Risk of fragility fracture is aggravated during bone regeneration processes in osteoporotic sheep

PLOS One | h ps://doi.o g/10.1371/jou nal.pone.0319910 May 2, 2025 1 / 24 OPEN ACCESS Ci a ion: Toscano-Angulo JJ, Mo a-Macías J, Blázquez-Ca mona P, Mo gaz J, Na a e e-Cal o R, Domínguez J, e al. (2025) Risk o agili y ac u e is agg a a ed du ing bone egene a ion p ocesses in os eopo o ic sheep. PLoS One 20(5): e0319910. h ps://doi.o g/10.1371/ jou nal.pone.0319910 Edi o : Fu qan A. Shah, Uni e si y o Go henbu g: Go ebo gs Uni e si e , SWEDEN Recei ed: Oc obe 22, 2024 Accep ed: Feb ua y 10, 2025 Published: May 2, 2025 Copy igh : © 2025 Toscano-Angulo e al . This is an open access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal au ho and sou ce a e c edi ed. Da a a ailabili y s a emen : All ele an da a a e wi hin he pape and i s Suppo ing In o ma ion iles. Funding: This s udy was suppo ed by he MICIU (Minis y o Science, Inno a ion and RESEARCH ARTICLE Risk o agili y ac u e is agg a a ed du ing bone egene a ion p ocesses in os eopo o ic sheep Juan J. Toscano-Angulo 1*, Juan Mo a-Macías2, Pablo Blázquez-Ca mona 3, Juan Mo gaz4, Rocío Na a e e-Cal o4, Jaime Domínguez1, Es he Reina-Romo1 1 Depa men o Mechanical and Manu ac u ing Enginee ing, Escuela Técnica Supe io de Ingenie ía, Uni e sidad de Se illa, Se illa, Spain, 2 Depa men o Mining, Mechanical, Ene gy and Building Enginee ing, Escuela Técnica Supe io de Ingenie ía, Uni e sidad de Huel a, Huel a, Spain, 3 Depa men o Mechanical Enginee ing and Indus ial Design, Escuela Supe io de Ingenie ía, Uni e sidad de Cádiz, Pue o Real, Spain, 4 Depa men o Animal Medicine and Su ge y, Facul ad de Ve e ina ia, Uni e sidad de Có doba, Có doba, Spain * [email p o ec ed] Abs ac In oduc ion Bone egene a ion p ocesses a e associa ed wi h a sys emic skele al change in bone quali y, inc easing he isk o agili y ac u es. This condi ion may be agg a a ed in os eopo o ic pa ien s due o hei limi ed os eogenic capaci y. This wo k e alua es he impai men o he bone quali y in os eopo o ic sheep du ing a bone egene a ion p o- cess. I p o ides a deepe unde s anding abou he complex mul iscale dynamics o bone mine al densi y, mic os uc u e and chemical composi ion ac oss di e en bone issues, loca ions and ime poin s. Ma e ials and Me hods Os eopo osis was induced in i een Me ino sheep. A c i ical-size de ec was hen c ea ed in he sheep’s igh hind me a a sus and subsequen ly egene a ed using dis ac ion os eogenesis. The animals we e andomly sac i iced du ing bone egene - a ion, ei he on days 40 o 100 a e su ge y. Compu ed omog aphy, mic o-compu ed omog aphy and chemical composi ion analyses we e conduc ed on di e en bone issues (co ical, abecula and wo en) a se e al skele al loca ions ( he ope a ed me a a sus, he con ala e al one and he iliac c es ) o assess he indi idual bone quali y changes ela i e o he non-os eopo o ic ime poin . Resul s A e os eopo osis induc ion, he abecula issue expe ienced a 6.4% educ ion in he bone mine al densi y, while no signi ican changes we e epo ed in co ical issue quali y. Du ing bone egene a ion, he ope a ed bone inc eased signi ican ly he PLOS One | h ps://doi.o g/10.1371/jou nal.pone.0319910 May 2, 2025 2 / 24 wo en ossi ica ion whils he co ical mine al densi y dec eased by 18.7%. Simul a- neously, an ea ly de e io a ion in he mic os uc u e and chemical composi ion o he abecula bone was obse ed in he iliac c es , pe sis ing o e ime in non-ope a ed abecula egions. Conclusions Os eopo osis causes une en deg ada ion o abecula issue quali y ac oss di e - en bone loca ions. Fu he mo e, he bone egene a ion p ocess ia bone anspo in os eopo o ic subjec s leads o a sys emic skele al diso de ha u he impai s he bone quali y, su passing he damage caused by os eopo osis alone. This impai men appea s o be in ensi ied by he p e-exis ing os eopo o ic condi ion. In oduc ion Os eopo osis is he mos common bone disease, a ec ing app oxima ely 500 million people wo ldwide [1]. This sys emic skele al diso de is cha ac e ized by mic oa chi- ec u al de e io a ion o he abecula bone issue, dec eased bone mine al densi y (BMD) and chemical composi ional changes, leading o inc eased skele al agili y and a highe isk o ac u es. The clinical ou come o os eopo osis is o en ac u es om low-ene gy auma, known as agili y ac u es, which would no ypically occu in heal hy bones. Globally, 37 million agili y ac u es occu annually, a ec ing one- hi d o women and one- i h o men o e he age o i y [2–4]. These ac u es esul in signi ican economic cos and majo heal h consequences o pa ien s such as se e e pain, dependency and disabili y. In his con ex , i has been shown ha he isk o agili y ac u es inc eases 5- old du ing he i s yea ollowing an ini ial ac u e a he same o di e en skele al si es [5], hen g adually dec eases om a ac o o 2.7 a e one yea o 1.4 a e en yea s [6]. Nume ous p eclinical and clin- ical s udies indica e ha , du ing he bone egene a ion, a signi ican sys emic bone quali y loss may occu h oughou he skele on, especially in os eopo o ic pa ien s, u he agg a a ing he disease [7–11]. In addi ion, he bone egene a ion p ocess is equen ly comp omised by he poo os eogenic capabili y o os eopo osis [7,12], esul ing in delayed healing, non-unions, bone de o mi ies, ch onic pain and e en pos -su gical complica ions such as in ec ions [13–15]. In hese challenging bone healing si ua ions, dis ac ion os eogenesis ia bone anspo is a ecognized ech- nique, clinically indica ed due o i s po en ial o p omo e bone healing [16–18]. This gold-s anda d o hopedic p ocedu e consis s o g adually displacing a bone ag- men , along an os eo omized gap [16,19]. Despi e i s medical