Oyster mantle-derived exosomes alleviate osteoporosis by regulating bone homeostasis.

Osteoporosis is a major public health problem with an urgent need for safe and effective therapeutic interventions. The process of shell formation in oysters is similar to that of bone formation in mammals, and oyster extracts have been proven to exert osteoprotective effects. Oyster mantle is the most crucial organ regulating shell formation, in which exosomes play an important role. However, the effects of oyster mantle-derived exosomes (OMEs) on mammalian osteoporosis and the underlying mechanisms remain unknown. The OMEs investigated herein was found to carry abundant osteogenic cargos. They could also survive hostile gastrointestinal conditions and accumulate in the bones following oral administration. Moreover, they promoted osteoblastic differentiation and inhibited osteoclastic differentiation simultaneously. Further mechanistic examination revealed that OMEs likely promoted osteogenic activity by activating PI3K/Akt/ß-catenin pathway in osteoblasts and blunted osteoclastic activity by inhibiting NF-κB pathway in osteoclasts. These favorable pro-osteogenic effects of OMEs were also corroborated in a rat femur defect model. Importantly, oral administration of OMEs effectively attenuated bone loss and improved the bone microstructure in ovariectomy-induced osteoporotic mice, and demonstrating excellent biosafety. The mechanistic insights from our data support that OMEs possess promising therapeutic potential against osteoporosis.

Structure driven bio-responsive ability of injectable nanocomposite hydrogels for efficient bone regeneration.

Injectable hydrogels are promising for treatment of bone defects in clinic owing to their minimally invasive procedure. Currently, there is limited emphasis on how to utilize injectable hydrogels to mobilize body's regenerative potential for enhancing bone regeneration. Herein, an injectable bone-mimicking hydrogel (BMH) scaffold assembled from nanocomposite microgel building blocks was developed, in which a highly interconnected microporous structure and an inorganic/organic (methacrylated hydroxyapatite and methacrylated gelatin) interweaved nano structure were well-designed. Compared with hydrogels lacking micro-nano structures or only showing microporous structure, the BMH scaffold enhanced the ingrowth of vessels and promoted the formation of dense cellular networks (including stem cells and M2 macrophages), across the entire scaffold at early stage after subcutaneous implantation. Moreover, the BMH scaffold could not only directly trigger osteogenic differentiation of the infiltrated stem cells, but also provided an instructive osteo-immune microenvironment by inducing macrophages into M2 phenotype. Mechanistically, our results reveal that the nano-rough structure of the BMH plays an essential role in inducing macrophage M2 polarization through activating mechanotransduction related RhoA/ROCK2 pathway. Overall, this work offers an injectable hydrogel with micro-nano structure driven bio-responsive abilities, highlighting harnessing body's inherent regenerative potential to realize bone regeneration.

Gradient matters via filament diameter-adjustable 3D printing.

Gradient matters with hierarchical structures endow the natural world with excellent integrity and diversity. Currently, direct ink writing 3D printing is attracting tremendous interest, and has been used to explore the fabrication of 1D and 2D hierarchical structures by adjusting the diameter, spacing, and angle between filaments. However, it is difficult to generate complex 3D gradient matters owing to the inherent limitations of existing methods in terms of available gradient dimension, gradient resolution, and shape fidelity. Here, we report a filament diameter-adjustable 3D printing strategy that enables conventional extrusion 3D printers to produce 1D, 2D, and 3D gradient matters with tunable heterogeneous structures by continuously varying the volume of deposited ink on the printing trajectory. In detail, we develop diameter-programmable filaments by customizing the printing velocity and height. To achieve high shape fidelity, we specially add supporting layers at needed locations. Finally, we showcase multi-disciplinary applications of our strategy in creating horizontal, radial, and axial gradient structures, letter-embedded structures, metastructures, tissue-mimicking scaffolds, flexible electronics, and time-driven devices. By showing the potential of this strategy, we anticipate that it could be easily extended to a variety of filament-based additive manufacturing technologies and facilitate the development of functionally graded structures.

A mechanical-assisted post-bioprinting strategy for challenging bone defects repair.

