PREPARATION AND BIOCOMPATIBILITY EVALUATION OF A FUNCTIONAL SELF-ASSEMBLING PEPTIDE NANOFIBER HYDROGEL DESIGNED WITH LINKING THE SHORT FUNCTIONAL MOTIF OF BONE MORPHOGENETIC PROTEIN 7_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

LIULonggang ^1,2 , WUYaohong ² , TAOHui ² , JIAZhiwei ² , LIXiaochuan ² , WANGDeli ² , HEQing ² ,  RUANDike ^1,2

1. The Fourth Military Medical University, Xi'an Shaanxi, 710032, P. R. China;
2. Department of Orthopaedics, Navy General Hospital;

Corresponding author：

RUANDike, Email: ruandikengh@163.com

Keywords：

RADKPS; Functionalization; Self-assembling peptide; Hydrogel scaffold; Biocompatibility

DOI：

10.7507/1002-1892.20160098

Video：

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Objective To prepare of a novel functional self-assembling peptide nanofiber hydrogel scaffold RADKPS designed with linking the short functional motif of bone morphogenetic protein 7 (BMP-7) and to evaluate its biocompatibility so as to provide the experimental basis for in vivo studies on regeneration of degenerated nucleus pulposus tissue. Method A functional self-assembling peptide RADA-KPSS was designed by linking the short functional motif of BMP-7 to the self-assembling peptide RADA16-I. And the novel functional self-assembling peptide RADKPS was finally prepared by isometric mixing RADA16-I with RADA-KPSS. The structure characteristic of the functional self-assembling peptide nanofiber hydrogel scaffold RADKPS was evaluated by general observation and atomic force microscopy. Bone marrow mesenchymal stem cells (BMSCs) were isolated from 3-month-old New Zealand white rabbits and cultured. After the 3rd generation BMSCs were seeded on the peptide nanofiber hydrogel scaffold RADKPS for 7 days, the cellular compatibility of RADKPS was evaluated through scanning electron microscopy assay, cellular fluorescein diacetate/propidium iodide staining, and MTT assay. 1%RADKPS was injected into isolated intervertebral disc organs from 6-month-old New Zealand white rabbits, then the organs were cultured and the cellular activity of the intervertebral disc organs was observed. The blood compatibility of RADKPS was evaluated with hemolytic assay. After RADKPS was implanted into subcutaneous part of Kunming mice (aged 6-8 weeks) for 28 days, general observation and HE staining were carried out to evaluate the tissue compatibility. Results The functional self-assembling peptide solution RADKPS presented a homogeneous transparent hydrogel-like. Atomic force microscopy revealed that the RADKPS could self-assemble into three-dimensional nanofiber hydrogel scaffolds; the fibre diameter was (25.68±4.62) nm, and the fibre length was (512.42±32.22) nm. After BMSCs cultured on RADKPS for 7 days, scanning electron microscopy showed that BMSCs adhered to the scaffolds. And cell viability was maintained over 90%. MTT assay revealed that RADKPS of 0.1%, 0.05%, and 0.025% could increase the proliferation of BMSCs. The result of hemolytic assay revealed that the hemolysis rates of the RADKPS solutions with different concentrations were less than 5%, indicating that it met the requirement of hemolytic assay standard for medical biomaterials. After subcutaneous implantation, no vesicle, erythema, and eschar formation around injection site were observed. Meanwhile, HE staining showed inflammatory cells infiltration (lymphocytes), substitution of hydrogel scaffold by fibrous tissue, and good tissue compatibility. Conclusions The novel functional self-assembling peptide nanofiber hydrogel scaffold RADKPS has good biocompatibility and biological reliability, which would be suitable for tissue engineering repair and regeneration of nucleus pulposus tissue.

Citation： LIULonggang, WUYaohong, TAOHui, JIAZhiwei, LIXiaochuan, WANGDeli, HEQing, RUANDike. PREPARATION AND BIOCOMPATIBILITY EVALUATION OF A FUNCTIONAL SELF-ASSEMBLING PEPTIDE NANOFIBER HYDROGEL DESIGNED WITH LINKING THE SHORT FUNCTIONAL MOTIF OF BONE MORPHOGENETIC PROTEIN 7. Chinese Journal of Reparative and Reconstructive Surgery, 2016, 30(4): 491-498. doi: 10.7507/1002-1892.20160098 Copy

