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[Yu Yusa](https://orcid.org/0009-0009-1395-8182), Yoshinaka Shimizu, Masanobu Hayashi, [Takayuki Aizawa](https://orcid.org/0000-0002-6231-0368), Takahiro Nakahara, Takahiro Ueno, Akimitsu Sato, Chieko Miura, [Akiko Yamamoto](https://orcid.org/0000-0002-9182-4886), Yoshimichi Imai

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[Effect of hematoma on early degradation behavior of magnesium after implantation](https://mdr.nims.go.jp/datasets/5f5b5bd9-776b-4bab-9176-9cd264495e81)

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Effect of hematoma on early degradation behavior of magnesium after implantationBiomed. Mater. 19 (2024) 055043 https://doi.org/10.1088/1748-605X/ad7085Biomedical MaterialsOPEN ACCESSRECEIVED1 May 2024REVISED25 June 2024ACCEPTED FOR PUBLICATION16 August 2024PUBLISHED27 August 2024Original content fromthis work may be usedunder the terms of theCreative CommonsAttribution 4.0 licence.Any further distributionof this work mustmaintain attribution tothe author(s) and the titleof the work, journalcitation and DOI.PAPEREffect of hematoma on early degradation behavior of magnesiumafter implantationYu Yusa1,∗, Yoshinaka Shimizu1,2, Masanobu Hayashi1, Takayuki Aizawa1, Takahiro Nakahara2,Takahiro Ueno2, Akimitsu Sato1, Chieko Miura1, Akiko Yamamoto3 and Yoshimichi Imai11 Department of Plastic andReconstructive Surgery, TohokuUniversityGraduate School ofMedicine, 2-1 Seiryo-machi, Aoba-ku, Sendai,Miyagi 980-8575, Japan2 Central Research Laboratories, Nihon Parkerizing Co., Ltd, 4-5-1 Ohkami, Hiratsuka, Kanagawa 254-0012, Japan3 Research Center for Functional Materials, National Institute for Materials Sciences, 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan∗ Author to whom any correspondence should be addressed.E-mail: yusayu.yusayu@gmail.comKeywords:magnesium, hematoma, Raman analysis, energy-dispersive x-ray spectroscopy, insoluble saltSupplementary material for this article is available onlineAbstractThe corrosion of magnesium (Mg)-based bioabsorbable implanting devices is influenced byimplantation environment which dynamically changes by biological response including woundhealing. Understanding the corrosion mechanisms along the healing process is essential for thedevelopment of Mg-based devices. In this study, a hematoma model was created in a rat femur toanalyze Mg corrosion with hematoma in the early stage of implantation. Pure Mg specimen(99.9%, φ1.2× 6 mm) was implanted in rat femur under either hematoma or non-hematomaconditions. After a designated period of implantation, the specimens were collected and weighed.The insoluble salts formed on the specimen surfaces were analyzed using scanning electronmicroscopy, energy-dispersive x-ray spectroscopy, and Raman spectroscopy on days 1, 3, and 7. Theresults indicate that hematomas promote Mg corrosion and change the insoluble salt precipitation.The weight loss of the hematoma group (27.31± 5.91 µg mm−2) was significantly larger than thatof the non-hematoma group (14.77± 3.28 µg mm−2) on day 7. In the non-hematoma group,carbonate and phosphate were detected even on day 1, but the only latter was detected on day 7. Inthe hematoma group, hydroxide was detected on day 1, followed by the formation of carbonate andphosphate on days 3 and 7. The obtained results suggest the hypoxic and acidic microenvironmentin hematomas accelerates the Mg corrosion immediately after implantation, and the subsequenthematoma resorption process leads to the formation of phosphate and carbonate with organicmolecules. This study revealed the risk of hematomas as an acceleration factor of the corrosion ofMg-based devices leading to the early implant failure. It is important to consider this risk in thedesign of Mg-based devices and to optimize surgical procedures controlling hemorrhage atimplantation and reducing unexpected bleeding after surgery.1. IntroductionBiomedical application of magnesium (Mg) alloysas fracture fixation devices has been researchedworldwide [1, 2]. Because Mg alloys have bioab-sorbability and adequate strength, their biomedicalapplication in orthopedic and maxillofacial areaswill change the design and function of existingosteosynthesis implants, replacing conventional Tiand absorbable polymers [2–4]. However, Mg alloysreact with water immediately after implantation;the highest corrosion rate is generally observedimmediately after the implantation [5]. The cor-rosion of Mg alloys depends on the surroundingenvironment, especially the pH of the electrolyte[6–9].Mg corrosion in the simulated body fluid resultsin the releases of Mg ions (Mg2+), hydroxide ions(OH−), and hydrogen (H2) gas. The corrosion of Mgare described by the following reactions [3, 10]© 2024 The Author(s). Published by IOP Publishing Ltdhttps://doi.org/10.1088/1748-605X/ad7085https://crossmark.crossref.org/dialog/?doi=10.1088/1748-605X/ad7085&domain=pdf&date_stamp=2024-8-27https://creativecommons.org/licenses/by/4.0/https://creativecommons.org/licenses/by/4.0/https://orcid.org/0009-0009-1395-8182https://orcid.org/0000-0002-6231-0368mailto:yusayu.yusayu@gmail.comhttp://doi.org/10.1088/1748-605X/ad7085Biomed. Mater. 19 (2024) 055043 Y Yusa et alMg→Mg2+ + 2e− (anodic reaction) . . . (1)2H2O+ 2e− →H2 + 2OH− (cathodic reaction) . . .