HETEROTOPIC CHONDROGENESIS OF CANINE MYOBLASTS ON POLY (LACTIDE-CO-GLYCOLIDE) SCAFFOLDS IN VIVO_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

GU Yanglin ¹ , WANG Yubin ¹ , SHI Keqin ¹ , YANG Yusheng ¹ , CHEN Peng ¹ , ZHU Wenhui ² , ZHU Guoxing ¹

1Department of Orthopedics, Wuxi Second Hospital Affiliated to Nanjing Medical University, Wuxi Jiangsu, 214002, P.R.China;;
2Department of Sports Medicine, Dongfang Hospital Affiliated to Tongji University. Corresponding author: ZHU Guoxing, E-mail: guoxing.zhu@yahoo.cn;

Corresponding author：

Keywords：

Tissue engineered cartilage; Myoblasts; Poly (lactide-co-glycolide); Canine Nude mouse

DOI：

10.7507/1002-1892.20130166

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Objective To explore heterotopic chondrogenesis of canine myoblasts induced by cartilage-derived morphogenetic protein 2 (CDMP-2) and transforming growth factor β1 (TGF-β1) which were seeded on poly (lactide-co-glycolide) (PLGA) scaffolds after implantation in a subcutaneous pocket of nude mice. Methods Myoblasts from rectus femoris of 1-year-old Beagle were seeded on PLGA scaffolds and cultured in medium containing CDMP-2 and TGF-β1 for 2 weeks in vitro. Then induced myoblasts-PLGA scaffold, uninduced myoblasts-PLGA scaffold, CDMP-2 and TGF-β1-PLGA scaffold, and simple PLGA scaffold were implanted into 4 zygomorphic back subcutaneous pockets of 24 nude mice in groups A, B, C, and D, respectively. At 8 and 12 weeks, the samples were harvested for general observation, HE staining and toluidine blue staining, immunohistochemical staining for collagen type I and collagen type II; the mRNA expressions of collagen type I, collagen type II, Aggrecan, and Sox9 were determined by RT-PCR, the glycosaminoglycans (GAG) content by Alician blue staining, and the compressive elastic modulus by biomechanics. Results In group A, cartilaginoid tissue was milky white with smooth surface and slight elasticity at 8 weeks, and had similar appearance and elasticity to normal cartilage tissue at 12 weeks. In group B, few residual tissue remained at 8 weeks, and was completely degraded at 12 weeks. In groups C and D, the implants disappeared at 8 weeks. HE staining showed that mature cartilage lacuna formed of group A at 8 and 12 weeks; no cartilage lacuna formed in group B at 8 weeks. Toluidine blue staining confirmed that new cartilage cells were oval and arranged in line, with lacuna and blue-staining positive cytoplasm and extracellular matrix in group A at 8 and 12 weeks; no blue metachromatic extracellular matrix was seen in group B at 8 weeks. Collagen type I and collagen type II expressed positively in group A, did not expressed in group B by immunohistochemical staining. At 8 weeks, the mRNA expressions of collagen type I, collagen type II, Aggrecan, and Sox9 were detected by RT-PCR in group A at 8 and 12 weeks, but negative results were shown in group B. The compressive elastic modulus and GAG content of group A were (90.79 ± 1.78) MPa and (10.20 ± 1.07) μg/mL respectively at 12 weeks, showing significant differences when compared with normal meniscus (P lt; 0.05). Conclusion Induced myoblasts-PLGA scaffolds can stably express chondrogenic phenotype in a heterotopic model of cartilage transplantation and represent a suitable tool for tissue engineering of menisci.

