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[Akiko Yamamoto](https://orcid.org/0000-0002-9182-4886), Akemi Kikuta

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[Development of a Model System for Gas Cavity Formation Behavior of Magnesium Alloy Implantation](https://mdr.nims.go.jp/datasets/607712b6-d6e5-4c0f-ba08-ba337bb95b98)

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Development of a Model System for Gas Cavity Formation Behavior of Magnesium Alloy ImplantationDevelopment of a Model System for Gas Cavity Formation Behaviorof Magnesium Alloy ImplantationAkiko Yamamoto* and Akemi KikutaCite This: ACS Biomater. Sci. Eng. 2022, 8, 2437−2444 Read OnlineACCESS Metrics & More Article Recommendations *sı Supporting InformationABSTRACT: Clinical applications of magnesium (Mg)-based screws havereported gas cavity formation in the surrounding tissue, which sometimesdelays the fixation of the bone fracture. The gas cavity formation isconsidered to depend on the balance between hydrogen generation by Mgcorrosion reacting with water in the body fluid and its diffusion into thesurrounding tissue by capillary flow. In order to understand the gas cavityformation behavior by Mg-based material implantation, we developed a newin vitro model system to recreate this cavity formation phenomenon: thehydrogen generation by corrosion and its diffusion into the medium. A modeltissue is prepared by gelation of the cell culture medium in a sterile condition.The immersion of Mg alloy samples was performed under 5% CO2atmosphere with periodic observation by X-ray computed tomography,which enabled us to observe gas cavity growth up to 28 d. For demonstratingthe usefulness of our model system, Mg alloy samples with different corrosionrates were prepared by a biodegradable polymer coating. AZ31 screws were spin-coated by poly-L-lactide (PLLA) and classified intothree groups by their coating thickness as 1.0 ± 0.0, 1.6 ± 0.2, and 2.0 ± 0.1 μm (ave. ± s.d.). Upon their immersion into the modeltissue, the gas cavity volumes formed were 1.57 ± 0.23, 1.06 ± 0.22, and 0.38 ± 0.09 mm3/mm2 for 1.0, 1.6, and 2.0 μm coatingsamples, having the weight loss of 20.2 ± 2.93, 18.5 ± 2.84, and 11.3 ± 3.54 μg/mm2, respectively (ave. ± s.d.). This result clearlyindicates the dependence of gas cavity formation on the corrosion rate of the sample. The gas cavity volume was only 3.3∼7.5% ofthe total hydrogen gas volume estimated based on the weight loss of the samples at 28 d, which is in the range of those calculatedfrom the clinical report (3.2∼9.4% at 4w). This system can be an effective tool to investigate the gas cavity formation behavior andcontribute to understand the mechanisms and controlling factors of this phenomenon.KEYWORDS: biodegradable metals, magnesium alloys, biocorrosion, hydrogen gas, in vitro evaluation■ INTRODUCTIONBiomedical applications of magnesium (Mg) and its alloys arewidely investigated for the success of biodegradable implantdevices such as coronary stents and bone fracture fixatives.1−3Mg is an essential element found in the human body at arelatively high content.4 Mg and its alloys have a Young’smodulus similar to that of the human cortical bone.5 They areeasily corroded by reacting with water in the body fluids andtissues, which is utilized as degradability inside the humanbody. Several reports on clinical studies of Mg alloy devices areavailable;6−12 some of them are about cannulated compressivescrews made of Mg-4wt %Y-3wt %RE (WE43, RE indicatesrare-earth elements)-based alloy.6−8,11,12 In case of halluxvalgus treatment, gas cavity formation in the tissue surroundingthe Mg alloy screws was observed in almost all (38 of 39)patients at 6w of follow up.7 Furthermore, early disintegrationand the failure of the implanted screws were found in 7 and 1cases out of 39, respectively.7 Applying this screw to unstablescaphoid fractures, extensive resorption cysts were observed in3 out of 5 patients, resulting in the retardation of fracturehealing.12 Implantation of two screws to reconstructscaphotrapeziotrapezoidal joint failed with osteolytic seams,cyst formation, and loosening of both screws, resulting inrevision by nondegradable screws.11 To avoid the clinicalcomplication or inconvenience caused by gas cavity formationaccompanying the corrosion of the Mg alloy devices, it isimportant to understand its mechanism and to evaluate its riskbefore clinical trial.The cyst or gas cavity formation is attributed to thehydrogen (H2) generation by the Mg corrosion reaction.13 Theamount of gas dissolved into the fluid is proportional to itspartial pressure, which is known as Henry’s law. Before theimplantation, the body fluid contains dissolved nitrogen (N2),Received: November 11, 2021Accepted: April 29, 2022Published: May 23, 2022Articlepubs.acs.org/journal/abseba© 2022 The Authors. Published byAmerican Chemical Society2437https://doi.org/10.1021/acsbiomaterials.1c01429ACS Biomater. Sci. Eng. 