(2)Mg + 2H2O→Mg2+ + 2OH−+H2 (overall reaction) . . .. (3)The released OH− ions increase the pH of theelectrolyte surrounding the Mg specimen [11, 12]and causes the precipitation of insoluble salts such ashydroxide, carbonate, and phosphate [12, 13]. In caseof a simple simulated body fluid such as 0.9% NaCl,which has a limited buffering capacity, Mg(OH)2 isprecipitated on the Mg surface. However, it is con-verted to highly water-solubleMgCl2 by chloride ions(Cl−) by the following reaction [14].Mg(OH)2 + 2Cl− →MgCl2 + 2OH−. . .. (4)Since body fluids contain calcium and phosphateions, the precipitation of calcium phosphate on thespecimen surface is reported [10]. The formationof an insoluble salt layer on the specimen surfacehindered the contact of water molecule to the Mgmetal substrate, resulting in the retardation of thecorrosion.As in the above reaction (3), the H2 generationrate is proportional to the Mg corrosion rate; it alsodecreases with increase in insoluble salt precipitation.The released ions and generated H2 diffuse into thetissue by dissolving into the body fluid and capillaryflow. When the H2 generation is larger than H2 dif-fusion, the gas cavity is formed in the tissue [15].Subsequently, the H2 generation decreases and capil-lary grows, which gradually results in the reduction ofgas cavity volume in the tissue.The corrosion of the implanted Mg alloys issusceptible for complex physiological environmentinfluenced by implantation site, ionic concentration,and diffusion capacity of the surrounding tissue [16].It is further complicated by the action of cells inwound and fracture healing at the implantation site,which induces the dynamic changes in the circulat-ory environment due to angiogenesis and granulation[17]. Surgical intervention and bleeding at fracturesites can also be one of the influencing factors; acertain amount of hemorrhage forms a hematoma,which generates a hypoxic microenvironment indu-cing anaerobic metabolism resulting in lactic acidproduction and an acidic environment [18–20]. Thisenvironment becomes evenmore acidic by osteoclastsactivities [21]. Moreover, an acidic environment atthe fracture site induces resorption of hydroxyapat-ite from the bone, which increases calcium and phos-phate ions in the body fluids [18].In clinical cases of fracture fixation, the hem-atoma formation occurs at a certain rate due toinsufficient hemostatic technique and unexpectedbleeding [22]. It may also be influenced by thefracture conditions and patient underlying disease.Unexpected bleeding is not considered to cause a ser-ious damage on conventional, non-absorbable metalfixtures, however, no information is available forMg-based, bioabsorbable fixtures. Clinically availableMg-based device is suspected to cause complicationssuch as infection and osteolysis in about 13.3% ofcases, which remains a problem that must be solved[15, 23, 24].It is mandatory for Mg-based devices to con-trol their corrosion to achieve the clinical success.Analysis of the corrosion mechanism of Mg in theearly implantation phases is essential for better con-trol of its corrosion behavior in physiological envir-onment. As most implantation studies were per-formed in normal tissues, such as marrow cavity,or intramuscular tissue adjacent to bone [25–27],the effects of hematoma on Mg corrosion remainunclear. A few in vitro studies reported the higherMg2+ release from the Mg alloys in contact to wholeblood for 6 h than in PBS(-) or plasma [28, 29].However, no report describes the detailed relation-ship between Mg corrosion behavior and hematomawithin one week of implantation in vivowith detailedanalysis of the insoluble salts formed on the specimensurface.This study aims to analyze the effect of hematomaon Mg corrosion and insoluble salt precipitation inthe early phase of implantation. Through the study,we can obtain useful and unprecedented knowledgeon themechanism ofMg corrosion in vivo, which willcontribute to the development and improvement ofthe quality of Mg-based devices for fracture fixation.2. Methods2.1. Test materialsCommercially available pure Mg wire (99.9%,φ1.2 mm, MACRW Co., Ltd, Shizuoka, Japan) wascut into 6 mm lengths and both ends were polishedwith JIS #2500 using a SiC paper. The nominal com-positions of theMgwires are listed in table 1. The nat-ural oxide film on the specimen surface was removedby immersion into a nital solution (5% nitric acid inethanol) at 20 ◦C in an ultrasonic cleaner, and thespecimen was washed with ultrapure water beforedrying under vacuum [30, 31]. The pretest weightof each specimen (W0 [mg]) was measured usingan electronic balance (BM-5; A&D Corporation,Tokyo, Japan). The specimen length was decidedbased on the W0, specimen radius (0.6 [mm]),and the density of pure Mg (1.738 [mg mm−3]) as2Biomed. Mater. 19 (2024) 055043 Y Yusa et alTable 1. Composition of the pure Mg wire (mass%).Al Mn Zn Si Cu Ni Fe Mgpure Mg (99.9%) 0.009 0.011 0.016 0.003 0.008 0.001 0.002 Bal.Figure 1. Sample placement in rat femur. Non-hematoma group (left) and hematoma group (right).W0/(π × 0.62 × 1.738), which giving the initial sur-face area of the specimen (S0 [mm2]) by equation (1),S0 = 2× 0.6× 0.6×π +2×W00.6× 1.738. (1)2.2. Implantation in ratsThe animal experimental protocol was reviewed andapproved by the Animal Care and Use Committee ofTohoku University (Sendai, Japan, approval number:2022MdA-009-01). It follows the Guide for the Careand Use of Laboratory Animals, 8th ed. (NationalResearch Council, revised in 2010). Eleven-week-oldWistar rats were purchased and acclimatized withnormal feed and tap water for one week. Six twelve-week-old rats (mean weight: 272 ± 10.4 [mg]) wereassigned to each of implantation periods (1, 3, 7 d)and implantation surgerywas performed. Three addi-tional animals per each implantation period weresubjected to implantation for analysis by scanningelectronmicroscopy (SEM) andRaman spectroscopy.2.3. Implantation procedureGeneral anesthesia was induced by inhalation of 5.0%isoflurane (isoflurane for animals, Intervet, Tokyo,Japan), and the concentration of isofluranewasmain-tained at 2.5 [vol.