in e es , he e is a lack o knowledge abou he skele al bone quali y al e a ions o applying such complex echniques in os eopo o ic pa ien s. In clinical s udies, dual ene gy X- ay abso p iome y (DXA) is he gold s an- da d me hod o assessing os eopo osis and os eopenia due o i s speed, clinical accessibili y and low- adia ion exposu e. Ne e heless, his me hod is limi ed by i s wo-dimensional assessmen o abecula mac oscale o appa en BMD and ends Uni e si ies o he Spanish Go e nmen ) and ERPF/EU (Eu opean Regional De elopmen Fund o he Eu opean Union) ia G an PID 2023-148828OB-I00 unded by MICIU/ AEI/10.13039/501100011033 and by he ERDF/ EU, ecei ed by Es he Reina-Romo and Juan Mo a-Macías. Compe ing in e es s: The au ho s ha e decla ed ha no compe ing in e es s exis . PLOS One | h ps://doi.o g/10.1371/jou nal.pone.0319910 May 2, 2025 3 / 24 o o e es ima e BMD due o i s poo di e en ia ion be ween abecula and co ical bone issues [20,21]. In his con ex , he e a e al e na i e echniques o assessing bone quali y ha a e less common o clinical applica ions due o hei cos , hei high le els o adia ion o hei in asi e na u e (e.g., bone biopsy ex ac ion). Howe e , hese echniques a e a g ea sou ce o knowledge abou bone quali y in expe imen al esea ch. In his ega d, compu ed omog aphy (CT) and mic o-compu ed omog aphy (μCT) a e wo widely used h ee-dimensional and high- esolu ion echniques o assessing, espec i ely, he mac oscale BMD [22–25], he mic oscale BMD, and he mic os uc u e o bone samples [26–28], hus o e ing a mul iscale analysis o he bone mine al issue. In addi ion, chemical composi ion analyses o bone samples can p o ide insigh s in o he bone olume ic composi ion and s oichiome y o bone issue as seen in s udies on co ical and wo en issues o heal hy subjec s [29,30]. These echniques could enhance he unde s anding o bone egene a ion in os eopo o ic pa ien s, aiding clinicians o de elop no el pa ien -speci ic ea men s and he apies. This could lead o a mo e e ec i e medical ca e and ollow-up o he disease, educing eco e y ime and imp o ing pa ien s’ quali y o li e, as well as con ibu ing o he de elopmen o p e en i e s a egies ocused on educing he isk o e ac u es o seconda y ac u es. Os eopo osis clinical and expe imen al esea ch p ima ily ocuses on unde s anding he biochemical and mechano-biological mechanisms unde lying he disease, iden i ying isk ac o s [31–33], imp o ing ea ly de ec ion echniques [1,34–39], de eloping be e d ug ea men s o he apies o pa ien s [40–43], o p e en ing he onse o he disease o agili y ac u e h ough nu i ion [44–46] o physical exe cise [47,48]. A smalle p opo ion o his e o is dedica ed o enhancing bone healing managemen . In his con ex , la ge animal models ai h ully ep esen human bone condi ions due o hei size and ana omy, enhancing ansla ional ele ance and assessmen o ea men e icacy in os eopo osis esea ch. Nume ous la ge animal s udies ha e alida ed o cha ac e ized he e ec o a ious os eo- po osis induc ion p o ocols on bone quali y [49–56], p o iding he empo al e olu ion da a on BMD o mic os uc u e o co ical o abecula issues, commonly by DXA and μCT [57–67]. Fo example, Zhang e al. [61] s udied he in luence o o a iec omy in sheep a e 12 mon hs om su ge y by μCT o he abecula issue a he lumba e eb a, emo al neck, mandibula angle and ib. They epo ed signi ican di e ences in abecula mic os uc u e deg ada ion depending on he skele al si e loca ion. Simila ly, Bisazza e al. [57] compa ed DXA and CT echniques o assess empo al changes in abecula and co ical BMD a lumba e eb ae in he sheep os eopo o ic model. They concluded ha CT p o ides g ea e accu acy in de ec ing changes in BMD and bone mic os uc u e. Ne e heless, ew la ge animals’ s udies ha e e alua ed he skele al bone quali y e olu ion expe ienced by indi iduals adding he complexi y o bone egene a ion [10,11]. In his challenging con ex , Lill e al. [11] assessed by CT he mac oscale BMD changes in co ical and abec- ula issues o he ibia be ween heal hy and os eopo o ic sheep g oups a e a mid-sha ibia1 os eo omy. They ound a signi ican appa en BMD down end, bu wi hou gi ing in o ma ion abou o he bone loca ions o mic os uc u e and chemical composi ion changes. In con as , Bindl e al. [10] s udied he in luence o os eopo osis and igh emo- al me aphysis bone gap healing on di e en skele al loca ions, p o iding insigh s in o he mic os uc u al bone quali y loss in ibial co ical and abecula issues be ween heal hy and os eopo o ic sheep g oups. Howe e , he e is a lack o knowledge ega ding he empo al bone quali y loss expe ienced by he subjec -speci ic no only in e ms o mic os uc- u e, bu also in e ms o mine al densi y and chemical composi ion, du ing he onse o os eopo osis and he subsequen bone egene a ion p ocess. Fu he mo e, as hese s udies p esen a simple ac u e healing model, hey do no p o ide in o ma ion abou how os eopo osis in luences on bone egene a ion p ocesses o majo complexi y bu equen ly used in cases o se e e auma, such as he dis ac ion os eogenesis model. In con as , he e a e s udies ha ha e assessed di e en bone egene a ion p ocesses in heal hy la ge animals, such as ac u e healing [68–72] o dis ac ion os eo- genesis [73–77]. The wo en bone gene a ed wi hin he callus has been cha ac e ized h ough di e en app oaches like nanoinden a ion [78,79], biomechanical es s [11,80,81], his ology [71,79,81,80], CT [11,73,75], ini e elemen analysis om CT econs uc ions [80,82] and gai analysis [74,75] among o he s. Howe e , hese s udies lack in o ma ion on he cha ac e iza ion o his imma u e issue in os eopo o ic subjec s. PLOS One | h ps://doi.o g/10.1371/jou nal.pone.0319910 May 2, 2025 4 / 24 F om all o his, i is essen ial