In vitro and in vivo studies on pure Mg, Mg-1Ca and Mg-2Sr alloys processed by equal channel angular pressing

In the present work,the biomedical as-cast pure Mg,Mg–1 Ca and Mg–2 Sr alloys were processed with equal channel angular pressing(ECAP)technique to develop ultrafine microstructure within the materials,and their microstructures,mechanical properties,degradation behavior,cytocompatibility in vitro and biocompatibility in vivo were studied comprehensively.Finer-gained microstructures and improved mechanical properties of these three materials after ECAP were confirmed compared to their as-cast counterparts.Moreover,after ECAP the degradation rate of pure Mg was increased while that of Mg–1 Ca or Mg–2 Sr alloys decreased compared to the ascast counterparts.Additionally,good in vitro cytocompatibility and in vivo biocompatibility of these three materials were revealed by cell cultural tests using osteoblastic MC3 T3-E1 and human mesenchymal stem cells(h MSC)and in vivo animal tests at the lateral epicondyle of SD-rats’femur.This study offers an alternative powerful avenue to achieve good comprehensive properties of magnesium-based biodegradable metals.It might also help to extend the applied range of magnesium-based biodegradable metals in orthopedic field.

Tailoring the multiscale mechanics of tunable decellularized extracellular matrix (dECM) for wound healing through immunomodulation

With the discovery of the pivotal role of macrophages in tissue regeneration through shaping the tissue immune microenvironment, various immunomodulatory strategies have been proposed to modify traditional biomaterials. Decellularized extracellular matrix (dECM) has been extensively used in the clinical treatment of tissue injury due to its favorable biocompatibility and similarity to the native tissue environment. However, most reported decellularization protocols may cause damage to the native structure of dECM, which undermines its inherent advantages and potential clinical applications. Here, we introduce a mechanically tunable dECM prepared by optimizing the freeze-thaw cycles. We demonstrated that the alteration in micromechanical properties of dECM resulting from the cyclic freeze-thaw process contributes to distinct macrophage-mediated host immune responses to the materials, which are recently recognized to play a pivotal role in determining the outcome of tissue regeneration. Our sequencing data further revealed that the immunomodulatory effect of dECM was induced via the mechnotrasduction pathways in macrophages. Next, we tested the dECM in a rat skin injury model and found an enhanced micromechanical property of dECM achieved with three freeze-thaw cycles significantly promoted the M2 polarization of macrophages, leading to superior wound healing. These findings suggest that the immunomodulatory property of dECM can be efficiently manipulated by tailoring its inherent micromechanical properties during the decellularization process. Therefore, our mechanics-immunomodulation-based strategy provides new insights into the development of advanced biomaterials for wound healing.

Puerarin@Chitosan composite for infected bone repair through mimicking the bio-functions of antimicrobial peptides

It is important to eliminate lipopolysaccharide(LPS)along with killing bacteria in periprosthetic joint infection(PJI)therapy for promoting bone repair due to its effect to regulate macrophages response.Although natural antimicrobial peptides(AMPs)offer a good solution,the unknown toxicity,high cost and exogenetic immune response hamper their applications in clinic.In this work,we fabricated a nanowire-like composite material,named P@C,by combining chitosan and puerarin via solid-phase reaction,which can finely mimic the bio-functions of AMPs.Chitosan,serving as the bacteria membrane puncture agent,and puerarin,serving as the LPS target agent,synergistically destroy the bacterial membrane structure and inhibit its recovery,thus endowing P@C with good antibacterial property.In addition,P@C possesses good osteoimmunomodulation due to its ability of LPS elimination and macrophage differentiation modulation.The in vivo results show that P@C can inhibit the LPS induced bone destruction in the Escherichia coli infected rat.P@C exhibits superior bone regeneration in Escherichia coli infected rat due to the comprehensive functions of its superior antibacterial property,and its ability of LPS elimination and immunomodulation.P@C can well mimic the functions of AMPs,which provides a novel and effective method for treating the PJI in clinic.

Enhancement of critical-sized bone defect regeneration by magnesium oxide-reinforced 3D scaffold with improved osteogenic and angiogenic properties

