3D printed silk-gelatin hydrogel scaffold with different porous structure and cell seeding strategy for cartilage regeneration

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.

Black phosphorus nanosheets-enabled DNA hydrogel integrating 3D-printed scaffold for promoting vascularized bone regeneration

The classical 3D-printed scaffolds have attracted enormous interests in bone regeneration due to the customized structural and mechanical adaptability to bone defects.However,the pristine scaffolds still suffer from the absence of dynamic and bioactive microenvironment that is analogous to natural extracellular matrix(ECM)to regulate cell behaviour and promote tissue regeneration.To address this challenge,we develop a black phosphorus nanosheets-enabled dynamic DNA hydrogel to integrate with 3D-printed scaffold to build a bioactive gel-scaffold construct to achieve enhanced angiogenesis and bone regeneration.The black phosphorus nanosheets reinforce the mechanical strength of dynamic self-healable hydrogel and endow the gel-scaffold construct with preserved protein binding to achieve sustainable delivery of growth factor.We further explore the effects of this activated construct on both human umbilical vein endothelial cells(HUVECs)and mesenchymal stem cells(MSCs)as well as in a critical-sized rat cranial defect model.The results confirm that the gel-scaffold construct is able to promote the growth of mature blood vessels as well as induce osteogenesis to promote new bone formation,indicating that the strategy of nano-enabled dynamic hydrogel integrated with 3D-printed scaffold holds great promise for bone tissue engineering.

Biomimetic three-layered membranes comprising(poly)-e-caprolactone,collagen and mineralized collagen for guided bone regeneration

The distinct structural properties and osteogenic capacity are important aspects to be taken into account when developing guided bone regeneration membranes.Herein,inspired by the structure and function of natural periosteum,we designed and fabricated using electrospinning a fibrous membrane comprising(poly)-e-caprolactone(PCL),collagen-I(Col)and mineralized Col(MC).The three-layer membranes,having PCL as the outer layer,PCL/Col as the middle layer and PCL/Col/MC in different ratios(5/2.5/2.5(PCM-1);3.3/3.3/3.3(PCM-2);4/4/4(PCM-3)(%,w/w/w))as the inner layer,were produced.The physiochemical properties of the different layers were investigated and a good integration between the layers was observed.The three-layeredmembranes showed tensile properties in the range of those of natural periosteum.Moreover,the membranes exhibited excellent water absorption capability without changes of the thickness.In vitro experiments showed that the inner layer of the membranes supported attachment,proliferation,ingrowth and osteogenic differentiation of human bone marrowderived stromal cells.In particular cells cultured on PCM-2 exhibited a significantly higher expression of osteogenesis-related proteins.The three-layered membranes successfully supported new bone formation inside a critical-size cranial defect in rats,with PCM-3 being the most efficient.The membranes developed here are promising candidates for guided bone regeneration applications.

Modulating Myofibroblastic Differentiation of Fibroblasts through Actin-MRTF Signaling Axis by Micropatterned Surfaces for Suppressed Implant-Induced Fibrosis

Myofibroblasts,the primary effector cells for implant-induced fibrosis,contribute to this process by secreting excessive collagen-rich matrix and contracting.Thus,approaches that suppress myofibroblasts may achieve desirable suppression effects in the fibrotic process.As one of the important physical properties of materials,material topographical structures have been proven to affect various aspects of cell behaviors,so is it possible to manipulate the formation of myofibroblasts by tailoring the topographical properties of medical devices?In this study,polycaprolactone(PCL)surfaces with typical micropatterns(micro column and micro pit)were fabricated.The regulatory effects of surface micropatterns on the myofibroblastic differentiation of fibroblasts were investigated.Compared to the flat surfaces and surfaces with micro pit,surfaces with micro columns triggered the F-to G-actin transition,inhibiting the nuclear transfer of myocardin-related transcription factor-A.Subsequently,the downstream geneα-smooth muscle actin,which is a marker of myofibroblasts,was suppressed.Further in vivo investigation showed that PCL implants with micro-column-patterned surfaces inhibited the formation of peri-implant fibrotic capsules.Our results demonstrate that surface topographical properties are a potent regulator of fibroblast differentiation into myofibroblasts and highlight the antifibrotic potential of modifying surfaces with micro-column patterns.

