Preconditioning of human dental pulp stem cells by H₂O₂ promotes functional recovery in a rat spinal cord contusion injury model

Abstract

Cell therapy is a potential therapeutic choice to treatment spinal cord injury (SCI). Studies proved that hypoxic precondition has protective effects on damaged cells. It sounds that a sub-lethal hypoxic exposure is likely enhance survival rates of transplanted cells. The aim of this study is the effect a novel strategy of preconditioning of human dental pulp stem cells (hDPSCs) via hydrogen peroxide (H₂O₂) on treatment rat SCI model. The hDPSCs were isolated from human third molar. The cells were cultured in various concentrations of H₂O₂ (0–400 μM) at different time points (2–48 hours). Then, the cell viability was estimated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. In rat SCI model, 0.5×10⁶ hDPSCs with and without H₂O₂ preconditioning were locally transplanted in contusion site. Behavioral assessment was rendered within 7 weeks after injection. Besides, the brain-derived neurotrophic factor (BDNF) was evaluated. The results of MTT showed that the cell viability rates were significantly lower at the higher concentrations of H₂O₂ at various times. However, the cell viability declined, followed by exposure to 50 μM H₂O₂ for a period of 2-12 hours compared with untreated cells. Basso, Beattie, and Bresnahan (BBB) findings revealed transplantation of the preconditioned hDPSCs increased BBB score significantly compared to the SCI group. Also, BDNF as a neurotrophic factor, dramatically increased in treated group. These finding suggest transplantation of sub-lethal concentration of H₂O₂ preconditioned hDPSCs would have protective roles through paracrine signals by enhancing survival capability transplanted cells which can cause motor neuron recovery in SCI.

Introduction

The spinal cord is a part of the central nervous system (CNS) responsible for interaction between the brain and the peripheral nerve system. Also, it controls the spinal reflex and regulates the functions of other organs [1]. Physical traumatic injuries and non-traumatic implications from some diseases (e.g., cancer and infection) can result in axonal degeneration, demyelination, and neural and glial cell dysfunction in the spinal cord, which are generally defined as spinal cord injury (SCI) [2, 3]. SCI is one of the most serious CNS damages adversely affecting people’s life quality and brings a burden to the family and healthcare system. SCI is characterized by temporary or permanent impairments in sensory and motor function, depending on the injury site and degree [2, 4]. Unfortunately, endogenous regeneration following SCI is limited due to inadequate numbers of neurotrophic factors, lack of neurite outgrowth, and some inhibitory factors existing in myelin debris, glial scars, and fibroblasts that prevent precursor cells from migrating to the injured site [5-7]. Among several therapeutic approaches, such as drug administration and surgical interventions, offered to deal with this issue, none has been hopefully useful for repairing the injured spinal cord. Therefore, developing novel strategies that focus on regeneration is very important [8, 9]. Currently, cell therapy has attracted the attention of researchers [3, 9, 10]. Among different stem cells, dental pulp stem cells (DPSCs) have been widely applied in neurodegenerative diseases. These cells have some unique characteristics, including simple accessibility, non-allogenic, and noninvasive isolation without ethical concern [11, 12]. In addition, several studies have shown that DPSCs have a high proliferation rate and multiple differentiation abilities to generate sources of different lineage cells. Therefore, they can hold a great promise for cell replacement in SCI [11, 13]. Despite many attempts made in this regard, the clinical outcomes of stem cell transplantation are barely successful due to the low proliferation and survival rate of transplanted stem cells [14]. Micro-environmental factors at the spinal cord lesion site, increased level of reactive oxygen species (ROS), and subsequent oxidative stress and cell apoptosis induction provide harmful conditions that interrupt cells migration, differentiation, and population [15]. Gene modification and preconditioning cells with chemical factors are some strategies proposed to overcome these obstacles and improve therapeutic efficacy [16, 17]. It has been reported that hydrogen peroxide (H₂O₂) plays a dual role in both physiological and experimental environments. H₂O₂ is not only non-harmful but also has a protective effect on the proliferation and differentiation of stem cells at specific concentrations [15, 18]. Previous animal model studies have confirmed that preconditioning the bone marrow mesenchymal stem cells with H₂O₂ restores decreased stem cells’ viability and proliferation after injury [14]. In addition to the protective role of hypoxia or ischemic preconditioning approaches, it has been shown that hormones especially estrogens positively affect the processes of stem cells’ proliferation, survival, and differentiation [19-22]. However, the effect of DPSCs preconditioned on SCI has not been studied yet. Thus, we hypothesized that the contribution of H₂O₂ can serve as a protective factor in regenerative therapy. The present study aims to evaluate whether hypoxia human dental pulp stem cells (hDPSCs) preconditioning with H₂O₂ can improve the viability of grafted cells in the SCI model.

