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. 2020 Jun 25;5(26):16064–16075. doi: 10.1021/acsomega.0c01379

Thermosensitive bFGF-Modified Hydrogel with Dental Pulp Stem Cells on Neuroinflammation of Spinal Cord Injury

Abdullkhaleg Albashari †, Yan He ‡,#, Yanni Zhang †, Jihea Ali ∥, Feiou Lin †, Zengming Zheng ⊥, Keke Zhang †, Yanfan Cao †, Chun Xu #,*, Lihua Luo †,*, Jianming Wang ‡, Qingsong Ye †,§,#,*
PMCID: PMC7346236  PMID: 32656428

Abstract

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Acute spinal cord injury (SCI) induces severe neuroinflammation, which increases intermediary filaments and neurodegeneration. Previous studies have shown that a basic fibroblast growth factor (bFGF) and dental pulp stem cells (DPSCs) contribute to a protective effect on injured neuronal cells, but the mechanism of SCI repair is still unclear. In this study, in situ heparin (HeP) hydrogel injection containing bFGF and DPSCs (HeP-bFGF-DPSCs), as well as in vitro studies of bFGF and DPSCs, proved an effective control over inflammation. The in vivo application of HeP-bFGF-DPSCs regulated inflammatory reactions and accelerated the nerve regeneration through microtubule stabilization and tissue vasculature. Our mechanistic investigation also showed that bFGF-DPSCs treatment inhibited microglia/macrophage proliferation and activation. Furthermore, HeP-bFGF-DPSCs prevented microglia/macrophage activation and reduced proinflammatory cytokine release. In this paper, we discovered that bFGF and DPSCs worked together to attenuate tissue inflammation of the injured spinal cord, resulting in a superior nerve repair. Our results indicated that a thermosensitive hydrogel delivering bFGF and DPSCs could serve as a promising treatment option for spinal cord injuries.

1. Introduction

Spinal cord injury (SCI) is a traumatic disease of the central nervous system, which causes poor quality of life and lifelong disability in people of all age groups.1 After the primary injury, a unique mechanism causes the interference of a lesion area and increases the release of bio-molecules such as IL-6 and TNF-α and other different cytokines. These are the factors that increase cellular functions such as inflammation and apoptosis close to the lesion site.2,3 Neural damage causes cystic formation around the lesion site, which is encircled by a reactive cellular tissue such as a glial scar. This scar is formed mostly by reactive astrocytes, which results in motor destruction.4 Different repair pathways depend upon the beneficial aspects of tissue inflammation. The nuclear factor-kappa B (NF-κB) is known as an important factor in inflammatory cytokine regulation during the spinal cord injury and also in the cytokine-induced production as a major factor at the inflammatory stage.5 IκB kinase β (IKKβ) also plays an important part in energizing NF-κB. It also serves as an essential catalyst of the IKK mechanism, which is a primary initiator of inflammation.6,7 Therefore, the inhibitory effect of NF-κB prevents the immune cells from releasing and expressing various proinflammatory cytokines, such as tumor necrosis factor α (TNF-α), interleukin (IL)-6, and interleukin 1β, in injury.8 Recently, it has been shown that inhibition of NF-κB may decrease inflammation in the lesion and it could be beneficial for nerve regeneration.9,10 However, the biomolecular pathways of NF-κB signals in SCI repair are not so much understood.

Stem cell therapy is to use unspecialized, undifferentiated self-renewal cells to differentiate into new tissues.11,12 Transplantation of stem cells to the lesion site has shown promising signs in the treatment of spinal cord injury. Stem cells have the capability to modulate the immune response after neural injury by elevating the release rate of anti-inflammatory and decreasing the release rate of proinflammatory cytokines.13 Stem cells also reduce the proinflammatory cytokine production in systemic inflammatory states such as blood and spleen.14,15 Some studies have measured the major changes in cytokine release of immune cells after injury (as tumor necrosis factor α and interferon β-1a).16,17 However, few studies have explained the direct relation between transplanted stem cells and immune cells in the SCI.18 Dental pulp stem cells (DPSCs), originated from a neural crest and enclosed in a dental pulp chamber, are considered an important source of stem/progenitor cells for nerve regeneration.19 DPSCs express IL-6, IL-8, and TGFβ via the Toll-like receptor (TLR) during the neural inflammation phase in treating central nerve diseases.20 The expression of the IL-8 of DPSCs increased due to the expression of the TLR4. This happens particularly in spinal cord injuries where IL-8 has been related to the maintenance of neural cell integrity and the reduction of the lesion.21 Co-culture of T cells and DPSCs has indicated that DPSCs could encourage the secretion of inflammatory factors of T cells including intracellular adhesion molecule-1, vascular adhesion molecule-1, human leukocyte antigen-G, intracellular adhesion molecule-1, IL-10, and hepatocyte growth factor (HGF), while it down regulates proinflammatory factors such as IL-6, IL-7, IL-17A, IL-12, and TNF-α.22 In treating SCI-induced dysfunction, evidence has pointed out that DPSCs together with a growth factor and a hydrogel could successfully restore motor and sensory functions of SCI-modelled rats.23

Pluronic F-127 (PF127, also known as poloxamer 407) is a biosynthetic hydrogel synthesized from amphiphilic copolymers containing ethylene oxide and units of polypropylene oxide.24 Recently, due to excellent biocompatibility and mechanical properties, heparin (HeP)-based biomaterials have been widely used in stem cell research and tissue regeneration such as nerves and cartilage. In vitro studies have shown that hydrogels could encapsulate stem cells and bio-molecules, encourage cell proliferation, and release bioactive molecules to the target.25,26 Our recent research showed that a hydrogel was able to form an injectable formulation.26 It functioned as an ideal carrier when mixed with a growth factor and stem cells, featuring in sustained release of bioactive growth factors and favorable accommodation for cell proliferation and differentiation.27,28 When mixed with DPSCs and basic fibroblast growth factor (bFGF) and applied in situ after spinal cord injury, it could facilitate the regeneration of damaged tissues and aid in the restoration of sensory and motor functions.23

In this work, we assessed the anti-inflammation capacity of bFGF and DPSCs in vitro. We delivered bFGF, DPSCs, and bFGF-DPSCs in a hydrogel on an SCI animal model and compared the inflammation control effects of bFGF, DPSCs, and bFGF-DPSCs in vivo. The hypothesis was that the combined use of bFGF and DPSCs could offer a low-inflammatory microenvironment to allow nerve repair after acute spinal cord injury.