%] intraoperatively. Local anes-thesia was performed by subcutaneous injection of1% lidocaine with epinephrine (<0.01 mg kg−1;Xylocaine®□ injection 1% epinephrine, AstraZeneca,Osaka) in the middle thigh. A 1 cm long skin incisionwas made in the middle of the thigh followed by care-ful dissection of the intermuscular space approach-ing to the femur mind avoiding unnecessary bleed-ing. The groupwithout hemorrhage from the femoralbone marrow was defined as the non-hematomagroup (figure 1, left). In the other group, after afemoral approach was prepared by the same mannerto the non-hematoma group, a hole of 1.6 mm dia-meter was drilled by a steel bar (ST1 HP 016, Hager &Meisinger GmbH, Germany) in the anterior aspect ofthe femur to create a hematoma. Then, the implanta-tion space of approximately 4–5 mm wide, 8–10 mmlong, and 3–4 mm in depth was filled with approx-imately 200 mm3 of hematoma, and the entire speci-men was inserted in the hematoma. The group withthis bone hole was defined as the hematoma group(figure 1, right). After insertion of the specimen, thefascia was closed using nylon thread to prevent thespecimen from displacement, and the wound wasclosed with skin sutures.Every rat received 2 specimens; 1 on its left femurfor the hematoma condition and 1 on its right forthe non-hematoma condition. For theweight loss andinsoluble salt quantification, 6 specimens are used pergroup per implantation period. For the scanning elec-tron microscope equipped with an energy-dispersivex-ray spectrometer (SEM-EDX) and Raman spectro-scopy, 3 specimens were employed per group perimplantation period. The weights of the specimensfrom the non-hematoma and hematoma groupswere 10.678 ± 0.085 mg and 10.843 ± 0.073 mgrespectively.After implantation, the experimental animalswere kept individually in cages; there was no signific-ant weight loss in all animals. No signs of infectionsuch as wound dehiscence or drainage were identi-fied in all cases, although no antibiotics were admin-istered. The animals were euthanized to collect thespecimen for analysis on days 1, 3, and 7. At the timeof specimen collection, all specimens were in contactwith the femur. Collected specimens were vacuumbagged to prevent oxidation and stored in vacuumdesiccators.2.4. Specimen weight measurementThe specimens were collected and washed withultrapure water before vacuum drying. The weightof the specimen after implantation (W1 [mg]) wasmeasured to calculate the weight of insoluble salt3Biomed. Mater. 19 (2024) 055043 Y Yusa et alper unit surface area (W inso [mg mm−2]) usingequation (2),Winso =W1 −W0S0. (2)Each of the six specimens per group per implant-ation period was immersed in 5 mL of a chromicacid mixture (20 g of CrO3, 1 g of AgNO3, and 2 gof Ba(NO3)2 in 100 ml distilled water) to removethe insoluble salts and rewashed with ultrapure waterbefore vacuum drying. The chromic acid mixtureafter specimen immersion was collected for furtheranalysis (section 2.5).The weight (Wr [mg]) of each specimen afterchromic acid cleaning was measured to calculate theweight loss per unit surface area (W loss [mg mm−2])using equation (3),Wloss =W0 −WrS0. (3)2.5. Quantitative analysis of insoluble saltsMg, calcium (Ca), and phosphorus (P) in the collec-ted acid mixture were quantitatively analyzed usinginductively coupled plasma-mass spectrometry (ICP-MS). The difference between the elemental concen-tration in the collected chromic acid mixture (Ccp[µg mL−1]) and that in the unused acid mixture(Ccontrol [µg ml−1]) gave the amount in precipita-tion per unit surface area Wcp [mg mm−2] usingequation (4),Wcp =(Ccp −Ccontrol)× 51000× S0. (4)2.6. Analysis of insoluble salts by SEM-EDXThe surface of the post-implantation specimens wasanalyzed using a SEM-EDX (S-4800, Hitachi High-Technologies Corporation, Tokyo, Japan). Prior to theSEM observation, the specimen surface was coatedwith platinum. Elemental analysis was performed atan acceleration voltage of 15 keV and resolution of512 × 384 [pixels]. The percentage elemental distri-bution of Mg, Ca, P, carbon (C), and oxygen (O) wasanalyzed on the surface of three post-implantationspecimens per group.2.7. Analysis of insoluble salts using micro-RamanspectroscopyThe post-implantation specimens were ana-lyzed by Raman spectroscopy (Laser RamanSpectrophotometer Automatic Imaging SystemNRS-5100; Japan Spectroscopy Corporation, Tokyo,Japan). One or two typical places on the surface wereanalyzed per specimen. The measurement conditionswere as follows; grating L = 600 nm, B = 500 nm,objective lens magnification = 100, excitationlaser = 532.13 nm, exposure time = 10 s, number ofintegrations = 5 times, laser power = 3.2 mW (50%in measurement), and wave number range = 100–3700 cm−1.2.8. Statistical processingAll data are presented as mean ± standard devi-ation. Statistical analysis of group differences was per-formed using JSTAT 6.9 for windows, with t-test andlinear regression analysis.3. Results3.1. Weight loss of magnesium specimenThe weight loss of the specimens was summarized infigure 