Application potential of bone marrow mesenchymal stem cell (BMSCs) based tissue-engineering for spinal cord defect repair in rat fetuses with spina bifida aperta

Abstract

Spina bifida aperta are complex congenital malformations resulting from failure of fusion in the spinal neural tube during embryogenesis. Despite surgical repair of the defect, most patients who survive with spina bifida aperta have a multiple system handicap due to neuron deficiency of the defective spinal cord. Tissue engineering has emerged as a novel treatment for replacement of lost tissue. This study evaluated the prenatal surgical approach of transplanting a chitosan-gelatin scaffold seeded with bone marrow mesenchymal stem cells (BMSCs) in the healing the defective spinal cord of rat fetuses with retinoic acid induced spina bifida aperta. Scaffold characterisation revealed the porous structure, organic and amorphous content. This biomaterial promoted the adhesion, spreading and in vitro viability of the BMSCs. After transplantation of the scaffold combined with BMSCs, the defective region of spinal cord in rat fetuses with spina bifida aperta at E20 decreased obviously under stereomicroscopy, and the skin defect almost closed in many fetuses. The transplanted BMSCs in chitosan-gelatin scaffold survived, grew and expressed markers of neural stem cells and neurons in the defective spinal cord. In addition, the biomaterial presented high biocompatibility and slow biodegradation in vivo. In conclusion, prenatal transplantation of the scaffold combined with BMSCs could treat spinal cord defect in fetuses with spina bifida aperta by the regeneration of neurons and repairmen of defective region.

Fig. 1

Operation process of fetal surgery and implantation. The black arrow indicate the cell-scaffold construct

Fig. 2

a The LM micrograph of BMSCs after adherent 24 h; b the LM micrograph of BMSCs to P3 generation showing the characteristic of plastic adherence and spindle shape. c The result of flow cytometry histograms demonstrating high expression of non-hematopoietic markers of CD90 and CD44 and low expression for CD34 and CD45. d,e The figure d, e was in the same perspective, after GFP adenovirus transfection, the efficiency rate can reach above 90 %. The scale of all the pictures are 100 μm

Fig. 3

Scaffold characterisation. a, b SEM photomicrographs of the chitosan–gelatin scaffold. c Macroscopic aspects similar to a sponge

Fig. 4

Characterization of the cell-scaffold construct. a The chitosan-gelatin scaffold seeded with GFP postive BMSCs. Before transplantation, the cell-scaffold construct was incubated for 48 h at 37 °C with 5 % CO₂. b FDA- Pl staining of the cell-scaffold construct. c–e Scanning electron microscopy of the cell-scaffold construct

Fig. 5

The repair of the defective spinal cord after cell-scaffold transplantation. a Stereomicroscopic imaging of fetus with spina bifida aperta showed the skin defect almost closed after cell-scaffold transplantation. The black object indicated by arrow was cell-scaffold construct. b Fluorescent stereomicroscopic imaging (same vision with figure a) showed the most of GFP labelled BMSCs were stayed in the scaffold (white arrow), and many GFP labelled BMSCs migrate outside to the scaffold and spread to the defective region of spina bifida aperta (red arrow). c Stereomicroscopic imaging of fetus with spina bifida aperta without cell-scaffold transplantation showed obvious defective spinal cord(view multiple was ×2). d Frozen transverse section of defective spinal cord with cell-scaffold (same fetus with Fig. 6a, the thickness of 20 μm), the red was the scaffold, the green was GFP labelled BMSCs (the scale was 100 μm)

Fig. 6

The differentiation of BMSCs in transplanted cell-scaffold construct in spinal column. The linear region is double positive cells, namely the GFP-BMSCs after transplantation in vivo differentiated into neural stem cells (Nestin) and neurons (Tubulin). (the vision are all in ×20)