2. Results

2.1. Micromorphology of Hydrogels and the Viability of DPSCs

Scanning electron microscopic (SEM) images were used to show the cross-sectional morphology of the hydrogels. Hydrogels with and without the growth factor bFGF demonstrated reticular porous network morphology. At low magnification, dense tubular-shaped cavities with interconnected pores of different diameters were seen in hydrogels PF127, PF127-bFGF, and HeP; the pore size was smaller, and the pore shape was more round in HeP-bFGF (Figure 1, upper panel). At high magnification, HeP-bFGF showed more and smaller pores compared to the other hydrogels. These characteristics contributed to its high-level permeability and biosolubility of gels, harmonious with its high degradability during the function. Other characteristic results are given in the Supporting Information.

Figure 1.

Figure 1

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Micromorphology of the lyophilized PF127, PF127-bFGF, HeP, and HeP-bFGF hydrogels. At low magnification, representative SEM images of hydrogels showed a porous reticular structure with interconnection in all hydrogels. The pores in hydrogels of PF127, PF127-bFGF, and HeP were shaped in flattened cavities, whereas pores in HeP-bFGF hydrogels were smaller and rounder. At high magnification, the pore structures of hydrogels of PF127, PF127-bFGF, and HeP showed no obvious difference, while HeP-bFGF hydrogels displayed a slightly denser and smaller porous structure. Scale bar: 1 mm (upper panel); 100 μm (lower panel).

2.2. Viability of Dental Pulp Stem Cells on Hydrogels In Vitro

DPSCs were co-cultured with all four types of hydrogels (PF127, PF127-bFGF, HeP, and HeP-bFGF) for 14 days. The protein expression of α-tubulin, a neural marker, indicated a good biocompatibility of all hydrogels (Figure 2A). DPSCs spread well in all hydrogels, indicating that the porous micromorphology of the materials facilitated the cellular penetration and proliferation. However, only DPSCs in HeP-bFGF showed a highly organized growth, where the cells were aligned in one direction. DPSCs in other three hydrogels organized themselves at a lesser degree. We assumed that this might be caused by the subtle difference observed in the micromorphology of the hydrogels. When the fluorescence intensity of α-tubulin was quantified and compared against that of the PF127 hydrogel group, there was a significant increase of α-tubulin expression. Addition of bFGF enhanced the α-tubulin expression of DPSCs. Cells in the HeP-bFGF hydrogel had the highest α-tubulin expression than in the other three hydrogels (Figure 2B). This might be caused by growth of more cells on HeP-bFGF due to a well-organized growth pattern that allowed highly efficient use of the growth space.

Figure 2.

Figure 2

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Evaluation of DPSCs encapsulated in various formulations of thermoreversible hydrogels. (A) Expression of α-tubulin (green), a neural marker, was chosen to stain the DPSCs cultured in hydrogels of PF127, PF127-bFGF, HeP, and HeP-bFGF for 14 days. The positive expression of α-tubulin presented both the neural feature and viability of the DPSCs. Cellular nuclei were visualized with 4′,6-diamidino-2-phenylindole (DAPI) (blue). DPSCs were well spread inside all hydrogels. DPSCs in HeP-bFGF showed a highly oriented pattern, where all of the cells were aligned in one direction. DPSCs in other hydrogels showed a less organized pattern. Scale bar: 200 μm. (B) Fluorescence intensity of α-tubulin was quantified and compared against that of the PF127 group. The α-tubulin expression was significantly high in PF127-bFGF, HeP, and HeP-bFGF groups with DPSCs in HeP-bFGF being the highest. These results indicated that PF127-bFGF, HeP, and HeP-bFGF were biocompatible to DPSCs proliferation and friendly to neural regeneration. P < 0.01. Results represent data recorded at least three times.

2.3. bFGF Reduced the Inflammation Expression of Lipopolysaccharide (LPS)-Challenged DPSCs

Studies have reported that bFGF plays a role in inhibiting the activation of inflammation. To evaluate this effect of bFGFs, LPS was applied as an inflammatory stimulus. The expression of IL-6 confirmed by Western blot (WB) and immunofluorescence was studied to investigate whether bFGF would palliate the inflammation. As shown in Figure 3A,B, a clear dose–effect relation was observed between the dosage of bFGF and the expression level of IL-6, with 120 and 160 ng/mL bFGF showing no difference in IL-6 expression when compared with the control, DPSCs not challenged by LPS. Moreover, the protein expression of IL-6 was considerably reduced as the bFGF concentration increased, which indicates that the high concentration of bFGF reduced proinflammation factors (Figure 3B). All groups presented an expanded stem cell body after being exposed to LPS (Figure 2C) despite that bFGF treatment had decreased the density of cells. Data proposed that high bFGF dosage attenuated the proinflammatory status of DPSCs through suppression of IL-6 expression. Furthermore, we studied the NF-κB activation and IκB-α degradation. After LPS stimulation, the expression of NF-κB was dramatically increased with the increase of bFGF concentration (Figure 3D–F). The results showed that bFGF inhibited the NF-κB activation and the bFGF also caused the degradation of IκB-α, an inhibitor of NF-κB (Figure 3E). All data suggested that bFGF played a role in reducing the inflammation activities by inhibiting the NF-κB pathway.

Figure 3.

Figure 3

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Effect of bFGF on LPS-stimulated DPSCs. (A, B) IL-6 expression determined by Western blot and densitometric analyses of IL-6 showed a clear dose–effect relation between bFGF and the IL-6 expression level. When 120 and 160 ng/mL bFGF were applied, IL-6 expression was restored to the same level as the control, where DPSCs were not exposed to LPS. (C) Immunofluorescence of IL-6 of DPSCs. Scale bar: 200 μm. (D, E) Western blot of NF-κB and IκB-α and densitometric analyses suggested that bFGF could play an active role, attenuating the irritated DPSCs through the NF-κB pathway. (F) Immunofluorescence of NF-κB of DPSCs. Scale bar: 50 μm. #P < 0.05, ##P < 0.01 versus the control group; *P < 0.05, **P < 0.01 versus LPS. Results represent data recorded at least three times.

