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Hydrogel scaffolds are attractive for tissue defect repair and reorganization because of their human tissue-like characteristics. However, most hydrogels offer limited cell growth and tissue formation ability due to their submicron- or nano-sized gel networks, which restrict the supply of oxygen, nutrients and inhibit the proliferation and differentiation of encapsulated cells. In recent years, 3D printed hydrogels have shown great potential to overcome this problem by introducing macro-pores within scaffolds. In this study, we fabricated a macroporous hydrogel scaffold through horseradish peroxidase (HRP)-mediated crosslinking of silk fibroin (SF) and tyramine-substituted gelatin (GT) by extrusion-based low-temperature 3D printing. Through physicochemical characterization, we found that this hydrogel has excellent structural stability, suitable mechanical properties, and an adjustable degradation rate, thus satisfying the requirements for cartilage reconstruction. Cell suspension and aggregate seeding methods were developed to assess the inoculation efficiency of the hydrogel. Moreover, the chondrogenic differentiation of stem cells was explored. Stem cells in the hydrogel differentiated into hyaline cartilage when the cell aggregate seeding method was used and into fibrocartilage when the cell suspension was used. Finally, the effect of the hydrogel and stem cells were investigated in a rabbit cartilage defect model. After implantation for 12 and 16 weeks, histological evaluation of the sections was performed. We found that the enzymatic cross-linked and methanol treatment SF5GT15 hydrogel combined with cell aggregates promoted articular cartilage regeneration. In summary, this 3D printed macroporous SF-GT hydrogel combined with stem cell aggregates possesses excellent potential for application in cartilage tissue repair and regeneration., Graphical abstract Image 1, Highlights • A novel macro-porous hydrogel scaffold for cartilage tissue engineering was optimized by enzymatically crosslinking of SF and GT molecules. • The advantages of this scaffold are excellent structural stability, suitable mechanical properties for cartilage regeneration. • Stem cells differentiated into hyaline cartilage by the cell aggregates seeding method while into fibrocartilage by the cell suspension seeding method. more...

The extracellular matrix microenvironment, including chemical constituents and topological structure, plays key role in regulating the cell behavior, such as adhesion, proliferation, differentiation, apoptosis, etc. Until now, to investigate the relationship between surface texture and cell response, various ordered patterns have been prepared on the surface of different matrixes, whereas almost all these strategies depend on advanced instruments or severe synthesis conditions. Herein, cell-mediated mineralization method has been applied to construct nanopattern on the surface of β-TCP scaffold. The formation process, morphology, and composition of the final pattern were characterized, and a possible mineralization mechanism has been proposed. Moreover, the cell behavior on the nanopattern has been investigated, and the results showed that the mouse bone marrow mesenchyme stem cells (mBMSCs) display good affinity with the nanopattern, which was manifested by the good proliferation and osteogenic differentiation status of cells. The synthetic strategy may shed light to construct advanced topological structures on other matrixes for bone repair. more...

Peripheral nerve damage can lead to loss of function or even complete disability, which brings about a huge burden on both the patient and society. Regulating nerve cell behavior and promoting nerve injury repair by bionic construction of a biological electric field to simulate natural nerve electrophysiological characteristics have attracted great attention, and many studies have shown that electroactive materials have a good repair effect on peripheral nerves. Here, we summarize the electroactive materials commonly used in peripheral nerve repair and their characteristics, especially for the design and application of a biodegradable non-invasive spontaneous electrical system. Besides, based on the fact that electroactive materials can provide electrical conductivity or a power generation performance, the specific role of electroactive materials in peripheral nerve repair was summed up from the following three aspects: cell behavior regulation, signaling pathway regulation and immune microenvironment regulation. This review provides a summary of the current research on the use of electroactive materials in peripheral nerve repair, which may help provide a more effective strategy for peripheral nerve injury (PNI) and allow more patients to benefit from artificial nerve catheter treatment. more...

Additive manufacture (AM) has been widely and rapidly applied in fabrication of 3D porous scaffolds for tissue engineering applications. For synthetic polymers of high melting temperature, the melting-extruding technique is the most applied AM method for such fabrication of polymer porous scaffolds. This results in a big challenge to directly process the scaffolds using the polymers and thermosensitive substances simultaneously because of deactivation under high temperature. In this article, the selective laser sintering (SLS) method was proposed to make a poly(l-lactic acid) (PLLA) porous scaffold containing dexamethasone (Dex) simultaneously. Dex was encapsulated in two groups of PLLA-bioactive glass (BG) composite microspheres with an average diameter of 115-120 μm and loading amounts of 0.68 ± 0.09 and 0.84 ± 0.10 μg/mg, respectively. The drug-loading composite microspheres were then fabricated into scaffolds under a laser fluence of 0.83-2.08 J/mm more...

Biocompatible and bioactive scaffolds with multiscale porosity, shape memory effect and drug delivery performance are highly promising in bone tissue engineering applications within the approach of minimally invasive surgical implantation. In this study, we report an effective method to fabricate multiscale porous bioactive (MPB) scaffolds with shape memory effect by freeze drying of 3D printed high internal phase Pickering emulsion (Pickering HIPE) templates stabilized by hydrophobically modified hydroxyapatite and silica nanoparticles. MPB scaffolds containing a shape memory polymer matrix of poly(D,L-lactide-co-trimethylene carbonate) display high porosity and fully interconnected filament networks with multiscale pore structures. The in vitro biomineralization study verifies that MPB scaffolds are bioactive with the apatite formation ability. The thermal property testing shows that the glass transition temperature of MPB scaffolds is easily tuned to fall between 45.9 and 49.1 °C by varying the HIPE composition. Moreover, MPB scaffolds exhibit observable shape memory properties with fast shape recovery. The antibacterial drug enrofloxacin can be efficiently loaded into MPB scaffolds and then released in a sustained manner displaying effective antimicrobial effect. Furthermore, cell culture assays confirm that MPB scaffolds could sustain the cell growth, proliferation and osteogenic differentiation, indicating the cytocompatibility and osteoinduction activity of MPB scaffolds. Therefore, combining freeze drying with 3D printing of Pickering HIPE templates paves the way towards MPB scaffolds with shape memory effect, which are promising as minimally invasive implantation scaffolds in bone tissue engineering applications. more...

