Evaluation of the Toxicity of Human Dental Pulp-Derived Mesenchymal Stem Cells on Animal Models: An Animal Study.

Introduction: Dental pulp remains one of the important sources of mesenchymal stem cells for most preclinical and clinical studies. Aim and
Objectives: To assess the safety after injecting human dental pulp-derived mesenchymal stem cells by intramucosal and intrabony routes in rabbits for clinical application. Materials and
Methods: Animal studies were carried out among 30 New Zealand male white rabbits (3-5 months old), weighing 1.5-2 kgs, which were divided into three groups with 10 animals in each group. Group 1: control group, Group 2: intramucosal route, Group 3: intrabony route. Data were analyzed using Student's t-test, and any P ≤ 0.05 was statistically significant.
Results: A total of 30 rabbits were selected for the study, among which significant statistical difference for Packed cell volume (PCV) (P < 0.05), MCHC (P < 0.05), platelet count (P < 0.05), and ESR (p < 0.001) has been reported in the hematological parameters. The results of the present study indicate that the transplantation of hDPSCs by intramucosal and intrabony routes into a rabbit is non-toxic without any detectable side effects or local or systemic rejection. The pre-clinical safety and toxicity of the hDPSCs in various human disease models need to be determined in future studies. Various pre-clinical studies to determine the safety and toxicity of hDPSCs in human disease models have to be done in the future.
Conclusion: This study showed that the intramucosal route and intrabony route of administration of stem cells were found to be non-toxic at 10 million per mL concentration. A further evaluation must be done for more definitive results. Copyright:

INTRODUCTION

At present, it is challenging to treat patients with oral mucosal lesions such as oral submucous fibrosis, ulcers, mucositis, carcinomas, and osteomyelitis. The dental pulp remains one of the important sources of Mesenchymal stem cells (MSCs) for most of the studies.[1] Both allogenic and autologous MSCs used for the treatment of human diseases have been proved to be safe in multiple clinical studies, which were conducted earlier.[2] This study was performed to evaluate, before the initiation of a clinical trial using the MSC Clinical Trial 2010,[3] whether MSCs are safe to be injected through the intramucosal and intrabony routes in healthy rabbits.

MATERIALS AND METHODS

Thirty New Zealand male white rabbits (3–5 months old), weighing 1.5–2 kgs were selected for the study. The experimental protocol was approved by the institutional animal ethics committee (IAEC) P1F/IAEC/SA/2018/YTR/Rabbits and experiments were conducted according to the guidelines of the Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA) 282/PO/Bt/S/2000/CPCSEA.

The experimental groups were randomly distributed (control group and test group).

Group 1: control group in which 10 rabbits received normal saline.

Group 2: 10 rabbits received high-dose MSCs group (10 million stem cells per animal in 50 μL of sterile saline) through the intramucosal route. After injection, they were sacrificed after 14 days.

Group: 3–10 rabbits received high-dose MSCs group (10 million stem cells per animal in 50 μL of sterile saline) through the intrabony route. After injection, they were sacrificed after 14 days.

This study used the cell number used by a dose optimization pilot study performed previously by Jin et al. (2018).[4] The period of study was 30 days. At the end of the experiment, all rabbits were physically observed for clinical signs and then dissected for examination. They were examined grossly for evidence of morphological abnormalities. Mucosal and bony samples were collected for histopathology [Figure 1]. Hematological and biochemical parameters were analyzed in all rabbits after euthanasia [Figure 2]. The student's t-test was used to analyze the data and any P ≤ 0.05 was statistically significant.

