Signal transduction at GPCRs: Allosteric activation of the ERK MAPK by ß-arrestin.

ß-arrestins are multivalent adaptor proteins that bind active phosphorylated G protein-coupled receptors (GPCRs) to inhibit G protein signaling, mediate receptor internalization, and initiate alternative signaling events. ß-arrestins link agonist-stimulated GPCRs to downstream signaling partners, such as the c-Raf-MEK1-ERK1/2 cascade leading to ERK1/2 activation. ß-arrestins have been thought to transduce signals solely via passive scaffolding by facilitating the assembly of multiprotein signaling complexes. Recently, however, ß-arrestin 1 and 2 were shown to activate two downstream signaling effectors, c-Src and c-Raf, allosterically. Over the last two decades, ERK1/2 have been the most intensely studied signaling proteins scaffolded by ß-arrestins. Here, we demonstrate that ß-arrestins play an active role in allosterically modulating ERK kinase activity in vitro and within intact cells. Specifically, we show that ß-arrestins and their GPCR-mediated active states allosterically enhance ERK2 autophosphorylation and phosphorylation of a downstream ERK2 substrate, and we elucidate the mechanism by which ß-arrestins do so. Furthermore, we find that allosteric stimulation of dually phosphorylated ERK2 by active-state ß-arrestin 2 is more robust than by active-state ß-arrestin 1, highlighting differential capacities of ß-arrestin isoforms to regulate effector signaling pathways downstream of GPCRs. In summary, our study provides strong evidence for a new paradigm in which ß-arrestins function as active "catalytic" scaffolds to allosterically unlock the enzymatic activity of signaling components downstream of GPCR activation.

A facile Immunoregulatory Constructional Design by Proanthocyanidin Optimizing Directional Chitosan Microchannel.

Improving the interconnected structure and bioregulatory function of natural chitosan is beneficial for optimizing its performance in bone regeneration. Here, a facile immunoregulatory constructional design is proposed for developing instructive chitosan by directional freezing and alkaline salting out. The molecular dynamics simulation confirmed the assembly kinetics and structural features of various polyphenols and chitosan molecules. Along with the in vitro anti-inflammatory, antioxidative, promoting bone mesenchymal stem cell (BMSC) adhesion and proliferation performance, proanthocyanidin optimizing chitosan (ChiO) scaffold presented an optimal immunoregulatory structure with the directional microchannel. Transcriptome analysis in vitro further revealed the cytoskeleton- and immune-regulation effect of ChiO are the key mechanism of action on BMSC. The rabbit cranial defect model (Φ = 10 mm) after 12 weeks of implantation confirmed the significantly enhanced bone reconstitution. This facile immunoregulatory directional microchannel design provides effective guidance for developing inducible chitosan scaffolds.

Nucleation Domains in Biomineralization: Biomolecular Sequence and Conformational Features.

Biomolecules play a vital role in the regulation of biomineralization. However, the characteristics of practical nucleation domains are still sketchy. Herein, the effects of the representative biomolecular sequence and conformations on calcium phosphate (Ca-P) nucleation and mineralization are investigated. The results of computer simulations and experiments prove that the line in the arrangement of dual acidic/essential amino acids with a single interval (Bc (Basic) -N (Neutral) -Bc-N-Ac (Acidic)- NN-Ac-N) is most conducive to the nucleation. 2α-helix conformation can best induce Ca-P ion cluster formation and nucleation. "Ac- × × × -Bc" sequences with α-helix are found to be the features of efficient nucleation domains, in which process, molecular recognition plays a non-negligible role. It further indicates that the sequence determines the potential of nucleation/mineralization of biomolecules, and conformation determines the ability of that during functional execution. The findings will guide the synthesis of biomimetic mineralized materials with improved performance for bone repair.

Advancements in Photothermal Therapy Using Near-Infrared Light for Bone Tumors.

