Ultrasound-Assisted Acellular Spinal Cord Scaffold for Spinal Cord Injury Treatment

Abstract

Spinal cord injury (SCI) treatment remains challenging globally, with limited breakthroughs. Tissue engineering offers promise, particularly using acellular spinal cord scaffolds. This study developed a 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)-crosslinked vascular endothelial growth factor (VEGF)-modified acellular spinal cord scaffold for sustained VEGF release. The results show sustained VEGF release over 20 days without altering the scaffold’s properties. Enhanced stability and mechanical properties were observed without increased cytotoxicity. In a rat SCI model, the system improved motor function, reduced glial scarring, and restored spinal cord morphology and histology, indicating potential for SCI therapy.

1. Introduction

Spinal cord injury (SCI) has high rates of disability and mortality, posing a heavy burden on society and families. Currently, no effective treatment measures have been found for SCI [1]. The challenges in SCI repair and treatment research include primary and secondary neuronal cell death, lack of trophic factors in the local microenvironment of the injury, and the formation of glial scars at the injury site [2]. In recent years, various strategies have been explored for SCI repair. These include pharmacological treatments, such as the combination of ceftriaxone and methylprednisolone [3]; cellular transplantation therapies, where stem cells and other cell types are used to promote tissue repair [4]; and gene therapy approaches, like the use of microRNA markers for diagnosis and treatment [5]. Existing tissue-engineering scaffolds include natural degradable materials, like chitosan; synthetic polymer materials, such as poly(lactic-co-glycolic acid); and composite materials. However, these scaffolds often face limitations, such as insufficient three-dimensional biomimetic structures or suboptimal biocompatibility, which restrict their application in SCI regeneration and repair. A recent trend in tissue engineering is the use of natural decellularized tissues to construct advanced scaffolds. This approach has shown promise across various applications, including engineered hearts [6], blood vessels [7], tendons [8], corneas [9], and bones [10]. With the development of tissue engineering in recent years, there is hope that scaffold materials can be used as carriers to load seed cells and neurotrophic factors and implanted into injured sites to achieve the purpose for repairing damaged spinal cord tissue [11]. Among the three essential elements of tissue engineering, the construction of scaffold materials is a critical factor. Our research group has previously constructed allogeneic acellular spinal cord scaffold materials. These scaffolds possess a three-dimensional mesh structure that is highly biomimetic and possesses good biomechanical properties, low immunogenicity, and good biocompatibility [12]. However, these scaffolds face the challenge of inadequate vascularization, which is critical for tissue survival and regeneration, especially in large cell masses. To overcome this, we have developed a novel approach by introducing the neurotrophic factor VEGF (vascular endothelial growth factor) to the acellular spinal cord scaffold. This innovation aims to enhance vascularization by constructing a VEGF-modified acellular spinal cord tissue-engineering composite.

However, with further research, it has been found that the use of acellular spinal cord scaffolds alone for the regenerative repair of SCI faces the problem of the insufficient vascularization of the regenerated tissue in the transplant area. In spinal cord regeneration research, vascularization is crucial. Studies have shown that scaffolds and seed cells in tissue-engineered composites significantly impact tissue regeneration because of inadequate blood supply. For effective regeneration, seed cells must remain within 150–200 μm of blood vessels; cells beyond 200 μm from capillaries cannot survive because of a lack of nutrients and oxygen. Cell masses larger than 1 mm³ are prone to necrosis if no vascular ingrowth occurs [13,14]. The EDC-crosslinked VEGF-modified acellular spinal cord scaffold developed in this study represents a significant advancement over existing approaches by addressing these vascularization challenges and ensuring a stable and sustained release of VEGF within the transplant microenvironment. This novel scaffold is expected to provide a more effective nutritional supply and a metabolite transport system at the transplantation site, thereby improving the prospects of SCI repair [15]. Simple physical methods, such as soaking or injection, cannot maintain the stable and sustained release of the VEGF in the transplant microenvironment. Studies have shown that 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) can covalently bind amino and carboxyl groups on protein molecules to form stable amide bonds and can slowly release bioactive proteins bound to three-dimensional porous collagen scaffolds into the microenvironment surrounding the scaffold [16].

Therefore, this study aims to use EDC as a crosslinker to stably bind VEGF to the collagen molecules of the acellular spinal cord scaffold through covalent bonds, enabling the slow release of the VEGF into the microenvironment surrounding the scaffold, thereby constructing a VEGF-modified acellular spinal cord scaffold sustained-release system. In vivo and in vitro experiments will be conducted to demonstrate the therapeutic effect of this sustained-release system on SCI repair, providing a theoretical basis for the design of novel tissue-engineered spinal cord scaffolds.

2. Materials and Methods

2.1. Materials

Sodium deoxycholate and Triton X-100 were purchased from Beijing Aoboxing Technology Co., Ltd. (Beijing, China). Rat neurotrophic factor-3 was purchased from BioVision (Milpitas, CA, USA), and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxy succinimide (NHS), and VEGF were purchased from Thermo Scientific (Waltham, MA, USA). Anhydrous ethanol, a streptavidin–biotin–peroxidase complex (SABC) kit, and a CCK-8 (cell-counting kit 8) assay kit were purchased from Beyotime (Shanghai, China). Trypsin was purchased from Amresco (Solon, OH, USA). An HE (hematoxylin and eosin) staining kit was purchased from Beyotime. The anti-CD4 (anti-cluster of differentiation 4) antibody, anti-CD8 (anti-cluster of differentiation 8) antibody, and fluorescent secondary antibody were purchased from EarthOx Technology Co., Ltd. (Burlingame, CA, USA). Pentobarbital sodium was sourced from the Educational and Scientific Support Office of the Army Medical University. SD rats were obtained from the Animal Experiment Center of the Army Medical University. All the above chemical reagents were used as received without further purification.

2.2. Methods

2.2.1. Construction of VEGF-Modified Decellularized Spinal Cord Scaffold Slow-Release System

Adult Sprague–Dawley (SD) rats, weighing 230–260 g, regardless of sex, were provided by the Experimental Animal Center of the Third Affiliated Hospital of the Army Medical University. A 0.3% sodium pentobarbital solution was prepared, and the SD rats were intraperitoneally anesthetized with a dosage of 40–50 mg/kg. After successful anesthesia, the SD rats were placed in a prone position, with their limbs fixed on a rat dissection board. The back was shaved, and, after routine disinfection and draping, the skin was longitudinally incised along the midline using dissection scissors. The paraspinal muscles were dissected away from the spinous processes to expose the spine. The spinal column was transected at the atlanto-occipital joint and the lumbosacral region. The laminae were progressively removed to expose the spinal cord, which was then extracted and cut into uniform 2 cm segments.

Freeze–Thaw + Ultrasonication + Chemical Extraction Method: The spinal cord specimens were stored in a deep freezer at −80 °C for 6 h and then thawed gradually in a −20 °C freezer, a 4 °C fridge, and a 37 °C water bath. The specimens were then soaked in distilled water for 6 h at room temperature. They were placed in a 2% Triton X-100/double-distilled water solution and treated with an ultrasonic disruptor (Sonics, Newtown, CT, USA, VCX-750) for 5 min, followed by continuous oscillation in a constant-temperature shaker for 3 h. After soaking and oscillating in PBS for 3 h, the specimens were placed in a 2% sodium deoxycholate/double-distilled water solution and treated with an ultrasonic disruptor for another 5 min, followed by continuous oscillation in the shaker for another 3 h. This process was repeated, and the spinal cord tissue was then dried in a freeze-dryer. The decellularized spinal cord scaffold was thus prepared [17].

