3D-printed collagen/silk fibroin/secretome derived from bFGF-pretreated HUCMSCs scaffolds enhanced therapeutic ability in canines traumatic brain injury model

Abstract

The regeneration of brain tissue poses a great challenge because of the limited self-regenerative capabilities of neurons after traumatic brain injury (TBI). For this purpose, 3D-printed collagen/silk fibroin/secretome derived from human umbilical cord blood mesenchymal stem cells (HUCMSCs) pretreated with bFGF scaffolds (3D-CS-bFGF-ST) at a low temperature were prepared in this study. From an in vitro perspective, 3D-CS-bFGF-ST showed good biodegradation, appropriate mechanical properties, and good biocompatibility. In regard to vivo, during the tissue remodelling processes of TBI, the regeneration of brain tissues was obviously faster in the 3D-CS-bFGF-ST group than in the other two groups (3D-printed collagen/silk fibroin/secretome derived from human umbilical cord blood mesenchymal stem cells (3D-CS-ST) group and TBI group) by motor assay, histological analysis, and immunofluorescence assay. Satisfactory regeneration was achieved in the two 3D-printed scaffold-based groups at 6 months postsurgery, while the 3D-CS-bFGF-ST group showed a better outcome than the 3D-CS-ST group.

Keywords: bFGF; collagen; human umbilical cord blood mesenchymal stem cells; secretome; silk fibroin; traumatic brain injury.

FIGURE 1

(A) HUCMSCs morphology under phase contrast microscopy. (B–B1) Expression of CD90 (green) and CD105 (red) in HUCMSCs under immunofluorescence. (C–J) Representative morphology of 3D-CS-bFGF-ST by using CLSM (C–C1), digital camera (D–E) and stereomicroscope (F–H1), SEM (I–I2), and HE staining (J). (K) The degradation test of 3D-CS-bFGF-ST. (L–N) Mechanical behavior of six kinds of scaffolds. The porosity ratio (L), water absorption ratio (M), and lastic modulus (N) of the six kinds of scaffolds. (O) DSC curves of 3D-CS, bFGF-ST and 3D-CS-bFGF-ST. (P) Cumulative secretome release of 3D-CS-bFGF-ST. ^* p < 0.05, ^** p < 0.01 vs. 3D-C. ^# p < 0.05, ^## p < 0.01 vs. 3D-C-ST. ^& p < 0.05, ^&& p < 0.01 vs. 3D-C-bFGF-ST.

FIGURE 2

Typical morphology of cocultures of the two kinds of scaffolds (3D-CS-ST (A,C,E) or 3D-CS-bFGF-ST (B,D,F) and HUCMSCs under phase-contrast microscopy, SEM and HE staining. (G) MTT results of cocultures of the two kinds of scaffolds and HUCMSCs. (H) Typical morphology under an inverted fluorescence microscope. (I–K) Identification of NSCs by Nestin staining. (L) Cell adhesion rate of the 3D-CS-ST group and the 3D-CS-bFGF-ST group. (M) MTT results of cocultures of the two kinds of scaffolds and NSCs. (N–S) Expression of NF (N–O), GAP43 (P–Q), and NeuN (R–S) in NSCs after coculture. ^* p < 0.05, ^** p < 0.01 vs. 3D-CS-ST.

FIGURE 3

mGCS score (A), Purdy score (B) and NDS score (C) of the four groups. ^* p < 0.05, ^** p < 0.01 vs. TBI. ^# p < 0.05, ^## p < 0.01 vs. 3D-CS-ST.

FIGURE 4

(A-E) Representative HE staining images (A-D1) in Sham group (A and A1), TBI group (B and B1), 3D-CS-ST group (C and C1), and 3D-CS-bFGF-ST group (D and D1), and their quantitative analysis (E). (F-J) Representative Bielschowsky's silver staining images (F-I1) in Sham group (F and F1), TBI group (G and G1), 3D-CS-ST group (H and H1), and 3D-CS-bFGF-ST group (I and I1), and their quantitative analysis (J). (K-O) Representative LFB staining images (K-N1) in Sham group (K and K1), TBI group (L and L1), 3D-CS-ST group (M and M1), and 3D-CS-bFGF-ST group (N and N1), and their quantitative analysis (O). (P-T) Representative Nissl staining images (P-S1) in Sham group (P and P1), TBI group (Q and Q1), 3D-CS-ST group (R and R1), and 3D-CS-bFGF-ST group (S and S1), and their quantitative analysis (T). (U-Y) Representative Masson staining images (U-X1) in Sham group (U and U1), TBI group (V and V1), 3D-CS-ST group (W and W1), and 3D-CS-bFGF-ST group (X and X1), and their quantitative analysis (Y). **P < 0.01 vs TBI. #P < 0.05, ##P < 0.01 vs 3D-CS-ST.

FIGURE 5

(A-D1) Typical picture of double-labelled immunofluorescence staining (NF (green) and MBP (red)) in Sham group (A and A1), TBI group (B and B1), 3D-CS-ST group (C and C1), and 3D-CS-bFGF-ST group (D and D1). (E-F) Quantitative analysis of NF and MBP. (G-J1) Typical immunofluorescence staining picture of GAP43 (green) in Sham group (G and G1), TBI group (H and H1), 3D-CS-ST group (I and I1), and 3D-CS-bFGF-ST group (J and J1). (K) Quantitative analysis of GAP43. *P < 0.05, **P < 0.01 vs TBI. ##P < 0.01 vs 3D-CS-ST.

FIGURE 6

(A-D1) Typical immunofluorescence staining picture of Tuj-1 (green) in Sham group (A and A1), TBI group (B and B1), 3D-CS-ST group (C and C1), and 3D-CS-bFGF-ST group (D and D1). (E) Quantitative analysis of Tuj-1. (F-I1) Typical immunofluorescence staining picture of PSD95 (green) in Sham group (F and F1), TBI group (G and G1), 3D-CS-ST group (H and H1), and 3D-CS-bFGF-ST group (I and I1). (J) Quantitative analysis of Tuj-1. (K-N) Typical immunofluorescence staining picture of MAP-2 (red) in Sham group (K and K1), TBI group (L and L1), 3D-CS-ST group (M and M1), and 3D-CS-bFGF-ST group (N and N1). (O) Quantitative analysis of MAP-2. **P < 0.01 vs TBI. ##P < 0.01 vs 3D-CS-ST.

FIGURE 7

(A-D1) Typical immunofluorescence staining picture of vWF (red) in Sham group (A and A1), TBI group (B and B1), 3D-CS-ST group (C and C1), and 3D-CS-bFGF-ST group (D and D1). (E) Quantitative analysis of MAP-2. (F-I) Typical TUNEL (green) staining picture. (J) Quantitative analysis of TUNEL in Sham group (F), TBI group (G), 3D-CS-ST group (H), and 3D-CS-bFGF-ST group (I). (K-N) The expression of IL-6 (K), TNF-α (L), IL-10(M), and IL-10/IL-6 (N) at 1 week after TBI. (O-R) The expression of IL-6 (O), TNF-α (P), IL-10 (Q), and IL-10/IL-6 (R) at 6 months after TBI. **P < 0.01 vs TBI. #P < 0.05, ##P < 0.01 vs 3D-CS-ST.