2.4. bFGF Reduced the Inflammation Expression of LPS-Induced Macrophages

To further unveil the bFGF’s role in the LPS-stimulated macrophages, we examined the expression of proinflammatory activities on RAW 264.7. We observed that the IL6 expression was mainly increased within LPS for 24 h (Figure 4A,C), which was identical to the excretion level analysis, (Figure 4B) though bFGF attenuated both the secretion and expression of IL6 with macrophage cells. These data suggest that bFGF might decrease the IL-6 activation by inhibiting proinflammatory cytokines on macrophage cells.

Figure 4.

Figure 4

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Effect of bFGF on RAW 264.7 stimulated by LPS. (A, B) 24 h LPS exposure resulted in elevated expression of IL-6 in macrophages significantly. 160 ng/mL bFGF could effectively calm the proinflammatory situation by attenuating the IL-6 expression significantly when compared with no addition of bFGF. (C) Immunofluorescence of IL-6 of RAW 264.7. Scale bar: 200 μm. (D, E) Western blotting of NF-κB and IκB-α and densitometric analyses suggested that LPS activated the proinflammatory NF-κB pathway of RAW 264.7. With the presence of bFGF, the irritated macrophages could be effectively relieved compared with no addition of bFGF, the RAW group. (F) Immunofluorescence of NF-κB of RAW 264.7. #P < 0.05, ##P < 0.01 versus the RAW group; *P < 0.05, **P < 0.01 versus LPS. Results represented data recorded at least three times. Scale bar: 50 μm.

To further prove the effect of bFGF on macrophage cells with LPS activation, we studied the NF-κB expression. As shown in Figure 4D,E, LPS remarkably increased NF-κB expression and decreased IκB-α expression and it was noted that bFGF could effectively reduce this increase in NF-κB expression (P < 0.01, Figure 4E,F). At the same time, bFGF also increased the IκB-α expression under LPS stimulation (Figure 4E). These data supplementarily proved the anti-inflammatory role of bFGF in LPS-treated macrophage cells.

2.5. DPCSs Polarized the LPS-Challenged Macrophages to M2 Type

In this study, we tested the polarization effect of DPSCs on inflamed macrophages, RAW 264.7 challenged by LPS. Macrophages were co-cultured with DPSCs for 24 h, and then cell functions and morphologies were studied by Western blot and immunofluorescence staining. We examined the expression level of proinflammatory factors of LPS-stimulated macrophages with or without the presence of DPSCs. We noticed that the IL-6 expression in macrophages was significantly increased when exposed to LPS for 24 h (Figure 5A,B). In addition, DPSCs could significantly reduce the expression of IL-6 compared to LPS-stimulated ones. Figure 5C showed that RAW cells cultured without DPSCs seem clustered and round-shaped; when co-cultured with DPSCs, they appeared more morphologically elongated.

Figure 5.

Figure 5

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Effect of DPSCs on RAW 264.7 stimulated by LPS. (A, B) Western blot and densitometric analyses of IL-6 expression of RWA264.7. The results indicated that LPS-challenged macrophages expressed significantly more IL-6 than macrophages without LPS. When co-cultured with DPSCs, the release of IL-6 of macrophages was significantly decreased. (C) Immunofluorescence staining of IL-6 of RWA264.7. Scale bar: 200 μm. RAW cells seemed to elongate with the presence of DPSCs. (D, E) Western blot and densitometric analyses of NF-κB and IκB-α. The results indicated that LPS-challenged macrophages expressed significantly more NF-κB than the macrophages without LPS. On the contrary, the expression of IκB-α in LPS-challenged macrophages was dramatically suppressed than that of the cells without LPS. Co-culture with DPSCs could significantly reverse the release of the proinflammatory factor and its inhibitor. (F) Immunofluorescence staining of NF-κB. #P < 0.05, ##P < 0.01 versus the RAW group; *P < 0.05, **P < 0.01 versus LPS. Results represented data recorded at least three times. Scale bar: 50 μm.

RAW 264.7 cells were analyzed by the expression of proinflammatory factors such as the NF-κB signal pathway and its inhibitor IκB-α. Cells were examined to identify DPSCs’ effect on the immune modulation of macrophages. The results indicated that LPS significantly disturbed the expressions of NF-κB and IκB-α compared with LPS-free cell culture and both were significantly modulated by the co-culture with DPSCs compared with LPS cell culture (Figure 5D,E). The representative fluorescence staining of NF-κB of RAW cells coincided with western blotting results, showing distinctive fluorescent intensities among treatment groups. These data suggested that DPSCs have the capacity to attenuate the activation of macrophages induced by LPS by inhibiting inflammatory factors.

2.6. DPSCs Treatment Prevented Microglia/Macrophages Activation and Reduced Proinflammatory Cytokine Release

To find out whether DPSCs influenced the activation of microglia/macrophages and the release of proinflammatory factors, we studied the proinflammatory expression of factors such as TNF-α and IL-6 in SCI animals. Three and seven days post SCI surgery, there was an increase in the expression levels of TNF-α and IL-6 of the SCI group in comparison to those of the control group, confirmed by Western blot and protein quantification (P < 0.05, Figure 6A–D). In the short term, 3 days post SCI, the HeP-bFGF hydrogel group showed the most significant decrease in the expression of IL-6 and TNF-α (Figure 6A,B). Neither HeP-DPSCs nor HeP-bFGF-DPSCs groups were able to influence the damaged tissue. However, 7 days post-SCI, Hep-DPSCs and HeP-bFGF-DPSCs groups presented an obvious decrease in the expression of IL-6 and TNF-α (P < 0.05).

Figure 6.

Figure 6

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(A) WB of the protein expression of TNF-α and IL-6 on 3rd day of treatment after spinal cord injury. (B) Quantification of the WB of TNF-α and IL-6 on 3rd day. These showed that HeP-bFGF treatment was the most effective intervention in short term post-SCI damage, where the expression of the proinflammatory factors, TNF-α and IL-6, was significantly decreased compared to the HeP-DPSCs and HeP-bFGF-DPSCs interventions. (C) WB of the protein expression of TNF-α and IL-6 on the 7th day of treatment after spinal cord injury. (D) Quantification of the WB intensity of TNF-α and IL-6 on the 7th day. These showed, contrary to the results on day 3, that the HeP-DPSCs and HeP-bFGF-DPSCs groups were the most effective interventions decreasing the expressions of the TNF-α and IL-6. (E) Quantitative analysis of TNF-α and IL-6-positive cells of the immunohistochemistry results on day 28 post-SCI damage. (F) Cross-sectioning results of the protein expression of TNF-α and IL-6 via the immunohistochemical staining on the 28th day of SCI. The quantification results were obtained by ImageJ. #P <0.05, ##P <0.01 versus the control group *P <0.05, **P <0.01 versus the SCI group. Scale bar: 50 μm.