Bone tumors, particularly osteosarcoma, are prevalent among children and adolescents. This ailment has emerged as the second most frequent cause of cancer-related mortality in adolescents. Conventional treatment methods comprise extensive surgical resection, radiotherapy, and chemotherapy. Consequently, the management of bone tumors and bone regeneration poses significant clinical challenges. Photothermal tumor therapy has attracted considerable attention owing to its minimal invasiveness and high selectivity. However, key challenges have limited its widespread clinical use. Enhancing the tumor specificity of photosensitizers through targeting or localized activation holds potential for better outcomes with fewer adverse effects. Combinations with chemotherapies or immunotherapies also present avenues for improvement. In this review, we provide an overview of the most recent strategies aimed at overcoming the limitations of photothermal therapy (PTT), along with current research directions in the context of bone tumors, including (1) target strategies, (2) photothermal therapy combined with multiple therapies (immunotherapies, chemotherapies, and chemodynamic therapies, magnetic, and photodynamic therapies), and (3) bifunctional scaffolds for photothermal therapy and bone regeneration. We delve into the pros and cons of these combination methods and explore current research focal points. Lastly, we address the challenges and prospects of photothermal combination therapy.

Hydrophobic Tetracycline Immobilized in Fibrous Hyaluronan Regulates Adhesive Collagen-Based Hydrogel Stability for Infected Wound Healing.

Collagen-based hydrogels have a significant impact on wound healing, but they suffer from structural instability and bacterial invasion in infected wounds. Here, electrospun nanofibers of esterified hyaluronan (HA-Bn/T) are developed to immobilize the hydrophobic antibacterial drug tetracycline by π-π stacking interaction. Dopamine-modified hyaluronan and HA-Bn/T are employed simultaneously to stabilize the structure of collagen-based hydrogel by chemically interweaving the collagen fibril network and decreasing the rate of collagen degradation. This renders it injectable for in situ gelation, with suitable skin adhesion properties and long-lasting drug release capability. This hybridized interwoven hydrogel promotes the proliferation and migration of L929 cells and vascularization in vitro. It presents satisfactory antibacterial ability against Staphylococcus aureus and Escherichia coli. The structure also retains the functional protein environment provided by collagen fiber, inhibits the bacterial environment of infected wounds, and modulates local inflammation, resulting in neovascularization, collagen deposition, and partial follicular regeneration. This strategy offers a new solution for infected wound healing.

Construction of Microfluidic Chip Structure for Cell Migration Studies in Bioactive Ceramics.

Cell migration is an essential bioactive ceramics property and critical for bone induction, clinical application, and mechanism research. Standardized cell migration detection methods have many limitations, including a lack of dynamic fluid circulation and the inability to simulate cell behavior in vivo. Microfluidic chip technology, which mimics the human microenvironment and provides controlled dynamic fluid cycling, has the potential to solve these questions and generate reliable models of cell migration in vitro. In this study, a microfluidic chip is reconstructed to integrate the bioactive ceramic into the microfluidic chip structure to constitute a ceramic microbridge microfluidic chip system. Migration differences in the chip system are measured. By combining conventional detection methods with new biotechnology to analyze the causes of cell migration differences, it is found that the concentration gradients of ions and proteins adsorbed on the microbridge materials are directly related to the occurrence of cell migration behavior, which is consistent with previous reports and demonstrates the effectiveness of the microfluidic chip model. This model provides in vivo environment simulation and controllability of input and output conditions superior to standardized cell migration detection methods. The microfluidic chip system provides a new approach to studying and evaluating bioactive ceramics.

Covalently Grafted Biomimetic Matrix Reconstructs the Regenerative Microenvironment of the Porous Gradient Polycaprolactone Scaffold to Accelerate Bone Remodeling.

Integrating a biomimetic extracellular matrix to improve the microenvironment of 3D printing scaffolds is an emerging strategy for bone substitute design. Here, a "soft-hard" bone implant (BM-g-DPCL) consisting of a bioactive matrix chemically integrated on a polydopamine (PDA)-coated porous gradient scaffold by polyphenol groups is constructed. The PDA-coated "hard" scaffolds promoted Ca2+ chelation and mineral deposition; the "soft" bioactive matrix is beneficial to the migration, proliferation, and osteogenic differentiation of stem cells in vitro, accelerated endogenous stem cell recruitment, and initiated rapid angiogenesis in vivo. The results of the rabbit cranial defect model (Φ = 10 mm) confirmed that BM-g-DPCL promoted the integration between bone tissue and implant and induced the deposition of bone matrix. Proteomics confirmed that cytokine adhesion, biomineralization, rapid vascularization, and extracellular matrix formation are major factors that accelerate bone defect healing. This strategy of highly chemically bonded soft-hard components guided the construction of the bioactive regenerative scaffold.

