ORIGINAL ARTICLE
Mechanics P edic s E ec i e C i ical-Size Bone Regene a ion
Using 3D-P in ed Bioce amic Sca olds
Pablo Bla
´zquez-Ca mona
1,4
•Juan Mo a-Macı
´as
2,4
•F ancisco J. Ma ı
´nez-Va
´zquez
1
•
Juan Mo gaz
3
•Jaime Domı
´nguez
1,4
•Es he Reina-Romo
1,4
Recei ed: 9 May 2023 / Re ised: 25 June 2023 / Accep ed: 11 July 2023 / Published online: 22 Augus 2023
ÓThe Au ho (s) 2023
Abs ac
BACKGROUND: 3D-p in ed bioce amic sca olds ha e gained popula i y due o hei con olled mic oa chi ec u e and
hei p o en biocompa ibili y. Howe e , hei high b i leness makes hei su gical implemen a ion complex o weigh -
bea ing bone ea men s. Thus, hey would equi e di icul - o-ins umen igid in e nal ixa ions ha limi a igo ous
e alua ion o he egene a ion p og ess h ough he analysis o mechanic-s uc u al pa ame e s.
METHODS: We in es iga ed he compa ibili y o lexible ixa ions wi h agile ce amic implan s, and i mechanical
moni o ing echniques a e applicable o bone issue enginee ing applica ions. Tissue enginee ing expe imen s we e pe -
o med on 8 o ine me a a si. A 15 mm bone segmen was di ec ly eplaced wi h a hyd oxyapa i e sca old and s abilized by
an ins umen ed Iliza o - ype ex e nal ixa o . Se e al in i o moni o ing echniques we e employed o assess he
mechanical and s uc u al p og ess o he issue.
RESULTS: The applied su gical p o ocol succeeded in combining ex e nal ixa o s and subjec -speci ic bioce amic
sca olds wi hou causing a al ac u es o he implan due o s ess concen a o . The bea ing capaci y o he ea ed limb
was ini ially al e ed, quan i ying a 28–56% educ ion o he g ound eac ion o ce, which g adually no malized du ing he
consolida ion phase. A as e eco e y was epo ed in he bea ing capaci y, s i ening and bone mine al densi y o he
callus. I acqui ed a p edominan mechanical ole o e he ixa o in he dis ibu ion o in e nal o ces a e one pos -
su gical mon h.
CONCLUSION: The bioce amic sca old signi ican ly accele a ed in i o he bone o ma ion compa ed o o he adi-
ional al e na i es in he li e a u e (e.g., dis ac ion os eogenesis). In addi ion, he implemen ed assessmen echniques
allowed an accu a e quan i a i e e alua ion o he bone egene a ion h ough mechanical and imaging pa ame e s.
Keywo ds Tissue enginee ing Bioce amic sca old Mechanobiology Compu e ized omog aphy Bone mine al
densi y
&Pablo Bla
´zquez-Ca mona
[email p o ec ed]
1
Escuela Te
´cnica Supe io de Ingenie ı
´a, Uni e sidad de
Se illa, A enida Camino de los Descub imien os s/n,
41092 Se ille, Spain
2
Escuela Te
´cnica Supe io de Ingenie ı
´a, Uni e sidad de
Huel a, Huel a, Spain
3
Depa amen o Medicina y Ci ugı
´a Animal, Uni e sidad de
Co
´ doba, Campus Uni e si a io de Rabanales, Co
´ doba,
Spain
4
Ins i u o de Biomedicina de Se illa (IBiS), Uni e sidad de
Se illa, Se ille, Spain
123
Tissue Eng Regen Med (2023) 20(6):893–904 Online ISSN 2212-5469
h ps://doi.o g/10.1007/s13770-023-00577-2
1 In oduc ion
When skele al issues ha e been c i ically de as a ed o
los by congeni al anomaly, poo ly co ec ed auma,
os eomyeli is o cance , he p e e ed p ocedu es o acu e
ca e su geons a e dis ac ion os eogenesis [1–4], massi e
o Papineau open cancellous bone g a ing [5], and as-
cula ized ee ibula ans e ence [6]. Despi e epo ed
egene a i e e ec i eness h ough op imiza ion o i s
biomechanical ac o s (e.g., a e and equency o dis ac-
ion), he Iliza o me hod is associa ed wi h high isks
ela ed o iscoelas ic and s uc u al changes in he su -
ounding so issues [7–9]. Rega ding bone subs i u es,
au og a s and allog a s a e cu en ly he mos implan ed
ma e ials due o hei unma ched os eoinduc i e, os eo-
conduc i e and biocompa ible p ope ies [10]. Ne e he-