o deepen unde s anding he skele al bone quali y changes in os eopo o ic pa ien s adding he complexi y o bone egene a ion. As a no el y, he p esen wo k will explo e his combined condi ion h ough in i o expe imen s using a la ge os eopo o ic animal model unde going a bone egene a ion p ocess ea ed by dis ac ion os eogenesis ia bone anspo . We hypo hesize ha he os eopo o ic bone egene a ion p ocess signi ican ly agg a a es he sys emic bone qual- i y de e io a ion beyond he baseline e ec s o os eopo osis, which could be es ed by measu ing he mine al densi y, mic os uc u e and chemical composi ion. In addi ion, he bone impai men may be in ensi ied due o he p esence o he disease. Thus, he s udy aims o elucida e and compa e he bone quali y changes in di e en bone loca ions o indi iduals du ing os eopo osis and os eopo o ic bone egene a ion. This will be achie ed h ough mul iscale imaging echniques (CT and μCT) and chemical composi ion analyses o explo e empo al e olu ion o speci ic subjec ’s skele ons. In his sense, he mac oscale and mic oscale BMD, mic os uc u e, and chemical composi ion o di e en bone issues (co ical, abec- ula and wo en bones) ac oss di e en skele al si es ( he ope a ed bone, i s con ala e al coun e pa and a bone a om he ope a ed bone) will be e alua ed empo ally and indi idually. Ma e ials and me hods This sec ion is o ganized as ollows: i s ly, he os eopo o ic animal model and he dis ac ion p ocedu e using bone anspo (BT) a e desc ibed. Nex , he s udy design is ou lined o quan i y he indi idual empo al impac o os eopo osis and he egene a ion o os eopo o ic bone de ec ac oss di e en bone loca ions and issues. Finally, he me hodology ollowed in he di e en analyses is desc ibed: he appa en o mac oscale BMD (BMDCT) da a measu ed by CT, he mic oscale BMD (BMDμCT) and mic os uc u e da a measu ed by μCT, and chemical composi ion analysis (ash ac ion, olume ic composi ion, and elemen al mass con en ). Os eopo o ic animal model and dis ac ion p ocedu e Os eopo osis was induced in a o al o i een emale me ino sheep (weigh 60.2 ± 5.6 kg), 2–4 yea s old using he p o ocol p o ided by Za inkalam e al. [54]. The animals we e selec ed om a esea ch a m and ma ked on he wool o a oid con ounde s. The andomly selec ion c i e ia ensu es ha he specimens a e heal hy and ha e p ope accina ion and dewo ming p o ocols. The sample size was calcula ed o educe he numbe o animals equi ed o a ele an minimum, ob aining signi ican di e ences. Fo his pu pose, he insigh s epo ed by Bindl e al. [10] was used as a e e ence, in which simila pa ame e s we e analyzed in he same os eopo o ic animal model. The sheep we e anspo ed o he Clin- ical Ve e ina y Hospi al o he Uni e si y o Co doba, whe e all in i o expe imen s we e conduc ed. This esea ch cen e has spacious, enced and pa ially oo ed ou doo acili ies, whe e he animals we e housed and ca ed o . The induc ion pe iod began wi h a bila e al o a iec omy. The o a iec omized sheep ecei ed pe iodic in amuscula injec ions o gluco- co icoid (500 mg Solu-Mode ín® + 7.8 ml injec able wa e ) e e y 3 weeks o 33 weeks. Addi ionally, hey we e exclusi ely ed wi h a calcium- ee die ill sac i ice, comp ising 12% c ude p o ein, 9% c ude ibe , 6.5% c ude ash, 2% c ude a , 0% calcium, 0.1% phospho us and 0.1% sodium. The BT expe imen s began wi h a su gical p ocedu e pe o med on he igh hind me a a sus o he o a iec omized ani- mal a week 33 ± 2.5. The sheep was unde gene al anes hesia and in uba ed du ing bo h su gical p ocedu es (BT su ge y and o a iec omy), while he body empe a u e, blood p essu e, oxygen le els, exhaled ca bon dioxide le els, and elec o- ca diog ams we e con inuously moni o ed. As illus a ed in Fig 1, an Iliza o - ype ex e nal ixa o [73–75,83] was ini ially implan ed using Ø4 mm Schanz sc ews. Once he me a a sus was s abilized, h ee c oss-sec ional os eo omies we e made using a guided oscilla ing saw (1.2 mm hickness). These os eo omies c ea ed wo diaphyseal bone agmen s: a 25 mm p oximal anspo able bone agmen (p e iously a ached o he dis ac o h ough wo Ø2.5 mm S einmann pins) and a dis al 15 mm bone segmen which was emo ed o gene a e a c i ical-size bone de ec (Fig 1). Following he bone su ge y, he calcium ee die was con inued h oughou he expe imen al pe iod, bu s e oid injec ions we e ceased o PLOS One | h ps://doi.o g/10.1371/jou nal.pone.0319910 May 2, 2025 5 / 24 p ese e pos su gical animal wel a e. A e one-week la ency pe iod, a 15-days dis ac ion phase was ca ied ou , apply- ing a 1 mm/day dis al displacemen o he anspo able bone agmen pe day along he 15 mm bone gap. In his way, he naï e issue o med wi hin he p oximal os eo omy was elonga ed (dis ac ion callus) while he issue o he c i ical-size bone de ec was comp essed (docking si e callus). Finally, he animals we e andomly sac i iced by an o e dose o sodium pen oba bi al IV Eu hasol® a wo di e en ime poin s du ing he os eopo o ic bone egene a ion, a day 40 (n = 5) o a day 100 (n = 5) a e he BT su ge y. Animal wel a e was gua an eed h oughou he induc ion pe iod, su gical in e en ions and expe imen al phases ol- lowing he ARRIVE guidelines, Eu opean (63/2010/EU) and na ional (RD 53/2013) egula ions on animal esea ch. The animal e hics o his s udy was app o ed by Uni e si y o Có doba (P o ocol Numbe : 2021PI/21). S udy design a di e en bone loca ions As shown in Fig 2, a ious bone issue ypes om h ee bone loca ions o he sheep we e analyzed: he igh hind me a- a sus (MO, co ical and wo en issues) as he ope a ed bone, he dis al le hind me a a sus me aphysis (MNO, abecula issue) as he con ala e al coun e pa , and he iliac c es (IC, abecula issue) as a bone dis an om he ope a ed bone. The bone samples