2022, 8, 2437−2444Downloaded via NATL INST FOR MATLS SCIENCE (NIMS) on January 24, 2024 at 07:54:11 (UTC).See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Akiko+Yamamoto"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdfhttps://pubs.acs.org/action/doSearch?field1=Contrib&text1="Akemi+Kikuta"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdfhttps://pubs.acs.org/action/showCitFormats?doi=10.1021/acsbiomaterials.1c01429&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsbiomaterials.1c01429?ref=pdfhttps://pubs.acs.org/doi/10.1021/acsbiomaterials.1c01429?goto=articleMetrics&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsbiomaterials.1c01429?goto=recommendations&?ref=pdfhttps://pubs.acs.org/doi/10.1021/acsbiomaterials.1c01429?goto=supporting-info&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsbiomaterials.1c01429?fig=abs1&ref=pdfhttps://pubs.acs.org/toc/abseba/8/6?ref=pdfhttps://pubs.acs.org/toc/abseba/8/6?ref=pdfhttps://pubs.acs.org/toc/abseba/8/6?ref=pdfhttps://pubs.acs.org/toc/abseba/8/6?ref=pdfpubs.acs.org/journal/abseba?ref=pdfhttps://pubs.acs.org?ref=pdfhttps://pubs.acs.org?ref=pdfhttps://doi.org/10.1021/acsbiomaterials.1c01429?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://pubs.acs.org/journal/abseba?ref=pdfhttps://pubs.acs.org/journal/abseba?ref=pdfhttps://creativecommons.org/licenses/by-nc-nd/4.0/https://creativecommons.org/licenses/by-nc-nd/4.0/https://creativecommons.org/licenses/by-nc-nd/4.0/https://creativecommons.org/licenses/by-nc-nd/4.0/https://acsopenscience.org/open-access/licensing-options/oxygen (O2), and carbon dioxide (CO2) proportionally to theirpartial pressure inside the body (Figure 1a). When a Mg alloydevice is implanted, H2 is generated by corrosion reaction,resulting in the increase in its partial pressure inside the body.At the beginning of the implantation period, however, thepartial pressure of H2 is much lower than those of N2, O2, andCO2; most of the generated H2 cannot dissolve into the bodyfluid and forms a gas cavity in the tissue surrounding theimplant (Figure 1b). Along the progress of the corrosionreaction, the partial pressure of H2 increases more, whereasthose of other gases decrease. Then, some of the H2 in the gascavity dissolves into the body fluid and diffuses through themicrocapillary network, whereas undissolved N2, O2, and CO2join to the cavity in the tissue, resulting in the exchange of H2in the cavity by N2, O2, and CO2 (Figure 1c).14 This issupported by the analysis of the gas composition in the cavityformed in animal tissue by Mg alloy implantation as mainlycomposed of N2, followed by O2 and H2.15 The quick decreasein H2 concentration in the gas cavity was also confirmed by anin vivo experiment with subcutaneous injection of H2 intohairless mice.14The partial pressure of H2 in the tissue near the Mg alloyimplant depends on the balance between its generation bycorrosion reaction and its diffusion by blood flow becauseevery tissue has a capillary network. If the diffusion rate byblood flow is faster than the generation rate, the partialpressure of H2 does not increase, and then, gas cavity does notform. Actually, no gas cavity formation is reported in theclinical study for vascular stents,16−18 where the blood flowrates in coronary arteries are much higher than those incapillary.19 In the clinical trial of pure Mg screws, the gas cavityformation depends on the implanting tissues; it was notobserved in the femoral head or neck but observed in themetatarsal bone.3 This dependence on the implanting tissuemakes it difficult to predict the gas cavity formation behavior ofthe Mg alloy implants because blood flow differs not only withthe types of tissues but also with patient conditions such as age,types of injury, pathology, underlying diseases, and so forth.In order to tackle the gas cavity formation by Mg alloyimplantation, a few in vivo experiments were recently carriedout focusing on this phenomenon in small animals.14,20−22These in vivo experiments are valuable to observe andunderstand the gas cavity formation behavior using a Mgalloy sample, but they are not applicable to risk assessment ongas cavity formation by a Mg alloy for a specific clinical use.Currently available Mg alloy screws had to be evaluated by invivo implantation tests prior to their clinical application, butthey failed to reveal the clinical risks reported in their clinicalstudies.7,11,12 This can be attributed to an indispensable issuelying under in vivo experiments, difference in species. Healthyanimal tissue can never be the same to injured human tissue,including simple factors such as the body weight and fluid/tissue amount. It is also difficult to control each ofphysiological factors such as blood flow separately in ananimal body. If we have a good in vitro evaluation methodwhich can simulate the human body environment and cancontrol the factors influencing gas cavity formation behavior, itis effective to