Preparation and <i>in vitro</i> evaluation of tissue engineered osteochondral integration of multi-layered scaffold _Chinese Journal of Reparative and Reconstructive Surgery

Authors：

LI Jianwei ¹ , ZHANG Xueliang ^1,2 , GUO Quanyi ² , ZHANG Jingchun ¹ , CAO Yanjie ¹ , ZHANG Xinguo ¹ , HUANG Jianjun ¹ , WANG Qi ¹ , LIU Xiaogang ¹ ,  HAO Chunxiang ²

1. Shanxi Institute of Traditional Chinese Medicine, Taiyuan Shanxi, 030012, P.R.China;
2. Institute of Orthopaedics, Beijing Key Laboratory of Regenerative Medicine in Orthopaedics, Key Laboratory of Musculoskeletal Trauma & War Injuries, Chinese PLA General Hospital, Beijing, 100853, P.R.China;

Corresponding author：

HAO Chunxiang, Email: haochunxiang@163.com

Keywords：

Osteochondral; tissue engineering; multi-layered scaffold; extracellular matrix; mechanical property; biocompatibility

DOI：

10.7507/1002-1892.201712038

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ObjectiveThe tissue engineered osteochondral integration of multi-layered scaffold was prepared and the related mechanical properties and biological properties were evaluated to provide a new technique and method for the repair and regeneration of osteochondral defect.MethodsAccording to blend of different components and proportion of acellular cartilage extracellular matrix of pig, nano-hydroxyapatite, and alginate, the osteochondral integration of multi-layered scaffold was prepared by using freeze-drying and physical and chemical cross-linking technology. The cartilage layer was consisted of acellular cartilage extracellular matrix; the middle layer was consisted of acellular cartilage extracellular matrix and alginate; and the bone layer was consisted of nano-hydroxyapatite, alginate, and acellular cartilage extracellular matrix. The biological and mechanics characteristic of the osteochondral integration of multi-layered scaffold were evaluated by morphology observation, scanning electron microscope observation, Micro-CT observation, porosity and pore size determination, water absorption capacity determination, mechanical testing (compression modulus and layer adhesive strength), biocompatibility testing [L929 cell proliferation on scaffold assessed by MTT assay, and growth of green fluorescent protein (GFP)-labeled Sprague Dawley rats’ bone marrow mesenchumal stem cells (BMSCs) on scaffolds].ResultsGross observation and Micro-CT observation showed that the scaffolds were closely integrated with each other without obvious discontinuities and separation. Scanning electron microscope showed that the structure of the bone layer was relatively dense, while the structure of the middle layer and the cartilage layer was relatively loose. The pore structures in the layers were connected to each other and all had the multi-dimensional characteristics. The porosity of cartilage layer, middle layer, and bone layer of the scaffolds were 93.55%±2.90%, 93.55%±4.10%, and 50.28%±3.20%, respectively; the porosity of the bone layer was significantly lower than that of cartilage layer and middle layer (P<0.05), but no significant difference was found between cartilage layer and middle layer (P>0.05). The pore size of the three layers were (239.66±35.28), (153.24±19.78), and (82.72±16.94) μm, respectively, showing significant differences between layers (P<0.05). The hydrophilic of the three layers were (15.14±3.15), (13.65±2.98), and (5.32±1.87) mL/g, respectively; the hydrophilic of the bone layer was significantly lower than that of cartilage layer and middle layer (P<0.05), but no significant difference was found between cartilage layer and middle layer (P>0.05). The compression modulus of the three layers were (51.36±13.25), (47.93±12.74), and (155.18±19.62) kPa, respectively; and compression modulus of the bone layer was significantly higher than that of cartilage layer and middle layer (P<0.05), but no significant difference was found between cartilage layer and middle layer (P>0.05). The osteochondral integration of multi-layered scaffold was tightly bonded with each layer. The layer adhesive strength between the cartilage layer and the middle layer was (18.21±5.16) kPa, and the layer adhesive strength between the middle layer and the bone layer was (16.73±6.38) kPa, showing no significant difference (t=0.637, P=0.537). MTT assay showed that L929 cells grew well on the scaffolds, indicating no scaffold cytotoxicity. GFP-labeled rat BMSCs grew evenly on the scaffolds, indicating scaffold has excellent biocompatibility.ConclusionThe advantages of three layers which have different performance of the tissue engineered osteochondral integration of multi-layered scaffold is achieved double biomimetics of structure and composition, lays a foundation for further research of animal in vivo experiment, meanwhile, as an advanced and potential strategy for osteochondral defect repair.

