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Transplantation of Autologous Bone Marrow Mesenchymal Stem Cells with Platelet-Rich Plasma Accelerate Distraction Osteogenesis in a Canine Model

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Transplantation of Autologous Bone Marrow Mesenchymal Stem Cells with Platelet-Rich Plasma Accelerate Distraction Osteogenesis in a Canine Model Original Article Transplantation of Autologous Bone Marrow Mesenchymal Stem Cells with Platelet-Rich Plasma Accelerate Distraction Osteogenesis in A Canine Model 0RKDPPDG0HKGL'HKJKDQ'903K'10RKDPDGUH]D%DJKDEDQ(VODPLQHMDG3K'2 1DGHU0RWDOOHEL]DGHK0'3-DYDG$VKUD¿+DODQ'903K'4/HLOD7DJL\DU06F2 6DUDQJ6RURRUL'903K'1$JELEL1LNPDK]DU06F20LUVHSHKU3HGUDP '903K'1$EGROKRVVHLQ6KDKYHUGL3K'2+RVVHLQ.D]HPL 0HKUMHUGL'903K'56DGUD,]DGL'901 'HSDUWPHQWRI&OLQLFDO6FLHQFHV)DFXOW\RI9HWHULQDU\0HGLFLQH8QLYHUVLW\ of Tehran7HKUDQ,UDQ 'HSDUWPHQWRI6WHP&HOOVDQG'HYHORSPHQWDO%LRORJ\DW&HOO6FLHQFHV5HVHDUFK&HQWHU5R\DQ,QVWLWXWHIRU 6WHP&HOO%LRORJ\DQG7HFKQRORJ\$&(&57HKUDQ,UDQ /LPE/HQJWKHQLQJRI,UDQ7HKUDQ,UDQ 'HSDUWPHQWRI&OLQLFDO6FLHQFHV)DFXOW\RI9HWHULQDU\0HGLFLQH7DEUL]8QLYHUVLW\ 7DEUL],UDQ 'HSDUWPHQWRI&OLQLFDO6FLHQFHV)DFXOW\RI9HWHULQDU\0HGLFLQH)HUGRZVL8QLYHUVLW\RI0DVKKDG 0DVKKDG,UDQ *Corresponding Address: P.O.Box: 16635-148, Department of Stem Cells and Developmental Biology at Cell Sciences Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran Email: [email protected] 5HFHLYHG'HF$FFHSWHG$SU Abstract 2EMHFWLYHDistraction osteogenesis (DO) is a surgical procedure used to generate large volumes of new bone for limb lengthening. 0DWHULDOVDQG0HWKRGV In this animal experimental study, a 30% lengthening of the left tibia (mean distraction distance: 60.8 mm) was performed in ten adult male dogs by callus GLVWUDFWLRQDIWHURVWHRWRP\DQGDSSOLFDWLRQRIDQ,OL]DURY¿[DWRU'LVWUDFWLRQZDVVWDUWHGRQ postoperative day seven with a distraction rate of 0.5 mm twice per day and carried out at a rate of 1.5 mm per day until the end of the study. Autologous bone marrow mesenchy- mal stem cells (BM-MSCs) and platelet-rich plasma (PRP) as the treatment group (n=5) or PRP alone (control group, n=5) were injected into the distracted callus at the middle and end of the distraction period. At the end of the consolidation period, the dogs were VDFUL¿FHGDIWHUZKLFKFRPSXWHUL]HGWRPRJUDSK\ &7 DQGKLVWRPRUSKRPHWULFHYDOXDWLRQV were performed. 