Berberine-Encapsulated Poly(lactic-co-glycolic acid)-Hydroxyapatite (PLGA/HA) Microspheres Synergistically Promote Bone Regeneration with DOPA-IGF-1 via the IGF-1R/PI3K/AKT/mTOR Pathway.

Polymer microspheres have recently shown outstanding potential for bone tissue engineering due to their large specific surface area, good porosity, injectable property, good biocompatibility, and biodegradability. Their good load-release function and surface modifiability make them useful as a carrier of drugs or growth factors for the repair of bone defects in irregularly injured or complex microenvironments, such as skull defects. In this study, berberine (BBR)-encapsulated poly(lactic-co-glycolic acid) (PLGA)/hydroxyapatite (HA) microspheres were fabricated using electrified liquid jets and a phase-separation technique, followed by modification with the 3,4-hydroxyphenalyalanine-containing recombinant insulin-like growth-factor-1 (DOPA-IGF-1). Both the BBR and the IGF-1 exhibited sustained release from the IGF-1@PLGA/HA-BBR microspheres, and the composite microspheres exhibited good biocompatibility. The results of the alkaline phosphatase (ALP) activity assays showed that the BBR and IGF-1 in the composite microspheres synergistically promoted the osteogenic differentiation of MC3T3-E1 cells. Furthermore, it was confirmed that immobilized IGF-1 enhances the mRNA expression of an osteogenic-related extracellular matrix and that BBR accelerates the mRNA expression of IGF-1-mediated osteogenic differentiation and cell mineralization. Further cellular studies demonstrate that IGF-1 could further synergistically activate the IGF-1R/PI3K/AKT/mTOR pathway using BBR, thereby enhancing IGF-1-mediated osteogenesis. Rat calvarial defect repair experiments show that IGF-1@PLGA/HA-BBR microspheres can effectively promote the complete bony connection required to cover the defect site and enhance bone defect repair. These findings suggest that IGF-1@PLGA/HA-BBR composite microspheres show a great potential for bone regeneration.

PLGA/BGP/Nef porous composite restrains osteoclasts by inhibiting the NF-κB pathway, enhances IGF-1-mediated osteogenic differentiation and promotes bone regeneration.

BACKGROUND: Novel bone substitutes are urgently needed in experimental research and clinical orthopaedic applications. There are many traditional Chinese medicines that have effects on bone repair. However, application of natural medicines in traditional Chinese medicine to bone tissue engineering and its mechanism were rarely reported. RESULTS: In this study, the osteogenic ability of bioactive glass particles (BGPs) and the osteogenic and osteoclastic ability of neferine (Nef) were fused into PLGA-based bone tissue engineering materials for bone regeneration. BGPs were prepared by spray drying and calcination. Particles and Nef were then mixed with PLGA solution to prepare porous composites by the phase conversion method. Here we showed that Nef inhibited proliferation and enhanced ALP activity of MC3T3-E1 cells in a dose- and time-dependent manner. And the composites containing Nef could also inhibit RANKL-induced osteoclast formation (p < 0.05). Mechanistically, the PLGA/BGP/Nef composite downregulated the expression of NFATC1 by inhibiting the NF-κB pathway to restrain osteoclasts. In the other hands, PLGA/BGP/Nef composite was first demonstrated to effectively activate the IGF-1R/PI3K/AKT/mTOR pathway to enhance IGF-1-mediated osteogenic differentiation. The results of animal experiments show that the material can effectively promote the formation and maturation of new bone in the skull defect site. CONCLUSIONS: The PLGA/BGP/Nef porous composite can restrain osteoclasts by inhibiting the NF-κB pathway, enhance IGF-1-mediated osteogenic differentiation and promotes bone regeneration, and has the potential for clinical application.

Tannin-bridged magnetic responsive multifunctional hydrogel for enhanced wound healing by mechanical stimulation-induced early vascularization.

