3D printed collagen/silk fibroin scaffolds carrying the secretome of human umbilical mesenchymal stem cells ameliorated neurological dysfunction after spinal cord injury in rats

Abstract Although implantation of biomaterials carrying mesenchymal stem cells (MSCs) is considered as a promising strategy for ameliorating neural function after spinal cord injury (SCI), there are still some challenges including poor cell survival rate, tumorigenicity and ethics concerns. The performance of the secretome derived from MSCs was more stable, and its clinical transformation was more operable. Cytokine antibody array demonstrated that the secretome of MSCs contained 79 proteins among the 174 proteins analyzed. Three-dimensional (3D) printed collagen/silk fibroin scaffolds carrying MSCs secretome improved hindlimb locomotor function according to the Basso–Beattie–Bresnahan scores, the inclined-grid climbing test and electrophysiological analysis. Parallel with locomotor function recovery, 3D printed collagen/silk fibroin scaffolds carrying MSCs secretome could further facilitate nerve fiber regeneration, enhance remyelination and accelerate the establishment of synaptic connections at the injury site compared to 3D printed collagen/silk fibroin scaffolds alone group according to magnetic resonance imaging, diffusion tensor imaging, hematoxylin and eosin staining, Bielschowsky’s silver staining, immunofluorescence staining and transmission electron microscopy. These results indicated the implantation of 3D printed collagen/silk fibroin scaffolds carrying MSCs secretome might be a potential treatment for SCI.

The ST of MSCs contains many proteins composed of growth factors and cytokines, as well as vesicle parts of exosomes and microvesicles [36,37]. The nature of the essential components of the therapeutic MSCs ST remains subtle. Sart et al. [38] indicated that the ST of MSCs could facilitate the endogenous secretion of the extracellular matrix of neural progenitor aggregates, as well as secreting transforming growth factor-b1 (TGF-b1), brainderived neurotrophic factor and fibroblast growth factor-2 to accelerate cell adhesion and proliferation of the cells. Threedimensional printed biomaterial not only controlled the release rate of ST in vivo, but also supported the adhesion, survival, migration and differentiation of neural stem cells (NSCs) [39,40].
A wide variety of biomaterials are widely used to facilitate SCI repair. By bridging the two ends of the SCI area, biomaterials can reconnect the nerve tissue in the injured area, hinder scar formation, reduce inflammatory processes, rebuild nerve conduction circuits and prevent spinal neuron apoptosis, and ultimately ameliorate locomotion function after SCI [41,42]. The basic parameters of biomaterials, such as biocompatibility, biodegradability, permeability and porosity, are critical in the study of neural tissue engineering [43]. Many natural and synthetic polymers are used as scaffold materials. Collagen, as a natural extracellular matrix component, is widely used in various tissue engineering studies because of its excellent biocompatibility, biodegradability, low immunogenicity and cell adhesion [11,22,44]. Some limitation, such as poor mechanical properties, weak manipulability and rapid degradation of collagen hinder its clinical application [45]. Silk fibroin possesses mechanical strength, significant elasticity and environmental stability. Silk fibroin is currently used as a biomaterial for soft tissue engineering and reconstruction [46,47]. The addition of silk fibroin in collagen can make up for the shortcomings of using a collagen scaffold alone.
Good mechanical properties of biomaterials should guide axon regeneration before being completely biodegraded, and be strong enough to withstand stress from the spine and surrounding muscles [48]. The rapid biodegradation rate and low mechanical strength of certain natural materials have hindered their use as tissue engineering materials, and 3D printing technology is required to ameliorate their mechanical properties and bridge the two ends of the SCI area and obtain the favorable effect for SCI treatment. Furthermore, the application of tissue engineering is limited by the preparation technology. In SCI animal experiments, the diameter of the scaffold implanted at the injury site is only about 3 mm [49]. Traditional manufacturing technologies, such as particle leaching technology, extrusion technology and thermal compression, are actually difficult to achieve in the large-scale manufacturing of scaffolds with accurate internal microstructures. As a solid free-form manufacturing technology in tissue engineering, the latest advent of 3D printing provides a novel and feasible method to ameliorate the properties of scaffolds [49,50]. During the 3D printing process, the print head prepares a 3D composite scaffold containing a series of 2D layers. Printing parameters can be continuously adjusted and optimized on the computer workstation [51,52]. Three-dimensional printing technology integrates mechanical engineering, computer-aided design (CAD), reverse engineering technology, layered manufacturing technology and numerical control technology, so that it has high accuracy and can achieve a multi-level structure with the coexistence of large holes and micro holes. Therefore, this technology can print scaffolds with complex internal structures, control the size of pores and ensure the biological activity of biomaterials.
In the present study, we try to fabricate 3D printed collagen/ silk fibroin scaffolds adsorbed with the ST of human umbilical mesenchymal stem cells (HUCMSCs) and aimed to investigate the repair effect of the implanted scaffold as well as their potential to reconstruct neural networks after SCI.