Electrospun Hyaluronan Nanofiber Membrane Immobilizing Aromatic Doxorubicin as Therapeutic and Regenerative Biomaterial.

Lesioned tissue requires synchronous control of disease and regeneration progression after surgery. It is necessary to develop therapeutic and regenerative scaffolds. Here, hyaluronic acid (HA) was esterified with benzyl groups to prepare hyaluronic acid derivative (HA-Bn) nanofibers via electrospinning. Electrospun membranes with average fiber diameters of 407.64 ± 124.8 nm (H400), 642.3 ± 228.76 nm (H600), and 841.09 ± 236.86 nm (H800) were obtained by adjusting the spinning parameters. These fibrous membranes had good biocompatibility, among which the H400 group could promote the proliferation and spread of L929 cells. Using the postoperative treatment of malignant skin melanoma as an example, the anticancer drug doxorubicin (DOX) was encapsulated in nanofibers via hybrid electrospinning. The UV spectroscopy of DOX-loaded nanofibers (HA-DOX) revealed that DOX was successfully encapsulated, and there was a π-π interaction between aromatic DOX and HA-Bn. The drug release profile confirmed the sustained release of about 90%, achieved within 7 days. In vitro cell experiments proved that the HA-DOX nanofiber had a considerable inhibitory effect on B16F10 cells. Therefore, the HA-Bn electrospun membrane could facilitate the potential regeneration of injured skin tissues and be incorporated with drugs to achieve therapeutic effects, offering a powerful approach to developing therapeutic and regenerative biomaterial.

[Role of Stem Cells and Their Biomimetic Matrix Microenvironment in Regenerative Repair of Articular Cartilage: A Review].

It is difficult for the articular cartilage to self-heal any damage it may incur due to its lack of nerves and blood vessels. Development in stem cell technology provides new prospects for articular cartilage regeneration. Currently, stem cells from different sources and their diverse applications have demonstrated different degrees of therapeutic effect and potential in articular cartilage repair. However, stem cells are highly sensitive to their microenvironment. Therefore, more and more researchers are focusing their attention on regulating stem cells and thus accelerating cartilage regeneration through the biomimetic microenvironment constructed by biologically functional scaffolds. We reviewed in this paper the sources of the stem cells used for cartilage repair, the application method of these stem cells, as well as the therapeutic effect, mechanism and limitations in the application of stem cells synergizing with the biomimetic microenvironment in promoting articular cartilage repair and regeneration. We hoped to provide suggestions for practical clinical research in the design and improvement of biofunctional cartilage repair scaffolds that synergize with stem cells.

Cellulose Nanocrystal Reinforced Collagen-Based Nanocomposite Hydrogel with Self-Healing and Stress-Relaxation Properties for Cell Delivery.

While injectable in situ cross-linking collagen hydrogels offer great potential for applying stem cell therapy to regenerate articular cartilage via minimally invasive procedures, the encapsulated cells experience high shear stress during injection, which results in limited cell survival. In this study, surface-modified cellulose nanocrystals (CNCs) have been investigated as green and biocompatible reinforcing agents for collagen hydrogel. Aldehyde-functionalized CNCs (a-CNCs) were produced through a facile one-pot oxidation. A nanocomposite a-CNC/collagen hydrogel cross-linked rapidly by dynamic Schiff base bonds based on a-CNCs and collagen under physiological conditions. The a-CNC/collagen hydrogel exhibited fast shear-thinning, self-healing characteristics, and improved elastic modulus compared with CNC/collagen hydrogel without Schiff base bonds. The a-CNC/collagen hydrogel was then investigated for mesenchymal stem cell (MSC) delivery. MSCs encapsulated in the a-CNC/collagen hydrogel showed high cell viability after extrusion in vitro. Subcutaneous injection of MSCs encapsulated in the a-CNC/collagen hydrogel showed improved implant integrity and higher cell retention. The proposed self-healing collagen-based hydrogel would not only protect cells during injection but also fit into the irregular cartilage defect, thus holding promise in delivering MSCs for cartilage regeneration through minimally invasive procedures.

Bioactive scaffolds based on collagen filaments with tunable physico-chemical and biological features.