Wound healing is a complex process. Wound-repair materials require multiple functionalities, such as anti-inflammatory, antibacterial, angiogenesis, pro-proliferation, and remodeling. To achieve rapid tissue regeneration, magnetic field-assisted therapy has become a promising means. In this study, a homogeneous magnetic responsive nanocomposite hydrogel with enhanced mechanical properties was obtained through a tannin (TA)-assisted bridge between magneto-deformable cobalt ferrite nanoparticles (CFO NPs) and polyvinyl alcohol (PVA) matrix. In the presence of an external static magnetic field (SMF), the TA bridge could efficiently transmit magnetically actuated deformation to the PVA, which originated from the CFO NPs, generating a larger topographic change on the surface. The change of topography provided a mechanical cue to increase cell adhesion and proliferation. Moreover, due to the synergistic effects of TA modification and CFO NPs, the obtained magnetic responsive hydrogel exhibited considerable antibacterial activity. Furthermore, the results of in vivo study confirmed the anti-inflammatory properties of the TA-CFO/PVA hydrogel. More importantly, the TA-CFO/PVA hydrogel accelerated wound healing under a SMF, which contributed to the early vascularization induced by mechanical stimuli generated from the TA-CFO/PVA nanocomposite hydrogel. As a proof-of-concept, we provided an optimizing strategy for magneto-controlled skin tissue regeneration, which may have important guiding significance for the clinical application of magnetic field-assisted therapy.

Bioorthogonal DOPA-NGF activated tissue engineering microunits for recovery from traumatic brain injury by microenvironment regulation.

Stem cell treatment is vital for recovery from traumatic brain injury (TBI). However, severe TBI usually leads to excessive inflammation and neuroinhibitory factors in the injured brain, resulting in poor neural cell survival and uncontrolled formation of glial scars. In this study, a bioorthogonal microenvironment was constructed on biodegradable poly(lactide-co-glycolide) (PLGA) microcarriers through immobilization of mussel-inspired bioorthogonal 3,4-dihydroxyphenylalanine-containing recombinant nerve growth factor (DOPA-NGF) and human umbilical cord mesenchymal stem cells (hUMSCs) for minimally invasive therapy of TBI. Cell culture and RNA-seq analysis revealed enhanced extracellular matrix (ECM) secretion and viability of hUMSCs on PLGA microcarriers compared to 2D culture. Immobilized DOPA-NGF further promoted adhesion, proliferation, and gene expression in RSC96 neurotrophic cells and hUMSCs. Specifically, the neurotrophin receptor of NT-3 (NTRK3) in hUMSCs was activated by DOPA-NGF, leading to MYC transcription and paracrine enhancement to build an adjustable biomimetic microenvironment. After transplantation of microunits in animal models, the motor and learning-memory ability of TBI mice were improved through rollbacks of overactivated inflammatory reaction regulation, neuronal death, and glial scar formation after injury. This was attributed to the paracrine enhancement of hUMSCs activated by the DOPA-NGF. Our study provides a neural regenerative microenvironment-based therapeutic strategy to advance the effects of transplanted hUMSCs in cell-based regenerative medicine for TBI therapy. STATEMENT OF SIGNIFICANCE: Extensive studies have demonstrated the importance of the microenvironment for posttraumatic brain injury recovery. However, an efficient method that can mimic the neural regenerative microenvironment to strengthen stem cell therapy and brain injury recovery is still absent. In this study, the minimally invasive transplantation of DOPA-NGF immobilized biodegradable microcarriers with mesenchymal stem cells was found to be an effective method for regeneration of injured brain. Moreover, transcriptome analysis revealed that neurotrophin receptor of NT-3 (NTRK3) was activated by DOPA-NGF for MYC transcription and paracrine enhancement to build a kind of adjustable biomimetic microenvironment for brain injury therapy. This study provides a neural regenerative microenvironment-based therapeutic strategy to advance the transplanted hUMSCs in cell-based regenerative medicine for neural recovery.

EDTMP ligand-enhanced water interactions endowing iron oxide nanoparticles with dual-modal MRI contrast ability.

