: :1001-9731(2014)14-14001-07 14001 * ( 200237) : 2-2 2.1 (HTCC) ; β-tcp 2.1.1 (CSM W =5.05 10 5 )23- (GTMAC ) β - : ; ; ( β -GP )BCA : R318.08 :A ( ) DOI:10.3969/j.issn.1001-9731.2014.14.001 (rhbmp-2 ) β - TCP 1 2.1.2 HTCC 2g 99 ml 1 ml 1mol/L NaOH ph 10 4h [1-4] Chenite [7-9] 23- / β - [5-6] 80mL 80 23-3.5h 3.5h ; 3 12h (HTCC) 3 24h HTCC/GP β- ( β -TCP) KBr HTCC 4000~500cm -1 2.2 2.2.1 HTCC/ β -GP/ β -TCP -2(rhBMP-2) HTCC HTCC β -GP (1.2g/mL) β-tcp β -GP HTCC * : (2012CB933600) :2014-06-10 :2014-07-15 : E-mail:liucs@ecust.edu.cn : (1987-)
14002 2014 14 (45) β-tcp/ β -GP HTCC HTCC/ β -GP/ β -TCP 20mg/mL ( β -TCP/HTCC);(3) β -TCP 40mg/mL ( β -TCP/HTCC) 20μg -20 12h rhbmp-2 3 48h HTCC/ β -GP/ β -TCP 5mLPBS(pH =7.2) 10mL 37 80r/min (1235710142128 35d) 2.2.2 HTCC/ β -GP/ β -TCP 5 ml PBS = (ph =7.2) BCA 10mmL=20 mm 3 2 mm/min 3 rhbmp-2-2.2.3 HTCC/ β -GP/ β -TCP 2.3.3 (PBS) (1) : 1mm 10mm 2.2. PBS 1 40 μlrhbmp-2 (0.5mg/mL) ;12 3 4 :1 HTCC/ β -GP+rh- BMP-2;2 HTCC/ β -GP/ β -TCP(c( β -TCP)=20 mg/ SD =W t /W 0 W t ;W 0 ml)+rhbmp-2;3 HTCC/ β -GP/ β -TCP(c( β -TCP) =40 mg/ml)+rhbmp-2 (2) : 2.2.4 HTCC/ β -GP/ β -TCP MTS (23~25g) 3% :(1) 3cm 2 1mL 37 24h ;(2) MC3T3-E1 3 mm 96 10000 150μL 37 2.3.4 5% CO 2 24 72h;(3) 20μL MTS 4 37 5h;(4) 490nm (OD ) (relativegrowthratergr) RGR = OD ( )/ 4% 3 OD ( ) 100% 600 6h : RGR 100 0 RGR=75~99 1 RGR=50 2.3.5 ~74 2 RGR=25~49 3 RGR=1~24 4 4% 4 RGR=0 5 0~1 2 24h ; 30% - 3~5 72h; 2.3 rhbmp-2 2.3.1 rhbmp-2 HTCC H-E rhbmp-2 HTCC/ β -GP/ β -TCP : HTCC/ β -GP/ β -TCP 3 rhbmp-2(0.5 mg/ml) 3.1 10min HTCC 24h 1 HTCC 1 2.3.2 FT-IR 1596.78cm -1 3 :(1) β -TCP 0;(2) β -TCP N H HTCC FT-IR
: 14003 1596.78cm -1 N H β -GP 1482.61cm -1 CH 3 HTCC β -GP HTCC 2 1103.23cm -1 1089.14 HTCC HTCC/ β -GP cm -1 HTCC NH 2 1 HTCC Fig1FT-IRspectraforchitosanand HTCC β-tcp HTCC/ β -GP/ HTCC/ β -GP β-tcp 3 2 HTCC HTCC/ β -GP Fig2ThephotoofHTCCsolutionand HTCC/ β -GP hydrogel 3.2 β-tcp 3 β -TCP HTCC/ β -GP/ β -TCP Fig3TheSEM graphsofhtcc/ β -GP/ β -TCPhydrogelcontainingdiferentamountsofβ-TCP 3 β-tcp β -TCP β -TCP 3000