Native tissues such as nerve bundles, blood vessels and tendons have extracellular matrices with a characteristic linear orientation, which cannot be fully achieved with the current technology for the development of regenerative biomaterials. In this study, bioactive and oriented collagen filaments have been fabricated using a combination of wet-spinning and carbodiimide-based crosslinking. The wet-spinning techniques, including extrusion and collection rates, and their influences on collagen filaments were studied and optimized. The diameter of the attained collagen filaments can be adjusted ranging from 30 µm to 650 µm. Further characterizations, such as circular dichroism, scanning electron microscopy, small-angle X-ray scattering and Fourier transform infrared spectra analysis, showed that the native structure of the collagen was greatly preserved after the filament preparation process. The measurements of weight swelling ratio and degradation rate indicate that the crosslinking method can efficiently regulate the physico-chemical properties of collagen filaments, including water absorption and degradation behaviors. In particular, the mechanical strength of collagen filaments can be greatly improved via crosslinking. In addition, cells can adhere and spread on collagen filaments in well-aligned patterns, showing appropriate biological features. It can be concluded that the bioactive collagen filaments with tunable properties are preferable for developing tissue engineering scaffolds with characteristic orientation features. With further study of the interactions between collagen filaments and cells, this work may shed light on the development of collagen based biomaterials that would be beneficial in the field of tissue engineering.

MutL traps MutS at a DNA mismatch.

DNA mismatch repair (MMR) identifies and corrects errors made during replication. In all organisms except those expressing MutH, interactions between a DNA mismatch, MutS, MutL, and the replication processivity factor (ß-clamp or PCNA) activate the latent MutL endonuclease to nick the error-containing daughter strand. This nick provides an entry point for downstream repair proteins. Despite the well-established significance of strand-specific nicking in MMR, the mechanism(s) by which MutS and MutL assemble on mismatch DNA to allow the subsequent activation of MutL's endonuclease activity by ß-clamp/PCNA remains elusive. In both prokaryotes and eukaryotes, MutS homologs undergo conformational changes to a mobile clamp state that can move away from the mismatch. However, the function of this MutS mobile clamp is unknown. Furthermore, whether the interaction with MutL leads to a mobile MutS-MutL complex or a mismatch-localized complex is hotly debated. We used single molecule FRET to determine that Thermus aquaticus MutL traps MutS at a DNA mismatch after recognition but before its conversion to a sliding clamp. Rather than a clamp, a conformationally dynamic protein assembly typically containing more MutL than MutS is formed at the mismatch. This complex provides a local marker where interaction with ß-clamp/PCNA could distinguish parent/daughter strand identity. Our finding that MutL fundamentally changes MutS actions following mismatch detection reframes current thinking on MMR signaling processes critical for genomic stability.

In vivo immunological properties research on mesenchymal stem cells based engineering cartilage by a dialyzer pocket model.

As the seed cells, the immune properties of the mesenchymal stem cells are important for the tissue engineering restoring effect. But the in vivo research model is lacking. In the study, based on a dialyzer pocket model, changes in immunological properties and the differentiation of seeded mesenchymal stem cells (MSCs) in collagen hydrogel were studied in muscle and articular cavity implantation, respectively. The results showed that collagen hydrogel can induce MSCs to form cartilage tissue, followed by alteration of immunological properties. In muscle implantation, relatively low expression of major histocompatibility complex (MHC) molecules and low level of one-way mixed lymphocyte reactions (MLR) on the seeded MSCs were observed, but only a little cartilage tissue formed. In articular cavity implantation, more cartilage tissue formed, but higher MHC expressions and MLR level were found. Results indicated that the immunomodulation and the cartilage formation of the seeded MSCs will be impacted by the scaffold and the environment of the in vivo implanted site. The dialyzer pocket model can be used for the in vivo research for the MSC-based strategy of the tissue engineering, especially for the optimization of the immunomodulation.

Bio-Functional Design, Application and Trends in Metallic Biomaterials.

Introduction of metals as biomaterials has been known for a long time. In the early development, sufficient strength and suitable mechanical properties were the main considerations for metal implants. With the development of new generations of biomaterials, the concepts of bioactive and biodegradable materials were proposed. Biological function design is very import for metal implants in biomedical applications. Three crucial design criteria are summarized for developing metal implants: (1) mechanical properties that mimic the host tissues; (2) sufficient bioactivities to form bio-bonding between implants and surrounding tissues; and (3) a degradation rate that matches tissue regeneration and biodegradability. This article reviews the development of metal implants and their applications in biomedical engineering. Development trends and future perspectives of metallic biomaterials are also discussed.

