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
.
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dis ibu ion, and ep oduc ion in any medium,
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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.
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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
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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
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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
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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.
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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.
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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
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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
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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
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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.
P ojec adminis a ion: Es he Reina-Romo.
Resou ces: Juan J. Toscano-Angulo, Juan Mo a-Macías, Es he Reina-Romo.
So wa e: Juan J. Toscano-Angulo.
Supe ision: Juan Mo a-Macías, Juan Mo gaz, Es he Reina-Romo.
Valida ion: Juan J. Toscano-Angulo, Juan Mo a-Macías, Es he Reina-Romo.
Visualiza ion: Juan J. Toscano-Angulo.
W i ing – o iginal d a : Juan J. Toscano-Angulo.
W i ing – e iew & edi ing: Juan Mo a-Macías, Pablo Blázquez-Ca mona, Juan Mo gaz, Jaime Domínguez, Es he
Reina-Romo.
Re e ences
1. Kanis JA, McCloskey EV, Johansson H, Oden A, Mel on LJ, Khal ae N. A e e ence s anda d o he desc ip ion o os eopo osis. Bone. 2008
Ma ;42:467–75. h ps://doi.o g/10.1016/j.bone.2007.11.001 PMID: 18180210
2. Kanis JA, Johnell O, Oden A, Se nbo I, Redlund-Johnell I, Dawson A, e al. Long- e m isk o os eopo o ic ac u e in Malmö. Os eopo os In .
2000;11(8):669–74. h ps://doi.o g/10.1007/s001980070064 PMID: 11095169
3. Cu is EM, an de Velde R, Moon RJ, an den Be gh JPW, Geusens P, de V ies F, e al. Epidemiology o ac u es in he Uni ed Kingdom 1988-
2012: Va ia ion wi h age, sex, geog aphy, e hnici y and socioeconomic s a us. Bone. 2016;87:19–26. h ps://doi.o g/10.1016/j.bone.2016.03.006
PMID: 26968752
4. Wu AM, Bisignano C, James SL, Abady GG, Abedi A, Abu-Gha bieh E, e al. Global, egional, and na ional bu den o bone ac u es in 204
coun ies and e i o ies, 1990–2019: a sys ema ic analysis om he Global Bu den o Disease S udy 2019. Lance Heal hy Longe . 2021 Sep
1;2(9):e580–92. h ps://doi.o g/10.1016/S2666-7568(21)00172-0 PMID: 34723233
5. Van Geel TACM, Van Helden S, Geusens PP, Winkens B, Dinan GJ. Clinical subsequen ac u es clus e in ime a e i s ac u es. Ann Rheum
Dis. 2009 Jan;68(1):99–102. h ps://doi.o g/10.1136/a d.2008.092775 PMID: 18677009.
6. Johansson H, Siggei sdó i K, Ha ey NC, Odén A, Gudnason V, McCloskey E, e al. Imminen isk o ac u e a e ac u e. Os eopo os In .
2017;28(3):775–80. h ps://doi.o g/10.1007/s00198-016-3868-0 PMID: 28028554
7. Fische V, Ha ne -Lun ze M, Amling M, Igna ius A. Calcium and i amin D in bone ac u e healing and pos - auma ic bone u no e . Eu Cell
Ma e . 2018 Jan 1; 35:365–85. h ps://doi.o g/10.22203/eCM. 035a25 PMID: 29931664
8. Osipo B, Emami AJ, Ch is iansen BA. Sys emic bone loss a e ac u e. Clin Re Bone Mine Me ab. 2018 Dec 1;16(4):116–30. h ps://doi.
o g/10.1007/s12018-018-9253-0 PMID: 31363348
9. Fische V, Ha ne -Lun ze M, P ys az K, Scheid A Vom Busse B, Schinke T, e al. Calcium and i amin-D de iciency ma ginally impai s ac u e healing
bu agg a a es pos auma ic bone loss in os eopo o ic mice. Sci Rep. 2017 Dec 1;7(1). h ps://doi.o g/10.1038/s41598-017-07511-2 PMID: 28775273.