Heparinized PGA host-guest hydrogel loaded with paracrine products from electrically stimulated adipose-derived mesenchymal stem cells for enhanced wound repair

The microenvironment of the wound bed is essential in the regulation of wound repair.In this regard,strategies that provide a repairing favorable microenvironment may effectively improve healing outcomes.Herein,we attempted to use electrical stimulation(ES)to boost the paracrine function of adipose-derived stem cells from rats(rASCs).By examining the concentrations of two important growth factors,VEGF and PDGF-AA,in the cell culture supernatant,we found that ES,especially 5𝜇A ES,stimulated rASCs to produce more paracrine factors(5𝜇A-PFs).Further studies showed that ES may modulate the paracrine properties of rASCs by upregulating the levels of TRPV2 and TRPV3,thereby inducing intracellular Ca^(2+) influx.To deliver the PFs to the wound to effectively improve the wound microenvironment,we prepared a heparinized PGA host-guest hydrogel(PGA-Hp hydrogel).Moreover,PGA-Hp hydrogel loaded with 5𝜇A-PFs effectively accelerated the repair process of the full-thickness wound model in rats.Our findings revealed the effects of ES on the paracrine properties of rASCs and highlighted the potential application of heparinized PGA host-guest hydrogels loaded with PFs derived from electrically stimulated rASCs in wound repair.

Microneedle-array patch with pH-sensitive formulation for glucoseresponsive insulin delivery

Glucose-responsive insulin delivery systems show great promise to improve therapeutic outcomes and quality of life for people with diabetes.Herein,a new microneedle-array patch containing pH-sensitive insulin-loaded nanoparticles(NPs)(SNP(I))together with glucose oxidase(GOx)-and catalase(CAT)-loaded pH-insensitive NPs(iSNP(G+C))is constructed for transcutaneous glucose-responsive insulin delivery.SNP(I)are prepared via double emulsion from a pH-sensitive amphiphilic block copolymer,and undergo rapid dissociation to promote insulin release at a mild acidic environment induced by GOx in iSNP(G+C)under hyperglycemic conditions.CAT in iSNP(G+C)can further consume excess H_(2)O_(2) generated during GOx oxidation,and thus reduce the risk of inflammation toward the normal skin.The in vivo study on type 1 diabetic mice demonstrates that the platform can effectively regulate blood glucose levels within normal ranges for a prolonged period.

A polyamidoamine(PAMAM)derivative dendrimer with high loading capacity of TLR7/8 agonist for improved cancer immunotherapy

Tumor associated macrophages(TAMs)tend to exhibit tumor-promoting M2 phenotype and contribute to the development of immunosuppressive microenvironment of solid tumors.Reprograming TAMs from M2 into tumoricidal M1 phenotype is robust for stimulating tumor immunosuppressive microenvironment(TIME).In this study,we developed a poly(amidoamine)(PAMAM)derivative dendrimer(denoted as fourth generation-N,N-diethylaminoethyl(G4-DEEA))for efficient loading of Toll-like receptor 7 and 8(TLR7/8)agonist(R848)to remodel the TIME for potent cancer immunotherapy,G4-DEEA exhibited a high loading capacity of R848 up to 35.9 wt%by taking advantage of its dendritic structure.The resulting formulation(designated as G4-DEEA@R848)effectively polarized M2 macrophages into M1 phenotype in vitro,and improved the maturation and activation of antigen-presenting cells.In the 4T1 orthotopic breast cancer model,G4-DEEA@R848 showed a stronger tumor inhibitory effect than free drug.The mechanistic studies suggested that G4-DEEA@R848 could significantly stimulate the TIME by repolarizing TAMs into M1 phenotype,reducing the presence of immunosuppressive myeloid cells and increasing the infiltration of tumor cytotoxic T cells.This study provides a simple but effective dendrimer-based strategy to improve the formulation of R848 for improved cancer immunotherapy.

Bioactive glass promotes the barrier functional behaviors of keratinocytes and improves the Re-epithelialization in wound healing in diabetic rats

Upon skin injury,re-epithelialization must be triggered promptly to restore the integrity and barrier function of the epidermis.However,this process is often delayed or interrupted in chronic wounds like diabetic foot ulcers.Considering that BG particles can activate multiple genes in various cells,herein,we hypothesized that bioactive glass(BG)might be able to modulate the barrier functional behaviors of keratinocytes.By measuring the transepithelial electrical resistance(TEER)and the paracellular tracer flux,we found the 58S-BG extracts substantially enhanced the barrier function of keratinocyte monolayers.The BG extracts might exert such effects by promoting the keratinocyte differentiation and the formation of tight junctions,as evidenced by the increased expression of critical differentiation markers(K10 and involucrin)and TJ protein claudin-1,as well as the altered subcellular location of four major TJ proteins(claudin-1,occludin,JAM-A,and ZO-1).Besides,the cell scratch assay showed that BG extracts induced the collective migration of keratinocytes,though they did not accelerate the migration rate compared to the control.The in vivo study using a diabetic rat wound model demonstrated that the BG extracts accelerated the process of re-epithelialization,stimulated keratinocyte differentiation,and promoted the formation of tight junctions in the newly regenerated epidermis.Our findings revealed the crucial effects of BGs on keratinocytes and highlighted its potential application for chronic wound healing by restoring the barrier function of the wounded skin effectively.

