Coating of bioactive glass on magnesium alloys to improve its degradation behavior: Interfacial aspects

The preferable mechanical properties of Mg alloys along with excellent compatibility with human bone have established their applicability as implant biomaterials.However,a higher corrosion/degradation rate of Mg alloys in body fluids limits its biomedical applications.In this direction,surface modification and coating are explored as appropriate strategies to mode the degradation rate of Mg alloys.The constituents of bioactive glass(BG)provide strength,bio-inertness and bone bonding capability.Hence,researchers have explored the coating of BG on Mg alloys and investigated chemical,mechanical and biological properties of the coated alloys.In this review,we have made an attempt to compile the literature works done on the coating of BG on Mg alloys and its features.Underlying interfacial aspects of the coated substrates towards the degradation behavior are highlighted.The way forward to further improve the coating characteristics of BG coated Mg alloys are remarked.

Preparation and Characterization of the System SiO_2-CaO-P_2O_5 Bioactive Glasses by Microemulsion Approach

The system of SiO2-CaO-P2O5 bioactive glasses （BG） were successfully synthesized by microemulsion approach. X-ray diffraction （XRD）,scanning electron micro scopy（SEM） and energy dispersive X-ray （EDX） analyses, transmission electron microscopy（TEM）,Fourier transform infrared spectroscopy （FTIR）, BET N2 gas adsorption analysis techniques were utilized in order to evaluate the phase composition, dimension, morphology, interconnectivity of pores and particle size of the synthesized BG respectiveely. The biocompatibility of BG was assessed by using dimethylthiazol diphenyl tetrazolium bromide （MTT）.The BG scaffolds were implanted in rabbit mandibles and studied histologically.The results showed that the BG with a particle size less than 100 nm was prepared successfully. The measured BET specific surface area and pore volume was 113.9 m2/g and 0.28 cm3/g respectively. Cell cultures revealed that BG has been shown to have good biocompatibility and is also beneficial to the survival of Schwann cells, which can promote cell proliferation in vivo assay indicating that the BG can promote osteoconductivity.

Broad-Spectrum Antibacterial Characteristics of Four Novel Borate-Based Bioactive Glasses

Bioactive glasses have been developed for medical applications in the body for bone and tissue repair and regeneration. We have developed a borate-containing bioactive glass (13-93B3, referred to as B3), which is undergoing clinical trials to assess its wound-healing properties. To complement the healing properties of B3, metal ion dopants have been added to enhance its antimicrobial properties. Bioactive glasses doped with silver, gallium or iodine ions were found to have broad spectrum antimicrobial effects on clinically relevant bacteria including MRSA. While the B3 glass alone was sufficient to produce antibacterial effects on select bacteria, adding dopants enhanced the broad-spectrum antibacterial properties: Live-Dead staining fluorescence microscopy suggests cell membrane integrity is disrupted in gram positive bacteria exposed to the glass compounds, but not gram negative bacteria, indicating multiple mechanisms of action for each glass formulation.

Stress Corrosion Crack Growth Behavior of Titanium Alloy/Bioactive Glasses Sandwiches in Simulated Human Physiological Environment

Based on a series of newly developed bioactive glasses having suitable thermo-mechanical properties to allow application as fixation agents between bone and titanium alloy biomedical implants, the stress corrosion crack growth (SCCG) behavior of their interfaces with Ti6AI4V was investigated in simulated body fluid (SBF) with the objective of discerning the salient mechanisms of crack advance and to assess the reliability of the bonds. Results indicated that crack growth rates in Ti6AI4V/glass/Ti6AI4V sandwich specimens were nearly the same as or slightly lower than those in the bulk glasses at comparable stress intensities; indeed, cracks would prefer to propagate off the interface, suggesting that the Ti6AI4V/glass interface has relatively good crack-growth resistance. Mechanistically, interfacial crack growth appears to be controlled by the classic stress corrosion mechanisms for silicate glasses, with no discernible effect of bioactivity on the SCCG behavior being observed.