5HVXOWV Radiographic evaluationsrevealed that the amount and quality of callus for- mations were significantly higher in the treatment group (P<0.05). As measured by CT scan, the healing parametersin dogs of the treatment group were significantly greater (P<0.05). New bone formation in the treatment group was significantly higher (P<0.05). &RQFOXVLRQThe present study showed that the transplantation of BM-MSCs positively affects early bony consolidation in DO. The use of MSCs might allow a shortened period of consolidationArchive and therefore permit earlier device removal.of SID Keywords: Distraction Osteogenesis, Bone Lengthening, Mesenchymal Stem Cells, Autologous Transplantation, Platelet-Rich Plasma Cell Journal(Yakhteh), Vol 17, No 2, Summer 2015, Pages: 243- 252 &LWDWLRQ'HKJKDQ00%DJKDEDQ(VODPLQHMDG00RWDOOHEL]DGHK1$VKUD¿+DODQ-7DJL\DU/6RURRUL61LNPDK]DU $3HGUDP06KDKYHUGL$.D]HPL0HKUMHUGL+,]DGL67UDQVSODQWDWLRQRIDXWRORJRXVERQHPDUURZPHVHQFK\PDOVWHP FHOOVZLWKSODWHOHWULFKSODVPDDFFHOHUDWHGLVWUDFWLRQRVWHRJHQHVLVLQDFDQLQHPRGHO&HOO- CELL JOURNAL(Yakhteh), Vol 17, No 2, Summer 2015 243 www.SID.ir MSCs with PRP Accelerate DO Introduction VLEOH DOWHUQDWLYH WR DFFHOHUDWH ERQH UHJHQHUDWLRQ 'XULQJ'2DVHULHVRIFHOOXODUSURFHVVHVDQG 'LVWUDFWLRQ RVWHRJHQHVLV '2 LV D VXUJLFDO FRPSOH[PROHFXODUHYHQWVLQGXFHWKHGLIIHUHQWLD procedure by which controlled displacement of WLRQRIPHVHQFK\PDOVWHPFHOOV 06&V LQWRERQH bone fragments is used to induce the generation IRUPLQJFHOOVDQGHYHQWXDOO\WKHIRUPDWLRQRID RIODUJHYROXPHVRIQHZERQH7KLVWHFKQLTXHKDV GLVWUDFWLRQFDOOXV EHHQ VXFFHVVIXOO\ XVHG IRU OLPE OHQJWKHQLQJ ,QWKLVELRPHFKDQLFDOSURFHVVIRUVNHOHWDOOHQJWK 06&V DUH DGXOW VWHP FHOOV WKDW FDQ EH GHULYHG HQLQJ WKH IRUPDWLRQ RI QHZ ERQH DORQJ WKH GLV IURPPDQ\WLVVXHVLQSDUWLFXODUERQHPDUURZ %0 WUDFWLRQVWUHVVOLQHLVVWLPXODWHGE\H[HUWLQJVWUDLQ WLVVXH%\IDUPXOWLSOHVWXGLHVKDYHFRQ¿UPHGWKH RQERWKHQGVRIWKHERQ\VHJPHQWV 7KLVWHFK JUHDWSRWHQWLDORI06&VLQSURPRWLQJUHJHQHUDWLRQ QLTXHZDV¿UVWGHVFULEHGE\&RGLYLOOD EXWGLG of bone defects both in animal models and humans 6FLHQWLVWVEHOLHYHWKDW06&VFDQKHOSUH not gain wide acceptance until the physiological generation by two ways: differentiation into tissue and mechanical aspects required for the success cells in order to restore lost morphology as well IXO UHJHQHUDWLRQ RI QHZ ERQH ZHUH LGHQWL¿HG E\ as function