The acellular spinal cord scaffold treated with freeze-drying was soaked in a PBS solution containing 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxy succinimide (NHS) at room temperature for 20 min, with a solution ratio of (EDC:NHS:PBS = 24 mg:60 mg:1 mL). The scaffold was then removed and rinsed in PBS buffer for 5 min. After rinsing, the scaffold was soaked in a VEGF-containing PBS solution for 1 h for coupling treatment, with a VEGF solution concentration of 1 μg/mL. A VEGF-releasing acellular spinal cord scaffold system was prepared, and its physicochemical properties were compared with those of the pure acellular spinal cord scaffold.

2.2.2. Testing the Sustained-Release Characteristics of VEGF-Modified Decellularized Spinal Cord Scaffolds

The VEGF content in the VEGF-modified decellularized spinal cord scaffold sustained-release system was determined under different experimental conditions using ELISA (Beyotime, Nantong, China). The impacts of different preparation protocols on the VEGF-loading capacity of the modified decellularized spinal cord scaffolds were evaluated. The data were fit based on the loading capacity and time; the loading capacity curve was drawn, and an optimized modification scheme that can effectively increase the loading capacity of bioactive proteins was established.

The balance point between a high VEGF-loading capacity and good sustained-release characteristics of the VEGF-modified decellularized spinal cord scaffolds was explored, and the preparation protocol of the VEGF-modified decellularized spinal cord scaffold sustained-release system was further optimized as follows: The in vitro drug release properties of the microspheres were determined using the paddle method. Six equally modified decellularized spinal cords were placed in the dissolution cups of a D-800 intelligent drug dissolution tester (Tianjin University Precision Instrument Factory, Tianjin, China), and 500 mL of double-distilled water was added to each cup. The mixtures were stirred at a constant speed of 100 rpm at 37 ± 0.5 °C. Then, 2 mL of the sample solution was collected from each dissolution cup at each time point from 12 h to day 21 and promptly replenished with an equal volume of double-distilled water. The amount of the VEGF released from the sample solution was measured using the Elisa method; the cumulative release rate of the scaffold was calculated; the data were fit based on the cumulative release rate and time, and the sustained-release curve was drawn. The decellularized spinal cords that were not treated with EDC were directly immersed in VEGF and used as a control.

2.2.3. Biocompatibility Study of VEGF-Modified Decellularized Spinal Cord Scaffolds In Vivo and In Vitro

In vitro: Three groups of spinal cord scaffolds (Group A: normal spinal cord tissue, Group B: decellularized spinal cord tissue, and Group C: EDC-crosslinked decellularized spinal cord tissue) were prepared by slicing into sections approximately 200 µm thick, as shown in Figure 1, Figure 2 and Figure 3. These sections were placed in EP tubes for sealed storage and then sterilized using Cobalt-60 irradiation (conducted at the Irradiation Center of the Army Medical University, with a dose of 20 kGy). A VEGF cell suspension with a concentration of 5 × 10⁵ cells/mL was prepared. The sterilized scaffolds from Groups A, B, and C were placed into sterile cell culture plates, and 100 µL of the cell suspension was seeded onto each scaffold. The scaffolds with the cell complexes were then incubated for 2 h, after which endothelial cell culture medium was added to each well. The medium was replaced every 2 days, and the culture was maintained for a total of 7 days. To assess the relative growth rates of the endothelial cells on the scaffolds, a CCK-8 assay was performed. The control group consisted of endothelial cells cultured without scaffolds. Each culture plate was removed, and 110 µL of a CCK-8 and serum-free medium mixture (1:10) was added. The plates were incubated at 37 °C for 2 h, and the optical density (OD) values at 450 nm were measured using a microplate reader (Thermo Scientific).

In vivo: Healthy SD rats (Third Military Medical University) were randomly divided into four groups, with six rats in each group. Group A was the normal spinal cord tissue group, Group B was the decellularized-scaffold-only group, Group C was the EDC-crosslinked decellularized-scaffold group, and Group D was the negative control group. The SD rats were anesthetized by the intraperitoneal injection of 2% pentobarbital sodium. After successful anesthesia, the backs of the rats were shaved and prepared for surgery and then fixed on the surgical operation board for routine disinfection and draping. A longitudinal incision was made along the posterior midline spinous process, and the subcutaneous and deep fascia were bluntly separated. The scaffolds of Groups A, B, and C were implanted under the deep fascia, and the wounds were closed layer by layer. In Group D, only surgical exposure was performed, and the wound was directly sutured. All the rats received an intramuscular injection of penicillin postoperatively. Specimens were collected from each group at four time points: one week, two weeks, seven weeks, and fourteen weeks. The paraffin sections of the scaffolds from Groups A, B, and C at each time point were stained with HE for histological observation under a microscope (Leica, Wetzlar, Germany). The immunogenicity of the scaffolds in Groups A, B, C, and D was evaluated by the number of CD4+ and CD8+ T-lymphocytes. Specimens from each group at 4 weeks after implantation were taken for CD4 and CD8 immunohistochemical staining (using the SABC kit as the secondary antibody and 3,3′-diaminobenzidine (DAB) for color development). The CD4+ and CD8+ T-lymphocytes (cluster-of-differentiation-4-positive and cluster-of-differentiation-8-positive T-lymphocytes) in each group were observed under a microscope. Four random 400× fields were selected to measure the number of positive cells, and the mean value was calculated.

2.2.4. VEGF-Modified Decellularized Spinal Cord Scaffold Sustained-Release System Transplantation for Spinal Cord Injury

A rat T10 semi-transection model was prepared, and the EDC-crosslinked VEGF-modified decellularized spinal cord scaffold sustained-release system was implanted (anatomical, isometric transplantation) to observe its repair effect on rat spinal cord injury and explore its possible mechanisms. SD rats were randomly divided into four groups: Group A (laminectomy group), Group B (simple spinal cord semi-transection group), Group C (simple decellularized spinal cord scaffold transplantation group), and Group D (VEGF-modified decellularized spinal cord scaffold transplantation group). The Basso, Beattie, and Bresnahan (BBB) scoring of the motor function; motor-evoked potential (MEP) measurements; gross morphological observations of postoperative specimens; HE-staining observations of postoperative specimens; and biotinylated dextran amine (BDA) anterograde tracing were performed at corresponding time points.

2.2.5. Porosity, Hydration Rate, and Enzymatic Degradation Rate

Porosity refers to the ratio of the total volume of small pores within a porous scaffold to the total volume of the scaffold. It is expressed by the formula φ = (pore volume within the scaffold (V1)/total volume of the scaffold (V2)) × 100%. To measure the porosity, the normal spinal cord tissue (Group A), decellularized spinal cord scaffold (Group B), and EDC-crosslinked decellularized spinal cord scaffold (Group C) were subjected to freeze-drying. Each scaffold was placed in a graduated test tube, and anhydrous ethanol was added (initial volume (V1)). After ensuring that the scaffolds were fully immersed, the new volume was recorded as V2. The volume of each scaffold was, thus, V2 − V1. The scaffolds were then removed, and the volume of the ethanol after removing the scaffolds was noted as V3. The volume of the pores within each scaffold was V1 − V3. The total volume of the scaffold was calculated as (V2 − V1) + (V1 − V3) = V2 − V3. The porosity of each scaffold was then determined using the following formula: Porosity = ((V1 − V3)/V2 − V3) × 100%.