Isolation, culture and analysis of HUCMSCs
According to existing methods [53,54], HUCMSCs were isolated from the Wharton's jelly of the umbilical cord provided by the Obstetrics and Gynecology Department of Characteristic Medical Center of People's Armed Police Forces, and the pregnant woman and her family members signed the informed consent. After cutting the umbilical cord Wharton's glue, it was treated with collagenase and 0.25% trypsin. The cell suspension was then obtained by filtering the digested mixture through a 100-lm filter. The extracted cells were cultured in an incubator with a volume fraction of 5% CO 2 at 37 C, and the culture medium was changed once after 3 days. After 70-80% cell fusion, the cells were digested with 0.25% trypsin and subcultured. An inverted phase-contrast microscope was used to observe the morphology of HUCMSCs. The immunophenotype of HUCMSCs (p3-generation) was analyzed using immunofluorescence staining [55]. Rabbit polyclonal antibody against CD90 (1:200, Bioss, Beijing, China) and mouse polyclonal antibody against CD105 (1:200, Bioss, Beijing, China) were used as primary antibodies. The secondary primary antibodies were used: Alexa Fluor 568-conjugated (1:1000, Invitrogen, Carlsbad, CA, USA) and Oregon Green 488-conjugated secondary antibodies (1:1000, Invitrogen, Carlsbad, CA, USA). The images were observed and captured by using a fluorescence microscope (Leica TCS SP5, Germany). According to the existing method [53,54], the flow cytometer was performed to analyze the surface antigen of HUCMSC (passage 3). The antibodies used were as follows: CD90, CD105, CD73, CD116, CD19, CD45 and HLA-DR antibodies (Abcam, Cambridge, UK). Cytomics FC500 flow cytometer (BD Biosciences, San Jose, CA) was used to analyze the positive rate of these antigens.

Isolation and collection of HUCMSCs-ST
The ST derived from HUCMSCs was obtained according to existing methods [56]. To generate the ST, primary HUCMSCs were seeded onto DMEM culture flasks (2 Â 10 6 cells/flask (75 cm 2 ) containing 10% fetal bovine serum and cultured for 24 h. The medium was then replaced with serum-free, low-glucose DMEM. The conditioned media was collected after 24 h of culture in serum-free low-glucose DMEM, and then centrifuged at 500 Â g for 10 min once, followed by centrifugation at 800 Â g for 15 min twice at 4 C. The supernatants were concentrated by ultrafiltration using a Minimate Tangential Flow Filtration (TFF) capsule system (PALL Corporation, Ann Arbor, MI) with a 100 kDa membrane. Subsequently, ST produced by 1 Â 10 7 cells was concentrated to 20 lL. A bicinchoninic acid (BCA) protein assay kit (Beyotime, China) was performed to measure the proteins concentration of ST.

Cytokine antibody array
An antibody-based cytokine array system (RayBio Human Cytokine Antibody Array C Series 2000, RayBiotech Inc. Norcross, GA) was implemented to analyze a panel of specified proteins in supernatant of HUCMSCs as previously described [56]. In brief, after dilution with blocking buffer (at a ratio of 1:50 (v/v)), the samples were put in a pre-blocked array overnight at 4 C. After subsequent incubation in streptavidin-conjugated peroxidase for 2 h, membranes were exposed to a peroxidase substrate for 5 min, followed by developing on X-ray film. A Kodak ImageStation 4000 M (Eastman Kodak, Rochester, NY) was performed to analyze optical density (OD) with the background subtracted from the edge of the spots. A positive control spot on each array was obtained by normalizing the spot data.