Bioprinting that can synchronously deposit cells and biomaterials has lent fresh impetus to the field of tissue regeneration. However, the unavoidable occurrence of cell damage during fabrication process and intrinsically poor mechanical stability of bioprinted cell-laden scaffolds severely restrict their utilization. As such, on basis of heart-inspired hollow hydrogel-based scaffolds (HHSs), a mechanical-assisted post-bioprinting strategy is proposed to load cells into HHSs in a rapid, uniform, precise and friendly manner. HHSs show mechanical responsiveness to load cells within 4 s, a 13-fold increase in cell number, and partitioned loading of two types of cells compared with those under static conditions. As a proof of concept, HHSs with the loading cells show an enhanced regenerative capability in repair of the critical-sized segmental and osteoporotic bone defects in vivo. We expect that this post-bioprinting strategy can provide a universal, efficient, and promising way to promote cell-based regenerative therapy.

3D-bioprinted anisotropic bicellular living hydrogels boost osteochondral regeneration via reconstruction of cartilage-bone interface.

Reconstruction of osteochondral (OC) defects represents an immense challenge due to the need for synchronous regeneration of special stratified tissues. The revolutionary innovation of bioprinting provides a robust method for precise fabrication of tissue-engineered OCs with hierarchical structure; however, their spatial living cues for simultaneous fulfilment of osteogenesis and chondrogenesis to reconstruct the cartilage-bone interface of OC are underappreciated. Here, inspired by natural OC bilayer features, anisotropic bicellular living hydrogels (ABLHs) simultaneously embedding articular cartilage progenitor cells (ACPCs) and bone mesenchymal stem cells (BMSCs) in stratified layers were precisely fabricated via two-channel extrusion bioprinting. The optimum formulation of the 7% GelMA/3% AlgMA hydrogel bioink was demonstrated, with excellent printability at room temperature and maintained high cell viability. Moreover, the chondrogenic ability of ACPCs and the osteogenic ability of BMSCs were demonstrated in vitro, confirming the inherent differential spatial regulation of ABLHs. In addition, ABLHs exhibited satisfactory synchronous regeneration of cartilage and subchondral bone in vivo. Compared with homogeneous hydrogels, the neo-cartilage and neo-bone in ABLHs were augmented by 23.5% and 20.8%, respectively, and more important, a more harmonious cartilage-bone interface was achieved by ABLHs due to their well-tuned cartilage-bone-vessel crosstalk. We anticipate that such a strategy of tissue-mimetic ABLH by means of bioprinting is capable of spatiotemporal cell-driven regeneration, offering insights into the fabrication of anisotropic living materials for the reconstruction of complex organ defects.

Small-molecule amines: a big role in the regulation of bone homeostasis.

Numerous small-molecule amines (SMAs) play critical roles in maintaining bone homeostasis and promoting bone regeneration regardless of whether they are applied as drugs or biomaterials. On the one hand, SMAs promote bone formation or inhibit bone resorption through the regulation of key molecular signaling pathways in osteoblasts/osteoclasts; on the other hand, owing to their alkaline properties as well as their antioxidant and anti-inflammatory features, most SMAs create a favorable microenvironment for bone homeostasis. However, due to a lack of information on their structure/bioactivity and underlying mechanisms of action, certain SMAs cannot be developed into drugs or biomaterials for bone disease treatment. In this review, we thoroughly summarize the current understanding of SMA effects on bone homeostasis, including descriptions of their classifications, biochemical features, recent research advances in bone biology and related regulatory mechanisms in bone regeneration. In addition, we discuss the challenges and prospects of SMA translational research.

Digital light processing-bioprinted poly-NAGA-GelMA-based hydrogel lenticule for precise refractive errors correction.