2(a) and table S1 in the supplementary materi-als. As shown in figure 2(a), the weight loss of bothgroups increased depending on the square root ofthe implantation period, indicating a decrease in theweight loss per day with an increase in the implant-ation period. The increasing trend of the weight loss(i.e. the slope of the regression line against the squareroot of the implantation period) of the hematomagroup is twice as large as that of the non-hematomagroup (p < 0.01 by the parallelism test of 2 regres-sion lines). This suggests the involvement of diffu-sion process on the corrosion rate of the Mg speci-men.After 7 d of implantation, the averageweight lossof the hematoma group was 27.31 ± 5.91 µg mm−2,which was 1.8 times larger than that of the non-hematoma group (14.77 ± 3.28 µg mm−2). Chenet al reported the weight loss rate of pure Mg spe-cimens (1.1 mmφ × 15 mm) implanted into rabbitmuscle tissue for 1 week was 4.7% [32], which equalsto 21.58 µg mm−2. This value is slightly larger thanthat of the non-hematoma group, probably due to thedifferences in the implantation tissue, animal species,specimen size and impurities.3.2. Weight of insoluble saltsThe weight of the insoluble salts was summarized infigure 2(b) and table S2 in the supplementary mater-ials. The parallelism test of the regression lines forboth hematoma and non-hematoma groups againstthe square root of the implantation period indicates asignificant difference not in the increasing rate of theinsoluble salt weight (i.e. a slope of the regression line)but in the initial weight of the insoluble salts (i.e. aintercept of the regression line, p < 0.01). This sug-gests that the specimens in the hematoma group havesignificantly larger amount of salt precipitation in thevery early stage of the implantation (within 24 h).In figure 2(c), W inso/W loss was plotted againstthe square root of the implantation period, indic-ating the clear difference between hematoma andnon-hematoma groups. For the latter group, it wasstable at around 0.9, whereas that for the hematomagroup decreased with an increase in the implantationperiod. The higher W inso/W loss ratio indicates moreprecipitation of insoluble salts. This can be achieved4Biomed. Mater. 19 (2024) 055043 Y Yusa et alFigure 2. Results of (a) weight loss per unit surface area, (b) weight of insoluble salts formed on the specimen surface afterimplantation, and (c) ratios of insoluble salt weight to weight loss plotted against the square root of the implantation period,√t.In (a), the increasing trend in the weight loss of the hematoma group is significantly larger than that of non-hematoma group(p< 0.05 by the parallelism test of 2 regression lines). In (b), there is no significant difference in the increasing trends of theinsoluble salt weight between the hematoma and non-hematoma groups, but their intercepts (the initial insoluble salt weight) aresignificantly different (p< 0.01 by the parallelism test of 2 regression lines).Figure 3. Results on quantification of Mg (a), Ca (b), and P (c) in insoluble salt on days 1, 3, and 7 after implantation by ICP-MS,plotted against the square root of the implantation period,√t. The ratios of Mg (d), Ca (e), and P (f) to insoluble salts weight arealso plotted against√t.by the higher concentration of released Mg2+ andhigher local pH, which reduces the solubility of insol-uble salts [33]. The concentration of released Mg2+near the specimen surface is influenced by the diffu-sion rate in the microenvironment at the implanta-tion site. The higherW inso/W loss at day 1 of the hem-atoma group may be attributed to its higher corro-sion rate and less diffusive environment. The shift inW inso/W loss may reflects the change in the microen-vironment in hematoma, such as recovery of micro-circulation and decrease in local pH due to the retard-ation of Mg corrosion.3.3. Elemental quantification of insoluble salts byinductively coupled plasma-mass spectroscopy3.3.1. MagnesiumThe Mg weight as insoluble salts was summarizedin figure 3(a) and table S3 of the supplementarymaterials. A significant increase in Mg weight wasobserved over time for both groups (F (1,5)= 21.416,p < 0.001). Both groups displayed a similar increaserate of Mg weight (p > 0.1 by Parallelism test of2 regression lines against the square root of theimplantation period). However, the intercepts of theregression lineswere significantly different (p< 0.01).This trend is similar to that of the insoluble saltweight as described in the previous chapter, indic-ating the significant difference was occurred in thevery early stage of the implantation (within 24 h). TheMg weight of the hematoma group was significantlyhigher than that of the non-hematoma group on days1 and 7.The ratio of Mg weight to W inso was plottedin figure 3(d), indicating its stability around 0.18through days 1–7 for both groups. This agreementin Mg/W inso also suggests that the same kinds of5Biomed. Mater. 