Surface Structure of Hydroxyapatite from Simulated Annealing Molecular Dynamics Simulations.

The surface structure of hydroxyapatite (HAP) is crucial for its bioactivity. Using a molecular dynamics simulated annealing method, we studied the structure and its variation with annealing temperature of the HAP (100) surface. In contrast to the commonly used HAP surface model, which is sliced from HAP crystal and then relaxed at 0 K with first-principles or force-field calculations, a new surface structure with gradual changes from ordered inside to disordered on the surface was revealed. The disordering is dependent on the annealing temperature, Tmax. When Tmax increases up to the melting point, which was usually adopted in experiments, the disordering increases, as reflected by its radial distribution functions, structural factors, and atomic coordination numbers. The disordering of annealed structures does not show significant changes when Tmax is above the melting point. The thickness of disordered layers is about 10 Å. The surface energy of the annealed structures at high temperature is significantly less than that of the crystal structure relaxed at room temperature. A three-layer model of interior, middle, and surface was then proposed to describe the surface structure of HAP. The interior layer retains the atomic configurations in crystal. The middle layer has its atoms moved and its groups rotated about their original locations. In the surface layer, the atomic arrangements are totally different from those in crystal. In particular for the hydroxyl groups, they move outward and cover the Ca(2+) ions, leaving holes occupied by the phosphate groups. Our study suggested a new model with disordered surface structures for studying the interaction of HAP-based biomaterials with other molecules.

Influences of the steam sterilization on the properties of calcium phosphate porous bioceramics.

The influences of steam sterilization on the physicochemical properties of calcium phosphate (Ca-P) porous bioceramics, including ß-tricalcium phosphate (ß-TCP), biphasic calcium phosphate (BCP) and hydroxyapatite (HA) are investigated. After being steam sterilized in an autoclave (121 °C for 40 min), the porous bioceramics are dried and characterized. The steam sterilization has no obvious effects on the phase composition, thermal stability, pH value and dissolubility of ß-TCP porous bioceramic, but changes its morphology and mechanical strength. Meanwhile, the steam sterilization leads to the significant changes of the morphology, phase composition, pH value and dissolubility of BCP porous bioceramic. The increase of dissolubility and mechanical strength, the decrease of pH value of the immersed solution and partial oriented growth of crystals are also observed in HA porous bioceramic after steam sterilization. These results indicate that the steam sterilization can result in different influences on the physicochemical properties of ß-TCP, BCP and HA porous bioceramics, thus the application of the steam sterilization on the three kinds of Ca-P porous bioceramics should be considered carefully based on the above changed properties.

Novel synthesis strategy for composite hydrogel of collagen/hydroxyapatite-microsphere originating from conversion of CaCO3 templates.

Inspired by coralline-derived hydroxyapatite, we designed a methodological route to synthesize carbonated-hydroxyapatite microspheres from the conversion of CaCO3 spherulite templates within a collagen matrix under mild conditions and thus constructed the composite hydrogel of collagen/hydroxyapatite-microspheres. Fourier transform infrared spectroscopy (FTIR) and x-ray diffraction (XRD) were employed to confirm the successful generation of the carbonated hydroxyapatite phase originating from CaCO3, and the ratios of calcium to phosphate were tracked over time. Variations in the weight portion of the components in the hybrid gels before and after the phase transformation of the CaCO3 templates were identified via thermogravimetric analysis (TGA). Scanning electron microscopy (SEM) shows these composite hydrogels have a unique multiscale microstructure consisting of a collagen nanofibril network and hydroxyapatite microspheres. The relationship between the hydroxyapatite nanocrystals and the collagen fibrils was revealed by transmission electron microscopy (TEM) in detail, and the selected area electron diffraction (SAED) pattern further confirmed the results of the XRD analyses which show the typical low crystallinity of the generated hydroxyapatite. This smart synthesis strategy achieved the simultaneous construction of microscale hydroxyapatite particles and collagen fibrillar hydrogel, and appears to provide a novel route to explore an advanced functional hydrogel materials with promising potentials for applications in bone tissue engineering and reconstruction medicine.