Fabrication of scaffolds
Silk fibroin was fabricated with silkworm silk (Jiaxing, Zhejiang, China) following the previously reported method [57][58][59]. Silkworm silk was placed in 0.5% anhydrous sodium carbonate solution (Solarbio Science & Technology Co., Ltd. China) and treated at 98-100 C for 30 min (the operation was repeated three times, the first two times were using tap water, and the third time using ionized water to degumming), and then oven dried at 60 C. The ternary solution of CaCl 2 .CH 3 CH 2 OH.H 2 O (substance concentration ratio of 1:2:8, the amount of each substance used is 188.7 g, 197.54 ml, 244.8 ml) (Solarbio Science & Technology Co., Ltd. China) was prepared at 60 C to dissolve 50 g silk, and then a mixed solution was obtained in 2 h. The mixed solution was cooled, centrifuged in an 8000 r/min centrifuge for 10 min, and the supernatant was taken and placed in a 3500D dialysis bag. Dialysis was performed with flowing tap water for 48 h, deionized water for 1 day, and water was changed once every 2 h. After dialysis, the solution was placed in 40% PEG (molecular weight 20 000 polyethylene glycol, deionized water), concentrated for 7-8 h and concentrated to about 15% in order to obtain an appropriate concentration of silk fibroin solution. Collagen was fabricated by using the fresh bovine tendon according to the previous protocols [58][59][60]. The fresh bovine tendon was peeled off the outer membrane, crushed and soaked in 0.05 Mtris buffer (Solarbio Science & Technology Co., Ltd. China) for 24 h to obtain the precipitate. An acetic acid solution (North China Pharmaceutical Factory, China) containing pepsin (Solarbio Science & Technology Co., Ltd. China) was then added and the supernatant was collected. The supernatant was added to a 3.5 mol/l NaCL solution, and the salted-out precipitate was collected and dialyzed at 4 C for 5 days to get the collagen gel.
To fabricate scaffolds, the silk fibroin protein and collagen were evenly mixed at a mass ratio of 1:2 for later preparation of 3D printing. For low temperature 3D printing technology, 3D bioprinter (Regenovo Biotechnology Co., Ltd., Hangzhou, China) was implemented to create constructs at -20 C. CAD was used to construct a multi-hole support template. Modeling software (Solidworks Software, Dassault Systèmes, V elizy, France) was performed to design the 3D printing model for this study, followed by converting the pre-designed STL 3D model into G code. The printing parameters of this study were as follows: nozzle diameter ¼ 160 lm, extrusion speed ¼ 0.17 mm/min, printing speed ¼ 12 mm/s, thickness ¼ 0.3 mm per layer, platform temperature ¼ -20 C. After the collagen/silk fibroin mixed solution was put into the printer cartridge, the material was squeezed out from the printing nozzle under the control of the computer and quickly cooled into a solid form. After printing, the 3D solid molding was placed at -80 C overnight and vacuum freeze-dried for 48-72 h to form 3D collagen/silk fibroin scaffold (3D-C/S) with a stable morphology. Cylinder-shaped 3D-C/S scaffolds (2 mm Â3 mm Â3 mm) were manufactured with a hole punch and sterilized by 60 Co (Jinpengyuan Irradiation Technology Co., Ltd., Tianjin, China). ST solution was sterilized by using a 0.22 lm filter (Millipore) at 4 C. Then to equilibrium binding, 0.1 g 3D printed collagen/silk fibroin scaffolds were soaked with 20 lL ST solution (200 lg) for 24 h at 4 C under sterile conditions to prepare 3D printed collagen/silk fibroin scaffolds adsorbed with ST derived from HUCMSCs (3D-C/SþST). D-Hank's solution was used to wash all scaffolds 30 min before use.

Determination of scaffold characteristics
As previously stated, ultrastructural observations of scaffolds were performed by using a scanning electron microscope (SEM) (Hitachi, Tokyo, Japan) [22]. The water absorption ratio and the porosity ratio were examined as previously described [22]. The dynamic mechanical properties of the scaffolds are used to represent the mechanical behavior of the support under cyclic load in wet state. The mechanical properties of the scaffolds were measured by Instron 5865 (Instron, Norwood, MA, USA) material testing machine. The scaffold was immersed in PBS (PH ¼ 7.4) at 37 C for 24 h until equilibrium, and the elastic modulus of the compression strain of the three groups of samples was measured. A 0.5 Hz sawtooth waveform was used, the preload was 0.1 N, the increment was 50%, the speed was 100%/min and the cycle was repeated three times. Ten scaffolds were measured in each group of samples, and the mean value and standard deviation (SD) of the obtained data were calculated to obtain the elastic modulus parameters of the scaffolds. An infrared spectrometer (Nicolet 870; Thermo Fisher Scientific, Waltham, MA, USA) was used to analyze Fourier transform infrared spectroscopy of 3D-C/S, 3D-C/ SþST and ST [60]. Differential scanning calorimetry (DSC) (DSC822e differential scanning calorimeter; Mettler Toledo, Greifensee, Switzerland) was carried out to analyze the phase transition temperatures of 3D-C/S, 3D-C/SþST and ST [60]. The X-ray diffractometer (D8 Advance, Germany) was used to perform crystallization analysis of 3D-C/S, 3D-C/SþST and ST. The relevant parameters for X-ray diffractometry were as follows: tube voltage ¼ 40 kV, tube current ¼ 30 mA, target material was CuKa (¼ 0.15406 nm), scanning speed ¼ 5 /min, step width ¼ 0.02 and the diffraction angle range ¼ 5 -90 .