In the preparation of bioactive bone graft materials, surface topography is essential for the ultimate stem cell response. However, the tunable fabrication of surface topography for 3D bioceramic scaffolds is still a technical problem because of the low processability and high brittleness of bioceramics. In this study, an evenly spaced hexagon-like column array surface was fabricated in situ via a hydrothermal method on 3D plotted hydroxyapatite scaffolds. Compared with the Control scaffolds, hydroxyapatite scaffolds with a hexagon-like column array topography possessed a higher crystal orientation degree and specific surface area, which further enhanced fibronectin adsorption. The array topography on the hydroxyapatite scaffolds also showed good biocompatibility with human adipose-derived stem cells (ADSCs). More importantly, the Array scaffolds significantly promoted the expression levels of osteogenic-related genes and proteins compared with the Control scaffolds. The results suggested that the construction of hexagon-like column array topography might be critical for the design of bone regeneration scaffolds with spontaneous stimulation capacity. more...

Background: Localized delivery of antimicrobial agents such as antimicrobial peptides (AMPs) by a biomaterial should be on-demand. Namely, AMPs should be latent and biocompatible in the absence of bacterial infection, but released in an amount enough to kill bacteria immediately in response to bacterial infection. Methods: To achieve the unmet goal of such on-demand delivery, here we turned a titanium implant with titania nanotubes (Ti-NTs) into a Pandora's box. The box was loaded with AMPs (HHC36 peptides, with a sequence of KRWWKWWRR) inside the nanotubes and "closed" (surface-modified) with a pH-responsive molecular gate, poly(methacrylic acid) (PMAA), which swelled under normal physiological conditions (pH 7.4) but collapsed under bacterial infection (pH ≤ 6.0). Thus, the PMAA-gated Ti-NTs behaved just like a Pandora's box. The box retarded the burst release of AMPs under physiological conditions because the gate swelled to block the nanotubes opening. However, it was opened to release AMPs to kill bacteria immediately when bacterial infection occurred to lowering the pH (and thus made the gate collapse). Results: We demonstrated such smart excellent bactericidal activity against a panel of four clinically important bacteria, including Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus. In addition, this box was biocompatible and could promote the osteogenic differentiation of human mesenchymal stem cells. Both in vitro and in vivo studies confirmed the smart "on-demand" bactericidal activity of the Pandora's box. The molecularly gated Pandora's box design represents a new strategy in smart drug delivery. more...

Injectable hydrogels have shown great potential for cartilage tissue engineering as three-dimensional cell culture scaffolds, owing to their similarity to the extracellular matrix (ECM), which endows them with the ability to match irregular defects, minimally invasive properties and convenience for cell transplantation and proliferation. However, injectable hyaluronic acid (HA) hydrogels have limited applications in cartilage tissue engineering due to their poor mechanical properties and fast enzymatic degradation. In this study, a double cross-linked (DC) hydrogel was designed and prepared by combining the Diels–Alder click reaction and a phenyl boronate ester bond, in which the DA reaction provides mechanical properties and the phenyl boronate bond provides versatility, such as injectability, adhesion and anti-degradation. Compared with the single cross-linked hydrogel, the DC hydrogel not only possessed good injectability and pH responsiveness, but also had repeatable strength and durability. In addition, in vitro cell experiments indicated that the injectable DC hydrogel was capable of supporting cell loading and proliferation, thus being a promising candidate for repairing cartilage defects. more...

Hydrogels are appealing biomaterials for regenerative medicine since biomimetic modifications of their polymeric network can provide unique physical properties and emulate the native extracellular matrix (ECM). Meanwhile, therapeutic metal ions, such as magnesium ions (Mg2+), not only regulate cellular behaviours but also stimulate local bone formation and healing. However, the absence of a meaningful macroporous structure and the uncompromising mechanical strength are still challenges. Herein, we designed a macroporous composite hydrogel based on mild and fast thiol-ene click reactions. The Pickering emulsion method was adopted to form a macroporous structure and introduce MgO nanoparticles (NPs). The results show that the composite hydrogel possesses good mechanical strength and an evenly distributed macroporous structure. MgO NPs stabilized at the oil/water interface not only function as effective emulsion stabilizers, but also enhance the mechanical properties of hydrogels and mediate the sustained release of Mg2+. In vitro cell experiments demonstrated that the composite hydrogel displays good biocompatibility. More importantly, the release of Mg2+ ions from hydrogels can effectively promote the osteogenic differentiation of BMSCs. Furthermore, an in vivo study showed that macroporous hydrogels can provide a good extracellular matrix microenvironment for in situ osteogenesis and accelerate bone tissue regeneration. more...

Cell-material interactions are important to tissue engineering. Inspired by the natural topographic structures on the extracellular matrix, a growing number of studies have integrated engineering topography into investigations of cell behavior on biomaterials. Engineering topography has a significant influence on cell behaviors. These cell-topography interactions play an important role in regenerative medicine and tissue engineering. Similarly, cell-topography interactions are important to corneal reconstruction and regeneration. In this review, we primarily summarized the effects of topographic cues on the behaviors of corneal cells, including cell morphology, adhesion, migration, and proliferation. Furthermore, the integration of engineering surface topography into corneal tissue engineering was also discussed., Graphical abstract Image 1, Highlights • This article aims to provide insight into the potential applications of topography on corneal reconstruction and regeneration. • Discussed the relation between surface topography and corneal repair. • Reviewed the effect of topographic cues on corneal cell behaviors. • Reviewed the applications of topographic cues in corneal tissue engineering. • Discussed the underlying mechanism of the topographic effect on cell behaviors. more...