Anti-biofilm Effect of Glass Ionomer Cements Incorporated with Chlorhexidine and Bioactive Glass

The effect of glass ionomer cement and resin-modified glass ionomer cement incorporated with chlorhexidine and bioactive glass on antimicrobial activity and physicochemical properties were investigated. The experimental results showed that groups incorporated with 1% chlorhexidine exhibited a significant reduction of optical density values of the bacterial suspension and increased the degradation of Streptococcus mutans biofilm. However, groups incorporated with 10% bioactive glass did not affect the optical density values and the biofilm formation. The mechanical properties of the materials and the polymerization were not influenced by the addition of chlorhexidine. Nevertheless, the compressive strength was lower when the materials were incorporated with bioactive glass. It can be concluded that glass ionomer cements incorporated with chlorhexidine can maintain its mechanical properties as well as reduce early S mutans biofilm formation. Controlled release/sustained release technology may be required to optimize the antibacterial activity of glass ionomer cements incorporated with bioactive glass.

Enhanced Healing of Full-thickness Diabetic Wounds Using Bioactive Glass and Yunnan Baiyao Ointments

In order to accelerate the chronic wounds healing, we investigated the healing effects of bioactive glass and Yuunan baiyao ointments in streptozotocin-induced diabetic rats. The ointments were prepared by mixing 45S5 bioactive glass powder （16% weight） with Vaseline and different weight percentages of Yurman baiyao. Full-thickness defect wounds were created on the back of 130 SD rats and were randomly divided into 8 groups. The wound healing rates were calculated at 4, 7, 10, 14 and 21 days after surgery. The samples were harvested for further observations. Considering the wound closure rate, group 6 （with 5% Yuunan baiyao） has better wound healing performance than other diabetic groups. The lower inflammatory response was observed by gross observation and confirmed by the results of H＆E staining and TEM observation. Besides, the proliferation of fibroblasts, the formation of granulation tissue, as well as the vascularization, were improved in group 6 compared to other diabetic groups. All results suggest that bioactive glass and Yunnan baiyao ointments can accelerate the recovery of diabetes-impaired skin wounds, and comparing to other diabetic groups, group 6 （with 5% Yunnan baiyao） has better healing effect.

Injectable bone cements:What benefits the combination of calcium phosphates and bioactive glasses could bring?

Out of the wide range of calcium phosphate(CaP)biomaterials,calcium phosphate bone cements(CPCs)have attracted increased attention since their discovery in the 1980s due to their valuable properties such as bioactivity,osteoconductivity,injectability,hardening ability through a low-temperature setting reaction and moldability.Thereafter numerous researches have been performed to enhance the properties of CPCs.Nonetheless,low mechanical performance of CPCs limits their clinical application in load bearing regions of bone.Also,the in vivo resorption and replacement of CPC with new bone tissue is still controversial,thus further improvements of high clinical importance are required.Bioactive glasses(BGs)are biocompatible and able to bond to bone,stimulating new bone growth while dissolving over time.In the last decades extensive research has been performed analyzing the role of BGs in combination with different CaPs.Thus,the focal point of this review paper is to summarize the available research data on how injectable CPC properties could be improved or affected by the addition of BG as a secondary powder phase.It was found that despite the variances of setting time and compressive strength results,desirable injectable properties of bone cements can be achieved by the inclusion of BGs into CPCs.The published data also revealed that the degradation rate of CPCs is significantly improved by BG addition.Moreover,the presence of BG in CPCs improves the in vitro osteogenic differentiation and cell response as well as the tissue-material interaction in vivo.

A bioactive glass functional hydrogel enhances bone augmentation via synergistic angiogenesis,self-swelling and osteogenesis

Bone augmentation materials usually cannot provide enough new bone for dental implants due to the material degradation and mucosal pressure.The use of hydrogels with self-swelling properties may provide a higher bone augmentation,although swelling is generally considered to be a disadvantage in tissue engineering.Herein,a double-crosslinked gelatin-hyaluronic acid hydrogels(GH)with self-swelling properties were utilized.Meanwhile,niobium doped bioactive glasses(NbBG)was dispersed in the hydrogel network to prepare the GH-NbBG hydrogel.The composite hydrogel exhibited excellent biocompatibility and the addition of NbBG significantly improved the mechanical properties of the hydrogel.In vivo results found that GH-NbBG synergistically promoted angiogenesis and increased bone augmentation by self-swelling at the early stage of implantation.In addition,at the late stage after implantation,GH-NbBG significantly promoted new bone formation by activating RUNX2/Bglap signaling pathway.Therefore,this study reverses the self-swelling disadvantage of hydrogels into advantage and provides novel ideas for the application of hydrogels in bone augmentation.