we e e alua ed a di e en ime poin s: on he day o o a iec omy as a non-os eopo o ic ime poin (Heal hy), on he day o he BT su ge y (week 33 a e he o a iec omy) ep esen ing he os eopo o ic ime poin (OP), and on he day o sac i ice, 40 o 100 days a e su ge y, ep esen ing he os eopo o ic bone egene a ion ime poin (OP + R40 o OP + R100, espec i ely). Table 1 shows he ime poin s a which he samples we e measu ed by he di e en analyses. The in luence o os eopo osis and os eopo o ic bone egene a ion on he bone ype and loca ion was e alua ed wi h BMDCT measu emen s in wo en, co ical and abecula bones by CT. And he analysis h ough scales and loca ions we e assessed wi h BMDμCT, mic os uc u e and chemical composi ion measu emen s o abecula bone (see Table 1). Fo his pu pose, μCT and chemical composi ion analyses we e pe o med in abecula biopsies ex ac ed om MNO and IC. MNO biopsies we e only ex ac ed a e sac i icing (OP + R40 o OP + R100), as pe o ming his p ocedu e in i o could comp omise animal wel a e. Meanwhile, he CT scans and he IC biopsy ex ac ions we e conduc ed in i o o Heal hy and OP ime poin s du ing he su gical p ocedu es (unde gene al anes hesia and in uba ed), and ex i o o OP + R40 and OP + R100 ime poin s. Unlike he MNO, he IC as dis an bone is sui able o in i o biopsy ex ac ions as i is easily accessible and wi hou majo ha es ing isk, wi h abundan abecula issue. The biopsies we e ex ac ed using a biopsy punch and we e p ese ed a -80ºC in PBS-soaked gauzes. Fig 1. Bone anspo (BT) su ge y p ocedu e in he igh hind me a a sus o an o a iec omized sheep. Fixa o implan a ion and bone de ec pe o mance: (A) p oximal c oss-sec ional os eo omy; (B) 25 mm anspo agmen ; (C) 15 mm c i ical-size bone de ec ; (D) Ø4 mm Schanz-sc ews; (E) Ø2.5 mm S einmann pins; (F) ex e nal ixa o ames; (G) non-ins umen ed ixed ba s. h ps://doi.o g/10.1371/jou nal.pone.0319910.g001 PLOS One | h ps://doi.o g/10.1371/jou nal.pone.0319910 May 2, 2025 6 / 24 Fo he di e en analysis pe o med on all bone samples, each sheep da a epo ed a he Heal hy ime poin is used as i s indi idual con ol da a. In his way, each sheep’s da a a subsequen ime poin s we e also no malized by i s espec i e con ol da a, hus p o iding an indi idualized empo al e olu ion o he animal. Mac oscale da a: BMDCT using CT As illus a ed in Fig 3A, CT scans we e pe o med in i o on he animal unde gene al anes hesia and in uba ed a he Clinical Ve e ina y Hospi al o he Uni e si y o Co doba. All CT images we e acqui ed using Re olu ion ACT (Gene al Elec ic, Pekin, China) CT scanne (XYZ oxel size 460–570 x 460–570 x 625 μm/px). A CT phan om QRM-BDC/6–200® (PTW, F eibu g, Ge many) was also included o linea ly co ela e BMDCT (0–800 mg HA/cm3) wi h he s ack’s Houns ield Uni s (Fig 3B). All he bone samples (MO, MNO and IC) and he phan om inse s (6 x Ø = 18 mm, h = 200 mm) we e indi id- ually segmen ed using he open-sou ce image p ocessing ool ImageJ-Fiji. F om each axial c oss-sec ional slice, a mean and s anda d de ia ion alue o he BMDCT was es ima ed a each ime poin . In e media e CT scans we e aken a weeks 10 and 20 a e o a iec omy (10w and 20w ime poin s, espec i ely) o assess he empo al e olu ion o BMDCT in MNO Fig 2. S udy design scheme. O a iec omized sheep analyses o he igh hind me a a sus co ical and wo en bone issue (MO), he dis al me aphysis abecula issue o he le hind me a a sus (MNO), and he iliac c es abecula issue (IC). h ps://doi.o g/10.1371/jou nal.pone.0319910.g002 Table 1. S udy design o e iew. Bone assessmen Time poin s Name In e es Tissue ype (a) Heal hy (b) OP (c) OP + R40 (d) OP + R100 Righ Hind Me a a sus (MO) Ope a ed bone Co ical & Wo en CT CT CT CT Le Hind Me a a sus (MNO) Con ala e al coun e pa T abecula CT CT CT μCT ChC CT μCT ChC Iliac C es (IC) Dis an bone om he ope a ed one T abecula μCT ChC μCT ChC CT μCT ChC CT μCT ChC Di e en bone loca ions (MO, MNO and IC) and bone issue ypes (co ical, abecula and wo en) analyses (CT: compu ed omog aphy, μCT: mic o-compu ed omog aphy and ChC: chemical composi ion) a each ime poin : (a) Heal hy, on he day o o a iec omy; (b) OP, on he day o he BT su ge y, 33 weeks a e he o a iec omy; (c) OP + R40 and (d) OP + R100, on day 40 and 100 a e he BT su ge y, espec i ely. h ps://doi.o g/10.1371/jou nal.pone.0319910. 001 PLOS One | h ps://doi.o g/10.1371/jou nal.pone.0319910 May 2, 2025 7 / 24 du ing he os eopo osis induc ion phase. The abecula issue was segmen ed wi hou including he co ical issue in he MNO (13% o o al leng h o he le hind me a a sus) and IC (comple e bone). As shown in Fig 3D, di e en ep esen a i e egions wi hin he ope a ed me a a sus (MO) we e moni o ed o quan i y he local and empo al e olu ion o he BMDCT in he co ical and wo en bone issues acco ding o each animal’s inal CT scan (OP + R40 o OP + R100). Mean and s anda d de ia ion alues o BMDCT we e calcula ed om h ee co ical ag- men s and wo bone calluses. The co ical BMDCT was quan i ied in he p oximal, anspo and dis al agmen s (13%, 9% and 12% o he o al leng h o MO, espec i ely). The wo en bone BMDCT was measu ed in he dis ac ion and docking si e calluses (5%, and 0.5% o he o al leng h o MO, espec i ely). Mic oscale da a: BMDμCT and mic os uc u e using μCT The μCT measu emen s we e aken wi h he μCT scanne (XYZ oxel size 22.17–34.61 μm/px) model Y. COUGAR SMT (YXLON, Hambu g, Ge many) a he Uni e si y o Se ille Resea ch, Technology and Inno a ion Cen e (CITIUS). As shown in Figs 4A and 4B, he μCT measu emen includes ha es ed abecula biopsies om he iliac c es (IC) o con- ala e al me a a sus (MNO), along wi h a cylind ical μCT phan om, bo h ixed o a 3D p in ed suppo and placed wi hin Fig 3. Mac oscale BMD cha ac e iza ion o he os eopo