In vitro study of the PLLA-Mg65Zn30Ca5 composites as potential biodegradable materials for bone implants

Tolei Wng ,Cho Lin ,Dn Btlu ,Lu Zhng ,Jingzhou Hu ,Wei Lu,?

aInstitute for Regenerative Medicine,Shanghai East Hospital,School of Materials Science and Engineering,Tongji University,Shanghai 200123,China

b Department of Oral &Maxillofacial-Head &Neck Oncology,Ninth People’s Hospital,Shanghai Key Laboratory of Stomatology,Shanghai Jiao Tong University School of Medicine,Shanghai Research Institute of Stomatology,National Clinical Research Center of Stomatology,Shanghai 200011,China

cUniversity Politehnica of Bucharest,Splaiul Independentei 313,Bucharest 060042,Romania

d Shanghai Key Lab of D&A for Metal Functional Materials,Institute for Regenerative Medicine,Shanghai East Hospital,School of Life Science and Technology,Tongji University,Shanghai 200123,China

e School of Stomatology,Weifang Medical University,Weifang 261000,China

Abstract PLLA-magnesium composites have been widely investigated as potential biodegradable materials for bone implants.Lower/higher corrosion resistance of the crystalized/amorphous magnesium alloys allows tailoring of biodegradability rate.In this work,the amorphous Mg65Zn30Ca5 was investigated versus traditional crystalized Mg65Zn30Ca5,and a PLLA-Mg65Zn30Ca5 composite has been successfully fabricated using hot injection process.Furthermore,the high corrosion resistance of the amorphous Mg65Zn30Ca5 prevented the high alkalization and deterioration of mechanical strength.In addition,the high Zn content intended to improve the glass forming ability,also enhances the anti-bacterial property of the PLLA-Mg65Zn30Ca5 composite.The remarkable performance of the PLLA-Mg65Zn30Ca5 composite shows its promising application in bone repair and tissue regeneration.

Keywords: Polymer-matrix composites(PMCs);Magnesium glasses;Corrosion;Mechanical properties.

1.Introduction

Biodegradable materials are extensively studied for bone implants and stents [1].Comparing to permanent implants,the biodegradable ones can be completely degraded in/by the human body,avoiding a secondary surgery [2,3].Numerous biodegradable materials,including polymers,magnesium alloys,zinc alloys,calcium alloys,and ceramics [1,4,5]have been developed for biomedical applications.Comparing to the metallic alloys or the ceramics,the polymer have low cost and can be easily shaped into complex geometries through hot injection [6]or 3D printing [7,8],technologies favorable for the medical applications.

Among numerous polymers,poly(L-lactic)acid(PLLA)is a promising and versatile biodegradable polymer [9].However,the application of PLLA in clinical practice has been limited by its hydrophobicity,causing inadequate biocompatibility and poor antibacterial activity [10].In addition,the mechanical strength of the PLLA requires further improvement to meet the clinical demand [11].In the past years,additives like hydroxyapatite [12,13],graphite oxide(GO)[14,15],zinc oxide [14],magnesium oxide [12,16]and magnesium [7,17–19]have been already used to improve the performance of PLLA or other polymers.The addition of magnesium shows improvement in the osteoconductivity[7,18,20]and the mechanical strength [21]of the polymers.For example,Wu et al.[20]prepared the AZ31 magnesium alloy fibers/ PLGA composites and found that addition of magnesium alloy fibers can enhance the cell attachment and neutralized the acid degradation products of PLGA.Yu et al.[17]prepared the PLLA-Mg composites and revealed the enhanced cell growth,adhesion and ALP activity with the addition of Mg particles.Additionally,the degradation of Mg can buffer the acid degradation of the polymer [18].