[Inhibitory Effect of Hydroxyapatite Particles with Different Size on Malignant Melanoma A375 Cells: A Preliminary Study].

To explore the inhibitory effect of hydroxyapatite (HA) particles with different sizes on malignant melanoma A375 cells in vitro, we synthesized 4 short rod-like HA particles using TIPS. Their mean diameters were 998.0 nm (HA1), 511.0 nm (HA2), 244.0 nm (HA3), and 71.6 nm (HA4), respectively. Malignant melanoma A375 cells were co-cultured with HA particles in vitro. Results showed that HA particles smaller than 511.0 nm in mean diameter could always inhibit proliferation of A375 cells, and nanometer-HA particles (HA4) had the strongest inhibitory effect on A375 cell proliferation and the strongest inducing effect on apoptosis. HA particles were distributed in plasma of A375 cells. The ultrastructure changes of A375 cells were found most significant in nanometer-HA particles (HA4) group. We conclude that particle size is a very important influencing factor on anti-tumor effects of HA and that nanometer-HA particle has the strongest inhibitory effect on tumor cell proliferation.

Nano-hydroxyapatite promotes cell apoptosis by co-activating endoplasmic reticulum stress and mitochondria damage to inhibit glioma growth.

Despite a growing body of studies demonstrating the specific anti-tumor effect of nano-hydroxyapatite (n-HA), the underlying mechanism remained unclear. Endoplasmic reticulum (ER) and mitochondria are two key players in intracellular Ca2+ homeostasis and both require Ca2+ to participate. Moreover, the ER-mitochondria interplay coordinates the maintenance of cellular Ca2+ homeostasis to prevent any negative consequences from excess of Ca2+, hence there needs in-depth study of n-HA effect on them. In this study, we fabricated needle-like n-HA to investigate the anti-tumor effectiveness as well as the underlying mechanisms from cellular and molecular perspectives. Data from in vitro experiments indicated that the growth and invasion of glioma cells were obviously reduced with the aid of n-HA. It is interesting to note that the expression of ER stress biomarkers (GRP78, p-IRE1, p-PERK, PERK, and ATF6) were all upregulated after n-HA treatment, along with the activation of the pro-apoptotic transcription factor CHOP, showing that ER stress produced by n-HA triggered cell apoptosis. Moreover, the increased expression level of intracellular reactive oxygen species and the mitochondrial membrane depolarization, as well as the downstream cell apoptotic signaling activation, further demonstrated the pro-apoptotic roles of n-HA induced Ca2+ overload through inducing mitochondria damage. The in vivo data provided additional evidence that n-HA caused ER stress and mitochondria damage in cells and effectively restrain the growth of glioma tumors. Collectively, the work showed that n-HA co-activated intracellular ER stress and mitochondria damage are critical triggers for cancer cells apoptosis, offering fresh perspectives on ER-mitochondria targeted anti-tumor therapy.

Osteo-immunomodulatory effects of macrophage-derived extracellular vesicles treated with biphasic calcium phosphate ceramics on bone regeneration.

Literature on osteoimmunology has demonstrated that macrophages have a great influence on biomaterial-induced bone formation. However, there are almost no reports clarifying the osteo-immunomodulatory capacity of macrophage-derived extracellular vesicles (EVs). This study comprehensively investigated the effects of EVs derived from macrophages treated with biphasic calcium phosphate (BCP) ceramics (BEVs) on vital events associated with BCP-induced bone formation such as immune response, angiogenesis, and osteogenesis. It was found that compared with EVs derived from macrophages alone (control, CEVs), BEVs preferentially promoted macrophage polarization towards a wound-healing M2 phenotype, enhanced migration, angiogenic differentiation, and tube formation of human umbilical vein endothelial cells, and induced osteogenic differentiation of mesenchymal stem cells. Analysis of 15 differentially expressed microRNAs (DEMs) related to immune, angiogenesis, and osteogenesis suggested that BEVs exhibited good immunomodulatory, pro-angiogenic, and pro-osteogenic abilities, which might be attributed to their specific miRNA cargos. These findings not only deepen our understanding of biomaterial-mediated osteoinduction, but also suggest that EVs derived from biomaterial-treated macrophages hold great promise as therapeutic agents with desired immunomodulatory capacity for bone regeneration.