19 (2024) 055043 Y Yusa et alFigure 4. Typical examples of the EDX analysis for the top surface (a) of pure Mg specimen implanted for 1 d undernon-hematoma condition. The EDX spectrum (b) was obtained by the point analysis of a red square indicated in the SEM imagein (a).insoluble salts containingMgwere formed on the spe-cimen surface for both groups.3.3.2. CalciumThe Ca weight as insoluble salts was summarized infigure 3(b) and table S4 in the supplementary materi-als. The hematoma group has a slightly larger increaserate of the Ca weight than the non-hematoma group,but no significant difference was observed betweenthe two groups (p > 0.1 by the Parallelism Testof 2 regression lines). However, the intercepts ofthe regression lines were different (p < 0.1 by theParallelism Test). This indicates that the hematomagroup has the significantly smaller precipitation of Casalts in the very early stage of implantation.The ratio of Ca weight to W inso was plotted infigure 3(e) against the square root of the implanta-tion period. For the non-hematoma group, Ca/W insois significantly greater than that of the hematomagroup at day 1. It decreased thereafter, whereas thatof the hematoma group slightly increased. These dataclearly indicate the different trend of Ca salt precipita-tion between non-hematoma and hematoma groups.3.3.3. PhosphorusThe P weight as insoluble salts was summarized infigure 3(c) and table S5 in the supplementary mater-ials. In the same manner to Ca, the hematoma grouphas a slightly larger increase rate of P weight than theother with no significance (p> 0.1 by the parallelismtest of the regression lines), but a significant differencewas observed in their intercepts (p > 0.1). As shownin figure 3(f), the non-hematoma group has the sig-nificantly greater P/W inso than the other on days 1,3, and 7. The P/W inso of the non-hematoma groupdecreased from day 1–3, while that of the hemat-oma group slightly increased through the implanta-tion period. These trends are similar to those of Ca,suggesting the co-precipitation of Ca and P on thespecimen surface.3.4. Surface analysis of implanted specimens bySEM-EDX3.4.1. Non-hematoma groupTypical SEM-EDX images of the post-implantationspecimen surface were shown in figure 4 (day 1), andsupplementary figures S2 (day 3) and S3 (day 7) withthose of the pre-implantation specimen surface (sup-plementary figure S1). A thin, homogeneous insol-uble salt layer with shallow cracks was formed on thespecimen surface on day 1. The distribution ofMg onthe specimen surface became less pronounced on day3, and the distribution of Ca and P was dominant onday 7. The formation of O, Ca, and P rich layer at spe-cimen surface was confirmed by the cross-sectionalanalysis of a pure Mg wire implanted into a rat aortawall [34], which agrees with the finding in this study.On the pre-implantation specimen, Mg was detectedas the main peak with negligible peaks assigned to C,O, and P (figure S1).The results of EDX point analysis are summar-ized in figure 5 and table S6 in the supplementary6Biomed. Mater. 19 (2024) 055043 Y Yusa et alFigure 5. Results of quantitative EDX analysis of pure Mg specimens implanted under non-hematoma and hematoma conditions(atm. %) for day1(a), day 3(b), and day 7(c). The statistical significance between the groups was decided by t-test (∗p< 0.05,∗∗p< 0.01).materials. It clearly shows the different pattern of ele-mental distributions at day 1 from those at day 3 and7. The relatively high Mg concentration with low Oconcentration at day 1 indicates the formation of athin insoluble salt layer on the specimen surface. Atday 3 and 7, the Mg concentration decreased whereasCa and P increased, suggesting the growth of theinsoluble salt layer. At day 1, concentrations of Caand P are much smaller than that of Mg, whereasthose by ICP-MS analysis are close to that of Mg.This suggests that the EDX results includes the sig-nals from the Mg substrate because the insolublesalt layer is thin as described. At day 3 and 7, theratios of Ca and P to Mg increased to be close to1, which is even larger than those by ICP-MS ana-lysis. This indicates the higher distribution of Ca andP on the surface of the insoluble salt layer at day 3and 7.3.4.2. Hematoma groupTypical SEM-EDX images of theMg specimen surfaceafter implantationwere shown in figure 6 (day 1), andsupplementary figures S4 (day 3) and S5 (day 7). Nocrack was observed on the specimen surface on day1 whereas shallow cracks were observed in the non-hematoma group. Moreover, the surface was gener-ally unevenwith bumps.Onday 3, surface crackswereobserved with a decrease inMg, an increase in Ca andP distribution; this trend was further enhanced onday 7.The results of EDX analysis are summarized infigure 5, and supplementary table S6 with thoseof the non-hematoma group. Similar to the non-hematoma group, the concentration of Mg decreasedwhile those of Ca and P increased with the increasein the implantation period. On day 1, the hemat-oma group has 1.7 times higher Mg and 40% lowerO than the other group, indicating the clear differ-ence in the specimen surface composition betweenthe two groups. Similar to the non-hematoma group,the relatively high Mg and low O concentration sug-gests the formation of a thin insoluble salt layer onthe specimen surface. However, the hematoma grouphas the larger W inso than the other (see figure 2(b)).This controversial result may be attributed to theinhomogeneity of insoluble salt formation on the spe-cimen surface of the hematoma group, as observed inSEM images (figure 6(a)). These findings suggest thatthe hematomas strongly influence the insoluble saltprecipitation at the initial stage of implantation. Onday 3 and 7, the surface compositions of the hemat-oma group were similar to those of non-hematomagroup, with significantly less Ca and P concentra-tions. Similar to the non-hematoma group, the ratiosof Ca and P to Mg are larger than those by ICP-MSanalysis, suggesting the higher distribution of it at thesurface of the insoluble salt layer. In