Still,it is worth to mention that the degradation of Mg is relatively fast and could lead to local alkalization,also producing hydrogen gas pockets,which is not favorable for the healing process [22].Additionally,the mechanical strength of the PLLA-Mg composites would decrease with degradation of magnesium [23],increasing the risk of implant failure.Extensive research was focused on improving the corrosion resistance of the magnesium and its alloys,including the alloying technique and the surface modification[22].Traditional alloying technique had limited improvement and the surface modification might not be efficient[24]during the implantation.However,some researches show that rapid quenching technique can produce Mg-base metallic glass with long-term corrosion resistance[25,26],which is better than the traditional alloying [27–29]and coating techniques [30–33].The main obstacle in the clinical application of the Mg-base glass is the small size,due to the limited formability [34]of complex shapes.However,the combination of the Mg-base glass and the polymers could avoid this limitation,the polymers being easily fabricated into bone implants with large size and complex shapes[7],hence the addition of the Mg-based glass will tailor the properties and behavior of the composite.

The magnesium glasses from the systems Mg-Zn-Ca,Mg-Cu-Y,Mg-Ni-Sn,Mg-Cu-Ag-Er and Mg-Cu-Sn were successfully developed[35].However,rare earth elements and heavy metals might be harmful to human body,therefore,MgZnCabased glass is a proper candidate for medical implants,since both Zn and Ca are traced elements in human body[35,36].In addition,the antibacterial activity of the PLLA is relatively poor and requires further improvement [14,37].The Zn2+ions released during the degradation of Mg-Zn-Ca alloys provide antibacterial activity for the composite [14,38].Furthermore,the existence of Zn forms a dense oxidation layer on the surface,which could prevents the later corrosion [25,39].Therefore,in this paper,MgZnCa-based magnesium glasses were prepared and assessed as additives for PLLA.Corrosion resistance,degradation behavior,mechanical strength during the degradation andin vitrobiological assessments were performed.

2.Materials preparation and characterization

2.1.Preparation of the amorphous Mg65Zn30Ca5 ribbons

Pure Mg ingots(>99.9%)and pure Zn ingots(>99.9%)were purchased from Northeast Nonferrous Metals Co.,Ltd,(Shenyang,China).Pure Ca powder(>99.0%)was purchased from Greagent,Shanghai,China.After polishing and ultrasonic wash in alcohol,the Mg ingots,Zn ingots and Ca particles were weighed and transferred into in a graphite crucible for the melting process.Mg65Zn30Ca5(at.%)alloy was obtained in an induction melting furnace,under argon atmosphere.During the melting process,the molten state was maintained for 15 min and then poured in to a copper mold.The obtained Mg65Zn30Ca5ingot was shattered and transferred into a quartz crucible for the melt-spin process.Amorphous ribbons of Mg65Zn30Ca5were fabricated by ejecting the molten alloy onto the surface of a rotating copper wheel under argon atmosphere.The copper wheel surface velocity was 30 m/s.The melt-spun ribbons were manually ground in an agate mortar,and the obtained powder was sieved through a 100-mesh sieve.A small quantity of the as-quenched ribbons was annealed for recrystallization at 350 °C for 2 h,and it was used as a control sample.

2.2.Preparation of the PLLA-Mg65Zn30Ca5 composites

The schematic preparation diagram of the composites is shown in Fig.1.Two compositions were obtained by mixing the sieved powder of Mg65Zn30Ca5(5 and 10 wt.%)with PLLA powder.The mixture was then dissolved in dichloromethane,with a 10:90 wt.% ratio of PLLA:dichloromethane.The obtained suspension was continuously stirred for 30 min to ensure the uniform distribution of the Mg65Zn30Ca5particles in the dissolved PLLA.After stirring,the colloid was immediately poured onto a 30×30 cm2square Teflon plate and placed in the fume hood for 12 h to evaporate the solvent and obtain a dry PLLA-MgZnCa film.Afterwards,the obtained film was cut into small pieces and transferred into a WZS10D Miniature Injection Molding Machine.The as-casted PLLA-Mg65Zn30Ca5pieces were porous due to the dichloromethane evaporation.The as-casted PLLA-Mg65Zn30Ca5pieces were transferred into the mold and heated to 180°C for 5 min.During the heating process,a stainless steel was used to eliminate the pores in the PLLAMg65Zn30Ca5composites.After that,the PLLA-Mg65Zn30Ca5composite was injected into copper molds(GB/T 1040–1BA and GB/T 9341)with an injection pressure of 0.6 MPa.Based on the addition amount of the Mg65Zn30Ca5powder,the obtained composites were denoted as AM5 and AM10.Pristine PLLA,and PLLA with 5 wt.% annealed powder of Mg65Zn30Ca5were also casted,and the samples were denoted as PLLA and AN5,respectively.