10. Bindl R, Oheim R, Pogoda P, Beil FT, G uchenbe g K, Rei maie S, e al. Me aphyseal ac u e healing in a sheep model o low u no e os eo-
po osis induced by hypo halamic-pi ui a y disconnec ion (HPD). J O hop Res. 2013 No ;31(11):1851–7. h ps://doi.o g/10.1002/jo .22416 PMID:
23813786
11. Lill CA, Hesseln J, Schlegel U, Eckha d C, Goldhahn J, Schneide E. Biomechanical e alua ion o healing in a non-c i ical de ec in a la ge animal
model o os eopo osis. J O hop Res. 2003;21(5):836–42. h ps://doi.o g/10.1016/S0736-0266(02)00266-8 PMID: 12919871
12. Go e EA, Reinde s CR, K ijnen P, Appelman-Dijks a NM, Schippe IB. The e ec o os eopo osis and i s ea men on ac u e healing a sys em-
a ic e iew o animal and clinical s udies. Bone Rep. 2021;15:101117. h ps://doi.o g/10.1016/j.bon .2021.101117 PMID: 34458509
13. Shih Y, Ba scha NI, Cheng EY. Sliding hip sc ew and side pla e o in e ochan e ic hip ac u es. JBJS Essen Su g Tech. 2022 Feb
16;12(1):e19.00038. h ps://doi.o g/10.2106/JBJS.ST.19.00038 PMID: 35685237
14. Hu S-J, Chang S-M, Du S-C, Zhang L-Z, Xiong W-F. Two-pa in e ochan e ic emu ac u es wi h bisec ion o he lesse ochan e : an i educ-
ible ac u e pa e n. Ge ia O hop Su g Rehabil. 2023;14:21514593231153827. h ps://doi.o g/10.1177/21514593231153827 PMID: 36712599
15. Leslie MP, Baumgae ne MR. In e ochan e ic emu ac u e. O hopedic T auma Call o he A ending Su geon. 2023. p. 259–68. h ps://doi.
o g/10.1201/9781003525493-33 PMID: 29630213
16. Iliza o GA The ension-s ess e ec on he genesis and g ow h o issues: Pa II. The in luence o he a e and equency o dis ac ion. Clin
O hop Rela Res 1989;239:263–85. h ps://pubmed.ncbi.nlm.nih.go /2912628/
17. Comp on J, F agomen A, Rozb uch S. Skele al epai in dis ac ion os eogenesis: Mechanisms and enhancemen s. JBJS Re . 2015 Aug 1;3(8):1–
12. h ps://doi.o g/10.2106/JBJS.RVW.N.00107 PMID: 27490473
PLOS One | h ps://doi.o g/10.1371/jou nal.pone.0319910 May 2, 2025 21 / 24
18. Maai N, F ank FA, Schildhaue TA, Königshausen M. Bone egene a ion a e nail dis ac ion os eogenesis: e iew o cu en knowledge and appli-
ca ion combined wi h a case epo wi h adiological, his ological, and elec on mic oscopic analysis. J. Clin. Med. 2024 Oc 30;13(21):6504. h ps://
doi.o g/10.3390/JCM13216504 PMID: 39518644.
19. Iliza o GA The ension-s ess e ec on he genesis and g ow h o issues. Pa I. The in luence o s abili y o ixa ion and so - issue p ese a ion.
Clin O hop Rela Res 1989;238:249–81. h ps://pubmed.ncbi.nlm.nih.go /2910611/
20. Bolo in HH. Inaccu acies inhe en in dual-ene gy X- ay abso p iome y in i o bone mine al densi ome y may law os eopenic/os eopo o ic in e -
p e a ions and mislead assessmen o an i eso p i e he apy e ec i eness. 2001;28(5):548–55. h ps://doi.o g/10.1016/s8756-3282(01)00423-9
PMID: 11344055
21. Seb o R, Ashok SS. A s a is ical app oach ega ding he diagnosis o os eopo osis and os eopenia om DXA: a e we unde diagnosing os eopo o-
sis? JBMR Plus. 2021;5(2). h ps://doi.o g/10.1002/jbm4.10444 PMID: 33615110.
22. Genan HK, Engelke K, P e hal S. Ad anced CT bone imaging in os eopo osis. Rheuma ology. 2008;47(SUPPL. 4):i 9–16. h ps://doi.
o g/10.1093/ heuma ology/ken180 PMID: 18556648
23. Link TM. Os eopo osis imaging: S a e o he a and ad anced imaging. Vol. 263, Radiology. 2012. p. 3–17. h ps://doi.o g/10.1148/ adiol.12110462
PMID: 22438439.