Materials and Methods

Human dental pulp stem cells isolation and culture

hDPSCs were extracted from human third molar of individuals (18–25 years old) at the dental clinic of Mazandaran Medical University. All procedures were conducted according to the standard guidelines and approval of the institutional ethics committee of Mazandaran Medical Sciences University and after receiving the informed consent from all individuals. Collected teeth were immediately disinfected by transferring them in phosphate-buffered saline (PBS) to the research laboratory and washing them in iodine (povidone iodine 10%) for 5 minutes. Under the sterilized condition, parts of the root and dentin were removed with a surgery cutter, and pink pulp tissue was gently separated by forceps. Then, the pulp tissue was minced into small pieces with a scalpel and enzymatically digested in a 0.25% trypsin solution (Gibco). Afterward, the solution containing pulp tissue, trypsin, and DMEM/F12 (Gibco-Life Technologies) was transferred into a falcon and incubated at 4°C. Then, the solution containing tissue was centrifuged at 1,800 rpm for 5 minutes. In the next step, the supernatant was discarded, and tissues were cultured in a T25 flask and incubated at 37°C, 5% CO2, and 95% humidity. The supplement was replaced with a fresh medium, and cultured tissue was observed every 2–3 days under an inverted microscope (Olympus) to monitor the proliferation and migration of cells out of the tissue. As the cells grew to optimal confluence, they were detached from the primary culture flask with trypsin and passaged for sub-culturing in other flasks to achieve purified hDPSCs. The cells from passages 3–5 were used for further experiments [23].

Ethical approval

This research was approved by Ethical Committees of Mazandaran University of Medical Sciences (No. IR.MAZUMS..REC.1399.8324).

Characterization of human dental pulp stem cells

FCM was used to analyze the mesenchymal markers CD73, CD90, and CD105 and hematopoietic stem/progenitor cell markers CD34 and CD45. Cells at P5 were detached using trypsin and centrifuged at 1,800 rpm for 5 minutes. Then, the supernatant was discarded, and 3×10⁵ of the prepared cells were re-suspended in 100 µl PBS. Cells were fixed in 1% paraformaldehyde for 15 minutes on ice, treated with triton for 10 minutes to be permeable for further reagents, and treated with a blocking solution for 45 minutes. Next, cells were incubated for 30 minutes at 4°C in a dark chamber with fluorescent FITC conjugated antibodies against antigens, including CD73, CD90, and CD105 as superficial mesenchymal markers CD34 and CD45 for hematopoietic stem/progenitor cell. After incubation, cells were washed 3 times with PBS, centrifuged at 1,800 rpm for 5 minutes, and incubated with conjugated secondary antibody for 30 minutes. Cells were passed through a flow cytometer, and marker expression was immediately measured on a BD FACSCalibur cell analyzer (Becton Dickinson). Data were analyzed using FlowJo software (flow cytometry [FCM]; BD FACSCalibur). During the process, cells were washed with cold PBS between each step, and untreated cells were considered negative controls [23].

Preconditioning of human dental pulp stem cells via H₂O₂

To investigate the effects of H₂O₂ on hDPSCs in vitro, we cultured cells in various concentrations of H₂O₂ (0–400 µM) at different time points (2–48 hours). Also, the cells’ viability was estimated using an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay.

Based on these findings, 50 µM H₂O₂ after 12 hours exposure was selected as the optimal concentration to model oxidative stress in these cells and was thus used in subsequent experiments. The hDPSCs were incubated with 50 µM H₂O₂ after 12 hours according to the IC50 values as the preconditioned group. Also, the nonpreconditioned cells were considered the control group.

MTT assay

Cell viability was assessed based on the mitochondrial function of living cells by reducing tetrazolium salt (MTT) at different concentrations of H₂O₂ (0–400 µM) at various time (2, 4, 8, 12, and 24 hours). Briefly, 5×10³ cells were seeded on 96-well plates. Then, 50 μl of 1 mg/ml MTT solution (Sigma-Aldrich) created in PBS was added to each well, and cells were incubated for 3 hours at 37°C in the dark. After incubation, the media was removed and replaced with 50 μl of 100% dimethyl sulfoxide (Sigma-Aldrich) and then on a shaker for 5–10 minutes to agitate and dissolve formazan crystals. Absorbance at 570 nm was measured by a Cytofluor 4000 plate reader (PerSeptive Biosystems). All experiments were performed in triplicate [24].

