in bone defects in aged mice. However, direct transfection of Usp26 in vivo is inefficient. Therefore, improving the efficient expression of USP26 in target cells is the key to promoting bone tissue repair. Herein, 3D printing combined with microfluidic technology is applied to construct a functional microunit (protein fate regulating functional microunit, denoted as PFFM), which includes GelMA microspheres loaded with BMSCs overexpressing Usp26 and seeded into PCL 3D printing scaffolds. The PFFM provides a microenvironment for BMSCs, significantly promotes adhesion, and ensures cell activity and Usp26 supplementation that stabilizes ß-catenin protein significantly facilitates BMSCs to express osteogenic phenotypes. In vivo experiments have shown that PFFM effectively accelerates intervertebral bone fusion. Therefore, PFFM can provide new ideas and alternatives for using USP26 for intervertebral fusion and other hard-to-repair bone defect diseases and is expected to provide clinical translational potential in future treatments.

IDG-SW3 Cell Culture in a Three-Dimensional Extracellular Matrix.

Osteocytes are considered to be nonproliferative cells that are terminally differentiated from osteoblasts. Osteoblasts embedded in the bone extracellular matrix (osteoid) express the Pdpn gene to form cellular dendrites and transform into preosteocytes. Later, preosteocytes express the Dmp1 gene to promote matrix mineralization and thereby transform into mature osteocytes.This process is called osteocytogenesis. IDG-SW3 is a well-known cell line for in vitro studies of osteocytogenesis. Many previous methods have used collagen I as the main or the only component of the culturing matrix. However, in addition to collagen I, the osteoid also contains a ground substance, which is an important component in promoting cellular growth, adhesion, and migration. In addition, the matrix substance is transparent, which increases the transparency of the collagen I-formed gel and, thus, aids the exploration of dendrite formation through imaging techniques. Thus, this paper details a protocol to establish a 3D gel using an extracellular matrix along with collagen I for IDG-SW3 survival. In this work, dendrite formation and gene expression were analyzed during osteocytogenesis. After 7 days of osteogenic culture, an extensive dendrite network was clearly observed under a fluorescence confocal microscope. Real-time PCR showed that the mRNA levels of Pdpn and Dmp1 continually increased for 3 weeks. At week 4, the stereomicroscope revealed an opaque gel filled with mineral particles, consistent with the X-ray fluorescence (XRF) assay. These results indicate that this culture matrix successfully facilitates the transition from osteoblasts to mature osteocytes.

Geometric Angles and Gene Expression in Cells for Structural Bone Regeneration.

Geometry and angles play crucial roles in cellular processes; however, its mechanisms of regulation remain unclear. In this study, a series of three dimensional (3D)-printed microfibers with different geometries is constructed using a near-field electrostatic printing technique to investigate the regulatory mechanisms of geometry on stem cell function and bone regeneration. The scaffolds precisely mimicked cell dimensions with high porosity and interoperability. Compared with other spatial topography angles, microfibers with a 90° topology can significantly promote the expression of osteogenic gene proteins in bone marrow-derived mesenchymal stem cells (BMSCs). The effects of different spatial structures on the expression profiles of BMSCs differentiation genes are correlated and validated using microRNA sequencing. Enrichment analysis shows that the 90° microfibers promoted osteogenesis in BMSCs by significantly upregulating miR-222-5p/cbfb/Runx2 expression. The ability of the geometric architecture to promote bone regeneration, as assessed using the cranial defect model, demonstrates that the 90° fiber scaffolds significantly promote new bone regeneration and neovascular neural network formation. This study is the first to elucidate the relationship between angular geometry and cellular gene expression, contributing significantly to the understanding of how geometric architecture can promote stem cell differentiation, proliferation, and function for structural bone regeneration.

A general method for endowing hydrophobic nanoparticles with water dispersion abilities.