The hydration rate refers to the abilities of the freeze-dried normal spinal cord tissue (Group A), simple acellular spinal cord scaffold (Group B), and EDC-crosslinked acellular spinal cord scaffold (Group C) to bind water molecules. After freeze-drying, Groups A, B, and C were weighed, i.e., dry weight (T1), and immersed in a PBS buffer solution at room temperature for 2 h. Then, the brackets of each group were placed on the absorbent filter paper and weighed after absorbing more water, that is, wet weight (T2). The hydration rate is as follows: α = (T2 − T1)/T1 × 100%.

The enzymatic hydrolysis rate refers to the freeze-dried normal spinal cord tissue (Group A), simple acellular spinal cord scaffold (Group B), and EDC-crosslinked acellular spinal cord scaffold (Group C) weighed (T1), immersed in a trypsin–PBS solution (concentration: 1 mg/mL) at 37 °C (room temperature), and placed in a constant-temperature generator. They were taken out at 5 phase points: 12 h, 24 h, 2 days, 4 days, 8 days, and 12 days and freeze-dried and weighed (T2). The formula of the enzymatic hydrolysis rate is as follows: β = ((T1 − T2)/T1) × 100%.

2.2.6. Biomechanical Property Testing

In the Biomechanics Laboratory at Chongqing University, a biomechanical testing device (Instron model 1011) was used to evaluate the normal spinal cord tissue (Group A), decellularized spinal cord scaffold (Group B), and EDC-crosslinked decellularized spinal cord scaffold (Group C). The freeze-dried scaffolds from Groups A, B, and C were fully hydrated by immersing them in a PBS buffer solution for 12 h. After hydration, the scaffolds were removed and fixed at both ends to the testing apparatus at room temperature. The initial tensile force was set at zero Newtons, and a caliper was used to measure the length and diameter of each scaffold. A maximum tensile force of 50 Newtons was applied at a constant rate of 1 mm/min. After the scaffold ruptured, the computer system automatically generated the maximum tensile stress, maximum tensile strain, and elastic modulus.

2.2.7. In Vivo Subcutaneous Implantation Experiment

The SD rats were anesthetized with an intraperitoneal injection of 2% sodium pentobarbital. Once successful, the rats were shaved on their backs, fixed on a surgical board, and prepared with routine disinfection and draping. A longitudinal incision was made along the midline of the back, and blunt dissection was performed to separate the subcutaneous tissue from the deep fascia. Scaffolds from Groups A, B, and C were implanted in a subfascial manner, and the incision was closed in layers. For Group D, only a surgical exposure was performed, and the wound was directly sutured. All the rats received intramuscular injections of penicillin postoperatively. Samples from each group were collected at four time points: one week, two weeks, seven weeks, and fourteen weeks. After retrieval, all the specimens were fixed in formalin, embedded in paraffin, and sectioned. And the specimens were collected for hematoxylin and eosin (HE) staining and to evaluate the therapy’s efficacy.

2.2.8. Immunohistochemical Observations

To evaluate the immunogenicity of scaffolds from Groups A, B, C, and D, the numbers of CD4+ and CD8+ T-lymphocytes were assessed. Sections from the subcutaneous implantation experiment at 4 weeks were used for this analysis. CD4 and CD8 immunohistochemical staining was performed using a secondary antibody from the SABC kit, with DAB as the chromogen. Microscopic (Leica) examination was conducted to observe the CD4+ and CD8+ T-lymphocyte distributions in each group. Four random fields at 400× magnification were selected, and the numbers of positive cells were counted and averaged. The paraffin sections were deparaffinized and hydrated and then treated with a 3% hydrogen peroxide solution for 10 min to quench the endogenous peroxidase activity. The sections were then rinsed with distilled water. Next, the sections were placed in a citrate buffer and heated to boiling in an oven and then cooled and washed in PBS buffer. The sections were blocked with 5% fetal bovine serum for 20 min, and excess liquid was removed. Primary antibodies (rabbit anti-CD4 and anti-CD8) were added to the slides and incubated overnight. The slides were washed with PBS for 2 min and then incubated with anti-rabbit IgG for 20 min, followed by another 2 min PBS wash. The slides were then incubated with SABC for 20 min and washed with PBS for 5 min. The DAB chromogen was added to the slides, and, after achieving satisfactory staining under the microscope, staining was terminated by rinsing the slides with distilled water. The slides were counterstained with hematoxylin, rinsed with distilled water, dehydrated through an alcohol gradient, cleared with xylene, and mounted with neutral resin.

2.2.9. Analysis of Statistical Results

The data were processed using the SPSS 13.0 statistical software package. The specimens were randomly selected from each group of stent data for measurements (N = 6). The results were expressed as means ± standard deviations (x ± s). The comparisons between the groups were performed using one-way analysis of variance and independent sample t-tests for statistical analysis. The test level was set at α = 0.05; (p < 0.05 indicates a significant difference, and p < 0.01 indicates a highly significant difference, both with statistical significance).

3. Results

3.1. Construction of VEGF-Modified Decellularized Spinal Cord Scaffold Sustained-Release System and Measurement of Its Physical and Chemical Properties

3.1.1. General Morphology

The general morphology of the normal spinal cord tissue is cylindrical, with a creamy white color and sparsely distributed capillary networks on the surface (Figure 1a). The simple scaffold group appears milky white and cylindrical, with a length slightly shorter than the normal spinal cord tissue (Figure 1b). The EDC-crosslinked decellularized spinal cord scaffold group appears yellowish brown and cylindrical, with a length slightly shorter than the normal spinal cord tissue (Figure 1c).

Figure 1. (a) Normal spinal cord tissue, magnification 2×; (b) purely acellular spinal cord tissue; (c) EDC-crosslinked acellular spinal cord scaffold.

Figure 1. (a) Normal spinal cord tissue, magnification 2×; (b) purely acellular spinal cord tissue; (c) EDC-crosslinked acellular spinal cord scaffold.

3.1.2. Histology

HE staining: The HE-staining sections of the normal spinal cord tissue in Group A showed a large number of nerve cells, myelin sheaths, and an extracellular matrix closely integrated, as shown in Figure 2a,b (Figure 2a is a cross-section; Figure 2b is a longitudinal section). The HE-staining sections of the acellular spinal cord scaffolds in Group B and EDC-crosslinked acellular spinal cord scaffolds in Group C showed that both scaffolds exhibited a network-like structure under optical microscopy, with the complete removal of cells and a relatively intact retention of the extracellular matrix components (Figure 2c–f).

Figure 2. (a) Normal spinal cord tissue from Group A (transverse section); (b) normal spinal cord tissue from Group A (longitudinal section); (c) simple acellular spinal cord scaffold in Group B (cross-section); (d) simple acellular spinal cord scaffold of Group B (longitudinal section); (e) cross-section of EDC-crosslinked acellular spinal cord scaffold in Group C; (f) crosslinked acellular spinal cord scaffold of Group C (longitudinal section); magnification: 400×.