Seeding of NSCs
NSCs isolated from embryonic Day 14 brains were cultured in the 3D-C/S and 3D-C/SþST to evaluate the cytocompatibility of scaffolds according to the method previously reported [60,61]. NSCs (passage 4) were seeded on 3D-C/S and 3D-C/SþST at a density of 1 Â 10 6 /ml. An inverted phase contrast phase-contrast microscope (Nikon, Tokyo, Japan) was performed to observe the length of cell protrusions and angle of cell protrusions of NSCs on 3D-C/ S and 3D-C/SþST at 7 days after co-culture. The morphology and growth of the cells on 3D-C/S and 3D-C/SþST were observed on Day 7 by using the SEM (Hitachi, Tokyo, Japan). Hematoxylin and eosin (HE) staining was implemented to assess the growth status of NSCs on 3D-C/S and 3D-C/SþST on Day 7; 3D-C/S and 3D-C/ SþST were put into 96-well plates separately to quantify the cell adhesion rate. Twenty microliters of NSCs were seeded at a density of 5 Â 10 6 /ml per scaffold. Cell adhesion rate of NSCs was measured after incubation for 1, 12, 24, 36, 48, 60 and 72 h. The formula for calculating the cell adhesion rate was as follows: cell adhesion rate ¼ (number of adherent cells/number of seeding cells) Â 100%.

In vitro NSCs viability
To evaluate the effect of the scaffold on the proliferation of neural stem cells, the viability of neural stem cells is analyzed by Cell Counting Kit-8 (CCK-8) assay (Solarbio Science & Technology Co., Ltd); 100 lL of NSC was seeded on the 3D-C/S and 3D-C/SþST in each well of the 96-well plate at a density of 1 Â 10 9 /L (five duplicate wells per group). At 1, 3, 5 and 7 days after co-culture, 10 lL CCK-8 solution was added to each well, followed by culture for 3 h. A multi-mode microplate reader (BioTek, Synergy2, USA) was implemented to detect the OD value at 450 nm. According to the method previously reported [60], a complete transection of the 2 mm spinal cord segment at the T10 spinal cord level was performed on adult female Sprague-Dawley rats (250-280 g) to establish the rat SCI model. Immediately after exact hemostasis, 3D-C/S and 3D-C/SþST were implanted to fill up the transected gap in the scaffold groups. According to different treatments method, all rats were randomized into four groups: (i) Sham group, (only laminectomy without SCI, n ¼ 20); (ii) SCI group, (SCI without any implantation, n ¼ 20); (iii) 3D-C/S group, (3D printed collagen/ silk fibroin scaffolds were implanted into the transected gap in the rats with spinal cord transection, n ¼ 20); (iv) 3D-C/SþST group, (3D printed collagen/silk fibroin scaffolds adsorbed ST derived from HUCMSCs were implanted into the transected gap in the rats with spinal cord transection, n ¼ 20). For postoperative care, the rats received penicillin for infection prevention within 7 days after surgery and manual bladder compression twice daily until reflex bladder control was restored.

Evaluation of locomotor function
The behavioral assessment on all rats in an open area was carried out by two observers blind to the experimental conditions to evaluate the degree of functional restoration after SCI according to the Basso-Beattie-Bresnahan (BBB) scale at 1 day before the operation, and at 1 day, 1, 2, 3, 4, 6 and 8 weeks after SCI (n ¼ 10 for each group). All rats were subjected to an inclined-grid climbing test at 1 day before the operation, and at 1 day, 1, 2, 4, 6 and 8 weeks after SCI (n ¼ 10 for each group). Rats could maintain the maximum angle of the board and the ground when the body was held for 5 s in two different positions on the board, and the angle to be measured was obtained. The bilateral hind limb movements of all rats were observed at 8 weeks after SCI.