o ic bone. CT assessmen o he BMDCT in co ical (ope a ed me a a sus, MO: p oximal, anspo and dis al agmen s), in wo en (MO: dis ac ion callus, docking si e callus) and in abecula (non-ope a ed me a a sus, MNO). (A) CT mea- su emen o an o a iec omized sheep. (B) CT phan om included in he CT measu emen . C) CT XZ ( op) and XY (down) measu emen p ojec ions. (D) C oss-sec ional BMDCT h oughou he bone dis al pe cen age o MO leng h (0% p oximal, 100% dis al) in an o a iec omized sheep a Heal hy, OP and OP + R100 ime poin s. h ps://doi.o g/10.1371/jou nal.pone.0319910.g003 PLOS One | h ps://doi.o g/10.1371/jou nal.pone.0319910 May 2, 2025 8 / 24 he μCT scanne . The μCT phan om (Ø = 4.5 mm, h = 5 mm) model Mic oCT-HAD4.5® (QRM, Moeh endo , Ge many) was included o es ablish a linea co ela ion be ween he BMDμCT o he phan om inse s (0–1200 mg HA/cm³) and he Houns ield Uni s o he μCT s ack. The μCT measu emen s we e ca ied ou using an open μCT mul i ocus ube o 25–160 kV ol age and cu en in ensi y o 0.01–1 mA. The comple e pos -p ocessing me hodology o he μCT measu emen s is illus a ed in Fig 4C, acco ding o he guide- lines o assessmen o bone mic os uc u e p o ided by Bouxsein e al. [84]. Fi s , he BMDμCT o he phan om inse s (5 x Ø = 0.8 mm, 4.5 mm heigh ) was measu ed using ImageJ-Fiji® by means o he plugging BoneJ. Using he Ami a-A izo® so wa e, all abecula samples we e segmen ed wi h a lowe h eshold o 400 mg HA/cm3 o no malized segmen a ion alue o Houns ield Uni s. The h eshold was confi med by isual inspec ion a e checking i s capabili y o adequa ely disc imina e be ween abeculae and o he componen s p esen ed in he di e en bone samples (po es, blood, ai , e c.). F om he 3D econs uc ion o he abecula olume, he abecula mic os uc u al pa ame e s we e calcula ed: he abecula hickness (Tb.Th), he abecula sepa a ion (Tb.Sp), he bone olume pe o al olume (BV/TV), he abecu- la numbe (Tb.N), and he s uc u e model index (SMI). Finally, he deg ee o aniso opy (DA), he connec i i y densi y (Conn.D), he ac al dimension (FD), and he mean c oss-sec ional mic oscale BMDμCT alue o he sample was mea- su ed using ImageJ-Fiji® by means o he plugging BoneJ. Chemical composi ion analyses Chemical composi ion analyses we e pe o med on he abecula biopsies om he iliac c es (IC) and he con ala e al me a a sus (MNO) a e he μCT measu emen s a he Uni e si y o Se ille Resea ch, Technology and Inno a ion Cen e (CITIUS). Fi s ly, a manual g inding p ocedu e was ca ied ou using a s e ilized pes le and mo a o educe he samples Fig 4. Mic oscale BMD and mic os uc u e cha ac e iza ion o he os eopo o ic abecula issue samples. (A) μCT se up. (B) XY μCT s ack: IC biopsy and μCT phan om ixed o a 3D p in ed suppo ( op). (C) μCT measu emen p ocedu e. h ps://doi.o g/10.1371/jou nal.pone.0319910.g004 PLOS One | h ps://doi.o g/10.1371/jou nal.pone.0319910 May 2, 2025 9 / 24 o a pa icle size o 1 mm. Subsequen ly, he o al mass m o he sample was measu ed. This mass comp ises he mass o wa e mw, he o ganic mass o o ganic phase mo and he mine al mass o mine al phase mm, as de ined in Eq. 1. m =mw+mo+mm (1) A e wa ds, a d ying p ocedu e was applied o he sample by hea ing i a 105ºC o 1 hou in a BINDER VD 23 acuum d ying chambe (BINDER GmbH, Tu lingen, Ge many). The samples we e weighed and hea ed again a 105ºC e e y 15 minu es un il a cons an d y mass md was achie ed. md=mo+mm=m –mw (2) Then, he samples unde wen an ashing p ocess in a Nabe he m Mu fle Fu nace (Nabe he m GmbH, Lilien hal Ge many), ollowing he p o ocol p o ided by Ma ínez-Reina e al. [29]: (A) a lineal inc ease o 250ºC o 30 min; (B) a cons an empe a u e o 250ºC o 1 hou ; (C) a lineal inc ease om 250ºC o 650ºC o 30 min; (D) a cons an empe a- u e o 650ºC o 2 hou s; (E) sample weighing and 650ºC cons an empe a u e o 30 min un il a cons an ash mass ma is achie ed. The p o ocol bu ns he o ganic phase o he sample, so ma co esponds wi h he mine al phase, as shown in Eq. 3. ma=mm=md–mo (3) To assess he mine al con en , he ash ac ion, α, was calcula ed using Eq. 4. α = m a md = m m mm+mo (4) The olume ic ac ions x o he mass x is ob ained by: ν x= Vx Vw+Vo+Vm = m x ρx mw ρ w +mo ρ o +mm ρ m (5) whe e he densi y o he mass is ρw = 1 g/cm3, ρo = 1.43 g/cm3 [85] and ρm = 3.12 g/cm3 [29]. La e , an elemen al analysis was ca ied ou using a T uSpec Chns Mic o analyze (LECO Co po a ion, S Joseph, MI, USA) o de e mine he mass pe cen age o ca bon. A e p e iously dissol ing he samples in hyd oalcoholic acid, he mass pe cen age o calcium, phospho us, magnesium, po assium, sodium and s on ium we e inally acqui ed h ough induc i ely coupled plasma a omic emission spec ome e Ul ima 2 (HORIBA Jobin Y on, Edison, NJ, USA). S a is ical analyses The s a is ical analyses we e ca ied ou using he so wa e ool MATLAB R2023b® (The Ma hWo ks Inc., Na ick, MA, USA). A mean and s anda d de ia ion alue om he indi iduals was ob ained o all pa ame e s measu ed a e e y ime g oup (Heal hy, OP, OP + 40 and OP + R100) in each analysis (CT, μCT and chemical composi ion). Simul aneously, a mean and s anda d de ia ion alue was calcula ed om he indi idual da a p e iously no malized by i s espec i e Heal hy ime poin . The unde lying absolu e and no malized indi idual da a a e compiled in he S1-S4 Tables. The ime g oups ou lie s we e checked and excluded using a G ubbs’ es [86] when p < 0.05. A Shapi o–Wilk es was pe o med o e i y he no mali y o ime g oups. The da a we e analyzed in sea ch o signi ican di e ences among ime g oups using non-pa ame ic es s due o he non-no mal dis ibu ion (p < 0.05) o he ime g oups p esen ed in he di e en analyses (absolu e and no malized da a). K uskal-Wallis es ollowed by Dunn–Sidak pos hoc and co