Introduction The physique of college students is very important. The physique of college students is the prerequisite for the country’s revitalization, and health is the prerequisite for the transformation of intellectual capital. Object Aiming at the current college students’ physical education class arrangements and curriculum reform, the paper uses exercise prescriptions to make a brief plan for students’ physical exercise. Method The thesis uses the exercise prescription teaching method to carry out health intervention education for middle school students, which is used to analyze the physical requirements of students. Results After five months of fitness exercise prescription, the students’ physical fitness has increased significantly. We use fitness exercise prescriptions to make students feel good about exercise. Conclusion The application of physical and healthy exercise prescriptions can effectively improve the systematic and scientific nature of students’ participation in sports and help students learn and master healthy physical and mental self-exercise skills. Level of evidence II; Therapeutic studies - investigation of treatment results. RESUMO Introdução O físico dos estudantes universitários é muito importante. O físico dos universitários é o pré-requisito para a revitalização do país, e a saúde é o pré-requisito para a transformação do capital intelectual. Objeto Visando os arranjos das aulas de educação física dos atuais estudantes universitários e a reforma curricular, o artigo usa prescrições de exercícios para fazer um breve plano para os exercícios físicos dos alunos. Método A tese utiliza o método de ensino da prescrição de exercícios para realizar educação de intervenção em saúde para alunos do ensino médio, que é utilizada para analisar as exigências físicas dos alunos. Resultados Após cinco meses de prescrição de exercícios físicos, a aptidão física dos alunos aumentou significativamente. Usamos prescrições de exercícios físicos para fazer os alunos se sentirem bem com os exercícios. Conclusão A aplicação de prescrições de exercícios físicos e saudáveis pode efetivamente melhorar a natureza sistemática e científica da participação dos alunos em esportes e ajudá-los a aprender e dominar habilidades saudáveis de auto-exercício físico e mental. Nível de evidência II; Estudos terapêuticos - investigação dos resultados do tratamento. RESUMEN Introducción El físico de los estudiantes universitarios es muy importante. El físico de los estudiantes universitarios es el requisito previo para la revitalización del país, y la salud es el requisito previo para la transformación del capital intelectual. Objeto Con el objetivo de los arreglos de las clases de educación física de los estudiantes universitarios actuales y la reforma del plan de estudios, el documento utiliza prescripciones de ejercicios para hacer un plan breve para el ejercicio físico de los estudiantes. Método La tesis utiliza el método didáctico de prescripción de ejercicios para realizar la educación de intervención en salud para estudiantes de secundaria, que se utiliza para analizar los requisitos físicos de los estudiantes. Resultados Después de cinco meses de prescripción de ejercicios físicos, la aptitud física de los estudiantes ha aumentado significativamente. Usamos recetas de ejercicios físicos para que los estudiantes se sientan bien con el ejercicio. Conclusión La aplicación de prescripciones de ejercicio físico y saludable puede mejorar de manera efectiva la naturaleza sistemática y científica de la participación de los estudiantes en los deportes y ayudar a los estudiantes a aprender y dominar habilidades saludables de autoejercicio físico y mental. Nivel de evidencia II; Estudios terapéuticos: investigación de los resultados del tratamiento. more...

There have been extensive studies on the application of topography in the field of tissue repair. A common feature of these studies is that the existence of topological structures in tissue repair scaffolds can effectively regulate a series of behaviors of cells and play a positive role in a variety of tissue repair and regeneration processes. This review focuses on the application of topography in the field of peripheral nerve repair. The integration of the topological structure and biomaterials to construct peripheral nerve conduits to mimic a natural peripheral nerve structure has an important role in promoting the recovery of peripheral nerve function. Therefore, in this review, we systematically analysed the structure of peripheral nerves and summarized the effects of topographic cues of different scales and shapes on the behaviors of nerve cells, including cell morphology, adhesion, proliferation, migration and differentiation. Furthermore, the application and performance of scaffolds with different topological structures in peripheral nerve repair are also discussed. This systematic summary may help to provide more effective strategies for peripheral nerve regeneration (PNR) and shed light on nervous tissue engineering and regenerative medicine. more...

Inspired by the natural topological structure of skeletal muscle tissue, the topological surface construction of bionic scaffolds for skeletal muscle repair has attracted great interest. Many previous studies have focused on the effects of the topological structure on myoblasts. However, these studies used only specific repeating sizes and shapes to achieve the myoblast alignment and myotube formation; moreover, the regulatory effects of the size of a topological structure on myogenic differentiation are often neglected, leading to a lack of guidance for the design of scaffolds for skeletal muscle tissue engineering. In this study, we fabricated a series of microgroove topographies with various widths and depths via a combination of soft lithography and melt-casting and studied their effects on the behaviors of skeletal muscle cells, especially myogenic differentiation, in detail. Microgrooved poly(lactic-co-glycolic acid) substrates were found to effectively regulate the proliferation, myogenic differentiation, and myotube formation of C2C12 cells, and the degree of myogenic differentiation was significantly dependent on signals in response to the size of the microgroove structure. Compared with their depth, the width of the microgroove structures can more strongly affect the myogenic differentiation of C2C12 cells, and the degree of myoblast differentiation was enhanced with increasing groove width. Microgroove structures with relatively large groove widths and small groove depths promoted the myogenic differentiation of C2C12 cells. In addition, the integrin-mediated focal adhesion kinase signaling pathway and MAPK signaling pathway were activated in cells in response to the external topological structure, and the size of the topological structure of the material surface effectively regulated the degree of the cellular response to the external topological structure. These results can guide the design of scaffolds for skeletal muscle tissue engineering and the construction of effective bionic scaffold surfaces for skeletal muscle regeneration. more...