1.	Liu X, Wang X, Wang X, et al. Functionalized self-assembling peptide nanofiber hydrogels mimic stem cell niche to control human adipose stem cell behavior in vitro. Acta Biomater, 2013, 9(6) :6798-6805.
2.	Koutsopoulos S, Zhang S. Long-term three-dimensional neural tissue cultures in functionalized self-assembling peptide hydrogels, matrigel and collagen I. Acta Biomater, 2013, 9(2) :5162-5169.
3.	Horii A, Wang X, Gelain F, et al. Biological designer self-assembling peptide nanofiber scaffolds significantly enhance osteoblast proliferation,differentiation and 3-D migration. PLoS One, 2007, 2(2) :e190.
4.	An HS, Takegami K, Kamada H, et al. Intradiscal administration of osteogenic protein-1 increases intervertebral disc height and proteoglycan content in the nucleus pulposus in normal adolescent rabbits. Spine (Phila Pa 1976) , 2005, 30(1) :25-32.
5.	Imai Y, Miyamoto K, An HS, et al. Recombinant human osteogenic protein-1 upregulates proteoglycan metabolism of human anulus fibrosus and nucleus pulposus cells. Spine (Phila Pa 1976) , 2007, 32(12) :1303-1310.
6.	Kawakami M, Hashizume H, Matsumoto T, et al. Safety of epidural administration of Osteogenic Protein-1 (OP-l/BMP-7) :behavioral and macroscopic observation. Spine (Phila Pa 1976) , 2007, 32(13) :1388-1393.
7.	Tao H, Zhang Y, Wang CF, et al. Biological evaluation of human degenerated nucleus pulposus cells in functionalized self-assembling peptide nanofiber hydrogel scaffold. Tissue Eng Part A, 2014, 20(11-12) :1621-1631.
8.	郑磊,王前,裴国献.可降解聚合物在骨组织工程中的应用进展. 中国修复重建外科杂志, 2000, 14(3) :175-180.
9.	ISO 10993.4-2002. Biological evaluation of medical devices-Part 4:Selection of tests for interactions with blood. ISO 10993.4-2002(E):1-30.
10.	刘晓峰, 张郑瑶, 邓旭明, 等. 纳米 TCP/明胶/鹿茸多肽复合材料的生物相容性评价. 中国修复重建外科杂志, 2009, 23(5) :598-601.
11.	张士杰, 裴庆国, 潘可风, 等. PLGA/HA支架材料生物相容性的动物实验研究. 口腔颌面外科杂志, 2007, 17(1) :40-45.
12.	Zhang S, Gelain F, Zhao X. Designer self-assembling peptide nanofiber scaffolds for 3D tissue cell cultures. Semin Cancer Biol, 2005, 15(5) :413-420.
13.	Ni N, Hu Y, Ren H, et al. Self-assembling peptide nanofiber scaffolds enhance dopaminergic differentiation of mouse plviripotent stem cells in 3-dimensional culture. PLoS One, 2013, 8(12) :e84504.
14.	Kisiday J, Jin M, Kurz B, et al. Self-assembling peptide hydrogel fosters chondrocyte extracellular matrix production and cell division:implications for cartilage tissue repair. Proc Natl Acad Sci U S A, 2002, 99(15) :9996-10001.
15.	Liu X, Wang X, Horii A, et al. In vivo studies on angiogenic activity of two designer self-assembling peptide scaffold hydrogels in the chicken embryo chorioallantoic membrane. Nanoscale, 2012, 4(8) :2720-2727.
16.	Guo HD, Cui GH, Yang JJ, et al. Sustained delivery of VEGF from designer self-assembling peptides improves cardiac function after myocardial mfarction. Biochem Biophys Res Cominun, 2012, 424(1) :105-111.
17.	Chaofeng W, Chao Z, Deli W, et al. Nucleus pulposus cells expressing hBMP7 can prevent the degeneration of allogenic IVD in a canine transplantation model. J Orthop Res, 2013, 31(9) :1366-1373.
18.	Chubinskaya S, Hurtig M, Rueger DC. OP-l/BMP-7 in cartilage repair. Int Orthop, 2007, 31(6) :773-781.
19.	Chen Y, Webster TJ. Increased osteoblast ftmctions in the presence ofBMP-7 short peptides for nanostructured biomaterial applications. J Biomed Mater Res A, 2009, 91(1) :296-304.
20.	Tao H, Wu Y, Li H, et al. BMP7-Based Functionalized Self-Assembling Peptides for Nucleus Pulposus Tissue Engineering. ACS Appl Mater Interfaces, 2015, 7(31) :17076-17087.
21.	Markova DZ, Kepler CK, Addya S, et al. An organ culture system to model early degenerative changes of the intervertebral disc Ⅱ:profiling global gene expression changes. Arthritis Res Ther, 2013, 15(5) :R121.
22.	Chan SC, Walser J, Ferquson SJ, et al. Duration-dependent influence of dynamic torsion on the intervertebral disc:an intact disc organ culture study. Eur Spine J, 2015, 24(11) :2402-2410.
23.	Jim B, Steffen T, Moir J, et al. Development of an intact intervertebral disc organ culture system in which degeneration can be induced as a prelude to studying repair potential. Eur Spine J, 2011, 20(8) :1244-1254.