The healing of critical-sized bone defects(CSD)remains a challenge in orthopedic medicine.In recent years,scaffolds with sophisticated microstructures fabricated by the emerging three-dimensional(3D)printing technology have lighted up the treatment of the CSD due to the elaborate microenvironments and support they may build.Here,we established a magnesium oxide-reinforced 3D-printed biocompos-ite scaffold to investigate the effect of magnesium-enriched 3D microenvironment on CSD repairing.The composite was prepared using a biodegradable polymer matrix,polycaprolactone(PCL),and the disper-sion phase,magnesium oxide(MgO).With the appropriate surface treatment by saline coupling agent,the MgO dispersed homogeneously in the polymer matrix,leading to enhanced mechanical performance and steady release of magnesium ion(Mg^(2+))for superior cytocompatibility,higher cell viability,advanced osteogenic differentiation,and cell mineralization capabilities in comparison with the pure PCL.The in-vivo femoral implantation and critical-sized cranial bone defect studies demonstrated the importance of the 3D magnesium microenvironment,as a scaffold that released appropriate Mg^(2+) exhibited remarkably increased bone volume,enhanced angiogenesis,and almost recovered CSD after 8-week implantation.Overall,this study suggests that the magnesium-enriched 3D scaffold is a potential candidate for the treatment of CSD in a cell-free therapeutic approach.

Corrigendum to“Magnesium cationic cue enriched interfacial tissue microenvironment nurtures the osseointegration of gamma-irradiated allograft bone”[Bioact.Mater.10C(April 2022)32-47]

The authors regret a mistake of funding numbers in the Acknowledgment Section failed to be corrected during proof reading.Below is the corrected funding statement in Acknowledgment SECTION This work was supported by the National Natural Science Foundation of China(NSFC)(Nos.81902189,81772354,82002303,31570980),Clinical Innovation Research Program of Guangzhou Regenerative Medicine and Health Guangdong Laboratory(2018GZR0201002),National Key Research and Development Plan(2018YFC1105103).

Corrigendum to‘Fabrication of a bio-instructive scaffold conferred with a favorable microenvironment allowing for superior implant osseointegration and accelerated in situ vascularized bone regeneration via type H vessel formation’[Bioactive Materials,Volume 9(March 2022)Page 491-507]

The authors regret a mistake of funding numbers in the Acknowledgment Section failed to be corrected during proofreading.Below is the corrected funding statement in ACKNOWLEDGMENT SECTION:This work was supported by the National Natural Science Foundation of China(NSFC)(Nos.82072415,81772354,81902189),Clinical Innovation Research Program of Guangzhou Regenerative Medicine and Health Guangdong Laboratory(2018GZR0201002),Science Technology Project of Guangzhou City(2019ZD15).

Bone grafts and biomaterials substitutes for bone defect repair:A review

Bone grafts have been predominated used to treat bone defects,delayed union or non-union,and spinal fusion in orthopaedic clinically for a period of time,despite the emergency of synthetic bone graft substitutes.Nevertheless,the integration of allogeneic grafts and synthetic substitutes with host bone was found jeopardized in long-term follow-up studies.Hence,the enhancement of osteointegration of these grafts and substitutes with host bone is considerably important.To address this problem,addition of various growth factors,such as bone morphogenetic proteins(BMPs),parathyroid hormone(PTH)and platelet rich plasma(PRP),into structural allografts and synthetic substitutes have been considered.Although clinical applications of these factors have exhibited good bone formation,their further application was limited due to high cost and potential adverse side effects.Alternatively,bioinorganic ions such as magnesium,strontium and zinc are considered as alternative of osteogenic biological factors.Hence,this paper aims to review the currently available bone grafts and bone substitutes as well as the biological and bio-inorganic factors for the treatments of bone defect.

Regulation of extracellular bioactive cations in bone tissue microenvironment induces favorable osteoimmune conditions to accelerate in situ bone regeneration

The design of orthopedic biomaterials has gradually shifted from“immune-friendly”to“immunomodulatory,”in which the biomaterials are able to modulate the inflammatory response via macrophage polarization in a local immune microenvironment that favors osteogenesis and implant-to-bone osseointegration.Despite the well-known effects of bioactive metallic ions on osteogenesis,how extracellular metallic ions manipulate immune cells in bone tissue microenvironments toward osteogenesis and subsequent bone formation has rarely been studied.Herein,we investigate the osteoimmunomodulatory effect of an extracellular bioactive cation(Mg^(2+))in the bone tissue microenvironment using custom-made poly lactic-co-glycolic acid(PLGA)/MgO-alendronate microspheres that endow controllable release of magnesium ions.The results suggest that the Mg^(2+)-controlled tissue microenvironment can effectively induce macrophage polarization from the M0 to M2 phenotype via the enhancement of anti-inflammatory(IL-10)and pro-osteogenic(BMP-2 and TGF-β1)cytokines production.It also generates a favorable osteoimmune microenvironment that facilitates the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells.The in vivo results further verify that a large amount of bony tissue,with comparable bone mineral density and mechanical properties,has been generated at an early post-surgical stage in rat intramedullary bone defect models.This study demonstrates that the concept of in situ immunomodulated osteogenesis can be realized in a controlled magnesium tissue microenvironment.