other words, Mgsalts are formed in the deeper part of the insolublesalt layer on days 3 and 7. The concentration of Cwas increased from day 3 to day 7, suggesting the co-precipitation of organic compounds in the insolublesalt layer.3.5. Analysis of the insoluble salts by micro-Ramanspectroscopy3.5.1. Raman spectra of the non-hematoma groupTypical Raman spectra of the specimens in the non-hematoma group are shown in figure 7 with theirmajor peaks summarized in table 2. For reference, theRaman spectrum of the pre-immersion specimen isshown in the supplementary figure S6, which does nothave a specific peak in the region of 1100–950 cm−1.On day 1, the peaks at 960 or 1085 cm−1 weredetected at the different sites on the specimen sur-face. The peak at 960 cm−1 is attributed to thev1 stretching vibration of PO43- [35, 36], while thepeak at 1080–1090 cm−1 is attributed to the stretch-ing vibration of CO32− [37, 38], confirming localprecipitation of phosphate and carbonate at theearly stage of implantation. The detection of CO32−and PO43− on the implanted specimen surface wasalso reported by the microscopic Fourie transforminfrared spectroscopy of the pure Mg wire surfaceimplanted into the rat aorta wall [34, 39]. On day3, peaks at 967 and 1083 cm−1, assigned to PO43−and CO32−, were observed at the same site, suggest-ing the co-precipitation of phosphate and carbon-ate. On day 7, however, the peak corresponding toCO32− disappeared while the peak assigned to PO43−7Biomed. Mater. 19 (2024) 055043 Y Yusa et alFigure 6. Typical examples of the EDX analysis for the top surface (a) of pure Mg specimen implanted for 1 d under hematomacondition. The EDX spectrum (b) was obtained by the point analysis of a red square indicated in the SEM image in (a).Figure 7. Raman spectra of the specimen surface in the non-hematoma group. (a) (b) Different sites on day 1, (c) day 3, and (d)day 7.8Biomed. Mater. 19 (2024) 055043 Y Yusa et alTable 2. Raman shifts of the major peaks observed for insoluble salts formed on the Mg specimen surface implanted undernon-hematoma condition.Wavenumaber (cm−1)PO43− CO32− CH3+ Appearance formDay1 960 1085 IndependentlyDay3 967 1083 2941 SimultaneouslyDay7 987 2929 SimultaneouslyFigure 8. Raman spectra of the specimen surface in the hematoma group. (a) (b) Different sites on day 1, (c) day 3, and (d) day 7.was observed at 987 cm−1. This indicates the domin-ance of phosphate precipitation at day 7. On days 3and 7, the peak at approximately 2930 cm−1 was alsoobserved, which is attributed to the stretching vibra-tion of CH3+ in organic substances [40, 41].3.5.2. Raman spectra of the hematoma groupTypical Raman spectra of the specimens in the hem-atoma group were shown in figure 8 with their majorpeaks summarized in table 3. Peaks at 1123 and3648 cm−1 were observed on day 1. Hydromagnesite,hydrated magnesium carbonate, has peaks corres-ponding to CO32− and OH− at 1120–1123 cm−1and 3648 cm−1, respectively [38]. Magnesium phos-phate is another candidate; its Raman spectrum hasa peak at 1122 cm−1 [42]. Therefore, hydromagnes-ite and/or magnesium phosphate may precipitate atthe early stage of implantation, whereas carbonateand phosphate are observed on day 1 for the non-hematoma group. On day 3, peaks were observedat 967 and 1093 cm−1 whereas those at 967, 1095,and 2934 cm−1 were detected on day 7. These obser-vations confirmed the co-precipitation of phosphateand carbonate on days 3 and 7 for the hematomagroup, while only phosphate was observed on day 7for the non-hematoma group. The co-precipitationor absorption of organic substances was also delayed9Biomed. Mater. 19 (2024) 055043 Y Yusa et alTable 3. Raman shifts of the major peaks observed for insoluble salts formed on the Mg specimen surface implanted under hematomacondition.Wavenumaber (cm−1)PO43− PO43− or CO32− CO32− CH3+ OH− Appearance formDay1 1123 3648 SimultaneouslyDay3 967 1093 SimultaneouslyDay7 967 1095 2934 Simultaneouslyfor the hematoma group; which was observed fromday 3 for the non-hematoma group.4. DiscussionMany reports describe the degradation behavior ofMg and its alloys, both in vitro and in vivo; however,they have different results in degradation rates andstructures of the insoluble salt layer formed on thespecimen surface [13, 43, 44]. In in-vitro studies, thecorrosion of Mg depends on the chemical composi-tion and pH of the immersion solution [8], in whichMg2+, OH− and H2 gas diffuse easily. However,when implanted in vivo, the degradation behavioris further complicated; because multiple factors areinvolved, including relatively high ionic strength,ion/gas diffusion by blood flow, cellular influence,and the presence of organic compounds such as gluc-ose, amino acids, and proteins [45]. The corrosionbehavior of implanted Mg specimen is more com-plex because surgical invasion and bleeding fromthe fracture site markedly affect the environment ofthe implantation site compared with that in normaltissue.In case of the specimen implantation into normaltissue with reasonable bleeding control, an intersti-tial fluid will contact to the specimen surface. Thisfluid has a buffering ability tomaintain its pHof 6.60–7.60, however, it ismore variable than the pHof blood[46]. In addition to chloride ion (Cl−), it containsHCO3− andHPO42− which contribute to pHbufferingin an equilibrium state [47, 48]. As components of theinterstitial fluid penetrate through lymphatic vesselsand capillary walls to circulate in the whole body, theMg2+ and OH− released