Inorganic nanoparticles with long-chain ligands are usually hydrophobic. However, simple and practical methods for converting hydrophobic nanoparticles to hydrophilic nanoparticles are still lacking. Herein, we developed a general method involving using dimercaptosuccinic acid (DMSA) for endowing hydrophobic nanoparticles with water dispersion abilities. By mixing a tetrahydrofuran solution of DMSA with a cyclohexane solution of hydrophobic nanoparticles, the long-chain ligands were replaced with DMSA, with the replacement due to the strong and broad-spectrum coordination abilities of sulphydryls and carboxyls. Four representative kinds of hydrophobic nanoparticles, namely Ag, NaGdF4, TiO2, and ZnS nanoparticles, were selected for verifying the performance of this DMSA-based modification method. Meanwhile, this method can also widen the applications of hydrophobic nanoparticles and facilitate their being subjected to further graft modifications. We hope that our research will increase the chances for applications of nanomaterials to be made.

In Situ Sustained Macrophage-Targeted Nanomicelle-Hydrogel Microspheres for Inhibiting Osteoarthritis.

There are still challenges in applying drug nanocarriers for in situ sustained macrophage targeting and regulation, due to the rapid clearance of nanocarriers and burst drug release in vivo. Herein, a nanomicelle-hydrogel microsphere, characterized by its macrophage-targeted nanosized secondary structure that allows it to accurately bind to M1 macrophages through active endocytosis, is employed for in situ sustained macrophage targeting and regulation, and addresses the insufficient osteoarthritis therapeutic efficacy caused by rapid clearance of drug nanocarriers. The 3-dimensional structure of a microsphere can prevent the rapid escape and clearance of a nanomicelle, thus keeping it in joints, while the ligand-guided secondary structure can carry drugs to accurately target and enter M1 macrophages, and release drugs via the transition from hydrophobicity to hydrophilicity of nanomicelles under inflammatory stimulation inside the macrophages. The experiments show that the nanomicelle-hydrogel microsphere can in situ sustainably target and regulate M1 macrophages for more than 14 days in joints, and attenuate local "cytokine storm" by continuous M1 macrophage apoptosis promotion and polarization inhibition. This micro/nano-hydrogel system shows excellent ability to sustainably target and regulate macrophage, realizes the improvement of drug utilization and efficacy inside the macrophage, and thereby can be a potential platform for treating macrophage-related diseases.

Click chemistry extracellular vesicle/peptide/chemokine nanocarriers for treating central nervous system injuries.

Central nervous system (CNS) injuries, including stroke, traumatic brain injury, and spinal cord injury, are essential causes of death and long-term disability and are difficult to cure, mainly due to the limited neuron regeneration and the glial scar formation. Herein, we apply extracellular vesicles (EVs) secreted by M2 microglia to improve the differentiation of neural stem cells (NSCs) at the injured site, and simultaneously modify them with the injured vascular targeting peptide (DA7R) and the stem cell recruiting factor (SDF-1) on their surface via copper-free click chemistry to recruit NSCs, inducing their neuronal differentiation, and serving as the nanocarriers at the injured site (Dual-EV). Results prove that the Dual-EV could target human umbilical vascular endothelial cells (HUVECs), recruit NSCs, and promote the neuronal differentiation of NSCs in vitro. Furthermore, 10 miRNAs are found to be upregulated in Dual-M2-EVs compared to Dual-M0-EVs via bioinformatic analysis, and further NSC differentiation experiment by flow cytometry reveals that among these miRNAs, miR30b-3p, miR-222-3p, miR-129-5p, and miR-155-5p may exert effect of inducing NSC to differentiate into neurons. In vivo experiments show that Dual-EV nanocarriers achieve improved accumulation in the ischemic area of stroke model mice, potentiate NSCs recruitment, and increase neurogenesis. This work provides new insights for the treatment of neuronal regeneration after CNS injuries as well as endogenous stem cells, and the click chemistry EV/peptide/chemokine and related nanocarriers for improving human health.

Circadian Clock Regulation via Biomaterials for Nucleus Pulposus.