Phosphorous-containing biopolymers have been applied to expedite the regeneration of damaged bone tissue by stimulating the function of phosphorous groups in natural bones. However, the underlying mechanism of phosphorous-containing biopolymers in promoting osteogenic differentiation is unclarified. Herein, we synthesized phosphorylated chitosan hydrogels by incorporating phosphocreatine into chitosan molecular chains under mild conditions. The introduction of phosphate groups improved properties of protein adsorption and calcium deposition without affecting the morphology of hydrogels. Our results showed that phosphorylated chitosan hydrogels could not only promote alkaline phosphatase activity and mineralization but also upregulate the expression of osteogenic-related genes and proteins. Meanwhile, application of c-Jun N-terminal kinase inhibitor SP600125 and p38 mitogen-activated protein kinase inhibitor SB203580 repressed the expression of osteogenic-related markers in gene and protein levels. To the best of our knowledge, it is reported for the first time that phosphorous-containing biopolymers promote osteogenic differentiation of osteoblasts via JNK and p38 signaling pathways. more...

Most of traditional injectable hydrogels based on light curing or enzyme crosslinking are difficult to control the crosslinking time accurately and lack tissue adhesion, which leads to difficult clinical application and poor tissue repair effect. In this study, a novel injectable DMEM (Dulbecco's Modified Eagle's Medium)-induced phenylboronic acid-modified hyaluronic acid self-crosslinking hydrogel was designed and prepared by combining the phenylboronic acid and a diol on hyaluronic acid as the main network, in which dynamically reversible phenylboronic acid esters imparted good self-healing properties and tissue adhesion properties to the hydrogels. Cell medium that induced the formation of the hydrogel could simulate the pH of the physiological environment and provide uniform nutrients for the encapsulated cells. In addition, in vitro cell experiments indicated that the DMEM-induced phenylboronic acid-modified hyaluronic acid self-crosslinking hydrogel was capable of supporting cell loading and proliferation, thus being a promising candidate for tissue repair materials. more...

As the main component of the natural cornea, collagen (COL) has been widely applied to the construction of corneal repair materials. However, the applications of collagen are limited due to its poor mechanical properties. Cellulose nanocrystals (CNCs) possess excellent mechanical properties, optical transparency and good biocompatibility. Therefore, in this study, we attempted to introduce cellulose nanocrystals into collagen-based films to obtain corneal repair materials with a high strength. CNCs were incorporated at 1, 3, 5, 7 and 10 wt%. The physical properties of these composite films were characterized, and in vitro cell-based analyses were also performed. The COL/CNC films possessed better mechanic properties, and the introduction of CNCs did not affect the water content and light transmittance. The COL/CNC films demonstrated good biocompatibility toward rabbit corneal epithelial cells and keratocytes in vitro. Moreover, the collagen films with appropriate ration of CNCs effectively induced the migration of corneal epithelial cells and inhibited the myofibroblast differentiation of keratocytes. A collagen film with 7 wt% CNCs displayed the best combination of physical properties and biological performance in vitro among all the films. This study describes a nonchemical cross-linking method to enhance the mechanical properties of collagen for use in corneal repair materials and highlights potential application in corneal tissue engineering. more...

Collagen is a promising material for corneal tissue engineering. The surface topography of collagen is critical for cornea reconstruction and regeneration. Herein, we fabricated collagen films with microgrooved surface to investigate the effect of collagen film topography on corneal cell migration and antifibrosis. We found that the patterned films with microscale grooves could greatly affect the orientation, proliferation, migration, and gene expression of rabbit corneal epithelial cells and keratocytes. Compared with the natural cornea, the optimized collagen films with microgroove pattern show similar swelling performance, optical clarity, and biodegradability, which could efficiently promote the epithelial cell migration, accelerate wound healing process, and inhibit the fibrosis of keratocytes. These results suggest that collagen films with microgroove pattern are promising in corneal tissue engineering. more...

Biocompatible, biodegradable and bioactive nanocomposite (NC) scaffolds with well-defined interconnected porous structures have attracted increasing attention in bone tissue engineering. In this work, we develop a facile method to fabricate poly(L-lactic acid)-modified hydroxyapatite (g-HAp)–poly(e-caprolactone) (PCL) NC porous scaffolds by solvent evaporation based on water-in-dichloromethane (W/O) Pickering high internal phase emulsion (HIPE) templates, which are stabilized using g-HAp nanoparticles. The resultant porous scaffolds demonstrate interconnected and rough pore structures, which can be adjusted readily by varying g-HAp nanoparticle concentration, PCL concentration and the internal phase volume fraction. Moreover, the investigation of mechanical properties and in vitro biomineralization activity shows that the Young's modulus, compressive stress and bioactivity of the fabricated porous scaffolds are significantly enhanced upon increasing the g-HAp nanoparticle concentration. In addition, in vitro drug release studies of the porous scaffolds using ibuprofen (IBU) as a model drug show that the loaded IBU displays a sustained release profile. In vitro cell culture assays confirm that mouse bone mesenchymal stem cells can adhere, spread, and proliferate on the porous scaffolds, indicating that the porous scaffolds are biocompatible. All these results suggest that the fabricated g-HAp–PCL NC scaffolds have a promising potential for bone tissue engineering application. more...

Polymeric porous microspheres can be used as functional vehicles in drug delivery and cell culture. However, the conventional porous microspheres are somewhat complicated with respect to preparation, and limited in composition and controllability. Here we report the preparation of poly(lactic-co-glycolic acid)/calcium carbonate (PLGA/CC) composite microspheres with uniform superficial macropores through a facile single-step method, where CC acts simultaneously as in situ pore-forming agent and reinforcing phase. The SEM images and quantified results from mercury intrusion porosimetry indicated that the size and density of the superficial macropores were highly controllable via changing the starting parameters such as size and content of CC particles and concentration of PLGA. Mouse bone mesenchymal stem cells were cultured on microspheres of different topographies to investigate the cell–substrate interaction. The results showed that cells adhered and grew well on all microspheres, while the topography with smaller and more discrete macropores exhibited the highest proliferation, indicating that cells responded to the topography of the microsphere. This work provides a novel approach to obtain diverse porous composite microspheres designed for tissue repair and study of cell–substrate interaction. more...