24. Oei L, Ko omani F, Ri adenei a F, Zillikens MC, Oei EHG. Quan i a i e imaging me hods in os eopo osis. Quan Imaging Med Su g. 2016;6:680–
98. h ps://doi.o g/10.21037/qims.2016.12.13 PMID: 28090446
25. Pin o EM, Ne es JR, Teixei a A, F ada R, A ilano P, Oli ei a F, e al. E icacy o houns ield uni s measu ed by lumba compu e omog aphy on
bone densi y assessmen : a sys ema ic e iew. Spine (Phila Pa 1976). 2022;47(9):702–10. h ps://doi.o g/10.1097/BRS.0000000000004211 PMID:
34468433
26. Kim YJ, Henkin J. Mic o-compu ed omog aphy assessmen o human al eola bone: bone densi y and h ee-dimensional mic o-a chi ec u e. Clin
Implan Den Rela Res. 2015;17(2):307–13. h ps://doi.o g/10.1111/cid.12109 PMID: 23837565
27. Se illa RS, C uz F, Chiu CS, Xue D, Be ano KA, Zhu J, e al. De elopmen and op imiza ion o a high- h oughpu mic o-compu ed omog aphy
imaging me hod inco po a ing a no el analysis echnique o e alua e bone mine al densi y o a h i ic join s in a oden model o collagen induced
a h i is. Bone. 2015 Ap 1;73:32–41. h ps://doi.o g/10.1016/j.bone.2014.11.014 PMID: 25482211
28. Gaê a-A aujo H, Nascimen o EHL, B asil DM, Madlum DV, Hai e -Ne o F, Oli ei a-San os C. In luence o econs uc ion pa ame e s
o mic o-compu ed omog aphy on he analysis o bone mine al densi y. Imaging Sci Den . 2020;50(2):153–9. h ps://doi.o g/10.5624/
isd.2020.50.2.153 PMID: 32601590
29. Ma ínez-Reina J, Ga cía-Rod íguez J, Mo a-Macías J, Domínguez J, Reina-Romo E. Compa ison o he olume ic composi ion o lamella bone
and he wo en bone o calluses. P oc Ins Mech Eng H. 2018 Jul 1;232(7):682–9. h ps://doi.o g/10.1177/0954411918784085 PMID: 29962326
30. Boskey AL. Bone composi ion: ela ionship o bone agili y and an ios eopo o ic d ug e ec s. Bonekey Rep. 2013 Dec 4;2. h ps://doi.o g/10.1038/
bonekey.2013.181 PMID: 24501681.
31. De Lae C, Odén A, Johansson H, Johnell O, Jönsson B, Kanis JA. The impac o he use o mul iple isk indica o s o ac u e on case- inding
s a egies: a ma hema ical app oach. Os eopo os In . 2005;16(3):313–8. h ps://doi.o g/10.1007/s00198-004-1689-z PMID: 15241584
32. Kanis JA, Johnell O, De Lae C, Johansson H, Oden A, Delmas P, e al. A me a-analysis o p e ious ac u e and subsequen ac u e isk. Bone.
2004 Aug;35(2):375–82. h ps://doi.o g/10.1016/j.bone.2004.03.024 PMID: 15268886
33. Kanis JA, Johnell O, Oden A, Johansson H, De Lae C, Eisman JA, e al. Smoking and ac u e isk: a me a-analysis. Os eopo os In .
2005;16(2):155–62. h ps://doi.o g/10.1007/s00198-004-1640-3 PMID: 15175845
34. Johnell O, Kanis JA, Oden A, Johansson H, De Lae C, Delmas P, e al. P edic i e alue o BMD o hip and o he ac u es. J Bone Mine Res.
2005;20(7):1185–94. h ps://doi.o g/10.1359/JBMR.050304 PMID: 15940371
35. Kanis JA, Ha ey N, Coope C, Johansson H, Odén A, McCloskey E, e al. A sys ema ic e iew o in e en ion h esholds based on FRAX: A epo
p epa ed o he Na ional Os eopo osis Guideline G oup and he In e na ional Os eopo osis Founda ion. A ch Os eopo os. 2016 Dec 1;11:25.
h ps://doi.o g/10.1007/s11657-016-0278-z PMID: 27465509
36. Kanis JA, Coope C, Rizzoli R, Regins e J-Y. Eu opean guidance o he diagnosis and managemen o os eopo osis in pos menopausal women.