Electrophysiological analysis
Neurophysiology monitoring setup was used to evaluate motor evoked potentials (MEP) with evoked potential equipment (Nicolet VikingQuest; Natus Medical Inc., Pleasanton, CA, USA) at 2, 4, 6 and 8 weeks after surgery (n ¼ 10 for each group). Changes in the MEP latency and amplitude were observed. MEP was recorded to assess the functional status of motor axonal conduction. The implementation of electrophysiological analysis was based on previously established methods [11,22]. Briefly, the cortical motor area at the intersection of the coronal and sagittal sutures was selected as the location of the stimulating electrode. The posterior tibial nerve was positioned to record the electrode. The parameters to be set were as follows: the stimulus intensity (46 V), the stimulation frequency (1 Hz) and the pulse width (0.2 ms). The MEP waveform can be obtained from the monitor and the amplitude and latency can be analyzed after electrical stimulation.

Magnetic resonance imaging and diffusion tensor imaging
To observe the morphology of the SCI area, magnetic resonance imaging (MRI) and diffusion tensor imaging (DTI) were carried out as previously described at 8 weeks after surgery (n ¼ 10 for each group) [60]. To evaluate the continuity of the nerve fiber bundles in the spinal cord, diffusion tensor tractography (DTT), the number of regenerated nerve fibers at the injury site and fractional anisotropy (FA) values were obtained, which cannot be examined by MRI [3,62]. Thirteen FA values were measured at 1.5mm intervals between the rostral and caudal of each rat by using Trackvis software, followed by drawing FA value distribution map.

Histological analysis
At 8 weeks after SCI, HE staining and Bielschowsky's silver staining were performed for pathological examination according to an established method (n ¼ 10 for each group) [11,[63][64][65]. Ten slices were selected from every fifth section of the posterior ventral surface of the spinal cord. After taking photos, Image-Pro Plus software was performed for quantitative statistics. For HE staining, the SCI area (width 2 mm, length 3 mm) was selected to calculate the cavity area. For Bielschowsky's silver staining, the SCI area (width 2 mm, length 3 mm) was selected to calculate Bielschowsky's silver staining area.

Immunofluorescence staining
At 8 weeks after SCI, the rats were transcardially perfused with isotonic saline and then 4% paraformaldehyde. The spinal cord sections were embedded, followed by cryosection longitudinally into 20 lm slices. The primary antibodies were incubated with the samples of the injury/graft site overnight at 4 C (n ¼ 10 for each group). The primary antibodies used were as follows:

Transmission electron microscopy
A Philips CM10 electron microscope (Eindhoven, Holland) was used to examine remyelination in the SCI area at Week 8 according to existing methods (n ¼ 5 for each group) [66]. The sections at the injury/graft site were cut into ultrathin sections (100 nm thickness) by an ultramicrotome (Reichert E, Co, Vienna, Austria). The number of myelinated axons at the injury site was quantified by selecting at least 10 random fields per sample at Â1500 magnification. The myelinated axons diameter and thickness of myelin sheath at the injury site were measured by using Image-Pro Plus software (Media Cybernetics).

Statistical analysis
IBM SPSS Statistics 22 was used for statistical analyses. The quantitative data were represented as mean 6 SD values. Two sets of data were compared by using an independent-samples ttest or two-way analysis of variance. When the P values < 0.05, a statistically significant difference was considered.

Morphology and characteristic protein identification of HUCMSCs
The fusiform HUCMSCs at p3-generation were observed by using a phase-contrast microscope (Fig. 1A). The expression of surface markers CD90 and CD105 in HUCMSCs was detected by a fluorescent microscope (Fig. 1B-D). The results of flow cytometry showed that the expression rates of CD90, CD105 and CD73 were all > 95%, which also proved that HUCMSCs had been successfully extracted and the purity of HUCMSCs could meet the requirements of this experiment (Fig. 1E-H).

The proteins of HUCMSCs-ST
The concentration of ST, measured by a BCA protein assay kit, was measured to be $ 10 mg/ml. To ascertain therapeutic molecular mediators in the HUCMSCs ST, high-density protein arrays were used to analyze the levels of 174 key signaling proteins in ST. The results of the cytokine antibody array indicated that ST contained 79 proteins among the 174 proteins analyzed. The 79 proteins of ST were presented in detail in previously published studies [56].