Controlled release and biocompatibility of polymer/titania nanotube array system on titanium implants

Bacterial infection and tissue inflammation are the major causes of early failure of titanium-based orthopedic implants;thus,surgical implants with tunable drug releasing properties represent an appealing way to address some of these problems of bacterial infection and tissue inflammation in early age of orthopedic implants.In this work,a hybrid surface system composed of biodegradable poly(lactic-coglycolic acid)(PLGA)and titania nanotubes(TNTs)has been successfully constructed on Ti implants with the aim of preventing bacterial infection via long-term drug release.By varying the size of the TNTs and the thickness of the polymer film,the drug release profile can be tuned to achieve the optimal therapeutic action throughout the treatment time.The size of TNTs plays a dominant role in the drug loading dose of TNTs/PLGA hybrid coatings.In this work,TNTs with an average size of 80 nm can achieve the largest loading dose.Depending on the polymer thickness,significant improvement in the drug release characteristics is attained,for instance,reduced burst release(from 84%to 27%)and overall release time extended from 5 to over 40 days.In addition,the PLGA layers may favor the proliferation and osteogenesis of MC3T3-E1 mouse cells at an earlier stage.Therefore,this TNT/PLGA hybrid surface system can be employed as an effective bioplatform for improving both self-antibacterial performance and biocompatibility of Ti-based biomaterials.

Stepwise 3D-spatio-temporal magnesium cationic niche: Nanocomposite scaffold mediated microenvironment for modulating intramembranous ossification

The fate of cells and subsequent bone regeneration is highly correlated with temporospatial coordination of chemical,biological,or physical cues within a local tissue microenvironment.Deeper understanding of how mammalian cells react to local tissue microenvironment is paramount important when designing next generation of biomaterials for tissue engineering.This study aims to investigate that the regulation of magnesium cationic(Mg^2+)tissue microenvironment is able to convince early-stage bone regeneration and its mechanism undergoes intramembranous ossification.It was discovered that moderate Mg^2+content niche(~4.11 mM)led to superior bone regeneration,while Mg^2+-free and strong Mg^2+content(~16.44 mM)discouraged cell adhesion,proliferation and osteogenic differentiation,thereby bone formation was rarely found.When magnesium ions diffused into free Mg zone from concentrated zone in late time point,new bone formation on free Mg zone became significant through intramembranous ossification.This study successfully demonstrates that magnesium cationic microenvironment serves as an effective biochemical cue and is able to modulate the process of bony tissue regeneration.The knowledge of how a Mg^2+cationic microenvironment intertwines with cells and subsequent bone formation gained from this study may provide a new insight to develop the next generation of tissuerepairing biomaterials.

Nanograins on Ti-25Nb-3Mo-2Sn-3Zr alloy facilitate fabricating biological surface through dual-ion implantation to concurrently modulate the osteogenic functions of mesenchymal stem cells and kill bacteria

Surface mechanical attrition treatment(SMAT)method is an effective way to generate nanograined(NG)surface on Ti-25 Nb-3 Mo-2 Sn-3 Zr(wt.%)(named as TLM),a kind ofβ-type titanium alloy,and the achieved nanocrystalline surface was proved to promote positive functions of osteoblastic cells.In this work,to further endow the NG TLM alloy with both good osteogenic and antibacterial properties,magnesium(Mg),silver(Ag)ion or both were introduced onto the NG TLM surface by ion implantation process,as a comparison,the Mg and Ag ions were also co-implanted onto coarsegrained(CG)TLM surface.The obtained results show that subsequent ion implantation does not remarkably induce the surface roughness and topography alteration of the SMAT-treated layers,and it also has little impact on the microstructure of the SMAT-derivedβ-Ti nanograins.In addition,the implanted Mg and Ag ions are observed to exist as MgO and metallic Ag na noparticles(NPs)embedding tightly in theβ-Ti matrix with grain size of about 15 and 7 nm,respectively.Initial cell adhesion and functions(including proliferation,osteo-differentiation and extracellular matrix mineralization)of rabbit bone marrow mesenchymal stem cells(rBMMSCs)and the bacterial colonization of Staphylococcus aureus(S.aureus)on the different surfaces were investigated.The in-vitro experimental results reveal that the Mg and Ag single-ion implanted NG surface either significantly promotes the rBMMSCs response or inhibits the growth ofS.aureus,whereas the Mg/Ag coimplanted NG surface could concurrently enhance the rBMMSCs functions as well as inhibit the bacterial growth compared to the NG surface,and this efficacy is more pronounced as compared to the Mg/Ag co-implantation in the CG surface.The SMAT-achieved nanograins in the TLM surface layer are identified to not only play a leading role in determining the fate of rBMMSCs but also facilitate fabricating dualfunctio nal surface with both good osteogenic and antibacterial activities through co-implantation of Mg and Ag ions.Our investigation provides a new strategy to develop high-performance Ti-based implants for clinical application.