A microarray platform designed for high-throughput screening the reaction conditions for the synthesis of micro/nanosized biomedical materials

Materials research usually relies on lengthy and largely trial-and-error methods,high-throughput technology has thereby emerged as an alternative method which is proven to be a simple,rapid,accurate and sensitive technique.Here,we presented a microfluidic platform with a set of 6×6 microarray chips for high-throughput synthesis and rapid screening the reaction conditions of biomedical materials.The core design of this platform is to generate concentration gradient inside microarray chips.Considering that calcium phosphates(CaP)are the most important inorganic constituents of biological hard tissues,different phases of calcium phosphates particles were synthesized with various morphogenesis when the reaction conditions such as Ca/P concentration ratio,NaOH concentration were screened using our platform.And this platform is universal and expected to apply to other systems for high-throughput screening and synthesis.

On-off-on fluorescence detection for biomolecules by a fluorescent cage through host-guest complexation in water

Detection of nucleoside derivatives has paramount importance because they are the essential biomolec-ular units for all life.Herein,we report a host-guest approach by using a fluorescent tetraphenylethene-based octacationic cage as host and 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt(HPTS)as guest and fluorescent indicator to form non-fluorescent 1:1:1 host-(endo-exo)guest complex in water.This new host-(endo-exo)guest complex can be successfully used for detecting nucleosides(e.g.,ATP and GTP),DNA(e.g.,sm-DNA),and antibiotics(e.g.,Penicillin G)with off-on fluorescence response via a competitive host-guest exchange with HPTS as exo-guest in water.Furthermore,this on-off-on fluorescent host-guest com-plex is also used for cell imaging based on ATP concentration in HeLa cells.Therefore,this study not only provides insight into the construction of a supramolecular probe with on-off-on fluorescence via host-guest complexation and exchange in solution,but also realizes a universal method for detecting and monitoring biomolecules.

Nanostructural origins of irreversible deformation in bone revealed by an in situ atomic force microscopy study

The structural origins of bone toughness at the nanoscale are not completely understood.Therefore,we performed in situ scanning using atomic force microscopy during macroscopic mechanical testing of antler and bovine bone,to reveal the origins of the irreversible plastic deformation at the mineralized collagen fibril(MCF)array and MCF levels.We found that the plastic deformation behavior at the nanoscale level could be divided into two stages.The first stage of plastic deformation at the nanoscale level was characterized by slippage between the MCF arrays,which contained mineral aggregate grains with regular shapes under load.In the second stage of nanoscale plastic deformation,the MCFs broke through the bonds of the extrafibrillar mineral aggregate grains and exhibited interfibrillar slippage.These nanoscale plastic deformation behaviors may thus be the origins of stress whitening and irreversible plastic deformation.Thus,the findings in this study not only shed light on the plastic deformation mechanisms of MCF arrays and MCFs,but also provide structural and mechanistic insights into bioinspired materials design and mechanisms of relevant bone diseases.

Modulation of Conformational Transition of Polypeptides under Slightly Acidic Environment

Controlling the conformational transition of polypeptides under slightly acidic environment is challenging.Herein,we report a class of pH-responsive helix-to-coil conformationally transitionable polypeptides(pCTPs)by simply conjugating tertiary amine groups(TAs)to polylysine.Their conformation is highly dependent on the charge state of TAs,showing a helical structure when most TAs are deprotonated and a non-helical structure when majority of TAs are protonated.The conformational transition pH can be modulated by tuning the hydrophobicity of TAs and the incorporation of hydrophobic monomers at a pH range of 7.2 to 6.0.Such pCTP showed a vesicle-to-micelle transition when their conformation transformed from helix to coil,facilitating controlled drug release.Our study provided an approach to control the conformational transition of polypeptides under slightly acidic condition.