Refractive disorder is the most prevalent cause of visual impairment worldwide. While treatment of refractive errors can bring improvement to quality of life and socio-economic benefits, there is a need for individualization, precision, convenience, and safety with the chosen method. Herein, we propose using pre-designed refractive lenticules based on poly-NAGA-GelMA (PNG) bio-inks photo-initiated by digital light processing (DLP)-bioprinting for correcting refractive errors. DLP-bioprinting allows PNG lenticules to have individualized physical dimensions with precision achievable to 10µm (µm). Material characteristics of PNG lenticules in tests included optical and biomechanical stability, biomimetical swelling and hydrophilic capability, nutritional and visual functionality, supporting its suitability as stromal implants. Cytocompatibility distinguished by morphology and function of corneal epithelial, stromal, and endothelial cells on PNG lenticules suggested firm adhesion, over 90% viability, phenotypic maintenance instead of excessive keratocyte-myofibroblast transformation.In-vitroimmune response analyzed by illumina RNA sequencing in human peripheral blood mononuclear cells indicated that PNG lenticules activated type-2 immunity, facilitating tissue regeneration and suppressing inflammation.In-vivoperformance assessed using intrastromal keratoplasty models in New Zealand white rabbits illustrated that implantation of PNG lenticules maintained stable optical pathway, induced controlled stromal bio-integration and regeneration, avoided complications such as stromal melt, interface scarring, etc, but exerted no adverse effects on the host. Postoperative follow-up examination on intraocular pressure, corneal sensitivity, and tear production remained unaffected by surgery up to 1-month post-implantation of PNG lenticules. DLP-bioprinted PNG lenticule is a bio-safe and functionally effective stromal implants with customizable physical dimensions, providing potential therapeutic strategies in correction of refractive errors.

Osteoprotegerin deficiency aggravates methionine-choline-deficient diet-induced nonalcoholic steatohepatitis in mice.

Clinical studies have shown that osteoprotegerin (OPG) is reduced in patients with nonalcoholic steatohepatitis (NASH), but the underlying mechanisms are unclear. The current study focuses on the role of OPG in the NASH pathogenesis. OPG knockout mice and wild-type control mice fed a methionine choline-deficient diet (MCD) for 4 weeks resulted in an animal model of NASH. Measurement of triglycerides (TG) in serum and liver to assess steatosis. Hematoxylin eosin (HE), Sirius Red and Masson staining were used to assess the liver damage. Transcriptome sequencing analysis, qPCR and western blot were to analyze changes in lipid metabolism and inflammation-related indicators in the liver. In vivo knockout of OPG resulted in a reduction of TG levels in the liver and a significant increase in serum ALT and AST. The expression of inflammatory factors and fibrosis genes was significantly upregulated in the livers of OPG knockout mice. Transcriptome sequencing analysis showed that OPG knockout significantly enhanced MCD diet-induced activation of the mitogen-activated protein kinase (MAPK) signaling pathway. Mechanistically, OPG may inhibit MAPK signaling pathway activity by upregulating the expression of dual specificity phosphatase 14 (DUSP14), thereby reducing inflammatory injury. OPG could regulate the activity of the MAPK signaling pathway via DUSP14, thus regulating the expression of some inflammatory factors in NASH, it may be a promising target for the treatment of NASH.

Synthetic Extracellular Matrices for 3D Culture of Schwann Cells, Hepatocytes, and HUVECs.

Synthetic hydrogels from polyisocyanides (PIC) are a type of novel thermoreversible biomaterials, which can covalently bind biomolecules such as adhesion peptides to provide a suitable extracellular matrix (ECM)-like microenvironment for different cells. Although we have demonstrated that PIC is suitable for three-dimensional (3D) culture of several cell types, it is unknown whether this hydrogel sustains the proliferation and passaging of cells originating from different germ layers. In the present study, we propose a 3D culture system for three representative cell sources: Schwann cells (ectoderm), hepatocytes (endoderm), and endothelial cells (mesoderm). Both Schwann cells and hepatocytes proliferated into multicellular spheroids and maintained their properties, regardless of the amount of cell-adhesive RGD motifs in long-term culture. Notably, Schwann cells grew into larger spheroids in RGD-free PIC than in PIC-RGD, while HL-7702 showed the opposite behavior. Endothelial cells (human umbilical vein endothelial cells, HUVECs) spread and formed an endothelial cell (EC) network only in PIC-RGD. Moreover, in a hepatocyte/HUVEC co-culture system, the characteristics of both cells were well kept for a long period in PIC-RGD. In all, our work highlights a simple ECM mimic that supports the growth and phenotype maintenance of cells from all germ layers in the long term. Our findings might contribute to research on biological development, organoid engineering, and in vitro drug screening.

