Cranioplasty with Adipose-Derived Stem Cells, Beta-Tricalcium Phosphate Granules and Supporting Mesh: Six-Year Clinical Follow-Up Results

C anioplas y wi h Adipose-De i ed S em Cells, Be a- T icalcium Phospha e G anules and Suppo ing Mesh: Six-Yea Clinical Follow-Up Resul s TUOMO THESLEFF , a KAI LEHTIMA ¨KI, a TERO NISKAKANGAS, a SANNA HUOVINEN, b BETTINA MANNERSTRO ¨M, c SUSANNA MIETTINEN, d RIITTA SEPPA ¨NEN-KAIJANSINKKO, c JUHA € OHMAN a Key Wo ds. Adipose s em cells •Tissue enginee ing •Bioma e ials •Be a- icalcium phospha e • C anial epai •Ou come ABSTRACT Se e al al e na i e echniques exis o econs uc skull de ec s. The complica ion a e o he c a- nioplas y p ocedu e is high and he sea ch o op imal ma e ials and echniques con inues. To epo long- e m esul s o pa ien s who ha e ecei ed a c anioplas y using au ologous adipose- de i ed s em cells (ASCs) seeded on be a- icalcium phospha e (be aTCP) g anules. Be ween 10/ 2008 and 3/2010, fi e c anioplas ies we e pe o med ( ou emales, one male; a e age age 62.0 yea s) using ASCs, be aTCP g anules and i anium o eso bable meshes. The a e age de ec size was 8.1 36.7 cm 2 . Pa ien s we e ollowed bo h clinically and adiologically. The ini ial esul s we e p omising, wi h no se ious complica ions. Ne e heless, in he long- e m ollow-up, h ee o he fi e pa ien s we e e-ope a ed due o g a ela ed p oblems. Two pa ien s showed ma ked eso p- ion o he g a , which led o e ision su ge y. One pa ien de eloped a la e in ec ion (7.3 yea s pos -ope a i e) ha equi ed e ision su ge y and emo al o he g a . One pa ien had a success- ully ossified g a , bu was e-ope a ed due o ecu ence o he meningioma 2.2 yea s pos - ope a i ely. One pa ien had an une en ul clinical ollow-up, and he cosme ic esul is sa is ac o y, e en hough skull x- ays show hypodensi y in he bo de s o he g a . Albei no se ious ad e se e en s occu ed, he 6-yea ollow-up esul s o he fi e cases a e unsa is ac o y. The clinical esul s a e no supe io o esul s achie ed by con en ional c anial epai me hods. The use o s em cells in combina ion wi h be aTCP g anules and suppo ing meshes in c anial de ec econs uc ion need o be s udied u he be o e con inuing wi h clinical ials. STEM CELLS TRANSLATIONAL MEDICINE 2017;6:1576–1582 SIGNIFICANCE STATEMENT In his a icle we epo long- e m esul s o i e pa ien s who ecei ed a c anioplas y using au ologous adipose-de i ed s em cells seeded on be a- icalcium phospha e g anules. The ini ial esul s we e p omising, wi h no se ious complica ions. Ne e heless, he 6-yea ollow-up esul s o he i e cases a e unsa is ac o y. The use o his s a egy o c anial de ec econs uc- ion mus be s udied u he be o e con inuing wi h clinical ials and be o e applying he me hod in clinical p ac ice. INTRODUCTION Skull es o a ion emains a challenge o neu osu - geons and plas ic su geons [1]. The numbe o pa ien s in need o c anioplas y is inc easing because o con inuous imp o emen s in neu osu - gical c i ical ca e and a g owing numbe o decom- p essi e c aniec omies pe o med [1]. Common complica ions in c anial epai su ge y include o example in ec ion, wound dehiscence, in ac anial hemo hage, eso p ion, and/o disloca ion o he g a [2–5]. The epo ed complica ion a e o c a- nioplas ies oday is 16%–40%, wi h a gene al eop- e a ion a e o 25% [6–10].Pa ien s’au ologous emo ed and s o ed bone has been conside ed as he gold s anda d in c anial aul econs uc ion, bu he e a e s udies epo ing supe io esul s achie