Biomineralization inspired 3D printed bioactive glass nanocomposite scaffolds orchestrate diabetic bone regeneration by remodeling micromilieu

TypeⅡdiabetes mellitus(TIIDM)remains a challenging clinical issue for both dentists and orthopedists.By virtue of persistent hyperglycemia and altered host metabolism,the pathologic diabetic micromilieu with chronic inflammation,advanced glycation end products accumulation,and attenuated biomineralization severely impairs bone regeneration efficiency.Aiming to“remodel”the pathologic diabetic micromilieu,we 3D-printed bioscaffolds composed of Sr-containing mesoporous bioactive glass nanoparticles(Sr-MBGNs)and gelatin methacrylate(GelMA).Sr-MBGNs act as a biomineralization precursor embedded in the GelMA-simulated extracellular matrix and release Sr,Ca,and Si ions enhancing osteogenic,angiogenic,and immunomodulatory properties.In addition to angiogenic and anti-inflammatory outcomes,this innovative design reveals that the nanocomposites can modulate extracellular matrix reconstruction and simulate biomineralization by activating lysyl oxidase to form healthy enzymatic crosslinked collagen,promoting cell focal adhesion,modulating osteoblast differentiation,and boosting the release of OCN,the noncollagenous proteins(intrafibrillar mineralization dependent),and thus orchestrating osteogenesis through the Kindlin-2/PTH1R/OCN axis.This 3D-printed bioscaffold provides a multifunctional biomineralization-inspired system that remodels the“barren”diabetic microenvironment and sheds light on the new bone regeneration approaches for TIIDM.

Bioadaptable bioactive glass-β-tricalcium phosphate scaffolds with TPMS-gyroid structure by stereolithography for bone regeneration

Bone defect repair remains a troubling problem in clinical orthopedics,which involves complex biological processes.Calcium phosphates(CaPs)have been widely used owing to their advantage of biocompatibility.However,single component and traditional fabrication methods cannot meet the requirements of bioadaptability during the tissue repair process.In this work,0%,5%,15%,25%wt%of BG-TCP(bioactive glass-β-tricalcium phosphate)bioresorbable scaffolds with triply-periodic minimal surfaces(TPMS)-gyroid structure were prepared by the stereolithography(SLA)technology.TPMS-gyroid structure provided an accurate mimicry of natural bone tissue,and the incorporation of BG improved the compressive strength ofβ-TCP matrix,matched with the defective bone(2–12 MPa).Rapid but tunable degradation kinetics(compared with pure TCP)of BG enabled the BG-TCP system to sh8ow adaptable biodegradability to new bone generation.In vitro studies have shown that composite scaffolds have better mechanical properties(7.82 MPa),and can released appropriate contents of calcium,phosphorous,and magnesium ions,which promoted the osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)and angiogenic ability of endothelial progenitor cells(EPCs).Moreover,the in vivo assessment of rat femoral defect revealed that TPMS-structure-based TCP scaffolds accelerated bone ingrowth to the pores.Moreover,BG-TCP scaffolds,especially 15BG-TCP group,exhibited superior bone regeneration capacity at both 4 and 8 weeks,which achieved an optimal match between the rate of material degradation and tissue regeneration.In summary,this study provides insight into influences of bioactive components(BG)and bionic structures(TPMS)on the physical-chemical properties of materials,cell behavior and tissue regeneration,which offers a promising strategy to design bioadaptive ceramic scaffolds in the clinical treatment of bone defects.

Treatment of critical bone defects using calcium phosphate cement and mesoporous bioactive glass providing spatiotemporal drug delivery

Calcium phosphate cements (CPC) are currently widely used bone replacement materials with excellent bioactivity, but have considerable disadvantages like slow degradation. For critical-sized defects, however, an improved degradation is essential to match the tissue regeneration, especially in younger patients who are still growing. We demonstrate that a combination of CPC with mesoporous bioactive glass (MBG) particles led to an enhanced degradation in vitro and in a critical alveolar cleft defect in rats. Additionally, to support new bone formation the MBG was functionalized with hypoxia conditioned medium (HCM) derived from rat bone marrow stromal cells. HCM-functionalized scaffolds showed an improved cell proliferation and the highest formation of new bone volume. This highly flexible material system together with the drug delivery capacity is adaptable to patient specific needs and has great potential for clinical translation.

