Dopamine modified and cartilage derived morphogenetic protein 1 laden polycaprolactone-hydroxyapatite composite scaffolds fabricated by three-dimensional printing improve chondrogenic differentiation of human bone marrow mesenchymal stem cells_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

XU Yan ¹ , WEI Bo ¹ , ZHOU Jin ¹ , YAO Qingqiang ¹ , WANG Liming ¹ ,  NA Jian ²

1. Department of Orthopedics, Nanjing Hospital Affiliated to Nanjing Medical University (Nanjing First Hospital), Nanjing Jiangsu, 210006, P.R.China;
2. Department of Orthopedics, Xuzhou Central Hospital, Xuzhou Jiangsu, 221009, P.R.China;

Corresponding author：

NA Jian, Email: Najian997@sina.com

Keywords：

Three-dimensional printing technology; dopamine; polycaprolactone; hydroxyapatite; composite scaffold; cartilage derived morphogenetic protein 1; bone marrow mesenchymal stem cells; cartilage tissue engineering

DOI：

10.7507/1002-1892.201708017

Video：

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ObjectiveTo prepare dopamine modified and cartilage derived morphogenetic protein 1 (CDMP1) laden polycaprolactone-hydroxyapatite (PCL-HA) composite scaffolds by three-dimensional (3D) printing and evaluate the effect of 3D scaffolds on in vitro chondrogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs).MethodsA dimensional porous PCL-HA scaffold was fabricated by 3D printing. Dopamine was used to modify the surface of PCL-HA and then CDMP-1 was loaded into scaffolds. The surface microstructure was observed by scanning electron microscope (SEM) and porosity and water static contact angle were also detected. The cytological experiment in vitro were randomly divided into 3 groups: group A (PCL-HA scaffolds), group B (dopamine modified PCL-HA scaffolds), and group C (dopamine modified and CDMP-1 laden PCL-HA scaffolds). The hBMSCs were seeded into three scaffolds, in chondrogenic culture conditions, the cell adhesive rate, the cell proliferation (MTT assay), and cell activity (Live-Dead staining) were analyzed; and the gene expressions of collagen type Ⅱ and Aggrecan were detected by real-time fluorescent quantitative PCR.ResultsThe scaffolds in 3 groups were all showed a cross-linked and pore interconnected with pore size of 400–500 μm, porosity of 56%, and fiber orientation of 0°/90°. For dopamine modification, the scaffolds in groups B and C were dark brown while in group A was white. Similarly, water static contact angle was from 76° of group A to 0° of groups B and C. After cultured for 24 hours, the cell adhesion rate of groups A, B, and C was 34.3%±3.5%, 48.3%±1.5%, and 57.4%±2.5% respectively, showing significant differences between groups (P<0.05). Live/Dead staining showed good cell activity of cells in 3 groups. MTT test showed that hBMSCs proliferated well in 3 groups and the absorbance (A) value was increased with time. The A value in group C was significantly higher than that in groups B and A, and in group B than in group A after cultured for 4, 7, 14, and 21 days, all showing significant differences (P<0.05). The mRNA relative expression of collagen type Ⅱ and Aggrecan increased gradually with time in 3 groups. The mRNA relative expression of collagen type Ⅱafter cultured for 7, 14, and 21 days, and the mRNA relative expression of Aggrecan after cultured for 14 and 21 days in group C were significantly higher than those in groups A and B, and in group B than in group A, all showing significant differences (P<0.05).ConclusionCo-culture of dopamine modified and CDMP1 laden PCL-HA scaffolds and hBMSCs in vitro can promote hBMSCs’ adhesion, proliferation, and chondrogenic differentiation.

Citation： XU Yan, WEI Bo, ZHOU Jin, YAO Qingqiang, WANG Liming, NA Jian. Dopamine modified and cartilage derived morphogenetic protein 1 laden polycaprolactone-hydroxyapatite composite scaffolds fabricated by three-dimensional printing improve chondrogenic differentiation of human bone marrow mesenchymal stem cells. Chinese Journal of Reparative and Reconstructive Surgery, 2018, 32(2): 215-222. doi: 10.7507/1002-1892.201708017 Copy

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16.	Lee YB, Shin YM, Lee JH, et al. Polydopamine-mediated immobilization of multiple bioactive molecules for the development of functional vascular graft materials. Biomaterials, 2012, 33(33): 8343-8352.
17.	Poh CK, Shi Z, Lim TY, et al. The effect of VEGF functionalization of titanium on endothelial cells in vitro. Biomaterials, 2010, 31(7): 1578-1585.
18.	Lai M, Cai K, Zhao L, et al. Surface functionalization of TiO2 nanotubes with bone morphogenetic protein 2 and its synergistic effect on the differentiation of mesenchymal stem cells. Biomacromolecules, 2011, 12(4): 1097-1105.
19.	Chien CY, Tsai WB. Poly (dopamine)-assisted immobilization of Arg-Gly-Asp peptides, hydroxyapatite, and bone morphogenic protein-2 on titanium to improve the osteogenesis of bone marrow stem cells. ACS Appl Mater Interfaces, 2013, 5(15): 6975-6983.
20.	Kim SE, Yun YP, Shim KS, et al. Effect of lactoferrin-impregnated porous poly (lactide-co-glycolide) (PLGA) microspheres on osteogenic differentiation of rabbit adipose-derived stem cells (rADSCs). Colloids Surf B Biointerfaces, 2014, 122: 457-464.
21.	Haeshin L, Dellatore SM, Miller WM, et al. Mussel-inspired surface chemistry for multifunctional coatings. Science, 2007, 318(5849): 426-430.
22.	Hotten GC, Matsumoto T, Kimura M, et al. Recombinant human growth/differentiation factor 5 stimulates mesenchyme aggregation and chondrogenesis responsible for the skeletal development of limbs. Growth Factors, 1996, 13(1-2): 65-74.
23.	Enochson L, Stenberg J, Brittberg M, et al. GDF5 reduces MMP13 expression in human chondrocytes via DKK1 mediated canonical Wnt signaling inhibition. Osteoarthritis Cartilage, 2014, 22(4): 566-577.