On the 28th day, immunohistochemical staining suggested that the HeP-bFGF-DPSCs group demonstrated a significant decrease in the density of IL-6 and TNF-α staining around the injury site when compared with the SCI and control groups (Figure 6F). This implied that HeP-bFGF-DPSCs treatment was not only able to effectively relieve the irritated tissue but it was also a far more effective treatment than the HeP-bFGF and HeP-DPSCs interventions. From the analysis of IL-6 and TNF-α staining (Figure 6E), there was little difference in samples between HeP-bFGF-DPSCs groups and the control group. The cell numbers of IL-6 and TNF-α positive in the HeP-bFGF-DPSCs hydrogel group were much lower than in the HeP-DPSCs and HeP-bFGF groups on the 28th day (Figure 6E,F). Therefore, they prevented the accumulation of IL-6 and TNF-α at the injury site. The HeP-bFGF-DPSCs group showed the lowest level of pro-inflammatory cytokines compare to HeP-DPSCs and HeP-bFGF groups, suggesting that HeP-bFGF-DPSCs hydrogel treatment might decrease the area of injury occupied by reducing the proinflammatory factor levels and block the microglia/macrophage activation and following injury.

2.7. DPSCs Treatment Suppressed NF-κB Signaling Pathway

To further investigate whether the HeP-bFGF-DPSCs intervention presents any anti-inflammatory effect in in vivo applications, we explored the NF-κB signaling pathway in a rat SCI model with treatment interventions by Western blotting and immunohistochemistry. On days 3 and 7, the expression amount of NF-κB and IκB-α was significant in the SCI group indicating an acute and irritated status of the damaged spinal cord tissue. On day 3, the HeP-bFGF group showed an obvious change in NF-κB and IκB-α expressions compared to the SCI group; the HeP-bFGF-DPSCs group showed an early decrease of NF-κB expression. On day 7, HeP-DPSCs and HeP-bFGF-DPSCs groups showed significant changes in the NF-κB and IκB-α expressions compared to the SCI group.

Immunohistochemistry staining and analyses, Figure 7E,F, displayed the NF-κB and IκB-α expression levels on day 28 post-SCI, where both were highly expressed in the SCI group, indicating that an active self-healing existed at the damaged tissue site. The HeP-bFGF-DPSCs group was the only group that showed decreased expression of NF-κB and increased expression of IκB-α, which constituted a tissue-regeneration-friendly microenvironment. The low expression of IκB-α in HeP-bFGF and HeP-DPSCs groups might serve well as a successful combined application of the biomaterials, growth factor, and stem cells.

Figure 7.

Figure 7

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(A) WB of the protein expression of NF-κB and IκB-α on 3rd day after SCI. (B) Quantification of the WB intensity of NF-κB and IκB-α on 3rd day after SCI. A typical activation of the NF-κB pathway, increased expression of NF-κB and decreased expression of IκB-α, an inhibitor of NF-κB, were observed in the SCI group on day 3 post SCI damage. It was obvious that HeP-bFGF and HeP-bFGF-DPSCs treatments showed an effective decrease in NF-κB expression. Only HeP-bFGF effectively elevated the expression of IκB-α. (C) WB of the protein expression of NF-κB and IκBα on the 7th day of treatment after spinal cord injury. (D) Quantification of the WB intensity of NF-κB and IκB-α on day 7. High level of NF-κB observed in the SCI group indicated that the spinal cord tissue was still at the irritated status on day 7 post SCI damage. HeP-DPSCs and HeP-bFGF-DPSCs interventions showed a significant decrease in NF-κB expression and an increase in IκB-α expression. (E) Quantitative analysis of the immunohistochemistry staining of NF-κB and IκBα on day 28 post SCI damage. The result indicated that the expression of NF-κB was decreased and the expression of IκB-α was increased by HeP-bFGF-DPSCs treatment significantly. The expression of both NF-κB and IκB-α in the SCI group was highly expressed, indicating that the self-healing existed in the damaged spinal cord tissue. In HeP-bFGF and HeP-DPSCs treatment groups, the expression of IκB-α was significantly low compared to that of the SCI group, indicating unsustained expression of IκB-α and weak anti-inflammatory power. On the contrary, the IκB-α level was still maintained at high level on day 28 in the HeP-bFGF-DPSCs group. In the HeP-bFGF-DPSCs, the low level of NF-κB and the high level of IκB-α served a regeneration-favoring microenvironment for SCI repair. (F) Cross-sectioning results of the protein expression of NF-κB and IκB-α via the immunohistochemical staining on day 28 after the SCI. The quantification results were obtained by ImageJ. #P < 0.05, ##P < 0.01 versus control; *P < 0.05, **P < 0.01 versus SCI. Results represent three independent samples. Scale bar: 50 μm.

2.8. DPSCs Promoted Neurite Repair and Improved Neural Cell Sprouting in SCI

To study the neural repair in SCI with and without interventions, we dissected the spinal cord tissue samples and explored the expression of acetylated tubulin (Ace-tubulin) and microtubule-associated protein 2 (MAP-2) on day 21 post-SCI, when regeneration was expected. Ace-tubulin might play a crucial role in the differentiation of microtubule structure and function; associated with microtubule stability,2929 MAP-2 is primarily expressed in neuron dendrites and stabilizes the microtubules. Western blot results displayed a decreased expression of both Ace-tubulin and MAP-2 on day 21 in the SCI group. In contrast, all intervention groups showed significantly high expression of Ace-tubulin and MAP-2 (P < 0.01), with the HeP-bFGF-DPSCs group showing the highest. Thus, HeP-bFGF-DPSCs presented a constructive augmentation in the expression of neural repair and cell- sprouting-related proteins and provided microtubule stabilization of functional proteins, providing continued inducement in neural growth (Figure 8B).