Figure 2. (a) Normal spinal cord tissue from Group A (transverse section); (b) normal spinal cord tissue from Group A (longitudinal section); (c) simple acellular spinal cord scaffold in Group B (cross-section); (d) simple acellular spinal cord scaffold of Group B (longitudinal section); (e) cross-section of EDC-crosslinked acellular spinal cord scaffold in Group C; (f) crosslinked acellular spinal cord scaffold of Group C (longitudinal section); magnification: 400×.

3.1.3. Scanning Electron Microscopy and Measurement of Pore Size

Under scanning electron microscopy observation (Crossbeam 340, ZEISS, Jena, Germany), we can see in Group A (normal spinal cord scaffold) that the longitudinal section (Figure 3a) shows significant wrinkling of the tissue, with no visible three-dimensional reticulate fibrous structure. The cross-section of the normal spinal cord scaffold (Figure 3b) has a compact structure, with no obvious porous reticulate structure visible. In contrast, comparing the longitudinal sections of Group B (purely decellularized spinal cord scaffold) and Group C (EDC-crosslinked decellularized spinal cord scaffold) (Figure 3c,d), there is no significant difference observed under scanning electron microscopy. Both groups show an internal structure, with irregularly intersecting pores that are interconnected to form a complex three-dimensional network. The cross-sections of Group B and Group C (Figure 3e,f) show similar scanning electron microscopy results, with the extracellular collagen matrix of the decellularized scaffolds preserved. Through measurements using the built-in analysis software of the scanning electron microscope, the average pore size in Group A was 0 µm, while the average pore sizes in Group B and Group C were 47.3 ± 18.3 µm and 42.8 ± 13.7 µm, respectively (Figure 3g). There was no statistically significant difference between the pore sizes of Group B (purely decellularized spinal cord scaffold group) and Group C (EDC-crosslinked decellularized spinal cord scaffold group) (p > 0.05).

Figure 3. (a) Longitudinal section of normal spinal cord tissue in Group A; (b) cross-section of normal spinal cord tissue in Group A; (c) longitudinal section of the simple acellular spinal cord scaffold in Group B; (d) longitudinal section of the EDC-crosslinked decellularized spinal cord scaffold in Group C; (e) cross-section of the acellular spinal cord scaffold in Group B; (f) cross-section of EDC-crosslinked decellularized spinal cord scaffold from Group C; (g) comparison of pore sizes in Groups A, B, and C; magnification: 400×.

Figure 3. (a) Longitudinal section of normal spinal cord tissue in Group A; (b) cross-section of normal spinal cord tissue in Group A; (c) longitudinal section of the simple acellular spinal cord scaffold in Group B; (d) longitudinal section of the EDC-crosslinked decellularized spinal cord scaffold in Group C; (e) cross-section of the acellular spinal cord scaffold in Group B; (f) cross-section of EDC-crosslinked decellularized spinal cord scaffold from Group C; (g) comparison of pore sizes in Groups A, B, and C; magnification: 400×.

3.1.4. Porosity, Hydration Rate, and Enzymatic Hydrolysis Rate

Porosity refers to the ratio of the total volume of tiny pores within the porous scaffold to the total volume of the porous scaffold. The calculation formula is φ = (pore volume within the scaffold (V1)/total volume of the scaffold (V2)) × 100%. Upon measurement, the porosity of Group A (normal spinal cord tissue group) ranged from a maximum of 14.8% to a minimum of 9.7%, with an average of (12.1 ± 2.2)%. Group B (acellular spinal cord scaffold group) had a porosity ranging from a maximum of 94.8% to a minimum of 82.5%, with an average of (89.4 ± 4.3)%. Group C (EDC-crosslinked acellular spinal cord scaffold group) had a porosity ranging from a maximum of 92.1% to a minimum of 80.4%, with an average of (87.4 ± 5.1)%. There was a statistically significant difference between Group A and Group B (p < 0.01), as well as between Group A and Group C (p < 0.01). However, there was no statistically significant difference between Group B and Group C (p > 0.05). It can be seen that the porosities of the acellular spinal cord scaffold group and the EDC-crosslinked acellular spinal cord scaffold group are significantly higher than that of the normal spinal cord group, and EDC crosslinking has no significant effect on the porosity of the acellular spinal cord scaffold (Figure 4a). The hydration rate refers to the ability of scaffolds in each group, including normal spinal cord tissue after the freeze-drying treatment (Group A), decellularized spinal cord scaffolds alone (Group B), and EDC-crosslinked decellularized spinal cord scaffolds (Group C), to bind water molecules. The calculation formula is as follows: α = (T2 − T1)/T1 × 100%. The measured results showed that the hydration rate of Group A (normal spinal cord tissue group) ranged from a minimum of 42.1% to a maximum of 62.5%, with an average of (52.3 ± 7.8)%. The hydration rate of Group B (decellularized spinal cord scaffold group alone) ranged from a minimum of 190.4% to a maximum of 212.3%, with an average of (199.4 ± 8.4)%. The hydration rate of Group C (EDC-crosslinked decellularized spinal cord scaffold group) ranged from a minimum of 170.1% to a maximum of 192.8%, with an average of (182.2 ± 8.3)%. It can be seen that the hydration rates of the Group B and Group C scaffolds were significantly higher than that of Group A, and this difference was statistically significant (p < 0.05). The hydration rate of the Group C scaffolds was slightly lower than that of Group B, and there was no statistical significance between the two groups (p > 0.05). It can be seen that the hydration rates of the decellularized spinal cord scaffolds and EDC-crosslinked decellularized spinal cord scaffolds were higher than that of the normal spinal cord scaffolds, and EDC crosslinking can cause a slight decrease in the hydration rate of the decellularized spinal cord scaffolds (Figure 4b). The enzymatic degradation rate refers to the rate of the dissolution and digestion of scaffolds from Groups A, B, and C after freeze-drying them in a trypsin solution. The calculation formula is β = ((t1 − t2)/t1) × 100%. The average enzymatic degradation rates of Group A (normal spinal cord tissue group) at various time points of 12 h, 24 h, 2 days, 4 days, 8 days, and 12 days under room-temperature conditions at 37 °C were as follows: (5.1 ± 1.3)%, (9.7 ± 0.7)%, (12.9 ± 1.5)%, (20.3 ± 1.5)%, (24.5 ± 1.0)%, and (31.1 ± 2.1)%, respectively. For Group B (purely acellular spinal cord scaffold group), the average enzymatic degradation rates at the same time points were (43.9 ± 5.9)%, (76.0 ± 7.8)%, (97.1 ± 3.3)%, 100%, 100%, and 100%, respectively. For Group C (EDC-crosslinked acellular spinal cord scaffold group), the average enzymatic degradation rates at the same time points were (5.8 ± 0.9)%, (8.3 ± 0.6)%, (11.1 ± 0.9)%, (16.1 ± 0.8)%, (21.8 ± 1.7)%, and (27.6 ± 1.4)%, respectively. As shown in the figure below (Figure 4c), the enzymatic degradation rate of Group B was significantly lower than those of Groups A and C, with statistical significance (p < 0.01). The enzymatic degradation rate of Group C was slightly lower than that of Group A, and there was no statistical significance between the two groups (p > 0.05). This suggests that EDC crosslinking can significantly improve the enzymatic resistance of the purely acellular spinal cord scaffolds, making these scaffolds’ enzymatic resistance close to that of the normal spinal cord tissue.

