In this study, recombinant human bone morphogenetic protein-2 (rhBMP-2) delivery system with slow mode was successfully developed in three-dimensional (3D) printing-based polycaprolactone (PCL)/poly(lactic-co-glycolic acid) (PLGA) scaffolds for bone formation of critical-sized rabbit segmental diaphyseal defect. To control the delivery of the rhBMP-2, collagen (for long-term delivery up to 28 days) and gelatin (for shor-term delivery within a week) solutions encapsulating rhBMP-2 were dispensed into a hollow cylinderical type of PCL/PLGA scaffold. An effective dose of 5μg/mL was determined by measuring the alkaline phosphatase and osteocalcin gene expression levels of human nasal inferior turbinate-derived mesenchymal stromal cells (hTMSCs) seeded on the PCL/PLGA/collagen scaffold in vitro. However, it was found that a burst release of rhBMP-2 from the PCL/PLGA/gelatin scaffold did not induce the osteogenic differentiation of hTMSCs in vitro at an equivalent dose. In the in vivo animal experiements, microcomputed tomography and histological analyses confirmed that PCL/PLGA/collagen/rhBMP-2 scaffolds (long-term delivery mode) showed the best bone healing quality at both weeks 4 and 8 after implantation without inflammatory response. On the other hand, a large number of macrophages indicating severe inflammation provoked by burst release of rhBMP-2 were observed in the vicinity of PCL/PLGA/gelatin/rhBMP-2 (short-term delivery mode) at week 4.
Get full access to this article
View all access options for this article.
References
1.
BurasteroG., ScarfiS., FerrarisC., FresiaC., SessaregoN., FruscioneF., MonettiF., ScarfoF., SchupbachP., PodestaM., GrappioloG., and ZocchiE.The association of human mesenchymal stem cells with BMP-7 improves bone regeneration of critical-size segmental bone defects in athymic rats. Bone, 47, 117, 2010.
2.
ReichertJ.C., SaifzadehS., WullschlegerM.E., EpariD.R., SchutzM.A., DudaG.N., SchellH., van GriensvenM., RedlH., and HutmacherD.W.The challenge of establishing preclinical models for segmental bone defect research. Biomaterials, 30, 2149, 2009.
3.
IacobellisC., BerizziA., and AldegheriR.Bone transport using the Ilizarov method: a review of complications in 100 consecutive cases. Strat Traum Limb Recon, 5, 17, 2010.
4.
SenM.K., and MiclauT.Autologous iliac crest bone graft: should it still be the gold standard for treating nonunions? Injury, 38, S75, 2007.
5.
DeieM., OchiM., AdachiN., NishimoriM., and YokotaK.Artificial bone grafting [calcium hydroxyapatite ceramic with an interconnected porous structure (IP-CHA)] and core decompression for spontaneous osteonecrosis of the femoral condyle in the knee. Knee Surg Sports Traumatol Arthrosc, 16, 753, 2008.
6.
SakurakichiK., TsuchiyaH., KabataT., YamashiroT., WatanabeK., and TomitaK.Correction of juxtaarticular deformities in children using the Ilizarov apparatus. J Orthop Sci, 10, 360, 2005.
7.
SchaafH., LendeckelS., HowaldtH.P., and StreckbeinP.Donor site morbidity after bone harvesting from the anterior iliac crest. Oral Surg Oral Med Oral Pathol Oral Radiol Endod, 109, 52, 2010.
HanL.H., SuriS., SchmidtC.E., and ChenS.Fabrication of three-dimensional scaffolds for heterogeneous tissue engineering. Biomed Microdevices, 12, 721, 2010.
10.
JungJ.W., KangH.W., KangT.Y., ParkJ.H., ParkJ., and ChoD.W.Projection image-generation algorithm for fabrication of a complex structure using projection-based microstereolithography. Int J Precis Eng Man, 13, 445, 2012.
11.
HutmacherD.W., SittingerM., and RisbudM.V.Scaffold-based tissue engineering: rationale for computer-aided design and solid free-form fabrication systems. Trends Biotechnol, 22, 354, 2004.
12.
SeolY.J., KangT.Y., and ChoD.W.Solid freeform fabrication technology applied to tissue engineering with various biomaterials. Soft Matter, 8, 1730, 2012.
13.
SchuckertK.H., JoppS., and TeohS.H.Mandibular defect reconstruction using three-dimensional polycaprolactone scaffold in combination with platelet-rich plasma and recombinant human bone morphogenetic protein-2: de novo synthesis of bone in a single case. Tissue Eng Part A, 15, 493, 2009.
14.
PeckJ.N., and Marcellin-LittleD.J.Advances in small animal total joint replacement. In: Marcellin-LittleD.J., and HarryssonO.L.A., eds. Custom Total Joint Arthroplasty. Hoboken, NJ: John Wiley and Sons, Inc., 2013, pp. 223–241.