by Mg corrosion are expec-ted to diffuse relatively quickly by microcirculation.Even so, the pH of the interstitial fluid near the spe-cimen surface can locally increase, depending on thecorrosion rate of Mg specimen, buffering ability ofthe fluid, and diffusion condition in the microenvir-onment as observed in the in vitro experiments [11,49]. Then, the concentrations of CO32−, HPO42−, andPO43− increase, resulting in the reaction of these ionswith Mg2+ and Ca2+ in the interstitial fluid to forminsoluble salts as MgCO3, Mg3(PO4)2, CaCO3, andCa3(PO4)2 in addition to Mg(OH)2 [10, 50]. Theseinsoluble salts precipitate and cover the specimen sur-face, retarding the corrosion rate.In clinical cases, unexpected bleeding from frac-ture surfaces or soft tissue after the surgery creates themicroenvironment different from that in the inter-stitial fluid. Hematomas cause primary hemostasisvia platelet aggregation, followed by an acceleratedcoagulation cascade and fibrin accumulation to forma fibrin network, resulting in an aggregate isolatedfrom the surrounding environment [51, 52]. Theloss of capillary and circulation inside the hematomareduces the oxygen supply, resulting in high concen-trations of lactic acid by anaerobic metabolism andCO2 exudation from dead cells [19, 53]. However, thegradual gathering of endothelial cells forms capillar-ies, and consequently resumes circulation and diffu-sion within the hematoma [54]. Thus, the pH withinthe hematoma drops to approximately 5.0 and recov-ers around pH 7.0 over about two weeks [18, 19, 55].This low-pH, low-circulation microenvironment inthe hematomas can be an accelerating factor for thecorrosion of the implanted Mg specimen. There area few in vitro studies investigating the effect of wholeblood onMg alloy corrosion for short time as 6 h [28,29], but no in vivo study was performed to investigatethe effect of hematoma on the corrosion behavior ofMg or Mg alloy specimens so far.In this study, pure Mg specimens were implantedinto rat thighs under hematoma or non-hematomaconditions up to 7 d. The volume of the hemat-oma formed at the implantation site is estimated200 mm3, which is equivalent to 20–30 ml hemor-rhage for a human adult weighing 60 kg. This volumeis an acceptable level in the clinical cases of fracturefixation as unexpected bleeding after surgery.The specimens were collected after the desig-nated period of implantation, followed by weightloss measurement and quantification of Mg, Ca,and P in insoluble salts by ICP-MS. The elementaldistributions and chemical structures of the insol-uble salts formed on the specimen surface were ana-lyzed by SEM-EDX and Raman spectroscopy. Asthe results, the hematoma accelerated the corro-sion of pure Mg with changes in the structure ofthe insoluble salt layer. The corrosion rate estim-ated by the weight loss for the hematoma group wasalmost doubled that for the non-hematoma groupafter 7 d of implantation (figure 2(a)). Wang et alreported that the pure Mg wire implanted into rataorta lumen was slightly less corroded than that10Biomed. Mater. 19 (2024) 055043 Y Yusa et alFigure 9.Mechanism of insoluble salt formation. The non-hematoma group (a) and the hematoma group (b).implanted in aorta wall [39], which is controversialto that obtained in this study. In their study, thespecimen implanted into aorta lumen was initiallyexposed to the dynamic blood flow but was even-tually covered by a fibrous capsule before 3 d [39].This suggests the difference in the microenvironmentbetween the aorta lumen and the hematoma in thepresent study. Initial non-dynamic, fibrous structureof hematomamay accelerate the corrosion rate of Mgspecimen than in non-hematoma tissue. The shift inW inso/W loss of the hematoma group with the increasein the implantation period indicates the change in themicroenvironment influencing the insoluble salt pre-cipitation, which may correspond to the resume ofmicrocirculation.The ICP-MS and EDX analysis revealed the dif-ference in the insoluble salt precipitation betweenthe hematoma and non-hematoma groups. The EDXanalysis found that the hematoma group had thesignificantly lower concentrations of Ca and P thanthose of the non-hematoma group (figure 5 and tableS2). In comparison to the results by ICP-MS, Ca andP tended to distribute at the surface of the insolublesalt layer on days 3 and 7. Raman spectroscopy con-firmed that the non-hematoma group had a phos-phate layer on the specimen surface from the earlystage of implantation, while the hematoma group hada less or delayed formation of it. In the latter group,carbonate is more dominant than phosphate, andhydroxide was observed on day 1.Differences in structures of the insoluble salt layerbetween the hematoma and non-hematoma groupsare possibly attributed to the difference in pH, ionsupply, and diffusion in the microenvironment [10].The schematic illustrations of the insoluble salt pre-cipitation processes are shown in figure 9. At the veryinitial moment of the implantation, the local concen-trations of Mg2+ and OH− increased near the speci-men surface due to a corrosion reaction. This guidesto temporal precipitation of Mg(OH)2, which even-tually disappears when the pH is lower than 11.5 [3].Instead, precipitation of MgCO3 likely occurs sinceHCO3− is abundant in the interstitial fluid. In thenon-hematoma group, Mg2+ and OH− diffuse rel-atively quickly with supplies of Ca2+ and PO43− bymicrocirculation, leading to precipitation of calciumphosphate even at