Single-modal magnetic resonance imaging (MRI) contrast agents sometimes cause signal confusion in clinical diagnosis. Utilizing ligands to endow iron oxide nanoparticles (IO NPs) with excellent dual-modal MRI contrast efficiency might be an effective strategy to improve diagnostic accuracy. This work presents the development of a special ligand-assisted one-pot approach for the preparation of super-hydrophilic magnetic NPs with excellent water dispersion, biocompatibility and T1-T2 dual-modal contrast enhancement properties. In addition, the strong binding capacity between the ethylenediamine tetramethylenephosphonic acid (EDTMP) ligand and water molecules induced by the presence of abundant hydrogen bonds significantly improves spin-lattice (T1) and spin-spin (T2) imaging of the IO core. After being modified with the EDTMP ligand, the T2 relaxation rate of the IO core is dramatically increased from 71.78 mM-1 s-1 to 452.38 mM-1 s-1, and a moderate T1 relaxation rate (11.61 mM-1 s-1) is observed simultaneously, implying that the NPs with an average size of 9.7 nm may be potential candidates as high-efficiency T1-T2 MRI contrast agents. This fundamental technique of using super-hydrophilicity ligands to endow IO NPs with dual-modal contrast properties without size change and damage in the T2 contrast effect may provide a useful strategy to facilitate the application of magnetic NPs in the field of medical diagnosis.

Porous polyetheretherketone microcarriers fabricated via hydroxylation together with cell-derived mineralized extracellular matrix coatings promote cell expansion and bone regeneration.

Porous microcarriers have aroused increasing attention recently by facilitating oxygen and nutrient transfer, supporting cell attachment and growth with sufficient cell seeding density. In this study, porous polyetheretherketone (PEEK) microcarriers coated with mineralized extracellular matrix (mECM), known for their chemical, mechanical and biological superiority, were developed for orthopedic applications. Porous PEEK microcarriers were derived from smooth microcarriers using a simple wet-chemistry strategy involving the reduction of carbonyl groups. This treatment simultaneously modified surface topology and chemical composition. Furthermore, the microstructure, protein absorption, cytotoxicity and bioactivity of the obtained porous microcarriers were investigated. The deposition of mECM through repeated recellularization and decellularization on the surface of porous MCs further promoted cell proliferation and osteogenic activity. Additionally, the mECM coated porous microcarriers exhibited excellent bone regeneration in a rat calvarial defect repair model in vivo, suggesting huge potential applications in bone tissue engineering.

Improved hemostatic effects by Fe3+ modified biomimetic PLLA cotton-like mat via sodium alginate grafted with dopamine.

The development of an excellent, bioabsorbable hemostatic material for deep wound remains a challenge. In this work, a biodegradable cotton-like biomimetic fibrous mat of poly (l-lactic acid) (PLLA) was made by melt spinning. Subsequently, SD composite was prepared by cross-linking sodium alginate (SA) with dopamine (DA). It was immobilized on the fibre surface, which inspired by mussel byssus. Finally, Fe3+ was loaded onto the 0.5SD/PLLA composite by chelation with the carboxyl of alginate and phenolic hydroxy of dopamine. The haemostasis experiment found that the hemostatic time 47 s in vitro. However, the bleeding volume was 0.097 g and hemostatic time was 23 s when 20Fe3+-0.5SD/PLLA was applied in the haemostasis of the rat liver. As a result of its robust hydrophilicity and bouffant cotton-like structure, it could absorb a large water from blood, which could concentrate the component of blood and reduce the clotting time. Furthermore, the addition of Fe3+ in the 0.5SD/PLLA had a significant effect on improve hemostatic property. It also displayed excellent antibacterial property for Escherichia coli and Staphylococcus aureus. Notably, it possesses superior hemocompatibility, cytocompatibility and histocompatibility. Hence, 20Fe3+-0.5SD/PLLA has high potential application in haemostasis for clinical settings due to its outstanding properties.

Biomimetic polyetheretherketone microcarriers with specific surface topography and self-secreted extracellular matrix for large-scale cell expansion.