2.3.Characterization

Mg65Zn30Ca5powders were characterized by X-ray diffraction(XRD,Cu Kαradiation).The current and the voltage of the X-ray tube were 30 mA and 30 kV,respectively.The samples were measured in the continuous registration mode at a rate of 2 °/min,with 2θ-angles ranging from 20 to 70° The microstructures of composites,the as-quenched and the annealed ribbons were investigated by optical microscopy(OM),after etching the surface of ribbons with 2 wt.% hydrochloric acid.Tensile and bending strength tests were conducted on Exceed Model E45 machine.The extension rate for the tensile strength tests was set at 2 mm/min.The bending strength of the samples was determined by the three-point bending method according to the standard GB/T 9341 for a span of 50 mm,and the crosshead speed of 2 mm/min.All the mechanical tests were replicated for three times.

Fig.1.Schematic diagram for preparation of PLLA-Mg65Zn30Ca5 composites.

2.4.Degradability tests in Hank’s solution

The as-quenched and the annealed ribbons were immersed in Hank’s solution(Hank’s balanced salts,H2387,Sigma–Aldrich,Kore)at 37 °C for 30 days.After first 14 days,the immersed ribbons were taken out for investigation by Field Emission Scanning Electron Microscopy(FE-SEM,Supra 40,Zeiss,Germany).The PLLA and PLLA-Mg65Zn30Ca5composites were also immersed in Hank’s solution for 30 days.During the immersion tests,the Hank’s solution was not refreshed before the tests were done.The pH values of the Hanks’ solution were measured using a pH-meter(PHSJ-6L,Leici,Shanghai).

2.5.Electrochemical test

The polarization tests were carried out with CHI660C electrochemical workstation and the experiments were performed in Hank’s solution at 37±0.5 °C.A conventional three electrode was set up with a saturated calomel electrode(SCE)and platinum wire working as a reference and counter electrode.Ribbons(0.5×0.5 mm of exposed area)were used as the working electrode.Samples were immersed in the solution for 20 min to establish the open-circuit potential(OCP),and subsequently,Tafel plot was obtained by polarization test with the potential swept from?2 V to 0 V in anodic direction under a sweep rate of 1 mV/s.The corrosion parameters were calculated using the Tafel extrapolation method.

2.6.Cell culture

4 weeks old male Specific-Pathogen Free Sprague Dawley(SPF SD)rats were provided by the experimental animal center of Shanghai No.9 People’s Hospital.Rats were sacrificed by neck dislocation;bone marrow was obtained by rising the bone marrow cavity by Dulbecco’s Modified Eagle’s(DMEM)medium after cutting off the epiphysis at both ends of bone.The collected bone marrow washing fluid was centrifuged in the centrifuge at 1800 rpm for 10 min.The cell suspension was seeded into the culture dish.The culture conditions were 37 °C,5% CO2,and the passage 2 to passage 4 of bone marrow mesenchymal stem cells were used in this research.The ratio of sample to culture medium was 1.25 cm2/mL according to ISO 10,993–5.After 24 h of culture,the culture medium was removed and rinsed with PBS.The cells were stained with propidium iodide(PI)(1:1000 buffer)and Calcein-AM(1:1000 buffer)for 10 min and then photographed under a fluorescence microscope.The Calcein-AM/PI Double Stain Kit was purchased from Yeasen,Shanghai,China.The dead cells were stained with PI and the live cells were stained with Calcein-AM.The cell number was measured by ImageJ 1.52a.

2.7.Alkaline phosphatase(ALP)activity

Mesenchymal stem cells(MSCs)were seeded on the samples in 24-well plates,at a density of 3×104cells/well and cultured for 7 days in osteogenic medium.Parallel sets of cells were cultured for 7 days with a mixture of 50% osteogenic induction medium and 50% Culture Medium(CM)of materials.As controls,cells were incubated only in osteogenic induction medium.CM was replaced in the 3rd day with an equal volume of fresh medium.ALP activity was assessed in cell layers by determining the release of pnitrophenol from p-nitrophenylphosphate(Sigma)at 37 °C and a pH of 10.5.The data were normalized to the total protein amounts in cell layers determined by the Bio-Rad protein assay.