Characterization of 3D-C/S1ST
3D-C/SþST were prepared using a 3D bioprinter ( Fig. 2A); 3D scaffolds were shown to be porous by light microscope (Fig. 2B), fluorescent microscope (Fig. 2C and D), SEM (Fig. 2E-G) and HE staining (Fig. 2H), facilitating cell adhesion and growth. Compared to C/S, the porosity ratio of 3D-C/S (P < 0.05) and 3D-C/ SþST (P < 0.05) was significantly increased, which promoted the diffusion of nutrient solution and tissue formation (Fig. 2I). The water absorption ratio of 3D-C/S (P < 0.01) and 3D-C/SþST (P < 0.01) was lower compared to C/S, which facilitated the maintenance of ST (Fig. 2J). 3D-C/S (P < 0.05) and 3D-C/SþST (P < 0.05) exhibited a significant increase in the elastic modulus compared to C/S, which provided support for tissue regeneration (Fig. 2K). 3D-C/S and 3D-C/SþST showed similar porosity ratio, water absorption ratio and elastic modulus. Infrared spectrum showed that ST was successfully added to 3D printed collagen/silk fibroin scaffolds (Fig. 2L). Through the combination of ST, the absorption peak at 3281.62 cm À1 in 3D-C/SþST distinctly increased compared to that in 3D-C/S alone (Fig. 2L). Moreover, compared with 3D-C/S, the absorption peak at 1536.09 cm À1 and 1637.53 cm À1 in 3D-C/SþST were sharper and turned to a lower wave number (Fig. 2L). The results of the infrared spectroscopy indicated that hydrogen bonds between collagen/silk fibroin and ST may be formed. For DSC, Tm was the apex of the endothermic change caused by temperature, which might be caused by the denaturation of ST. The Tm value of 3D-C/SþST (Fig. 2M) was higher than that of pure ST, which suggested that the interaction between ST and 3D printed collagen/silk fibroin might augmented the stability of ST. X-ray diffraction analysis suggested a sharp diffraction peak at 24.54 was observed and the crystallinity of the sample changed with the amount of silk fibroin added to collagen (Fig. 2N). In this experiment, 3D-C/S and 3D-C/SþST showed favorable crystallinity, which was beneficial to the formation of the stability of the scaffold and the control of the degradation rate.