3.1.5. Biomechanical Characterization

The biomechanical results of Group A (normal rat spinal cord tissue), Group B (purely acellular spinal cord scaffold), and Group C (EDC-crosslinked acellular spinal cord scaffold) are shown in Figure 5. According to the maximum tensile stress diagrams of the three groups of scaffolds, the maximum tensile stress of both Group B and Group C scaffolds is lower than that of Group A, with statistically significant differences (p < 0.01). The maximum tensile stress of the Group C scaffolds is greater than that of Group B, with statistically significant differences (p < 0.05). This indicates that the maximum tensile stress of the normal spinal cord tissue is significantly greater than those of both the purely acellular spinal cord scaffold and the EDC-crosslinked acellular spinal cord scaffold. The maximum tensile stress of the EDC-crosslinked acellular spinal cord scaffold is significantly improved compared to that of the purely acellular spinal cord scaffold (Figure 5a). The maximum tensile strain diagrams of the three groups of scaffolds show that there is no significant difference in the maximum tensile strain among Groups A, B, and C, with no statistically significant differences (p > 0.05). This suggests that the maximum tensile strain of the normal spinal cord tissue does not change significantly after acellular treatment, and, after EDC-crosslinking, the maximum tensile strain of the Group C scaffolds does not change significantly compared to that of the normal spinal cord tissue (Figure 5b). The elastic modulus diagrams of the three groups of scaffolds reveal that the elastic moduli of the Group B and Group C scaffolds are lower than that of Group A, with statistically significant differences (p < 0.01). The elastic modulus of the Group C scaffolds is greater than that of Group B, with statistically significant differences (p < 0.05). This indicates that the elastic modulus of the normal spinal cord tissue is significantly greater than those of both the purely acellular spinal cord scaffold and the EDC-crosslinked acellular spinal cord scaffold. The elastic modulus of the EDC-crosslinked acellular spinal cord scaffold is significantly improved compared to that of the purely acellular spinal cord scaffold (Figure 5c).

3.2. Sustained-Release Characteristics of VEGF-Modified Decellularized Spinal Cord Scaffolds

From the single sustained-release curve graphs of the two scaffold groups (Figure 6a), it can be seen that Group A (EDC-crosslinked decellularized spinal cord scaffold group) has the highest release amounts at 12 h and 24 h, with the concentration of the free VEGF in the sustained-release solution decreasing over time. Detected using ELISA kits, the sustained-release of the VEGF can last for more than 20 days. Group B (non-crosslinked decellularized spinal cord scaffold group) has significantly higher release amounts than Group A at 12 h and 24 h, with a sharp decrease in the release amount on the second day, which is significantly lower than that of Group A, forming a clear burst-release phenomenon. On the eighth day, almost no free VEGF can be detected in the sustained-release solution through ELISA kits.

From the cumulative sustained-release curve graphs of the two scaffold groups (Figure 6b), it can be seen that the curve of Group A (EDC-crosslinked decellularized spinal cord scaffold group) shows a steady upward trend in the cumulative release over time during the sustained-release process. The curve of Group B (non-crosslinked decellularized spinal cord scaffold group) reaches a plateau after 24 h, and negligible free VEGF is released later. It can be seen that almost all the VEGF in the Group B scaffolds is burst-released within the first day. Therefore, the EDC-crosslinked decellularized spinal cord scaffold can maintain a slow and sustained release of VEGF for a certain period of time, demonstrating certain sustained-release characteristics.

3.3. Biocompatibility and Biosafety of VEGF-Modified Decellularized Spinal Cord Scaffolds

The relative growth rates of the rat vascular endothelial cells cultured with scaffolds from Groups A, B, and C for one, three, five, and seven days were determined using the CCK-8 method. The results are presented in Figure 7a. It can be seen that after the coculture of the vascular endothelial cells with the normal spinal cord (Group A), decellularized spinal cord scaffolds alone (Group B), and EDC-crosslinked decellularized spinal cord scaffolds (Group C), the relative growth rates of Group A were slightly higher than those of Group B and Group C at each time point, but the differences were not statistically significant (p > 0.05). The relative growth rates of Group B were slightly higher than those of Group C at each time point, but the differences were also not statistically significant (p > 0.05). Therefore, both the pure decellularized spinal cord scaffolds and the EDC-crosslinked decellularized spinal cord scaffolds exhibited a cytotoxicity similar to that of normal spinal cord tissue cells.

The HE staining was observed in subcutaneous embedding experiments for the scaffolds in Groups A, B, and C. The specimens removed from the embedding experiments of Group A (normal spinal cord tissue group), Group B (acellular spinal cord scaffold group), and Group C (EDC-crosslinked acellular spinal cord scaffold group) at different time points were observed after paraffin sectioning and HE staining to observe the tissue responses of the different scaffolds under the skin. As shown in Figure 7b below, one week after the subcutaneous embedding of the scaffolds in Groups A, B, and C, many reactive inflammatory cells were observed under the microscope, similar to the body’s foreign body response. And, two weeks after the subcutaneous embedding of the scaffolds in the three groups, the reactive inflammatory cells under the microscope were significantly reduced in Groups A, B, and C. The tissue structure of Groups A and B was loose, and proliferative fibroblasts and new blood vessels were visible. Similarly, proliferative fibroblasts and new blood vessels were observed around the scaffolds in Group C. Seven weeks after the subcutaneous embedding of the scaffolds in the three groups, the scaffolds in Groups A and B were partially organized and absorbed, and a large number of proliferative fibrous tissues separated and wrapped the scaffolds. This organized absorption of scaffolds in Group C was not obvious, and the tissue structure was still tight, but there was still fibrous tissue surrounding it. Fourteen weeks after the subcutaneous embedding of the scaffolds in the three groups, the scaffolds in Groups A and B were almost completely organized and absorbed and replaced by proliferative fibrous tissues. Some scaffolds in Group C also began to be organized and absorbed, separated, and wrapped by proliferative fibrous tissues and were completely organized and absorbed within half a year after embedding. It can be seen that the scaffolds in Groups A, B, and C showed a certain degree of foreign body response in vivo, but no significant rejection immune reaction occurred. The scaffolds in Groups A and B were absorbed rapidly in vivo, while the scaffolds in Group C, crosslinked with EDC, could slow down the absorption process of the scaffolds in vivo.

3.4. Immunohistochemical Observation of Subcutaneous Implantation of EDC-Crosslinked Decellularized Spinal Cord Scaffolds

Four groups of implantation experiments, including Group A (normal spinal cord tissue group), Group B (decellularized spinal cord scaffold group), Group C (EDC-crosslinked decellularized spinal cord scaffold group), and Group D (negative control group), were performed. Four weeks after surgery, the transplanted specimens from all four groups were retrieved and subjected to immunohistochemical detection of CD4 and CD8. The cytoplasm of the CD4- and CD8-positive T-cells appeared brownish. The immunohistochemical results of the normal spinal cord group (Group A) showed a large number of CD4- and CD8-positive T-cells (Figure 8a). In the decellularized spinal cord scaffold group (Group B), the numbers of CD4- and CD8-positive T-cells were significantly lower than in Group A (Figure 8a). The immunohistochemical results of the EDC-crosslinked decellularized spinal cord scaffold group were similar to those of the negative control group (Figure 8a), showing only a small number of CD4- and CD8-positive T-cells.