less, he ising numbe o auma pa ien s wi h long bone
de ec s is inc easing he global demand [11]. To sa is y
hei insu icien a ailabili y, issue enginee ing (TE) has
apidly eme ged as he ul ima e o hopaedical hope o
unlimi edly eplace p e ious na u al app oaches. They
o e a i icial linkage s uc u es called sca olds o p o-
mo e os eogenesis while educing o he g a ing sho -
comings, including dono si e complica ions and mo bidi y
[12] o po en ial dono pa hogen ansmission [13]. Mos
ecen in es iga ions a e ocused on in i o and in silico
models ying o each he ideal manu ac u ing echnology,
con igu a ions, in e nal a chi ec u e and, indeed, he bio-
ma e ial subs i u e. Speci ically, bioce amic ma e ials,
composed o pa ially o non-c ys alline ce amics (e.g.,
hyd oxyapa i e, calcium phospha e cemen o glass–ce-
amics), a e gaining popula i y in clinical esea ch due o
hei demons a ed key mechanisms leading o enhanced
new ha d issue g ow h by egula ing os eoblas p oli e a-
ion, di e en ia ion, and gene exp ession [14]. Unlike
commonly employed me allic ma e ials ha could inhe -
en ly obs uc he in asion o blood essel walls o equi e
a pe iodic eplacemen [15], bioce amics allow a ‘‘lea e
no hing behind’’ app oach h ough a g adual eso p ion as
he new bone issue g ows. Mo eo e , bioce amic sca olds
a e gene ally buil h ough addi i e manu ac u ing ech-
nologies, which allow p o o yping and compa ing pa ien -
speci ic sca olds wi h di e en mic oa chi ec u es and
con olled po e size dis ibu ions [16,17]. So a , bioce-
amic sca olds ha e been es ed p ima ily on animal
s udies wi h limi ed success [18–21] and mo e in i o
in es iga ions a e needed.
Du ing bone egene a ion p ocesses, con inuous moni-
o ing is essen ial in he ea ly healing s ages when ejec ion
o g a s and implan s o non-unions a e mo e likely o
occu . The adi ional in i o clinical e alua ions, such as
plain ilm adiology, clinical signs o in lamma ion o pain,
and mobili y examina ions o he ope a ed limb, a e no
accu a e enough o ensu e ha a bone callus is o ming
p ope ly o o make ele an decisions, including su gical
ein e en ion [22–24]. In he las decades, enginee s ha e
de eloped new angled acking al e na i es h ough he
ins umen a ion o medical de ices. Among hese s a egies
a e using acous ic emissions [25], ib a ional es s ana-
lyzing he wa e p opaga ion h ough he issue and i s
esonan equency [26], o os eoin eg a ion moni o ing
using ul asounds [27]. None heless, hese echniques a e
di icul o s anda dize o ans e o ou ine clinical use
oday due o hei signi ican dependence on hei expe i-
men al p o ocol and es ing bounda y condi ions [28]. By
cons, he ins umen ed ex e nal ixa ions helped con ol
os eogenesis based on indi ec ly es ima ing appa en
mechanical p ope ies and loading capaci y o he callus
issue om he o ce, s ess, o aw s ain da a
[1–4,29–35]. This app oach has been adi ionally applied
in i o o dis ac ion os eogenesis o ac u e healing
ea men s, hus p o iding addi ional p ac ical in o ma ion:
compa ison o he e ec i eness o di e en p o ocols o
he op imiza ion o he ime-poin o he ixa o ’s emo al.
Fo ins ance, Liu e al. [32] ecen ly ound ha an axial
load-sha e a io below 10% is sa e o disassemble a
monola e al ex e nal ixa o when ea ing human auma.
As a as he au ho s’ knowledge, no p e ious esea ch has
explo ed he applicabili y o hese moni o ing echnologies
o he mechano-s uc u al e olu ion o he TE callus
compa ed o o he clinical al e na i es (e.g., bone anspo
o bone g a ing). In TE, hey would be pa icula ly in e -
es ing due o he limi ed imaging ollow-up esul ing om
he high adiopaci y o many sca olding base ma e ials
and hei ypical leng h scale.