Figure 8.

Figure 8

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(A) WB result of the expressions of Ace-tubulin and MAP-2 on the 21st day after SCI damage. (B) Quantification of the expression levels of Ace-tubulin and MAP-2. There was an increase in cell-sprouting-related protein expressions. The injured SCI group, with no active intervention applied, represented natural disease prognosis where little cell sprouting resulting in limited neural reconnection. In contrast, in experimental intervention groups, cell-sprouting-related protein expression was significantly elevated. The expression level of Ace-tubulin of HeP-DPSCs and HeP-bFGF-DPSCs was restored to the control level; the expression level of MAP-2 of HeP-bFGF, HeP-DPSCs, and HeP-bFGF-DPSCs was significantly higher than that of the control. #P < 0.05, ##P < 0.01 versus control, **P < 0.01 versus SCI, the injury group. All results were recorded three times.

2.9. HeP-bFGF-DPSCs Hydrogel Stimulated Axonal Regeneration

To study the axonal regeneration after SCI damage with and without interventions, we investigated the spinal cord at various levels on day 28 post-injury. Hematoxylin–eosin (HE) slides of the injured spinal cord (injury group) showed separated cells as well as voids on day 28. Intervention groups provided tissue regeneration to different levels with increasing order of HeP-bFGF, HeP-bFGF-DPSCs, and HeP-DPSCs (Figure 9A,B). At higher magnifications in HE slides, voids were reduced and a lumen was formed (Figure 9A). In the HeP-DPSCs-bFGF group, red blood cells were observed inside the lumen, indicating that vasculature had commenced by day 28. Consistent with HE results, a quantitative analysis of the volume of the preserved tissue indicated the HeP-DPSCs group showed the highest value (Figure 9B). All of these tests showed that HeP-bFGF-DPSCs could maximize the DPSCs’ impact on nerve repair. This indicated that HeP-bFGF-DPSCs had the capacity to maintain neural restoration and tissue recovery after the SCI. In representative magnetic resonance imaging (MRI) images, in the cross-sectional view of the injured region of the spinal cord, there was barely a visible lesion in the HeP-DPSCs group, suggesting that excellent tissue recovery had happened compared to other groups (Figure 9C).

Figure 9.

Figure 9

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Repair effect of nerve fibers and axonal regeneration of SCI-modeled rats on 28th day after treatments. (A) Cross-section of HE staining of the injured spinal cord tissue on the 28th day after the damage. The upper panel displays the cord at low magnification, where scar tissue and voids were seen in the SCI group and voids in HeP-bFGF and HeP-DPSCs groups. In the HeP-bFGF-DPSCs group, there were mostly solid tissues and cells at the injured site. At higher magnification (lower panel) in contrast to the control, where cells were intricately connected, the majority of cells in the SCI group were separated from each other. Cell density was increased, and the lumen structure was more often observed in the order of HeP-bFGF, HeP-DPSCs, and HeP-bFGF-DPSCs. In the HeP-bFGF-DPSCs group, red blood cells (black arrows) were seen inside the lumen (white arrows), indicating that vascular structures were formed on day 28. Scale bar: 500 μm (upper panel) and 100 μm (lower panel). (B) Preserved spinal cord tissue on day 28 after SCI. Quantitative analysis of HE slides indicated that there was more tissue on day 28 in HeP-DPSCs and HeP-bFGF-DPSCs groups compared to the injury group. **P < 0.01 versus the SCI group; ##P < 0.01 versus the HeP-bFGF group. All results were recorded at least three times. (C) Representative MRI images of the cross-sectional view of the spinal cords at T9 in control (red arrow), injury, HeP-bFGF, HeP-DPSC and HeP-DPSC-bFGF groups at 28 days post damage. Compared to the spinal cord of the SCI (injury), the cord tissue integrity was restored in HeP-bFGF, HeP-DPSC, and HeP-DPSC-bFGF groups. Scale bar: 1 cm.

3. Discussion

Tissue and cell engineering is a contemporary area of medical science that is based on the principles of cell biology, biomaterials, bioengineering, biochemistry, and biophysics, and its definitive goal is to help in treating diseases related to tissue loss and organ damage.13,30 Specifically, stem cell biotherapy has been considered as one of the potential treatment strategies for a chronic disease. This holds true for neural regeneration in inflammatory spinal cord injury.31,32 In fact, in our previous study, it was shown that the application of ex vitro, which develops pulp stem cells in models of spinal cord injury, can rather restore diseased and reconstructed tissues and DPSCs have been observed as a promising strategy for stem cell treatment.23,33 It is considered that transplanted DPSCs do not only participate directly in nerve repair but they can also recruit host neural cells by releasing different trophic factors,34 which are developed as their primary mechanisms to promote neural regeneration. However, some recent studies on the paracrine secretion of MSCs have concentrated on recruiting neural host cells having anti-inflammatory and immunoregulatory potential.35,36 Few studies have considered the paracrine secretion of MSCs on inflamed cells and neural injury cell differentiation during central nerve and peripheral nerve regeneration.21,22 In this study, a direct trans-well system was utilized to explore the biomolecular activity of DPSCs on macrophages cells, which allowed the diffusion of biomolecules between cells. Thereby, it can distinguish the paracrine effects from the direct cell-to-cell interactions and eliminate the cell contamination from DPSCs in the subsequent analyses. The results have shown that co-cultures with DPSCs in the presence of LPS induced low expression of IL-6 and NF-κB of macrophage cells in vitro. First, we showned that the production of IL6 in macrophages stimulated by LPS was mainly inhibited by DPSCs. Then, we found that NF-κB was extremely expressed after LPS was induced on RAW 264.7 and most macrophages cells displayed an expanded cell morphology and an increase in fluorescence intensity. The NF-κB and IL-6 expression was significantly decreased. Last, we also showed that HeP-bFGF-DPSCs hydrogel treatment could decrease the area of tissue occupied by reducing proinflammatory cytokines following an injury.

