BIO-MODIFICATION OF POLYHYDROXYALKANOATES AND ITS BIOCOMPATIBILITY WITH CHONDROCYTES_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

GAOTianxi ¹ , CHANGHuimin ² , FANMinjie ³ , LUXiaoyun ⁴ ,  WANGZhenghui ¹ , ZHANGXianghong ¹ , JINGXiaohong ¹ , SHIYanxia ¹ , LIZhihui ¹

1. Department of Otolaryngology-Head and Neck Surgery, the Second Affiliated Hospital, Xi'an Jiaotong University, Xi'an Shaanxi, 710004, P. R. China;
2. Department of Otorhinolaryngology, Affiliated Hospital of Xi'an Medical College;
3. Department of Otorhinolaryngology, People's Hospital of Hancheng, Xi'an Shaanxi, 710004, P. R. China;
4. School of Life Science and Technology, Xi'an Jiaotong University, Xi'an Shaanxi, 710004, P. R. China;

Corresponding author：

WANGZhenghui, Email: ehui4298@163.com

Keywords：

Cartilage tissue engineering; Polyhydroxyalkanoates; Poly (3-hydroxybutyrate-co-3-hydroxyvalerate); Poly (3-hydroxybutyrate-co-3-hydroxyhexanoate); Polyhydroxyalkanoates granule binding protein fused with Arg-Gly-Asp peptide fusion proteins; Biocompatibility

DOI：

10.7507/1002-1892.20140224

Video：

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Abstract Full text Figures/Tables Video References Cited by

Objective To study the hydrophilicity and the cell biocompatibility of the poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) coated with a fusion protein polyhydroxyalkanoates granule binding protein (PhaP) fused with Arg-Gly-Asp (RGD) peptide (PhaP-RGD). Methods PHBV and PHBHHx films were fabricated by solvent evaporation.Scanning electronic microscope (SEM) was used to study the morphology of the films.PhaP-RGD fusion proteins were expressed and purified by the technology of protein engineering; PHBV and PHBHHx films were immersed in the PhaP-RGD with an amount of 3.5 mg/mL protein/per sample respectively.The hydrophilicity of the surface were detected by the contact angle measurements.Septal cartilage cells obtained from human septal cartilage were cultured in vitro.The 2nd passage chondrocytes were incubated on PHBV unmodified with PhaP-RGD in group A1,PHBV modified with PhaP-RGD in group A2,PHBHHx unmodified with PhaP-RGD in group B1,PHBHHx modified with PhaP-RGD in group B2,and on the cell culture plates in group C.After cultured for 3 days,the proliferation of cells was detected by the DAPI staining; the proliferation viability of cells was detected by the MTT assay after cultured for 3 and 7 days; after cultured for 7 days,the adhesion and morphology of the cells on the surface of the biomaterial films were observed by SEM and the matrix of the cells was detected through the toluidine blue staining. Results SEM observation showed that PHBV and PHBHHx films had porous structures.The contact angle of the surface of the PHBV and PHBHHx films modified with PhaP-RGD fusion proteins were significantly reduced when compared with the films unmodified with PhaP-RGD fusion proteins (P<0.05).Chondrocytes of human nasal septal cartilage incubated on the films could grow in all groups.After 3 days of cultivation in vitro,the cell proliferation and viability of group B2 were the strongest among all groups (P<0.05); the cell proliferation after cultured for 7 days was significantly stronger than that after cultured for 3 days in groups A1,A2,B1,and B2 (P<0.05); and the cell proliferation was significantly stronger in groups B1 and B2 than groups A1,A2 and C,in group B2 than group B1,and in group A1 than group A2 (P<0.05).The results of toluidine blue staining showed that blue metachromasia matrixes were observed in groups A1,A2,B1,and B2; group A1 and group A2 had similar staining degree,and the staining of group B2 was deeper than that of group B1.The adhesion of cells in all groups was good through SEM observation; and the connection of cells formed and stretched into the pores of the materials. Conclusion The biomaterial films of PHBHHx modified with PhaP-RGD fusion protein can promote its biocompatibility with chondrocytes.

