Abstract
A composite construct comprising of a bone mesenchymal stem cell (BMSC) sheet, plasmid DNA, encoding human bone morphogenic protein-2 (hBMP-2), and poly(lactide-co-glycolide)/hydroxyapatite (PLGA/HA) sponge was designed and employed in the restoration of rat calvarial defects. To improve gene transfection efficiency, a cationic chitosan derivative, N,N,N,-trimethyl chitosan chloride (TMC), was employed as the vector. The TMC/DNA complexes had a transfection efficiency of 13% in rat BMSCs, resulting in heterogeneous hBMP-2 expression in a 10-d culture period in vitro. In vivo culture of the composite constructs was performed by implantation into rat full-thickness calvarial defects, using constructs lacking pDNA-hBMP-2 or BMSC sheets as controls. Significantly higher heterogeneous expression of hBMP-2 was detected in vivo at 2 weeks for the cell sheet/DNA complex/scaffold constructs, compared with the constructs lacking pDNA-hBMP-2 or BMSC sheets. New bone formation was evident as early as 4 weeks in the experimental constructs. At 8 weeks, partial bridging of calvarial defects was observed in the experimental constructs, which was significantly better than the constructs lacking pDNA-hBMP-2 or BMSC sheets. Therefore, the combination of the PLGA/HA scaffold with BMSC sheets and gene therapy vectors is effective at enhancing bone formation.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
Yamamoto M, Takahashi Y, Tabata Y. Enhanced bone regeneration at a segmental bone defect by controlled release of bone morphogenetic protein-2 from a biodegradable hydrogel. Tissue Eng, 2006, 12: 1305–1311
Chen Y. Orthopedic applications of gene therapy. J Orthop Sci, 2001, 6: 199–207
Rutherford R B, Moalli M, Franceschi R T, et al. Bone morphogenetic protein-transduced human fibroblasts convert to osteoblasts and form bone in vivo. Tissue Eng, 2002, 8: 441–452
Aviles M O, Lin C H, Zelivyanskaya M, et al. The contribution of plasmid design and release to in vivo gene expression following delivery from cationic polymer modified scaffolds. Biomaterials, 2010, 31: 1140–1147
Bonadio J, Smiley E, Patil P, et al. Localized, direct plasmid gene delivery in vivo: Prolonged therapy results in reproducible tissue regeneration. Nat Med, 1999, 5: 753–759
Heo S J, Kim S E, Wei J, et al. Fabrication and characterization of novel nano- and micro-HA/PCL composite scaffolds using a modified rapid prototyping process. J Biomed Mater Res A, 2009, 89A: 108–116
Nie H, Wang C H. Fabrication and characterization of PLGA/HAp composite scaffolds for delivery of BMP-2 plasmid DNA. J Control Release, 2007, 120: 111–121
Li D, Ye C, Zhu Y, et al. Fabrication of poly(lactide-co-glycolide) scaffold embedded spatially with hydroxyapatite particles on pore walls for bone tissue engineering. Polym Adv Tech, 2011, doi: 10.1002/pat.2066
Ouyang H W, Cao T, Zou X H, et al. Mesenchymal stem cell sheets revitalize nonviable dense grafts: Implications for repair of large-bone and tendon defects. Transplantation, 2006, 82: 170–174
Miyahara Y, Nagaya N, Kataoka M, et al. Monolayered mesenchymal stem cells repair scarred myocardium after myocardial infarction. Nat Med, 2006, 12: 459–465
Iwata T, Yamato M, Tsuchioka H, et al. Periodontal regeneration with multi-layered periodontal ligament-derived cell sheets in a canine model. Biomaterials, 2009, 30: 2716–2723
Yu M, Zhou W, Song Y, et al. Development of mesenchymal stem cell-implant complexes by cultured cells sheet enhances osseointegration in type 2 diabetic rat model. Bone, 2011, 49: 387–394
Nakamura A, Akahane M, Shigematsu H, et al. Cell sheet transplantation of cultured mesenchymal stem cells enhances bone formation in a rat nonunion model. Bone, 2010, 46: 418–424
Ma D, Ren L, Chen F, et al. Reconstruction of rabbit critical-size calvarial defects using autologous bone marrow stromal cell sheets. Ann Plast Surg, 2010, 65: 259–265
Ma D, Yao H, Tian W, et al. Enhancing bone formation by transplantation of a scaffold-free tissue-engineered periosteum in a rabbit model. Clin Oral Implants Res, 2011, 22: 1193–1199
Abbah S A, Lam C X, Ramruttun K A, et al. Autogenous bone marrow stromal cell sheets-loaded mPCL/TCP scaffolds induced osteogenesis in a porcine model of spinal interbody fusion. Tissue Eng Part A, 2011, 17: 809–817