understand the critical conditions for gas cavityformation and contribute to the appropriate estimation of therisks. It is also beneficial on the following points as low costs,relatively short testing periods, and no animal uses (no issue onanimal welfare). However, most of in vitro evaluation methodsare carried out in a simple liquid environment; no method isestablished to observe gas cavity formation behavioraccompanying the implantation of biodegradable metal devicesso far.Based on the described background, we developed a new invitro method to observe the gas cavity formation behavior of abiodegradable metal sample under a controllable condition.We employed a gel of cell culture medium by adding thethickener (gelation reagent) to form a model tissue anddemonstrated the observation of gas cavity formation using anAZ31 screw up to 28 d using mircofocus X-ray computedtomography (μCT).■ MATERIALS AND METHODSSample Preparation. Commercially available industrial screws(MG-0204, M2 and 4 mm in length, WILCO Co. Ltd.) made of Mg-3wt %Al-1wt %Zn (AZ31) were employed as a model device. Itschemical composition is shown in Table 1. The screws wereimmersed in 1 M NaOH at 80 °C for 5 min and rinsed thoroughlywith ultrapure water to clean their surface. Then, surface activationwas carried out by 1 M HNO3 at room temperature for 10 s, followedby rinsing with ultrapure water. The screws were dried in vacuo, andtheir initial weight (W0) was measured. Biodegradable polymercoating was employed to control the initial degradation rate of thescrew samples without seriously changing their surface area, as studiedin our previous work.23,24 Poly-L-lactide (PLLA), one of therepresentative biodegradable polymers, was applied because it wasalready used as a clinical implant. The screws were spin-coated with aFigure 1. Schematic explanation of gas cavity formation and H2diffusion in the tissue.Table 1. Chemical Composition of the AZ31 Alloy (Wt.%)Al Zn Mn Fe Si Cu Ni Mg3.00 1.00 0.40 <0.01 0.01 <0.01 <0.01 Bal.ACS Biomaterials Science & Engineering pubs.acs.org/journal/abseba Articlehttps://doi.org/10.1021/acsbiomaterials.1c01429ACS Biomater. Sci. Eng. 2022, 8, 2437−24442438https://pubs.acs.org/doi/10.1021/acsbiomaterials.1c01429?fig=fig1&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsbiomaterials.1c01429?fig=fig1&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsbiomaterials.1c01429?fig=fig1&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsbiomaterials.1c01429?fig=fig1&ref=pdfpubs.acs.org/journal/abseba?ref=pdfhttps://doi.org/10.1021/acsbiomaterials.1c01429?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-as20 μL portion of 0.5, 1, and 2% (w/v) solution of PLLA (Mw 300,000,Polysciences, Inc.) in chloroform at 100 rpm for 10 s, followed by1000 rpm for 60 s. These coated samples were dried in vacuo, andtheir weight with coating (Wc) was measured to determine theamount of the coated polymer. The coating thickness of the polymer,hcoat, was calculated by the following equation:ρ=−hW WSccoat0p 0 (1)where ρp and S0 indicate the density of the polymer (1.15 mg/mm3)and the initial surface area of the screw, respectively. S0 wasdetermined based on the μCT observation described in the nextsection. The coated screws were categorized into three groupsdepending on their coating thickness: thick (2.0 ± 0.1 μm on average± standard deviation), intermediate (1.6 ± 0.2 μm), and thin (1.0 ±0.0 μm). Each group has three samples. Prior to gel immersion, eachsample was sterilized by ethylene oxide gas (EOG) and stored invacuo.Gel Immersion. Eagle’s minimum essential medium (Eagle’sMEM “Nissui” 1, Nissui Pharmaceutical Co Ltd.) supplemented with10% (v/v) fetal bovine serum (E-MEM + FBS) was employed as asimulated body fluid. The major chemical components of E-MEM areshown in Table 2 in comparison with those of human blood plasma.25To simulate the living tissue, a thickener (gellan gum, Wako PureChemical Corporation) was added to E-MEM + FBS to be 0.4%(w/v) as a final concentration. In detail, an appropriate amount of thethickener was dissolved in water at 90 °C, followed by the addition ofan appropriate amount of 10 times concentrated E-MEM solutionwithout supplements. After sterilization by autoclaving, the solutionwas maintained at 55 °C, while appropriate amounts of supplements(3% L-glutamate, 7.5% NaHCO3, and FBS) were aseptically added.A schematic illustration of the gel immersion procedure is shown inFigure 2. A 3 mL portion of the prepared immersion medium waspoured into a sterile polystyrene vessel and solidified at roomtemperature for 10 min. Then, a Mg alloy screw was half-buried intothe gel (keeping its head out of the gel) to control its depth in the gel.Then, an additional 5 mL portion of the immersion medium waspoured over the screw, which was completely immersed into the gel.The vessel was placed in the CO2 incubator (37 °C, 5% CO2) looselycapped for gas exchange. Within a few hours, the gel