ec ion was pe o med in PLOS One | h ps://doi.o g/10.1371/jou nal.pone.0319910 May 2, 2025 16 / 24 sheep ibia ac u e healing model, wi h BMDCT educ ions o 1.3% and 2.2% a he p oximal and dis al bone agmen s ends, espec i ely. The g ea e educ ions in he cu en s udy a e likely due o he highe size o ou de ec and he in lu- ence o induced os eopo osis. Auga e al. [68] p oposed ha his phenomenon migh esul om educed load-bea ing capaci y in he ope a ed limb ollowing su ge y, leading o bone issue eadap a ion and eso p ion [74,83]. This condi ion induces a bone issue eadap a ion in he in e ened limb esul ing in bone eso p ion. Ano he possible explana ion ha also could lead o an ope a ed bone dec eased os eoblas ic ac i i y, is he educ ion o ascula iza ion in he dis al di ec ion due o he os eo omies and he size o he de ec [88]. This hypo hesis can be suppo ed by he mo e p onounced deg ada- ion o he co ical issue in he dis al di ec ion o he me a a sus (Fig 5A). Auga e al. [68] also obse ed BMDCT di e ences al hough no signi ican , p obably due o he simplici y o he de ec gene a ed (2 mm os eo omy). One las hypo hesis could be he need o bone mine al o epai he bone de ec s, which is in line o he men ioned inc eased le el o ossi ica ion epo ed in bo h calluses a bo h bone egene a ion, especially ea ly in he docking si e callus egene a ion, as mechani- cally cha ac e ized by Mo a-Macías e al. [78] in BT heal hy sheep model. In he p esen s udy, he men ioned mine al need becomes e en mo e c i ical due o he poo os eogenic capabili y caused by he pa hology [7,12]. In his con ex , bone de ec egene a ion p ocesses could p oduce a sys emic skele al diso de , making he a eas closes o he de ec a po en- ial sou ce o mine al esou ces o con ibu e o he bone egene a ion [68]. Howe e , he anspo agmen does no seem o ha e a signi ican mine al con ibu ion du ing he i s 40 days a e he bone in e en ion (OP o OP + R40 in Table 2 and Fig 5A). This ou come is p obably due o he ascula disconnec ion o his anspo agmen which p e en s i s mine als om being used as a esou ce o bo h adjacen wo en mine aliza ion. In con as , he e was a signi ican loss o BMDCT a 100 days a e su ge y (OP o OP + R100), sugges ing he need o p io angiogenesis and wo en b idging o con ibu e o bo h callus os eogenesis. Concluding wi h he mac oscale cha ac e iza ion, in he ope a ed bone, he signi ican co ical BMDCT loss o e ime and wo en mine aliza ion indica es ha he mine al has been mobilized o he bone de ec s egen- e a ion ea ed by dis ac ion os eogenesis. This need appea s o be e en mo e c i ical due o os eopo osis. In con as , he in luence o his disease alone in he BMDCT seems o be limi ed o he abecula issue o he con ala e al bone (Fig 5C). Rega ding he mic oscale analysis o he abecula issue pe o med in he iliac c es by μCT (Table 4 and Figs 6A and 6B), no signi ican di e ences we e epo ed a e 33 weeks o os eopo osis induc ion in any o he compu ed pa ame e s (Heal hy o OP). This inding suppo s p e ious s udies indica ing ha os eopo osis does no uni o mly a ec abecula issues a di e en loca ions [89–91]. In con as , he iliac c es abecula mic os uc u e was shown o emodel du ing he i s 40 days a e he BT su ge y (OP o OP + R40), wi h a signi ican inc ease in he Tb.Sp (67%), and a dec ease in he BV/TV (31%), in he Tb.N (28%), and in he Conn.D (54%) in ela ion o Heal hy alue. Consequen ly, he iliac c es abeculae a e he in e en ion a e no ably less dense and in e connec ed, wi h ewe abeculae. As a as he au ho s a e conce ned, he e a e no s udies assessing he indi idual mic os uc u al changes o he abecula issue h oughou a bone de ec egene a ion p ocess in an os eopo o ic sheep model o compa e. Howe e , ou indings a e in line wi h Bindl e al. [10], who compa ed os eopo o ic and heal hy sheep g oups, he os eopo o ic igh emo al ac u e healing also induces a dec ease in he BV/TV o 32% and he Tb.N o 31%, and a 57% inc ease in he Tb.Sp a he igh ibial a- becula issue a 56 days pos - ac u e. In addi ion, he μCT analysis conduc ed by Fische e al. [4] in os eopo o ic mice e ealed a signi ican deg ada ion o abecula mic os uc u e (25% educ ion in BV/TV and Tb.N) in he lumba e eb ae o he emu ac u ed g oup compa ed o he non- ac u ed g oup. Rega ding he BMDμCT measu ed 40 days a e BT su ge y (OP o OP + R40), no signi ican di e ences we e epo ed. Unlike CT, which compu es an appa en BMD (BMDCT) wi h abeculae and po es, μCT has he esolu ion o analyze he abeculae ocusing on he mine al phase, which appea s o main ain he mean BMDμCT while impai ing he abecula iliac c es mic os uc u e. By 100 days pos -su ge y, he absence o signi ican di e ences in BMDμCT and mic os uc u e pa ame e s o he iliac c es wi h espec o 40 days a e su ge y (OP + R40 o OP + R100, 60 days di e ence) sugges s ha abecula issue a enough om he egene - a ion ocus is no a ec ed by he bone egene a ion in an os eopo o ic subjec (Table 4 and Figs 6A and 6B). This is in line wi h he mul iscale BMD esul s o bo h abecula bones (Figs 7A and 7B) and he abecula mic os uc u e esul s PLOS One | h ps://doi.o g/10.1371/jou nal.pone.0319910 May 2, 2025 17 / 24 o he con ala e al me a a sus (Table 4). This mic os uc u e was obse ed o be weake (highe Tb.Sp, lowe Tb.N and Conn.D) han ha epo ed by Blázquez-Ca mona e al. [77] in he ope a ed me a a sus abecula issue o heal hy sheep unde going a bone leng hening p ocess using dis ac ion os eogenesis. Acco ding o his, he os eoclas ic ac i i y seems o inc ease in he os eopo o ic abecula issue o a non- ac u ed bones a he ea ly s age, du ing he i s 40 days a e he bone su gical in e en ion (OP o OP + R40). This ac is p obably a sys emic esponse o he os eopo o ic skele on o u he con