Thymosin alpha 1 provides short-term and Long-term benefits in the reimplantation of avulsed teeth:

a double-blind randomized control pilot study

Wings T.Y. Loo DDS, MSc, PhD^a, Y.D. Dou DDS, MDS^a, W.K.J. Chou BSc (Pharmacy)^b,

Min Wang DDS, MDS^c,?

^aJinhua Dentistry, Kowloon, Hong Kong, People‘s Republic of China

^bLeslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada

^cState Key Laboratory of Oral Diseases, West China Stomatology College, Sichuan University, Chengdu, People‘s Republic of China

Received 30 July 2007; accepted 13 September 2007

Abstract

Background: This study investigates the short-term and Long-term effects of thymosin alpha 1 (Talpha1) on reimplantation of avulsed teeth.

Methods and Material: Seventy-three patients with avulsed teeth were double-blind randomly assigned to a control group and group that used Talpha1. The teeth were reimplanted after treatment with Talpha1 or saline. Patients were monitored for both short-term and long-term parameters.

Results: The thymosin group demonstrated a lower interferon, tumor necrosis factor ? and interleukin-6 levels (P b .05) and higher white blood cell levels than the control group (P b .05). The thymosin group demonstrated greater periodontal healing (P b .05), less ankylosis (P b .05), less tooth movement, and greater lifetime of the replanted teeth.

Conclusion: The use of Talpha1 has both short-term and long-term beneficial effects during reimplantation of avulsed teeth.

(C) 2008

Introduction

Thymosin alpha 1 (Talpha1) is a synthetic 28-amino acid peptide with multiple biological activities primarily directed toward immune response enhancement [1]. It was originally

* Corresponding author. State Key Laboratory of Oral Diseases Engineering, Ministry of Education, West China of Stomatology College, Sichuan University, Chengdu 610041, People’s Republic of China. Tel.: +86 852 2345 7671; fax: +86 852 3176 7247.

E-mail address: [email protected] (M. Wang).

developed for the treatment of hepatitis B virus infection, but is currently used for the treatment of hepatitis B virus and Hepatitis C virus infections [1] and being tested for non- small cell Lung cancer [2], Hepatocellular carcinoma [3], AIDS [1], and malignant melanoma [4].

As an immune stimulant, Talpha1 may improve period- ontal healing in dental patients with avulsed teeth. This study is the first double-blind randomized control trial to determine the short-term and long-term effects of Talpha1 on the reimplantation of avulsed teeth. Short-term effects include the level of pain, the level of C-reactive protein (CRP) expression, the level of certain pro- and anti-inflammatory

0735-6757/$ - see front matter (C) 2008 doi:10.1016/j.ajem.2007.09.007

T alpha 1 improves re-implanted teeth after avulsion

cytokines and the white blood cell count. Long-term effects include teeth movement, periodontal healing, devel- opment of ankyolosis, and lifetime of the replanted teeth.

Method and materials

Sample selection

Patients with avulsed teeth presented within 45 minutes of the injury to the West China Stomatology College of Sichuan University, Department of Prosthetics, between June 2000 and June 2007. At this time, the avulsed teeth, including the portion attached to the alveolar socket, were stored in Dulbecco/Vogt modified Eagle’s Minimal Essential Med- ium. Patients were then given a full explanation of our double-blind randomized control trial and were invited to join the study. Exclusion criteria included unstable systemic signs, fractures, or contamination of the avulsed tooth, fractures of the alveolar bone or socket, inflammation, or periodontal disease.

Informed consent was obtained from all qualified patients and the study protocol was approved by the Hong Kong University ethics committee. The qualified subjects ranged from 12 to 26 years old and included 73 patients of whom 16 were female and 29 were double-blind randomly assigned to the control group whereas the others were assigned to the thymosin group (Table 1). The avulsed teeth included 63 upper incisors and 10 lower incisors.

Preparation of avulsed teeth

The dentist washed the avulsed tooth in 0.9% normal saline for 5 minutes. Afterwards, pulp tissue was extracted from the tooth before conventional gutta-percha points root canal obturation was completed. The teeth of the thymosin group were then immersed in Talpha1 1.6 mg (SciClone Pharmaceuticals, Hong Kong) for 10 minutes. During the entire treatment, the root surface was kept moist with saline [5]. The teeth of the patients in the control group followed the same procedures, but the use of Talpha1 was replaced with 0.9% normal saline.