Figure 1

Buccal mucosa, sublingual tissue, and jawbone samples were collected

Figure 2

Blood sample collected

RESULTS

A total of 30 rabbits were divided into three groups, among which histopathological scoring concluded that hDPSCs are slight irritants at the site with the proliferation of fibrocytes/fibroblasts and migration of round epithelial cells and chondrocytes near the site of injection. The transplantation of high-dose human dental pulp-derived MSCs in rabbits was non-toxic in terms of the development of tumor and mortality. Bar diagrammatic representation of the hematological parameters represents the mean value variation in PCV, MCHC, and ESR which represent the anemic changes [Figure 3]. High platelet counts were found to occur more frequently than low counts in bar diagrammatic representation of platelets. It shows that there was variation between the control and implanted sites [Figure 4]. Serum biochemical parameters show mild variations in creatinine, bilirubin, alkaline phosphatase, serum albumin, globulin, which suggest that spontaneous changes in the kidneys and liver are seen [Figure 5]. When compared statistically, the control and experiment animals revealed a statistical difference for PCV (P < 0.05), MCHC (P < 0.05), platelet count (P < 0.05), and ESR (p < 0.001). Mild anemic changes were seen in all rabbits [Table 1]. The mean values of biochemical variables that were studied in [Table 2] reported that the glucose (P = 0.03), serum creatinine (P = 0.001), serum bilirubin (P = 0.01), serum alkaline phosphatase (P < 0.001), serum albumin (P < 0.001), globulin (P < 0.001) increased in all animals after the administration of stem cells. Spontaneous changes in the kidneys and liver were observed in the biochemical parameters.

Figure 3

Bar diagrammatic representation of the hematological parameters

Figure 4

Bar diagrammatic representation of platelets

Figure 5

Bar diagrammatic representation of the serum biochemical parameters

Table 1

Hematological parameters showing the statistical comparison between control and experiment animals

Student’s t-test	Group	N	Mean	Std. deviation	P
White blood count	Implanted	10	7.650	1.1683	0.82 (NS)
	Control	10	7.740	0.4575
Red blood count	Implanted	10	6.156000	0.3719379	0.55 (NS)
	Control	10	5.994000	0.7543238
Hemoglobin (Hb%)	Implanted	10	12.430	0.6929	0.10 (NS)
	Control	10	13.030	0.8603
Hematocrit (PCV)	Implanted	10	41.670000	2.2745207	0.03* (S)
	Control	10	44.300000	2.7640550
Mean RBC volume (MCV)	Implanted	10	68.670000	1.7359275	0.77 (NS)
	Control	10	68.490000	0.8425490
Mean RBC Hemoglobin (MCH)	Implanted	10	20.820000	0.6033241	0.40 (NS)
	Control	10	21.010000	0.3725289
Mean RBC Hemoglobin concentration (MCHC)	Implanted	10	30.350	0.4743	0.03* (S)
	Control	10	30.740	0.2221
Platelet count	Implanted	10	434.40	60.089	0.01* (S)
	Control	10	369.60	42.526
Neutrophils	Implanted	10	26.70	4.739	0.46 (NS)
	Control	10	25.20	4.237
Lymphocytes	Implanted	10	64.10	4.977	0.36 (NS)
	Control	10	62.30	3.592
Monocytes	Implanted	10	4.60	0.966	0.47 (NS)
	Control	10	4.90	0.876
Eosinophils	Implanted	10	1.80	0.789	0.43 (NS)
	Control	10	2.10	0.876
Basophils	Implanted	10	0.10	0.316	0.33 (NS)
	Control	10	0.00	0.000
Reticulocyte count	Implanted	10	1.8700	0.25303	0.07 (NS)
	Control	10	2.0340	0.10916
Bleeding time	Implanted	10	3:10:00.00	0:12:14.847	0.86 (NS)
	Control	10	3:09:00.00	0:14:29.483
Clotting time	Implanted	10	6:21:00.00	0:20:14.907	0.73 (NS)
	Control	10	6:24:00.00	0:18:58.420
ESR	Implanted	10	7.50	2.068	0.001* (S)
	Control	10	4.30	1.418
Prothrombin time	Implanted	10	14.20	2.573	0.05 (NS)
	Control	10	16.00	0.816
Activated partial thromboplastin time (APTT)	Implanted	10	42.80	3.765	0.48 (NS)
	Control	10	41.90	1.370

Table 2

Biochemical parameters showing the statistical comparison between control and implanted animals