3D-C/S1ST showed favorable cytocompatibility
Immunofluorescence staining showed that Nestin was expressed in the neurospheres (Fig. 3A). After 7 days of co-culture, NSCs grew on the surface and in the pores of 3D-C/SþST and 3D-C/S using phase-contrast microscope, SEM, HE staining and immunofluorescence staining (Fig. 3B-E). Simultaneously, the number of NSCs adhering to 3D-C/SþST was significantly higher than that of 3D-C/S (Fig. 3B-E). The implantation of 3D-C/SþST increased cell adhesion rate of NSCs compared to 3D-C/S at the same time point (Fig. 3F). Compared with implanting 3D-C/S, implanting 3D-C/SþST showed a significant increase in the absorbance of NSCs based on CCK-8 assay (P < 0.05) (Fig. 3G). These results indicated that both 3D-C/SþST and 3D-C/S scaffolds had cytocompatibility and 3D-C/SþST group was more conducive to cell growth compared to 3D-C/S group. 3D-C/SþST and 3D-C/S exhibited good cytocompatibility and therefore have the potential to be implanted in vivo for biomedical study.
HUCMSCs-ST released from the implant of 3D-C/ S1ST facilitated locomotor function and electrophysiological activity after SCI Three to eight weeks after surgery, BBB scores of the left and right hindlimbs in the 3D-C/SþST group were significantly higher than those of the SCI group and 3D-C/S group (P < 0.05) (Fig. 4A and B). Eight weeks after surgery, the 3D-C/SþST group showed a significantly increased BBB scores compared with SCI group (P < 0.01)  (Fig. 4C and D). Consistent with the BBB scoring experiment, the results of the inclined-grid climbing experiment indicated that the slope angle was increased in the 3D-C/SþST group compared to SCI group and the 3D-C/S group (Fig. 4E). Furthermore, implanting 3D-C/SþST exhibited a significant increase in the slope angle compared to implanting 3D-C/S (P < 0.05) or without implanting scaffolds (P < 0.01) at 8 weeks after surgery (P < 0.05) (Fig. 4F). Compared with SCI group and 3D-C/S group, the hindlimb walking ability of 3D-C/SþST group showed significantly better recovery (Fig. 4G).
MRI and DTI indicated implanting 3D-C/S1ST enhanced nerve fibers tracts regeneration after SCI Eight weeks after surgery, rats received the implantation of 3D-C/ SþST exhibited more connections at the injury area by T2 weighted imaging (T2WI )and more regenerative nerve fiber tract at the injury area, when compared with those received 3D-C/S and those that did not received any scaffolds (Fig. 6A). After implantation of 3D-C/SþST for 8 weeks, the number of regenerated nerve fibers (83 6 13.166) at the injury area was markedly more than that in the SCI group (23.26 6 3.017, P < 0.01) and the 3D-C/S group (52.37 6 7.158, P < 0.01) (Fig. 6B). These results indicated that implantation of 3D-C/SþST might contribute to the regeneration of nerve fibers tracts in the host spinal cord.
There were prominently larger FA values at these locations (9 mm, 7.5 mm, 6 mm, 4.5 mm, 3 mm, 1.5 mm, -3 mm, -4.5 mm, -6 mm, -7.5 mm and -9 mm from the epicenter of the injury site) in the in the 3D-C/S group than in the SCI group and the 3D-C/S group (P < 0.05) (Fig. 6C). Consistent with the results of the MRI and DTT images, the 3D-C/SþST group exhibits better repair than the SCI group and the 3D-C/S group at these locations (9 mm, 7.5 mm, 6 mm, 4.5 mm, 3 mm, 1.5 mm, -3 mm, -4.5 mm, -6 mm, -7.5 mm and -9 mm from the epicenter of the injury site) based on the FA value.
The implantation of 3D-C/S1ST markedly reduced cavity formation, facilitate nerve fiber regeneration and remyelination at the injury site The improvement of locomotor function may be related to the histology of the SCI site. Histological analysis of the injured spinal cord was performed at 8 weeks postoperatively. Macroscopic observation indicated that the damaged area of 3D-C/SþST group exhibited better connection and integration with host tissue than the SCI group and the 3D-C/S group (Fig. 7A). The average cavity area were markedly reduced in the 3D-C/SþST group (2.458 6 0.429 mm 2 ) compared with the SCI group (4.202 6 0.406 mm 2 ) (P < 0.01) and the 3D-C/S group (3.059 6 0.356 mm 2 ) (P < 0.05) (Fig. 7B and C), which suggested implanting 3D-C/SþST provided tissue support that integrated well with both ends of the SCI area. Implanting 3D-C/SþST (2.26 6 0.22 mm 2 ) exhibited a significant increase in the Bielschowsky's silver staining area at the injury site compared to implanting 3D-C/S (1.96 6 0.23 mm 2 ) (P < 0.05) or without implanting scaffolds (1.38 6 0.24 mm 2 ) (P < 0.01) at 8 weeks after surgery (Fig. 7E), suggesting that implanting 3D-C/SþST could facilitate nerve fiber regeneration at the injury site. The implantation of 3D-C/SþST markedly increased the number of NF-positive nerve fibers wrapped by MBPpositive myelin sheath structures at the injury site compared to the implantation of 3D-C/S or without any implantation (Fig. 8A), which indicated that implanting 3D-C/SþST could facilitate nerve fiber regeneration and remyelination at the injury site. At 8 weeks after surgery, rats that received the implantation of 3D-C/SþST significantly exhibited more myelinated axons, bigger diameter of myelinated axons and thicker myelin sheath at the injury site by transmission electron microscopy (TEM), when compared with those received 3D-C/S and those that did not received any scaffolds (Fig. 8B-E), which suggesting that implanting 3D-C/SþST could facilitate remyelination at the injury site.
The implantation of 3D-C/S1ST could increase the formation of synaptic connections at the injury site Double-labeled immunofluorescence staining of PSD95 and SYP at the injury/graft site were performed to assess whether implanting 3D-C/SþST increase the formation of synaptic connections at the injury site at 8 weeks after SCI. The expression of PSD95 and SYP of the 3D-C/SþST group was significantly higher compared with SCI group (P < 0.01) and 3D-C/S group (P < 0.01), which suggested that implanting 3D-C/SþST could facilitate the formation of synaptic connections at the injury site ( Fig. 9A-C).