Citation： GU Yanglin,WANG Yubin,SHI Keqin,YANG Yusheng,CHEN Peng,ZHU Wenhui,ZHU Guoxing. HETEROTOPIC CHONDROGENESIS OF CANINE MYOBLASTS ON POLY (LACTIDE-CO-GLYCOLIDE) SCAFFOLDS IN VIVO. Chinese Journal of Reparative and Reconstructive Surgery, 2013, 27(6): 749-754. doi: 10.7507/1002-1892.20130166 Copy

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2.	Gu YL, Wang YB. Treatment of meniscal injury: a current concept review. Chin J Traumatol, 2010, 13(6): 370-376.
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6.	Asakura A, Komaki M, Rudnicki M. Muscle satellite cells are multipotential stem cells that exhibit myogenic, osteogenic and adipogenic differentiation. Differentiation, 2001, 68(4-5): 245-253.
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9.	Freymann U, Endres M, Neumann K, et al. Expanded human meniscus-derived cells in 3-D polymer-hyaluronan scaffolds for meniscus repair. Acta Biomater, 2012, 8(2): 677-685.
10.	Koning M, Harmsen MC, van Luyn MJ, et al. Current opportunities and challenges in skeletal muscle tissue engineering. J Tissue Eng Regen Med, 2009, 3(6): 407-415.
11.	Liu Y, Li C, Wang HY, et al. Synthesis of Thermo- and pH-Sensitive Polyion Complex Micelles for Fluorescent Imaging. Chemistry, 2012, 18(8): 2297-2304.
12.	Asadi H, Rostamizadeh K, Salari D, et al. Preparation and characterization of tri-block poly (lactide)-poly (ethylene glycol)-poly (lactide) nanogels for controlled release of naltrexone. Int J Pharm, 2011, 416(1): 356-364.
13.	Singh D, Nayak V, Kumar A. Proliferation of myoblast skeletal cells on three-dimensional supermacroporous cryogels. Int J Biol Sci, 2010, 6(4): 371-381.
14.	Steinert AF, Ghivizzani SC, Rethwilm A, et al. Major biological obstacles for persistent cell-based regeneration of articular cartilage. Arthritis Res Ther, 2007, 9(3): 213.
15.	Tian H, Yang S, Xu L, et al. Chondrogenic differentiation of mouse bone marrow mesenchymal stem cells induced by cartilage-derived morphogenetic protein-2 in vitro. J Huazhong Univ Sci Technolog Med Sci, 2007, 27(4): 429-432.
16.	Bobick BE, Chen FH, Le AM, et al. Regulation of the chondrogenic phenotype in culture. Birth Defects Res C Embryo Today, 2009, 87(4): 351-371.
17.	Garg T, Singh O, Arora S, et al. Scaffold: a novel carrier for cell and drug delivery. Crit Rev Ther Drug Carrier Syst, 2012, 29(1): 1-63.
18.	Subhash HM, Xie H, Smith JW, et al. Optical detection of indocyanine green encapsulated biocompatible poly (lactic-co-glycolic) acid nanoparticles with photothermal optical coherence tomography. Opt Lett, 2012, 37(5): 981-983.
19.	Espandar L, Bunnell B, Wang GY, et al. Adipose-derived stem cells on hyaluronic Acid-derived scaffold: a new horizon in bioengineered cornea. Arch Ophthalmol, 2012, 130(2): 202-208.
20.	Cui L, Wu Y, Cen L, et al. Repair of articular cartilage defect in non-weight bearing areas using adipose derived stem cells loaded polyglycolic acid mesh. Biomaterials, 2009, 30(14): 2683-2693.