Zou X H, Zhi Y L, Chen X, et al. Mesenchymal stem cell seeded knitted silk sling for the treatment of stress urinary incontinence. Biomaterials, 2010, 31: 4872–4879
Hong Y, Song H, Gong Y, et al. Covalently crosslinked chitosan hydrogel: Properties of in vitro degradation and chondrocyte encapsulation. Acta Biomater, 2007, 3: 23–31
Mao Z W, Ma L, Jiang Y, et al. N,N,N-trimethylchitosan chloride as a gene vector: Synthesis and application. Macromol Biosci, 2007, 7: 855–863
Karageorgiou V, Kaplan D. Porosity of 3D biomaterial scaffolds and osteogenesis. Biomaterials, 2005, 26: 5474–5491
Pfaffl M W. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res, 2001, 29: e45
Zong C, Xue D, Yuan W, et al. Reconstruction of rat calvarial defects with human mesenchymal stem cells and osteoblast-like cells in poly-lactic-co-glycolic acid scaffolds. Eur Cell Mater, 2010, 20: 109–120
Usas A, Ho A M, Cooper G M, et al. Bone regeneration mediated by BMP4-expressing muscle-derived stem cells is affected by delivery system. Tissue Eng Part A, 2009, 15: 285–293
Zhou W, Han C, Song Y, et al. The performance of bone marrow mesenchymal stem cell-implant complexes prepared by cell sheet engineering techniques. Biomaterials, 2010, 31: 3212–3221
Datta N, Pham Q P, Sharma U, et al. In vitro generated extracellular matrix and fluid shear stress synergistically enhance 3D osteoblastic differentiation. Proc Natl Acad Sci USA, 2006, 103: 2488–2493
Kimura Y, Miyazaki N, Hayashi N, et al. Controlled release of bone morphogenetic protein-2 enhances recruitment of osteogenic progenitor cells for de novo generation of bone tissue. Tissue Eng Part A, 2010, 16: 1263–1270
Lecanda F, Avioli L V, Cheng S L. Regulation of bone matrix protein expression and induction of differentiation of human osteoblasts and human bone marrow stromal cells by bone morphogenetic protein-2. J Cell Biochem, 1997, 67: 386–396
Guo R, Xu S, Ma L, et al. Enhanced angiogenesis of gene-activated dermal equivalent for treatment of full thickness incisional wounds in a porcine model. Biomaterials, 2010, 31: 7308–7320
Thanou M, Florea B I, Geldof M, et al. Quaternized chitosan oligomers as novel gene delivery vectors in epithelial cell lines. Biomaterials, 2002, 23: 153–159
Fang J, Zhu Y Y, Smiley E, et al. Stimulation of new bone formation by direct transfer of osteogenic plasmid genes. Proc Natl Acad Sci USA, 1996, 93: 5753–5758
Uchiyama H, Yamato M, Sasaki R, et al. In vivo 3D analysis with micro-computed tomography of rat calvaria bone regeneration using periosteal cell sheets fabricated on temperature-responsive culture dishes. J Tissue Eng Regen Med, 2011, 5: 483–490
Chew S A, Kretlow J D, Spicer P P, et al. Delivery of plasmid DNA encoding bone morphogenetic protein-2 with a biodegradable branched polycationic polymer in a critical-size rat cranial defect model. Tissue Eng Part A, 2010, 17: 751–763
Kasper F K, Young S, Tanahashi K, et al. Evaluation of bone regeneration by DNA release from composites of oligo(poly(ethylene glycol) fumarate) and cationized gelatin microspheres in a criticalsized calvarial defect. J Biomed Mater Res A, 2006, 78: 335–342
Govender S, Csimma C, Genant H K, et al. Recombinant human bone morphogenetic protein-2 for treatment of open tibial fractures: A prospective, controlled, randomized study of four hundred and fifty patients. J Bone Joint Surg Am, 2002, 84-A: 2123–2134
Glassman S D, Carreon L, Djurasovic M, et al. Posterolateral lumbar spine fusion with INFUSE bone graft. Spine J, 2007, 7: 44–49
Author information
Authors and Affiliations
Corresponding author
Additional information
This article is published with open access at Springerlink.com
Rights and permissions
This article is published under an open access license. Please check the 'Copyright Information' section either on this page or in the PDF for details of this license and what re-use is permitted. If your intended use exceeds what is permitted by the license or if you are unable to locate the licence and re-use information, please contact the Rights and Permissions team.
About this article
Cite this article
Li, D., Wang, W., Guo, R. et al. Restoration of rat calvarial defects by poly(lactide-co-glycolide)/hydroxyapatite scaffolds loaded with bone mesenchymal stem cells and DNA complexes. Chin. Sci. Bull. 57, 435–444 (2012). https://doi.org/10.1007/s11434-011-4914-0
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11434-011-4914-0