Reattachment of avulsed teeth

Patients were locally anesthetized for the procedure. The avulsed tooth was implanted into the socket with the help of

Table 1 Subject sex and randomization into control and thymosin (test) groups
	Control	Test	Total
Female	6	10	16
Male	23	34	57
Total	29	44	73

575

Table 2 Mean levels of CRP between thymosin and control groups
	Group	Mean (pg/mL)	SD	SEM
Day 0	Thymosin	0.3045	0.02132	0.00455
	Control	0.3150	0.06364	0.01500
Day 1	Thymosin	1.6968	0.58802	0.12537
	Control	2.3089 ?	1.02187	0.24086
Day 2	Thymosin	1.6859	0.76329	0.16273
	Control	2.7522 ?	1.32781	0.31297
Day 5	Thymosin		0.58880	0.12553

finger pressure then sutures were placed. Intermediate restorative material (Dentsply, DeTrey, Germany) was used as a sealer. The reimplanted teeth were splinted with the neighboring teeth using a semiridged splint (fiber splint) for 10 days [6]. The occlusion was adjusted during the Healing process to prevent contact with opposing teeth.

0.9291

Control 2.2078 ?

* Significant P value b .05.

0.28491 0.06715

Patients were prescribed amoxicillin 250 mg to be taken

3 times a day and 0.12% chlorhexidine mouthwash (Peridex, Zila, Phoenix, AZ) to be taken every 4 hours for 7 days. During these 7 days, the thymosin group had 0.2 mL of Talpha1 injected into the periodontal sockets and the control group had the same volume of sterile 0.9% normal saline injected.

Patient monitoring and follow-up

Short-term monitoring began on the day of reimplantation and again on days 1, 2, and 5. The patient’s temperature, level of pain, the level of CRP, the levels of tumor necrosis factor ? (TNF-?), interferon (INF), interleukin 4 and 6 (IL-4, IL-6), and the WBC count were measured.

Long-term monitoring began on the third month post reimplantation and again on months 6, 12, 18, 24, 30, 36, 42, and 48. X-rays and periodontal radiographs were taken to access movement of the reimplanted tooth as well as ankyolysis and periodontal healing. The regeneration of periodontal tissue was measured at 6 points by periodontal probe at 0.5 mm.

Statistical analysis

The 2 group’s pain level was analyzed with Spearman’s nonparametric correlation. White blood cell, cytokine, and CRP levels were analyzed with linear mean comparison. All statistical tests were preformed with SPSS 11.5 for Windows (SPSS, Inc, Chicago, Ill).

Results

Short-term monitoring measured the pain score, CRP level, cytokine level, and WBC level. Both control and

576 W.T.Y. Loo et al.

IFN indicates interferon; D, day.

Control Thymosin

* P b .05.

4/29

34/44 ?

25/29

10/44 ?

		(P b .05)
IFN D0	Thymosin	-	22.1446	2.49068
	Control		20.6754	2.46968
IFN D1	Thymosin	*	19.1702	1.56059
	Control		28.9131	2.57830
IFN D2	Thymosin	*	20.3106	2.01050
	Control		28.2877	2.12256
IFN D5	Thymosin	*	19.1694	3.16206
	Control		28.9696	2.79543
TNF D0	Thymosin	-	31.9985	5.27782
	Control		23.9160	1.58532
TNF D1	Thymosin	*	28.6558	3.09073
	Control		44.4057	2.48212
TNF D2	Thymosin	*	25.3481	2.66155
	Control		43.3542	3.04656
TNF D5	Thymosin	*	26.0498	2.74142
	Control		41.9150	3.09912
IL-6 D0	Thymosin	-	0.8203	0.14244
	Control		1.0541	0.28636
IL-6 D1	Thymosin	*	3.2983	0.53964
	Control		8.3680	2.33283
IL-6 D2	Thymosin	*	1.8204	0.30082
	Control		3.0237	0.33156
IL-6 D5	Thymosin	*	1.2860	0.24795
	Control		3.8135	1.21070
IL-4 D0	Thymosin	-	2.2958	0.23016
	Control		2.1177	0.09255
IL-4 D1	Thymosin	-	2.7524	0.41937
	Control		2.5204	0.26901
IL-4 D2	Thymosin	-	2.6742	0.33486
	Control		2.2251	0.12081
IL-4 D5	Thymosin	-	2.2669	0.16191
	Control		2.1411	0.09542

Table 3 Mean level of pro- and anti-inflammatory cytokines in the thymosin and control groups					Table 5 Long-term (48 mo) outcome of thymosin and control group
Group	Significant	Mean	SEM		Periodontal healing	Ankyolosis

The thymosin group demonstrated similar CRP levels as the control group on day 0, but a statistically significant decrease in CRP levels was observed on days 1 to 5 (Table 2).