Citation： LI Jianwei, ZHANG Xueliang, GUO Quanyi, ZHANG Jingchun, CAO Yanjie, ZHANG Xinguo, HUANG Jianjun, WANG Qi, LIU Xiaogang, HAO Chunxiang. Preparation and in vitro evaluation of tissue engineered osteochondral integration of multi-layered scaffold . Chinese Journal of Reparative and Reconstructive Surgery, 2018, 32(4): 434-440. doi: 10.7507/1002-1892.201712038 Copy

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9.	Lin HY, Tsai WC, Chang SH. Collagen-PVA aligned nanofiber on collagen sponge as bi-layered scaffold for surface cartilage repair. J Biomater Sci Polym Ed, 2017, 28(7): 664-678.
10.	Su JY, Chen SH, Chen YP, et al. Evaluation of magnetic nanoparticle-labeled chondrocytes cultivated on a type Ⅱ collagen-chitosan/poly (lactic-co-glycolic) acid biphasic scaffold. Int J Mol Sci, 2017, 18(1): pii: E87.
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12.	Nooeaid P, Salih V, Beier JP, et al. Osteochondral tissue engineering: scaffolds, stem cells and applications. J Cell Mol Med, 2012, 16(10): 2247-2270.
13.	Izadifar Z, Chen X, Kulyk W. Strategic design and fabrication of engineered scaffolds for articular cartilage repair. J Funct Biomater, 2012, 3(4): 799-838.
14.	Ruan SQ, Yan L, Deng J, et al. Preparation of a biphase composite scaffold and its application in tissue engineering for femoral osteochondral defects in rabbits. Int Orthop, 2017, 41(9): 1899-1908.
15.	Sartori M, Pagani S, Ferrari A, et al. A new bi-layered scaffold for osteochondral tissue regeneration: In vitro and in vivo preclinical investigations. Mater Sci Eng C Mater Biol Appl, 2017, 70(Pt 1): 101-111.
16.	Kon E, Roffi A, Filardo G, et al. Scaffold-based cartilage treatments: with or without cells? A systematic review of preclinical and clinical evidence. Arthroscopy, 2015, 31(4): 767-775.
17.	Levingstone TJ, Ramesh A, Brady RT, et al. Cell-free multi-layered collagen-based scaffolds demonstrate layer specific regeneration of functional osteochondral tissue in caprine joints. Biomaterials, 2016, 87: 69-81.
18.	Levingstone TJ, Matsiko A, Dickson GR, et al. A biomimetic multi-layered collagen-based scaffold for osteochondral repair. Acta Biomater, 2014, 10(5): 1996-2004.
19.	Shi W, Sun M, Hu X, et al. Structurally and functionally optimized silk-fibroin-gelatin scaffold using 3D printing to repair cartilage injury in vitro and in vivo. Adv Mater, 2017, 29(29). doi: 10.1002/adma.201701089.
20.	Filardo G, Kon E, Roffi A, et al. Scaffold-based repair for cartilage healing: a systematic review and technical note. Arthroscopy, 2013, 29(1): 174-186.
21.	Holmes B, Zhu W, Li J, et al. Development of novel three-dimensional printed scaffolds for osteochondral regeneration. Tissue Eng Part A, 2015, 21(1-2): 403-415.
22.	M JC, Reardon PJ, Konwarh R, et al. Mimicking hierarchical complexity of the osteochondral interface using electrospun silk-bioactive glass composites. ACS Appl Mater Interrfaces, 2017, 9(9): 8000-8013.
23.	Vindas Bolaños RA, Cokelaere SM, Estrada McDermott JM, et al. The use of a cartilage decellularized matrix scaffold for the repair of osteochondral defects: the importance of long-term studies in a large animal model. Osteoarthritis Cartilage, 2017, 25(3): 413-420.
24.	Vikingsson L, Claessens B, Gómez-Tejedor JA, et al. Relationship between micro-porosity, water permeability and mechanical behavior in scaffolds for cartilage engineering. J Mech Behav Biomed Mater, 2015, 48: 60-69.
25.	Amadori S, Torricelli P, Panzavolta S, et al. Multi-layered scaffolds for osteochondral tissue engineering: in vitro response of co-cultured human mesenchymal stem cells. Macromol Biosci, 2015, 15(11): 1535-1545.