Os eopo os In . 2019 Jan 18;30(1):3–44. h ps://doi.o g/10.1007/s00198-018-4704-5 PMID: 30324412
37. McCloskey E V., Odén A, Ha ey NC, Leslie WD, Hans D, Johansson H, e al. A me a-analysis o abecula bone sco e in ac u e isk p edic ion
and i s ela ionship o FRAX. J Bone Mine Res. 2016 May 1;31(5):940–8. h ps://doi.o g/10.1002/jbm .2734 PMID: 26498132.
38. The kildsen J, Thygesen J, Win he S, S ensson M, Hauge E-M, Bö che M, e al. Ve eb al bone mine al densi y measu ed by quan i a i e
compu ed omog aphy wi h and wi hou a calib a ion phan om: a compa ison be ween 2 di e en so wa e solu ions. J Clin Densi om. 2018 Jul
1;21(3):367–74. h ps://doi.o g/10.1016/j.jocd.2017.12.003 PMID: 29680671
39. Sheps one L, Lenaghan E, Coope C, Cla ke S, Fong-Soe-Khioe R, Fo dham R, e al. Sc eening in he communi y o educe ac u es in olde
women (SCOOP): a andomised con olled ial. The Lance . 2018 Feb 24;391(10122):741–7. h ps://doi.o g/10.1016/S0140-6736(17)32640-5
PMID: 29254858.
40. Roelo s AJ, Thompson K, Go don S, Roge s MJ, Boyce, Roodman, e al. Molecula mechanisms o ac ion o bisphosphona es: cu en s a us. Clin
Cance Res. 2006 Oc 15;12:6222s–30s. h ps://doi.o g/10.1158/1078-0432.CCR-06-0843 PMID: 17062705
PLOS One | h ps://doi.o g/10.1371/jou nal.pone.0319910 May 2, 2025 22 / 24
41. Bone HG, Wagman RB, B andi ML, B own JP, Chapu la R, Cummings SR, e al. 10 yea s o denosumab ea men in pos menopausal women
wi h os eopo osis: esul s om he phase 3 andomised FREEDOM ial and open-label ex ension. Lance Diabe es Endoc inol. 2017 Jul
1;5(7):513–23. h ps://doi.o g/10.1016/S2213-8587(17)30138-9 PMID: 28546097
42. Cummings SR, Ma in JS, McClung MR, Si is ES, Eas ell R, Reid IR, e al. Denosumab o p e en ion o ac u es in pos menopausal women wi h
os eopo osis. N Engl J Med. 2009;361(8):756–65. h ps://doi.o g/10.1056/NEJMoa0809493 PMID: 19671655
43. Kuźnik A, Paździe niok-Holewa A, Jewula P, Kuźnik N. Bisphosphona es—much mo e han only d ugs o bone diseases. Eu J Pha macol. 2020
Jan 5;866:172773. h ps://doi.o g/10.1016/j.ejpha .2019.172773 PMID: 31705903
44. Ha ey NC, Bi e E, Kau man JM, Baue J, B anco J, B andi ML, e al. The ole o calcium supplemen a ion in heal hy musculoskele al ageing:
An expe consensus mee ing o he Eu opean Socie y o Clinical and Economic Aspec s o Os eopo osis, Os eoa h i is and Musculoskele al
Diseases (ESCEO) and he In e na ional Founda ion o Os eopo osis (IOF). Os eopo os In . 2017;28(2):447–62. h ps://doi.o g/10.1007/s00198-
016-3773-6 PMID: 27761590
45. Tang BM, Eslick GD, Nowson C, Smi h C, Bensoussan A. Use o calcium o calcium in combina ion wi h i amin D supplemen a ion o p e en
ac u es and bone loss in people aged 50 yea s and olde : a me a-analysis. Lance . 2007;370(9588):657–66. h ps://doi.o g/10.1016/S0140-
6736(07)61342-7 PMID: 17720017
46. Wea e CM, Alexande DD, Boushey CJ, Dawson-Hughes B, Lappe JM, LeBo MS, e al. Calcium plus i amin D supplemen a ion and isk o
ac u es: an upda ed me a-analysis om he Na ional Os eopo osis Founda ion. Os eopo os In . 2016 Jan 1;27(1):367–76. h ps://doi.o g/10.1007/
s00198-015-3386-5 PMID: 26510847
47. Howe TE, Shea B, Dawson LJ, Downie F, Mu ay A, Ross C, e al. Exe cise o p e en ing and ea ing os eopo osis in pos menopausal women.