ed wi h syn he ic ma e ials such as hyd oxy- apa i e, bioac i e fibe - ein o ced composi e, poly- me hylme hac yla e, o i anium [9, 11, 12]. Ou aim was o o e come common p oblems associa ed wi h con en ional c anioplas ies by c ea ing bone om pa ien s’ own adipose-de i ed s em cells (ASCs), be a- icalcium phospha e (be aTCP) g anules and suppo ing meshes [13, 14]. In ecen yea s, ASCs ha e been unde ex en- si e s udy in egene a i e medicine [15–18]. They a e easily e ie ed in high numbe s om a Au ho ed by a membe o a Depa men o Neu oscience and Rehabili a ion, Tampe e Uni e si y Hospi al, Tampe e, Finland; b Depa men o Pa hology, Fimlab Labo a o ies, Tampe e Uni e si y Hospi al, Finland; c Depa men o O al and Maxillo acial Diseases, Uni e si y o Helsinki and Helsinki Uni e si y Hospi al, Finland; d Adul S em Cells, BioMediTech, Facul y o Medicine and Li e Sciences, Uni e si y o Tampe e, Finland Science Cen e , Tampe e Uni e si y Hospi al, Finland Co espondence: Tuomo Thesle , M.D., Depa men o Neu oscience and Rehabili a ion, Tampe e Uni e si y Hospi al, P.O. Box 2000, FI-33521 Tampe e, Finland. Telephone: 1358-3- 311-63023; e-mail: uomo. [email p o ec ed] Recei ed Sep embe 17, 2016; accep ed o publica ion Ma ch 27, 2017; fi s published May 15, 2017. O cAlphaMed P ess 1066-5099/2017/$30.00/0 h p://dx.doi.o g/ 10.1002/sc m.16-0410 This is an open access a icle unde he e ms o he C ea i e Commons A ibu ion- NonComme cial-NoDe i s License, which pe mi s use and dis ibu ion in any medium, p o ided he o iginal wo k is p ope ly ci ed, he use is non- comme cial and no modifica ions o adap a ions a e made. STEM CELLS TRANSLATIONAL MEDICINE 2017;6:1576–1582 www.S emCellsTM.com O c2017 The Au ho s STEM CELLS TRANSLATIONAL MEDICINE published by Wiley Pe iodicals, Inc. on behal o AlphaMed P ess H UMAN C LINICAL A RTICLE issue and expanded in i o, which makes hem an a ac i e ool in issue econs uc ion [14, 19, 20]. Howe e , scien ific e idence in e ms o sa e y and e ficacy o ASC use in egene a i e medi- cine is s ill limi ed [14, 20–24]. We pe o med fi e c anioplas ic su ge ies using ASCs seeded in be aTCP g anules suppo ed by eso bable o i anium meshes o pa ien s who had mode a e-sized c anial de ec s o a ying e i- ologies. The g a s seemed o be success ul in sho - e m ollow- up, bo h clinically and adiologically, bu al eady he mid- e m esul s showed inc eased g a eso p ion [13, 14]. In he cu en s udy, ou objec i e is o epo he long- e m esul s (6.1–7.4 yea s) o his me hod. MATERIALS AND METHODS Fi e pa ien s, ou emales and one male, a e age age 62.0 yea s ( ange, 54–75 yea s), unde wen c anioplas y using his no el echnique be ween 10/2008 and 3/2010. The a e age de ec size was 8.1 36.7 cm 2 ( ange, 6.5 33.7 – 9.0 39.3; Table 1). P i- ma y diagnoses and indica ions o c anioplas ies a e p esen ed in Table 1. Adipose issue ha es s om subcu aneous abdominal a (app ox. 100 ml) we e pe o med app oxima ely 3 weeks p io o he c anioplas y p ocedu e. The a oge he wi h 60 ml o au olo- gous se um was anspo ed o Regea Ins i u e o egene a i e medicine, Uni e si y o Tampe e o s em-cell isola ion and expan- sion. None o he c anioplas y cons uc s was supplemen ed wi h hBMP-2. ASC Isola ion and P epa a ion o T ansplan a ion ASCs we e isola ed and expanded in i o in clean ooms acco d- ing o s anda d ope a ing p ocedu es and ollowing Good Manu- ac u ing P ac ice guidelines a Regea. De ails o he isola ion and expansion p ocedu