1. Liu X, Wang X, Wang X, et al. Functionalized self-assembling peptide nanofiber hydrogels mimic stem cell niche to control human adipose stem cell behavior in vitro. Acta Biomater, 2013, 9(6) :6798-6805.
2. Koutsopoulos S, Zhang S. Long-term three-dimensional neural tissue cultures in functionalized self-assembling peptide hydrogels, matrigel and collagen I. Acta Biomater, 2013, 9(2) :5162-5169.
3. Horii A, Wang X, Gelain F, et al. Biological designer self-assembling peptide nanofiber scaffolds significantly enhance osteoblast proliferation,differentiation and 3-D migration. PLoS One, 2007, 2(2) :e190.
4. An HS, Takegami K, Kamada H, et al. Intradiscal administration of osteogenic protein-1 increases intervertebral disc height and proteoglycan content in the nucleus pulposus in normal adolescent rabbits. Spine (Phila Pa 1976) , 2005, 30(1) :25-32.
5. Imai Y, Miyamoto K, An HS, et al. Recombinant human osteogenic protein-1 upregulates proteoglycan metabolism of human anulus fibrosus and nucleus pulposus cells. Spine (Phila Pa 1976) , 2007, 32(12) :1303-1310.
6. Kawakami M, Hashizume H, Matsumoto T, et al. Safety of epidural administration of Osteogenic Protein-1 (OP-l/BMP-7) :behavioral and macroscopic observation. Spine (Phila Pa 1976) , 2007, 32(13) :1388-1393.
7. Tao H, Zhang Y, Wang CF, et al. Biological evaluation of human degenerated nucleus pulposus cells in functionalized self-assembling peptide nanofiber hydrogel scaffold. Tissue Eng Part A, 2014, 20(11-12) :1621-1631.
8. 郑磊,王前,裴国献.可降解聚合物在骨组织工程中的应用进展. 中国修复重建外科杂志, 2000, 14(3) :175-180.
9. ISO 10993.4-2002. Biological evaluation of medical devices-Part 4:Selection of tests for interactions with blood. ISO 10993.4-2002(E):1-30.
10. 刘晓峰, 张郑瑶, 邓旭明, 等. 纳米 TCP/明胶/鹿茸多肽复合材料的生物相容性评价. 中国修复重建外科杂志, 2009, 23(5) :598-601.
11. 张士杰, 裴庆国, 潘可风, 等. PLGA/HA支架材料生物相容性的动物实验研究. 口腔颌面外科杂志, 2007, 17(1) :40-45.
12. Zhang S, Gelain F, Zhao X. Designer self-assembling peptide nanofiber scaffolds for 3D tissue cell cultures. Semin Cancer Biol, 2005, 15(5) :413-420.
13. Ni N, Hu Y, Ren H, et al. Self-assembling peptide nanofiber scaffolds enhance dopaminergic differentiation of mouse plviripotent stem cells in 3-dimensional culture. PLoS One, 2013, 8(12) :e84504.
14. Kisiday J, Jin M, Kurz B, et al. Self-assembling peptide hydrogel fosters chondrocyte extracellular matrix production and cell division:implications for cartilage tissue repair. Proc Natl Acad Sci U S A, 2002, 99(15) :9996-10001.
15. Liu X, Wang X, Horii A, et al. In vivo studies on angiogenic activity of two designer self-assembling peptide scaffold hydrogels in the chicken embryo chorioallantoic membrane. Nanoscale, 2012, 4(8) :2720-2727.
16. Guo HD, Cui GH, Yang JJ, et al. Sustained delivery of VEGF from designer self-assembling peptides improves cardiac function after myocardial mfarction. Biochem Biophys Res Cominun, 2012, 424(1) :105-111.
17. Chaofeng W, Chao Z, Deli W, et al. Nucleus pulposus cells expressing hBMP7 can prevent the degeneration of allogenic IVD in a canine transplantation model. J Orthop Res, 2013, 31(9) :1366-1373.
18. Chubinskaya S, Hurtig M, Rueger DC. OP-l/BMP-7 in cartilage repair. Int Orthop, 2007, 31(6) :773-781.
19. Chen Y, Webster TJ. Increased osteoblast ftmctions in the presence ofBMP-7 short peptides for nanostructured biomaterial applications. J Biomed Mater Res A, 2009, 91(1) :296-304.
20. Tao H, Wu Y, Li H, et al. BMP7-Based Functionalized Self-Assembling Peptides for Nucleus Pulposus Tissue Engineering. ACS Appl Mater Interfaces, 2015, 7(31) :17076-17087.
21. Markova DZ, Kepler CK, Addya S, et al. An organ culture system to model early degenerative changes of the intervertebral disc Ⅱ:profiling global gene expression changes. Arthritis Res Ther, 2013, 15(5) :R121.
22. Chan SC, Walser J, Ferquson SJ, et al. Duration-dependent influence of dynamic torsion on the intervertebral disc:an intact disc organ culture study. Eur Spine J, 2015, 24(11) :2402-2410.
23. Jim B, Steffen T, Moir J, et al. Development of an intact intervertebral disc organ culture system in which degeneration can be induced as a prelude to studying repair potential. Eur Spine J, 2011, 20(8) :1244-1254.

Chinese Journal of Reparative and Reconstructive Surgery

PREPARATION AND BIOCOMPATIBILITY EVALUATION OF A FUNCTIONAL SELF-ASSEMBLING PEPTIDE NANOFIBER HYDROGEL DESIGNED WITH LINKING THE SHORT FUNCTIONAL MOTIF OF BONE MORPHOGENETIC PROTEIN 7

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