Inspired by the constitution of things in the natural world, three-dimensional (3D) nanofiber scaffold/cells complex was constructed via the combination of electrospinning technology and origami techniques. The nanofiber boxes prepared by origami provided a limited space for the layer-by-layer nanofiber films, and the human fetal osteoblasts (hFOBs) seeded on the both sides of the nanofiber films were expected to facilitate the bonding of the adjacent nanofiber films through the secretion of extracellular matrix. Specifically, the hFOBs presented 3D distribution in the nanofiber scaffold, and they can stretch across the gaps between the adjacent nanofiber films, forming the cell layers and filling the whole 3D nanofiber scaffold. Eventually, a 3D block composed of electrospun nanofiber scaffold and cells was obtained, which possesses potential applications in bone tissue engineering. Interestingly, we also created 3D nanofiber structures that range from simple forms to intricate architectures via origami, indicating that the combination of electrospinning technology and origami techniques is a feasible method for the 3D construction of tissue engineering scaffolds. more...

By mimicking the role of phosphate groups in native tissues and encouraging tissue formation, phosphate-functionalized biopolymers have been widely employed to accelerate the formation of bone injury. In this study, phosphocreatine is screened as a phosphate source and applied in the preparation of phosphocreatine-modified chitosan (CS-P) and porous scaffolds. CS-P exhibits much better hydrophilicity, solubility, protein adsorption and mineralization than pure chitosan (CS). Meanwhile, in comparison to CS scaffold, the CS-P scaffold significantly enhances the in vitro osteogenic differentiation of hFOB cells, indicated by the higher alkaline phosphatase (ALP) activity, denser mineralization deposition and up-regulated osteogenesis-related genes including runt-related transcription factor-2, ALP, osteocalcin and osteopontin at both mRNA and protein levels. The in vivo results indicate an enhancing effect of the CS-HP scaffold on the bone regeneration ability. We further confirm that phosphocreatine modification promotes the osteogenic differentiation of hFOB cells by activating the MAPK signalling pathway. The phosphocreatine-modified chitosan holds great potential for bone repair and regeneration. more...

In this study, we developed a simple approach for the controllable growth of whitlockite (WH) on a β-tricalcium phosphate surface and investigated its cell viability via CCK-8, its live-dead staining and its alkaline phosphatase activity. Herein, WH with controllable morphologies was prepared by regulating the hydrothermal reaction conditions. The results of scanning electron microscopy, X-ray diffraction and X-ray photoelectron spectroscopy indicated that pure hexagonal plates of WH were prepared successfully. In vitro cell experiments showed that WH possessed excellent biocompatibility and effectively promoted the adhesion and proliferation of mouse bone mesenchymal stem cells. The osteogenesis of the WH was also enhanced. The obtained WH was expected to be utilized for promising applications as implantable block materials for bone repair. more...

Death from uncontrollable hemorrhage remains a worldwide concern, especially for deep noncompressible wounds caused by gunshot and sharp objects. However, traditional hemostatic methods such as gauze, tourniquets and direct pressure are not powerful enough for these extreme bleeding and promoting wound healing. In this study, a shape memory cryogel was prepared through mixing quaternized chitosan (QCS) and mesoporous bioactive glass (MBG) under −20 °C, in which MBG could activate intrinsic coagulation pathway and endow the cryogel with the function for promoting wound healing. The QCS/MBG cryogels possessed highly interconnected macroporous structure between 100 and 200 μm and robust mechanical property, which could maintain stable even after undergoing 10 cycles compression test. The hemostatic results demonstrated that QSC/MBG cryogel exhibited desirable hemostasis effect for liver prick injury model (71.58 ± 9.11 s) and liver noncompressible hemorrhage model (46.94 ± 0.92 s) compared to commercial gelatin sponge. Meanwhile the cryogels also showed efficient antibacterial properties for both gram-positive and negative bacteria. Furthermore, MBG loaded cryogel significantly accelerated wound healing rate in infected full-thickness wound. In summary, the shape memory cryogel can quickly control bleeding as well as avoid bacterial infection and accelerate wound healing, which had great potential for clinical application as a novel multifunctional hemostatic. more...

A series of novel bacterial cellulose/C60 (BCC60) composites was prepared using a original dehydration-rehydration method. The composites were characterized to demonstrate their potential in multifunctional wound dressings for skin cancer treatment using photodynamic therapy. Raman spectroscopy revealed that the C60 nanoparticles were successfully incorporated into the bacterial cellulose (BC) network. Scanning electron microscopy was used to examine the morphology and distribution of the C60 particles as photosensitizers in the bacterial cellulose network, and the C60 particles were uniformly distributed in the hyperfine three-dimensional BC network with diameters less than 100 nm. Reactive oxygen species (ROS) measurements indicated that the BCC60 composites possessed a high ROS generation ability when exposed to light. The antibacterial assessment of the BCC60 composites revealed their ability to inhibit the growth of E. coli and S. aureus and their relationship with light irradiation. In vitro cell experiments also confirmed that the BCC60 composites had low cytotoxicity in the dark, while they exhibited significant cancer cell damage activity under visible light. more...

CaCO3 crystals with different hierarchical structure were fabricated by the precipitation of CaCO3 in an aqueous solution containing guar gum (GG). Through adjusting a range of parameters, the size distribution and micro-structure of CaCO3 were well controlled. Results showed that GG facilitated the formation of the hierarchical hollow sphere through assembling of nanocrystals, while inhibited the growth of CaCO3 crystals and restrained phase transition from calcite to vaterite at high concentration of GG (CGG) condition; Moreover, increasing reaction temperature led to larger particles and stable crystalline phase formed. According to the results, a morphology map was presented. Furthermore, Drug loading and releasing results ensured porous CaCO3 microspheres a promising vector for drug delivery. more...