Unlike he abo e, he c i ical-size and load-bea ing bone
de ec s eplaced by sca olding a e usually s abilized by
means o in e nal splin s in he li e a u e [18–21]. Thei
s i e assembly me hods p o ide be e bony alignmen
and o a ion, mo e accu a e con ol o in e agmen a y
mo emen s and, consequen ly, less signi ican mechanical
es ic ions o he s uc u al in eg i y o b i le hos
implan s. Fo example, Vidal e al. [20] eso ed o in e nal
ixa ions when ea ing a 35 mm sheep me a a sal de ec
wi h calcium phospha e sca olds. Despi e he epo ed
high damage- ole an beha io o he biomo phic ce amic
sca olds’ hie a chical a chi ec u e, Kon e al.[18] also
en us ed wi h s abilizing hei me a a sal de ec s on di ec
sc ewing me allic ixa ion plaques. Fu he mo e, beyond
he mo e easible ins alla ion o measu ing senso s, all o
hem o ei he ex e nal app oaches’ ad an ages o
equi ing less signi ican body in asion ha bene i s he
pa ien by a oiding ex ensi e so issue damage, educing
blood loss du ing su ge ies, and simpli ying he associa ed
so issue managemen [36,37]. All his leads o ea ly
894 Tissue Eng Regen Med (2023) 20(6):893–904
123
mobiliza ion o he pa ien and a sho e hospi aliza ion
[27]. Poblo h e al. [38] succeeded in combining a hyb id-
ing ex e nal ixa o wi h a ubula -shaped sca old made o
b-TCP g anules a ached o a eso bable polyme empla e
in an o ine ibia model. The ce ain de o mabili y o i s
base ma e ial allowed a be e dissipa ion o he mechanical
o ces in i o. Oh e al. [39] designed an ex e nal ixa o
adap able o di e en axial s i nesses aimed a issue
enginee ing applica ions in oden models, as a as he
au ho s a e conce ned, i s e ec i eness has no been es ed
in i o. Conside ing he p e ious echnical di icul ies, a
sa e su gical con igu a ion and egene a ing e icacy a e
s ill a huge challenge in implemen ing hese ce amic
sca olds as a comme cial p oduc o ea human o ho-
pedic de ec s unde a mechanical en i onmen con olled
by a lexible ex e nal ixa ion.
Based on he p e ious backg ound, he goals o his
wo k a e: (1) o es ablish a su gical p ocedu e and an
implan a ion p o ocol ha allow sol ing he clinical chal-
lenge o combining subjec -speci ic bioce amic sca olds
unde a lexible mechanical en i onmen ; (2) o es o he
i s ime me hods o mechanical assessmen o he
egene a ion p ocess in issue enginee ing and discuss i s
use ulness o clinical decision making; and (3) o discuss
he eco e y o he analyzed mechano-s uc u al pa ame-
e s in compa ison wi h o he al e na i e egene a ion
p ocesses al eady moni o ed in he li e a u e (e.g., dis-
ac ion os eogenesis o ac u e healing).
2 Ma e ials and me hods
Tissue enginee ing su ge ies we e pe o med in he ipsi-
la e al hind me a a sus o emale Me ino sheep aged
be ween 3 and 5 yea s (n= 8). Speci ically, 15 mm in e -
media e bone de ec s we e egene a ed aided by subjec -
speci ic bioce amic sca olds s abilized wi h lexible ixa-
ions. Animal e hics o he Uni e si y o Co
´ doba app o ed
his s udy (2021PI/21). The wel a e o he sheep was
gua an eed du ing he comple e su gical in e en ions and
expe imen al phases ollowing he HIPAA, Eu opean
(2010/63/UE) and na ional (RD 1201/2005) no ms on
animal expe imen a ion. As shown in Table 1, animals
we e slaugh e ed a di e en ime-poin s o he consoli-
da ion phase so as o analyze he e olu ion o compu ed
omog aphy (CT) pa ame e s, whose egula app aisal was
limi ed by he complex segmen a ion esul ing om he
e lec ions o he ixa o ’s me allic componen s. The
manu ac u ing p ocess o he implan s, he su gical in e -
en ion, and he se o in i o and ex i o expe imen s a e
speci ied as ollows, including images in Fig. 2. Th ough-
ou he ollowing subsec ions, i ing unc ions will be buil
o illus a e he ends o mos o he pa ame e s.