The results of the CD4- and CD8-positive T-cell counts are shown in Figure 8b. It can be seen that the CD4- and CD8-positive T-cell counts in the acellular spinal cord scaffold group (Group B), the EDC-crosslinked acellular spinal cord scaffold group (Group C), and the negative control group (Group D) were significantly lower than those in the normal spinal cord tissue group (Group A), with statistically significant differences (p < 0.01). The CD4- and CD8-positive T-cell counts in the EDC-crosslinked acellular spinal cord scaffold group (Group C) were significantly lower than those in the acellular spinal cord scaffold group (Group B), with statistically significant differences (p < 0.05). This suggests that the immunogenicity of both the acellular spinal cord scaffold and the EDC-crosslinked acellular spinal cord scaffold is significantly lower than that of normal spinal cord tissue, and the immunogenicity of the EDC-crosslinked acellular spinal cord scaffold is lower than that of the acellular spinal cord tissue.

3.5. Experimental Study on VEGF-Modified EDC-Crosslinked Acellular Spinal Cord Scaffold Transplantation for the Treatment of Spinal Cord Injury

3.5.1. BBB Scores of Motor Function in Rats from Groups A, B, C, and D after Surgery

Observations were made from two weeks postoperatively to the fourth month postoperatively. All the rats in Group A (laminectomy group) survived, and there was no significant change in the BBB scores of the motor function in the right hindlimb before and after surgery, which were both normal. In Group B (simple spinal cord hemisection group, N = 24), one rat died on the day after surgery, and two rats died three weeks postoperatively. After death, the number of rats in this group was replenished using modeling. Except for two rats with slight hip flexion activity in the right lower limb, all the other rats in this group exhibited complete flaccid paralysis of the right lower limb. In Group C (simple acellular spinal cord scaffold transplantation group, N = 24), two rats died on the day after surgery, one rat died two weeks postoperatively, and three rats died six weeks postoperatively. After death, the number of rats in this group was replenished using modeling. In Group D (VEGF-modified acellular spinal cord scaffold transplantation group, N = 24), one rat died on the day after surgery, two rats died one week postoperatively, two rats died three weeks postoperatively, and three rats died eight weeks postoperatively. After death, the number of rats in this group was replenished using modeling. In Groups C and D, partial joint activity (mainly in the right hip joint) in the right lower limbs of some rats was observed two weeks postoperatively, and non-weight-bearing sliding of the right lower limb appeared over time. According to the BBB score results, the scores of Groups C and D were higher than those of Group B but lower than those of Group A, with statistical significance (p < 0.05). The results of Group C were lower than those of Group D, and there was no statistical significance at two weeks, one month, and two months postoperatively (p > 0.05). However, at four months postoperatively, the results of Group C were significantly lower than those of Group D, with statistical significance (p < 0.05). The results are shown in Figure 9a.

3.5.2. Measurement of Motor-Evoked Potentials (MEPs) in Rats of Groups A, B, C, and D after Surgery

Literature reports indicate that in rats with spinal cord injury, there is a decrease in the amplitude of motor-evoked potentials (MEPs) accompanied by the prolongation of latency [18]. Observations were made at two weeks, one month, two months, and four months postoperatively. All the rats in Group A (laminectomy group) survived, and the MEP amplitudes of the right lower limbs were (28.45 ± 1.47) ms, (27.92 ± 1.13) ms, (28.05 ± 1.05) ms, and (28.14 ± 0.98) ms, respectively, and the latency results were (6.24 ± 0.43) μV, (6.18 ± 0.27) μV, (6.58 ± 0.53) μV, and (6.69 ± 0.38) μV, respectively. In Group B (simple spinal cord hemisection group), no MEP amplitudes or latencies were elicited in the right lower limbs of the rats, with results of 0 ms and 0 μV. In Group C (simple acellular spinal cord scaffold transplantation group), the MEP amplitudes of the right lower limbs were (7.56 ± 0.48) ms, (8.47 ± 0.79) ms, (10.45 ± 0.97) ms, and (13.21 ± 0.54) ms, respectively, and the latency results were (24.59 ± 1.35) μV, (22.73 ± 1.27) μV, (20.47 ± 1.42) μV, and (19.25 ± 1.19) μV, respectively. In Group D (VEGF-modified acellular spinal cord scaffold transplantation group), the MEP amplitudes of the right lower limbs were (8.13 ± 0.21) ms, (14.78 ± 0.41) ms, (16.27 ± 0.88) ms, and (19.02 ± 0.48) ms, respectively, and the latency results were (20.19 ± 1.08) μV, (18.43 ± 0.92) μV, (15.69 ± 1.13) μV, and (14.58 ± 1.26) μV, respectively. The MEP amplitudes of the rats in Groups C and D were significantly lower than those in Group A and higher than those in Group B at each postoperative time point (p < 0.01) (Figure 9b). The MEP latencies of the rats in Groups C and D were significantly longer than those in Group A and shorter than those in Group B at each postoperative time point (p < 0.01) (Figure 9c). At each postoperative time point, the amplitudes in Group D were greater than those in Group C, and the latencies were slightly shorter in Group D compared to Group C, with statistical significance (p < 0.05).

3.5.3. Gross Morphological Observation of Rats in Groups A, B, C, and D after Surgery

Group A (laminectomy group): There were no abnormal gross morphological findings in the specimens at two months and four months after surgery (Figure 10). The spinal cord continuity was good, and no hyperemia or edema was observed on the surface of the dura mater;

Group B (simple spinal cord hemisection group): Gross morphological observation of the specimens at two months and four months after surgery showed that the defect in the right hemisection area persisted, and no spinal cord tissue growth was observed at the upper and lower ends of the transection side (Figure 10);

Group C (simple acellular spinal cord scaffold transplantation group): Gross morphological observation of the specimens at two months after surgery showed that the acellular spinal cord scaffold in the right hemitransplantation area had restored morphological connection with the spinal cord tissue at the upper and lower ends, and the scaffold was significantly adhered to the surrounding soft tissue, making separation difficult (Figure 10). Gross morphological observation of the specimens at four months after surgery showed that the right hemitransplantation area had restored morphological connection with the upper and lower ends, and the scaffold in the transplantation area appeared thinner, tougher, and darker in color compared to the spinal cord at the cephalic and caudal ends. The scaffold was not significantly adhered to the surrounding soft tissue, but separation was still difficult (Figure 10);

Group D (VEGF-modified acellular spinal cord scaffold transplantation group): Gross morphological observation of the specimens at two months after surgery showed that the acellular spinal cord scaffold in the right hemitransplantation area had restored morphological connection with the spinal cord tissue at the upper and lower ends, and the scaffold was not significantly adhered to the surrounding soft tissue (Figure 10). Gross morphological observation of the specimens at four months after surgery showed that the right hemitransplantation area had restored morphological connection with the upper and lower ends, and the scaffold in the transplantation area appeared thinner, tougher, and darker in color compared to the spinal cord at the cephalic and caudal ends. The scaffold was not significantly adhered to the surrounding soft tissue (Figure 10).