Levels of pro-inflammatory cytokines (INF, TNF, and IL-6) were lower in the thymosin group compared to the control group after day 0, whereas levels of anti-inflammatory cytokines (IL-4) were not statistically different between the 2 groups on all days (Table 3).

The thymosin group demonstrated a statistically signifi- cant increase in WBC levels compared to the control group since day 1 (Table 4).

A larger number of patients in the thymosin group demonstrated long-term periodontal healing and less anky- losis on x-ray observation when compared to the control group (Table 5). Less tooth movement was observed for patients in the thymosin group as well. The lifetime of the replanted teeth was greater in the thymosin group. Among the reimplanted teeth of the control group, 10 were lost by the 12-month mark and 12 more were lost by the 36-month mark with only 7 still lasting at the 48 month mark. Comparatively, fewer teeth were lost in the thymosin group; 2 were lost by the 12-month mark and 4 more were lost by the 36-month mark. There were still 38 teeth at the 48-month mark.

thymosin groups demonstrated similar body temperatures from day 0 to day 5, at around 37?C.

The thymosin group demonstrated a statistically signifi- cant decrease in pain score on day 1, as compared to the control group as calculated by Spearman’s nonparametric correlation (P = .009). The pain score for the subsequent days was similar between the 2 groups.

Table 4 White blood cell count in thymosin and control

groups

6.7168

Thymosin 8.7000 ? 1.74693 0.41176

	Group	Mean	SD	SEM
D1 WBC	Control	7.6000	1.59463	0.33998
	Thymosin	9.5011 ?	2.40865	0.56772
D2 WBC	Control		1.08458	0.23123

D5 WBC	Control	7.8982	2.56142	0.54610
	Thymosin	6.6906	3.09659	0.72987
* P b .05.

Discussion

Avulsion is the complete detachment of a tooth from its socket [7]. Most commonly injured teeth are the maxillary central incisors and protruding teeth [7]. Avulsion involves severed periodontal ligament and possible alveolar fracture [7]. The avulsion of permanent teeth is a true dental emergency. The optimal time to reimplant the tooth is less than 30 minutes after avulsion [8]. The traditional protocol for reimplantation involves soaking the tooth in sterile salt solution for 20 to 30 minutes then splinting the tooth [8].

Talpha1 is a synthetic 28-amino acid peptide with multiple biological activities primarily directed toward immune response enhancement [9]. This study investigated the effects that Talpha1 had on reimplantation of avulsed teeth in terms of level of pain, the level of CRP, the level of pro- and anti-inflammatory cytokines, and the WBC count (short-term effects), and also in terms of teeth movement, periodontal healing, development of ankyolosis, and lifetime of the replanted teeth (long-term effects).

T alpha 1 improves re-implanted teeth after avulsion 577

Our study demonstrated both short-term and long-term benefits of Talpha1 in reimplantation of avulsed teeth.

Pain was significantly less in patients of the thymosin group on day 1. This may be attributed to the accompanied change in cytokine levels observed on around the same day. Pro-inflammatory cytokines, such as INF [10], TNF-? [10], and IL-6, mediate the Inflammatory process. The difference in the levels of these 3 cytokines between the 2 study groups were the lowest on day 1 (Table 3), leading to less inflammation and pain. The levels of anti-inflammatory cytokine, IL-4, did not differ between the 2 study groups. Thus this showed that Talpha1 reduces pain by decreasing the levels of pro-inflammatory cytokines, but not by increasing the levels of anti-inflammatory cytokines.

C-Reactive protein is known as an acute phase protein and is considered a marker of acute inflammation and a potential contributor to inflammatory diseases [11]. Our study demonstrated a significant decrease in CRP levels after Talpha1 use, showing a positive effect at controlling the inflammation caused by avulsion. The greatest effect was on day 5, with a mean CRP level of 0.9291 pg/mL (Table 2).

The significantly higher levels of WBC in the thymosin group demonstrated Talpha1’s immune potentiating proper- ties [12] (Table 4). The greatest difference in WBC levels between the 2 groups was on day 2. By day 5, there was no longer a statistically significant difference in WBC levels between the 2 groups.

The patients of the thymosin group had stronger and longer lasting reimplanted teeth than the control group as demonstrated by less teeth movement, less development of ankyolosis, higher levels of periodontal healing, and longer lifetime of replanted teeth.

In conclusion, this double-blind randomized control trial is a pilot study involving the effects of Talpha1 on the reimplantation of avulsed teeth. We conclude that the use of Talpha1 has both short-term and long-term

beneficial effects when used during reimplantation of avulsed teeth.

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