15.
ShorL., GuceriS., ChangR., GordonJ., KangQ., HartsockL., AnY., and SunW.Precision extruding deposition (PED) fabrication of polycaprolactone (PCL) scaffolds for bone tissue engineering. Biofabrication, 1, 015003, 2009.
16.
YeoA., SjuE., RaiB., and TeohS.H.Customizing the degradation and load-bearing profile of 3D polycaprolactone-tricalcium phosphate scaffolds under enzymatic and hydrolytic conditions. J Biomed Mater Res B, 87, 562, 2008.
17.
LiR.H., and WozneyJ.M.Delivering on the promise of bone morphogenetic proteins. Trends Biotechnol, 19, 255, 2001.
18.
SethiA., CraigJ., BartolS., ChenW., JacobsonM., CoeC., and VaidyaR.Radiographic and CT evaluation of recombinant human bone morphogenetic protein-2-assisted spinal interbody fusion. Am J Roentgenol, 197, W128, 2011.
19.
WeiS., CaiX., HuangJ., XuF., LiuX., and WangQ.Recombinant human BMP-2 for the treatment of open tibial fractures. Orthopedics, 35, e847, 2012.
20.
LeeJ., DeckerJ.F., PolimeniG., CortellaC.A., RohrerM.D., WozneyJ.M., HallJ., SusinC., and WikesjoU.M.Evaluation of implants coated with rhBMP-2 using two different coating strategies: a critical-size supraalveolar peri-implant defect study in dogs. J Clin Periodontol, 37, 582, 2010.
21.
LiB., YoshiiT., HafemanA.E., NymanJ.S., WenkeJ.C., and GuelcherS.A.The effects of rhBMP-2 released from biodegradable polyurethane/microsphere composite scaffolds on new bone formation in rat femora. Biomaterials, 30, 6768, 2009.
22.
VoT.N., KasperF.K., and MikosA.G.Strategies for controlled delivery of growth factors and cells for bone regeneration. Adv Drug Deliver Rev, 64, 1292, 2012.
23.
ParkJ.K., ShimJ.H., KangK.S., YeomJ., JungH.S., KimJ.Y., LeeK.H., KimT.H., KimS.Y., ChoD.W., and HahnS.K.Solid free-form fabrication of tissue-engineering scaffolds with a poly(lactic-co-glycolic acid) grafted hyaluronic acid conjugate encapsulating an intact bone morphogenetic protein-2/poly(ethylene glycol) complex. Adv Funct Mater, 21, 2906, 2011.
24.
LeeJ.W., KangK.S., LeeS.H., KimJ.Y., LeeB.K., and ChoD.W.Bone regeneration using a microstereolithography-produced customized poly(propylene fumarate)/diethyl fumarate photopolymer 3D scaffold incorporating BMP-2 loaded PLGA microspheres. Biomaterials, 32, 744, 2011.
25.
SawyerA.A., SongS.J., SusantoE., ChuanP., LamC.X., WoodruffM.A., HutmacherD.W., and CoolS.M.The stimulation of healing within a rat calvarial defect by mPCL-TCP/collagen scaffolds loaded with rhBMP-2. Biomaterials, 30, 2479, 2009.
26.
KimJ.Y., YoonJ.J., ParkE.K., KimD.S., KimS.Y., and ChoD.W.Cell adhesion and proliferation evaluation of SFF-based biodegradable scaffolds fabricated using a multi-head deposition system. Biofabrication, 1, 015002, 2009.
27.
KimJ.Y., and ChoD.W.Blended PCL/PLGA scaffold fabrication using multi-head deposition system. Microelectron Eng, 86, 1447, 2009.
28.
HwangS.H., KimS.Y., ParkS.H., ChoiM.Y., KangH.W., SeolY.J., ParkJ.H., ChoD.W., HongO.K., RhaJ.G., and KimS.W.Human inferior turbinate: an alternative tissue source of multipotent mesenchymal stromal cells. Otolaryngol Head Neck Surg, 147, 568, 2012.
29.
HwangS.H., KimS.W., KimS.Y., ParkS.H., LeeH.J., ChoiM.Y., OhH.J., KimS.H., RhieJ.W., and SunD.I.Human turbinate mesenchymal stromal cells as a potential option for cartilage tissue engineering. Tissue Eng Regen Med, 8, 536, 2011.
30.
HwangS.H., KimS.Y., ParkS.H., ChoiM.Y., BackS.A., KimY.I., SunD.I., and KimS.W.Osteogenic differentiation of human turbinate mesenchymal stromal cells. Tissue Eng Regen Med, 8, 544, 2011.
31.