ibu e o he supply o mine als o he bone epai o he ope a ed me a a sus. In addi ion, he main mic oscale changes in he abecula issue in he non- ac u ed bones a e due o he combina ion o os eopo osis wi h he bone cal- lus egene a ion p ocess, a he han solely os eopo osis. In e ms o chemical composi ion analyses o he iliac c es abecula issue, he ash and olume ic composi ion empo al e olu ion esul s a e illus a ed in Table 5 and Fig 8A. As a as he au ho s know, he e a e no s udies in which he olume ic composi ion is assessed du ing os eopo osis and os eopo o ic bone egene a ion ( he same o he ash ac ion) a he non- ac u ed abecula issue o compa e. Conside ing he di e ences, he esul s could be compa ed wi h hose o Ma ínez-Reina e al. [29], who analyzed he olume ic composi ion and elemen al mass con en pe cen age o he lamella (co ical) and wo en issue om he me a a sus o heal hy sheep unde going a BT egene a ion p ocess. Acco ding o Table 5 and Fig 8A, he pa ame e s measu ed did no epo signi ican changes a he end o he os eopo o- sis induc ion (Heal hy o OP), consis en wi h Bloebaum e al. [92], who ound no di e ences in he ash ac ion be ween young and os eopo o ic women g oups a di e en abecula si es. Du ing he i s 40 days a e he bone su gical in e - en ion (OP o OP + R40), a signi ican loss o he ash ac ion ook place (31.6% wi h espec o Heal hy alue). Wi h espec o he olume ic composi ion o he iliac c es du ing he bone egene a ion (OP o OP + R40 o OP + R100), he signi ican opposi e ends epo ed in wa e olume ic ac ion (down end) and o ganic phase olume ic ac ion (up end) seems o indica e a olume ic change compa able o ha epo ed by Ma ínez-Reina e al. [29] in heal hy wo en bone. In his imma u e bone issue, a high olume ic ac ion o o ganic phase means ha i is unde going a p o- cess o mine al change, ei he abso p ion o eso p ion. In addi ion, a mine al phase loss o olume ic ac ion occu ed in he ea ly pe iod o os eopo o ic bone egene a ion, which appea s o eco e o e ime. All his in o ma ion seems o indi- ca e ha he os eopo o ic bone egene a ion s imula es he p oduc ion o o ganic ma ix in dis an non- ac u ed bones, which sugges he displacemen o blood olume o con ibu e wi h he in lamma ion and he anspo o mine als. This ea ly loss o mine al phase mass in ela ion o he mine al phase plus o ganic phase mass (ash ac ion) and wa e olu- me ic ac ion e lec s, once again, a mine al mass eso p ion and mobiliza ion om he abecula issue o he iliac c es o con ibu e o he os eopo o ic egene a ion o he ope a ed me a a sus. This hypo hesis is consis en wi h he abecula iliac c es mic os uc u e pa ame e s deg ada ion discussed abo e (Table 4 and Figs 6A and 6B). The pe cen age o he elemen al mass con en om he mine al phase o he iliac c es abecula issue is shown in Table 5 and Fig 8B. While no di ec compa isons a e a ailable in he li e a u e, Ma ínez-Reina e al. [29] measu ed he mass pe cen ages o C, P, and Ca in non-os eopo o ic co ical and wo en issue. The compa isons be ween his s udy and he cu en wo k a e limi ed due o he os eopo osis in luence and he di e ences be ween hese bone issues wi h he abecula issue, wi hou also analyzing o he elemen s such as Mg, K, Na and S . As shown in Fig 8B, he pe - cen age o elemen al mass con en o C and Mg expe ienced an inc ease du ing he os eopo osis induc ion (Heal hy o OP). An inc ease in he mass pe cen age o C (p obably om a deg ada ion o he o ganic ma ix) and Mg, indica es an inc ease in he ca bona e con en o he hyd oxyapa i e, which may make i less s able and mo e agile and suscep ible o an os eopo o ic inc eased eabso p ion. Howe e , he elemen al mass pe cen ages o Ca, P, K, Na and S emained cons an , showing ha in his abecula bone he pe cen age o hyd oxyapa i e has no changed signi ican ly due o os eopo osis. The mos abundan me allic elemen s in he mass pe cen age o hyd oxyapa i e (Ca and P) appea no o change signi ican ly du ing he os eopo o ic bone egene a ion (OP o OP + R40 o OP + R100) o he ope a ed me a a sus. Howe e , i canno be assumed ha he mine al mass is no being los , since he esul s a e desc ibed as mass pe cen - age. The same applies o h ee o he less abundan elemen s in he bone mine al, C, Na and S . In con as , he mass PLOS One | h ps://doi.o g/10.1371/jou nal.pone.0319910 May 2, 2025 18 / 24 pe cen age o K and Mg epo ed a signi ican dec easing and inc easing end, espec i ely, du ing he os eopo o ic bone egene a ion p ocess, no epo ed du ing he os eopo o ic induc ion pe iod. This ac sugges s a me abolic and homeo- s a ic adap a ion h ough he edis ibu ion o seconda y elemen s. In his ega d, he inc ease in Mg could imp o e he o ma ion o new bone issue and cellula ac i i y [93], while he dec ease in K could indica e i s less di ec in ol emen in mine aliza ion, hus ocusing on he needs o os eopo o ic bone egene a ion o he ope a ed bone. Wi h espec o he compa ison o he os eopo o ic bone egene a ion poin s o bo h abecula issues o non- ac u ed bones (iliac c es and con ala e al me a a sus) in all he analyses (CT, μCT and chemical composi ion) pe o med in his wo k (Tables 4 and 5), no signi ican di e ences we e ound. This ac may indica e ha bo h abecula issues ha e beha ed in a simila way be ween days 40 and 100 a e he bone su ge y (OP + R40 o OP + R100), p io i izing he os eogenesis o ope a ed bone wo en issues o e he eco e y o non- ac u ed abecula os eopo o ic issue. As o he limi a ions o he wo k, i should be no ed ha no all he analyses p esen ed ha e been ca ied ou in all he bone issues and loca ions conside ed h oughou all he ime poin s (Heal hy, OP, OP + R40 o OP + R100) o measu e- men o he sheep. In he case o μCT scanning and chemical composi ion analysis, he