In vitro scratch assay

Scratch assay was performed according to Amiri et al. [18] and Malih et al. [25] protocol to evaluate in vitro cell migration. Briefly, preconditioned or non-preconditioned cells were seeded at 5×10⁴ into a 6-well plate and incubated in DMEM containing 5% (v/v) fetal bovine serum for 24 hours. A sterile pipette tip was used to scrape the cell monolayer. Cells were then incubated at 37°C for 24 hours to allow the cells to migrate into the scratch area. Finally, the scratch area was analyzed using the ImageJ software (National Institutes of Health).

Real-time PCR

Total mRNA of the preconditioned and non-preconditioned cells and spinal cord tissue samples was extracted using phenol/chloroform. Also, cDNA was synthesized according to the manufacturer’s instructions (Thermo-Fisher Scientific). Real-time PCR was conducted using the SYBR Green Master Mix and primers for SOD1, SOD2, Nrf2, TNF-α, and IL-6. PCRs consisted of an initial denaturation at 95°C for 10 minutes, followed by 40 cycles with 95°C for 15 seconds, 60°C for 30 seconds, and 72°C for 30 seconds. Finally, 18S ribosomal RNA was used for mRNA expression normalization, and quantitative analysis was assessed by ΔCt.

Experimental groups and transplantation procedure

A contusive spinal cord model was performed to induce SCI using the New York University (NYU) impactor device, as described previously [14]. A total of 24 male Sprague–Dawley rats weighing between 200 g and 250 g with normal locomotor performance were used in the study. The rats were randomly divided into 4 groups as follows (n=6 per group):

(1) The sham-operated group had only laminectomy without any SCI.

(2) SCI group, which underwent laminectomy with SCI, received no manipulations.

(3) SCI+hDPSCs group received 0.5×10⁶ hDPSCs suspensions in 5 µl PBS following SCI.

(4) SCI+preconditioned hDPSCs received 0.5×10⁶ H₂O₂-preconditioned hDPSCs in 5 µl PBS following SCI.

Briefly, after anesthetizing animals with 40 mg/kg of ketamine, they were fixed on the surgical table facilitated with a thermal blanket to maintain their body temperature. The hair on the back was shaved, and the skin was disinfected with ethanol. Then, an incision was made on the mid-thoracic area, and paravertebral muscle layers were separated. Subsequently, laminectomy was performed, and the spinal cord was exposed at vertebral level T8–T9. Using the NYU impactor, spinous processes of was fixed by clamps, impactor rod was placed 2.5 cm above the spinal cord and dropped on the exposed position of the spinal cord to produce severe contusion. The cells used for the in vivo study were labeled with DiI (Sigma-Aldrich), and the viability of cells before injection was more than 96% by trypan blue exclusion dye assay (Sigma-Aldrich). Next, the cells were injected into the rostral and caudal (the epicenter) of the contusion site using a Hamilton Syringe.

After surgery, muscle layers and skin were gently sutured. Post-surgical care was implemented to improve recovery. These measures included treating with cefazolin and solution twice a day for 3 days to avoid infection and corneal dehydration, respectively, and manual depletion expression of urine bladder 3 times a day until the voluntary function of the bladder was restored. During operation, physiological normal saline serum was injected to compensate for bleeding.

After transplantation, all animals received cyclosporine (10 mg/kg; Chong Kun Dang Pharm) every day.

Western blot analysis

The brain-derived neurotrophic factor (BDNF) expression in the spinal cord was evaluated by sacrificing the rats and taking their injured site. Total protein was extracted using a protease inhibitor cocktail and RIPA buffer. Protein was separated by 10% (v/v) sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The membrane was incubated with anti-BDNF and anti-beta actin antibodies overnight and horseradish peroxidase-conjugated secondary antibody for 1 hour. Bands were detected using a chemiluminescence enhanced chemiluminescence western blot substrate kit and measured by ImageJ software.

Behavioral assessment

The Basso, Beattie, and Bresnahan (BBB) test—often called the BBB locomotor rating scale—is a behavioral assessment used mainly in rodent models of SCI. Before SCI, animals were trained to adapt to the test environment. After surgery, the animals were placed on the open field (75–120 cm) and allowed to move across the field. Each of the right and left hind limbs’ functional movements in each animal was monitored by a digital video recorder. Next, they were measured by two independent examiner observers blinded to SCI identity for 4 minutes. Each hind limb was given a score of 0 (for no hind limb movement) to 21 (for normal function). The test was first examined daily for 7 consecutive days and then once a week within 7 weeks after surgery, and the average of the scores was considered the final result.