sample wasobserved by μCT (SMX-90CT, Shimadzu Corporation) at an optimalcondition with the resolution of 21 μm/voxel to confirm the initialcondition of the immersion medium. S0 was determined based on theμCT images at 0 d using image analysis software (VG Studio Max 3.0,Volume Graphics GmbH). The gas cavity formed around the screwwas observed by μCT at 1, 3, 5, 7, 10, 14, 21, and 28 d of immersion.The volume of the gas cavity was analyzed by the image analysissoftware and divided by S0 to give gas cavity volume per initial samplesurface area, Vcavity [mm3/mm2].After the immersion of 28 d, the screws were collected from theimmersion medium and dried in vacuo. An insoluble salt layer formedon the screw surface was removed by a chromic acid solution [20 g ofCrO3, 1 g of AgNO3, and 2 g of Ba (NO3)2 dissolved in 100 mL ofultrapure water], followed by rinsing with ultrapure water and dryingin vacuo. The remaining weight (Wr) was measured to determine theweight loss per unit surface area (Wloss [g/mm2]) by the followingequation:=−WW WSloss0 r0 (2)In order to calculate the total gas volume generated by corrosionduring the immersion period, generated H2 was assumed as the idealgas for simplicity. The total gas volume per unit surface area (Vtotal[mm3/mm2]) was calculated by the following equation:=·− VRTp10WW2total104lossm(3)where Wm, R, T, and p indicate the molecular weight of AZ31 (24.54[g/mol]), gas constant (0.0821 [L·atm/K·mol]), absolute temper-ature (310 [K]), and pressure, respectively. When p is assumed as 1[atm], the previous equation gives the following one:= ×V W1.037 10total6loss (4)which is used to estimate Vtotal generated during 28 d of gelimmersion. Then, the capture ratio of the generated gas in the gascavity is determined by dividing Vcavity by Vtotal.Real gases are known to have different behavior from that of idealgas, especially at high pressure and low temperature. In the presentstudy, however, the pressure and temperature are 1 [atm] and 310[K], respectively, and the difference from the ideal gas behavior isrelatively small.26 When Vtotal is calculated assuming H2 and applyingthe van der Waals equation, the difference from the value calculatedby the ideal gas law is less than 0.1%.■ RESULTSThe examples of macroscopic images of gel immersion samplesare shown in Figure 3. Gas cavity formation around the screwin the immersion medium was observed, suggesting thesuccessful recreation of the gas cavity formation phenomenonby the corrosion of a Mg alloy sample. It also shows that theimmersion medium could maintain the gas cavity during 28 dof immersion. The growth of gas cavity can be easily observedeven in the macroscopic images. However, to give a furtherdetailed analysis, μCT observation is performed at various timeTable 2. Major Chemical Components of Blood Plasma andE-MEMcomposition blood plasma25 E-MEMaNa+(mM) 142 143.5K+(mM) 5 5.37Mg2+(mM) 1.5 0.81Ca2+(mM) 2.5 1.80Cl−(mM) 103 124.7HCO3−(mM) 27 26.2HPO42−(mM) 1 0.90SO42−(mM) 0.5 0.81Glucose (g/L) ∼1.1 1amino acids and vitamins (g/L) 0.25−0.4 0.81proteins (g/L) 63−80phenol red (g/L) 0.006aEagle’s minimum essential medium “Nissui” 1, Nissui Pharmaceut-ical Co Ltd., Japan.Figure 2. Schematic illustration of the gel immersion procedure.ACS Biomaterials Science & Engineering pubs.acs.org/journal/abseba Articlehttps://doi.org/10.1021/acsbiomaterials.1c01429ACS Biomater. Sci. Eng. 2022, 8, 2437−24442439https://pubs.acs.org/doi/10.1021/acsbiomaterials.1c01429?fig=fig2&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsbiomaterials.1c01429?fig=fig2&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsbiomaterials.1c01429?fig=fig2&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsbiomaterials.1c01429?fig=fig2&ref=pdfpubs.acs.org/journal/abseba?ref=pdfhttps://doi.org/10.1021/acsbiomaterials.1c01429?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-aspoints. Figure 4 shows the examples of the two-dimensional(2D) μCT images of gas cavities and an immersed screw. Thisfigure clearly demonstrates the growth of the gas cavities withan increase in the immersion period, but it does not representthe whole volume and behavior of gas cavity formation.Therefore, the volume of the gas cavity was measured usingimage analysis software and summarized in Figure 5. It is clearthat Vcavity increased with an increase in the immersion periodfor all samples. The growth rate depends on the thickness ofthe coating; the screws with thinner coating have higher gascavity volumes.After 28 d of gel immersion, theWloss values of PLLA-coatedscrews were 20.2 ± 2.93, 18.5 ± 2.84, and 11.3 ± 3.54 μg/mm2for coating thicknesses of 1.0, 1.6, and 2.0 μm, respectively (seeSupplemental Table S1). Figure 6a indicates the correlationbetween Vcavity and Vtotal, which was estimated usingWloss by eq4. As Vtotal decreased with an increase in hcoat, Vcavity decreased.In other words, the higher corrosion rate of the screw resultedin the larger gas cavity