es wi h mino modifica ions a e desc ibed in a p e ious publica ion [20]. Recombinan collagenase NB 6 (In i o- gen, Paisley, Sco land, Uni ed Kingdom; Good Manu ac u ing P ac- ice g ade; SERVA Elec opho esis GmbH, Heidelbe g, Ge many) was u ilized o mince and diges he adipose issue. The isola ed ASCs we e expanded o app oxima ely 22 days in basal media con aining Dulbecco’s modified Eagle medium/F-12 (Gibco In i o- gen, Paisley, Sco land, Uni ed Kingdom) wi h 15% o au ologous Table 1. Cha ac e is ics o pa ien s and p ocedu es Pa ien Pa ien 1 Pa ien 2 Pa ien 3 Pa ien 4 Pa ien 5 Sex F M F F F Age a p ima y c anioplas y 60 59 62 75 54 Diagnosis Skull hemangioma F on al meningioma Acu e SDH C anial base meningioma Skull meningioma Indica ion o c anioplas y P ima y ope a ion Loosening o he ac yl g a Bone flap in ec ion Bone flap in ec ion P ima y ope a ion De ec size (leng h 3 heigh , cm) 6.9 36.4 9.0 37.5 8.9 36.8 6.5 33.7 9.0 39.3 Inne mesh No No Reso bable Reso bable Ti anium Ou e mesh Ti anium Reso bable Reso bable Reso bable Reso bable To al numbe ASCs implan ed (ASCs pe ml) 4,140,000 (37,637) 8,712,000 (108,900) 3,982,500 (49,781) 7,821,000 (391,050) 2,812,500 (28,125) Indica ion o e- ope a ion La e in ec ion Pa ial eso p ion and loosening o he g a — To al eso p ion Recu ence o he meningioma in he du a Time be ween c anio- plas y and e-ope a ion (yea s) 7.3 0.9 — 2.0 2.2 Re-ope a ion echnique Re ision, emo al o he g a Ti anium mesh applied on he de ec — Ti anium c anioplas y Re-c anio omy and excision o he du al meningioma His ological analysis o he g a Os eonec osis and acu e o subacu e os eomyeli is Pa ly nec o ic isle s con aining loose collagen and poo ly main ained os eoblas s. Focally some well- o med bone abeculae con aining i al os eocy es —— — No ice F on al sinus opened in he p ima y ope a ion and filled wi h bone cemen Remaining g a was ha d and fixa ed wi h sc ews in o a i a- nium mesh. Reso p ion o he g a in he skull x- ay Abdominal hema oma a he adipose issue ha es si e G a was success ully ossified and had g own in o he su ounding bone Clinical ollow-up (yea s) 7.4 6.8 6.2 6.5 6.1 Abb e ia ions: —, no da a; ASC, adipose-de i ed s em cell. Thesle , Leh im€ aki, Niskakangas e al. 1577 www.S emCellsTM.com O c2017 The Au ho s STEM CELLS TRANSLATIONAL MEDICINE published by Wiley Pe iodicals, Inc. on behal o AlphaMed P ess se um, wi hou an ibio ics. Subsequen ly he cells we e passaged on confluence and de ached mechanically wi h a cell sc ape and p epa ed o cell ansplan a ion. To allow cell a achmen , app ox- ima ely 15 310 6 cells o passages 3 o 4 we e combined wi h 60 ml o be aTCP g anules (Ch onos1, po osi y 60%, g anule size 1.4–2.8 mm; Syn hes, Obe do , Swi ze land) 48 hou s be o e he ope a ion.The cells we e es ed nega i e o Mycoplasma con am- ina ion as de e mined by a Mycoplasma PCR ki (Veno Gem; Mine a Biolabs GmbH, Be lin, Ge many) and cell s e ili y and endo oxins we e es ed by Bio ian L d. (Tu ku, Finland) acco ding o me hods desc ibed in he Eu opean Pha macopoeia (Council o Eu ope, S asbou g, F ance) [25]. In Vi o Analyses and Li e/Dead S aining Fo he in i o analyses, ASCs om all pa ien s we e expanded in basal media, excep in he os eogenic di e en ia ion capaci y assessmen , in which comme cially a ailable human se um (Bio- Whi ake ; Lonza, Walke s ille, Ma yland), was used because o he limi ed amoun o au ologous se um. Cell a achmen o he be aTCP g anules and he cell