Footprint test

Footprint assessment was expressed by the sciatic function index. The rat’s hind feet were dipped in ink and were allowed to walk through a tunnel.

Statistical analysis

Data are either expressed as individual data points in a vertical scatter dot plot with a line to indicate the mean or as bar graphs showing mean±SD. Comparisons between two groups were analyzed using the two-tailed Student’s t-test. For multiple comparisons, one-way analysis of variance was used, followed by Tukey’s post hoc test for analyzing parametric data. All statistical analyses were performed using GraphPad Prism (GraphPad Software Inc.). *P<0.05, **P<0.01, and ***P<0.001 are considered statistically significant.

Results

Some adherent hDPSCs cells with elongated contours were observed after 3 days of culture initiation. The cultured cells exhibited a heterogeneous morphology before reaching 70%–80% confluences at the end of the first passage (Fig. 1A). Confluent cells gradually became more spindle-shaped and displayed fibroblast-like morphology. Also, confluent cells were formed morphologically as a homogeneous population during subsequent passages, and their fibroblastic morphology was kept throughout the subcultures (Fig. 1B).

Fig. 1.

Characterization of human dental pulp stem cells (hDPSCs). Morphology of hDPSCs at (A) day 3 and (B) passage 3. (C) Flow cytometry analysis of hDPSCs were positive for CD73, CD90, and CD105 and negative for CD45 and CD34 (conjugated, red/FITC-conjugated, green). (D, E) Multilineage differentiation of hDPSCs. (D) Oil Red O–positive intracellular lipid droplets and (E) Alizarin Red S staining of mineralization.

The FCM analysis exhibited that hDPSCs expressed mesenchymal CD markers (namely, CD73, CD90, and CD105) with high percentages, while the cells were negative for CD34 and CD45 as hematopoietic SC markers (Fig. 1C). Adiposeness was identified by visualizing lipid vacuoles recolored with Oil Ruddy O and osteogenesis with Alizarin Red S to evaluate calcium statement (Fig. 1D, E).

The MTT assay of the H₂O₂ preconditioning on hDPSCs revealed that the cell viability rates were significantly lower at the higher concentrations of H₂O₂ (100 μM and 400 μM) at various times. However, the cell viability declined, followed by exposure to 50 μM H₂O₂ for a period of 2–12 hours compared with untreated cells. No statistically significant difference was observed between them. So, we considered 50 µM H₂O₂ after 12 hours exposure for the further experiment (Fig. 2).

Fig. 2.

Viability percentage of human dental pulp stem cells in various concentrations of hydrogen peroxide (H₂O₂) (0–400 µM) at different time points (2–48 hours); 50 µM H₂O₂ after 12 hours exposure was significantly different at 2–12 hours. MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide. *P<0.05; **P<0.01.

Moreover, as shown in Fig. 3, the gap area analysis proved that the migration of H₂O₂ preconditioning cells was dominantly lower than the untreated cells at a time period of 24 hours (Fig. 3).

Fig. 3.

The effect of hydrogen peroxide (H₂O₂) on migration evaluated by scratch assay; the migration of preconditioning cells was dominantly lower than the untreated cells at the time period of 24. ***P<0.001.

The results of real-time PCR indicated that the expression of SOD1 and SOD2 genes, which are considered scavenger ROS, was significantly upregulated in H₂O₂ preconditioning hDPSCs than in untreated cells (Fig. 4). Moreover, the expression level of Nrf2 was dramatically increased in treated cells compared with untreated cells (Fig. 4). The expression of IL-6 and TNF-α after preconditioned cell engraftment dramatically decreased compared to the untreated cells (Fig. 4).

Fig. 4.

Real-time PCR analysis of (A) SOD1, SOD2 and (B) Nrf2 genes; SOD1, SOD2, and Nrf2 were significantly upregulated in hydrogen peroxide (H₂O₂)-preconditioning human dental pulp stem cells (hDPSCs) than untreated cells. Also, the expression of (C) TNF-α and (D) IL-6 after preconditioned cell engraftment was dramatically decreased compared to untreated cells (n=3 biological samples; mean±SD). SCI, spinal cord injury. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Our western blot data showed that the BDNF, as a neurotrophic factor, dramatically increased in SCI+preconditioned hDPSCs compared to the unpretreated group (SCI+hDPSCs group), SCI group, and vehicle-transplanted group (Fig. 5).

Fig. 5.