14004 2014 14 (45) 3000 β-tcp HTCC/ β -GP 3.4 ; β -TCP 20 mg/ml 5 HTCC/ β -GP/ β -TCP (ph β-tcp ; β -TCP 40 mg/ml =6.0) (ph =7.0) (ph =8.0) β-tcp β -TCP 5(a) PBS HTCC/ β -GP 15.8; β -TCP 3.3 β-tcp ; β -TCP 20 mg/ml 4 HTCC/ β -GP/ β -TCP - 13.9; β -TCP 40 mg/ml β -TCP 11.2 3 β -TCP β -TCP HTCC/ β -GP 90 min β-tcp β -TCP 20 mg/ml 130 min β -TCP 40mg/mL 220 min 5(b) PBS 3 β - TCP β -TCP 02040mg/mL ph =7.0 PBS 4 β-tcp 02040 mg/ml HTCC/ 13.411.9 9.3 β-gp/ β -TCP - 5(c) PBS(pH =8.0) 3 Fig4Thecompressionstress-straincurvesofHTCC/ β-gp/ β -TCP containing diferent amount of β-tcp β -TCP β -TCP 0 20 40 mg/ml ph =8.0 PBS β -TCP = 40mg/mL 1.25 MPa 11.310.1 7.2 3.36 MPa β -TCP 5 HTCC/ β -GP/ β -TCP ph =6.07.0 8.0 Fig5Swelingdegree(SD)ofHTCC/ β -GP/ β -TCPlyophilizedhydrogelscafoldscontainingdiferentcontentof β-tcp (02040 mg/ml)atdiferentph:ph=6.07.0and8.0 HTCC/ β -GP/ β -TCP ph β-tcp β -GP 3.5 rhbmp-2 β -TCP 6(a) β -TCP 3 β-tcp rhbmp-2 β-tcp HTCC β -TCP rhbmp-2 HTCC β -GP 7d β -TCP 02040mg/mL 3 β -TCP rhbmp-2 93%82%74%; 6 (b) : HTCC β -GP : β -TCP β -TCP β-tcp HTCC β-gp HTCC/ β -GP/ β -TCP β-tcp
: 14005 β-tcp rhbmp-2 β -TCP rhbmp-2 rhbmp-2 β -TCP HTCC/ β -GP/ β-tcp β-tcp rhbmp-2 rhbmp-2 6 HTCC/ β -GP/ β -TCP rhbmp-2 Fig6ReleaseprofilesofrhBMP-2forHTCC/ β -GP/ β -TCPscafoldsanditsschematicilustration 3.6 HACC/ β -GP MTS 7(a) 24h β -TCP 02040 mg/ ml 3 RGR 78% 3.8 8 3 rhbmp-2 82%95% 75%~100% 1 4 β-tcp rhbmp-2 HTCC/ β -GP/ β-tcp 8 β-tcp rhbmp-2 HTCC/ β -GP β -TCP 7 HTCC/ β -GP/ β -TCP MC3T3-E1 K Na Ca Fig7 Cytotoxicityof HTCC/ β -GP/ β -TCP scafolds Mg by MTSanalysisafterincubationfor24and72 β -TCP h β-tcp β-tcp HTCC/ 7(b) 72h β -TCP 0 β-gp β -TCP 2040mg/mL 3 RGR 3.9 85%88%97% 1 HTCC/ β -GP/ β -TCP/rhBMP- 3 2 10 11 β -TCP β-tcp HTCC/ β -GP/ β -TCP rhbmp-2 Fig 8 Ectopic boneformation ofrhbmp-2-loaded- HTCC/ β -GP/ β -TCPscafold 9 4
14006 2014 14 (45) 9 4 rhbmp-2 (*P<0.05) Fig9 Wetandashweightofnewbonefor4weeks(*P<0.05) 10 HTCC/ β -GP/ β -TCP/rhBMP-2 ( 4 )HE NB F Fig10HEofHTCC/ β -GP/ β -TCP/rhBMP-2scafoldscontainingdiferentcontentofβ-TCP (02040mg/mL) implantedintorathindlimbmusclefor4weeks 11 HTCC/ β -GP/ β -TCP/rhBMP-2 ( 4 ) NB F Fig11 Masson'strichromestainingofHTCC/ β -GP/ β -TCP/rhBMP-2scafoldscontainingdiferentcontentof β-tcp (02040 mg/ml)implantedintorathindlimbmusclefor4weeks
