Elsevier

Oral Oncology

Volume 49, Issue 2, February 2013, Pages 136-143
Oral Oncology

Intraglandular transplantation of bone marrow-derived clonal mesenchymal stem cells for amelioration of post-irradiation salivary gland damage

https://doi.org/10.1016/j.oraloncology.2012.08.010Get rights and content

Summary

Objectives

External irradiation in head and neck cancers may induce irreversible hyposalivation and consequent xerostomia, stemming from radiation damage to salivary glands (SGs). As cell-based therapy has been reported to be able to repair or restore damaged SG tissues, we attempted to determine whether bone marrow-derived clonal mesenchymal stem cells (BM-cMSCs) can ameliorate irradiation-induced salivary gland damage via a murine model.

Methods

External irradiation at a dose of 15 Gy was delivered to the neck fields of C57BL/6 mice. We directly administered either homologous mouse BM-cMSCs labeled with PKH26 (treatment group) or PBS (control group) into SGs 24 h after irradiation. Salivary flow rate (SFR) and lag time of salivation were measured at 12 weeks after transplantation. At 4 and 12 weeks post-transplantation, we performed morphological, histological, and immunofluorescent examinations. Transdifferentiation of administered BM-cMSCs into salivary epithelial cells was observed by confocal microscopy.

Results

SFR was significantly increased in BM-cMSCs-transplanted mice compared with PBS-injected mice at 12 weeks after transplantation. Administration of BM-cMSCs preserved the microscopic morphologies of SGs, with more functional acini in BM-cMSC-transplanted SGs than in PBS-injected SGs. Immunofluorescent staining revealed less apoptotic cells and increased microvessel density in BM-cMSC-transplanted SGs compared with PBS-injected SGs. PKH-26 labeled BM-cMSCs were detected in transplanted SGs at 4 weeks after transplantation and in vivo transdifferentiation of BM-cMSCs into acinar cells was also observed.

Conclusion

This study suggests that BM-cMSCs can ameliorate salivary damage following irradiation and can be used as a source of cell-based therapy for restoration of irradiation-induced salivary hypofunction.

Introduction

Salivary hypofunction and consequent xerostomia are common and significant complications of irradiation treatment in head and neck cancers. Nearly 64% of long-term survivors of head and neck cancers after radiation therapy experience moderate to severe xerostomia.1 Irradiation-induced salivary hypofunction usually occurs shortly after radiotherapy: salivary flow decreases 50–60% in the first week and finally declines to approximately 20% at 7 weeks after conventional radiotherapy.2 This significant change is typically irreversible and a life-long problem.3

Salivary hypofunction may contribute to a variety of oral sequelae, as well as general health problems. Patients with xerostomia commonly experience swallowing difficulty, loss of taste, difficulty in speech, oral candidiasis, and dental caries. In the end, these complications may lead to nutritional insufficiency and weight loss.[4], [5], [6] Moreover, irradiation-induced salivary hypofunction not only affects quality of life of patient, but also interrupts the course of radiotherapy, posing tumor control.7

While there have been many of advances in SG-sparing radiation delivery techniques, it is still impossible to successfully prevent irradiation-induced salivary hypofunction in all patients, indicating a need for new biologic insights for which to prevent or restore the obstinate complications of radiotherapy. Recently, an increasing number of preclinical and clinical trials using stem cells for tissue regeneration have been conducted. For SGs, bone marrow-derived cells (BMCs) were recently reported to be a potential candidate for the treatment of salivary hypofunction.[8], [9], [10], [11] While the exact mechanisms by which BMCs improve organ functions remain unclear, BMCs are considered to exert positive effects on salivary function restoration via several mechanisms including paracrine effect, vasculogenesis, cell fusion and cell transdifferentiation.

However, these previous studies used a mixed population of mononuclear cells, which may have contained many different types of stem/progenitor cells, such as bone marrow mesenchymal stem cells (BM-MSCs), hematopoietic stem cells, or endothelial progenitor cells, because BMCs isolated by the conventional method usually contain different types of stem and progenitor cells in an isolated cell mixture.12 BM-MSCs enhance tissue repair by secreting paracrine factors and also exhibit the potential for site-specific multilineage differentiation.13 However, the effect of highly homogenous BM-MSCs on SG regeneration is still not yet documented in the literature.

This led us to hypothesize that BM-cMSCs might have the potential to ameliorate irradiation-induced salivary hypofunction by protecting against irradiation-induced cell damage or transdifferentiation into salivary epithelial cells after direct transplantation into post-irradiation SGs. In the present study, we used a subfractionation culturing method to obtain single cell-derived MSC lines, called clonal MSCs, from bone marrow. Using this method, we were able to isolate a homogeneous group of MSCs without any enzymatic digestion or artificial manipulation. We then attempted to document whether homogeneous mouse BM-cMSCs could survive and differentiate into salivary epithelial cells in recipient SGs after direct transplantation. Additionally, we also measured the apoptosis, proliferation, and microvessel density of salivary cells to explore the mechanisms of salivary function restoration as a result of BM-cMSCs post-transplantation, including paracrine effect and vasculogenesis.

Section snippets

Preparation of BM-cMSCs

A subfractionation culturing method was performed to isolate BM-cMSC as previously described.14 Briefly, mouse BM samples were harvested from the tibias and femurs of 5-week-old C57/BL6 mice. The bone marrow cavity was flushed with 5 ml of culture medium comprising Dulbecco’s modified Eagle’s Medium (DMEM)-low glucose (GIBCO-BRL, Life Technologies, Gaithersburg, MD), 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. This crude BM cell mixture was plated onto a 100 mm culture dish and

Macromorphological and functional improvements after BM-cMSC transplantation

Macromorphological measurements were performed at 12 weeks after transplantation. Body weights were not significantly different among all groups (Fig. 2A), but irradiation significantly reduced glandular weights in irradiated, untreated mice compared to those of non-irradiated normal mice (P < 0.05, Fig. 2B). BM-cMSC administration appeared to protect glandular weight loss or restore glandular weights in irradiated mice, despite a lack of statistical significance.

Saliva production and salivation

Discussion

In this study we demonstrated that highly homogenous BM-cMSCs can preserve the acinoductal integrity with secretary function of acinar cells after intraglandular transplantation following irradiation. We speculate that these favorable results are related to the mechanisms by which BM-cMSCs inhibit apoptosis of a variety of cells, such as salivary stem/progenitor cells and endothelial cells, and that BM-cMSCs also have the potential to transdifferentiate into salivary epithelial cells.

Lombaert

Conflict of interest statement

None declared.

Financial disclosures

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 2011-001847). This study was also supported by the Bio & Medical Technology Development Program (2011-0019634) of the National Research Foundation by the Korean government (MEST).

Disclosure

This paper was presented at the 3rd World Congress of the International Academy of Oral Oncology on July 14–17, 2011, in Singapore.

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    These authors contributed equally to this manuscript.

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