Implantation of regenerative complexes in traumatic brain injury canine models enhances the reconstruction of neural networks and motor function recovery

Abstract

Rationale: The combination of medical and tissue engineering in neural regeneration studies is a promising field. Collagen, silk fibroin and seed cells are suitable options and have been widely used in the repair of spinal cord injury. In this study, we aimed to determine whether the implantation of a complex fabricated with collagen/silk fibroin (SF) and the human umbilical cord mesenchymal stem cells (hUCMSCs) can promote cerebral cortex repair and motor functional recovery in a canine model of traumatic brain injury (TBI). Methods: A porous scaffold was fabricated with cross-linked collagen and SF. Its physical properties and degeneration rate were measured. The scaffolds were co-cultured with hUCMSCs after which an implantable complex was formed. After complex implantation to a canine model of TBI, the motor evoked potential (MEP) and magnetic resonance imaging (MRI) were used to evaluate the integrity of the cerebral cortex. The neurologic score, motion capture, surface electromyography (sEMG), and vertical ground reaction force (vGRF) were measured in the analysis of motor functions. In vitro analysis of inflammation levels was performed by Elisa while immunohistochemistry was used in track the fate of hUCMSCs. In situ hybridization, transmission electron microscope, and immunofluorescence were used to assess neural and vascular regeneration. Results: Favorable physical properties, suitable degradation rate, and biocompatibility were observed in the collagen/SF scaffolds. The group with complex implantation exhibited the best cerebral cortex integrity and motor functions. The implantation also led to the regeneration of more blood vessels and nerve fibers, less glial fibers, and inflammatory factors. Conclusion: Implantation of this complex enhanced therapy in traumatic brain injury (TBI) through structural repair and functional recovery. These effects exhibit the translational prospects for the clinical application of this complex.

Keywords: Collagen; Hemiplegic limb; Mesenchymal stem cell; Silk fibroin; Traumatic brain injury.

Figure 1

Timeline of the experiment.

Figure 2

Physical property test of collagen/SF scaffolds along with the schematic profile of complexes implantation and TBI repair. (A) The fabrication process of collagen/SF scaffolds (B) General view and microstructure of collagen/SF scaffolds. Scale bars: 2 mm (a-b), 1mm (c-d) 20 µm (e), 2 µm (f). (C) X-ray diffraction of collagen/SF scaffolds. (D) Infrared spectrum detection of collagen/SF scaffolds. (E) Evaluation of collagen fibroin scaffold by differential scanning calorimetry. (F) Compressive stress detection of collagen/SF scaffolds. (G) The degradation rate of collagen/SF scaffolds (H-I) Morphological changes of collagen/SF scaffolds at the time of 1 week, 2 weeks, 4 weeks and 8 weeks under HE staining and immunofluorescence. Scale bars: 100 µm. (J) Experimental grouping and corresponding interventions. (K-M) The schematic profile of complexes implantation and TBI repair.

Figure 3

Biocompatibility and toxicity test of collagen SF scaffold (A) Observation of hUCMSCs cultured on the surface and inside of collagen/SF scaffolds under inverted phase contrast microscopy and SEM. Scale bars: 100 µm (a-c), 20 µm (d). (B) HE staining of blank scaffolds and hUCMSCs co-cultured with collagen/SF scaffolds. Scale bars: 2.5 mm (a-b), 1 mm (b1), 200 µm (b2). (C) Identification of hUCMSCs with CD90 and CD105. Scale bars: 200 µm (a1-a4), 50 µm (a4'). (D) Immunofluorescence staining of surface markers of hUCMSCs when co-cultured with collagen/SF scaffolds. Scale bars: 200 µm (a1-a4), 50 µm (a4''). (E) CCK-8 test of hUCMSCs when co-cultured with collagen/SF scaffolds.

Figure 4

Electrophysiological examination and nuclear magnetic resonance evaluation. (A) MEP of four limbs under different levels of constant pressure stimulation (X1: left forelimb, X2: right forelimb, X3: left hindlimb, X4: right hindlimb) (B) Comparisons of MEP latency among groups under the levels of 90-120 V constant pressure stimulation (C) Comparisons of MEP amplitude among groups under the levels of 90-120 V constant pressure stimulation. (D) Observations on the integrity of corticospinal tract in TBI group, SC group, CS group, and CB group. (E) MR scanning of cerebral cortex in each group. (F) The results of spectroscopy imaging test on cerebral cortex lesions and comparisons of NAA/Cr, NAA/Cho and NAA/(Cho+Cr) values among groups. (*p < 0.05, **p < 0.01, compared with TBI group; ^#P < 0.05, ^##p < 0.01, compared with SC group; ⁺P < 0.05, ⁺⁺P < 0.01, compared with CS group).

Figure 5

Limb behavioral evaluation and neurological function assessment. (A) The motion details of left limbs in each group. (B-D) Comparisons of mGCS, Purdy, and NDS scores among groups. (E) Schematic profile of gait detection with motion capture, sEMG and vGRF system (*p < 0.05, **p < 0.01, compared with TBI group; ^#p < 0.05, ^##p < 0.01, compared with SC group; ⁺p < 0.05, ⁺⁺p < 0.01, compared with CS group).

Figure 6

Assessment of joint motion details under motion capture system. (A) Angle changes of joint B, C along with motion amplitude and trajectories of the left hindlimbs in each group. (B) Real-time trajectory tracking and height changes of each joint for left hindlimbs in each group. (LHL: left hindlimb, RHL: right hindlimb) (*p < 0.05, **p < 0.01, compared with TBI group; ^#p < 0.05, ^##p < 0.01, compared with SC group; ⁺p < 0.05, ⁺⁺p < 0.01, compared with CS group)

Figure 7

Detection of sEMG and vGRF. (A) Real-time sEMG changes of left and right hindlimbs in each group. (B) Changes in vGRF values of left and right hindlimbs during a single kinematic cycle in each group. (LHL: left hindlimb, RHL: right hindlimb) (**p< 0.01).

Figure 8

Gross and staining observation of cerebral cortex repair along with vascular regeneration. (A) General view of lesion filling and cerebral cortex repair in each group. (B) Glial hyperplasia detection of cerebral cortex lesion by Masson staining in each group. Scale bars: 1 cm (a-d), 0.2 cm (a1-d1) (C) LFB staining of cerebral cortex lesion in each group. Scale bars: 1 cm (a-d), 0.2 cm (a1-d1). (D) Silver staining of cerebral cortex lesion in each group. Scale bars: 1 cm (a-d), 100 µm (a1-d1). (E) mRNA and protein expression of vWF in the cerebral cortex lesion of each group. Scale bars: 200 µm (a1-d1 & a2-d2), 50 µm (a1'-d1' & a2'-d2').

Figure 9

Detection of neural regenerative markers among groups. (A) mRNA and protein expression of MBP and NEFM along with TEM scanning in the cerebral cortex lesion of each group. Scale bars: 200 µm (a1-d1 & a2-d2), 50 µm (a1'-d1' & a2'-d2'), 5 µm (a3-d3). (B) mRNA and protein expression of Syn and MAP-2 along with TEM scanning in the cerebral cortex lesion of each group. Scale bars: 200 µm (a1-d1 & a2-d2), 50 µm (a1'-d1' & a2'-d2'), 5 µm (a3-d3).