formation. It also indicates that Vcavity ismuch smaller than Vtotal, even though it has a good correlation.As described in the Materials and Methods Section, Vtotal isestimated by ideal gas law, which may have a difference of 0.1%from the estimation based on the real gas behavior (van derWaals equation, data not shown). However, this 0.1%difference is sufficiently low compared to that between Vcavityand Vtotal shown here.The capture ratio of the generated gas into the cavity isestimated by Vcavity/Vtotal and shown in Figure 6b. The captureratios are 3.3∼7.5% after 28 d of gel immersion and increasedwith the increase in Vtotal. This indicates that most of the gasgenerated by corrosion diffuses into the model tissue withoutforming the gas cavity. The extrapolation of the linearregression curve indicates the limit of the Vtotal, which doesnot form a gas cavity as ca. 4 mm3/mm2 for 28 d in thisexperimental condition. In order to estimate the gas diffusionrate (vd), the following equation is applied:= −v V V t( )d total cavity (5)where t is the immersion period of 28 d. Then, vd is plottedagainst the gas generation rate (vg), which is calculateddividing Vtotal by t (28 d). As shown in Figure 7, the cleardependence of vd on vg was observed, suggesting that 94% ofthe generated gas diffused into the model tissue.Figure 3. Examples of the macroscopic observation of gel immersionof a PLLA-coated screw at 3 d (a), 7 d (b), and 28 d (c).Figure 4. Examples of the cross-section images of gas cavity formationaround an immersed screw observed by μCT.Figure 5. Gas cavity growth curve during gel immersion (ave ± s.d, n= 3).Figure 6. (a) Correlation of the gas cavity volume per initial surfacearea, Vcavity, and the total gas volume generated by corrosion per initialsurface area, Vtotal. (b) Correlation of the gas capture ratio, Vcavity/Vtotaland Vtotal (ave ± s.d, n = 3).ACS Biomaterials Science & Engineering pubs.acs.org/journal/abseba Articlehttps://doi.org/10.1021/acsbiomaterials.1c01429ACS Biomater. Sci. Eng. 2022, 8, 2437−24442440https://pubs.acs.org/doi/suppl/10.1021/acsbiomaterials.1c01429/suppl_file/ab1c01429_si_001.pdfhttps://pubs.acs.org/doi/10.1021/acsbiomaterials.1c01429?fig=fig3&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsbiomaterials.1c01429?fig=fig3&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsbiomaterials.1c01429?fig=fig3&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsbiomaterials.1c01429?fig=fig3&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsbiomaterials.1c01429?fig=fig4&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsbiomaterials.1c01429?fig=fig4&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsbiomaterials.1c01429?fig=fig4&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsbiomaterials.1c01429?fig=fig4&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsbiomaterials.1c01429?fig=fig5&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsbiomaterials.1c01429?fig=fig5&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsbiomaterials.1c01429?fig=fig5&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsbiomaterials.1c01429?fig=fig5&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsbiomaterials.1c01429?fig=fig6&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsbiomaterials.1c01429?fig=fig6&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsbiomaterials.1c01429?fig=fig6&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsbiomaterials.1c01429?fig=fig6&ref=pdfpubs.acs.org/journal/abseba?ref=pdfhttps://doi.org/10.1021/acsbiomaterials.1c01429?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-as■ DISCUSSIONBiodegradable metals represented by Mg and its alloys havedifferent degradation mechanism from those of biodegradablepolymeric and ceramic materials. The total corrosion reactionof Mg is described below:27+ → + ++ −Mg 2H O Mg 2OH H222 (6)where the pH of the solution will increase and H2accumulation will occur with the progress of the corrosionreaction. Inside the living body, however, the vascular networksupplies blood circulation, which supports the diffusion ofcorrosion products such as Mg2+, OH−, and H2. Thiscontributes to maintain the pH of the blood and interstitialfluid near the implant ca. 7.4 and to reduce the gas cavityformation. Because Mg and its alloys are the very firstbiodegradable materials that generate H2 accompanying itsdegradation, no in vitro method is established to evaluate thegas cavity formation behavior of the material and the effect ofgas cavity in the surrounding tissue.In vivo implantation of Mg and its alloys was carried out toevaluate the material corrosion rate and biocompatibilityincluding gas cavity formation in the tissue. Most of thesestudies reported no specific adverse reaction caused by theimplantation, but they often observed gas cavity formation inthe surrounding tissue of the implant.20−22,28−30 As describedbefore, gas cavity formation depends on the balance betweenthe H2 