Fabrication of a bio-instructive scaffold conferred with a favorable microenvironment allowing for superior implant osseointegration and accelerated in situ vascularized bone regeneration via type H vessel formation

The potential translation of bio-inert polymer scaffolds as bone substitutes is limited by the lack of neovascularization upon implantation and subsequently diminished ingrowth of host bone,most likely resulted from the inability to replicate appropriate endogenous crosstalk between cells.Human umbilical vein endothelial cell-derived decellularized extracellular matrix(HdECM),which contains a collection of angiocrine biomolecules,has recently been demonstrated to mediate endothelial cells(ECs)-osteoprogenitors(OPs)crosstalk.We employed the HdECM to create a PCL(polycaprolactone)/fibrin/HdECM(PFE)hybrid scaffold.We hypothesized PFE scaffold could reconstitute a bio-instructive microenvironment that reintroduces the crosstalk,resulting in vascularized bone regeneration.Following implantation in a rat femoral bone defect,the PFE scaffold demonstrated early vascular infiltration and enhanced bone regeneration by microangiography(μ-AG)and micro-computational tomography(μ-CT).Based on the immunofluorescence studies,PFE mediated the endogenous angiogenesis and osteogenesis with a substantial number of type H vessels and osteoprogenitors.In addition,superior osseointegration was observed by a direct host bone-PCL interface,which was likely attributed to the formation of type H vessels.The bio-instructive microenvironment created by our innovative PFE scaffold made possible superior osseointegration and type H vessel-related bone regeneration.It could become an alternative solution of improving the osseointegration of bone substitutes with the help of induced type H vessels,which could compensate for the inherent biological inertness of synthetic polymers.

Magnesium cationic cue enriched interfacial tissue microenvironment nurtures the osseointegration of gamma-irradiated allograft bone

Regardless of the advancement of synthetic bone substitutes,allograft-derived bone substitutes still dominate in the orthopaedic circle in the treatments of bone diseases.Nevertheless,the stringent devitalization process jeopardizes their osseointegration with host bone and therefore prone to long-term failure.Hence,improving osseointegration and transplantation efficiency remains important.The alteration of bone tissue microenvironment(TME)to facilitate osseointegration has been generally recognized.However,the concept of exerting metal ionic cue in bone TME without compromising the mechanical properties of bone allograft is challenging.To address this concern,an interfacial tissue microenvironment with magnesium cationc cue was tailored onto the gamma-irradiated allograft bone using a customized magnesium-plasma surface treatment.The formation of the Mg cationic cue enriched interfacial tissue microenvironment on allograft bone was verified by the scanning ion-selective electrode technique.The cellular activities of human TERT-immortalized mesenchymal stem cells on the Mg-enriched grafts were notably upregulated.In the animal test,superior osseointegration between Mg-enriched graft and host bone was found,whereas poor integration was observed in the gamma-irradiated controls at 28 days post-operation.Furthermore,the bony in-growth appeared on magnesium-enriched allograft bone was significant higher.The mechanism possibly correlates to the up-regulation of integrin receptors in mesenchymal stem cells under modified bone TME that directly orchestrate the initial cell attachment and osteogenic differentiation of mesenchymal stem cells.Lastly,our findings demonstrate the significance of magnesium cation modified bone allograft that can potentially translate to various orthopaedic procedures requiring bone augmentation.

In vitro and in vivo antibacterial performance of Zr&O PIII magnesium alloys with high concentration of oxygen vacancies

The effects of dual Zr and O plasma immersion ion implantation(Zr&O PIII)on antibacterial properties of ZK60 Mg alloys are systematically investigated.The results show that a hydrophobic,smooth,and ZrO_(2)-containing graded film is formed.Electrochemical assessment shows that the corrosion rate of the plasma-treated Mg alloy decreases and the decreased degradation rate is attributed to the protection rendered by the surface oxide.In vitro and in vivo antibacterial tests reveal Zr&O PIII ZK60 presents higher antibacterial rate compared to Zr PIII ZK60 and untreated control.The hydrophobic and smooth surface suppresses bacterial adhesion.High concentration of oxygen vacancies in the surface films are determined by X-ray photoelectron spectroscopy(XPS),UV-vis diffuse reflectance spectra(UV-vis DRS)and electron paramagnetic resonance(EPR)and involved in the production of reactive oxygen species(ROS).The higher level of ROS expression inhibits biofilm formation by down-regulating the expression of icaADBC genes but up-regulating the expression of icaR gene.In addition,Zr&O PIII improves cell viability and initial cell adhesion confirming good cytocompatibility.Dual Zr&O PIII is a simple and practical means to expedite clinical acceptance of biodegradable magnesium alloys.