iabili y we e s udied using Li e/Dead s aining be o e he ope a ion. B iefly, he cell-bioma e ial combina ion was incuba ed wi h a mix u e o CellT acke g een (5-chlo ome hylfluo escein diace a e) and e hidium homodime (Molecula P obes, Eugene, O egon). The iable cells (g een fluo escence) and dead cells ( ed fluo es- cence) we e de ec ed wi h a fluo escence mic oscope. Flow Cy ome ic Su ace Ma ke Exp ession Analysis ASCs we e u he expanded in i o and we e analyzed a passages 4 o 5 by flow cy ome y (FACSA ia; BD Biosciences, E embodegem, Belgium). Monoclonal an ibodies agains CD9-PE, CD10-PECy7, CD13-PE, CD14-PECy, CD19-PECy7, CD29-APC, CD49d-PE, CD73-PE, CD90-APC, CD106-PE-Cy5, CD146-PE, and CD166-PE (BD Bioscien- ces); CD45-FITC (Mil enyi Bio ech, Be gisch Gladbach, Ge many); CD31-FITC, CD34-APC, CD44-FITC, HLA-ABC-PE, and HLA-DR-PE (Immuno ools GmbH, F iesoy he, Ge many); and CD105-PE (R&D Sys ems Inc., Minneapolis, Minneso a) we e used. Analysis was pe o med on 10,000 cells pe sample. The posi i e exp ession was defined as he le el o fluo escence g ea e han 99% o he co e- sponding uns ained cell sample. Os eogenic Di e en ia ion Capaci y Assessmen Fo he in i o os eogenic di e en ia ion analyses, su plus cells we e main ained in os eogenic media con aining basal media supplemen ed wi h 15% human se um, 50 mML-asco bic acid 2-phospha e (Sigma-Ald ich, S . Louis, Missou i), 10 mM be a- glyce ophospha e (Sigma-Ald ich), and 100 nM dexame hasone (Sigma-Ald ich). Con ol cell cul u es we e main ained in basal media supplemen ed wi h human se um. The cells we e seeded a a densi y o 2.5 310 3 cells/cm 2 and cul u es we e subse- quen ly analyzed by alkaline phospha ase s aining a day 14. C anioplas y P ocedu e All pa ien s ecei ed ou ine p eope a i e ce u oxime an ibio ic 1.5 g in a enously (Zinace , GlaxoSmi hKline, Uni ed Kingdom). A fi s he du a was exposed in all cases. In Pa ien 1, a hemangioma was emo ed wi h he ou e and inne diploe o bone, and he opened on al sinus was c anialized and filled wi h bone cemen (NORIAN Rein o ced Fas Se Pu y, Syn hes, Obe do , Swi ze - land, h p://www.syn hes.com). In Pa ien 2, a loosened ac yl g a was emo ed. In Pa ien s 3 and 4, he skin was ca e ully dis- sec ed loose om he du a. In Pa ien 5, a bone meningioma was esec ed. Nex , ASCs combined wi h be aTCP g anules we e applied in he de ec (Fig. 1). The numbe s o implan ed ASCs a e p esen ed in Table 1. To keep he be aTCP g anules in place, eso bable cus om-molded mesh (PLGA, 85:15 poly (L-lac ide-co- glycolide)) (RapidSo b, Syn hes, Obe do , Swi ze land, h p:// www.syn hes.com) o i anium mesh was used ei he in single o bilamina e ashion (Table 1). Meshes we e secu ed wi h ei he i anium o eso bable sc ews. Follow-Up Pa ien s we e ollowed a 1 week, 1, 6, and 12 mon hs a e su ge y and annually he ea e . Du ing he fi s yea , Figu e 1. In aope a i e pho og aph o a meningioma esec ion si e filled wi h au ologous adipose s em cell-seeded be a- icalcium phos- pha e (b-TCP) g anules (Pa ien 5). 