Variables	Implant		Control		P	Significance
	Mean	SD	Mean	SD
Serum glucose	108.70	8.23	117.40	8.67	0.03*	S
Blood urea nitrogen	22.80	2.04	23.40	2.07	0.52	NS
Serum creatinine	1.00	0.00	1.10	0.06	0.001*	S
Serum total bilirubin	0.00	0.00	0.05	0.05	0.01*	S
SGPT (ALT)	52.80	4.16	52.10	3.76	0.69	NS
SGOT (AST)	44.50	6.28	44.30	6.38	0.94	NS
Serum alkaline phosphatase	304.90	105.06	144.20	34.73	<0.001*	S
Serum total protein	5.20	0.42	5.37	0.19	0.26	NS
Serum albumin	2.00	0.00	2.30	0.05	<0.001*	S
Globulin	3.00	0.00	3.23	0.07	<0.001*	S
Serum total cholesterol	42.80	7.55	43.00	7.23	0.95	NS
serum triglycerides	54.30	6.27	54.00	6.43	0.91	NS
HDL	23.00	1.63	22.80	1.62	0.78	NS
LDL	22.40	1.71	22.80	1.93	0.63	NS
Serum phosphorus	5.80	0.92	5.99	0.94	0.66	NS
Serum calcium	13.30	0.82	13.33	0.78	0.93	NS
Serum sodium	140.20	0.92	140.02	0.55	0.60	NS
Serum potassium	4.00	0.00	3.94	0.41	0.65	NS
GGT	12.20	1.32	12.16	1.31	0.94	NS

DISCUSSION

Stem cells could be used for the treatment of incurable diseases, with the effectiveness of bone marrow stem cell therapy in various oral mucosal disorders such as submucous fibrosis, ulcers, mucositis, and carcinomas as suggested by Suma et al. (2015).[5] Stem cells were carried from the stem cell bank (Transcell Biolife) to the National institute of nutrition (NIN) in a cryovial, safely placed in the storage tank, containing dry ice (–80° C) to maintain the viability of the stem cells. Friedenstein et al. (1970),[6] Aithal et al. (2017),[7] and Elsaadany et al. (2015)[8] had used bone marrow-derived mesenchymal stem cells (BMMSCs) in their studies in contrast to the present study. In the present study, the administration of high-dose hDPSCs (10 × 106 cells/kg b.w.) were found to be non-toxic in rabbits. Lalu et al. (2012)[9] conducted a 14-day acute toxicity study in rats and rabbits using pooled hBMMSCs. In this study, pooled hBMMSCs (252 × 106 cells/kg b.w.) were injected through intravenous and intramucosal routes into Fischer rats. Similarly, the cells (130 × 106 cells/kg b.w.) were administered to New Zealand white rabbits. Rabbits were observed for mortality and clinical changes for 14 days. After euthanizing the rabbits, organs were sent to a pathological laboratory for examination. They concluded that hBMMSCs did not elicit abnormal clinical signs at a dose of 252 × 106 cells/kg b.w. in rats following intravenous and intramucosal administration. Similarly, the intramucosal administration of these cells (130 × 106 cells/kg b.w.) was also found to be non-toxic in rabbits. Naik et al. (2008),[10] Intix Pvt. Ltd. (2008)[10] conducted a similar study by injecting hBMMSCs derived from a single donor at a dose of 15 × 106 cells/kg b.w. or vehicle to rats. The organs were examined for gross pathological changes, and tissue samples were collected for histopathological examination. No abnormality was detected histologically in either vehicle or hBMMSC-treated animals. In the present study, similar results were observed after administering hDPSCs into rabbits. Studies such as Weissman (2000),[11] Keller (1995) suggest that MSCs mediate their therapeutic effects by either differentiating into functional reparative cells that replace injured tissues or by secreting paracrine factors that promote repair.[12] Overall, histopathology findings demonstrated proliferation of fibrocytes/fibroblasts and migration of stem cells after injecting high-dose hDPSCs by the intramucosal route and migration of round epithelial cells and chondrocytes at the site of injection by the intrabony route. These results were compared with the histopathological findings by Zychouski et al. (2013)[13] Baptista et al. (2008).[14] In a previous study by Yun et al. (2016), mesenchymal progenitor cells derived from the umbilical cord were administered intravenously in mice, no histological changes were observed in the euthanized animals.[15]

CONCLUSION

Within the limitations of this study, the following conclusions were drawn:

The intramucosal route of administration of human dental pulp-derived mesenchymal stem cells was non-toxic in 10 million per mL concentration.

The intrabony route of administration of stem cells was found to be non-toxic in 10 million per mL concentration.

Further evaluation has to be done for more definitive results.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.