Theranostics 2020; 10(18):8227-8249. doi:10.7150/thno.44276

Research Paper

Self-assembling peptide hydrogels functionalized with LN- and BDNF- mimicking epitopes synergistically enhance peripheral nerve regeneration

Shuhui Yang1*, Chong Wang2*, Jinjin Zhu3*, Changfeng Lu2,4, Haitao Li2, Fuyu Chen2, Jiaju Lu1, Zhe Zhang1, Xiaoqing Yan1,5, He Zhao1, Xiaodan Sun1, Lingyun Zhao1, Jing Liang6, Yu Wang2✉, Jiang Peng2✉, Xiumei Wang1✉

1. State Key Laboratory of New Ceramics and Fine Processing, Key Laboratory of Advanced Materials of Ministry of Education, School of Materials Science and Engineering, Tsinghua University, Beijing 100084, China.
2. Institute of Orthopedics, Chinese PLA General Hospital, Beijing 100853, China; Co-innovation Center of Neuroregeneration, Nantong University, Nantong, Jiangsu Province 226007, China.
3. Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine & Key Laboratory of Musculoskeletal System Degeneration and Regeneration Translational Research of Zhejiang, Hangzhou 310016, China.
4. Department of Orthopaedics and Trauma, Peking University People's Hospital, Beijing 100191, China.
5. School of Clinical Medicine, Tsinghua University, Beijing 100084, China.
6. Department of Pediatrics, Tianjin Hospital, Tianjin University, No. 406 Jiefang Nan Road, Tianjin 300211, China.
*Contributed equally to this work.

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Citation:
Yang S, Wang C, Zhu J, Lu C, Li H, Chen F, Lu J, Zhang Z, Yan X, Zhao H, Sun X, Zhao L, Liang J, Wang Y, Peng J, Wang X. Self-assembling peptide hydrogels functionalized with LN- and BDNF- mimicking epitopes synergistically enhance peripheral nerve regeneration. Theranostics 2020; 10(18):8227-8249. doi:10.7150/thno.44276. Available from http://www.thno.org/v10p8227.htm

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Abstract

The regenerative capacity of the peripheral nervous system is closely related to the role that Schwann cells (SCs) play in construction of the basement membrane containing multiple extracellular matrix proteins and secretion of neurotrophic factors, including laminin (LN) and brain-derived neurotrophic factor (BDNF). Here, we developed a self-assembling peptide (SAP) nanofiber hydrogel based on self-assembling backbone Ac-(RADA)4-NH2 (RAD) dual-functionalized with laminin-derived motif IKVAV (IKV) and a BDNF-mimetic peptide epitope RGIDKRHWNSQ (RGI) for peripheral nerve regeneration, with the hydrogel providing a three-dimensional (3D) microenvironment for SCs and neurites.

Methods: Circular dichroism (CD), atomic force microscopy (AFM), and scanning electron microscopy (SEM) were used to characterize the secondary structures, microscopic structures, and morphologies of self-assembling nanofiber hydrogels. Then the SC adhesion, myelination and neurotrophin secretion were evaluated on the hydrogels. Finally, the SAP hydrogels were injected into hollow chitosan tubes to bridge a 10-mm-long sciatic nerve defect in rats, and in vivo gene expression at 1 week, axonal regeneration, target muscular re-innervation, and functional recovery at 12 weeks were assessed.

Results: The bioactive peptide motifs were covalently linked to the C-terminal of the self-assembling peptide and the functionalized peptides could form well-defined nanofibrous hydrogels capable of providing a 3D microenvironment similar to native extracellular matrix. SCs displayed improved cell adhesion on hydrogels with both IKV and RGI, accompanied by increased cell spreading and elongation relative to other groups. RSCs cultured on hydrogels with IKV and RGI showed enhanced gene expression of NGF, BDNF, CNTF, PMP22 and NRP2, and decreased gene expression of NCAM compared with those cultured on other three groups after a 7-day incubation. Additionally, the secretion of NGF, BDNF, and CNTF of RSCs was significantly improved on dual-functionalized peptide hydrogels after 3 days. At 1 week after implantation, the expressions of neurotrophin and myelin-related genes in the nerve grafts in SAP and Autograft groups were higher than that in Hollow group, and the expression of S100 in groups containing both IKV and RGI was significantly higher than that in groups containing either IKV or RGI hydrogels, suggesting enhanced SC proliferation. The morphometric parameters of the regenerated nerves, their electrophysiological performance, the innervated muscle weight and remodeling of muscle fibers, and motor function showed that RAD/IKV/RGI and RAD/IKV-GG-RGI hydrogels could markedly improve axonal regeneration with enhanced re-myelination and motor functional recovery through the synergetic effect of IKV and RGI functional motifs.

Conclusions: We found that the dual-functionalized SAP hydrogels promoted RSC adhesion, myelination, and neurotrophin secretion in vitro and successfully bridged a 10-mm gap representing a sciatic nerve defect in rats in vivo. The results demonstrated the synergistic effect of IKVAV and RGI on axonal regrowth and function recovery after peripheral nerve injury.

Keywords: peripheral nerve regeneration, self-assembling peptide, neurotrophic peptide, laminin, nanofiber hydrogel