1578 C anioplas y wi h Adipose-De i ed S em Cells O c2017 The Au ho s STEM CELLS TRANSLATIONAL MEDICINE published by Wiley Pe iodicals, Inc. on behal o AlphaMed P ess STEM CELLS TRANSLATIONAL MEDICINE Houndsfield uni s (HU) we e measu ed om CT s udies o assess bone densi y. A e he fi s yea , imaging s udies we e done based on clinical conside a ion, and HU measu emen was no sys ema ically con inued. His ological samples o he g a we e ob ained om wo e-ope a ed pa ien s (Pa ien s 1 and 2). The o malin-fixed, pa a fin-embedded sample sec ions we e s ained wi h hema oxylin and eosin and pe iodic acid- Schi , and his ologically e alua ed. E hical Conside a ions The s udy was app o ed by he boa d o he local hospi al dis ic , and Finnish Medicines Agency (FIMEA) was in o med abou he p ocedu es. Be o e su ge y, he pa ien s we e in o med abou he p ocedu es, which hey app o ed and o which hey ga e hei w i en consen . RESULTS In Vi o Resul s The iabili y and adhe ence o he ASCs ansplan ed wi h he be aTCP g anules o he pa ien s was confi med by Li e/Dead s aining. The flow cy ome y showed ha he cells we e homogenous in mo phology by side-sca e and o wa d-sca e pa ame e s. Howe e he immunopheno ype showed a iabili y be ween pa ien s. Ne e heless, s ong posi i e exp ession (>90%) was seen in su ace ma ke s CD10, CD13, Cd29, CD44, CD49d, CD73,CD90, CD105, CD106, and CD166. Mode a e posi i e exp ession (>10%, <90%) was seen o ma ke s CD 9, CD106 and CD146. No exp ession, o low exp ession (<10%) was seen o ma ke s CD31, CD34, CD45, and majo his ocompa ibili y complex class I iso ype human leukocy e an igen DR, sugges ing low de ec- ion o cells o hema opoie ic and angiogenic lineages. Mo eo e , CD14 and CD 19 (hema opoie ic ma ke s) showed g ea a iabili y in su ace ma ke exp ession, anging om low o mode a e. Acco ding o alkaline phospha ase s aining, all pa ien s’ cell samples possessed he capaci y o di e en ia e owa d he os eo- genic lineages. Clinical Resul s Du ing he ollow-up pe iod (a e age 6.6 yea s, ange 6.1–7.4), all pa ien s excep one we e e-ope a ed. We obse ed signs o eso p ion o he g a in h ee cases ou o fi e (Table 1). One g a (Pa ien 1) which was su gically explo ed due o la e in ec ion was ela i ely so and was suppo ed o a la ge deg ee by he i anium mesh. Mic obiological cul u es o he in ec ed g a we e nega i e and he in ec ion was success ully ea ed by su gical emo al o he g a combined wi h ce u oxime and le ofloxacin (Ta anic, Sanofi, Uni ed Kingdom) an ibio ics. Two pa ien s had clinical loosening o he g a (Pa ien s 2 and 4). The g a in one pa ien showed adiological eso p ion a he bo de s bu he clin- ical esul was good (Pa ien 3; Fig. 2A, 2B). One pa ien was e- ope a ed due o ecu ence o he meningioma in he du a ma e unde lying he g a (Pa ien 5), bu he g a as such was success- ul (Table 1). His ological Resul s A his ological sample o he g a was ob ained om wo e- ope a ed pa ien s. The findings o he g a om Pa ien 1 we e consis en wi h os eonec osis and acu e o subacu e Figu e 2. (A): Re o ma ed CT image o he head 13 mon hs pos - ope a i ely (Pa ien 3). Pa ial eso p ion o he g a is seen a he basal a ea. (B): Skull x- ay 6 yea s pos -ope a i ely (Pa ien 3) shows subs an ial eso p ion o he g a a he bo de s. Figu e 3. Low-powe his ological appea ance o he biopsy speci- men om he skull lesion o pa ien 1 shows hypocellula degene - a ed collagenous ma e ial and loose fib inous issue. Some o he sphe ical nec o ic issue islands a e pa ially mine alized. Fungus hyphae a e p esen in he fib ous issue (a ow). The e is an inflam- ma o y polymo phonuclea leukocy e infil a e a he op o he pho omic og aph. Hema oxylin and eosin (H&E) s aining. O iginal magnifica ion 340. Scale ba 5100 lm. Thesle , Leh im€ aki, Niskakangas e al. 1579 www.S emCellsTM.com O c2017 The Au ho s STEM CELLS TRANSLATIONAL MEDICINE published by Wiley Pe