Discussion
Increasing studies demonstrated that the ST derived from MSCs can be an ideal substitute for MSCs transplantation. ST derived from HUCMSCs contains multiple proteins. Therefore, we hypothesized that the ST could have a good therapeutic potential on the treatment of SCI. In this study, we fabricated 3D printed collagen/silk fibroin scaffolds adsorbed with ST of HUCMSCs and implanted the scaffolds into the lesioned area of the rat spinal   The poor spontaneous regeneration ability after SCI is attributed to many complex biological processes. Microenvironment formed after SCI inhibits effective axon regeneration [67]. After complete spinal cord transection, the loss of all motor and sensory axonal tracts eliminates the possibility of lesion regeneration and recovery within a week, which is a huge challenge in current treatment [68]. The combination of biomaterials and neurotrophic factors can exert their respective advantages and provide excellent favorable effects.
Tissue engineering scaffolds should exhibit favorable biocompatibility, proper mechanical properties and good neuroinduction [69]. Collagen is widely used in the treatment of SCI thanks to its good cytocompatibility. Dai demonstrated that collagen scaffolds combined with neurotrophic factors could facilitate nerve regeneration after SCI in rats and Beagle dogs [70,71]. However, the poor biomechanics and rapid degradation in vivo of collagen limit its use. Silk fibroin possesses some advantages, such as good biocompatibility, stable structure, high mechanical properties and slow degradation in vitro. Therefore, the combination of collagen and silk fibroin can greatly reduce each other's shortcomings. An increasing number of studies indicated that MSCs and their ST can ameliorate the microenvironment of neurogenesis [72]. The growth factors of the ST have been shown to have a significant effect on neuron survival, differentiation and neurite growth [29,38,73]. The results of the cytokine antibody array demonstrated that ST derived from HUCMSCs contained 79 proteins among the 174 proteins analyzed. Treatment of CNS disease in the ST is related to multiple factors rather than a single factor. In our study, we fabricated porous 3D printed collagen/silk fibroin scaffold adsorbed with ST. The infrared spectroscopy data revealed that 3D-C/SþST had suitable fat-soluble and water-soluble chemical bonds that were suitable for the adhesion and growth of nerve cells, hydrogen bonds between collagen/silk fibroin and ST may be formed. The results of DSC indicated that the interaction between ST and 3D printed collagen/silk fibroin might augmented the stability of ST. For X-ray diffraction analysis, 3D-C/ SþST owned ideal crystallinity, which facilitated the formation of the stability of the scaffold and the control of the degradation rate. After co-culturing with NSCs, the 3D-C/SþST showed favorable cytocompatibility.
We established a rat T10 complete spinal cord transection model [74] and used BBB scores, inclined-grid climbing test, MEP, MRI and DTT to estimate neuroregeneration and function recovery. The results of behavioral assessment and electrophysiological studies could reveal the recovery of the 3D-C/SþST group was significantly improved compared to SCI group and 3D-C/S group, suggesting that 3D-C/SþST was conducive to the reconstruction of locomotor function after SCI. Locomotor function recovery was consistent with changes in imaging. MRI qualitatively describes the morphological changes of the tissue [75,76], while DTI is a quantitative observation of the microstructure of the tissue. The combination of MRI and DTI in this study provides solid data to estimate repairing after SCI. FA values can reveal white matter integrity, such as damage to axon structures and demyelination. In our study, high FA values reflect favorable repair after SCI. DTT can track local fibers in the area of SCI and visualize the morphological changes of the spinal cord. Compared with traditional MRI, DTI is more sensitive to detect the repair of SCI. The results of MRI and DTI indicated the repair of SCI was markedly enhanced in the 3D-C/SþST group compared to SCI group and 3D-C/S group. Histological analysis could provide more precise evidence for locomotor function repair. For the macroscopic histology images HE staining, we found implanting 3D-C/SþST could markedly reduce cavity formation and facilitate spinal tissue regeneration. Bielschowsky's silver staining revealed that implanting 3D-C/SþST could further promote the regeneration of spinal nerve fibers compared to implanting 3D-C/S, indicating the ST derived from HUCMSCs created a favorable microenvironment for the repair of SCI. As a critical protein on the myelin sheath, MBP is a necessary structure for nerve impulses to conduct along axons. Immunofluorescence staining showed that the implantation of 3D-C/SþST significantly increased NF-positive nerve fibers and NF-positive nerve fibers associated with myelin sheath at the injury/graft site, suggesting the implantation of 3D-C/SþST could provide a suitable microenvironment to facilitate the regeneration of nerve fibers and myelin sheath. TEM analysis indicated transplantation of 3D-C/SþST enhanced remyelination at the injury site. PSD95 is a post-synaptic marker and SYP is a presynaptic marker. The implantation of 3D-C/SþST promoted the establishment of synaptic connections at the injury site, which is anatomically crucial for the recovery of neurological function.
However, our study has some limitations. Many functions of 3D-C/SþST have not been explored in our study. Therefore, we need to investigate whether 3D-C/SþST can promote cell differentiation, remove reactive oxygen species and reduce inflammation in the injured microenvironment in the future.

Conclusion
We demonstrated that 3D printed collagen/silk fibroin scaffolds carrying MSCs ST ameliorated neurological dysfunction by bridging spinal cord lesions to partially reconstruct neuronal circuits after SCI in rats, suggesting the implantation of 3D printed collagen/silk fibroin scaffolds carrying MSCs ST have the potential to become a novel and safer treatment for SCI repair.