Circadian clock disorder during tissue degeneration has been considered the potential pathogenesis for various chronic diseases, such as intervertebral disc degeneration (IVDD). In this study, circadian clock-regulating biomaterials (ClockMPs) that can effectively activate the intrinsic circadian clock of nucleus pulposus cells (NPCs) in IVDD and improve the physiological function of NPCs for disc regeneration are fabricated via air-microfluidic technique and the chemical cross-linking between polyvinyl alcohol and modified-phenylboronic acid. In vitro experiments verified that ClockMPs can scavenge reactive oxygen species to maintain a stable microenvironment for the circadian clock by promoting the binding of BMAL1 and CLOCK proteins. ClockMPs can regulate the expression of core circadian clock genes by activating the PI3K-AKT pathway in NPCs to remodel the intrinsic circadian clock and promote extracellular matrix synthesis. Furthermore, in vivo experiments of IVDD treated with ClockMPs proved that ClockMPs can promote disc regeneration by regulating the circadian clock of NPCs. In conclusion, ClockMPs provided a novel and promising strategy for circadian clock regulation during tissue regeneration.

3D Biomimetic Calcified Cartilaginous Callus that Induces Type H Vessels Formation and Osteoclastogenesis.

The formation of a calcified cartilaginous callus (CACC) is crucial during bone repair. CACC can stimulate the invasion of type H vessels into the callus to couple angiogenesis and osteogenesis, induce osteoclastogenesis to resorb the calcified matrix, and promote osteoclast secretion of factors to enhance osteogenesis, ultimately achieving the replacement of cartilage with bone. In this study, a porous polycaprolactone/hydroxyapatite-iminodiacetic acid-deferoxamine (PCL/HA-SF-DFO) 3D biomimetic CACC is developed using 3D printing. The porous structure can mimic the pores formed by the matrix metalloproteinase degradation of the cartilaginous matrix, HA-containing PCL can mimic the calcified cartilaginous matrix, and SF anchors DFO onto HA for the slow release of DFO. The in vitro results show that the scaffold significantly enhances angiogenesis, promotes osteoclastogenesis and resorption by osteoclasts, and enhances the osteogenic differentiation of bone marrow stromal stem cells by promoting collagen triple helix repeat-containing 1 expression by osteoclasts. The in vivo results show that the scaffold significantly promotes type H vessels formation and the expression of coupling factors to promote osteogenesis, ultimately enhancing the regeneration of large-segment bone defects in rats and preventing dislodging of the internal fixation screw. In conclusion, the scaffold inspired by biological bone repair processes effectively promotes bone regeneration.

Visualizable and Lubricating Hydrogel Microspheres Via NanoPOSS for Cartilage Regeneration.

The monitoring of tissue regeneration is particularly important. However, most materials do not allow direct observation of the regeneration process in the cartilage layer. Here, using sulfhydryl polyhedral oligomeric silsesquioxane (POSS-SH) as a nano-construction platform, poly(ethylene glycol) (PEG), Kartogenin (KGN), hydrogenated soya phosphatidylcholine (HSPC), and fluorescein are linked through the "click chemistry" method to construct nanomaterial with fluorescence visualization for cartilage repair: POSS linked with PEG, KGN, HSPC, and fluorescein (PPKHF). PPKHF nanoparticles are encapsulated with hyaluronic acid methacryloyl to prepare PPKHF-loaded microfluidic hyaluronic acid methacrylate spheres (MHS@PPKHF) for in situ injection into the joint cavity using microfluidic technology. MHS@PPKHF forms a buffer lubricant layer in the joint space to reduce friction between articular cartilages, while releasing encapsulated positively charged PPKHF to the deep cartilage through electromagnetic force, facilitating visualization of the location of the drug via fluorescence. Moreover, PPKHF facilitates differentiation of bone marrow mesenchymal stem cells into chondrocytes, which are located in the subchondral bone. In animal experiment, the material accelerates cartilage regeneration while allowing monitoring of cartilage layer repair progression via fluorescence signals. Thus, these POSS-based micro-nano hydrogel microspheres can be used for cartilage regeneration and monitoring and potentially for clinical osteoarthritis therapy.