Biophysical cues (especially mechanical cues) embedded in cellular microenvironments show a critical impact on stem cell fate. Despite the capability of traditional hydrogels to mimic the feature of extracellular matrix (ECM) and tune their physicochemical properties via diverse approaches, their relatively large size not only induces biased results, but also hinders high-throughput screening and analysis. In this paper, a microgel model is proposed to recapitulate the role of 3D mechanical microenvironment on stem cell behaviors especially chondrogenesis in vitro. The small diameter of microgels brings the high surface area to volume ratio and then the enlarged diffusion area and shortened diffusion distance of soluble molecules, leading to uniform distribution of nutrients and negligible biochemical gradient inside microgels. To construct ECM-like microenvironment with tunable mechanical strength, three gelatin/hyaluronic acid hybrid microgels with low, medium and high crosslinking densities, i.e., Gel-HA(L), Gel-HA(M) and Gel-HA(H), are fabricated in microfluidic devices by Michael addition reaction between thiolated gelatin (Gel-SH) and ethylsulfated hyaluronic acid (HA-VS) with different substitution degrees of vinyl sulfone groups. Our results show that mouse bone marrow mesenchymal stem cell (BMSC) proliferation, distribution and chondrogenesis are all closely dependent on mechanical microenvironments in microgels. Noteworthily, BMSCs show a clear trend of differentiating into hyaline cartilage in Gel-HA(L) and fibrocartilage in Gel-HA(M) and Gel-HA(H). Whole transcriptome RNA sequencing reveals that mechanical microenvironment of microgels affects BMSC differentiation via TGF-β/Smad signaling pathway, Hippo signaling pathway and Integrin/YAP/TAZ signaling pathway. We believe this microgel model provides a new way to further explore the interaction between cells and 3D microenvironment. STATEMENT OF SIGNIFICANCE: In recent years, hydrogels have been frequently used to construct 3D microenvironment for cells. However, their relatively large size not only brings biased experimental results, but also limits high-throughput screening and analysis. Herein we propose a gelatin/hyaluronic acid microgel model to explore the effects of 3D cellular mechanical microenvironment (biophysical cues) on BMSC behaviors especially chondrogenesis, which can minimize the interference of biochemical gradients. Our results reveal that BMSC differentiation into either hyaline cartilage or fibrocartilage can be regulated via tailoring the mechanical properties of microgels. Whole transcriptome RNA sequencing proves that "TGF-β/Smad signaling pathway", "Hippo signaling pathway" and "Integrins/YAP/ TAZ signaling pathway" are activated or inhibited in this process. more...

The remarkable difference in cell type and matrix composition between two connected parts of a joint (cartilage and subchondral bone) makes it challenging to simultaneously regenerate both parts for joint repair. Thus we chemically designed a biphasic hydrogel made of two well-bonded shape-tunable hydrogel phases, termed bone-regenerating hydrogel (BRH) and cartilage-regenerating hydrogel (CRH). The BRH and CRH, encapsulating stem cells, were produced by photo-crosslinking bone and cartilage matrix-mimicking biopolymers and a nanobox (β-cyclodextrin) in situ in the subchondral bone defect and cartilage defect, respectively. The nanoboxes in BRH and CRH were loaded with osteogenic and chondrogenic differentiation inducers (melatonin and kartogenin) by host−guest interactions, respectively. Such interactions directed the sustained phase- and defect site-specific release of the inducers and subsequent site-specific stem cell differentiation into cartilage and bone forming cells for joint repair. The strategy opens up a new chemical approach to biomaterials with phase-specific drug release for tissue repair. more...

A novel bacterial cellulose/collagen–hydroxypropyltrimethyl ammonium chloride chitosan composite (BCC–H), as an ideal artificial hernia mesh, was synthesized by combining solution impregnation with the EDC/NHS chemical crosslinking method. Scanning electron microscopy was used to examine the morphology of the bacterial cellulose/collagen (BCC) and BCC–H composites. Infrared spectroscopy demonstrated that collagen and hydroxypropyltrimethyl ammonium chloride chitosan (HACC) were incorporated into the bacterial cellulose network successfully. Mechanical testing of the materials showed that the addition of HACC made the tensile strength decrease while the elongation at break was increased. Antibacterial assessment of the BCC–H composites revealed distinct growth inhibition against Escherichia coli and Staphylococcus aureus. In vitro cell experiments confirmed that the BCC–H composites had low cytotoxicity and good biocompatibility. more...

Nanosized celluloses are attractive building blocks to generate hierarchically advanced materials and have been gradually explored in emulsion applications. Here we report a high internal phase emulsion (HIPE) prepared by using supramolecular cellulose nanocrystals (CNCs) as Pickering stabilisers via one-step emulsification, and interconnected macroporous hybrid hydrogels were produced by utilizing this HIPE as a template. A quadruple hydrogen bonding moiety 2-ureido-4[1H]-pyrimidone (UPy) was firstly grafted onto the surface of cellulose nanocrystals through simple free radical polymerization. The polymer grafting was confirmed by elemental analysis and thermogravimetry. The UPy modified CNCs (CNC-UPy) exhibited superior emulsion stabilising ability compared to the pristine CNCs, and the oil-in-water emulsions with an internal phase volume ratio of 80% showed good long-term stability. The properties of resulting macroporous polyHIPE hydrogels, such as swelling behaviours, porous structures and mechanical strength, were investigated on the dependence of CNC-UPy concentrations. In addition, the macroporous hybrid hydrogel exhibits excellent cytocompatibility and cell adhesion as demonstrated by mouse bone mesenchymal stem cell (mBMSC) culture. With these promising properties, the developed hydrogels demonstrate great potential as active biological scaffolds for tissue engineering. more...