1. Grande DA, Schwartz JA, Brandel E, et al. Articular Cartilage Repair: Where We Have Been, Where We Are Now, and Where We Are Headed. Cartilage, 2013, 4(4): 281-285.
2. 裴福兴. 中国髋、膝关节置换的现状及展望. 中国骨与关节杂志, 2012, 1(1): 4-8.
3. Lee CH, Cook JL, Mendelson A, et al. Regeneration of the articular surface of the rabbit synovial joint by cell homing: a proof of concept study. Lancet, 2010, 376(9739): 440-448.
4. Yao Q, Wei B, Liu N, et al. Chondrogenic regeneration using bone marrow clots and a porous polycaprolactone-hydroxyapatite scaffold by three-dimensional printing. Tissue Eng Part A, 2015, 21(7-8): 1388-1397.
5. Poh CK, Shi Z, Lim TY, et al. The effect of VEGF functionalization of titanium on endothelial cells in vitro. Biomaterials, 2010, 31(7): 1578-1585.
6. Park K, Huang J, Azar F, et al. Scaffold-free, engineered porcine cartilage construct for cartilage defect repair in vitro and in vivo study. Artif Organs, 2006, 30(8): 586-596.
7. Steadman JR, Rodkey WG, Briggs KK. Microfracture to treat full-thickness chondral defects: surgical technique, rehabilitation, and outcomes. J Knee Surg, 2002, 15(3): 170-176.
8. Li WJ, Tuli R, Okafor C, et al. A three dimensional nanofibrous scaffold for cartilage tissue engineering using human mesenchymal stem cells. Biomaterials, 2005, 26(6): 599-609.
9. Lee M, Wu BM. Recent advances in 3D printing of tissue engineering scaffolds. Methods Mol Biol, 2012, 868: 257-267.
10. 吴天琦, 杨春喜. 可用于骨修复的 3D 打印多孔支架研究进展. 中国修复重建外科杂志, 2016, 30(4): 509-513.
11. Oh SH, Park IK, Kim JM, et al. In vitro and in vivo characteristics of PCL scaffolds with pore size gradient fabricated by a centrifugation method. Biomaterials, 2007, 28(9): 1664-1671.
12. Hardingham T, Tew S, Murdoch A. Tissue engineering: chondrocytes and cartilage. Arthritis Res, 2002, 4 Suppl 3: S63-68.
13. Heng BC, Cao T, Lee EH. Directing stem cell differentiation into the chondrogenic lineage in vitro. Stem Cells, 2004, 22(7): 1152-1167.
14. Place ES, Evans ND, Stevens MM. Complexity in biomaterials for tissue engineering. Nat Mater, 2009, 8(6): 457-470.
15. Sharon JL, Puleo DA. Immobilization of glycoproteins, such as VEGF, on biodegradable substrates. Acta Biomater, 2008, 4(4): 1016-1023.
16. Lee YB, Shin YM, Lee JH, et al. Polydopamine-mediated immobilization of multiple bioactive molecules for the development of functional vascular graft materials. Biomaterials, 2012, 33(33): 8343-8352.
17. Poh CK, Shi Z, Lim TY, et al. The effect of VEGF functionalization of titanium on endothelial cells in vitro. Biomaterials, 2010, 31(7): 1578-1585.
18. Lai M, Cai K, Zhao L, et al. Surface functionalization of TiO2 nanotubes with bone morphogenetic protein 2 and its synergistic effect on the differentiation of mesenchymal stem cells. Biomacromolecules, 2011, 12(4): 1097-1105.
19. Chien CY, Tsai WB. Poly (dopamine)-assisted immobilization of Arg-Gly-Asp peptides, hydroxyapatite, and bone morphogenic protein-2 on titanium to improve the osteogenesis of bone marrow stem cells. ACS Appl Mater Interfaces, 2013, 5(15): 6975-6983.
20. Kim SE, Yun YP, Shim KS, et al. Effect of lactoferrin-impregnated porous poly (lactide-co-glycolide) (PLGA) microspheres on osteogenic differentiation of rabbit adipose-derived stem cells (rADSCs). Colloids Surf B Biointerfaces, 2014, 122: 457-464.
21. Haeshin L, Dellatore SM, Miller WM, et al. Mussel-inspired surface chemistry for multifunctional coatings. Science, 2007, 318(5849): 426-430.
22. Hotten GC, Matsumoto T, Kimura M, et al. Recombinant human growth/differentiation factor 5 stimulates mesenchyme aggregation and chondrogenesis responsible for the skeletal development of limbs. Growth Factors, 1996, 13(1-2): 65-74.
23. Enochson L, Stenberg J, Brittberg M, et al. GDF5 reduces MMP13 expression in human chondrocytes via DKK1 mediated canonical Wnt signaling inhibition. Osteoarthritis Cartilage, 2014, 22(4): 566-577.

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Dopamine modified and cartilage derived morphogenetic protein 1 laden polycaprolactone-hydroxyapatite composite scaffolds fabricated by three-dimensional printing improve chondrogenic differentiation of human bone marrow mesenchymal stem cells

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