2.1 Fab ica ion and p epa a ion o he sca old
The subjec -speci ic geome y o he sca olds was shaped
om omog aphy scans pe o med on he o ine me a a si
be o e su ge y. DICOM images ( oxel size
0.115 90.115 90.600 mm) we e in e ac i ely h esh-
olded and econs uc ed using he open-sou ce so wa e
InVesaliusÒ(Rena o A che In o ma ion Technology
Cen e , Ama ais, B azil). As shown in Fig. 1, an in e me-
dia e 13 mm bony agmen was c opped om he me a-
a sal 3D geome y. Then, he inne medulla y ca i y was
illed. A cylind ical couple (4 mm Ø; 2 mm heigh ) was
subsequen ly buil in he 3D geome y a he bone ma ow
loca ion o one end ace o es ic he sca old’s mobili y
wi hin he de ec (see Fig. 1). On he o he end, a hole
(4 mm Ø; 10 mm heigh ) was c ea ed o be s u ed by
g a ing issue du ing he su ge y, hus s imula ing he
implan ’s biological esponse. The inne a chi ec u e was
nume ically op imized in a p e ious wo k o maximize he
os eoinduc i e and os eoconduc i e p ope ies while
ensu ing he mechanical in eg i y unde he physiological
loads o his bone model [40]. The eby, unde he
mechanical limi s imposed by hese physiological loads,
he po osi y, speci ic su ace a ea and po e size we e
maximized o gua an ee cell di usion, adhesion and p o-
li e a ion in he implan . The esul ing mic os uc u e
Table 1 Indi idual da a o each
specimen: consolida ion days
be o e eu hanasia, ime-poin s
a which omog aphic scans
we e pe o med, es ima ion o
he appa en s i ness o he
p oximal (Kb;p), dis al (Kb;d)o
bone- ixa o sys em, and
equi alen co ical s i ness (Kb)
Sheep Days CT ime-poin s Kb;p[kN/mm] Kb;d[kN/mm] Kb¼Kb;dKb;d
Kb;dþKb;d
S1 365 365, 285, 176 309.85 356.74 165.82
S2 219 219 144.51 138.36 70.68
S3 194 194, 158 199.86 206.13 101.47
S4 152 152 173.25 190.27 90.68
S5 123 123 236.96 333.16 138.13
S6 65 65 160.67 136.69 73.86
S7 51 51 206.71 219.49 106.45
S8 30 30 218.43 236.98 113.67
Tissue Eng Regen Med (2023) 20(6):893–904 895
123
p esen s 59.30% o po osi y, 5768.91 m
-1
o speci ic su -
ace a ea, and 360.80 lm o po e size [40].
Implan s we e manu ac u ed ollowing a laye -wise
long-pile pa e n o ods, using obo ic deposi ion de ice
(3-D Inks S ill-wa e Ò, Tulsa, Oklahoma, USA) and a
clinically p o en 45 ol. % hyd oxyapa i e (HA) ink, as
shown in Fig. 2A. The selec ed base ma e ial mimics he
bone chemical composi ion and mo phology [41,42].
Mo eo e , i s biodeg adabili y does no equi e su gical
emo al o he sca old a e he consolida ion p ocess.
Once manu ac u ed, he s uc u es we e d ied a oom
empe a u e, hea ed a 400 °C o 1 h o elimina e o ganic
componen s, and sin e ed a 1300 °C o 2 h. Implan s we e
chemically s e ilized unde a high o maldehyde concen-
a ion a 60 °C and wi h a ela i e humidi y o 75 o 100%.
2.2 Su gical p ocedu e
Sheep we e ini ially immobilized. An ibio e apy and
in lamma ion we e p e- ea ed wi h Amoxiciline 15 mg/kg
ClamoxylÒand Meloxicam 0.2 mg/kg Me acamÒ,
espec i ely. Animals we e seda ed wi h de omidine 20 lg/
kg De ogesicÒand mo phine 0.2 mg/kg Mo ina
B.B aunÒ. A e 10 min, he sheep we e posi ioned in
s e nal decubi us induc ion, and endo acheal in uba ion
was ca ied ou . Gene al anes hesia was main ained wi h
iso lu ane IsoVe Ò o expi a o y concen a ions o 1–1.2%.
The oxygen sa u a ion, elec oca diog am, espi a o y
gases, body empe a u e, and blood p essu e (ca he e izing
pedal o au icula a e ies) we e con olled du ing he
su ge y.
The su ge ies consis ed mainly o assembling an ex e -
nal ixa o (s i ness K o 593 N/mm) on he bone and
subs i u ing a agmen o he sca old. An insigh in o his
ixa o , al eady implan ed, is gi en in Fig. 2C. The ixa ion
is an Iliza o - ype de ice composed o wo ci cula alu-
minum ames ixed indi idually o he bone by six d illed
4 mm Ø Schanz pins, wo bico ical and one unico ical pe
ame. The ames a e addi ionally ex e nally in e con-
nec ed by ba s ins umen ed wi h Bu s e Ò8431–6001
load cells (Bu s e , Ge nsbach, Ge many), which ole a e
bending momen s while eal- ime measu ing o ces
h ough he ixa ion. A p e ious s udy p o ides mo e
de ails abou he ixa ion design, as well as he in i o and
in i o calib a ion o he wi eless acquisi ion pla o m [43].