Coch ane Da abase Sys Re . 2011;(7):CD000333. h ps://doi.o g/10.1002/14651858.cd000333.pub2 PMID: 21735380
48. Sinaki M, I oi E, Wahne HW, Wollan P, Gelzce R, Mullan BP, e al. S onge back muscles educe he incidence o e eb al ac u es: a p ospec-
i e 10 yea ollow-up o pos menopausal women. Bone. 2002;30(6):836–41. h ps://doi.o g/10.1016/s8756-3282(02)00739-1 PMID: 12052450
49. Kennedy OD, B ennan O, Racka d SM, S aines A, O’B ien FJ, Taylo D, e al. E ec s o o a iec omy on bone u no e , po osi y, and biomechan-
ical p ope ies in o ine compac bone 12 mon hs pos su ge y. J O hop Res. 2009 Ma ;27(3):303–9. h ps://doi.o g/10.1002/jo .20750 PMID:
18853433.
50. Eschle A, Röpenack P, He lyn PKE, Roesne J, Pille K, Büsing K, e al. The s anda dized c ea ion o a lumba spine e eb al comp ession ac-
u e in a sheep os eopo osis model induced by o a iec omy, co icos e oid he apy and calcium/phospho us/ i amin D-de icien die . Inju y. 2015
Oc 1;46 Suppl 4:S17–23. h ps://doi.o g/10.1016/S0020-1383(15)30014-0 PMID: 26542861
51. Lill CA, Ge lach UV, Eckha d C, Goldhahn J, Schneide E. Bone changes due o glucoco icoid applica ion in an o a iec omized animal model o
ac u e ea men in os eopo osis. Os eopo os In . 2002;13(5):407–14. h ps://doi.o g/10.1007/s001980200047 PMID: 12086352
52. Lill CA, Lill CA, Fluegel AK, Schneide E. E ec o o a iec omy, malnu i ion and glucoco icoid applica ion on bone p ope ies in sheep: a pilo
s udy. Os eopo os In . 2002;13(6):480–6. h ps://doi.o g/10.1007/S001980200058 PMID: 12107662.
53. New on BI, Coope RC, Gilbe JA, Johnson RB, Za diackas LD. The o a iec omized sheep as a model o human bone loss. J Comp Pa hol.
2004;130(4):323–6. h ps://doi.o g/10.1016/j.jcpa.2003.12.007 PMID: 15053937.
54. Za inkalam MR, Bea d H, Schul z CG, Moo e RJ. Valida ion o he sheep as a la ge animal model o he s udy o e eb al os eopo osis. Eu
Spine J. 2009 Feb;18(2):244–53. h ps://doi.o g/10.1007/s00586-008-0813-8 PMID: 19015899
55. Scho lemme S, Igna ius A, Claes L, Auga P. Inhibi ion o co ical and cancellous bone o ma ion in glucoco icoid- ea ed OVX sheep. Bone.
2005;37(4):491–6. h ps://doi.o g/10.1016/j.bone.2005.05.002 PMID: 16046208
56. Schulze F, Malhan D, El Khassawna T, Heiss C, Seckinge A, Hose D, e al. A issue-based app oach o selec ion o e e ence genes o quan-
i a i e eal- ime PCR in a sheep os eopo osis model. BMC Genomics. 2017 Dec 19;18(1). h ps://doi.o g/10.1186/s12864-017-4356-4 PMID:
29258442.