3.5.4. HE-Staining Observation of Rats in Groups A, B, C, and D after Surgery

Group A (laminectomy group): The HE-staining results of the specimens at two months and four months after surgery were the same as those of the normal spinal cord tissue, showing a large number of nerve cells, nuclei, myelin sheath tissue, and extracellular matrix closely combined (Figure 11). In Group B (simple spinal cord hemitransection group), HE staining of the specimens at two months and four months after surgery showed that there was still a defect in the right hemitransection area, and a large number of nerve cells, nuclei, myelin sheath tissue, and extracellular matrix were closely combined in the left non-transected area (Figure 11). In Group C (simple acellular spinal cord scaffold transplantation group), HE staining of the specimens at two months after surgery showed that the spinal cord stump had been combined with the scaffold, and inflammatory cell infiltration, liquefaction, and necrotic cavities were visible in the scaffold area, accompanied by fiber encapsulation (Figure 11). At four months after surgery, HE staining showed that the spinal cord stump had been combined with the scaffold, and a large number of glial scars and cavities were formed in the scaffold area (Figure 11). In Group D (VEGF-modified acellular spinal cord scaffold transplantation group), HE staining of the specimens at two months after surgery showed that the spinal cord stump had been combined with the scaffold, and a large number of vascular tissues and cellular components were visible in the transplant area, with some fiber encapsulation (Figure 11). At four months after surgery, the specimens showed that the spinal cord stump had been combined with the scaffold, and some fiber encapsulation and scar tissue were visible in the transplant area, with an area significantly smaller than that in Group C (Figure 11).

3.5.5. Retrograde-Tracing Observation of Biotinylated Dextran Amine (BDA) in Rats of Groups A, C, and D after Surgery

In group A (laminectomy group), biotinylated dextran amine staining showed the general direction of the spinal cord nerves two months and four months after surgery, with red fluorescence indicating the direction of the nerve travel (Figure 12). In group C (simple acellular spinal cord scaffold transplantation group), scattered punctate red fluorescence was observed two months and four months after surgery (Figure 12). In group D (VEGF-modified acellular spinal cord scaffold transplantation group), a small amount of red fluorescence was visible two months and four months after surgery (Figure 12). Among the three groups, the fluorescence imaging of groups C and D was significantly weaker than that of group A, and the fluorescence imaging of group C was weaker than that of group D.

4. Discussion

In recent years, with the rapid development of modern industrial and transportation systems, the incidence of high-energy injuries, such as falls from heights and traffic accidents, has increased, leading to a gradual rise in the prevalence of spinal cord injuries [19]. The treatment of spinal cord injuries remains a global challenge, and because of the complexity and uniqueness of the pathophysiological changes that occur after spinal cord injury, there has been no breakthrough progress in research on the treatment of spinal cord injuries [20]. Basic research on the use of tissue-engineering techniques to repair spinal cord injuries is currently a hot topic [21]. Our research team has successfully prepared allogeneic acellular spinal cord scaffolds and optimized the preparation process. We have also conducted preliminary investigations into the physicochemical properties and biocompatibilities of these scaffolds [12,17]. However, as our research continues to deepen, we have found that the use of acellular spinal cord scaffolds for the regenerative repair of spinal cord injuries still faces the severe challenge of insufficient vascularization of the regenerated tissue [22]. In this part of the experiment, we inoculated vascular endothelial cells onto the EDC-crosslinked VEGF-modified acellular spinal cord scaffold slow-release system and transplanted this scaffold–cell complex into a rat model of spinal cord hemisection to observe the recovery of the spinal cord function in the rats. Our study explored the use of an EDC-crosslinked VEGF-modified acellular spinal cord scaffold, designed to enhance vascularization and promote spinal cord regeneration. The sustained release of the VEGF from the scaffold played a crucial role in improving the biological environment within the injury site, leading to better recovery outcomes. Specifically, VEGF’s role in angiogenesis facilitated the formation of new blood vessels, ensuring an adequate supply of nutrients and oxygen to the regenerating tissue. This vascularization is critical because previous studies have shown that the survival and function of transplanted cells are severely limited without proper blood circulation, leading to necrosis and inadequate tissue regeneration.

The BBB motor function scoring is currently an objective and noninvasive method for studying the degree of recovery from spinal cord injury. This scoring system covers the behavioral changes in the recovery of the hindlimb motor function after surgery in rats and can reflect the details of spinal cord injury repair [23]. The results of this part of the experiment confirmed that four time points were selected: two weeks, one month, two months, and four months. We observed the recovery of the motor function in the right hindlimb at each time point in the following four groups: the simple laminectomy group (Group A), the simple spinal cord hemisection group (Group B), the simple acellular spinal cord scaffold group (Group C), and the VEGF-modified acellular spinal cord scaffold group (Group D). Starting from two weeks postoperatively, the motor function of the right hindlimb in Groups C and D recovered to a certain extent. When comparing Groups C and D, the scores in Group C were lower than those in Group D at two weeks, one month, and two months, but these differences were not statistically significant (p > 0.05). At four months postoperatively, the score in Group C was significantly lower than that in Group D, with statistical significance (p < 0.05). The scores in Groups B, C, and D were significantly lower than those in Group A, with statistical significance (p < 0.05). Group B, the simple spinal cord hemisection group, showed some recovery of the motor function in the right hindlimb after surgery in some rats. This recovery of the hindlimb motor function is attributed to the spinal cord compensatory function inherent in lower animals. Some scholars have reported that when performing spinal cord transection, if about 5% of the white matter fibers are preserved, some rats will still show recovery of the hindlimb motor function postoperatively [24]. Therefore, we disregarded the spontaneous recovery of the hindlimb motor function in Group B as an error. It can be seen that the VEGF-modified acellular spinal cord scaffold combined with vascular endothelial cells can promote the recovery of the spinal cord motor function to a certain extent and is significantly better than the simple acellular spinal cord scaffold group. This enhancement is likely related to the roles of VEGF in promoting angiogenesis, improving blood supply to the transplant area, and ensuring nutrient and oxygen supply to the regenerating tissue. Additionally, VEGF may further promote nerve regeneration by modulating the local inflammatory response and reducing glial scar formation.

Evoked-potential measurements are a commonly used auxiliary method to assess the recovery of spinal cord injuries, including somatosensory-evoked potential (SEP) and motor-evoked potential (MEP). SEP can be used to detect the pathway for the ascending spinal cord sensory fiber conduction, while MEP can be used to detect the pathway for the descending spinal cord motor fiber conduction [25]. Because of practical issues, such as operational difficulties and large errors in SEP detection in animals, in this part of the experiment, we chose to use MEP to detect the changes in the amplitude and latency at various time points in the simple laminectomy group (Group A), the simple spinal cord hemisection group (Group B), the simple acellular spinal cord scaffold group (Group C), and the VEGF-modified acellular spinal cord scaffold group (Group D). The amplitude changes of the MEP reflect the intensity of the potential, while the latency changes reflect the neural conduction function. After spinal cord injury, the amplitude of the MEP decreases, and the latency increases. In this part of the experiment, the amplitude and latency changes in rats in Groups A, B, C, and D were measured separately at two weeks, one month, two months, and four months postoperatively. The results showed that the amplitudes of Groups C and D were both lower than that of the normal group (Group A) and higher than that of the simple hemisection group, with statistically significant differences (p < 0.01). Among them, the amplitude of Group C was lower than that of Group D, with statistical significance (p < 0.05). It can be seen that the VEGF-modified acellular spinal cord scaffold combined with vascular endothelial cells can restore the conduction function of the spinal cord to a certain extent, and it is significantly better than the simple acellular spinal cord scaffold group. This suggests that the vascularization properties of the VEGF-modified decellularized spinal cord scaffold help to reduce tissue degeneration and glial scar formation in the transplantation area, improving the structural integrity of the spinal cord tissue.