Our previous work has shown that DPSCs with growth factors could restore the sensory and motor neural cells of SCI-modeled rats.23 In this work, we explored the mechanism of DPSCs combining with bFGF in neuroinflammation. We found that in an acute SCI status, proinflammatory factors and the NF-κB signal pathway were activated. Microglia in the spinal cord injury help as primary immune cells and sensitive receptors to the environment.37 In the spinal cord, immune cells are in direct contact with neural cells. Hence, microglia are in an active state. Any changes in the neural cells lead to stress and will damage neural cells.38,39 The immune cells have the ability to release specific immune factors that stimulate proinflammatory cytokine activity.40 We observed that bFGF inhibits the NF-KB and IL-6 expression in LPS-stimulated DPSCs. Also, bFGF (160 ng/mL) significantly regulated LPS-stimulated macrophages by decreasing the proinflammatory expression of NF-κB and IL-6 and increasing the anti-inflammatory expression of IκB-α. In this study, we found that the expression of proinflammatory factors (IL-6 and TNF-α) was significantly different in all groups. Proinflammatory factors were higher in SCI than in the other groups. The protein expression of proinflammatory factors (TNF-α and IL-6) slowly decreased on days 3 and 7 in HeP-DPSCS and HeP-bFGF-DPSCs compared to the SCI during the acute stage (Figure 6A,C), and the decreasing expression of IL-6 and TNF-α was continued till the 28th day (Figure 6E,F). The HeP-bFGF treatment was the most effective in attenuating inflammation on the 3rd day of the injury compared to the other groups (Figure 6A). This suggested that the anti-inflammatory role of bFGF proved by an in vitro cell study (Figures 3 and 4, bFGF-treated LPS stimulated DPSC and RAW) also proved to be effective in in vivo application (Figure 6, HeP-bFGF-treated SCI). However, this bFGF’s influence was not obvious compared to interventions that contained DPSCs in the long-term application, e.g., 7 and 28 days (Figure 7E,F), where a relatively late but persistent anti-inflammation was observed. In this study, we unveiled the anti-inflammatory role of different therapeutic interventions in a timely manner. This series of studies evidenced that the combined application of scaffold materials, growth factors, and cells is the most feasible solution to tissue engineering.41

The SCI modeled in this study had been confirmed with damaged neural cells (Figure 8, Ace-tubulin and MAP-2 figure) with high inflammatory reaction. Since many receptors promote proinflammatory factors including TNF-α and IL-6 in microglia,42 we tested the inflammation levels of TNF-α and IL-6. In the HeP-bFGF, HeP-DPSCs, and HeP-bFGF-DPSCs groups, a neural-cell-protecting and damage-repairing microenvironment was observed (Figures 7 and 8 Ace-tubulin and NF-κB). The injured cells were less stressed and displayed anti-inflammatory factors and decreased expression of inflammatory factors on the 3rd and 7th day in the acute stage of the injury. In addition, from the results, we found a difference in the proinflammation effects between the HeP-bFGF and HeP-DPSCs at the 3rd day. The expression of IκB-α protein in the HeP-bFGF-DPSCs and HeP-DPSCs was higher than in HeP-bFGF on the 7 day. This meant that the bFGF played an important role in the DPSCs during the first 3 days after injury. DPSCs proliferated and differentiated into the neural stem cells at the injury site and decreased inflammatory factors through different paracrine mechanisms.43,44 Also, the DPSCs’ response to the injury is due to the immunomodulatory effect of stem cells. Still, the DPSCs group had a minor anti-inflammatory effect during the first stage. Moreover, in SCI, the microglial activation is documented. Consequently, on the 3rd day of the injury we speculate that there is high activity of microglia (NF-κB) in DPSCs and bFGF-DPSCs and low activity of microglia in the bFGF (Figure 7D). Hence, HeP-DPSCs and HeP-bFGF-DPSCs interventions had low microglial activity on the 7th day.

The NF-κB pathway plays a critical role in proinflammatory-cytokine-induced inflammation of neural cells and a central role in proinflammatory-cytokine-induced cellular apoptosis.45 In this paper, it has been proven that proinflammatory-cytokine-induced inflammation played a role in the responses of injury. The protein expression level of NF-κB increased obviously in the SCI group and decreased by the treatment of HeP-bFGF-DPSCs after 7 and 28 days post-injury. Our results additionally revealed that on the 3rd day, the expression of IκB-α was downregulated and that of NF-κB was upregulated in HeP-bFGF-DPSCs and HeP-DPSCs groups.

The rationale of co-administrating dental stem cells and bFGF is based on the fact that a single factor is in general less effective because the neural repair is a complex process requiring different variables, including different types of cells and growth factors as well as their concentration and controlled release.26 Here, we were the first to dissect the anti-inflammation mechanism of the combined application of hydrogel scaffold materials, growth factor, and stem cells in central nerve repair. We proved that both bFGF and DPSCs were effective in inflammation control in vivo and thus greatly enhance the nerve repair. It has been established that bFGF has regenerative and neuroprotective abilities in stem cell proliferation and differentiation after injury.30,46 It participates in the colony formation of stem cells of the human exfoliated deciduous teeth (SHEDs).47 Moreover, the growth factor also stimulates vessel formation (Figure 9A) and cell sprouting (Figure 8) around the nerve injury site, which may assist stem cells with nerve repair and growth by decreasing toxic metabolites and transiting nutrients in cells.27,28 In this study, we have proved that the in situ injection of bFGF and DPSCs via a HeP hydrogel encouraged the neural regeneration responses in spinal cord injury. Reduced protein expression of MAP-2 and Ace-tubulin is attributed to neural death and dysfunction after SCI.48 Our studies found that HeP-bFGF-DPSCs played an important role in maintaining the microtubule structure, which indicated significant neurites repairing. MRI scanning and tissue staining results displayed various levels of repair done by therapeutic interventions to the spinal cord tissue after SCI. HeP-bFGF-DPSCs showed the best restoration of the spinal cord tissue (Figure 9). Contrary to the other groups, the injured site on the spinal cord of the HeP-bFGF-DPSCs group had almost disappeared and was restored by cells and newly reconstructed tissues. HE staining proposed that the HeP-DPSCs group had more preserved tissue than HeP-bFGF-DPSCs and HeP-bFGF groups did.

4. Conclusions

In summary we combined bFGF with dental pulp stem cells and encapsulated them in a thermosensitive hydrogel for neuronal regeneration. It was a promising technique to improve treatment by decreasing the inflammation in the spinal cord injury site. HeP hydrogel co-application with bFGF and DPSCs resulted in positive effects on nerve repair and neural microtubule stabilization in SCI. Furthermore, our mechanistic investigation also showed that NF-κB was expressed the least in HeP-bFGF-DPSCs but the most in SCI, which was relatively similar to the level in the control group. Therefore, the controlled in situ delivery of DPSC through temperature-sensitive HeP and bFGF can be a promising therapeutic solution for spinal cord injury.

