Effectiveness of stem cells and platelet rich plasma in the treatment of oral submucous fibrosis

ABSTRACT

Introduction:

Medical management of oral submucous fibrosis which is a common precancerous condition affecting population of Asian descent is still under investigations. Various modalities of medical management are detailed in the literature with varying degree of success. The major aim of the study is to evaluate the outcomes of platelet rich plasma and stem cells in the medical management of oral sub mucous fibrosis.

Materials and Methods:

Ten patients diagnosed with stage II, III and IV oral submucous fibrosis were included in the study. The stem cells and PRP derived from patients peripheral blood were injected intraorally into the areas of fibrosis. Mouth opening were recorded at the follow up visits.

Results:

All patients showed satisfactory results at the end of 6 months follow up interval. Mouth opening has improved post treatment; however we suggest a long term study with more number of patients.

Conclusion:

Stem cells and PRP can be a new effective modality for medical management of oral submucous fibrosis which requires detailed scientific studies.

INTRODUCTION

Oral submucous fibrosis (OSMF) is a chronic, progressive, disabling, scarring, precancerous condition of the oral mucosa, which was first described by Schwartz in 1952 among five East African women of Indian origin under the term atropica idiopathica mucosae oris.[1]

The most common symptom is a burning sensation of the oral mucosa, which is aggravated by spicy food. As the disease progresses, gradual stiffening of oral mucosa with tough and leathery consistency associated with fibrous bands in the buccal mucosa appears, which causes varying degrees of difficulty in mouth opening.

Advances in stem cell technology have opened new vistas for treatment of these lesions. Stem cell therapy is primarily aimed at neoangiogenesis by releasing cytokines and growth factors paracrine effect. This may result in increased free radical scavenging by antioxidants either naturally occurring or extraneous.[2] Neoangiogenesis may also facilitate the removal of senescent cells from the lesions by supplying more number of scavenging defense cells and reversal of hypoxia in the diseased tissue.[2]

Stem cell therapy may help to stimulate resident tissue stem cells to transform into new fibroblasts, which may help in the removal of disintegrated biochemically and morphologically altered collagen fibers.

Platelet-rich plasma (PRP) is a new approach to tissue regeneration: it is widely used in various surgical fields.[3,4] Commonly, PRP is used as a gel formed by mixing PRP (derived from the centrifugation of autologous whole blood) with thrombin and calcium chloride. PRP gel includes a high concentration of platelets and a native concentration of fibrinogen.[4] Besides their procoagulant effects, platelets form a rich source of important growth factors, such as platelet-derived growth factor (PDGF), transforming growth factor-b (TGF-b) 1 and 2, and vascular endothelial growth factor (VEGF); all of these are involved in the angiogenic cascade which along with stem cells aids in reduction of fibrosis in OSMF cases.[5,6]

MATERIALS AND METHODS

Ten patients who are diagnosed with OSMF with Grades II, III, and IV (with more than 10 mm mouth opening) who have not undergone any previous treatment for the same, reporting to Department of Oral and Maxillofacial Surgery were included in the study. Patients diagnosed with Grade IV OSMF with less than 10 mm mouth opening. Patients with immune compromised conditions were excluded from the study.

After explaining the procedure and the preoperative incisional biopsy was taken to confirm, only patients with OSMF were included in the study. Patients were shifted to the procedure room, and peripheral venous blood was withdrawn with the amount of 9 ml, respectively, for both the stem cell and PRP using 10 cc under aseptic precautions. The amount of blood withdrawn was then transferred to the respective stem cell kit and the PRP kits. Both the kits were then centrifuged at 3000 rpm in the centrifugation machine [Figure 1] for 10 min. Post 10 min, the tubes were taken out, which had the following layers that are being the lower layer comprising of the RBCs with the thixotropic gel in between and with the upper layer containing the raw stem cells and the PRP, respectively. Once the stem cells and PRP were ready [Figure 2], the tubes were opened, and the top layer of approximately 2 ml of the solution or the supernatant layer was discarded, and the remaining 4 ml of the solution comprising of the stem cells and the PRP were withdrawn using 5 cc disposable syringe. The contents then were mixed with 2 ml of 2% lignocaine with 1:800,00 adrenaline and were injected into the buccal mucosa [Figure 3], the retro molar areas and the labial mucosa. The same technique was followed and was repeated at an interval of 1^st, 2^nd, and 3^rd week. Postinjection, follow-ups were done at intervals of 1^st, 3^rd, and 6^th months. Post 6 months, mouth opening was assessed [Figures 4 and 5].

Figure 1.

Centrifugation Machine

Figure 2.

Prepared stem cells and PRP for Injection

Figure 3.

Injecting PRP on the buccal mucosa

Figure 4.