1. Kalff S, Meachem S, Preston C. Incidence of medial meniscal tears after arthroscopic assisted tibial plateau leveling osteotomy. Vet Surg, 2011, 40(8): 952-956.
2. Gu YL, Wang YB. Treatment of meniscal injury: a current concept review. Chin J Traumatol, 2010, 13(6): 370-376.
3. Eli N, Oragui E, Khan W. Advances in meniscal tissue engineering. Ortop Traumatol Rehabil, 2011, 13(4): 319-326.
4. 顾羊林, 朱文辉, 王予彬, 等. 犬成肌细胞体外向纤维软骨细胞表型分化的实验研究. 同济大学学报: 医学版, 2012, 33(2): 1-6.
5. Gu Y, Wang Y, Dai H, et al. Chondrogenic differentiation of canine myoblasts induced by cartilage-derived morphogenetic protein-2 and transforming growth factor-β1 in vitro. Mol Med Rep, 2012, 5(3): 767-772.
6. Asakura A, Komaki M, Rudnicki M. Muscle satellite cells are multipotential stem cells that exhibit myogenic, osteogenic and adipogenic differentiation. Differentiation, 2001, 68(4-5): 245-253.
7. Matsushita T, Matsui N, Fujioka H, et al. Expression of transcription factor Sox9 in rat L6 myoblastic cells. Connect Tissue Res, 2004, 45(3): 164-173.
8. Tuli R, Seghatoleslami MR, Tuli S, et al. p38 MAP kinase regulation of AP-2 binding in TGF-beta1-stimulated chondrogenesis of human trabecular bone-derived cells. Ann NY Acad Sci, 2002, 961: 172-177.
9. Freymann U, Endres M, Neumann K, et al. Expanded human meniscus-derived cells in 3-D polymer-hyaluronan scaffolds for meniscus repair. Acta Biomater, 2012, 8(2): 677-685.
10. Koning M, Harmsen MC, van Luyn MJ, et al. Current opportunities and challenges in skeletal muscle tissue engineering. J Tissue Eng Regen Med, 2009, 3(6): 407-415.
11. Liu Y, Li C, Wang HY, et al. Synthesis of Thermo- and pH-Sensitive Polyion Complex Micelles for Fluorescent Imaging. Chemistry, 2012, 18(8): 2297-2304.
12. Asadi H, Rostamizadeh K, Salari D, et al. Preparation and characterization of tri-block poly (lactide)-poly (ethylene glycol)-poly (lactide) nanogels for controlled release of naltrexone. Int J Pharm, 2011, 416(1): 356-364.
13. Singh D, Nayak V, Kumar A. Proliferation of myoblast skeletal cells on three-dimensional supermacroporous cryogels. Int J Biol Sci, 2010, 6(4): 371-381.
14. Steinert AF, Ghivizzani SC, Rethwilm A, et al. Major biological obstacles for persistent cell-based regeneration of articular cartilage. Arthritis Res Ther, 2007, 9(3): 213.
15. Tian H, Yang S, Xu L, et al. Chondrogenic differentiation of mouse bone marrow mesenchymal stem cells induced by cartilage-derived morphogenetic protein-2 in vitro. J Huazhong Univ Sci Technolog Med Sci, 2007, 27(4): 429-432.
16. Bobick BE, Chen FH, Le AM, et al. Regulation of the chondrogenic phenotype in culture. Birth Defects Res C Embryo Today, 2009, 87(4): 351-371.
17. Garg T, Singh O, Arora S, et al. Scaffold: a novel carrier for cell and drug delivery. Crit Rev Ther Drug Carrier Syst, 2012, 29(1): 1-63.
18. Subhash HM, Xie H, Smith JW, et al. Optical detection of indocyanine green encapsulated biocompatible poly (lactic-co-glycolic) acid nanoparticles with photothermal optical coherence tomography. Opt Lett, 2012, 37(5): 981-983.
19. Espandar L, Bunnell B, Wang GY, et al. Adipose-derived stem cells on hyaluronic Acid-derived scaffold: a new horizon in bioengineered cornea. Arch Ophthalmol, 2012, 130(2): 202-208.
20. Cui L, Wu Y, Cen L, et al. Repair of articular cartilage defect in non-weight bearing areas using adipose derived stem cells loaded polyglycolic acid mesh. Biomaterials, 2009, 30(14): 2683-2693.

Chinese Journal of Reparative and Reconstructive Surgery

HETEROTOPIC CHONDROGENESIS OF CANINE MYOBLASTS ON POLY (LACTIDE-CO-GLYCOLIDE) SCAFFOLDS IN VIVO

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