and secretion of a wide spectrum of ,OL]DURY 7KLVWHFKQLTXHLVFXUUHQWO\DVWDQGDUG ELRDFWLYHIDFWRUVWKDWKHOSWRFUHDWHDUHSDLUHQYL DQGDFFHSWDEOHPHWKRGIRUERQHOHQJWKHQLQJ'2 ronment by possessing immunoregulatory func is usually separated into three phases: i. the latency WLRQDQWLDSRSWRWLFHIIHFWVDQGWKHVWLPXODWLRQRI SKDVHLPPHGLDWHO\IROORZLQJWKHRVWHRWRP\DQG HQGRWKHOLDO SURJHQLWRU FHOO SUROLIHUDWLRQ 2Q EHIRUHGLVWUDFWLRQLL7KHGLVWUDFWLRQSKDVHFKDU WKH RWKHU KDQG QXPEHUV RI UHVHDUFK ZRUNV KDYH DFWHUL]HGE\WKHDFWLYHGLVWUDFWLRQRIWKHVHJPHQWV UHSRUWHGWKDWSODWHOHWULFKSODVPD 353 FDQHQKDQ IRUDFHUWDLQWLPHDWVSHFL¿FUDWHVDQGIUHTXHQFLHV FHWKHKHDOLQJSURFHVVLQERQHLQMXULHV 7KH DQGLLLWKHFRQVROLGDWLRQSKDVHFKDUDFWHUL]HGE\ ERQHUHJHQHUDWLYHHIIHFWVRI353KDYHDOVREHHQ ERQ\ XQLRQ DQG PLQHUDOL]DWLRQ 7KH GXUDWLRQ RI UHSRUWHGDWHDUO\SKDVHVRI'2 6RPHUH WKHFRQVROLGDWLRQGHSHQGVRQWKHGLVWUDFWLRQVLWH VHDUFKKDVLQGLFDWHGWKDWWKHDGGLWLRQRI06&LQWR WKHVWDWXVRIYDVFXODUL]DWLRQDQGWKHDJHRIWKHSD D353VFDIIROGZRXOGEHEHQH¿FLDOIRULQFUHDVHG WLHQW $OWKRXJK'2FDQSURGXFHUHOLDEOHFDOOXV QHZERQHIRUPDWLRQPLQHUDOL]DWLRQDQGPHFKDQL IRUPDWLRQZLWKRXWJUDIWLQJPDQ\VXUJHRQVSUHIHU FDOSURSHUW\ to graft autologous can cellous bone (the current JROGVWDQGDUG IRUDEHWWHURXWFRPH '2LVXVHG 7KHSUHVHQWVWXG\HYDOXDWHGWKHSRVVLELOLW\RIDQ IRUFRPSOLFDWLRQVRIIUDFWXUHVVXFKDVQRQXQLRQV increased distraction rate and decreased consolida FKURQLFRVWHRP\HOLWLVVKRUWHQHGH[WUHPLWLHVMRLQW WLRQSKDVHE\XVLQJ06&VDVDQH[RJHQRXVVRXUFH FRQWUDFWXUHVGHIRUPLWLHVDQGERQHORVVGXHWRWUDX RIRVWHRJHQLFSURJHQLWRUFHOOVIRU'27KLVH[SHUL PDLQIHFWLRQVRUWXPRUUHVHFWLRQV ,WVKRXOG PHQWDOVWXG\LQYHVWLJDWHGZKHWKHUWKHWUDQVSODQWD be mentioned that the use of this method has some WLRQRI06&VDQG353FRXOGDFFHOHUDWHWKHHYHQWV OLPLWDWLRQVVXFKDVWKHQHHGIRUH[SHQVLYHGHYLFHV RI '2 RU LI WKHVH FHOOV FRXOG LQÀXHQFH GLIIHUHQW ORQJWHUP WUHDWPHQW UHVXOWLQJ LQ D KLJK UDWH DVSHFWVRI'2 RIFRPSOLFDWLRQVVXFKDVLQIHFWLRQSLQORRVHQLQJ IUDFWXUH DGMDFHQW MRLQW FRQWUDFWXUHV VRIW WLVVXH Materials and Methods VZHOOLQJDQGSDLQ 0DQ\DWWHPSWVKDYHEHHQ Animals made to promote boneArchive formation by increasing the of SID distraction rate and shortening the consolidation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sue engineering in combination with osteogenic Mesenchymal stem cells isolation cells such as stem cells has been accepted as a pos &DQLQH 06&V ZHUH LVRODWHG DFFRUGLQJ WR WKH CELL