Pretreatment mouth opening 24 mm

Figure 5.

Post-treatment mouth opening 25 mm

RESULTS

In this study, the mouth opening at baseline was mean of 21.80 with standard deviation (SD) of 3.75 (mean rank, 1.60). Postintervention, the mouth opening of the participants after 1 month was mean of 22.50 with SD of 3.58 (mean rank, 2.10). At 3^rd month follow-up, the mouth opening was 23.20 with SD of 3.96 (mean rank, 2.70), and at 6^th month follow-up, it was found to be 24.20 with SD of 3.91 (mean rank, 3.60) [Table 1]. The difference was found to be statistically significant [Table 2].

Table 1.

Evaluation of mouth opening at postoperative 1^st, 3^rd, and 6^th months, burning sensation, mucosal changes, and postbiopsy changes

Age	Sex	Diagnosis	Stage	Prebiopsy Diagnosis	Mouth Opening (mm)	Stem Cells and Prp Injected (ml)	1st Month Mouth Opening (mm)	3rd Month Mouth Opening (mm)	6th Month Mouth Opening (mm)	Burning Sensation	Mucosal Changes	Postbiopsy Changes
30 years	M	OSMF	III	OSMF	23	6	23	25	26	Reduced	Yes	No
35 years	M	OSMF	II	OSMF	26	6	26	26	28	Reduced	Yes	No
36 years	M	OSMF	III	OSMF	16	6	19	19	19	Reduced	Yes	No
22 years	M	OSMF	III	OSMF	23	6	26	26	26	Reduced	Yes	No
35 years	F	OSMF	IV	OSMF	14	6	14	14	16	Reduced	No	No
36 years	M	OSMF	III	OSMF	24	6	24	25	26	Reduced	Yes	No
35 years	M	OSMF	III	OSMF	23	6	24	26	27	Reduced	Yes	No
26 years	M	OSMF	III	OSMF	24	6	24	26	26	Reduced	Yes	No
40 years	M	OSMF	III	OSMF	23	6	23	23	26	Reduced	Yes	No
24 years	M	OSMF	III	OSMF	22	6	22	22	22	Reduced	Yes	No

Table 2.

Friedman test

	n	Mean Rank	Mean	Standard Deviation	Minimum	Maximum
Mouth opening	10	1.60	21.80	3.765	14	26
1st month mouth opening	10	2.10	22.50	3.598	14	26
3rd month mouth opening	10	2.70	23.20	3.967	14	26
6th month mouth opening	10	3.60	24.20	3.910	16	28
Test Statistics
n				10
Chi-square				20.182
df				3
Asymp. Sig.				0.000

The comparison of mouth opening at baseline with other time intervals postintervention shows that there was no statistically significant difference between baseline and 1 month postintervention. However, there was statistically significant difference seen between baseline and 3 months (0.02), baseline and 6 months (0.007), between 1 month and 3 months (0.059), 1 month and 6 months (0.015), and 3 months and 6 months (0.026) follow-up [Table 3].

Table 3.

Wilcoxon signed ranks test^[a,b]

Test Statistics
	1st Month Mouth Opening-Mouth Opening	3rd Month Mouth Opening-Mouth Opening	6th Month Mouth Opening-1st Month Mouth Opening	6th Month Mouth Opening-3rd Month Mouth Opening	6th Month Mouth Opening-Mouth Opening	3rd Month Mouth Opening-1st Month Mouth Opening
Z	−1.633	−2.232	−2.428	−2.232	−2.714	−1.890
Asymp. Sig. (2-tailed)	0.102	0.026	0.015	0.026	0.007	0.059

^aWilcoxon Signed Ranks Test. ^bBased on negative ranks

DISCUSSION

OSMF can be “a chronic, progressive premalignant condition, with juxtaepithelial deposition of fibrotic tissue followed by muscular degeneration and limitation in oral opening.”[1]

The pathogenesis and treatment of OSMF have been the subject of controversy, ever since Schwartz first described the condition in 1952.[7] No satisfactory treatment has been described for OSMF. The use of local injection of dexamethasone, hyaluronidase, and placental extract has been described in literature.[1]

Steroids act as immunosuppressive agents by opposing the action of soluble factors released by sensitized lymphocytes following activation by specific antigens.[1] Steroids also prevent or suppress inflammatory reactions, thereby preventing fibrosis by decreasing fibroblastic proliferation and deposition of collagen.[1] Although these methods are effective, they provide only temporary relief to the patients.[1]

Stem cells are generically defined as undifferentiated cells that are capable of self-renewal through replication as well as differentiation into specific cell lineages.[8] Stem cells can be obtained from tissues of endodermal, mesodermal, or ectodermal lineages.[8]

Hematopoietic stem cells are well characterized cells of mesodermal origin and provide the basis for bone marrow transplant procedures.[8]

Mesenchymal stem cells (MSC) obtained from bone marrow have been reported to differentiate into several cell types such as those in muscle, fat, bone, cartilage, liver, lung, and nerve.