Once he bone was s abilized, wo os eo omies we e pe -
o med in he in e media e pa o he me a a sus, emo ing
he esul ing 15 mm bone agmen . The eupon, cancellous
au og a was ha es ed om he la e al side o he
hume us head. A 7 cm incision was pe o med wi h elec-
ical scalping, and a d ill was used o access he eques ed
issue. A e illing he sca old’s g a ing ca i y wi h pa
o he spongy issue, he implan was inally inse ed in o
he induced de ec (see Fig. 2B, C). The sca old couple
was i ed in he dis al bone ma ow. As a consequence o
he leng h o he main sca old body, a 1–2 mm gap
emained a i s p oximal in e ace wi h he me a a sal
agmen . This space was illed wi h he emaining can-
cellous issue, hus connec ing bo h su aces and p e en -
ing unexpec ed s ess concen a ions in he me a a sal-
implan con ac ha could ac u e he s uc u e. In addi-
ion, ope a ed animals we e eco e ed wi h oxygen unde
assis ance. Pos -ope a i e analgesia was also p o ided
(meloxicam Me acamÒand opioids) acco ding o pain
scales in sheep.
Fig. 1 Design o he pa ien -speci ic ou e sca old geome y: (1)
c opping an in e media e 13 mm bone segmen om compu ed
omog aphy images o each o ine me a a sus; and (2) addi ion o a
cylind ical hole and a couple o g a he s uc u e wi h cancellous
bone and o immobilize he implan inside he bone ma ow,
espec i ely
896 Tissue Eng Regen Med (2023) 20(6):893–904
123
2.3 In i o mechanical moni o ing
The e olu ion o se e al mechanical pa ame e s in ol ed
in bone egene a ion was assessed. A e one la ency week,
s ance analysis was pe o med wo o h ee imes weekly
h ough walking es s in a ci cula gai ci cui ins umen ed
wi h a wi eless load pla o m Pasco PS-2141Ò(PASCO,
Rose ille, CA, USA), as illus a ed in Fig. 2D. The g ound
eac ion cu es o a minimum 8 eads a an amble speed
(2–4 km/h) we e cap u ed in e e y expe imen , hus ana-
lyzing he da a as a daily a e age. This analysis ocused on
he maximum g ound eac ion o ce (GRF) due o i s
p o en in e ela ionship wi h he epai o bone de ec s in
o he egene a ion p ocesses [30,33]. Fu he mo e, he
o ces h ough he ixa o du ing he analyzed animal’s
s ance phases (F ) we e simul aneously acqui ed using he
senso s speci ied abo e. Following a simila s a egy o
p e ious wo ks [9,30,44], he in e nal o ce h ough he
me a a sus (Fi) was es ima ed by assuming a cons an a io
wi h he GRF. Using he bone- ixa o mechanical sys em
speci ied in Fig. 3A, his ela ionship was ixed du ing he
ea ly egene a ion s age o e e y specimen, he i s 2 pos -
su gical weeks, when he e is ha dly any ma u e b idging
connec ion be ween he sca old and he emaining ag-
men s. This ac allows conside ing ha he ixa ion loads
a e app oxima ely equal o he in e nal o ce (FiF ). In
he ollowing consolida ion phase, he e olu ion o he
bone callus loading capaci y (FC) was assessed acco ding
o Fig. 3B as:
FC¼FiF ð1Þ
Finally, he appa en callus s i ness Kcwas de e mined
by assuming homogeneous mechanical p ope ies in he
issue. F om he bone- ixa o model (Fig. 3B), hese s i -
ness alues we e easily es ima ed om he Fidis ibu ion
h ough he me a a sus, he s i ness o he ex e nal ixa o
K , and he appa en s i ness o he emaining p oximal
and dis al me a a sal agmen s wi hin he ixa ion, Kb;pand
Kb;d. These co ical s i ness alues di e ed om each
o he due o hei geome ical in e di e ences, and hey
we e es ima ed om he CT images in he sac i ice speci-
ied in he ollowing sec ions and supposing an elas ic
modulus o 21 GPa [9]. Thei alues pe animal a e also
de ailed in Table 1. Bea ing in mind all his and consid-
e ing an equi alen sys em displacemen du ing he sheep’s
h ead in lines, he Kcwas es ima ed using Eq. 2:
Kc¼K FcKb
F KbþFcK
ð2Þ
whe e Kbis he equi alen s i ness o Kb;pand Kb;dsp ings
in line (see Fig. 3B). The mechanical analysis was limi ed
o he in i o calib a ion ange o he measu emen
Fig. 2 A–D: P o ocol s eps o
ou o ine issue enginee ing
s udy: AManu ac u ing o he
ce amic me a a sal sca old ia
obocas ing; BSca old illing
wi h hume us cancellous
au og a ; CImplan a ion o he
ex e nal ixa o and eplacemen
o he bone agmen by he
biologically enhanced sca old;
DRou ine gai expe imen s in
he ins umen ed walking ci cui
Tissue Eng Regen Med (2023) 20(6):893–904 897
123
equipmen wi h a low le el o e o s and unce ain ies in
he es ima ion o s i ness [43]. F om ha ime-poin , gai
analysis con inued o be pe o med less equen ly o check
he gene al endency o hese pa ame e s.