Reusable microcarriers with appropriate surface topography, mechanical properties, as well as biological modification through decellularization facilitating repeated cell culture are crucial for tissue engineering applications. Herein, we report the preparation of topological polyetheretherketone (PEEK) microcarriers via gas-driven and solvent exchange method followed by hydrothermal treatment at high temperature and pressure. After hydrothermal treated for 8 h, the resulting topological PEEK microcarriers exhibit walnut-like surface topography and good sphericity as well as uniform size distribution of 350.24 ± 19.44 µm. And the average width between ravine-patterned surface of PEEK microcarriers is 780 ± 290 nm. After repeated steam sterilization by autoclaving for three times, topological PEEK microcarriers show nearly identical results compared with previous ones indicating strong tolerance to high temperature and pressure. This is a unique advantage for large-scale cell expansion and clinical applications. Moreover, PEEK microcarriers with special topography possess higher protein adsorption efficiency. In addition, the reutilization and biofunctionalization with repeated decellularization of topological PEEK microcarriers show highly beneficial for cell adhesion and proliferation. Therefore, our study is of great importance for new generation microcarriers with micro-and nano-scale surface feature for a broad application prospect in tissue engineering.

3D-printing of solvent exchange deposition modeling (SEDM) for a bilayered flexible skin substitute of poly (lactide-co-glycolide) with bioorthogonally engineered EGF.

Biodegradable polyesters have been widely used as rigid biomedical apparatus because of high mechanical properties but few flexible implants. Herein, we report a flexible poly(lactide-co-glycolide) (PLGA) scaffold using a rapid in situ formation system based on phase separation by solvent exchange deposition modeling (SEDM), which was different from traditional 3D printing of fused deposition modeling (FDM). The FDM printed product was rigidity, its Young's modulus was approximate 2.6 times higher than that of SEDM printed sample. In addition, the thickness of the solidified ink would not shrink during the SEDM printing process, its surface had nano-/micro pores in favor of protein immobilization and cell adhesion. Then a flexible bilayered scaffold with nano-/microstructure was constructed combing SEDM with electrospinning technology for skin substitute, wherein the SEDM printed sample acted as a sub-layer for cell and tissue ingrowth, the densely packed electrospun nanofibers served as an upper-layer improving the sub-layer's tensile strength by 57.07% and preventing from bacteria as physical barrier. Ultimately, the bilayered scaffold immobilized epidermal growth factor (EGF) by a bioorthogonal approach was successfully applied to facilitate full-thickness wound healing of rats.

A Novel Approach via Surface Modification of Degradable Polymers With Adhesive DOPA-IGF-1 for Neural Tissue Engineering.

The highly damaging state of spinal cord injuries has provided much inspiration for the design of surface modification of the implants that can promote nerve regeneration and functional reconstruction. DOPA-IGF-1, a new recombinant protein designed in our previous study, exhibited strong binding affinity to titanium and significantly enhanced the growth of NIH3T3 cells on the surface of titanium with the same biological activity as IGF-1. In this article, surface modification of poly(lactide-co-glycolide) (PLGA) films with recombinant DOPA-IGF-1 was performed to promote the paracrine activity of human umbilical cord mesenchymal stem cells (hUCMSCs) by secreting neurotrophic factors. DOPA-IGF-1 exhibited the strongest binding ability to PLGA films than commercial IGF-1 and nonhydroxylated YKYKY-IGF-1. In vitro cultures of hUCMSCs on the modified PLGA films showed that DOPA-IGF-1@PLGA substrates significantly improved the proliferation, adhesion, and neurotrophic factors secretion of hUCMSCs, especially for nerve growth factor, as confirmed by qRT-PCR and western blot analysis. Subsequently, the acquired neurotrophic factors secreted by the hUCMSCs cultured on the DOPA-IGF-1@PLGA films obviously enhanced neurite outgrowth of PC12 cells. Taken together, PLGA substrates with DOPA-IGF-1 immobilization is a promising platform for neural tissue engineering via neurotrophic factors secretion from MSCs and should be further tested in vivo.