This work unveiled the SCI repair process achieved by the combined use of a hydrogel, growth factors, and stem cells with an emphasis on inflammation control. Active ingredients of the formula, bFGF and DPSCs, were independently assessed for their anti-inflammatory effects in cell studies. Delivered with a hydrogel (HeP), the anti-inflammatory effects of bFGF, DPSCs, and bFGF-DPSCs were re-assessed in an SCI rat model, with the HeP-bFGF-DPSCs being the most effective. Although HeP-DPSCs showed better microtubule stability and more tissue preservation after SCI, an early inflammation control and obvious vasculature of HeP-bFGF earned it a place in the formula, not to mention its protective role in stem cell application. As a continuation of our previous research in this paper, this work has provided an in-depth observation of the repair mechanism of the spinal cord and offered insight into the relationship between inflammation and complex-formula-assisted nerve repair.

5. Experimental Section

5.1. Preparation of Hydrogels

Four types of hydrogels were prepared: PF127, PF127-bFGF, HeP, and HeP-bFGF. PF127 was commercially purchased (P2443, Sigma-Aldrich). The synthesis of monoamine-terminated poloxamer (MATP) was performed according to a previously described method by a two-step reaction.49 First, PF127 (1.0 mM) was reacted with 4-nitrophenyl chloroformate (1.3 mM) and methylene chloride (20 mL) was dissolved in triethylamine with stirring for 4 h at room temperature. Next, diaminoethylene (3.0 mM) and the intermediate (1.0 mM) were dissolved in methylene chloride (20 mL) overnight at room temperature. The solution was mixed four times through petroleum ether and then dialyzed with distilled water by a membrane at room temperature for 3 days. Heparin salt (0.5 mM)-coupled MATP (0.5 mM) with N-hydroxysuccinimide (NHS) (0.25 mM) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (0.5 mM) was added in 5.6 pH 2-(N-morpholino)ethanesulfonic acid (MES) buffer (0.5 M) for 1 day at room temperature. An amide functional group of PF127 was combined with the carboxyl bond of heparin salt purposely and participated in amide functional formation. Then a modified-PF127 solution was dialyzed with distilled water using a dialysis bag (molecular weight cut-off (MWCO) 14 000) for 3 days and then lyophilized to get the final product, the HeP hydrogel. To prepare the PF127-bFGF and HeP-bFGF hydrogels, bFGF was mixed with lyophilized PF127 or HeP with mild stirring and incubated at 4 °C overnight to form the bFGF-supplemented hydrogels.50 The characteristic features of all four hydrogels were studied by scanning electron microscopy (SEM), rheological hybrid rheometer amplitude sweep measurements, transition temperature evaluation, DPSCs co-culture, etc. Some features are presented in the Supporting Information.

5.2. Isolation and Culture of DPSCs

Third molars without dental caries and periodontal lesions were extracted from healthy patient aged between 15 and 20 years from the Stomatology Hospital of Wenzhou Medical University, Wenzhou, China. All experiments were approved by the Ethics Committee at the School of Stomatology, Wenzhou Medical University. Alcohol (70% v/v) was used to sterilize the teeth. The process of pulp tissue removal was as previously described.19 Briefly, the pulp tissue was removed and washed five times with phosphate-buffered saline (PBS) containing 2.5% streptomycin/penicillin (S/P). The pulp tissue was cut into small pieces and kept in an Eppendorf tube. Pulp pieces were digested with 3 mg/mL collagenase type I (Gibco, Gaithersburg, MD) and 4 mg/mL dispase (Sigma-Aldrich, Steinheim, Germany) for 15 min at 37 °C. Pulp pieces and cell suspension were resuspended and cultured in α-modified Eagle’s medium (α-MEM, Gibco) supplemented with 20% fetal bovine serum (FBS) and 1% S/P at 37 °C, 5% CO2 in a humidified incubator. This primary culture was routinely maintained, passaged, and stored with regular medium change. DPSCs in passage 5 were used for this study.

5.3. Viability of DPSCs Cultured in Hydrogels

HeP-bFGF was gradually added to the complete cell culture medium (α-MEM supplemented with 15% FBS and 1% S/P) in an ice bath for 12 h. Then, the solution was filtered by a bottle-top filter (0.22 μm pore size, Biofil). Next, DPSCs (1 × 106 cells/mL) were mixed in hydrogels in an ice bath. After culturing, the culture plate was put in a 37 °C and 5% CO2 incubator for 20 min till gel formation occurred. A total of 180 μL/well medium was added to prevent the gel from dehydrating. Light microscopy was used to affirm the homogeneous cell encapsulation. The regular medium was changed every 3 days. The stem cells were confirmed by immunofluorescence staining with α-tubulin (ab179484, Abcam).

5.4. Regulatory Roles of bFGF and DPSCs in LPS-Stimulated Cells

To determine the anti-inflammatory role of bFGF in LPS-challenged DPSCs, 4 × 104 cells/well DPSCs were inoculated in six-well plates and cultured for 24 h at 37 °C followed by the addition of 1 μg/mL LPS to continue the culture for another 24 h. Then, culture media supplemented with a series of concentrations of bFGF (0, 20, 40, 80, 120, 160 ng/mL) were used to culture the DPSCs for 24 h. The cells were assessed by western blotting and immunofluorescence staining with IL-6 (DF6087, Affbiotech), NF-κB (8242S, CST), and IκB-α (4814, CST).

To determine the anti-inflammatory role of bFGF in LPS-challenged macrophages, 5 × 106 macrophages were first seeded in 6-well plates and cultured for 24 h followed by the addition of 1 μg/mL LPS to continue the culture for another 24 h. Then, 160 ng/mL bFGF was added on cells for 24 h. The mouse monocyte macrophages cells (RAW 264.7), gifted by Dr. Junjie Deng of the Wenzhou Institute of Biomaterials and Engineering, were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% FBS and 1% S/P at 37 °C, 5% CO2 in a humidified incubator.