: 14007 10 11 HE tionsofrecentadvancesincarboxymethylchitosanbased 3 4 targeteddrugdeliveryandtissueengineeringapplications [J].JournalofControledRelease2014186:54-87. [2] CroisierFJér me C.Chitosan-basedbiomaterialsfor 3 4 tissueengineering[j].europeanpolymerjournal2013 49:780-792. [3] BhardwajNDavidQ TKaplanLetal.Potentialof3-3 β-tcp Dtissueconstructsengineeringedform bovinechondro- β -TCP cytes/silkfibroin-chitosanforinvitrocartilagetissueen- β -TCP gineering [J].Biomaterials201132:5773-5781. β -TCP [4] Tian MengYangZhiWanChangxiuetal.Deliveryof β-tcp demineralizedbone matrixpowderusingathermogeling chitosancarrier [J].Actabiomaterialia20128:753-762. β -TCP [5] BergerJReistACheniteAetal.Pseudo-thermoset- ; tingchitosanhydrogelsforbiomedicalapplication[j].in- β -TCP ternationaljournalof Pharmaceutics2005288:197- rhbmp-2 rhbmp-2 206. HTCC/ β -GP/ β - [6] CheniteABuschmann MKandaniNetal.Rheological TCP/rhBMP-2 characterisationofthermogelingchitosan/glycerol-phos- phatesolutions [J].CarbohydratePolymers200146: 4 tentivenessof2-hydroxyl-3-trimethyl β-tcp (ammonia)propyl chitosanchloride[j].chinesejournalofappliedchemis- HTCC/ β -GP/ β -TCP try199613:94-96. rhbmp-2 [8] VivianeA SMauroC M L.Preparationandcharacter- izationofquaternarychitosansalt:adsorptionequilibrium HTCC ofchromium(vi)ion [J].Reactive & FunctionalPoly- HTCC/ β -GP/ β -TCP mers200461 (6):347-352. [9] WuJieZhaoZhiguoMaGuanghui.Athermo-andpH- sensitivehydrogelcomposedofquaternizedchitosan/glyc- : [1] UpadhyayaLSinghJAgarwalVetal.Theimplica- 39-47. [7] XuchenLu Canhui.Hydroscopicityand moisturere- tics2006315:1-11. Researchofquaternizedchitosanbasedhybridscaffold asadeliverysystemforrhbmp-2 HU Yuan-yuanWANGJingLIU Chang-sheng (EngineeringResearchCenterforBiomedicalMaterialsofMinistryofEducation EastChinaUniversityofScienceandTechnologyShanghai200237China) Abstract:Quaternizedchitosan wassynthesizedbythereactionofchitosanandglycidyltrimethylammonium chloride(gtmac)andnamedasn-[(2-hydroxy-3-trimethylammonium)propyl]chitosanchloride (HTCC). AhydrogelsystemcomposedofHTCCandβ-glycerophosphate( β -GP)wasobtainedbyintermolecularforces. erophosphate [J].InternationalJournalofPharmaceu- Introductionofβ-TCPgranulesimprovedthemechanicalstrengthandcontroledtheswelingbehaviorandpro- longedthereleasingperiodforrhbmp-2ofthehydrogelscafold.moreoverrhbmp-2loadedscafoldswereas- sessedinectopicboneformation.theresultsindicatethatporoushtcc/ β -GP/ β -TCP/rhBMP-2scafoldsare promisingcandidatesforapplicationintissueengineeringofbone. Keywords:boneregeneration;chitosan;bonemorphogeneticprotein;calciumphosphate