Fig.2.SEM images of the grounded particles((a)as-quenched,(b)annealed),EDX results(c)and element mapping images of the as-quenched ribbon.

2.8.Antibacterial activity

E.coli(ATCC25922)were diluted with Mueller-Hinton Broth(MHB,Haibo Biotech)to a concentration of 5×105CFU/mL,and 100 μL of the bacterial suspension was added to a 96-well plate(Corning Costar).Samples with the size of 3×3×1 mm3were then placed in the plate.The strains were cultured in the MHB for 3 days at 37 °C.The absorbance at 600 nm was measured using a microplate reader.

2.9.Statistical analysis

Results were averaged and expressed as mean±standard deviation.Statistical analysis was performed using ANOVA.A value ofP <0.05 was considered statistically significant.

3.Results and discussion

3.1.Microstructural properties of Mg65Zn30Ca5 ribbons

As shown in Fig.2(a,b),after grinding and sieving,both the particles are flakes with a particle size of about 250 μm.The EDX results in Fig.2(c)show the actual composition of the as-quenched ribbon,and the atomic ration of Mg:Zn:Ca was about 64:31:5.The element mapping of the as-quenched ribbon(Fig.2(d))shows the uniform distribution of all elements.The XRD patterns of the as-quenched and annealed ribbons are shown in Fig.3.For the as-quenched ribbons,only one characteristic broad peak at around 2θ=38° can be observed,but no sharp peaks,a typical aspect for amorphous materials.On the other hand,the annealed ribbons show sharp peaks corresponding to recrystallized Mg65Zn30Ca5alloy.Some broad peaks at large angles indicate the presence of nano-crystallites.In addition,the OM images show elongated grains with visible grain boundaries(Fig.4.c and d),while the as-quenched ribbons show no obvious grain boundaries(Fig.4.a and b),even after etched with 2 wt.% hydrochloric acid.The results also confirm that the as-quenched ribbons are amorphous,and the annealed ribbons has recrystallized.

Fig.3.XRD patterns of the as-quenched and annealed Mg65Zn30Ca5 ribbons.

Fig.4.Metallographs of the as-quenched(a and b)and annealed ribbons(c and d).

3.2.Degradability assessment of the ribbons in Hank’s solution

The as-quenched and the annealed ribbons were immersed in Hank’s solution for 30 days.The evolution of pH in the first 14 days is displayed in Fig.5.a.For the annealed ribbons,the pH value of Hank’s solutions sharply increased from 7.4 to 10.5 within first 3 days,because of the fast degradation of the less corrosion resistant annealed alloy,that generates OH?ions and cause rapid alkalization of solution [22].On the other hand,the pH value of the Hank’s solutions containing the as-quenched ribbons only slightly increased up to 7.8 units,even after 14 days.The low pH value indirectly indicates that the degradation of the as-quenched ribbons is very slow,l showing much more corrosion resistance,a typical characteristic of the amorphous materials.

Fig.5.(a)Variation of the pH value of the Hank’s solutions containing as-quenched/annealed ribbons;(b)Typical polarization curves of the as-quenched ribbon and the annealed ribbon.

Fig.6.SEM images of the annealed ribbons(a,b)and the as-quenched ribbons(c,d)after 14 days of immersion.

Fig.5.b.shows the typical polarization curves of the as-quenched ribbon and the annealed ribbon.According to the Tafel extrapolation method,the corrosion potential(Ecorr)of both ribbons was ～1.31V,and the corrosion current density(icorr)of the annealed ribbon was ～3.56 ×10?5A/cm?2,while theicorrvalue of the as-quenched ribbon was ～1.10×10?5A/cm?2,which was much lower than that of the annealed ribbon.The lowericorrvalue of the as-quenched ribbon confirms that the amorphous ribbon has better corrosion resistance than the crystalized ribbon.