generating rate (=corrosion rate) and its diffusion rateby blood flow. However, in in vivo experiments, it is difficult toprepare the same environment of the human, injured tissue.Even though a sample can be implanted to the same type ofthe tissue, the environmental factors such as blood flow aredifferent between human and animal tissues. Alternatively, ifwe have an in vitro system to simulate the environment of thehuman tissue with individual controls of the environmentalparameters such as blood flow, we can use it to obtainsupportive data for the risk assessment of the gas cavityformation by biodegradable metal implants prior to theirclinical application.Having said so, most of the in vitro degradation/corrosiontests of Mg alloys were performed in physiological solution,which could not achieve the recreation of the gas cavityformation phenomenon. This is because diffusion of thegenerated gas in the solution is faster than that in the tissue.Therefore, as a first step to mimic in vivo implantationcondition, a model tissue is prepared by adding a thickener to acell culture medium to slow down the diffusion rate of the gas.E-MEM is employed as a physiological solution because it isdeveloped based on the composition of human blood plasma.Gellan gum, known as one of the alternatives of an agar formicrobiological culture, is selected as a gelation reagentbecause it forms a transparent gel and can be sterilized byautoclaving. As shown in Figure 3, the immersion of an AZ31screw into this gel offers macroscopic observation of the gascavity formation behavior during 28 d of immersion. This isthe very first in vitro evaluation method that enables us toobserve the gas cavity formation behavior in a model tissue.Using μCT, the gas cavity volume can be measured andanalyzed through the immersion period, as summarized inFigure 5. PLLA-coating was employed to control the H2generation rate by suppressing the corrosion of the screw;the thicker coating results in the lower corrosion rate (seeSupplemental Table S1). As shown in Figure 6, the gas cavitygrowth depends on the corrosion rate of the screw. Thisfinding demonstrates that our developed system can detect thedifference in the gas cavity formation behavior, which isderived from the difference in the material corrosion rate.Inside the human body, H2 generated by the corrosionreaction of the implanted Mg-based device will diffuse byblood flow. Therefore, the generated gas exceeding its diffusionlimits forms the gas cavity in the surrounding tissue. Thatmeans, the condition to form gas cavity in the tissue can bewritten as vg > vd, where vg and vd are the H2 generation rate bycorrosion and as the H2 diffusion rate, respectively. Then,Vcavity can be described as follows:= −V v v t( )cavity g d (7)where t indicates the immersion period. When (vg − vd) isconstant through the immersion period, Vcavity keeps growingat a constant rate. As shown in Figure 5, Vcavity increasesthrough the immersion period of 28 d in the present study, butthe slope of the growth curve is larger at the beginning of theimmersion than at the latter. This suggests that (vg − vd) is notconstant, but slightly decreases along the incubation period.Vcavity/Vtotal was plotted against Vtotal in Figure 6b. Gas captureratios were 3.3∼7.5% after 28 d of immersion, suggesting thatmore than 90% of the generated gas diffused via the immersionmedium.In order to find Vcavity/Vtotal, we analyzed the data in theclinical reports in which Mg-5wt %Ca (Mg-5Ca)22 or WE43-based7 screws were implanted to the metatarsal/midfootfractures or osteotomies. In ref 7, the gas cavity formationwas only reported as the maximum width of radiolucency areasaround the screw, and summarized as the average, theminimum, and the maximum values. Therefore, the gas cavityvolume is calculated assuming a sphere with a diameter of themaximum width of radiolucency areas. The average degrada-tion periods of Mg-5Ca or WE43-based screws were alsoassumed as 1231 and 18 months,32 respectively, with a constantdegradation rate through the entire implantation period for thesimplest model. As summarized in Tables 3 and 4, the Vcavity ofthe clinical cases decreases with increases in the implantationperiod. The increasing phase of Vcavity is missing because of thelack of follow-up at the early stage of implantation. In otherwords, the increase phase of Vcavity is relatively short in clinicalcases. This trend agrees with those of in vivo implantationexperiments.20−22 The decrease in Vcavity indicates vd > vg,Figure 7. Correlation of the gas diffusion rate, vd, and the gasgeneration rate, vg (ave ± s.d, n = 3).ACS Biomaterials Science & Engineering pubs.acs.org/journal/abseba Articlehttps://doi.org/10.1021/acsbiomaterials.1c01429ACS Biomater. Sci. Eng. 2022, 8, 2437−24442441https://pubs.acs.org/doi/suppl/10.1021/acsbiomaterials.1c01429/suppl_file/ab1c01429_si_001.pdfhttps://pubs.acs.org/doi/10.1021/acsbiomaterials.1c01429?fig=fig7&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsbiomaterials.1c01429?fig=fig7&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsbiomaterials.1c01429?fig=fig7&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsbiomaterials.1c01429?fig=fig7&ref=pdfpubs.acs.org/journal/abseba?ref=pdfhttps://doi.org/10.1021/acsbiomaterials.1c01429?