indi idual empo al e olu ion is only comple ed a he iliac c es , as any in i o biopsy ex ac ions a he ope a ed o con ala e al me a a sus would ha e comp omised he wel a e o he animals. In addi ion, wo sheep su e ed heal h di icul ies and had o be slaugh e ed be o e he BT su ge y. Meanwhile, h ee animals expe ienced pos BT su ge y complica ions ha made i necessa y o slaugh e hem be o e he bone egene a ion ime poin . Thus, hese specimens did no p o ide he comple e indi idual empo al e olu ion da a. The CT scans we e limi ed o a single ime poin du ing bone egene a ion due o he p esence o me al elemen s in he implan ed ex e nal ixa o , which equi ed pos -sac i ice CT scans a e ixa o emo al. Conse- quen ly, he compa ison be ween he OP + R40 and OP + R100 ime poin s e e o animals om di e en sac i ice g oups, he e o e i is no possible o alk abou an indi idual empo al e olu ion when compa ing bo h ime poin s. Finally, he in luence o os eopo osis du ing he bone egene a ion p ocess could no be de e mined explici ly since he s udy does no conside a heal hy con ol g oup unde going he same bone egene a ion p ocess. In conclusion, his s udy p o ides no el quan i a i e insigh s in o he al e a ions in bone quali y expe ienced by indi id- ual subjec s du ing he onse o os eopo osis combined wi h bone egene a ion p ocesses ea ed by dis ac ion os eogen- esis. Speci ically, mul iscale BMD, mic os uc u e and chemical composi ion we e assessed in di e en bone issues and loca ions in os eopo o ic la ge animal subjec s unde going a c i ical-size bone egene a ion p ocess. I is de e mined i s ly ha os eopo osis alone only seems o signi ican ly impai he bone quali y o abecula issue he e ogeneously depend- ing on he bone localiza ion. Secondly, he os eopo o ic bone egene a ion p ocess ollowing bone anspo in e en ions esul s in a sys emic skele al diso de , which is g ea e han os eopo osis i sel , and appea s o be in ensi ied by his dis- ease. A he ope a ed bone, his combined condi ion caused a signi ican loss o co ical bone quali y ha was agg a a ed o e ime in he dis al di ec ion. In addi ion, he abecula issues o a bones we e also especially a ec ed du ing he ea ly s age o bone egene a ion, de e io a ing hei mic os uc u e and al e ing hei chemical composi ion. This gene al- ized skele al bone quali y impai men seems o be a quick esponse o he o ganism o egula e he blood mobiliza ion o bone mine al sou ces ha can con ibu e o enhance he wo en issue mine aliza ion o he ope a ed bone. This mine al equi emen is e en mo e c i ical in os eopo o ic indi iduals in whom he os eogenic capaci y is se e ely diminished by es ogen de iciency, accele a ing bone eso p ion and dec easing os eoblas ic ac i i y. In his sense, he p esen wo k e lec s he need o s udy he bone quali y by di e en measu emen app oaches (mul iscale BMD, mic os uc u e, and chemical composi ion) a di e en bone loca ions o e idence he agg a a ion o he os eopo o ic bone egene a ion p ocess. In os eopo o ic pa ien s who need dis ac ion os eogenesis p ocedu e, i is impo an o p io i ize he con inuous moni o ing o he bones s a is ically mo e p one o a new ac u e, especially he al eady h ea ed bone unde egene a ion, whose ixa ion is a high isk o being mechanically and a ally comp omised. The e o e, i is c ucial he con inuous bone quali y ca e and clinical ollow-up, especially du ing he ea ly s age o bone egene a ion, o ensu e adequa e healing, conside ing he se e al isks o e ac u es o seconda y ac u es. PLOS One | h ps://doi.o g/10.1371/jou nal.pone.0319910 May 2, 2025 19 / 24 Suppo ing in o ma ion S1 Table. Unde lying da a poin s o Table 2. Sheep da a poin s o he mac oscale BMD cha ac e iza ion in os eopo o ic ope a ed bone. (XLSX) S2 Table. Unde lying da a poin s o Table 3. Sheep da a poin s o he mac oscale BMD cha ac e iza ion in os eopo o ic non- ac u ed abecula bones. (XLSX) S3 Table. Unde lying da a poin s o Table 4. Sheep da a poin s o he mic oscale BMD and mic os uc u e cha ac e i- za ion in os eopo o ic non- ac u ed abecula bones. (XLSX) S4 Table. Unde lying da a poin s o Table 5. Sheep da a poin s o he chemical composi ion cha ac e iza ion in os eo- po o ic non- ac u ed abecula bones. (XLSX) S5 Table. S a is ical analyses o Table 2. P- alues o he mac oscale BMD cha ac e iza ion in os eopo o ic ope a ed bone. (XLSX) S6 Table. S a is ical analyses o Table 3. P- alues o he mac oscale BMD cha ac e iza ion in os eopo o ic non- ac u ed abecula bones. (XLSX) S7 Table. S a is ical analyses o Table 4. P- alues o he mic oscale BMD and mic os uc u e cha ac e iza ion in os eo- po o ic non- ac u ed abecula bones. (XLSX) S8 Table. S a is ical analyses o Table 5. P- alues o he chemical composi ion cha ac e iza ion in os eopo o ic non- ac u ed abecula bones. (XLSX) S9 File. Full ARRIVE 2.0 Guidelines checklis . (PDF) Acknowledgmen s G an PID 2020–113790RB-I00 unded by MICIU/AEI/10.13039/501100011033. The au ho s would like o hank e e ina y echnicians Juan Ramón Ríos Pé ez, Ana So o Fe nández and Ál a o Rincón Panade o o hei commi men , e o and in ol emen in his wo k. Au ho con ibu ions Concep ualiza ion: Juan J. Toscano-Angulo, Juan Mo a-Macías, Pablo Blázquez-Ca mona, Es he Reina-Romo. Da a cu a ion: Juan J. Toscano-Angulo. Fo mal analysis: Juan J. Toscano-Angulo, Pablo Blázquez-Ca mona, Es he Reina-Romo. Funding acquisi ion: Juan Mo a-Macías, Es he Reina-Romo. In es iga ion: Juan J. Toscano-Angulo, Juan Mo a-Macías, Pablo Blázquez-Ca mona, Juan Mo gaz, Es he Reina-Romo. PLOS One | h ps://doi.o g/10.1371/jou nal.pone.0319910 May 2, 2025 20 / 24 Me hodology: Juan J. Toscano-Angulo, Juan Mo gaz, Rocío Na a e e-Cal o. 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