iodicals, Inc. on behal o AlphaMed P ess os eomyeli is. Non iable degene a ed issue wi h polymo phonu- clea inflamma o y infil a es and o ganizing g anula ion issue as well as adia ing sep a e hyphae cha ac e is ic o Aspe gillus in ec- ion we e seen (Fig. 3). The g a om Pa ien 2 showed mainly degene a ed os eoid-like issue isle s con aining loose collagen and poo ly main ained os eoblas s. Howe e , ocally he e we e some well- o med bone abeculae con aining i al os eocy es (Fig. 4). The e we e no ea u es sugges i e o neoplas ic change. DISCUSSION Bone issue enginee ing echniques wi h o wi hou ASCs ha e been s udied ac i ely du ing pas decades [26–28]. ASCs a e mul i- po en cells ha a e ecognized as po en ially beneficial in wide a ie y o medical he apies in econs uc i e su ge y [27–30]. The po en ial o hese cells has e oked g ea en husiasm and high expec a ions in bo h scien ific and comme cial ci cles. Howe e , in gene al clinical applica ions o s em cells in econs uc i e su ge y ha e been limi ed o small case se ies wi h a ying esul s and/o sho ollow-up pe iods [14, 31–33]. Among he published s udies, he e is a g ea a iabili y in, o example, s em cell sou ces, de ec si es and sizes, indica ions, cell ca ie s, and use o g ow h ac o s ha makes i di ficul o d aw a solid conclusion on s em cell e ec- i eness in bone issue enginee ing [26]. Addi ionally, he e has been conce n abou he sa e y and e ficacy o s em cells in clinical applica ions [21, 24, 33, 34]. This long- e m ollow-up epo on fi e pa ien s ecei ing c a- nioplas y using ASCs, be aTCP g anules and suppo ing meshes sugges s ha he me hod as such is sa e. The sho - e m esul s (less han a yea ) we e p omising and he bone densi y o he g a s (measu ed by HUs) ended o inc ease [13]. Howe e , in long ollow-up, he ou come o he c anioplas ies was unsa is ac- o y in mos cases pa ially due o poo ossifica ion and pa ially due o in ec ion o umo ecu ence. Two o he fi e pa ien s showed ma ked clinical and adiologi- cal eso p ion o he g a ha led o e-ope a ion (Pa ien s 2 and 4). Ne e heless, his ology o he e-fixa ed g a in Pa ien 2 showed ocally well- o med bone abeculae con aining i al os eocy es. One pa ien had adiological g a eso p ion, bu he g a was fi m on palpa ion, and no e-ope a ion was needed in 7 yea s ollow-up (Pa ien 3; Fig. 2A, 2B). One pa ien had a la e in ec ion o he g a (>7 yea s pos -ope a i ely) ha is no likely o be ela ed o he c anioplas y me hod (Pa ien 1). The la e in ec ion was mos likely due o unsuccess ul c anializa ion o he on al sinus. He on al sinus was opened in he p ima y ope a- ion and filled wi h a ificial ma e ial, which may ha e been he cause o he in ec ion. One pa ien (Pa ien 5), who had ecu - ence o he meningioma 2.2 yea s a e he p ima y ope a ion, was ound o ha e a clinically ossified g a ha had a ached well o de ec ma gins and p e en ed un as ening o he g a in he e-ope a ion. This pa ien had a i anium inne mesh, which p ob- ably played a ole in he success o he case. The e a e se e al ques ions ha mus be sol ed be o e clinical ials ega ding he e ec i eness o his s a egy a e con inued. Fi s , how could ossifica ion be induced and enhanced, and how can he bone o ming capaci y o he ASCs be s imula ed in his se up? ASCs hemsel es may s imula e bone o ma ion no only by di e en ia ing in o bone cells bu also by sec e ing pa ac ine ac o s ha enhance ossifica ion and ascula iza ion. We decided o use ASCs mainly because o hei g ea