Binding efficiency of recombinant collagen-binding basic fibroblast growth factors (CBD-bFGFs) and their promotion for NIH-3T3 cell proliferation.

The recombinant basic fibroblast growth factor (bFGF) containing collagen-binding domain (CBD) has been found to be a potential therapeutic factor in tissue regeneration. However, its binding efficiency and quantification remain uncertain. In this research, massive recombinant bFGFs with good bioactivity for enhancing the proliferation of NIH-3T3 cells were achieved. An ELISA-based quantitative method was set up to investigate the binding efficiency of CBD-bFGFs on collagen films. It indicated that the CBDs significantly increased the collagen-binding ability of bFGF (P < .05), with the optimum binding condition first determined to be in the pH range of 7.5-9.5 (P < .05). Then, the relevant equations to calculate the binding density of bFGF, C-bFGF, and V-bFGF were acquired. Analysis confirmed that the bioactivity of immobilized bFGFs was well correlated with the density of growth factor on collagen films. Based on this research, the density of growth factor is a logical and applicable dosage unit for quantification of binding efficiency of growth factors, rather than traditional concentration of soluble growth factors in tissue engineering applications.

A chitin film containing basic fibroblast growth factor with a chitin-binding domain as wound dressings.

Basic fibroblast growth factor (bFGF) can stimulate wound healing. However, consistent delivery of bFGF has many disadvantages. To decrease their instability and diffusible, we introduced chitin-binding domain (ChtBD) into bFGF. Two expression plasimids were constructed. The first one (named bFGF) contained bFGF (154 amino acids), the second (named ChtBD-bFGF) contained bFGF and the ChtBD (54 amino acids). ChtBD was derived from chitinase A1 (ChiA1) of Bacillus circulans WL-12. The recombinant protein ChtBD-bFGF had the same biological activity as bFGF in promoting fibroblast proliferation. Chitin powder was dissolved in 11wt% NaOH and 4wt% urea aqueous solution via the freezing/thawing method. A chitin solution was spread on a glass plate and coagulated with anhydrous alcohol. The chitin binding ability of ChtBD-bFGF was 11.4-fold higher (up to 286µg/cm2) than bFGF in vitro. The immunofluorescence data indicated that the ChtBD-bFGF@chitin film promoted cell adhesion and proliferation. The ChtBD-bFGF@chitin film and bFGF@chitin films were implanted subcutaneously. Histological analysis showed that ChtBD-bFGF promoted vascularization at the implanted site more effectively than bFGF. These results suggest that the ChtBD-bFGF@chitin film is a stabile delivery vehicle for accelerating wound healing.

Modulation of Osteogenesis in MC3T3-E1 Cells by Different Frequency Electrical Stimulation.

Electrical stimulation (ES) is therapeutic to many bone diseases, from promoting fracture regeneration to orthopedic intervention. The application of ES offers substantial therapeutic potential, while optimal ES parameters and the underlying mechanisms responsible for the positive clinical impact are poorly understood. In this study, we assembled an ES cell culture and monitoring device. Mc-3T3-E1 cells were subjected to different frequency to investigate the effect of osteogenesis. Cell proliferation, DNA synthesis, the mRNA levels of osteosis-related genes, the activity of alkaline phosphatase (ALP), and intracellular concentration of Ca2+ were thoroughly evaluated. We found that 100 Hz could up-regulate the mRNA levels of collagen I, collagen II and Runx2. On the contrary, ES could down-regulate the mRNA levels of osteopontin (OPN). ALP activity assay and Fast Blue RR salt stain showed that 100 Hz could accelerate cells differentiation. Compared to the control group, 100 Hz could promote cell proliferation. Furthermore, 1 Hz to 10 Hz could improve calcium deposition in the intracellular matrix. Overall, these results indicate that 100Hz ES exhibits superior potentialities in osteogenesis, which should be beneficial for the clinical applications of ES for the treatment of bone diseases.