57. Bisazza KT, Nelson BB, Sikes KJ, Nakamu a L, Easley JT. Compu ed omog aphy p o ides imp o ed quan i ica ion o abecula lumba spine
bone loss compa ed o dual-ene gy x- ay abso p iome y in o a iec omized sheep. JBMR Plus. 2023 Dec 1;7(12):e10807. h ps://doi.o g/10.1002/
jbm4.10807 PMID: 38130759
58. Wu Z, Lei W, Hu Y, Wang H, Wan S, Ma Z, e al. E ec o o a iec omy on BMD, mic o-a chi ec u e and biomechanics o co ical and cancellous
bones in a sheep model. Med Eng Phys. 2008 No ;30(9):1112–8. h ps://doi.o g/10.1016/j.medengphy.2008.01.007 PMID: 18595763
59. B ennan O, Kuliwaba JS, Lee TC, Pa kinson IH, Fazzala i NL, McNama a LM, e al. Tempo al changes in bone composi ion, a chi ec u e, and
s eng h ollowing es ogen de iciency in os eopo osis. Calci Tissue In . 2012;91(6):440–9. h ps://doi.o g/10.1007/s00223-012-9657-7 PMID:
23076448
60. Oheim R, Simon MJK, S eine M, Ve o azzi E, Ba encik F, Igna ius A, e al. Sheep model o os eopo osis: The e ec s o pe iphe al ho mone
he apy on cen ally induced sys emic bone loss in an os eopo o ic sheep model. Inju y. 2017 Ap 1;48(4):841–8. h ps://doi.o g/10.1016/j.
inju y.2017.02.009 PMID: 28237183
61. Zhang Y, Li Y, Gao Q, Shao B, Xiao J, Zhou H, e al. The a ia ion o cancellous bones a lumba e eb a, emo al neck, mandibula angle and ib
in o a iec omized sheep. A ch O al Biol. 2014;59(7):663–9. h ps://doi.o g/10.1016/j.a cho albio.2014.03.013 PMID: 24769217.
62. K eipke TC, Ri e a NC, Ga ison JG, Easley JT, Tu ne AS, Niebu GL. Al e a ions in abecula bone mic oa chi ec u e in he o ine spine and
dis al emu ollowing o a iec omy. J Biomech. 2014 Jun 3;47(8):1918–21. h ps://doi.o g/10.1016/j.jbiomech.2014.03.025 PMID: 24720887
PLOS One | h ps://doi.o g/10.1371/jou nal.pone.0319910 May 2, 2025 23 / 24
63. And easen CM, Ding M, O e gaa d S, Bollen P, Ande sen TL. A e e sal phase a es uncoupling he bone o ma ion and eso p ion con ibu es
o he bone loss in glucoco icoid ea ed o a iec omised aged sheep. Bone. 2015;75:32–9. h ps://doi.o g/10.1016/j.bone.2015.02.014 PMID:
25689083
64. K eipke TC, Ga ison JG, Easley J, Tu ne AS, Niebu GL. The oles o a chi ec u e and es ogen deple ion in mic odamage isk in abecula bone.
J Biomech. 2016;49(14):3223–9. h ps://doi.o g/10.1016/j.jbiomech.2016.08.009 PMID: 27544617
65. Heiss C, Ke n S, Malhan D, Böcke W, Engelha d M, Daghma DES, e al. A new clinically ele an -sco e s anda d o in e p e bone s a us in a
sheep model. Med Sci Moni Basic Res. 2017;23:326–35. h ps://doi.o g/10.12659/msmb .905561 PMID: 28966340
66. Cab e a D, Wolbe FM, Di me K, Roge s C, Ridle A, Abe dein D, e al. Glucoco icoids a ec bone mine al densi y and bone emodelling in OVX
sheep: A pilo s udy. Bone Rep. 2018 Dec 1;9:173–80. h ps://doi.o g/10.1016/j.bon .2018.11.001 PMID: 30480061
67. Mülle R, Henss A, Kampschul e M, Rohnke M, Langhein ich AC, Heiss C, e al. Analysis o mic oscopic bone p ope ies in an os eopo o ic
sheep model: a combined biomechanics, FE and ToF-SIMS s udy. J R Soc In e ace. 2019;16(151). h ps://doi.o g/10.1098/ si .2018.0793 PMID:
30958193
68. Auga P, Claes L. Inc eased co ical emodeling a e os eo omy causes pos auma ic os eopenia. Bone. 2008;43(3):539–43. h ps://doi.
o g/10.1016/j.bone.2008.05.017 PMID: 18586599
69. Malizos KN, Papach is os AA, P o opappas VC, Fo iadis DI. T ansosseous applica ion o low-in ensi y ul asound o he enhancemen and mon-
i o ing o ac u e healing p ocess in a sheep os eo omy model. Bone. 2006 Ap ;38(4):530–9. h ps://doi.o g/10.1016/j.bone.2005.10.012 PMID:
16361120
70. Gao J, Gong H, Huang X, Fang J, Zhu D, Fan Y. Rela ionship be ween mic os uc u e, ma e ial dis ibu ion, and mechanical p ope ies o sheep
ibia du ing ac u e healing p ocess. In J Med Sci. 2013 Sep 7;10(11):1560–9. h ps://doi.o g/10.7150/ijms.6611 PMID: 24046532.