the early stage of implantation asday 1. Under non-hematoma condition, this calciumphosphate formation at the early phase of implant-ation may efficiently retard the corrosion of the Mgspecimen.In the hematoma group, however, the lower pHin hematomas induces initial rapid corrosion of theMg specimen, which results in the higher local pHat the specimen surface. This leads to the largeramount of Mg(OH)2 precipitation than the othercondition, as detected by Raman spectroscopy on day1. Furthermore, low ion diffusion with low Ca2+ andPO43− supplies in the hematomas guided to hydro-magnesite precipitation at the early stage of implant-ation. Eventually, the carbonate becomes dominantalong with the progress of fibrinolysis. The precip-itation of calcium phosphate delays to day 3 andthereafter, which may be one of the causes of thehigher corrosion rate of Mg specimen under hemat-oma condition.The fibrinolysis as well as the recovery processof hematomas may influence the microenvironmentand thus insoluble salt precipitation on the implantedspecimen surface. In the hematoma group, both Caand P ratios againstW inso increased with the increasein implantation period (figures 3(e) and (f)) whileW inso/W loss decreased (figure 2(c)). These trendsmayreflect the recovery of local pH at the specimen sur-face in the hematoma along with the decrease in thecorrosion rate of the Mg specimen.The insoluble salts layer formed on the speci-men surface at the early stage of implantation can11Biomed. Mater. 19 (2024) 055043 Y Yusa et alinfluence the following tissue reaction against the spe-cimen. The formation of a stable calcium phosphatelayer on the Mg alloy specimen at the early stage ofimplantation is beneficial for bone formation [56].Mg-based device with localized contact to the hem-atomamay have localized corrosion, potentially lead-ing to its unintended failure [57]. Unintended break-age of the implanted device fails to achieve the expec-ted bone fusion, which causes fistula formation andsoft tissue damage leading to prolonged inflammation[58]. The results obtained in this paper indicate thehematoma as an acceleration factor for the corro-sion of Mg-based devices. That means, the Mg-baseddevices need to be designed and developed with theconsideration of hemorrhage and hematoma risks atthe implantation site. It also suggests the importanceof the optimization of surgical procedures to controlthe hemorrhage at implantation and to reduce theunexpected bleeding after the surgery.A limitation of the present study is that the hem-atoma was formed via a small bony foramen; themicroenvironment in the hematoma may be differ-ent from that formed via soft tissues or fracture sur-faces. In the general implantation condition for bonefracture fixation, the bone marrow was also exposedto the fixture device to some extent, leading to sup-pression of Ca and P supply due to demineraliza-tion in bone marrow. In this study, implantation testswere conducted as the entire specimen was insertedinto a hematoma. However, we believe that hemat-oma can accelerate localized corrosion even with itspartial contact to the specimen surface. In vivo exper-iments of a specimen having a similar shape to clinicaldevices are recommended for the accurate evaluationof the effect of hemorrhage in clinical conditions.5. ConclusionIn this study, the corrosion behavior of pure Mgwas investigated during the early stages of in-vivoimplantation under hematoma and non-hematomaconditions. As the results, hematoma promotes thecorrosion of the Mg specimen, influencing the struc-ture of the insoluble salt layer formed on the specimensurface. These effects were attributed to the microen-vironment in hematoma; lower pH and less microcir-culation. Obtained results indicate the risk of hem-atoma by unexpected bleeding after implant surgerythat accelerates the corrosion of Mg-based devicesleading to their failure in fracture fixation. It also sug-gests the importance of considering the risk of hemat-oma on the development and design of newMg-basedfixtures.Data availability statementAll data that support the findings of this study areincluded within the article (and any supplementaryfiles).AcknowledgmentsWe thankMs. Miho Oikawa of the Technical Divisionof Tohoku University Graduate School of Dentistryand Ms. Yoko Nakano, Mr. Takuya Takanashi, andMr. Syunsuke Kayamori of the Technical Division ofTohoku University Graduate School of Engineeringfor their technical supportConflict of interestThis study was funded by Nihon Parkerizing Co., Ltd.Funding statementPart of this research was supported by Grants-in-Aid for Scientific Research (issue numbers 22H03988,K23K159480, and S23310203) from the Japan Societyfor Promotion of Science.ORCID iDsYu Yusa https://orcid.org/0009-0009-1395-8182Takayuki Aizawa https://orcid.org/0000-0002-6231-0368References[1] Niranjan C A et al 2023 Magnesium alloys as extremelypromising alternatives for temporary orthopedicimplants—A review J. 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Weight of insoluble salts 3.3. Elemental quantification of insoluble salts by inductively coupled plasma-mass spectroscopy 3.3.1. Magnesium 3.3.2. Calcium 3.3.3. Phosphorus 3.4. Surface analysis of implanted specimens by SEM-EDX 3.4.1. Non-hematoma group 3.4.2. Hematoma group 3.5. Analysis of the insoluble salts by micro-Raman spectroscopy 3.5.1. Raman spectra of the non-hematoma group 3.5.2. Raman spectra of the hematoma group 4. Discussion 5. Conclusion References