After 14 days of immersion,the ribbons were taken out and observed by SEM.As shown in Fig.6a,b,many cracks formed on the surface of the annealed ribbon surface of the annealed ribbon,indicating low corrosion resistance.During the immersion,though the Mg(OH)2formed on the surface may protect the ribbon,the existence of Cl?ions would dissolve it,and the protection and adhesion of the layer is poor[32].On the other hand,dense flakes-like layer is observed on the dried surface of the as-quenched ribbon(Fig.6c,d).Much fewer and smaller cracks can be observed on the surface of the as-quenched ribbons,showing that the flakes-like layer provides a better protection and adhesion to the ribbons,confirming that the amorphous ribbons have a better corrosion resistance than the crystalline ones.

3.3.Degradability assessment of the PLLA and PLLAMg65Zn30Ca5 composites

The pH values of Hank’s solution containing PLLA or PLLA-Mg65Zn30Ca5composites are displayed in Fig.7.The pH value of the Hank’s solution containing the PLLA decreased to 6.5 units within ～25 days.Decreasing of pH value is a result of the acid reaction products generated during the degradation of the PLLA [23].On the other hand,the degradation of Mg65Zn30Ca5generates alkaline reaction products[40].Therefore,since the pH values of Hank’s solution containing PLLA-Mg65Zn30Ca5composites also decreased in the first 3 days,we can conclude that the degradation of PLLA from the composite is more pregnant in the beginning,but we should also consider the low content of alloy(5 and 10 wt.%).Also,Mg65Zn30Ca5particles are covered by polymer,hence the polymer shall degrade firstly dictating the pH tendency in the initial stage.On the other hand,after 5 days of immersion,the pH value of the Hank’s solution containing the composites increased above 7.4,as a result of reaction between solution and Mg65Zn30Ca5particles [18].The annealed Mg65Zn30Ca5has a poor corrosion resistance and thus the pH value of the Hank’s solution containing AN5 increased faster and at higher values.On the contrary,due to the high corrosion resistance of the amorphous Mg65Zn30Ca5,alkalization is significantly lowered.Even for 10 wt.% amorphous Mg65Zn30Ca5(sample 10 AM,Fig.7)the pH value of the Hank’s solution can be maintained at low values(8.2)after 30 days of immersion.

Fig.7.pH values of Hank’s solution containing the PLLA or PLLAMg65Zn30Ca5 composites.

Fig.8(a)and(b)shows the stress-strain curve of the tensile strength tests,calculated elastic modulus and elongation(b)of the PLLA and PLLA-Mg65Zn30Ca5composites before(0 days)and after 30,and 60 days of immersion.The addition of the Mg65Zn30Ca5particles reduced the elongation and the tensile strength.After the immersion,for the AM5 and AM10,the elongation decreased and the elastic modulus increased,which consisted with the pristine PLLA.On the contrary,for the AN5 samples,the elongation increased and the elastic modulus decreased.These results show that the rapid degradation of the crystallized Mg65Zn30Ca5particles would impact the mechanical strength of the composite while the slow degradation of the amorphous Mg65Zn30Ca5barely had any influence.Fig.8(c)and(d)shows the tensile and bending strength of the pristine PLLA and PLLA-Mg65Zn30Ca5composites after 0,30,and 60 days of immersion.Pristine PLLA shows better tensile strength than composites,even after 60 days of immersion.Tensile strength decreased ～16 and～23%for AM5/AN5 and AM10 composites,respectively.An accelerated decrease of tensile strength was noticed after 30 days of immersion.A stable behavior shall be noticed for AM5 sample in the first 30 days.On the other hand,sample AN5 shows the fastest and continuous decrease of tensile strength,especially after 60 days.The tensile strength of AN5 decreased ～20% while the tensile strength of AM5 and AM10 only decreased ～5%.In addition,the addition of the Mg65Zn30Ca5particles reduced the elongation,the elongation for the pristine PLLA was ～15% while the elongation decreased to ～4% and ～3% for AN5/AM5 and AN10 composites.A different mechanical behavior was observed at bending tests.All composites show a better bending strength than the pristine PLLA,even after ～40 days of immersion.Among all three composites,sample AM10 shows the highest bending strength up to ～58 days,after which decreases below AM5,while AN5 has the lowest bending strength,and after ～42 days is decreasing below pristine PLLA.It has been reported that the mechanical properties of the PLA/Mg composites only retained about 60% after 28 days of immersion in Hank’s solution due to the degradation of the Mg particles[41].Thus,during the immersion,owing to the slower degradation rate of the Mg65Zn30Ca5glass,the mechanical properties of the AM5 and AM10 was more stable comparing to that of the AN5,which is essential for the healing process.