Specimens from the simple laminectomy group (Group A), the simple acellular spinal cord scaffold group (Group C), and the VEGF-modified acellular spinal cord scaffold group (Group D) were removed for gross morphological observation at two months and four months postoperatively. In Group A, the specimens at two and four months postoperatively were similar to normal spinal cord tissue in rats, with good continuity of the spinal cord and no congestion or edema on the surface of the dura mater. In Group B, at two months postoperatively, the right half of the specimen still showed a defect; at four months postoperatively, the right half of the specimen still showed a defect, and no new spinal cord tissue growth was observed at the head and tail ends of the transection area. At the same time, the left half of the spinal cord and the tail end became thinner. In Group C, at two and four months postoperatively, the transplant area had restored the continuity between the head and tail ends of the transected spinal cord, and the transplant area was significantly thinner than the head and tail segments of the spinal cord. At two months postoperatively, there was severe adhesion between the right half of the transplanted spinal cord and the surrounding soft tissue, making separation difficult; at four months postoperatively, the adhesion between the right half of the transplanted spinal cord and the surrounding soft tissue had been reduced. In Group D, at two and four months postoperatively, the transplant area also restored the continuity between the head and tail ends of the transected spinal cord, and the transplant area was also thinner than the head and tail segments. However, the degree of thinning was less severe than in Group C. From the results of the HE staining, it can be seen that the transplant areas in Groups C and D have restored histological connections with the head and tail segments of the spinal cord. In Group C, a large amount of inflammatory infiltration and liquefaction necrosis cavities of varying sizes were observed in the transplant area at two months postoperatively; at four months postoperatively, a large number of glial scars and cavities were formed in the transplant area. In Group D, a large number of vascular tissues and cellular components were observed in the transplant area at two months postoperatively, along with partial fibrous encapsulation; at four months postoperatively, necrosis cavities, partial fibrous encapsulation, and scar tissue were observed, with significantly smaller areas and sizes compared to those in Group C. Therefore, transplantation of VEGF-modified acellular spinal cord scaffolds combined with vascular endothelial cells for the treatment of spinal cord injury can restore the gross morphological and histological continuity of the transected spinal cord. The areas and sizes of the liquefaction necrosis, fibrous encapsulation, and scar tissue in the transplant area are significantly smaller than those in the simple acellular spinal cord scaffold transplantation group. These results further support the critical role of VEGF-induced angiogenesis in SCI repair and suggest that regulating VEGF release can effectively reduce tissue degeneration and promote nerve regeneration.

Biotinylated dextran amine (BDA) is currently the most commonly used neural tracer, which has the advantages of long-term preservation and the ability to be combined with various immunohistochemical techniques or fluorescent tracers [26]. BDA can be injected extracellularly and taken up by neuronal cells and then propagated anterogradely or retrogradely along axons to distal sites. By performing corresponding immunofluorescence staining on the distal specimens, it is possible to observe whether there are nerve fibers in that area. This method is often used as an indicator to assess the recovery of the neural conduction in the injured area after spinal cord injury [26]. Therefore, in this part of the experiment, the recovery of the neural conduction in the postoperative specimens of rats in the four groups was observed through the technique of anterograde tracing using BDA in the simple laminectomy group (Group A), the simple acellular spinal cord scaffold group (Group C), and the VEGF-modified acellular spinal cord scaffold group (Group D). The results of the BDA fluorescent staining in this part showed that there was a large amount of red fluorescence along the corticospinal tract in the normal spinal cord specimens of Group A; sporadic punctate red fluorescence was visible in the transplant area specimens of Group C; a small amount of red fluorescence was also observed in the transplant-area specimens of Group D, with significantly more than in Group C. It can be seen that the transplantation of VEGF-modified acellular spinal cord scaffolds complexed with vascular endothelial cells can restore a certain degree of neural conduction in fiber bundles in the treatment of spinal cord injury, and the treatment effect is significantly better than that of the simple acellular spinal cord scaffold group. These results indicate that the VEGF-modified decellularized spinal cord scaffold promotes angiogenesis and nerve regeneration, showing significant advantages in enhancing SCI repair outcomes.

The broader implications of these findings suggest that the VEGF-modified acellular spinal cord scaffold not only enhances the structural repair of the spinal cord but also promotes functional recovery by creating a more conducive environment for neuronal regeneration. The ability of the scaffold to release VEGF in a controlled manner over time ensures that the regenerative processes are supported during the critical phases of healing. When considering the potential clinical applications, the VEGF-modified acellular spinal cord scaffold represents a promising approach for treating SCI. However, translating these findings into clinical practice requires further investigation into the long-term effects, scalability of scaffold production, and validation in larger animal models and human trials. Comparing this approach with other emerging therapies, such as gene therapy [5] or stem cell transplantation [4], our scaffold offers a unique advantage in its ability to simultaneously support vascularization and neuronal repair without the need for genetic modification or complex cell culture procedures.

5. Conclusions

In this study, simple acellular spinal cord scaffolds and VEGF-modified acellular spinal cord scaffolds were transplanted into a rat spinal cord hemitransection model to observe the recovery of the spinal cord function in rats. BBB scores were used to evaluate the recovery of the motor function in the right lower limbs of rats at different time points after surgery; motor-evoked potentials (MEPs) were also used to assess the motor function recovery. Specimens from the transplant areas were taken at different time points after surgery for gross morphological observation, histological observation (HE staining), and anterograde-tracing detection using BDA. The spinal cord repair situation in the VEGF-modified acellular spinal cord scaffold transplantation treatment of the rat spinal cord hemitransection model was comprehensively evaluated. The results showed that the VEGF-modified acellular spinal cord scaffold group had significantly better results in motor function BBB scores and MEP than the simple acellular spinal cord scaffold group and the negative control group. Moreover, the VEGF-modified acellular spinal cord scaffold group demonstrated superior restoration of both the gross morphological and histological connections of the spinal cord. This was evidenced by reduced liquefaction necrosis, smaller cavities, and less fibrous encapsulation, as seen in HE staining, as well as stronger BDA anterograde tracing compared to that of the simple acellular spinal cord scaffold group. These findings indicate that the VEGF-modified acellular spinal cord scaffold can better promote the repair of spinal cord injury, providing a new approach for tissue-engineered spinal cord research.

In addition to these key findings, this study highlights this scaffold’s abilities to enhance vascularization, reduce glial scarring, and support neuronal regeneration, which are critical for functional recovery. Future research should focus on long-term studies to assess the sustained effects of the VEGF-modified acellular spinal cord scaffold, explore the scalability of scaffold production, and conduct trials in larger animal models or human subjects. These steps are essential to translate these promising findings into clinical applications.