ShimJ.H., KimJ.Y., ParkM., ParkJ., and ChoD.W.Development of a hybrid scaffold with synthetic biomaterials and hydrogel using solid freeform fabrication technology. Biofabrication, 3, 034102, 2011.
32.
ShimJ.H., KimJ.Y., ParkJ.K., HahnS.K., RhieJ.W., KangS.W., LeeS.H., and ChoD.W.Effect of thermal degradation of SFF-based PLGA scaffolds fabricated using a multi-head deposition system followed by change of cell growth rate. J Biomat Sci Polym E, 21, 1069, 2010.
33.
KimJ.Y., JinG.Z., ParkI.S., KimJ.N., ChunS.Y., ParkE.K., KimS.Y., YooJ., KimS.H., RhieJ.W., and ChoD.W.Evaluation of solid free-form fabrication-based scaffolds seeded with osteoblasts and human umbilical vein endothelial cells for use in vivo osteogenesis. Tissue Eng Part A, 16, 2229, 2011.
34.
ShieldsL.B., RaqueG.H., GlassmanS.D., CampbellM., VitazT., HarpringJ., and ShieldsC.B.Adverse effects associated with high-dose recombinant human bone morphogenetic protein-2 use in anterior cervical spine fusion. Spine, 31, 542, 2006.
35.
FriessW., UludagH., FoskettS., BironR., and SargeantC.Characterization of absorbable collagen sponges as rhBMP-2 carriers. Int J Pharm, 187, 91, 1999.
36.
Kirker-HeadC.A.Potential applications and delivery strategies for bone morphogenetic proteins. Adv Drug Deliver Rev, 43, 65, 2000.
37.
AbarrategiA., CivantosA., RamosV., Sanz CasadoJ.V., and Lopez-LacombaJ.L.Chitosan film as rhBMP2 carrier: delivery properties for bone tissue application. Biomacromolecules, 9, 711, 2008.
38.
UludagH., D'AugustaD., PalmerR., TimonyG., and WozneyJ.Characterization of rhBMP-2 pharmacokinetics implanted with biomaterial carriers in the rat ectopic model. J Biomed Mater Res, 46, 193, 1999.
39.
ZhouH., QianJ., WangJ., YaoW., LiuC., ChenJ., and CaoX.Enhanced bioactivity of bone morphogenetic protein-2 with low dose of 2-N, 6-O-sulfated chitosan in vitro and in vivo. Biomaterials, 30, 1715, 2009.
40.
JeonO., SongS.J., YangH.S., BhangS.H., KangS.W., SungM.A., LeeJ.H., and KimB.S.Long-term delivery enhances in vivo osteogenic efficacy of bone morphogenetic protein-2 compared to short-term delivery. Biochem Biophys Res Commun, 369, 774, 2008.
41.
MaegawaN., KawamuraK., HiroseM., YajimaH., TakakuraY., and OhgushiH.Enhancement of osteoblastic differentiation of mesenchymal stromal cells cultured by selective combination of bone morphogenetic protein-2 (BMP-2) and fibroblast growth factor-2 (FGF-2). J Tissue Eng Regen Med, 1, 306, 2007.
42.
MaP.X.Biomimetic materials for tissue engineering. Adv Drug Deliver Rev, 60, 184, 2008.
43.
ItohS., NakamuraS., KobayashiT., ShinomiyaK., YamashitaK., and ItohS.Effect of electrical polarization of hydroxyapatite ceramics on new bone formation. Calcified Tissue Int, 78, 133, 2006.
44.
SchlichtingK., SchellH., KleemannR.U., SchillA., WeilerA., DudaG.N., and EpariD.R.Influence of scaffold stiffness on subchondral bone and subsequent cartilage regeneration in an ovine model of osteochondral defect healing. Am J Sports Med, 36, 2379, 2008.
45.
HutmacherD.W.Scaffolds in tissue engineering bone and cartilage. Biomaterials, 21, 2529, 2000.
46.
Van den BergT.K., and KraalG.A function for the macrophage F4/80 molecule in tolerance induction. Trends Immunol, 26, 506, 2005.
47.
JulkaA., ShahA.S., and MillerB.S.Inflammatory response to recombinant human bone morphogenetic protein-2 use in the treatment of a proximal humeral fracture: a case report. J Shoulder Elb Surg, 21, e12, 2012.
48.
LaceyD.C., SimmonsP.J., GravesS.E., and HamiltonJ.A.Proinflammatory cytokines inhibit osteogenic differentiation from stem cells: implications for bone repair during inflammation. Osteoarthritis Cartilage, 17, 735, 2009.
49.
MountziarisP.M., and MikosA.G.Modulation of the inflammatory response for enhanced bone tissue regeneration. Tissue Eng Part B, 14, 179, 2008.
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