Fig.8.Stress-strain curve(a)of the tensile strength tests,calculated elastic modulus and elongation(b)of the PLLA and PLLA-Mg65Zn30Ca5 composites vs.immersion time,tensile(c)and bending(d)strength of the PLLA and PLLA-Mg65Zn30Ca5 composites vs.immersion time.

To point out the mechanism of the mechanical strength decline,other bending strength tests were conducted on samples AN5 and AM5 after 7 days of immersion,and the fracture surface morphology was investigated by SEM(Fig.9).Two reasons of mechanical degradation can be noticed.First one,as described in Section 3.3,is regarding the cracks generated by corrosion of the Mg65Zn30Ca5ribbons during the immersion period.Similarly,microcracks can be observed on Mg65Zn30Ca5particles(Fig.9a),due to the water permeability of the PLLA,allowing the Hanks’s solution to diffuse ad corrode/degrade Mg65Zn30Ca5particles [42].The second reason,larger cracks formed at the interface between the Mg65Zn30Ca5particles and PLLA matrix.These cracks can be induced either by the release of the hydrogen during the degradation of the Mg65Zn30Ca5particles,by poor adhesion between metallic powder and polymer,or by the stress concentration generated by pre-existing cracks,that lead to the deterioration of the mechanical strength,as in case of AN5 [43].On the other hand,for sample AM5 no obvious cracks were noticed on particles surface.Cifuentes [41]also reported that the hydrogen release during the degradation of the Mg/PLDA composites induced cracks.The amorphous Mg65Zn30Ca5particles avoid the rapid degradation and the accompanying cracks.The higher corrosion resistance of the amorphous alloys prevented the generation of the microcracks and allowed a slower mechanical strength deterioration.PLLA surface fracture shows a typical fracture aspect,and some propagated cracks at the particle-polymer interface(Fig.9b).

Fig.9.Fracture morphologies of the AN5(a,b)and AM5(c,d)after 7 days of immersion in Hank’s solution.

3.4.Cell viability and cytotoxicity assessment

The cell viability of the cultured MSCs is shown in Fig.10(a).The cell viability is high for all samples after 24 h.However,after 3 days the cell viability of the AN5 drastically decreased to ～60%,comparing to the other three samples.After 7 days,the cell viability of the AN5 decreased to 40%,while the other three samples still have a high cell viability,above 80%.The biological response to the magnesium-based alloy is closely connected with the degradation.The rapid degradation of magnesium would increase the risks of hemolysis,cytotoxicity or bone resorption due to the high alkaline circumstance[28].As described in Section 3.2,after 5 days of immersion,serious alkalization occurred in Hank’s solution containing AN5,which significantly contributed to inhibition of cell viability [44].On the other hand,the slow degradation of the amorphous Mg65Zn30Ca5could neutralize the acid character of PLLA and provide a milder environment(with a closer pH value to the neutral one)for the cells to grow,and thus the cell viability of the AM5 and AM10 remained high.

Alkaline phosphatase(ALP)staining is a conventional method to detect whether cells differentiate into osteogenic direction.The expression of ALP is shown in Fig.10b.The results indicate that the addition of the Mg65Zn30Ca5particles promoted ALP expression.Many researches had shown that the release of the Mg2+ions could promote the bone formation and differentiation of bone marrow mesenchym stem cells(BMSCs)[12,17].Zhao et al.[45]reported that the degradation of Mg particles endows the promoting bone-forming ability of the Mg/PLA composite.Similarly,in this work,the Mg65Zn30Ca5particles added to PLLA can be a promising method to modulate the degradation and osteogenesis ability.It is worth to mention that even though the degradability tests showed that the amorphous Mg65Zn30Ca5degrades slowly,the PLA expression for the AN5 and AM5 was quite similar,indicating that the amorphous Mg65Zn30Ca5can also promote the bone formation.