71. Ve e A, Epa i DR, Seidel R, Schell H, F a zl P, Duda GN, e al. Tempo al issue pa e ns in bone healing o sheep. J O hop Res.
2010;28(11):1440–7. h ps://doi.o g/10.1002/jo .21175 PMID: 20872579
72. P eininge B, Checa S, Molna FL, F a zl P, Duda GN, Raum K. Spa ial- empo al mapping o bone s uc u al and elas ic p ope ies in a sheep
model ollowing os eo omy. Ul asound Med Biol. 2011 Ma ;37(3):474–83. h ps://doi.o g/10.1016/J.ULTRASMEDBIO.2010.12.007 PMID:
21256668.
73. Mo a-Macías J, Reina-Romo E, López-Pliego M, Gi áldez-Sánchez MA, Domínguez J. In i o mechanical cha ac e iza ion o he dis ac ion callus
du ing bone consolida ion. Ann Biomed Eng. 2015 No 1;43(11):2663–74. h ps://doi.o g/10.1007/s10439-015-1330-7 PMID: 25956927
74. Mo a-Macías J, Reina-Romo E, Mo gaz J, Domínguez J. In i o gai analysis du ing bone anspo . Ann Biomed Eng. 2015 Sep 29;43(9):2090–
100. h ps://doi.o g/10.1007/s10439-015-1262-2 PMID: 25650097
75. Blázquez-Ca mona P, Mo a-Macías J, Mo gaz J, Fe nández-Sa mien o JA, Domínguez J, Reina-Romo E. Mechanobiology o bone consolida ion
du ing dis ac ion os eogenesis: bone leng hening s. bone anspo . Ann Biomed Eng. 2021 Ap 1;49(4):1209–21. h ps://doi.o g/10.1007/s10439-
020-02665-z PMID: 33111968.
76. Blázquez-Ca mona P, Mo a-Macías J, Sanz-He e a JA, Mo gaz J, Na a e e-Cal o R, Domínguez J, e al. Mechanical in luence o su ound-
ing so issue on bone egene a ion p ocesses: a bone leng hening s udy. Ann Biomed Eng. 2021 Feb 1; 49(2):642–52. h ps://doi.o g/10.1007/
s10439-020-02592-z PMID: 32808118
77. Blázquez-Ca mona P, Mo a-Macías J, Paja es A, Má mol Á, Reina-Romo E. On he in luence o s uc u al and chemical p ope ies on he elas ic
modulus o wo en bone unde healing. F on Bioeng Bio echnol. 2024 Oc 1;12:1476473. h ps://doi.o g/10.3389/ bioe.2024.1476473 PMID:
39411059
78. Mo a-Macías J, Paja es A, Mi anda P, Domínguez J, Reina-Romo E. Mechanical cha ac e iza ion ia nanoinden a ion o he wo en bone de el-
oped du ing bone anspo . J Mech Beha Biomed Ma e . 2017 Oc 1;74:236–44. h ps://doi.o g/10.1016/j.jmbbm.2017.05.031 PMID: 28623826
79. Manjubala I, Liu Y, Epa i DR, Roschge P, Schell H, F a zl P, e al. Spa ial and empo al a ia ions o mechanical p ope ies and mine al con en o
he ex e nal callus du ing bone healing. Bone. 2009;45(2):185–92. h ps://doi.o g/10.1016/j.bone.2009.04.249 PMID: 19414072
80. Inglis B, Schwa zenbe g P, Klein K, on Rechenbe g B, Da wiche S, Dailey HL. Biomechanical duali y o ac u e healing cap u ed using i ual
mechanical es ing and alida ed in o ine bones. Sci Rep. 2022 Dec 1;12(1):2492. h ps://doi.o g/10.1038/s41598-022-06267-8 PMID: 35169187
81. Alagboso FI, Budak M, Somme U, Ray S, Kaise A, Kampschul e M, e al. Es ablishmen o a clinically ele an la ge animal model o assess he
healing o me aphyseal bone. Eu Cell Ma e . 2019 Jan 1;37:444–66. h ps://doi.o g/10.22203/ECM.V037A27 PMID: 31219613
82. Mo a-Macías J, Gi áldez-Sánchez MÁ, López M, Domínguez J, Reina-Romo ME. Compa ison o me hods o assigning he ma e ial p ope ies o
he dis ac ion callus in compu a ional models. In J Nume Me hod Biomed Eng. 2019 Sep 1;35(9):e3227. h ps://doi.o g/10.1002/cnm.3227 PMID:
31197959
83. Blázquez-Ca mona P, Mo a-Macías J, Mo gaz J, G anados MDM, Domínguez J, Reina-Romo E. Gai analysis: An e ec i e ool o mechani-
cally moni o he bone egene a ion o c i ical-sized de ec s in issue enginee ing applica ions. PLoS One. 2023;18(12):e0296510. h ps://doi.
