Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Jun 27:9:rbac043.
doi: 10.1093/rb/rbac043. eCollection 2022.

Three-dimensional-printed collagen/chitosan/secretome derived from HUCMSCs scaffolds for efficient neural network reconstruction in canines with traumatic brain injury

Affiliations

Three-dimensional-printed collagen/chitosan/secretome derived from HUCMSCs scaffolds for efficient neural network reconstruction in canines with traumatic brain injury

Xiaoyin Liu et al. Regen Biomater. .

Abstract

The secretome secreted by stem cells and bioactive material has emerged as a promising therapeutic choice for traumatic brain injury (TBI). We aimed to determine the effect of 3D-printed collagen/chitosan/secretome derived from human umbilical cord blood mesenchymal stem cells scaffolds (3D-CC-ST) on the injured tissue regeneration process. 3D-CC-ST was performed using 3D printing technology at a low temperature (-20°C), and the physical properties and degeneration rate were measured. The utilization of low temperature contributed to a higher cytocompatibility of fabricating porous 3D architectures that provide a homogeneous distribution of cells. Immediately after the establishment of the canine TBI model, 3D-CC-ST and 3D-CC (3D-printed collagen/chitosan scaffolds) were implanted into the cavity of TBI. Following implantation of scaffolds, neurological examination and motor evoked potential detection were performed to analyze locomotor function recovery. Histological and immunofluorescence staining were performed to evaluate neuro-regeneration. The group treated with 3D-CC-ST had good performance of behavior functions. Implanting 3D-CC-ST significantly reduced the cavity area, facilitated the regeneration of nerve fibers and vessel reconstruction, and promoted endogenous neuronal differentiation and synapse formation after TBI. The implantation of 3D-CC-ST also markedly reduced cell apoptosis and regulated the level of systemic inflammatory factors after TBI.

Keywords: canines; chitosan; collagen; low temperature extrusion 3D printing; secretome; traumatic brain injury.

PubMed Disclaimer

Figures

None
Graphical abstract
Figure 1.
Figure 1.
Characterization of HUCMSCs. Phase-contrast microscope (A). Immunofluorescence images of the biomarkers CD90 (green) and CD105 (red) in HUCMSCs (B and B1). Characterization of 3D-CC-ST. Representative morphological images of 3D-CC-ST under a confocal laser scanning microscope (CD1), stereomicroscope (FH1), SEM (IJ1) and HE (K and K1). Degradation curve of 3D-CC-ST with 5 five mass ratios (L). Porosity ratio (M), water absorption (N) and elastic modulus (O) for the five groups of scaffolds. Secretome release (P) and cumulative secretome release (Q) from 3D-CC-ST. *P <0.05, **P <0.01 vs C. #P <0.05, ##P <0.01 vs CC. &&P <0.01 vs 3D-C.
Figure 2.
Figure 2.
Cytocompatibility of the scaffold (AO). The effects of 3D-CC (A, C, E–H, M) and 3D-CC-ST (B, D, I–L, N) scaffolds on the adhesion and proliferation of HUCMSCs under the SEM images (A–D) (yellow arrow), immunofluorescence staining images (E–L) (yellow arrow) and HE staining images (M–N) (yellow arrow). MTT assay of HUCMSCs cultured on 3D-CC and 3D-CC-ST scaffolds at 1, 3, 5 and 7 days (O). *P <0.05, **P <0.01 vs 3D-CC.
Figure 3.
Figure 3.
Locomotor function assessment and electrophysiological analysis of the four groups after TBI. mGCS (A), purdy (B) and NDS (C) scores at 1 day, 1, 2, 4, 8, 12, 16, 20 and 24 weeks after TBI. (D) Typical MEP schematic diagrams of the left forelimb (LFL), right forelimb (RFL), left hindlimb (LHL), and right hindlimb (RHL) at 6 months after TBI. Quantitative analysis of amplitude (E) and latency (F) of MEP at 6 months after TBI. *P <0.05, **P <0.01 vs TBI. #P <0.05, ##P <0.01 vs 3D-CC.
Figure 4.
Figure 4.
Histological analysis of brain tissue formation and vessel reconstruction around and within the scaffolds at month 6 post-surgery in the four groups. (AT) HE staining (A–E), Bielschowsky’s silver staining (F–J), Nissl staining (K–O), Masson staining (P–T). (VU) Immunostaining with vWF (red) at 6 months after TBI for the four groups: Sham group (V and V1), TBI group (W and W1), 3D-CC group (X and X1), and 3D-CC-ST group (Y and Y1). There were a few regenerated blood vessels and few vWF-positive cells inside the TBI group and 3D-C/C group (W and X1), while a larger number of regenerating vessels and many vWF-positive cells were detected in the 3D-C/C+ST group (Y and Y1). Quantification of regenerated brain tissue and vessel reconstruction in the peri-injured area after TBI (E, J, O, T and U). **P <0.01 vs TBI. #P <0.05, ##P <0.01 vs 3D-CC.
Figure 5.
Figure 5.
Regeneration of nerve fibers, myelin sheaths and axons in vivo at 6 months after TBI. Expression of NF (green) and MBP (red) in the four groups: Sham group (A and A1), TBI group (B and B1), 3D-CC group (C and C1) and 3D-CC-ST group (D and D1). The number of NF- and MBP-positive cells in the 3D-CC-ST group was significantly increased compared with that in the TBI group and the 3D-CC group. Quantification of NF- and MBP-positive cells in the peri-injured area after TBI (E and F). Expression of GAP43 (green) in the four groups: Sham group (G and G1), TBI group (H and H1), 3D-C/S group (I and I1) and 3D-CC-ST group (J and J1). The 3D-CC-ST group showed significantly more positive cells of GAP43 than the TBI group and the 3D-CC group. Quantification of GAP43-positive cells in the peri-injured area after TBI (K). **P <0.01 vs TBI; ##P <0.01 vs 3D-CC.
Figure 6.
Figure 6.
Endogenous neuronal differentiation and synapse formation in the four groups at 6 months after TBI. (AD1) Typical images of tuj-1 (green). (E) Quantification of tuj-1-positive cells in the peri-injured area after TBI. (FI1) Typical images of SYN (green) and MAP2 (red). Quantification of SYN (J) and MAP2 (K)-positive cells in the peri-injured area after TBI. SYN (green)- and MAP2 (red)-positive cells in the 3D-CC-ST group were increased significantly compared with those in the TBI group and the 3D-CC group. (LO) Typical images of PSD95. (P) Quantification of PSD95-positive cells in the peri-injured area after TBI. **P <0.01 vs TBI. ##P <0.01 vs 3D-CC.
Figure 7.
Figure 7.
TUNEL Staining of brain tissue at 6 months after TBI and measurement of plasma inflammatory factors at 1 week and 6 months after TBI. Apoptosis in neuronal cells. TUNEL immunostaining in the injured area for the four groups (AD). Quantification of TUNEL-positive cells (E). Investigation of inflammatory factors at the acute stage (FI) and chronic stage (JM) in the peri-injured tissue. The expression of IL-6 (F, J) and TNF-α (I, M) was significantly increased in the 3D-CC-ST group at 1 week and 6 months post-surgery compared with that in the TBI group and the 3D-CC group at 1 week and 6 months post-surgery, whereas the expression of IL-10 (G, K) and IL-10/IL-6 (H, L) was significantly increased in the 3D-CC-ST group at 1 week and 6 months post-surgery compared with that in the TBI group and the 3D-CC group at 1 week and 6 months post-surgery. *P <0.05, **P <0.01 vs TBI; #P <0.05, ##P <0.01 vs 3D-CC.