Multi-layer-structured bioactive glass nanopowder for multistage-stimulated hemostasis and wound repair

Current treatments for full-thickness skin injuries are still unsatisfactory due to the lack of hierarchically stimulated dressings that can integrate the rapid hemostasis,inflammation regulation,and skin tissue remodeling into the one system instead of single-stage boosting.In this work,a multilayer-structured bioactive glass nanopowder(BGN@PTE)is developed by coating the poly-tannic acid andε-polylysine onto the BGN via facile layer-by-layer assembly as an integrative and multilevel dressing for the sequential management of wounds.In comparison to BGN and poly-tannic acid coated BGN,BGN@PTE exhibited the better hemostatic performance because of its multiple dependent approaches to induce the platelet adhesion/activation,red blood cells(RBCs)aggregation and fibrin network formation.Simultaneously,the bioactive ions from BGN facilitate the regulation of the inflammatory response while the poly-tannic acid and antibacterialε-polylysine prevent the wound infection,promoting the wound healing during the inflammatory stage.In addition,BGN@PTE can serve as a reactive oxygen species scavenger,alleviate the oxidation stress in wound injury,induce the cell migration and angiogenesis,and promote the proliferation stage of wound repair.Therefore,BGN@PTE demonstrated the significantly higher wound repair capacity than the commercial bioglass dressing Dermlin™.This multifunctional BGN@PTE is a potentially valuable dressing for full-thickness wound management and may be expected to extend to the other wounds therapy.

Superior biocompatibility and comparable osteoinductive properties: Sodium-reduced fluoride-containing bioactive glass belonging to the CaO–MgO–SiO2 system as a promising alternative to 45S5 bioactive glass

Bioactive glasses(BGs)are promising bone substitute materials.However,under certain circumstances BGs such as the well-known 45S5 Bioglass®(composition in wt%:45.0 SiO2,24.5 Na2O,24.5 CaO,6.0 P2O5)act cytotoxic due to a strong increase in pH caused by a burst release of sodium ions.A potential alternative is a sodiumreduced fluoride-containing BG belonging to the CaO–MgO–SiO2 system,namely BG1d-BG(composition in wt%:46.1 SiO2,28.7 CaO,8.8 MgO,6.2 P2O5,5.7 CaF2,4.5 Na2O),that has already been evaluated in-vitro,in-vivo and in preliminary clinical trials.Before further application,however,BG1d-BG should be compared to the benchmark amongst BGs,the 45S5 Bioglass®composition,to classify its effect on cell viability,proliferation and osteogenic differentiation of human mesenchymal stem cells(MSCs).Therefore,in this study,the biocompatibility and osteogenic potential of both BGs were investigated in an indirect and direct culture setting to assess the effect of the ionic dissolution products and the BGs’physical presence on the cells.The results indicated an advantage of BG1d-BG over 45S5 Bioglass®regarding cell viability and proliferation.Both BGs induced an earlier onset of osteogenic differentiation and accelerated the expression of late osteoblast marker genes compared to the control group.In conclusion,BG1d-BG is an attractive candidate for further experimental investigation.The basic mechanisms behind the different impact on cell behavior should be assessed in further detail,e.g.by further alteration of the BG compositions.

Incorporation of bioactive glass nanoparticles in electrospun PCL/chitosan fibers by using benign solvents

The use of bioactive glass(BG)particles as a filler for the development of composite electrospun fibers has already been widely reported and investigated.The novelty of the present research work is represented by the use of benign solvents(like acetic acid and formic acid)for electrospinning of composite fibers containing BG particles,by using a blend of PCL and chitosan.In this work,different BG particle sizes were investigated,namely nanosized and micron-sized.A preliminary investigation about the possible alteration of BG particles in the electrospinning solvents was performed using SEM analysis.The obtained composite fibers were investigated in terms of morphological,chemical and mechanical properties.An in vitro mineralization assay in simulated body fluid(SBF)was performed to investigate the capability of the composite electrospun fibers to induce the formation of hydroxycarbonate apatite(HCA).