Adaptive Chirality of an Achiral Cage:Chirality Transfer,Induction,and Circularly Polarized Luminescence through Aqueous Host-Guest Complexation

Chirality transfer,induction,and circularly polarized luminescence(CPL)using supramolecular hosts,such as macrocycles and cages,have been explored for wide-ranging applicationsin chiral recognition,sensing,catalysis,and chiroptical functional materials.Herein,we report the adaptive chirality of an achiral tetraphenylethene(TPE)-basedoctacationic cage(1)inducedby bindingwith enantiopure deoxynucleotides(A,T,C,and G)throughhost-guest(H-G)complexationinwater.The hydrophobic cavity of the cage efficiently stabilizes the hydrogen-bonded dimerization of deoxynucleotides(A_(2),T_(2),C_(2),and G_(2))to form H-G complexes in 1∶2 ratios.Given the photophysical properties and dynamic rotational conformation of the TPE units of the cage,cage⊃deoxynucleotide complexes exhibited excellent chiroptical propertiesbased on chirality transfer andinduction from the chiral guest to the achiral host.For this supramolecular system,the cage showed a unique adaptive chirality of the double clockwise-typed(PP)rotational conformation of the two TPE units,which was induced by chiral guests(e.g.,A_(2),T_(2),C_(2),and G_(2))through H-G complexation in water.Furthermore,the adaptive chirality of the cage⊃deoxynucleotide complexes successfully induced CPL signals in homogeneous aqueous solutions.This study provides insights for the construction of adaptive chirality from an achiral TPE-based octacationic cage with dynamic conformational nature,and might facilitate further design of chiral functional materials for several applications,such as chiral recognition,sensing,displays,catalysis,and other chiral fluorescent supramolecular systems based on aqueous H-G complexation.

An Intracellular pH-Actuated Polymer for Robust Cytosolic Protein Delivery

Robust cytosolic protein delivery requires both efficient protein binding with delivery vehicles and effective protein release after cell internalization.Although a variety of stimuli-responsive carriers have been designed,simultaneously integrating these two functions in one versatile carrier is challenging.Herein,we developed a polyamidoamine(PAMAM)-based polymer with an intracellular pHactuated hydrophobic-to-hydrophilic transition for this purpose.

Mechanical properties of AM Ti6Al4V porous scaffolds with various cell structures

Porous scaffolds as succedaneum of natural bone were investigated and applied in medical field.In this work, we carried out studies on mechanical properties of solid parts and porous scaffolds obtained by additive manufacturing(AM) technique.It is found that productions of AM process have a higher yield strength and higher microhardness compared to commercial Ti6Al4V.Roughened surface was observed for layer-by-layer process of AM and sticking of powder particles.The machining accuracy is affected by both dimensions and angles.Meanwhile, mechanical properties of porous scaffolds are influenced by machining accuracy and microdefects.In addition, the unit cell structures also impact the mechanical properties of porous scaffolds in terms of elastic modulus, yield strength and failure mode.Overall,considering the mechanical properties and biological properties, scaffolds with cube(CB) crystal cells are the best choice in our study.

内源性电场调控抗菌离子传输以提高植入材料抗菌性能

组织植入材料相关感染是临床治疗的一大顽疾,基于抗菌离子释放系统的抗菌植入材料是经济高效的抗生素治疗替代手段.但过量释放的抗菌离子在杀灭细菌的同时也可能引起生物毒性.因此,如何提高释放离子的抗菌效率是急需解决的根本问题.本研究设计了一种负载低剂量抗菌离子的铁电植入材料,该植入材料表面电势可在不改变自身成份的前提下通过外源电场极化进行调控.研究发现上述铁电植入材料释放的抗菌离子将在植入材料自身与带电细菌之间形成的内源性电场作用下定向输运到达细菌.研究结果表明在12 h内,高表面电势植入材料向细菌输运的离子量达到低表面电势植入材料输运量的2.4倍,从而使抗菌效率从65%提高到100%,并展现出较低的细胞毒性.本研究表明植入材料与生命体间的内源性电场在介导物质传输中发挥重要作用,为设计高性能抗菌生物材料提供了一个新的视角.

MicroRNA-activated hydrogel scaffold generated by 3D printing accelerates bone regeneration

Bone defects remain a major threat to human health and bone tissue regeneration has become a prominent clinical demand worldwide.The combination of microRNA(miRNA)therapy with 3D printed scaffolds has always posed a challenge.It can mimic physiological bone healing processes,in which a biodegradable scaffold is gradually replaced by neo-tissue,and the sustained release of miRNA plays a vital role in creating an optimal osteogenic microenvironment,thus achieving promising bone repair outcomes.However,the balance between two key factors-scaffold degradation behavior and miRNA release profile-on osteogenesis and bone formation is still poorly understood.Herein,we construct a series of miRNA-activated hydrogel scaffolds(MAHSs)generated by 3D printing with different crosslinking degree to screened the interplay between scaffold degradation and miRNA release in the osteoinduction activity both in vitro and in vivo.Although MAHSs with a lower crosslinking degree(MAHS-0 and MAHS-0.25)released a higher amount of miR-29b in a sustained release profile,they degraded too fast to provide prolonged support for cell and tissue ingrowth.On the contrary,although the slow degradation of MAHSs with a higher crosslinking degree(MAHS-1 and MAHS-2.5)led to insufficient release of miR-29b,their adaptable degradation rate endowed them with more efficient osteoinductive behavior over the long term.MAHS-1 gave the most well-matched degradation rate and miR-29b release characteristics and was identified as the preferred MAHSs for accelerated bone regeneration.This study suggests that the bio-adaptable balance between scaffold degradation behavior and bioactive factors release profile plays a critical role in bone regeneration.These findings will provide a valuable reference about designing miRNAs as well as other bioactive molecules activated scaffold for tissue regeneration.