In-situmineralized homogeneous collagen-based scaffolds for potential guided bone regeneration.

For guided bone regeneration (GBR) in clinical orthopedics, the importance of a suitable scaffold which can provide the space needed for bone regeneration and simultaneously promotes the new bone formation cannot be overemphasized. Due to its excellent biocompatibility, mechanical strength, and similarity in structure and composition to natural bone, the mineralized collagen-based scaffolds have been increasingly considered as promising GBR scaffolds. Herein, we propose a novel method to fabricate anin-situmineralized homogeneous collagen-based scaffold (IMHCS) with excellent osteogenic capability for GBR by electrospinning the collagen solution in combination with essential mineral ions. The IMHCS exhibited homogeneous distribution of apatite crystals in electrospun fibers, which helped to achieve a significantly higher tensile strength than the pure collagen scaffold (CS) and the scaffold with directly added nano-hydroxyapatite particles (HAS). Furthermore, the IMHCS had significantly better cell compatibility, cell migration ratio, and osteogenic differentiation property than the HAS and CS. Therefore, the IMHCS not only retains traditional function of inhibiting fibroblast invasion, but also possesses excellent osteogenic differentiation property, indicating a robust alternative for GBR applications.

In-situ formed elastin-based hydrogels enhance wound healing via promoting innate immune cells recruitment and angiogenesis.

Harnessing the inflammation and angiogenesis is extremely important in wound healing. In this study, we developed bioactive elastin-based hydrogels which can recruit and modulate the innate immune cells and accelerate angiogenesis in the wound site and subsequently improve wound regeneration. These hydrogels were formed by visible-light cross-linking of acryloyl-(polyethylene glycol)-N-hydroxysuccinimide ester modified elastin with methacrylated gelatin, in order to mimic dermal microenvironment. These hydrogels showed highly tunable mechanical properties, swelling ratios and enzymatic degradation profiles, with moduli within the range of human skin. To mimic the in vivo degradation of the elastin by elastase from neutrophils, in vitro co-culture of the hydrogels and neutrophils was conducted. The derived conditioned medium containing elastin derived peptides (EDP-conditioned medium) promoted the expression of both M1 and M2 markers in M1 macrophages in vitro. Additionally, the EDP-conditioned medium induced superior tube formation of endothelia cells in Matrigel. In mice wound model, these elastin-based hydrogels attracted abundant neutrophils and predominant M2 macrophages to the wound and supported their infiltration into the hydrogels. The outstanding immunomodulatory effect of the elastin-based hydrogels resulted in superior angiogenesis, collagen deposition and dermal regeneration. Hence, these elastin-based hydrogels can be a promising regenerative platform to accelerate wound repair.

Progranulin regulates the development and function of NKT2 cells through EZH2 and PLZF.

T helper 2 (Th2) cytokine production by invariant natural killer T (iNKT) cells is involved in the development of asthma, but the regulation of Th2 cytokines in iNKT cells remains unknown. Although it is known that progranulin (PGRN) induces the production of Th2 cytokines in iNKT cells in vivo, the underlying mechanism is not clear. This study aims to investigate the role of PGRN in iNKT cells. The effects of PGRN on the differentiation of iNKT cells was detected by flow cytometry. Then stimulation of iNKT cells and airway resistance were carried out to evaluate the function of PGRN on iNKT cells. Furthermore, the mechanisms of PGRN in regulating iNKT cells was investigated by RT-PCR, WB, confocal and luciferase reporter assays. The absolute number of iNKT cells decreased in PGRN KO mice despite an increase in the percentage of iNKT cells. Furthermore, analyzing the subsets of iNKT cells, we found that NKT2 cells and their IL-4 production were reduced. Mechanistically, the decrease in NKT2 cells in the PGRN KO mice was caused by increased expression of enhancer of zeste homolog 2 (EZH2), that in turn caused increased degradation and altered nuclear localization of PLZF. Interestingly, PGRN signaling decreased expression of EZH2 and treatment of the PGRN KO mice with the EZH2 specific inhibitor GSK343 rescued the defect in NKT2 differentiation, IL-4 generation, and PLZF expression. Altogether, We have revealed a new pathway (PGRN-EZH2-PLZF), which regulates the Th2 responses of iNKT cells and provides a potentially new target for asthma treatment.