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-aswhich was not observed in our model tissue in the presentstudy. Because the gas cavity is formed in the clinical cases, vgshould be larger than vd at the initial phase of implantation.Then, at some point, vg becomes smaller than vd, resulting in adecrease in Vcavity. Unfortunately, detailed information on thedegradation of Mg alloy screws were not available in clinicalcases, but the decrease in the corrosion rate of Mg alloysamples was reported by in vitro corrosion tests in simulatedbody fluids even within the first 24 h.33,34 Therefore, in clinicalcases, it is considered that vg decreases below vd in fairly earlystage of implantation. In the present study, however, it doesnot occur in our model tissue within 28 d. This might beattributed to the slow decrease in vg or relatively low vd of ourmodel system. The resolution of the μCT images we obtainedis not enough to find reduction in the volume of the immersedscrew for the shorter immersion period. Further improvement/investigation is necessary to obtain sequential data on Mg alloysample degradation during the incubation period.The gas capture ratios, Vcavity/Vtotal, of the clinical cases wereless than 15%, except that in the soft tissue at 1 week of followup, as reported in ref 22. This simple calculation based on theclinical data also indicates that most of the generated gasdiffuses through the tissue and blood flow after 4 w ofimplantation. The estimation in Tables 3 and 4 suggests thedifference in the gas capture ratio between the tissue types.The Vcavity/Vtotal of both bone and soft tissue are plottedagainst the incubation period, as shown in Figure 8. Theestimation in ref 22 is higher than that in ref 7, but maximumdata in ref 7 showed a similar level of Vcavity/Vtotal to those inref 22. For both cases, Vcavity/Vtotal of the soft tissue was higherthan that of bone.The Vcavity/Vtotal in the model tissue is those between the softtissue and bone of ref 22. Because the concentration of thegelation reagent in the model tissue is constant, to giving thesame diffusion condition, the higher corrosion rate (of the 1.0μm coating group than that of 2.0 μm coating group) results inthe larger overflow of H2, that is, (vg − vd). This suggests that,in clinical cases, the soft tissue has a larger H2 overflowcompared to those of the bone tissue. In our model tissue, thegelation reagent and its concentration are changeable, whichenables us to control the diffusion rate in the model tissuecloser to those in the actual human tissue. This brings us a newchallenge to recreate/simulate the environment in varioushuman tissues and conditions. Our model tissue can be usefulto observe the initial process of gas cavity formation, tounderstand its mechanism, and to elucidate the factorscontrolling this process. This will contribute to the successof new biodegradable medical devices made of biodegradablemetals with reducing risks of gas cavity formation.■ CONCLUSIONSIn the present study, we developed a new in vitro evaluationmethod for the gas cavity formation behavior by Mg alloycorrosion under a similar environment inside the human body.We demonstrate the recreation of the gas cavity formationphenomenon by immersing a Mg alloy sample into a modeltissue, which is prepared by the gelation of a cell culturemedium. The obtained results proved that our developedmethod can successfully detect the difference in the gas cavitygrowth rate, which is controlled by PLLA coating of thesample. At this condition, it confirmed that the highercorrosion rate gives the higher cavity growth rate. Thissuggests the dependency of gas cavity formation on the balancebetween the hydrogen generation rate by corrosion and itsTable 3. Estimation of the Gas Cavity Volume and the TotalGas Volume from the Literature22follow-upperiod (w) tissuegas cavityvolume (mm3)22gas captureratio (%)atotal gasvolume (mm3)b1 bone 79.32 ± 19.00 15.0 ± 3.6 531softtissue341.95 ± 126.42 64.5 ± 23.84 bone 67.95 ± 15.73 3.20 ± 0.74 2122softtissue198.50 ± 84.47 9.35 ± 3.988 bone 38.86 ± 7.55 0.92 ± 0.18 4244softtissue164.23 ± 70.21 3.87 ± 1.6512 bone 34.86 ± 6.07 0.55 ± 0.01 6367softtissue85.00 ± 42.95 1.34 ± 0.6726 bone 32.23 ± 5.61 0.23 ± 0.04 13,794softtissue35.59 ± 37.79 0.26 ± 0.2752 bone 26.43 ± 3.21 0.10 ± 0.01 27,589softtissue20.84 ± 31.17 0.08 ± 0.11aGas capture ratio is defined as the gas cavity volume divided by thetotal gas volume. bTotal gas volume is calculated as an ideal gas basedon the following assumptions: the weight of the Mg-5Ca screw as26.97 mg, and its degradation period as 12 months (the average valuesof the degradation period appear on the implant brochure31) with aconstant degradation rate through the period.Table 4. Estimation of the Gas Cavity Volume and the Total Gas Volume from the Literature7follow-up period (w) tissue ave./max radiolucency areas (mm)7 gas cavity volume (mm3)a gas capture ratio (%)b total gas volume (mm3)c6 bone ave 2.0 ± 1.3 4.19 ± 8.2 0.04 ± 0.07 11,390max 6 113 0.99soft tissue ave 3.0 ± 3.0 14.1 ± 42.4 0.12 ± 0.37max 11 697 6.112 bone ave 1.5 ± 0.9 1.77 ± 3.2 0.008 ± 0.014 22,780max 2.8 11.5 0.05soft tissue ave 1.0 ± 1.8 0.524 ± 2.8 0.002 ± 0.012max 5.7 97.0 0.43aGas cavity volume is calculated assuming a sphere with a diameter of the average and the maximum value of the maximum length of X-raytransparency reported.7 bGas capture ratio is defined as the gas cavity volume divided by the total gas volume. cTotal gas volume is calculated as anideal gas based on the following assumptions: the weight of the WE43-based compression screw as 150 mg, and its degradation period as 18months (the average values of the degradation period appears on the implant brochure32) with a constant degradation rate through the period.ACS Biomaterials Science & Engineering pubs.acs.org/journal/abseba Articlehttps://doi.org/10.1021/acsbiomaterials.1c01429ACS Biomater. Sci. Eng. 2022, 8, 2437−24442442pubs.acs.org/journal/abseba?ref=pdfhttps://doi.org/10.1021/acsbiomaterials.1c01429?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-asdiffusion rate in the biological environment. The gas captureratios of the model tissue were in the range of those in theclinical reports. Our developed method can be an effective toolto study the gas cavity formation behavior by Mg alloycorrosion under the similar environment to the actualimplantation condition.■ ASSOCIATED CONTENT*sı Supporting InformationThe Supporting Information is available free of charge athttps://pubs.acs.org/doi/10.1021/acsbiomaterials.1c01429.Wloss of the AZ31 screws after 28 d of gel immersion(PDF)■ AUTHOR INFORMATIONCorresponding AuthorAkiko Yamamoto − Research Center for Functional Materials,National Institute for Materials Sciences, Tsukuba, Ibaraki305-0044, Japan; orcid.org/0000-0002-9182-4886;Email: yamamoto.akiko@nims.go.jpAuthorAkemi Kikuta − Research Center for Functional Materials,National Institute for Materials Sciences, Tsukuba, Ibaraki305-0044, JapanComplete contact information is available at:https://pubs.acs.org/10.1021/acsbiomaterials.1c01429Author ContributionsThe manuscript was written through contributions of allauthors. All authors have given approval to the final version ofthe manuscript.FundingThis research was partially supported by JSPS KAKENHI(Grant Number JP 17H02116).NotesThe authors declare no competing financial interest.■ ACKNOWLEDGMENTSThe authors appreciate Ms. Y. Kohyama from NIMS, Mr. M.Gwiazda from the Warsaw University of Technology, and Ms.Y. Mukai from Tsukuba University for their participation tothe prepractice to establish this evaluation method. Thisresearch was partially supported by JSPS KAKENHI (GrantNumber JP 17H02116).■ ABBREVIATIONSAZ31, Mg-3wt %Al-1wt %Zn alloy; EOG, ethylene oxide gas;FBS, fetal bovine serum; hcoat, coating thickness; MEM,minimum essential medium; Mw, molecular weight; PLLA,poly-L-lactide; R, gas constant; RE, rare earth; S0, initial surfacearea; Vcabity, gas cavity volume per initial surface area; Vtotal,total gas volume per initial surface area; vd, diffusion rate of thegas; vg, generation rate of the gas; WE43, Mg-4wt %Y-3wt %REalloy; Wc, weight after coating; Wloss, weight loss; Wm,molecular weight (of an alloy); Wr, remaining weight; W0,initial weight; μCT, micro focus X-ray computed tomography;ρp, density of polymer; 2D, two dimensional.■ REFERENCES(1) Liu, Y.; Zheng, Y. F.; Chen, X. H.; Yang, J. A.; Pan, H.; Chen, D.;Wang, L.; Zhang, J.; Zhu, D.; Wu, S.; Yeung, K. W. K.; Zeng, R. C.;Han, Y.; Guan, S. Fundamental theory of biodegradable metalsDefinition, criteria, and design. Adv. Funct. 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B Biointerfaces 2015, 126, 603−606.(34) Witecka, A.; Yamamoto, A.; Świąszkowski, W. Influence ofSaOS-2 cells on corrosion behavior of castMg-2.0Zn0.98Mnmagnesium alloy. Colloids Surf., B Biointerfaces 2017, 150, 288−296.ACS Biomaterials Science & Engineering pubs.acs.org/journal/abseba Articlehttps://doi.org/10.1021/acsbiomaterials.1c01429ACS Biomater. Sci. Eng. 2022, 8, 2437−24442444 Recommended by ACSControllable Damping Magnetorheological ElastomerMeniscusXuhui Liu, Xiaoxue Wu, et al.DECEMBER 29, 2022ACS BIOMATERIALS SCIENCE & ENGINEERING READ Polymeric Microneedle Arrays with Glucose-SensingDynamic-Covalent Bonding for Insulin DeliveryZhou Ye, Matthew J. Webber, et al.SEPTEMBER 29, 2022BIOMACROMOLECULES READ Cell Adhesion Motif-Functionalized Lipopeptides:Nanostructure and Selective Myoblast CytocompatibilityElisabetta Rosa, Ian W. 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