Cell-seeded porous silk fibroin scaffolds promotes axonal regeneration and myelination in spinal cord injury rats

https://doi.org/10.1016/j.bbrc.2019.04.137Get rights and content

Highlights

  • PSFSs promote functional recovery of defected nerve.

  • BMSCs and PSFSs are very compatible.

  • PSFSs seeded with BMSCs improve the recovery of SCI rats.

Abstract

Accumulating evidence indicates that a suitable scaffold designed for the spinal cord injury (SCI) was needed to enhance the survival of transplanted Bone mesenchymal stem cells (BMSCs) and to promote nerve regeneration. The current study was aimed to evaluate the effect of the porous silk fibroin scaffold (PSFSs) seeded with BMSCS on nerve regeneration, myelination and functional recovery after SCI. We previously demonstrated that the PSFSs could bridge defected nerve with nerve fibers when applied to the transected spinal cord. And we found that BMSCs were adhered to the scaffold closely and have good biological compatibility with PSFSs. PSFSs seeded with BMSCs exhibited significant improvement in complete transverse thoracic SCI rat models. Flow cytometric assay also indicated that BMSCs grew well and adhered closely to the surface of the scaffold. The Basso-Beattie-Bresnehan (BBB) scores at each time point showed that the hindlimb motor function of each transplant group was also significantly restored. Meanwhile, growth associated protein 43 (GAP-43)marker of damaged axons regeneration and myelin basic protein (MBP) marker of maintaining the myelin structural and functional integrity, all markedly increased in PSFSs seeded with BMSCs models. Together, these results demonstrated that transplantation of PSFSs seeded with BMSCS could promote the nerve regeneration, myelination and functional recovery after SCI.

Introduction

Spinal cord injury (SCI) caused by a variety of direct and indirect reasons such as traffic accidents, infections, genetics, diving accidents and so on, bringing huge property damage and mental damage to patients, and also seriously restricts social stability [[1], [2], [3], [4]]. Although researches on the pathologic mechanisms of SCI made great progress, the prevention and treatment of SCI remains a major problem which troubles the medical profession [4]. Therefore, effective drugs and new therapeutic strategies have become the focus on SCI. Recent studies showed that natural products or components such as resveratrol and deferoxamine provide a new direction for adjuvant treatment of SCI [5,6], even have researches reported that delivery zonisamide could be a great way to cure SCI through loaded on a biosynthesis of gold nanoparticles. However, drug treatment alone often does not achieve satisfactory therapeutic efficacy and many drugs also affect the prognosis of the disease due to its poor selectivity and often accompanied by serious side effects. Thus, development of cell-specific therapy solutions will become a new strategy for SCI treatment.

After SCI, serious spinal cord tissue injury and nerve cells death are mainly manifested, followed by local ischemia, inflammation, a large numbers of neurons and oligodendrocytes deaths, then formation of glial scar and cystic cavities. Finally, an inhibitory microenvironment was formed in the damaged area that to blocked axon regeneration [7]. In addition, the poor internal growth potential of adult neurons leads to low spontaneous regeneration of spinal cord, which eventually leads to permanent nerve defects [[8], [9], [10], [11]]. Newly studies have shown that some adult neurons and axons are still capable of regeneration by regulating the intrinsic growth potential of nerve cells or by removing certain inhibiting factors in the microenvironment of SCI [[12], [13], [14], [15]]. We previous studies on the biocompatibility of seed cells such as olfactory sheath cells [16] and bone marrow mesenchymal stem cells (BMSCS) showed that they had good biocompatibility with the fibroin protein material. However, through stem cell transplantation only does not achieve good results in treatment of SCI cause lack of structural basis [17]. Meanwhile, there is no ideal that scaffold for treating SCI in consideration of the preparation cost, mechanical properties and degradation products. Therefore, combined with an auxiliary bionic organization structure, will increase BMSCs growth, reduce social burden and facilitate the recovery of SCI.

In recent years, stem cell therapy for SCI has been developed from simple cell transplantation to a combined therapy based on biological scaffold combining various therapeutic factors and stem cells, and has been achieved certain curative effect [18]. As one of the seed cells for tissue engineering, BMSCs are easy to obtain and amplify in vitro without ethical issues, which makes it be a promising cells to treatment SCI [19]. We previously reported that silk fibroin scaffold has good compatibility with olfactory ensheathing cells [[16], [17], [18], [19], [20], [21]]. These results implied silk fibroin scaffold could be a suitable scaffold for SCI repair. Attractively, whether fibroin material scaffold regulates BMSCs growth by modulating the above or another potential signaling pathway in SCI is interesting.

In the present study, we evaluated the effect of porous silk fibroin scaffold (PSFSs) seeded with BMSCS on Axon regeneration, myelination, functional recovery, and further elucidated the underlying mechanisms. We found that seeded with BMSCS, Porting PSFSs could alleviate SCI via GAP-43 and MBP dependent promotion pathway in total spinal cord excision rats. Our study provided a rationale for the identification of PSFSs seeded with BMSCS as a potent therapeutic agent for the treatment of SCI.

Section snippets

BMSC proliferation and identification

Primary BMSCs were separated from the femurs and tibias of 6–8 week-old Sprague-Dawley (SD) rats and cultured under sterile 10 cm petridish [22]. Antibodies such as CD34, CD45, CD90, and CD105 (BD PharMingen, USA) were used to detect the third passage BMSCs by flow cytometry (FCM; Germany) [23].

Preparation of the PSFSs

The Scaffolds were obtained as previously reported [24]. The structure was observed by scanning electron microscopy (SEM) after the scaffold surface was sprayed with gold.

Labeling of BMSCs with 5-Bromodeoxyuridine (BrdU)

The BMSCs were cultured with

PSFSs were observed by SEM

Previous studies have demonstrated that PSFSs has the characteristics of uniform gap and reasonable space, which is beneficial for BMSCs to growing in it [27]. Here we firstly prepared PSFSs by using silk protein aqueous solution. Results from ECM assay showed that the internal structure of the scaffold is three-dimensional, porous, with pores arranged along the longitudinal, transverse direction and with certain connectivity. The average diameter of uniformly distributed pores is about

Discussion

The goal of tissue engineering is to repair damaged or defective tissue by combining cell transplantation and biological scaffolds [29]. After SCI the support of extracellular matrix disappeared and the local mechanical microenvironment was damaged, therefore, the ideal scaffold should provide non-cellular mechanical support for the regeneration of nerve tissue and simulate the local microenvironment to promote its regeneration in vivo. The PSFSs which prepared in our researches not only

Disclosure

The authors have no conflicts to disclose.

Authors' contributions

KY and XL made substantial contributions to the study conception and design. KY, HC, PZ, FZ and XL collected and analyzed the data. KY, YZ, FC, LL and XL interpreted the data. All authors were involved in drafting the manuscript and provided final approval of this manuscript.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (81271723, 65985117 and 65982875), Youth Talent Development Program and International cooperation and exchange projects (65983652), Technological Innovation and Popular Science SITP Project (65982406), Think tank and academic influence arts project (65978660), Interdisciplinary pre-research projects and other projects (65980475).

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