Citation： GAOTianxi, CHANGHuimin, FANMinjie, LUXiaoyun, WANGZhenghui, ZHANGXianghong, JINGXiaohong, SHIYanxia, LIZhihui. BIO-MODIFICATION OF POLYHYDROXYALKANOATES AND ITS BIOCOMPATIBILITY WITH CHONDROCYTES. Chinese Journal of Reparative and Reconstructive Surgery, 2014, 28(8): 1023-1029. doi: 10.7507/1002-1892.20140224 Copy

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13.	闫虎,苏友新,林学义,等.Ⅱ型胶原酶消化法培养兔关节软骨细胞.中国组织工程研究,2013,17(50):8647-8653.
14.	Xie H,Li J,Li L,et al.Enhanced proliferation and differentiation of neural stem cells grown on PHA films coated with recombinant fusion proteins.Acta biomater,2013,9(8):7845-7854.
15.	Wu CS,Liao HT.The mechanical properties,biocompatibility and biodegradability of chestnut shell fibre and polyhydroxyalkanoate composites.Polymer Degradation and Stability,2014,99:274-282.
16.	Lomas AJ,Webb WR,Han JF,et al.Poly (3-hydroxybutyrate-co-3-hydroxyhexanoate)/collagen hybrid scaffolds for tissue engineering applications.Tissue Eng Part C Methods,2013,19(8):577-585.
17.	Choi JS,Kim BS,Kim JD,et al.In vitro cartilage tissue engineering using adipose-derived extracellular matrix scaffolds seeded with adipose-derived stem cells.Tissue Eng Part A,2011,18(1-2):80-92.
18.	Wang Y,Jiang XL,Peng SW,et al.Induced apoptosis of osteoblasts proliferating on polyhydroxyalkanoates.Biomaterials,2013,34(15):3737-3746.
19.	Puppi D,Chiellini F,Piras A M,et al.Polymeric materials for bone and cartilage repair.Progress in Polymer Science,2010,35(4):403-440.
20.	You M,Peng G,Li J,et al.Chondrogenic differentiation of human bone marrow mesenchymal stem cells on polyhydroxyalkanoate (PHA) scaffolds coated with PHA granule binding protein PhaP fused with RGD peptide.Biomaterials,2011,32(9):2305-2313.
21.	Yang X,Zhao K,Chen GQ.Effect of surface treatment on the biocompatibility of microbial polyhydroxyalkanoates.Biomaterials,2002,23(5):1391-1397.
22.	Rowland CR,Lennon DP,Caplan AI,et al.The effects of crosslinking of scaffolds engineered from cartilage ECM on the chondrogenic differentiation of MSCs.Biomaterials,2013,34(23):5802-5812.
23.	Appelman TP,Mizrahi J,Elisseeff JH,et al.The influence of biological motifs and dynamic mechanical stimulation in hydrogel scaffold systems on the phenotype of chondrocytes.Biomaterials,2011,32(6):1508-1516.
24.	徐磊,叶朝阳,周燕,等.体外传代培养兔关节软骨细胞的去分化现象.中国组织工程研究,2013,17(20):3626-3634.