A well-designed scaffold with accurately controlled structure, pore size as well as interconnection plays a key role in tissue engineering. In this study, porous Li-containing biphasic calcium phosphate scaffolds (Li-BCP) with expected structure were fabricated successfully by 3D plotting technique. The results of scanning electron microscopy (SEM) and micro-CT indicated that scaffolds from 3D plotting possessed an elaborate pore structure, porosity and interconnection. Besides, Li-BCP scaffolds also possessed excellent mechanical strength. Furthermore, in vitro cell experiments confirmed that mouse bone mesenchymal stem cells (mBMSCs) cultured on Li-BCP scaffolds exhibited good adhesion and spreading. Moreover, Li-BCP scaffolds could efficaciously promote the proliferation and osteogenic differentiation of mBMSCs, demonstrating their potential applicability as a candidate for bone repair applications. more...

Light weight and mechanically strong α-chitin aerogels were fabricated using the sol-gel/self-assembly method from α-chitin in different aqueous alkali hydroxide (KOH, NaOH and LiOH)/urea solutions. All of the α-chitin solutions exhibited temperature-induced rapid gelation behavior. 13C nuclear magnetic resonance (NMR) spectra revealed that the aqueous alkali hydrox- ide/urea solutions are non-derivatizing solvents for α-chitin. Fourier transform infrared (FT-IR), X-ray diffraction (XRD) and cross-polarization magic angle spinning (CP/MAS) 13C NMR confirmed that α-chitin has a stable aggregate structure after undergoing dissolution and regeneration. Subsequently, nanostructured α-chitin aerogels were fabricated by regeneration from the chitin solutions in ethanol and then freeze-drying from t -BuOH. These α-chitin aerogels exhibited high porosity (87% to 94%), low density (0.09 to 0.19 g/cm3), high specific surface area (419 to 535 m2/g) and large pore volume (2.7 to 3.8 cm3/g). Moreover, the α-chitin aerogels exhibited good mechanical properties under compression and tension models. In vitro studies showed that mBMSCs cultured on chitin hydrogels have good biocompatibility. These nanostructured α-chitin aerogels may be useful for various applications, such as catalyst supports, carbon aerogel precursors and biomedical materials. more...

Modifying substrates through mineralization is a popular way to improve the osteogenic performance. Screening of the best mineralization characteristics on specific substrates for stem cells is meaningful but not fully studied. In this paper, poly(lactic-co-glycolic acid)/hydroxyapatite (PLGA/HA, PH) microsphere scaffolds with superficial pores were fabricated by a low-temperature fusion method. After the mineralization in the 5× stimulated body fluid (SBF) for 0, 7, 12 and 24 h, four mineralized scaffolds (MPH-0, MPH-7, MPH-12 and MPH-24) with different apatite topographies were obtained. It was found that the surface of MPH-7 was evenly decorated with abundant micro-pores, MPH-12 with dense and plain apatite layer, and MPH-24 with small spherical bumps. The responses of mouse bone mesenchymal stem cells (mBMSCs) to the four scaffolds were further studied. The results showed that MPH-7 and MPH-24 had more obvious effects on mBMSCs attachment, proliferation and differentiation than MPH-0 and MPH-12. This work indicated that to obtain the maximum improvement, the mineralization characteristics had to be carefully chosen. This was noteworthy in the chemical modification of surfaces to form the functionalized scaffolds for bone repair. more...

In this paper, we fabricated three kinds of 3D microgrooves with different depth on biocompatible poly(lactic-co-glycolic acid) (PLGA) substrate via combination of soft-lithography and melt-casting methods, and investigated in detail their influence on C2C12 cell behaviors. It is found that cell proliferation, migration, alignment, spatial distribution, F-actin protein expression and gene expression are all remarkably distinct on these microgrooved samples and the smooth control PLGA substrate. The associated underlying mechanisms were further analyzed and discussed using real-time living cell monitoring, confocal laser scanning microscopy and gene microarray. Our preliminary results suggested that 3D microstructure could affect cell behaviors in a much more extensive manner than what we had understood before. more...

Precise control of stem cells, such as human bone marrow-derived mesenchymal stem cells (hMSCs), is critical for the development of effective cellular therapies for tissue engineering and regeneration medicine. Emerging evidence suggests that several miRNAs act as key regulators of diverse biological processes, including differentiation of various stem cells. In this study, we have described a delivery system for miR-29b using PEI-capped gold nanoparticles (AuNPs) to synergistically promote osteoblastic differentiation. The cell proliferation assay revealed that AuNPs and AuNPs/miR-29b exert negligible cytotoxicity to hMSCs and MC3T3-E1 cells. With the assistance of AuNPs as a delivery vector, miR-29b could efficiently enter the cytoplasm and regulate osteogenesis. AuNPs/miR-29b more effectively promoted osteoblast differentiation and mineralization through induced the expression of osteogenesis genes (RUNX2, OPN, OCN, ALP) for the long-term, compared to the widely used commercial transfection reagent, Lipofectamine. With no obvious cytotoxicity, PEI-capped AuNPs showed great potential as an adequate miRNA vector for osteogenesis differentiation. Interestingly, we observed loading of AuNPs as well as AuNPs/miR-29b into the lumen of the endoplasmic reticulum (ER). Our findings collectively suggest that AuNPs, together with miR-29b, exert a synergistic promotory effect on osteogenic differentiation of hMSCs and MC3T3-E1 cells. more...

Nanocomposite hydrogels consisting of a synthetic matrix reinforced by nanosized crystalline polysaccharides offer significant potential in various fields. Different from nanocellulose, the combination of nanochitin with synthetic polymers to obtain nanocomposite hydrogels has not been extensively and systematically studied. Herein, a physically and chemically dual crosslinked nanocomposite hydrogel was successfully synthesized, where chitin nanowhiskers (ChNWs) and Zn2+ were incorporated within polyacrylamide (PAAm) matrix. Nanochitin/metal ion dual reinforcement imparts increased elasticity, enhanced mechanical properties, and improved recovery performance to PAAm network. The PAAm/ChNWs/Zn2+ hydrogel could be stretched to over 13 times its original length with tensile strength of 321.9 ± 8.2 kPa, and restore its original shape rapidly even when compressed at a strain of 95% with a corresponding compressive strength of 6.95 ± 0.20 MPa. The multiple crosslinks and interactions among ChNWs, Zn2+ and synthetic polymeric network were investigated. Moreover, the hydrogel was applied in drug release and soft bioelectronics. more...