Bioactive glass coatings on metallic implants for biomedical applications

Metallic implant materials possess adequate mechanical properties such as strength,elastic modulus,and ductility for long term support and stability in vivo.Traditional metallic biomaterials,including stainless steels,cobalt-chromium alloys,and titanium and its alloys,have been the gold standards for load-bearing implant materials in hard tissue applications in the past decades.Biodegradable metals including iron,magnesium,and zinc have also emerged as novel biodegradable implant materials with different in vivo degradation rates.However,they do not possess good bioactivity and other biological functions.Bioactive glasses have been widely used as coating materials on the metallic implants to improve their integration with the host tissue and overall biological performances.The present review provides a detailed overview of the benefits and issues of metal alloys when used as biomedical implants and how they are improved by bioactive glass-based coatings for biomedical applications.

Multifunctional zinc ion doped sol-gel derived mesoporous bioactive glass nanoparticles for biomedical applications

Mesoporous bioactive glasses have been widely investigated for applications in bone tissue regeneration and,more recently,in soft tissue repair and wound healing.In this study we produced mesoporous bioactive glass nanoparticles(MBGNs)based on the SiO2-CaO system.With the intention of adding subsidiary biological function,MBGNs were doped with Zn2+ions.Zn-MBGNs with 8 mol%ZnO content were synthesized via microemulsion assisted sol-gel method.The synthesized particles were homogeneous in shape and size.They exhibited spherical shape,good dispersity,and a size of 130±10 nm.The addition of zinc precursors did not affect the morphology of particles,while their specific surface area increased in comparison to MBGNs.The presence of Zn2+ions inhibited the formation of hydroxycarbonate apatite(HCAp)on the particles after immersion in simulated body fluid(SBF).No formation of HCAp crystals on the surface of Zn-MBGNs could be observed after 14 days of immersion.Interestingly,powders containing relatively high amount of zinc released Zn2+ions in low concentration(0.6-1.2 mg L^−1)but in a sustained manner.This releasing feature enables Zn-MBGNs to avoid potentially toxic levels of Zn2+ions,indeed Zn-MBGNs were seen to improve the differentiation of osteoblast-like cells(MG-63).Additionally,Zn-MBGNs showed higher ability to adsorb proteins in comparison to MBGNs,which could indicate a favourable later attachment of cells.Due to their advantageous morphological and physiochemical properties,Zn-MBGNs show great potential as bioactive fillers or drug delivery systems in a variety of applications including bone regeneration and wound healing.

In-vitro bioactivity evaluation and physical properties of an epoxy-based dental sealer reinforced with synthesized fluorine-substituted hydroxyapatite, hydroxyapatite and bioactive glass nanofillers

The purpose of this study was to evaluate the physical properties and bioactivity potential of epoxy-based dental sealers modified with synthesized bioactive glass(BAG),hydroxyapatite(HA)and fluorine substituted hydroxyapatite(FHA)nanoparticles.The synthesized powders were incorporated at 10%and 20%into the epoxy-based dental sealer.The setting time,flow and solubility and microhardness of the modified and unmodified samples were examined.The bioactivity was evaluated using FESEM-EDX and elemental mapping,ATR-FTIR and XRD.The flow value of all of the experimental groups except the FHA modified samples,was greater than 20 mm.Concerning solubility,no specimens exhibited more than 1%weight loss.The solubility value of the FHA groups was statistically significant lower than other groups(p≤0.001).The mean hardness values of all of the modified samples were significantly higher than the unmodified group(p≤0.001).Regarding bioactivity,in vitro study revealed that after 3 days immersion in SBF a compact and continuous calcium phosphate layer formed on the surface of epoxy sealers containing BAG and HA nanoparticles.Based on these results,the addition of BAG and HA nanoparticles did not adversely alter the physical properties of epoxy sealers.Additionally,they improved the in vitro bioactivity of the epoxy sealer.

Effect of solution condition on hydroxyapatite formation in evaluating bioactivity of B2O3 containing 45S5 bioactive glasses