1. Cucchiarini M, de Girolamo L, Filardo G, et al. Basic science of osteoarthritis. J Exp Orthop, 2016, 3(1): 22.
2. Boushell MK, Hung CT, Hunziker EB, et al. Current strategies for integrative cartilage repair. Connect Tissue Res, 2017, 58(5): 393-406.
3. Bowland P, Ingham E, Jennings L, et al. Review of the biomechanics and biotribology of osteochondral grafts used for surgical interventions in the knee. Proc Inst Mech Eng H, 2015, 229(12): 879-888.
4. Fischer S, Kisser A. Single-step scaffold-based cartilage repair in the knee: A systematic review. J Orthop, 2016, 13(4): 246-253.
5. Frenkel SR, Bradica G, Brekke JH, et al. Regeneration of articular cartilage--evaluation of osteochondral defect repair in the rabbit using multiphasic implants. Osteoarthritis Cartilage, 2005, 13(9): 798-807.
6. Sun K, Li R, Jiang W, et al. Comparison of three-dimensional printing and vacuum freeze-dried techniques for fabricating composite scaffolds. Biochem Biophys Res Commun, 2016, 477(4): 1085-1091.
7. Shi X, Zhou J, Zhao Y, et al. Gradient-regulated hydrogel for interface tissue engineering: steering simultaneous osteo/chondrogenesis of stem cells on a chip. Adv Healthc Mater, 2013, 2(6): 846-853.
8. Camarero-Espinosa S, Cooper-White J. Tailoring biomaterial scaffolds for osteochondral repair. Int J Pharm, 2017, 523(2): 476-489.
9. Lin HY, Tsai WC, Chang SH. Collagen-PVA aligned nanofiber on collagen sponge as bi-layered scaffold for surface cartilage repair. J Biomater Sci Polym Ed, 2017, 28(7): 664-678.
10. Su JY, Chen SH, Chen YP, et al. Evaluation of magnetic nanoparticle-labeled chondrocytes cultivated on a type Ⅱ collagen-chitosan/poly (lactic-co-glycolic) acid biphasic scaffold. Int J Mol Sci, 2017, 18(1): pii: E87.
11. Pouran B, Arbabi V, Bleys RL, et al. Solute transport at the interface of cartilage and subchondral bone plate: Effect of micro-architecture. J Biomech, 2017, 52: 148-154.
12. Nooeaid P, Salih V, Beier JP, et al. Osteochondral tissue engineering: scaffolds, stem cells and applications. J Cell Mol Med, 2012, 16(10): 2247-2270.
13. Izadifar Z, Chen X, Kulyk W. Strategic design and fabrication of engineered scaffolds for articular cartilage repair. J Funct Biomater, 2012, 3(4): 799-838.
14. Ruan SQ, Yan L, Deng J, et al. Preparation of a biphase composite scaffold and its application in tissue engineering for femoral osteochondral defects in rabbits. Int Orthop, 2017, 41(9): 1899-1908.
15. Sartori M, Pagani S, Ferrari A, et al. A new bi-layered scaffold for osteochondral tissue regeneration: In vitro and in vivo preclinical investigations. Mater Sci Eng C Mater Biol Appl, 2017, 70(Pt 1): 101-111.
16. Kon E, Roffi A, Filardo G, et al. Scaffold-based cartilage treatments: with or without cells? A systematic review of preclinical and clinical evidence. Arthroscopy, 2015, 31(4): 767-775.
17. Levingstone TJ, Ramesh A, Brady RT, et al. Cell-free multi-layered collagen-based scaffolds demonstrate layer specific regeneration of functional osteochondral tissue in caprine joints. Biomaterials, 2016, 87: 69-81.
18. Levingstone TJ, Matsiko A, Dickson GR, et al. A biomimetic multi-layered collagen-based scaffold for osteochondral repair. Acta Biomater, 2014, 10(5): 1996-2004.
19. Shi W, Sun M, Hu X, et al. Structurally and functionally optimized silk-fibroin-gelatin scaffold using 3D printing to repair cartilage injury in vitro and in vivo. Adv Mater, 2017, 29(29). doi: 10.1002/adma.201701089.
20. Filardo G, Kon E, Roffi A, et al. Scaffold-based repair for cartilage healing: a systematic review and technical note. Arthroscopy, 2013, 29(1): 174-186.
21. Holmes B, Zhu W, Li J, et al. Development of novel three-dimensional printed scaffolds for osteochondral regeneration. Tissue Eng Part A, 2015, 21(1-2): 403-415.
22. M JC, Reardon PJ, Konwarh R, et al. Mimicking hierarchical complexity of the osteochondral interface using electrospun silk-bioactive glass composites. ACS Appl Mater Interrfaces, 2017, 9(9): 8000-8013.
23. Vindas Bolaños RA, Cokelaere SM, Estrada McDermott JM, et al. The use of a cartilage decellularized matrix scaffold for the repair of osteochondral defects: the importance of long-term studies in a large animal model. Osteoarthritis Cartilage, 2017, 25(3): 413-420.
24. Vikingsson L, Claessens B, Gómez-Tejedor JA, et al. Relationship between micro-porosity, water permeability and mechanical behavior in scaffolds for cartilage engineering. J Mech Behav Biomed Mater, 2015, 48: 60-69.
25. Amadori S, Torricelli P, Panzavolta S, et al. Multi-layered scaffolds for osteochondral tissue engineering: in vitro response of co-cultured human mesenchymal stem cells. Macromol Biosci, 2015, 15(11): 1535-1545.

Chinese Journal of Reparative and Reconstructive Surgery

Preparation and in vitro evaluation of tissue engineered osteochondral integration of multi-layered scaffold

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