o g/10.1371/jou nal.pone.0296510 PMID: 38157369
84. Bouxsein ML, Boyd SK, Ch is iansen BA, Guldbe g RE, Jepsen KJ, Mülle R. Guidelines o assessmen o bone mic os uc u e in oden s using
mic o-compu ed omog aphy. J Bone Mine Res. 2010 Jul;25(7):1468–86. h ps://doi.o g/10.1002/JBMR.141 PMID: 20533309
85. Lees S, Heeley JD, Clea y PF. A s udy o some p ope ies o a sample o bo ine co ical bone using ul asound. Calci Tissue In . 1979;29(2):107–
17. h ps://doi.o g/10.1007/BF02408065 PMID: 116750
PLOS One | h ps://doi.o g/10.1371/jou nal.pone.0319910 May 2, 2025 24 / 24
86. G ubbs FE. P ocedu es o de ec ing ou lying obse a ions in samples. Technome ics. 1969;11(1):1–21. h ps://doi.o g/10.1080/00401706.1969.10
490657
87. Ren T, Klein K, on Rechenbe g B, Da wiche S, Dailey HL. Image-based adiodensi y p o ilome y measu es ea ly emodeling a he bone-callus
in e ace in sheep. Biomech Model Mechanobiol. 2022 Ap 1;21(2):615–26. h ps://doi.o g/10.1007/s10237-021-01553-2 PMID: 34997398
88. Melnyk M, Henke T, Claes L, Auga P. Re ascula isa ion du ing ac u e healing wi h so issue inju y. A ch O hop T auma Su g.
2008;128(10):1159–65. h ps://doi.o g/10.1007/s00402-007-0543-0 PMID: 18094982
89. Jee WSS, Yao W. O e iew: animal models o os eopenia and os eopo osis. J Musculoskel Neu on In e ac 2001;1.
90. Schulz MC, Kowald J, Es en elde S, Jung R, Kuhlisch E, Eckel U, e al. Si e-speci ic a ia ions in bone mine al densi y unde sys emic condi ions
inducing os eopo osis in Minipigs. F on Physiol. 2017;8:426. h ps://doi.o g/10.3389/ phys.2017.00426 PMID: 28676766
91. Lelo as TT, Xan hos SE, Thoma GP, Ly i is IA. The labo a o y a as an animal model o os eopo osis esea ch. ingen aconnec .
comPP Don asCompa a i e medicine, 2008•ingen aconnec .com. 2008. A ailable om: h ps://www.ingen aconnec .com/con en /aalas/
cm/2008/00000058/00000005/a 00001
92. Bloebaum RD, Lundeen GA, Shea JE, Whi ake EL. Age- ela ed mine aliza ion he e ogenei y changes in abecula bone o he p oximal emu .
Ana Rec A Disco Mol Cell E ol Biol. 2004 Dec;281(2):1296–302. h ps://doi.o g/10.1002/a .a.20079 PMID: 15386275
93. Leidi M, Delle a F, Ma io i M, Maie JAM. High magnesium inhibi s human os eoblas di e en ia ion in i o. Magnes Res. 2011;24(1):1–6. h ps://
doi.o g/10.1684/MRH.2011.0271 PMID: 21421455