Nanomedicine-mediated ubiquitination inhibition boosts antitumor immune response via activation of dendritic cells

Tumor immunotherapy as a promising method for tumor treatment received tremendous attention. However, the problem of low clinical response rate still needs to be solved, especially in the poorly immunogenic tumors. The enhancement of tumor antigens presentation can effectively activate dendritic cells (DCs) and improve the tumor immunotherapy. In this work, TAK-243 as an inhibitor of the ubiquitin activating enzyme (UAE), was fabricated into cationic lipid-assisted nanoparticle (CLANTAK-243). The obtained CLANTAK-243 could act as an effective tumor immunotherapy enhancer to promote the maturation of DCs as well as antigen presentation, which obviously stimulated the T cells activation and proliferation. Such CLANTAK-243 injected intravenously could well trigger immune response to tumor cells in vivo. Importantly, mice treated with CLANTAK-243 could obtain a long immune memory effect to protect themselves from re-challenged tumor cells. Therefore, this work presented an effective immunotherapy strategy for poorly immunogenic tumor.

Phospholipid-grafted PLLA electrospun micro/nanofibers immobilized with small extracellular vesicles from rat adipose mesenchymal stem cells promote wound healing in diabetic rats

Small extracellular vesicles(sEVs)derived from mesenchymal stem cells(MSCs)can deliver a variety of bioactive factors to create a favorable local microenvironment,thereby holding huge potential in chronic wound repair.However,free sEVs administrated intravenously or locally are usually cleared rapidly,resulting in an insufficient duration of the efficacy.Thus,strategies that enable optimized retention and release profiles of sEVs at wound sites are desirable.Herein,we fabricated novel functional phosphoethanolamine phospholipid-grafted poly-L-lactic acid micro/nanofibers(DSPE-PLLA)to carry and retain sEVs from rat adipose MSCs,enabling the slow local release of sEVs.Our results showed that sEVs@DSPE-PLLA promoted the proliferation,migration and gene expression(ColⅠ,ColⅢ,TGF-β,α-SMA,HIF-1α)of fibroblasts.It also promoted keratinocyte proliferation.In addition,sEVs@DSPE-PLLA helped polarize macrophages toward the M2 phenotype by increasing the expression of anti-inflammatory genes(Arginase 1,CD 206,IL-10)and inhibiting the expression of pro-inflammatory genes(IL-1β,TNF-α).Further in vivo study in diabetic rat models showed that sEVs@DSPE-PLLA improved the wound-healing process by alleviating the inflammatory responses,stimulating cell proliferation,collagen deposition and angiogenesis.These results highlight the potential of using DSPE-grafted scaffolds for extracellular vesicle immobilization and suggest sEVs@DSPE-PLLA micro/nanofibers as promising functional wound dressings for diabetic wounds.

Visualizing phase transition of upper critical solution temperature (UCST) polymers with AIE

The stimuli-responsive polymers with upper critical solution temperatures(UCST) are highly attractive for drug delivery applications. However, the phase transition process of UCST polymer is usually characterized by turbidity measurement and electron microscopy, which are significantly restricted by low sensitivity and static observation. In contrary, the fluorescence technique has significant advantages in terms of high sensitivity, easy operation, and dynamic observation. However, the conventional fluorophores suffer from the drawbacks of aggregation-caused quenching(ACQ) after being encapsulated by UCST polymers, which are not suitable for direct visualization of the phase transition process. To tackle this challenge, we herein developed a series of UCST polymers based on polyacrylamides decorated with bile acid and aggregation-induced emission(AIE)-active tetraphenylethene(TPE) groups, which can be used for direct fluorescence monitoring of the phase transition process. Moreover, the AIE-active UCST polymers can serve as drug carriers, which can not only monitor the drug release process under thermal stimuli, but also verify the drug release by fluorescence recovery after thermal stimuli. It is expected that the AIE-active UCST polymers with self-monitoring ability are promising for biomedical applications.