3.5.Cell morphology and spreading assessment

The BMSCs were cultured on the samples for 6 h and stained with DAPI and FITC phalloidin.As shown in Fig.10c,after 6 h fewer cells appeared on AN5,which should be due to the local alkalization caused by the crystalline magnesium alloys.In addition,cells on AM5 and AM10 samples appeared well spread and extended in numerous directions,exhibiting a rearrangement,while cells on PLLA and AN5 became elongated.The results revealed that the addition of the amorphous alloys is favorable for the cells at the early stage to spread and propagate,which is the base for the cells’propagation and differentiation [17].According to Zhao et al.[45],Mg2+ions stimulate biomineralization and osteogenesis.However,the rapid corrosion was accompanied by local alkalization,high Mg2+concentration and hydrogen bubbles.High pH values could denature membrane proteins,thereby destroying the disabling active ionic transport[46].Therefore,the AM5 and AM10 shows promoting cell growth and spreading,owing to the moderate Mg degradation.

Fig.10.(a).BMSCs cell viability after cultured with PLLA and PLLA composites for 1,3 and 7 days.(b).Expression of ALP after 3 days of culture with PLLA and PLLA-based composites.(c).Fluorescent images of BMSCs stained with DAPI and FITC-phalloidin after cultured for 6h.(d).Optical density of E.coli after inhibited on each sample for 3 day.(n=3,?represents p <0.05,??represents p <0.01 compared with other groups,#represents p <0.05 compared with the other two groups).

3.6.Antibacterial activity assay

The antibacterial behavior of the pristine PLLA,and AN5,AM5 and AM10 composites was investigated against E.coli.The result is shown in Fig.10d.Comparing to the PLLA,the AN5,AM5 and AM10 have much lower optical density(OD)values,indicating that anti-bacterial ability of the composites was enhanced by the addition of the Mg65Zn30Ca5particles.It has been reported that Zn can reduce bacterial adhesion and growth by generating reactive oxygen species(ROS),and Zn ions can restrain bacteria growth by inhibiting multiple activities of bacteria [29].The AN5 has better anti-bacterial ability than AM5,as the crystalline Mg65Zn30Ca5particles have a faster degradation rate,releasing a higher concentration of Zn ions.Also,the alkaline environment can play a certain role in the antibacterial behavior.The improved anti-bacterial ability is also favorable for the healing process,since the poor anti-bacterial ability of PLLA might allow host infection[14].

4.Conclusion

The PLLA-Mg65Zn30Ca5composites have been successfully prepared and assessed forin vitrodegradation kinetics.Our results show that the degradation of the Mg65Zn30Ca5causes alkalization of immersion solution and accelerate the decrease of the mechanical strength of the composites.The amorphous Mg65Zn30Ca5has high corrosion resistance and provides a milder environment for cells to grow,as well as their addition to PLLA avoids drastically deterioration of the mechanical strength,an important aspect for the healing process.Though the degradation of the amorphous Mg65Zn30Ca5is relatively slow,it can still promote the bone formation and differentiation of the composite like the Mg65Zn30Ca5alloy.In addition,the high-Zn content in Mg65Zn30Ca5endows the composites a good antibacterial property.The great performance of the PLLA-Mg65Zn30Ca5composite recommends it as a promising candidate for biodegradable materials for bone implants.Two compositions of PLLA-Mg65Zn30Ca5composite(5 and 10 wt.% added alloy)allows the tailoring of mechanical and biological behavior of materials.Mechanical tests are useful to envision different designs of pgr implants.

Declaration of Competing Interest

We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted.

Acknowledgments

The present work was supported by National Natural Science Foundation of China(Grant No.51471120).The authors are grateful to Tongjiecheng New Material Co.,Ltd.for providing the hot ejection equipment.