1. Lu XY,Wang LL,Yang ZQ,et al.Strategies of polyhydroxyalkanoates modification for the medical application in neural regeneration/nerve tissue engineering.Advances in Bioscience & Biotechnology,2013,4(6):731.
2. Jacobs T,Morent R,De Geyter N,et al.Plasma surface modification of biomedical polymers:influence on cell-material interaction.Plasma Chem Plasma Process,2012,32(5):1039-1073.
3. Chen G,Wang Y.Medical applications of biopolyesters polyhydroxyalkanoates.Chinese Journal of Polymer Science,2013,31(5):719-736.
4. Yang Q,Wang J,Zhang S,et al.The properties of poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) and its applications in tissue engineering.Curr Stem Cell Res Ther,2014,9(3):215-222.
5. Sudesh K.Potential Applications of PHA//Polyhydroxyalkanoates from palm oil:biodegradable plastics.Berlin:Springer,2013:79-100.
6. Dong CL,Li SY,Wang Y,et al.The cytocompatability of polyhydroxyalkanoates coated with a fusion protein of PHA repressor protein (PhaR) and Lys-Gln-Ala-Gly-Asp-Val (KQAGDV) polypeptide.Biomaterials,2012,33(9):2593-2599.
7. He Y,Hu Z,Ren M,et al.Evaluation of PHBHHx and PHBV/PLA fibers used as medical sutures.J Mater Sci Mater Med,2014,25(2):561-571.
8. Liu J,Zhao B,Zhang Y,et al.PHBV and predifferentiated human adipose-derived stem cells for cartilage tissue engineering.J Biomed Mater Res A,2010,94(2):603-610.
9. Wang YY,Lu? LX,Shi JC,et al.Introducing RGD peptides on PHBV films through PEG-containing cross-linkers to improve the biocompatibility.Biomacromolecules,2011,12(3):551-559.
10. Dong Y,Li P,Chen CB,et al.The improvement of fibroblast growth on hydrophobic biopolyesters by coating with polyhydroxyalkanoate granule binding protein PhaP fused with cell adhesion motif RGD.Biomaterials,2010,31(34):8921-8930.
11. 王朝强,田丰德,谢辉,等.犬关节软骨细胞的体外分离与培养.中国组织工程研究,2012,16(46):8593-8598.
12. Qing C,Wei-ding C,Wei-min F.Co-culture of chondrocytes and bone marrow mesenchymal stem cells in vitro enhances the expression of cartilaginous extracellular matrix components.Braz J Med Biol Res,2011,44(4):303-310.
13. 闫虎,苏友新,林学义,等.Ⅱ型胶原酶消化法培养兔关节软骨细胞.中国组织工程研究,2013,17(50):8647-8653.
14. Xie H,Li J,Li L,et al.Enhanced proliferation and differentiation of neural stem cells grown on PHA films coated with recombinant fusion proteins.Acta biomater,2013,9(8):7845-7854.
15. Wu CS,Liao HT.The mechanical properties,biocompatibility and biodegradability of chestnut shell fibre and polyhydroxyalkanoate composites.Polymer Degradation and Stability,2014,99:274-282.
16. Lomas AJ,Webb WR,Han JF,et al.Poly (3-hydroxybutyrate-co-3-hydroxyhexanoate)/collagen hybrid scaffolds for tissue engineering applications.Tissue Eng Part C Methods,2013,19(8):577-585.
17. Choi JS,Kim BS,Kim JD,et al.In vitro cartilage tissue engineering using adipose-derived extracellular matrix scaffolds seeded with adipose-derived stem cells.Tissue Eng Part A,2011,18(1-2):80-92.
18. Wang Y,Jiang XL,Peng SW,et al.Induced apoptosis of osteoblasts proliferating on polyhydroxyalkanoates.Biomaterials,2013,34(15):3737-3746.
19. Puppi D,Chiellini F,Piras A M,et al.Polymeric materials for bone and cartilage repair.Progress in Polymer Science,2010,35(4):403-440.
20. You M,Peng G,Li J,et al.Chondrogenic differentiation of human bone marrow mesenchymal stem cells on polyhydroxyalkanoate (PHA) scaffolds coated with PHA granule binding protein PhaP fused with RGD peptide.Biomaterials,2011,32(9):2305-2313.
21. Yang X,Zhao K,Chen GQ.Effect of surface treatment on the biocompatibility of microbial polyhydroxyalkanoates.Biomaterials,2002,23(5):1391-1397.
22. Rowland CR,Lennon DP,Caplan AI,et al.The effects of crosslinking of scaffolds engineered from cartilage ECM on the chondrogenic differentiation of MSCs.Biomaterials,2013,34(23):5802-5812.
23. Appelman TP,Mizrahi J,Elisseeff JH,et al.The influence of biological motifs and dynamic mechanical stimulation in hydrogel scaffold systems on the phenotype of chondrocytes.Biomaterials,2011,32(6):1508-1516.
24. 徐磊,叶朝阳,周燕,等.体外传代培养兔关节软骨细胞的去分化现象.中国组织工程研究,2013,17(20):3626-3634.

Chinese Journal of Reparative and Reconstructive Surgery

BIO-MODIFICATION OF POLYHYDROXYALKANOATES AND ITS BIOCOMPATIBILITY WITH CHONDROCYTES

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