accessibili y and low pa ien mo bidi y when compa ed wi h o he mesenchymal cell sou ces such as bone ma ow o pe ios eum. The in i o, in i o and clinical bone- o ming capaci y o ASCs in combina ion wi h a ious sca old ma e ials ha e been epo ed by many au ho s [14, 20, 35–41]. In his se ies, he flow cy ome ic cha ac e iza ion o he ASCs expanded in au ologous se um co esponded o p e i- ously published esul s o ASCs [42, 43]. Acco ding o flow cy om- e y, and alkaline phospha ase s aining esul s, he cells we e o mesenchymal o igin and had a capaci y o di e en ia e in o he os eoblas ic lineage. The e was indi idual a iabili y in he num- be s o implan ed cells anging om 28,125 cells/ml o 391,050 cells/ml (a e age 123,098 cells/ml). In he u u e, i is i al o find he op imal doses o ASCs and bioma e ial g anules o achie e e ec i e ossifica ion. Also, he ole o g ow h ac o s such as hBMP-2 in econs uc i e su ge y is inde e mina e and sa e y conce ns ha e eme ged in clinical applica ions [44]. In his se ies g ow h ac o s we e no used. The e ec i eness o he cons uc migh be u he inc eased by soaking i in enous blood p io o implan a ion [45, 46]. Mo eo e , an in ac pe ios eum would be beneficial o bone o ma ion bu i s ole in his se up emains unclea [47]. The pu pose o he sca old in econs uc i e issue enginee - ing is o p o ide mechanical suppo o cell p oli e a ion, mig a- ion, and ac i i y by mimicking ex a cellula ma ix [48, 49]. Ideally, he sca old p o ides a empla e o bone o ma ion, and calcium phospha e based ma e ials a e conside ed ideal in his ega d. So a , dozens o calcium phospha e ma e ials including be aTCP ha e been in es iga ed bo h in i o and in i o [50–52]. We used g anula be aTCP pa ly because o i s bio eso babili y and la ge su ace a ea compa ed o solid sca olds. G anules also molded well o he de ec s. Mo eo e , he e is p io e idence o he use o be aTCP in c aniomaxillo acial su ge y [20, 53, 54]. The ailu e o he g a ossifica ion o Pa ien s 2 and 4 may be due o oo apid eso p iono be aTCPg anuleswhichin u nmaybe ela ed o he use o eso bable and less igid suppo ing meshes in hei cases. In u u e s udies, i is pi o al o find an op imal sca - old ma e ial o suppo ossifica ion in c anial a ea [55, 56]. The suppo ing mesh (mono/bilamina e) plays p obably a pi - o al ole in he success o he g a in his se up. The main Figu e 4. The biopsy specimen om pa ien 2 shows mainly sphe i- cal islands o degene a ed collagenous os eoid-like issue whe e smudgy appea ing nuclei a e isible. Howe e , wo well- o med a- beculae o wo en bone con aining iable os eocy es a e seen (a ows). Hema oxylin and eosin (H&E) s aining. O iginal magnifica ion 3100. Scale ba 5100 lm. 1580 C anioplas y wi h Adipose-De i ed S em Cells O c2017 The Au ho s STEM CELLS TRANSLATIONAL MEDICINE published by Wiley Pe iodicals, Inc. on behal o AlphaMed P ess STEM CELLS TRANSLATIONAL MEDICINE ad an age o a eso bable mesh, such as RapidSo b is ha he e will be no a ificial ma e ial le in he g a si e in he long un. Howe e , eso bable mesh may lose i s s eng h a he apidly; o example RapidSo b has 85% s eng h le a e 8 weeks, 40% a 15 weeks, and <10% a e 20 weeks [57], which may esul in inc eased mic omo ion, which po en ially p ohibi s sound bone b idging due o con inuous dis up ion o he agile ini ial bone s u s and ea ly essel o ma ion. On he o he hand, apidly eso bing PLGA cons uc s may accumula e acidi y due o hyd oly- sis o he mesh which in u n