2.4 Imaging ollow-up
A adiog aphic ollow-up was pe iodically pe o med o
e i y he egene a ion p og ess h ough do soplana and
la e al iews quali a i ely. As p e iously s a ed, CT images
o he ea ed limbs we e addi ionally aken a he sac i ice
ime-poin s o he consolida ion phase. Fu he mo e, he
ixa ion was emo ed p io o sac i ice in some long- e m
specimens, enabling in e media e CT images. The non-
ope a ed con ala e al limbs we e included in he images
and p ocessed as con ol da a. The esolu ion o he images
was a ound 300 lm/ oxel, and hei manual segmen a ion
was pe o med using he medical so wa e InVesaliusÒ.
Figu e 4A shows a scheme o he segmen a ion p ocess and
he gene a ion o he h ee-dimensional geome y. The
a e age callus c oss-sec ional a ea wi hin he de ec (CSA)
and o al callus olume (TV), including 1 cm o ex e nal
issue a ound he dis al and p oximal co ical agmen ,
we e calcula ed. Finally, he appa en bone callus mine al
densi y (BMD) was es ima ed as an indi ec measu e o he
sca old ossi ica ion. The phan oms QRM-BDC/6–200Ò
(PTW, F eibu g, Ge many) we e used as a sc eening ool o
co ela e he mine al densi y (0–0.8 g HA/cm
3
) wi h he
CT Houns ield Uni s linea ly. The con ala e al limbs we e
simul aneously included in he scans as a con ol. An
example o he moni o ed e olu ion o CSA and BMD
along he axial me a a sal c oss-sec ion in one o he CT
scans o he s udy is shown in Fig. 4B–C, including a
compa ison wi h he con ala e al con ol limb.
3 Resul s
3.1 Gai analysis and mechanical healing
The e olu ion o analyzed gai pa ame e s h ough he
consolida ion phase is shown in Fig. 5A. Acco ding o he
GRF da a, TE su ge y dec eased he animals’ bea ing
unc ional capaci y in he ope a ed limb a a ious le els.
The week a e he la ency pe iod, he s ance phases gen-
e ally epo ed a eac ion o ce be ween 28 and 56% o
heal hy alues (42–43% o he body weigh in his animal
model) [30,33]. This gai pa ame e ended o a p og es-
si e eco e y o app oxima ely 56–85% a mon h 4 a e
su ge y o up o 93% a e hal a yea .
F om he mechanical poin o iew, he Fidis ibu ion
p o ided in Fig. 5B indica ed an ini ial lack o mine alized
connec ion be ween he sca old and he o iginal diaphysis.
The eby, he ixa o loaded mos o he physiological o ces
(F [90% o Fi) du ing he i s healing o nigh . F om
hen on, he ea ly bone calluses acqui ed g ea e mechan-
ical ele ance un il he dis ibu ion o o ces was o e -
u ned a e an a e age o 30 consolida ion days, hus being
Fchighe han F . In 70 days, he eco e y o hei bea ing
capaci y was comple ed, and he o ces h ough he ex e -
nal ixa o s abilized a ound 0. A he same ime, he bone
calluses expe ienced a apid exponen ial inc ease in hei
mechanical p ope ies, as shown in Fig. 5C. While hei
mean appa en s i ness was quan i ied up o 650 N/mm on
day 40 a e su ge y, hey eached an a e age 7 kN/mm by
20 days la e .
Fig. 3 A–B: Mechanical
models o he in e nal o ce (Fi)
dis ibu ion be ween he bone
(FCÞand he ex e nal ixa o
(F ) as a unc ion o he g ound
eac ion o ce (GFR), muscle
ac i i y, and he componen s’
s i ness: p oximal and dis al
diaphysis agmen s (Kb;pand
Kb;d), bone callus (KC), and
ixa ion (K ). AScheme du ing
he i s weeks a e su ge y
wi hou ma u e connec ion
be ween he co ical bone and
he sca old; BRegula
scheme con ains he callus
s i ness KCdu ing he
consolida ion phase
898 Tissue Eng Regen Med (2023) 20(6):893–904
123
3.2 Callus geome y and mine aliza ion
The p og ess o he mine aliza ion in he same sheep a
di e en ime-poin s o he egene a ion p ocess is com-
pa ed in Fig. 6. Pos -su gical x- ay was included as a e -
e ence o he ini ial callus s a us. Despi e he ac ha he
implan was clea ly isible adiog aphically a any poin o
he expe imen , i was impossible o app ecia e he i s
s ages o egene a ion due o he ce amic opaci y. Al hough
he sca old was designed o wi hs and he daily loads o
he animal, i was logis ically impossible o ensu e he
absence o ins an aneous peaks o ex eme loads due o
unexpec ed mo emen s o beha io . Fo example, he la -
e al iews in Fig. 6show a local ac u e in he dis al le
co ne o he implan in he images ob ained a days 120
and 210 a e su ge y. These mino b eakages occu ed in
app oxima ely hal o he specimens in line wi h wha
occu ed in simila s udies [20]. Fo una ely, hey did no
a ec he gene al in eg i y o he s uc u e. In mo e
ad anced s ages o he consolida ion phase, ossi ica ion
a ound he sca old was gene ally obse ed, especially in
he pos e io in e zone, whe e physiologically mo e sig-
ni ican ascula iza ion is ound due o he loca ion o he
lexo endons. Signs o wo en bone emodeling in he
global callus geome y we e also epo ed a e one yea o
egene a ion.