Similar articles

Cited by

References

    1. Salehi A, Zhang JH, Obenaus A.. Response of the cerebral vasculature following traumatic brain injury. J Cereb Blood Flow Metab 2017;37:2320–39. - PMC - PubMed
    1. van Vliet EA, Ndode-Ekane XE, Lehto LJ, Gorter JA, Andrade P, Aronica E, Gröhn O, Pitkänen A.. Long-lasting blood-brain barrier dysfunction and neuroinflammation after traumatic brain injury. Neurobiol Dis 2020;145:105080. - PubMed
    1. Qian F, Han Y, Han Z, Zhang D, Zhang L, Zhao G, Li S, Jin G, Yu R, Liu H.. In situ implantable, post-trauma microenvironment-responsive, ROS depletion hydrogels for the treatment of traumatic brain injury. Biomaterials 2021;270:120675. - PubMed
    1. Sultan MT, Choi BY, Ajiteru O, Hong DK, Lee SM, Kim HJ, Ryu JS, Lee JS, Hong H, Lee YJ, Lee H, Suh YJ, Lee OJ, Kim SH, Suh SW, Park CH.. Reinforced-hydrogel encapsulated hMSCs towards brain injury treatment by trans-septal approach. Biomaterials 2021;266:120413. - PubMed
    1. Maas A, Menon DK, Adelson PD, Andelic N, Bell MJ, Belli A, Bragge P, Brazinova A, Büki A, Chesnut RM, Citerio G, Coburn M, Cooper DJ, Crowder AT, Czeiter E, Czosnyka M, Diaz-Arrastia R, Dreier JP, Duhaime AC, Ercole A, van Essen TA, Feigin VL, Gao G, Giacino J, Gonzalez-Lara LE, Gruen RL, Gupta D, Hartings JA, Hill S, Jiang JY, Ketharanathan N, Kompanje E, Lanyon L, Laureys S, Lecky F, Levin H, Lingsma HF, Maegele M, Majdan M, Manley G, Marsteller J, Mascia L, McFadyen C, Mondello S, Newcombe V, Palotie A, Parizel PM, Peul W, Piercy J, Polinder S, Puybasset L, Rasmussen TE, Rossaint R, Smielewski P, Söderberg J, Stanworth SJ, Stein MB, von Steinbüchel N, Stewart W, Steyerberg EW, Stocchetti N, Synnot A, Te Ao B, Tenovuo O, Theadom A, Tibboel D, Videtta W, Wang K, Williams WH, Wilson L, Yaffe K; InTBIR Participants and Investigators. Traumatic brain injury: integrated approaches to improve prevention, clinical care, and research. Lancet Neurol 2017;16:987–1048. - PubMed