An optogenetic approach for regulating human parathyroid hormone secretion.

Parathyroid hormone (PTH) plays crucial role in maintaining calcium and phosphorus homeostasis. In the progression of secondary hyperparathyroidism (SHPT), expression of calcium-sensing receptors (CaSR) in the parathyroid gland decreases, which leads to persistent hypersecretion of PTH. How to precisely manipulate PTH secretion in parathyroid tissue and underlying molecular mechanism is not clear. Here, we establish an optogenetic approach that bypasses CaSR to inhibit PTH secretion in human hyperplastic parathyroid cells. We found that optogenetic stimulation elevates intracellular calcium, inhibits both PTH synthesis and secretion in human parathyroid cells. Long-term pulsatile PTH secretion induced by light stimulation prevented hyperplastic parathyroid tissue-induced bone loss by influencing the bone remodeling in mice. The effects are mediated by light stimulation of opsin expressing parathyroid cells and other type of cells in parathyroid tissue. Our study provides a strategy to regulate release of PTH and associated bone loss of SHPT through an optogenetic approach.

Development of Graphene-Based Materials in Bone Tissue Engineaering.

Bone regeneration-related graphene-based materials (bGBMs) are increasingly attracting attention in tissue engineering due to their special physical and chemical properties. The purpose of this review is to quantitatively analyze mass academic literature in the field of bGBMs through scientometrics software CiteSpace, to demonstrate the rules and trends of bGBMs, thus to analyze and summarize the mechanisms behind the rules, and to provide clues for future research. First, the research status, hotspots, and frontiers of bGBMs are analyzed in an intuitively and vividly visualized way. Next, the extracted important subjects such as fabrication techniques, cytotoxicity, biodegradability, and osteoinductivity of bGBMs are presented, and the different mechanisms, in turn, are also discussed. Finally, photothermal therapy, which is considered an emerging area of application of bGBMs, is also presented. Based on this approach, this work finds that different studies report differing opinions on the biological properties of bGBMS due to the lack of consistency of GBMs preparation. Therefore, it is necessary to establish more standards in fabrication, characterization, and testing for bGBMs to further promote scientific progress and clinical translation.

Cell-matrix reciprocity in 3D culture models with nonlinear elasticity.

Three-dimensional (3D) matrix models using hydrogels are powerful tools to understand and predict cell behavior. The interactions between the cell and its matrix, however is highly complex: the matrix has a profound effect on basic cell functions but simultaneously, cells are able to actively manipulate the matrix properties. This (mechano)reciprocity between cells and the extracellular matrix (ECM) is central in regulating tissue functions and it is fundamentally important to broadly consider the biomechanical properties of the in vivo ECM when designing in vitro matrix models. This manuscript discusses two commonly used biopolymer networks, i.e. collagen and fibrin gels, and one synthetic polymer network, polyisocyanide gel (PIC), which all possess the characteristic nonlinear mechanics in the biological stress regime. We start from the structure of the materials, then address the uses, advantages, and limitations of each material, to provide a guideline for tissue engineers and biophysicists in utilizing current materials and also designing new materials for 3D cell culture purposes.

Advances in 3D bioprinting technology for functional corneal reconstruction and regeneration.

Corneal transplantation constitutes one of the major treatments in severe cases of corneal diseases. The lack of cornea donors as well as other limitations of corneal transplantation necessitate the development of artificial corneal substitutes. Biosynthetic cornea model using 3D printing technique is promising to generate artificial corneal structure that can resemble the structure of the native human cornea and is applicable for regenerative medicine. Research on bioprinting artificial cornea has raised interest into the wide range of materials and cells that can be utilized as bioinks for optimal clarity, biocompatibility, and tectonic strength. With continued advances in biomaterials science and printing technology, it is believed that bioprinted cornea will eventually achieve a level of clinical functionality and practicality as to replace donated corneal tissues, with their associated limitations such as limited or unsteady supply, and possible infectious disease transmission. Here, we review the literature on bioprinting strategies, 3D corneal modelling, material options, and cellularization strategies in relation to keratoprosthesis design. The progress, limitations and expectations of recent cases of 3D bioprinting of artifial cornea are discussed. An outlook on the rise of 3D bioprinting in corneal reconstruction and regeneration is provided.