Stem cells possess autologous properties and hence show minimal host reactions. Raw stem cells consumes less time for being prepared, has more patient compliance, and can be done on OPD basis and hence is more easier. They primarily aim for neoangiogenesis and revascularization and thus can be effective in treating patients with premalignant conditions like OSMF.

Neoangiogenesis also aims at the removal of senescent cells from the lesions by supplying more number of scavenging defense cells and reversal of hypoxia of the diseased tissue. Stem cells also helps to stimulate resident tissue stem cells to host fibroblasts, which may help in removal of disintegrated biochemically and morphologically altered collagen fibers in cases like OSMF.[9]

Platelets are known for their role in hemostasis where they help prevent blood loss at sites of vascular injury. To do this, they adhere, aggregate, and form a procoagulant surface leading to thrombin generation and fibrin formation.

Platelets also release substances that promote tissue repair and influence the reactivity of vascular and other blood cells in angiogenesis and inflammation. They contain storage pools of growth factors including PDGF, TGF-β, and VEGF, as well as cytokines including proteins such as PF4 and CD40L.[10] Hence, PRP was being used as an adjunct with the raw stem cells to facilitate the better results and also to promote neoangiogenesis.

Hence, this study was undertaken at our institute to evaluate the effectiveness of both stem cells and PRP in the treatment of OSMF.

Adalja and Adalja concluded that human stem cell applications are more focused on the MSCs, which are found in marrow stroma, and can be easily isolated.[11] These cells are also found to be precursor to the human tissues, which are undifferentiated and have replicated by its own.[11] The same has been confirmed by Seshadri et al. where they isolated the stem cells form bone marrow.[12]

In our study, the mouth opening of all the participants with OSMF at baseline was mean of 21.80 with SD of 3.75. Postintervention, the mouth opening of the participants after 1 month was mean of 22.50 with SD of 3.58. At 3^rd month follow-up, the mouth opening was 23.20 with SD of 3.96. At the end of 6^th month follow-up, it was found to be 24.20 with SD of 3.91. The difference was found to be significant. These measures have correlated with the study by Seshadri et al. where they concluded the improvement in the mouth opening after the treatment.[12]

In our study, there was reduction in the burning sensation for all the patients, which again can be correlated to the study by Seshadri et al.[12] We also found that there were significant changes in the mucosa affected by OSMF post-treatment. There was clinically noticeable reduction of the blanching, and the mucosa appeared near normal except for one patient where no changes were appreciated.

The main disadvantage of our study was the limited sample size and short-term follow-ups. Another disadvantage of our study being it was not compared with any other control group which might have allowed us to arrive at a better conclusion.

In our study, stage II OSMF resulted better results compared to stages III and IV where surgical modality can be combined to produce the desired results. The advantage of our study is that it can be carried out on OPD basis with minimal equipment, and since the stem cells used were autologous, there were no reported complications or allergies.

OSMF treatment is still under controversy as there are various treatment modalities being proposed by various authors. Raw stem cells and PRP is one of the new treatment modality, which can be used for the patients with chronic diseases like OSMF. However, more clinical studies with stem cells is required to obtain more significant and better results.

The drawbacks of our study is that the sample size is less to arrive at a specific conclusion. In addition, the follow-up period is insufficient. This modality of treatment can provide better results in patients with stages I and II OSMF compared to patients with advanced disease.

CONCLUSION

Presently, the use of stem cells have become popular and an upcoming tool in the treatment of various diseases, especially with the use of autologous ones. The stem cells emphasize on the mechanism that is neoangiogenesis, which, along with the use in combination with PRP, can enhance the treatment of chronic diseases like OSMF. In addition, the use of raw stem cells instead of processed ones can minimize the working time, less postoperative care as they are done under local anesthesia, and also can lead to more compliance of the patients.

In our study, we used raw stem cells from peripheral venous blood and thus results were significant only at the end of 6 month follow-up. The study is statistically significant but to be clinically more significant we need long-term follow-ups. Results were significant in patients with stage II OSMF specifically. Further burning sensation can be reduced along with surgical intervention by using processed stem cells.

Ethical clearance

Ethical Clerance certificate was obtained from Institutional Ethics Committee B.V.V. Sangh’s PMNM Dental College and Hospital, Bagalkot with reference no. PMNMDCH//801/2017-18 Letter dated 17 Nov 2017.

Consent

Taken consent from all participants.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form the patients have given their consent for their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.