JOURNAL(Yakhteh), Vol 17, No 2, Summer 2015 244 www.SID.ir Dehghan et al. PHWKRGRI.DGL\DODHWDO ZLWKVRPHPRGL¿FD 3ODWHOHWULFKSODVPDSUHSDUDWLRQ WLRQIURPWKHGRJV¶%0WKUHHZHHNVEHIRUHWKH¿UVW 353ZDVSUHSDUHGXVLQJDWHFKQLTXHGHVFULEHG WUDQVSODQWDWLRQRIVWHPFHOOV%ULHÀ\WKHQXFOHDWHG E\2NXGDHWDO %ULHÀ\P/RIDXWRORJRXV cell fraction of the marrow was enriched by gradi blood withdrawn from each dog was initially cen HQWFHQWULIXJDWLRQDQGFXOWXUHGLQFPÀDVNVDW WULIXJHG DW USP IRU PLQXWHV WR VHSDUDWH îFHOOVPOLQPOORZJOXFRVH'XOEHFFR 353DQG333SRUWLRQVIURPWKHUHGEORRGFHOOIUDF PRGL¿HG HDJOH PHGLXP '0(0 *LEFR *HU WLRQ7KH353DQG333SRUWLRQVZHUHDJDLQFHQWUL PDQ\ WKDWFRQWDLQHGIHWDOFDOIVHUXP )&6 IXJHGDWUSPIRUPLQXWHVWRVHSDUDWH353 *LEFR*HUPDQ\ 8POSHQLFLOOLQ*DQG IURP3337KHUHVXOWLQJSHOOHWRISODWHOHWVZDVUH 8PO VWUHSWRP\FLQ *LEFR *HUPDQ\ 7KH FHOOV VXVSHQGHGLQPORIUHVLGXDOSODVPD7KH353 ZHUHLQFXEDWHGDWÛ&LQDKXPLGL¿HG&2 ZDVDFWLYDWHGDWWKHWLPHRIDSSOLFDWLRQZLWKD DWPRVSKHUH2QGD\VHYHQWKHQRQDGKHUHQWFHOOV FDOFLXPFKORULGHVROXWLRQ 6LJPD86$ DQG ZHUHUHPRYHGDORQJZLWKWKHFXOWXUHPHGLD7KH 8RIERYLQHWKURPELQ 6LJPD86$ FXOWXUHVZHUHIHGWZLFHDZHHNSDVVDJHGRQGD\V E\OLIWLQJWKHFHOOVZLWK7U\SVLQ Ilizarov application P0 (WK\OHQHGLDPLQHWHWUDDFHWLF DFLG ('7$ *LEFR*HUPDQ\ H[SRVXUHIRU¿YHPLQXWHVDQG The lengthening procedure was performed by VSOLWLQDUDWLRLQWRQHZFPFXOWXUHÀDVNV DSSO\LQJDQ,OL]DURYV\VWHPWRWKHOHIWWLELDRIHDFK animal. All frames were identical and consisted of Differentiation potential RQHSUR[LPDODQGRQHGLVWDOULQJZLWKD PPGLDPHWHUFRQQHFWHGE\WKUHHWUHDGHGURGV 7RHYDOXDWHWKHQDWXUHRIWKH06&VWKHLVRODWHG PPGLDPHWHU ZLWKDPPSLWFK8QGHUJHQHUDO FHOOVZHUHLQGXFHGWRGLIIHUHQWLDWHLQWRRVWHRJHQLF anesthesia and sterile conditions a distractor was FKRQGURJHQLFDQGDGLSRJHQLFFHOOOLQHDJHV DWWDFKHGWRWKHOHIWWLELDXVLQJWZRPPGLDP )RU RVWHRJHQLF GLIIHUHQWLDWLRQ FRQÀXHQW SDV HWHUWUDQVRVVHRXVZLUHVRQERWKWKHSUR[LPDODQG VDJH FHOOV ZHUH FXOWXUHG LQ '0(0 PHGLXP distal portion of the tibia. The distractor was placed VXSSOHPHQWHGZLWKPJPO/DVFRUELFSKRV SDUDOOHOWRWKHORQJD[LVRIWKHWLELDDQGVHFXUHGE\ SKDWH 6LJPD86$ Q0GH[DPHWKDVRQHDQG wires. All wires were tensioned with a tensioner. In P0 ȕJO\FHURSKRVSKDWH 6LJPD 86$ IRU DOOGRJVRVWHRWRP\RIWKHWLELDDQG¿EXODZDVSHU ZHHNV )RU DGLSRJHQHVLV '0(0 PHGLXP WKDW IRUPHGLQWKHGLDSK\VLVDWWKHOHYHORIWZRWKLUGVRI FRQWDLQHGQ0GH[DPHWKDVRQHDQGPJPO the tibial length fromits
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