Fractal Design Boosts Extrusion-Based 3D Printing of Bone-Mimicking Radial-Gradient Scaffolds.

Although extrusion-based three-dimensional (EB-3D) printing technique has been widely used in the complex fabrication of bone tissue-engineered scaffolds, a natural bone-like radial-gradient scaffold by this processing method is of huge challenge and still unmet. Inspired by a typical fractal structure of Koch snowflake, for the first time, a fractal-like porous scaffold with a controllable hierarchical gradient in the radial direction is presented via fractal design and then implemented by EB-3D printing. This radial-gradient structure successfully mimics the radially gradual decrease in porosity of natural bone from cancellous bone to cortical bone. First, we create a design-to-fabrication workflow with embedding the graded data on basis of fractal design into digital processing to instruct the extrusion process of fractal-like scaffolds. Further, by a combination of suitable extruded inks, a series of bone-mimicking scaffolds with a 3-iteration fractal-like structure are fabricated to demonstrate their superiority, including radial porosity, mechanical property, and permeability. This study showcases a robust strategy to overcome the limitations of conventional EB-3D printers for the design and fabrication of functionally graded scaffolds, showing great potential in bone tissue engineering.

3D-bioprinted BMSC-laden biomimetic multiphasic scaffolds for efficient repair of osteochondral defects in an osteoarthritic rat model.

Osteochondral defect repair in osteoarthritis (OA) remains an unsolved clinical problem due to the lack of enough seed cells in the defect and chronic inflammation in the joint. To address this clinical need, we designed a bone marrow-derived mesenchymal stem cell (BMSC)-laden 3D-bioprinted multilayer scaffold with methacrylated hyaluronic acid (MeHA)/polycaprolactone incorporating kartogenin and ß-TCP for osteochondral defect repair within each region. BMSC-laden MeHA was designed to actively introduce BMSCs in situ, and diclofenac sodium (DC)-incorporated matrix metalloproteinase-sensitive peptide-modified MeHA was induced on the BMSC-laden scaffold as an anti-inflammatory strategy. BMSCs in the scaffolds survived, proliferated, and produced large amounts of cartilage-specific extracellular matrix in vitro. The effect of BMSC-laden scaffolds on osteochondral defect repair was investigated in an animal model of medial meniscectomy-induced OA. BMSC-laden scaffolds facilitated chondrogenesis by promoting collagen II and suppressed interleukin 1ß in osteochondral defects of the femoral trochlea. Congruently, BMSC-laden scaffolds significantly improved joint function of the injured leg with respect to the ground support force, paw grip force, and walk gait parameters. Therefore, this research demonstrates the potential of 3D-bioprinted BMSC-laden scaffolds to simultaneously inhibit joint inflammation and promote cartilage defect repair in OA joints.

AMPK Signaling in Energy Control, Cartilage Biology, and Osteoarthritis.

The adenosine monophosphate (AMP)-activated protein kinase (AMPK) was initially identified as an enzyme acting as an "energy sensor" in maintaining energy homeostasis via serine/threonine phosphorylation when low cellular adenosine triphosphate (ATP) level was sensed. AMPK participates in catabolic and anabolic processes at the molecular and cellular levels and is involved in appetite-regulating circuit in the hypothalamus. AMPK signaling also modulates energy metabolism in organs such as adipose tissue, brain, muscle, and heart, which are highly dependent on energy consumption via adjusting the AMP/ADP:ATP ratio. In clinics, biguanides and thiazolidinediones are prescribed to patients with metabolic disorders through activating AMPK signaling and inhibiting complex I in the mitochondria, leading to a reduction in mitochondrial respiration and elevated ATP production. The role of AMPK in mediating skeletal development and related diseases remains obscure. In this review, in addition to discuss the emerging advances of AMPK studies in energy control, we will also illustrate current discoveries of AMPK in chondrocyte homeostasis, osteoarthritis (OA) development, and the signaling interaction of AMPK with other pathways, such as mTOR (mechanistic target of rapamycin), Wnt, and NF-κB (nuclear factor κB) under OA condition.