The effects of testing solutions and conditions on hydroxyapatite(HAp)formation as a means of in vitro bioactivity evaluation of B2O3 containing 45S5 bioactive glasses were systematically investigated.Four glass samples prepared by the traditional melt and quench process,where SiO2 in 45S5 was gradually replaced by B2O3(up to 30%),were studied.Two solutions:the simulated body fluid(SBF)and K2HPO4 solutions were used as the medium for evaluating in vitro bioactivity through the formation of HAp on glass surface as a function of time.It was found that addition of boron oxide delayed the HAp formation in both SBF and K2HPO4 solutions,while the reaction between glass and the K2HPO4 solution is much faster as compared to SBF.In addition to the composition and medium effects,we also studied whether the solution treatments(e.g.,adjusting to maintain a pH of 7.4,refreshing solution at certain time interval,and no disturbance during immersion)affect HAp formation.Fourier transform infrared spectrometer(FTIR)equipped with an attenuated total reflection(ATR)sampling technique and scanning electron microscopy(SEM)were conducted to identify HAp formation on glass powder surfaces and to observe HAp morphologies,respectively.The results show that refreshing solution every 24 h produced the fastest HAp formation for low boron-containing samples when SBF was used as testing solution,while no significant differences were observed when K2HPO4 solution was used.This study thus suggests the testing solutions and conditions play an important role on the in vitro bioactivity testing results and should be carefully considered when study materials with varying bioactivities.

Reducing relapse and accelerating osteogenesis in rapid maxillary expansion using an injectable mesoporous bioactive glass/fibrin glue composite hydrogel

Rapid maxillary expansion(RME),as a common treatment for craniomaxillofacial deformity,faces the challenge of high relapse rates and unsatisfactory therapeutic effects.In this study,a standardized Sprague-Dawley(SD)rat RME model was first established with a modified expander as well as retainer design and optimized anterior maxillary expanding force of 100 g which exerted the most synchronized mobility of mid-palatal suture and incisors.Via the standardized model,the high relapse rate was proven to be attributed to insufficient osteogenesis in expanded suture,requiring long-term retainer wearing in clinical situations.To reduce the relapse rate,mesoporous bioactive glass/fibrin glue(MBG/FG)composite hydrogels were developed for an in situ minimal invasive injection that enhance osteogenesis in the expanded palate.The component of 1 wt%MBG was adopted for enhanced mechanical strength,matched degradation rate and ion dissolution,excellent in vitro biocompatibility and osteoinductivity.Effects of 1%MBG/FG composite hydrogel on osteogenesis in expanded mid-palatal sutures with/without retention were evaluated in the standardized model.The results demonstrated that injection of 1%MBG/FG composite hydrogel significantly promoted bone formation within the expanded mid-palatal suture,inhibited osteoclastogenesis and benefited the balance of bone remodeling towards osteogenesis.Combination of retainer and injectable biomaterial was demonstrated as a promising treatment to reduce relapse rate and enhance osteogenesis after RME.The model establishment and the composite hydrogel development in this article might provide new insight to other craniomaxillofacial deformity treatment and design of bone-repairing biomaterials with higher regenerative efficiency.

Antibacterial,pro-angiogenic and pro-osteointegrative zein-bioactive glass/copper based coatings for implantable stainless steel aimed at bone healing

Stainless steel implants are suitable candidates for bone replacement due to their cytocompatibility and mechanical resistance,but they suffer from lack of bioactivity and are prone to bacterial infections.Accordingly,to overcome those limitations,in this study we developed by electrophoretic deposition(EPD),an innovative surface coating made of(i)zein,a natural fibroblast-friendly polymer,(ii)bioactive glass,a pro-osteogenic inorganic material and(iii)copper containing bioactive glass,an antibacterial and pro-angiogenic material.FESEM images confirmed that porous,uniform and free of cracks coatings were obtained by EPD;moreover,coatings were resistant to mechanical stress as demonstrated by the tape test,resulting in a 4B classification of adhesion to the substrate.The coatings were cytocompatible as indicated by metabolism evaluation of human fibroblasts,endothelial cells and mature or progenitor osteoblasts cultivated in direct contact with the specimens.They also maintained pro-osteogenic properties towards undifferentiated progenitor cells that expressed osteogenic genes after 15 days of direct cultivation.Copper conferred antibacterial properties as biofilm formation of the joint pathogens Staphylococcus aureus,Staphylococcus epidermidis and Escherichia coli was significantly reduced in comparison with copper-free coatings(p<0.05).Moreover,this anti-infective activity resulted as targeted towards bacteria while the cells viability was preserved when cells and bacteria were cultivated in the same environment by a co-culture assay.Finally,copper ability to recruit blood vessels and to inhibit bacterial infection was confirmed in vivo where the growth of S.aureus biofilm was inhibited and the formation of new(<50μm diameter spread)blood vessels was observed.