Western blot analysis of brain-derived neurotrophic factor (BDNF). BDNF was significantly upregulated in hydrogen peroxide (H₂O₂) preconditioning human dental pulp stem cells (hDPSCs) than untreated cells (Fig. 4A) (n=3 biological samples; mean±SD). SCI, spinal cord injury. *P<0.05, **P<0.01.

The results of the BBB test showed that the control and sham groups attained a score of 21. A serious hind limb locomotor dysfunction was observed in the SCI group compared to the control and sham groups at weeks 1, 2, 3, 4, and 5 after SCI (Fig. 6). Transplantation of the preconditioned and non-preconditioned hDPSCs increased BBB score significantly compared to the SCI group. However, there was no significant difference between the preconditioned and non-preconditioned treated groups except in the fourth and fifth weeks (Fig. 6).

Fig. 6.

The effect of human dental pulp stem cells (hDPSCs) and hydrogen peroxide (H₂O₂) preconditioned cells on Basso, Beattie and Bresnahan (BBB) score, in sham-operated group, spinal cord injury (SCI) group, SCI+hDPSCs group and SCI+H₂O₂ group within 7 weeks after implantation.

The results of the footprint assessment illustrated an improved footprint of the animals in the treatment groups, especially in preconditioned hDPSCs compared with the SCI group, 7 weeks after cell transplantation (Fig. 7).

Fig. 7.

The effect of human dental pulp stem cells (hDPSCs) and hydrogen peroxide (H₂O₂) preconditioned cells on footprints, in sham-operated group, spinal cord injury (SCI) group, SCI+hDPSCs group, and SCI+H₂O₂ group within 7 weeks after implantation.

Discussion

The present study aimed to assess the preconditioning of hDPSCs by H₂O₂ and the effect of preconditioning on transplanted cell survival in vivo and measuring the functional recovery in rat spinal cord injuries. Previous studies recommend that H₂O₂ is essential for effective self-renewal in stem cells. These studies have explored the direct levels of H₂O₂ as signals to promote cell proliferation and survival that play a growth factor-like part in cells. In this respect, hDPSCs were cultured with an optimum dose of 50 μM H₂O₂ [14]. At that point, we inspected whether preconditioning hDPSCs with H₂O₂ could enhance the viability of the cells by increasing the SOD1, SOD2, and Nrf2 gene expression [26]. Our finding revealed that the Nrf2 (a transcription factor that regulates the expressions of antioxidant proteins and activates them) increased following the H₂O₂ group treatment in hDPSCs. This result is contrary to the findings by Rahimi et al. [27]. It appears that free radicals play reciprocal parts in cell development and proliferation. As a result, intracellular ROS levels and the related signal transduction pathways depend on long-term self-renewal, actuation, expansion, and differentiation of hematopoietic stem cells [27]. Furthermore, we expected that preconditioned hDPSCs migration would be increased significantly, although the migration rate in scratch assay and migration assay distinguished the considerable decrease in the treated group in comparison of an untreated group. In this respect, our study is in line with the previous works.

For the recognization of hDPSc stem cells, FCM was used for analyzing the CD markers. The data showed that CD 90 and CD 105 (special markers for stem cells) and CD 73 markers were expressed in hDPSCs cells, while CD 34 and CD 45 (specific for hematopoietic stem cells) were not expressed. This finding confirms the result that these cells were hDPSCs [13].

To confirm the multipotency of stem cells, we induced them into adipogenic and osteogenic lineages. The results showed the multipotency of the stem cells extracted and cultured in this study [14, 23, 28].

Our discoveries demonstrated that the cell’s viabilities and resistance against apoptosis increased compared to untreated cells against the H₂O₂ preconditioned culture [23]. Consistent with the present result, hypoxic preconditioning mesenchymal stem cells (MSCs) enhanced the expression of BDNF and cell viability and resistance against apoptosis [29].

The measurement of BDNF growth factor expression using western blot and the effect of transplanted preconditioned hDPSCs with H₂O₂ showed that it was higher in the preconditioned hDPSCs-treated group than in the untreated group. Also, it was found that BDNF can foster the hDPSCs migration and differentiation into neurons [30].

Finally, we used some behavioral tests such as the BBB test and foodprint test to assess locomotor functional recovery and measuring the efficacy of pre-conditional and hDPSCs in SCI rats. The outcomes showed that in the BBB test, transplantation of the preconditioned and non-preconditioned MSCs caused a significant increase in BBB score compared to the SCI group. This result confirms those reported in other works [30].

In conclusion, these finding suggest transplantation of sub-lethal concentration of H₂O₂ preconditioned hDPSCs would have protective roles through paracrine signals by enhancing survival capability transplanted cells which can cause motor neuron recovery in SCI.