The e olu ion o he pa ame e s quan i ied by CT
imaging a e shown in Fig. 5D, including he o al olume
(TV), he de ec c oss-sec ional a ea (CSA), and he bone
mine al densi y (BMD). They a e p esen ed as a pe cen age
o hei 15 mm non- ea ed con ala e al alue, being
2.17 ±0.26 cm
3
, 144.79 ±14.42 mm
2
, 1.25 ±0.07 g
HA/cm
3
, espec i ely. Bo h TV and CSA s a om alues
close o heal hy alues due o he subjec -speci ic na u e o
he implan . Howe e , acco ding o he i ing unc ion,
bo h pa ame e s apidly inc ease h oughou he i s hal o
he yea . The maximum peak alues we e epo ed a e
app oxima ely 200 days om su ge y, a e aging 347.88%
o he con ol alue o TV and 229.49% o CSA. Du ing
he second hal o he yea , he e is a emodeling sligh ly
ending o heal hy alues. A e one yea , he TV and CSA
we e s ill 280.82% and 156.13% o e con ol. Fo i s pa ,
Fig. 4 A–C: CT analysis o he ope a ed limbs o analyze he
geome y and he mine aliza ion s a e o he bone callus a di e en
ime-poin s o he egene a ion p ocess. ASegmen a ion o he
co ical, callus issue and phan oms o u he calcula e he o al
olume (TV), c oss-sec ional a ea (CSA) and bone mine al densi y
(BMD) e olu ion in he 3D gene a ed geome y; BExample o he
moni o ed e olu ion o he CSA along he ea ed and con ol
me a a sus; CExample o he BMD e olu ion along he ea ed and
con ol me a a sus. Peak alues ou side he de ec limi s a e due o he
wo en bone o med a ound he pins o he ex e nal ixa o
Tissue Eng Regen Med (2023) 20(6):893–904 899
123
he BMD was ini ially 36% o he heal hy mine al densi y
due o he appa en mine al con en o he sca old, which
was measu ed indi idually wi h he same CT scan. This
pa ame e expe ienced a con inuous eco e y o up o 95%
o he con ala e al alue a he end o he expe imen al
yea .
4 Discussion
The cu en wo k p o ides an in e disciplina y cha ac e i-
za ion o he TE callus consolida ion h oughou he e o-
lu ion o di e en mechanical and imaging pa ame e s
measu ed in i o and ex i o. The o e all h us was
owa ds a g adual eco e y o hei heal hy condi ions by
means o a lexible ixa ion and subjec -speci ic hyd ox-
yapa i e sca olds ha emained s uc u ally s u dy
h oughou he en i e p ocess. Speci ically, he su gical
me hodology o lea ing an app op ia e gap be ween he
b i le po ous subs i u e and he p oximal loading agmen ,
illed wi h spongy issue, success ully p e en ed a al s ess
concen a ions ia local con ac . No was i equi ed o
eso o bone mo phogene ic p o eins o o he bioac i e
molecules o suppo and cho eog aph bone issue o ma-
ion. Thus, he op imized sca old seems o mee by i sel
he clinical equi emen s o mechanical suppo and
os eoinduc i i y, and alida es in i o he nume ical op i-
miza ion o i s mic os uc u e [40]. This esea ch o e s
uncomplica ed s a egies o in es iga e u he he combi-
na ion o bioce amic ma e ials and ins umen ed ex e nal
ixa o s, as well as he need o no o biologically i a-
minize he sca old wi h g ow h ac o s o o he agen s ha
induce egene a ion.