Topographical cues provided by micropatterns on material surfaces have been demonstrated to control multiple cell functions. However, the majority of currently studied micropatterns fail to recapitulate the key characteristics of an osteon, which is the structural unit of natural cortical bone. Thus, in the present study, a micropatterned polycaprolactone (PCL) surface comprising a series of concentric circular microgrooves was fabricated by combining photolithography with the melt-casting method to mimic the concentric structure of an osteon in a two-dimensional setting. By culturing mouse mesenchymal stem cells (mMSCs) and osteoclast progenitor cells (RAW264.7 cells) on the osteon-like concentric microgrooved surface, the effects of this micropatterned surface on the osteogenesis of mMSCs and the osteoclastogenesis of RAW264.7 cells were systematically investigated. Osteoclastogenic differentiation was significantly inhibited in RAW264.7 cells on the fabricated osteon-like concentric microgrooved surface compared to that on the parallel linear microgrooved and flat surfaces, as indicated by the downregulated expression of key osteoclast-specific function genes (TRAP, CATK and MMP9), the lower activity of TRAP and less formation of TRAP-positive multinucleated giant osteoclasts. Further investigation indicated that RANK-NFκB signaling may have been involved in mediating the inhibited osteoclastogenesis of RAW264.7 cells by the osteon-like concentric microgrooved surface. In addition, the osteon-like concentric microgrooved surface greatly modulated the osteoclastogenic-related paracrine secretion of mMSCs (RANKL, M-CSF and OPG), despite its small effect on the osteogenesis of mMSCs. This secretory profile was found to be able to effectively inhibit osteoclastogenesis in RAW264.7 cells, confirming the enhanced osteoclastogenesis inhibitory functions of mMSCs on the osteon-like concentric microgrooved surface. Our findings demonstrate the importance of the microgroove orientation and arrangement in affecting cellular behaviors and highlight the potential benefits of incorporating osteon-like concentric microgrooved patterns on the surface of scaffolds for bone repair. more...

Modification of implants by antimicrobial peptides (AMPs) can improve the antimicrobial activity of the implants. However, AMPs have some cytotoxicity in vivo when they are exposed at body temperature. To tackle this challenge, we propose to develop a new approach to generating a smart antimicrobial surface through exposure of AMPs on the surface. A polydopamine film was first formed on the substrates, followed by the conjugation of a temperature-sensitive polymer, poly( N-isopropylacrylamide) (pNIPAM), to the film through atom transfer radical polymerization (ATRP). Then, AMPs were conjugated to the NIPAM on the resultant pNIPAM-modified surface through a click chemistry reaction. Because of the temperature-sensitive property of pNIPAM, the AMPs motif was more exposed to the external environment at room temperature (25 °C) than at body temperature (37 °C), making the surface present a higher antimicrobial activity at room temperature than at body temperature. More importantly, such a smart behavior is accompanied with the increased biocompatibility of the surface at body temperature when compared to the substrates unmodified or modified by AMPs or pNIPAM alone. Our in vivo study further verified that pNIPAM-AMP dual modified bone implants showed increased biocompatibility even when they were challenged with the bacteria at room temperature before implantation. These results indicate that the implants are antibacterial at room temperature and can be safely employed during surgery, resulting in no infection after implantations. Our work represents a new promising strategy to fully explore the antimicrobial property of AMPs, while improving their biocompatibility in vivo. The higher exposure of AMPs at room temperature (the temperature for storing the implants before surgery) will help decrease the risk of bacterial infection, and the lower exposure of AMPs at body temperature (the temperature after the implants are placed into the body by surgery) will improve the biocompatibility of AMPs. more...

Incorporation of nanofillers into aliphatic polyesters is a convenient approach to create new nanomaterials with significantly reinforced mechanical properties compared to the neat polymers or conventional composites. Nanoporous cellulose gels (NCG) prepared from aqueous alkali hydroxide/urea solutions can act as alternative reinforcement nanomaterials for polymers with improved mechanical properties. We report a simple and versatile process for the fabrication of NCG/poly(L-lactide-co-caprolactone) (NCG/P(LLA-co-CL) nanocomposites through in situ ring-opening polymerization of L-lactide (LLA) and ε-caprolactone (ε-CL) monomers in the NCG. The volume fraction of the NCG in the nanocomposites was tunable and ranged from 4.5% to 37%. Fourier transform infrared (FT-IR), X-ray diffraction (XRD), and differential scanning calorimetry (DSC) results indicated that P(LLA-co-CL) were synthesized within the NCG and partially grafted onto the surface of the cellulose nanofibrils. The glass-transition temperature (Tg) of the NCG/P(LLA-co-CL) nanocomposites could be altered by varying the molar ratio of LLA/ε-CL and was affected by the volume fraction of NCG. Atomic force microscopy (AFM) and scanning electron microscopy (SEM) images confirmed that the interconnected nanofibrillar cellulose network structure of the NCG was finely distributed and preserved in the P(LLA-co-CL) matrix after polymerization. The dynamic mechanical analysis (DMA) results showed remarkable reinforcement of the tensile storage modulus (E') of the P(LLA-co-CL) nanocomposites in the presence of NCG, especially above the Tg of the P(LLA-co-CL). The modified percolation model agreed well with the mechanical properties of the NCG/P(LLA-co-CL) nanocomposites. The introduction of NCG into the P(LLA-co-CL) matrix improved the mechanical properties and thermal stability of the NCG/P(LLA-co-CL) nanocomposites. Moreover, the NCG/P(LLA-co-CL) nanocomposites have tunable biodegradability and biocompatibility and potential applications in tissue engineering repair, biomedical implants, and packing. more...