To determine the role of DPSCs in LPS-challenged macrophages, RAW 264.7 cells were cultured and challenged. DPSCs and macrophages were co-cultured at a 1:0.2 ratio for 24 h in 6-well plates. DPSCs were inoculated in the upper compartment of trans wells (0.4 μM pore size, Corning, Lowell) with the macrophage on the basal well.

In the end, the expression of inflammatory factors of the challenged cells was assessed by Western blotting and immunofluorescence tests with IL-6 (DF6087, Affbiotech), NF-κB (8242S, CST), and IκB-α (4814, CST).

5.5. Recovery Effect of HeP-bFGF-DPSCs Hydrogel on an SCI Model

Seventy-five female adult Sprague–Dawley rats (210–260 g) were purchased from the Chinese Academy of Sciences Animal House (Shanghai, China). Before surgery, the animals were acclimated at the animal center for 10 days on a 12 h dark–light cycle. Animals were provided with free access to nutrients and water. All experimental procedures were approved and performed according to the Guide of Chinese National Institutes of Health and the Animal Care and Use Committee of Wenzhou Medical University.

The animals were randomly partitioned into five groups, including HeP-bFGF, HeP-DPSCs, HeP-bFGF-DPSCs, SCI, and control groups. The bFGF concentration in the intervention groups was prepared as 3 μg/μL.23 Rats were anesthetized through intraperitoneal injection of 8% chloral hydrate. Animals were placed on a prone position and fixed on a cork plate. The surgical area was routinely prepared with hair shaving and skin sterilization. A vertical incision was made between T8 and T10 of the spine along the midline to expose the vertebral column. T9 of the spine was located, and laminectomy was performed where the bone was carefully removed to expose the spinal cord. A moderate spinal cord injury was created by clamping a vascular clip on the spinal cord tissue at T9 for 2 min (30 g). Then, 10 μL of hydrogels of HeP-bFGF, HeP-DPSCs, and HeP-bFGF-DPSCs was injected in situ separately using a microsyringe. Animals in the SCI group received in situ injection of 10 μL of a sterile saline solution. Postsurgical care was delivered including pain control, prophylaxis infection control, individual housing, hydration and dietary needs, etc. The urinary bladder was manually emptied three times per day till autourination was restored whenever possible. Animals were euthanized on days 3, 7, 21, and 28 days within a CO2 chamber.

5.6. Western Blot Analysis

The spinal cord tissue at the T9 level was collected, dissected and stored at −80 °C on days 3, 7, and 21. To extract the protein in the spinal cord, the cord tissue was lysed in radioimmunoprecipitation assay (RIPA) buffer with protease inhibitor cocktail (Beyotime, Shanghai, China) and then centrifuged at 12 000 rpm. Protein concentration was quantified by a bicinchoninic acid kit. Proteins (80 μg) were prepared in 10% Bio-Tris polyacrylamide gel and then transferred onto a poly(vinylidene difluoride) (PVDF) membrane. The membranes were blocked with 5% skim milk (BD) in 0.05% BST containing 0.05% Tween for 2 h and then stored in primary antibodies for 16 h at 4 °C: TNF-α (ab6671, Abcam), IL-6 (21865-1-AP, Proteintech), NF-κB (8242S, CST), IκB-α (4814, CST), acetyl-α-tubulin (3971S, CST), and MAP-2 (Sigma-Aldrich). Then, the samples were treated with secondary antibodies for another 1 h at room temperature. The exposure of specific proteins was determined via the ChemiDoc XRS imaging system (Bio-Rad Laboratories, Hercules, CA). All experiments were done in triplicate.

5.7. Immunohistochemistry Analysis

Immunohistochemistry assessment of the injured spinal cords was performed on day 28 when animals were euthanized and perfused with paraformaldehyde (4%, pH = 7.4). A slice of the cord tissue (T9) was dehydrated by different concentrations of alcohol. Then, it was embedded in paraffin and cut into sections (4–6 mm). The slides for the immunohistochemistry analysis were treated with the primary antibodies of TNF-α (60291-1-IG, Proteintech), NF-κB (8242S, CST), IκB-α (4814, CST), and IL-6 (21865-1-AP, Proteintech) overnight (4 °C), followed by incubation with secondary antibodies for 1 h at 37 °C. Prior to being sealed, the slides were stained with hematoxylin (8 min). The slides were observed using a fluorescence microscope (Nikon, TS100, Japan).

5.8. Histological and Magnetic Resonance Imaging (MRI) Assessments of the Injured Spinal Cord

On day 28, the hematoxylin–eosin (HE)-stained section of the injured spinal cord at the T9 level was prepared and observed under a light microscope (Nikon TS100, Japan). Also, on day 28, rats were anesthetized and MRI was used to determine the restoration of tissues. The models were created by superconducting MRI imagers (GE Medical Systems) GE Signa (HDxT 3.0T) in the Wenzhou Second Affiliated Hospital. Super-resolution images were recorded from all animals by spin-echo T2-weighted MRI (acquisition matrix: 320 × 256, width: 41.67 kHz, NEX: 4.0, FOV: 9 cm, slice thickness: 1.5 mm, band TR/TE: 2560/92 ms).

5.9. Statistical Analysis

All data are presented as mean ± standard deviation. All statistical analyses were done by the SPSS software (SPSS 21.0, Chicago, IL). The data from immunofluorescent and immunoblotting were assessed using one-way analysis of variance (ANOVA) with the Tukey tests. GraphPad Prism (version 5, GraphPad Inc., CA) was used to display the quantitative results. P < 0.01 and 0.05 were used to determine statistical significance.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (81701032 and 81871503), the Wenzhou Medical University grant (QTJ16026), the Wenzhou Science and Technology Association Project, the Wenzhou Basic Research Project (Y20180131), the Zhejiang Province Program of the Medical and Health Science and Technology (2018KY537), and the Zhejiang Xinmiao Talents Program (2018R413186). The authors would like to thank Dr. Junjie Deng (Wenzhou Institute of Biomaterials and Engineering) for generously donating the macrophage cell line used in this study.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.0c01379.

  • Physichemical properties of the hydrogels (PDF)

Author Contributions

∇ A.A. and Y.H. contributed equally to this work.

The authors declare no competing financial interest.

Supplementary Material

ao0c01379_si_001.pdf (206.2KB, pdf)

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