The healing o he moni o ed pa ame e s showed di -
e en speeds. In he sho e m (0–3 mon hs a e su ge y),
a bone callus began o g ow be ween he diaphysis and he
sca old, as shown in Fig. 6(pos -su ge y and day 60). This
was e lec ed in a clea eco e y o he mechanical unc-
ionali y, loading capaci y and appa en s i ness o he
de ec (see Fig. 5B, C). Compa ed o o he bone egene -
a ion p ocesses in he same animal model [9,30,31], he
na u al s i ness o he implan con ibu ed o as e
mechanical go e nance o he in e nal o ce dis ibu ion.
Fo ins ance, he ex e nally s abilized 15 mm bone
Fig. 5 A–D: The e olu ion o he analyzed mechanical and imaging
pa ame e s o e he egene a ion ime, including A he g ound ec ion
o ce (GRF) as a pe cen age o heal hy alues (do ed lines); he B
ixa ion (F ) and callus o ce (Fc); C he bone callus s i ness (KCÞ;
and D he bone callus o al olume (TV), de ec c oss-sec ional a ea
(CSA), and bone callus mine al densi y (BMD), as a pe cen age o he
heal hy alues (do ed line)
900 Tissue Eng Regen Med (2023) 20(6):893–904
123
anspo calluses o Mo a-Macı
´as e al. [9] needed an
a e age o 10 addi ional days o o e each he ixa o ’s
loading (F Fc) in he same bone model. Mo eo e , while
bone anspo de ec s eco e ed comple ely hei capaci y
o sus aining he me a a sal o ce (Fc*80–90% o he Fi)
a e 70 days om su ge y, TE achie es i in jus 50 days.
A simila healing ad ance is ound when compa ing wi h a
15 mm leng hening callus Bla
´zquez-Ca mona e al. [30]
using he same ixa ion employed in he cu en s udy. Fo
i s pa , G asa e al. [31] quan i ied a s abiliza ion o he
o ces h ough hei unila e al ixa o in 30 consolida ion
days. Ne e heless, hey mechanically in es iga ed he
healing o a sho e and mo e ascula ized 2 mm ibial
ac u e. Rega ding he appa en callus s i ness KC,TE
callus also p esen ed a mo e exponen ial beha io han he
dis ac ion al e na i es by eaching s i ness quo es o e 15
kN/mm in less han 70 days a e su ge y. In he same
pe iod, he dis ac ion calluses o Bla
´zquez-Ca mona e al.
[30] and Mo a-Macı
´as e al. [4,45] did no e en each hal
o ha s i ness. Howe e , he calib a ion limi s o he
measu emen equipmen p e en ed us om explo ing i he
inclusion o he ce amic sca old al e s he p edic able s a-
biliza ion a ound he s i ness o an in ac me a a sus in he
sho e m [46].
In he medium e m (3–7 mon hs a e su ge y), once
he naı
¨ e b idging callus ended mine alizing (Fig. 6, days
120 and 210), GRF e u ned o heal hy condi ions a e he
undeniable su gical e ec (see Fig. 5A). This eco e y was
epo ed o be sho e in o he bone egene a ion p ocesses.
Fo ins ance, Mo a-Macı
´as e al. [33] quan i ied app oxi-
ma ely heal hy GRF alues a e abou 3–4 mon hs a e
su ge y owing o a less p onounced lameness in an
equi alen me a a sal de ec ea ed by bone anspo : a
loss be ween 0 o 10% o he GRF in he dis ac ion g oup
s. he 60% in TE. An in e media e ini ial lameness was
ob ained in he o ine ac u e healing s udy o Seebeck
e al. [47]. They quan i ied he GRF in 70% o he con ol
alue a e dis ac ing a 3 mm ibial gap. The signi ican
TE lameness could be associa ed wi h a mo e p olonged
lack o bone con inui y, especially compa ed o ac u e
healing app oaches. In bone anspo , once dis ac ed, he
hema oma o med du ing he la ency phase ensu es an
ea ly connec i e empla e be ween he dis al and p oximal
agmen s [8,48]. Consequen ly, TE animals could
ins inc i ely de elop gai compensa ion mechanisms by
elying on non-ope a ed limbs.
The p og essi e eco e y o he gai pa ame e seemed
o shea simila upwa d beha io o ha expe ienced by
BMD, being o e 80% o he co ical mine al densi y a e
six mon hs (see Fig. 5D). In he li e a u e, Poblo h e al.
[38] measu ed he same pa ame e in hei 40 mm o ine
ibia gaps. A e 12 weeks, hey ound a mine al deg ee o
Fig. 6 X- ay ollow-up o he ope a ed me a a si a di e en ime-poin s o he consolida ion phase, days 0, 60, 120, 210, 365 days a e su ge y.
Fi s ow co esponds o he do soplan a iew and he second ow o he la e al iew o he imaging assessmen in he same sheep
Tissue Eng Regen Med (2023) 20(6):893–904 901
123