may cause hyd olysis o he newly o med mine al deposi s and s ong ac i a ion o os eoclas s. Based on ou cu en expe ience, we belie e ha he use o a igid mesh such as i anium is necessa y o damping du al pulsa ions o o e op imal ci cums ances o bone o ma ion when g anula sca old ma e ial is used. On he o he hand, i anium and o he ma e ials may p o ide su aces suppo ing he mig a ion o os eo- blas s [58, 59]. In ou se ies, he i anium-con aining cons uc s (Pa ien s 1 and 5) had good in e media e ollow-up esul s and he obse ed os eolysis was due o non- econs uc ion ela ed causes (Pa ien 1) and ecu en meningioma (Pa ien 5). In pa ien 2, a single laye o eso bable mesh may ha e p o ided insu ficien s a- bili y o a oid mic omo ion su ficien ly, hence ailu e o app op i- a e in eg a ion o g a in ime which led o eso p ion and loosening o he g a . Bo h Pa ien s 3 and 4 had a double laye o eso bable mesh. The disc epancy o he esul s be ween Pa ien 3 ( eso p ion only a he bo de s) and Pa ien 4 (comple e eso p ion o he g a ) may be due o ma kedly highe age o he Pa ien 4, and lowe unc ionali y o he ASCs. Howe e , he limi ed numbe o pa ien s and high numbe o con ounding ac o s makes i impossible o d aw solid conclusions in ega d o op imal mesh ma e ial o echnique. The c i ical size o he de ec o his me hod emains inde e - mina e. La ge size p obably hinde s blood supply o cen al pa s o he g a , which may be c ucial o bone o ma ion. The de ec size in ou se ies was ela i ely la ge (a e age 8.1 36.7 cm 2 ); howe e , i was conside ably smalle han he a e age de ec size ollowing decomp essi e hemic aniec omy, which is a common neu osu gical p ocedu e leading o cal a ial de ec s [60, 61]. Ob i- ously, he e a e se e al addi ional issues o conside be o e he me hod is aken in o clinical use. CONCLUSION Con en ional c anioplas ies ha e high complica ion a es, which encou age sea ching o no el echniques. We pe o med fi e c a- nioplas ies using ASCs, be aTCP g anules and i anium o eso b- able mesh. The 6-yea esul s o mos o hese cases a e unsa is ac o y. The e we e no clea indica ions ha his p ocedu e s imula ed ossifica ion o ha ACSs o hei p ogeny p oduced new bone. Ne e heless, he e we e no se ious ad e se e en s, and all pa ien s eco e ed well om he su ge ies. The use o his s a egy o c anial de ec econs uc ion mus be s udied u he be o e con inuing wi h clinical ials and be o e applying he me hod in clinical p ac ice. AUTHOR CONTRIBUTIONS T.T.: Concep ion and design, Collec ion and/o assembly o da a, P o ision o s udy ma e ial, Da a analysis and in e p e a ion, Manusc ip w i ing, Final app o al o manusc ip ; K.L., T.N.: Con- cep ion and design, P o ision o s udy ma e ial, Da a analysis and in e p e a ion, Manusc ip w i ing, Final app o al o manusc ip ; S.H., B.M., S.M., R.S.-K., J.O.: P o ision o s udy ma e ial, Da a analysis and in e p e a ion, Manusc ip w i ing, Final app o al o manusc ip . DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST The au ho s indica ed no po en ial conflic s o in e es . REFERENCES 1Fe oze AH, Walmsley GG, Choudh i O e al. E olu ion o c anioplas y echniques in neu osu ge y: his o ical e iew, pedia ic con- side a ions, and cu en ends. J Neu osu g 2015;123:1098–1107. 2Klinge DR, Madden C, Beshay J e al. Au ologous and ac ylic c anioplas y: a e iew o 10 yea s and 258 cases. Wo ld Neu osu g 2014;82:e525–e530. 3Lee CH, Chung YS, Lee SH e al. 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