**12. Studies on the Salivary Gland and Oral Mucosa**

In a mouse model, the ability of ADSCs to minimize and/or repair single dose radiation-induced oral mucositis was demonstrated after 18 Gy single-dose of orthovoltage X-ray [98]. It was shown that intraperitoneal transplantation of 5 doses of 2.5 million freshly cultured syngenic ADSCs significantly and reproducibly reduced the duration of radiation-induced oral mucositis from 5.6 ± 0.3 days to 1.6 ± 0.3 days. The therapeutic benefits were shown to be significantly dependent on dose, frequency, and the start of cell transplantation.

Effects of BM-MSCs on irradiated salivary gland was assessed by mobilisation of autologus BM-MSC by administration of granulocyte stimulating factor (G-CSF) [99]. It was shown that the mobilised MSCs promoted regeneration of irradiated salivary glands and increased gland weight, number of ancinar cells, and salivary flow rate.

In another study [100], it was shown that the local transplantation of human ADSCs resulted in tissue remodelling with a greater number of salivary epithelial cells in a rat model of salivary gland irradiation. This indicated that local transplantation of ADSCs alleviated radiation-induced cell death. It was also shown that when an injectable porcine small intestinal submucosa matrix was used as a cell delivery carrier, the anti-apoptotic and anti-oxidative effects of ADSCs and salivary protein synthesis were enhanced.

Protective and regenerative effects of ADSCs on radiation induced salivary gland was also studied in rats [101]. These authors reported statistically significant improvements in the salivary gland of rats treated with ADMSc, 48 h after irradiation. The efficacy of stem cell transplantation and mobilisation in the treatment of radiation-induced xerostomia was discussed and reviewed [102,103].

Clinically, in a randomised placebo-controlled phase 1/2 trial [48], 30 patients were studied. In this study ADSCs or placebo were transplanted in submandibular glands of patients who had had previously received radiotherapy for oropharyngeal squamous cell carcinoma. No adverse events were detected from ADSC transplantation, indicating its safety. Unstimulated whole salivary flow rates in the transplanted group significantly increased compared to the placebo-arm. The xerostomia symptom scores significantly decreased and salivary gland function improved in the ADSC group.

#### **13. Discussion**

In a living body, cell loss and regeneration takes place continually as a natural process. Tissues function takes place as a result of a continued cell loss and replacement with new cells. Cells are lost due to ageing, wear and tear, or other insults such as radiation, and are replaced by new cells produced by indigenous stem cells or tissue-specific progenitor cells that differentiate into functional cells. Target cell theory of radiation damage [1] was developed exactly on this basis. According to this theory, cell loss is the cardinal cause of development of radiation lesions or tumour eradication by irradiation. In fact, radiation disturbs the usually continuous process of cell loss and cell replacement. The cells killed or damaged by radiation fail to produce sufficient progenies to replace the lost cells, therefore, the number of lost cells exceed the number of cells produced. When the deficit goes beyond a critical level where the number of progenies become so low that it cannot produce sufficient differentiated cells to maintain the tissue function, radiation lesion manifests. In early responding tissues, the latency period of the development of radiation lesion corresponds to the turnover time of the cells. For example, radiation mucositis, and radiation-induced moist desquamation of the skin are considered as a result of sterilisation of epithelia and their latency period corresponds to the turnover time of the target cells. However, late radiation damage cannot be described by turnover time of a certain cell type; however, it develops as a result of loss of a number of cells and subsequent events. For example, in the development of radiation-induced late dermal damage or late submucosal damage, loss or damage to endothelial cells play an important role. Loss of endothelial cells and damage to the vasculature impair the circulation and loss of parenchyma ensues. This is also true of radiation damage in two central nervous tissues, where late radiation damage manifests as demyelination of axons and necrosis. Some believe that the reproductive death of glial cells is the cardinal cause and demyelination and necrosis develop as a consequence of gradual loss of these cells [104]. However, some authors [105,106] consider vascular damage and lack of sufficient blood supply as the cardinal cause of the development of radiation-induced demyelination and necrosis of nervous tissue. Whatever the cause, both schools of thought agree that the demyelination and subsequent necrosis of nervous tissue is initiated by cell death, reproductive sterilisation of vascular or glial cells. The severity and duration of radiation-induced lesions are dose-dependent. This implies that the more cell loss, the more severe and long lasting the lesion. Besides radiation dose, radiation quality is another determinant factor on the degree of cell loss and consequently lesion development. However, treatment of radiation lesions, particularly treatment with stem cells, is in its infancy and there is not much data to be discussed.

Not all radiation lesions are fatal. Radiation lesions heal after sublethal doses; when surviving cells in the irradiated region regenerate or healthy cells from the margin of the irradiated region migrate to the irradiated area and revive the damaged tissue. However, when the cell loss is extensive or the number of surviving/migrating cells is not sufficient, the lesions remain unhealed. On this basis, replacement of lost cells by stem cell transplantation was a plausible attempt to modify radiation induced tissue damage.

Regeneration of irradiated salivary gland by mobilizing endogenous stem cells [99] supports the idea that there is always a number of stem cells in the damaged tissue and whole body, and their stimulation and mobilisation either by secretory factors from other stem cells or by cytokines could rescue damaged tissue. Protection of salivary glands from radiation-induced apoptosis and preservation of acinar structure and function were attributed to the activation of FGFR-PI3K signalling via actions of ADSC-secreted factors, including FGF10 [107].

The effectiveness of cell transplantation in amelioration of radiation lesions is supported by the works reviewed in this paper. Radiation lesions that develop due to lack

or insufficiency of functional cells are modified by the transplantation of exogenous cells [90,108–111]. Besides, amelioration of radiation-induced lesions and subcutaneous tissue formation in patients who received fat grafting after mastectomy of breast-irradiated patients can be attributed to the stem cell component of the fat graft [96,97].

However, it is not certain that the beneficial effect of stem cell transplantation is the result of direct integration of transplanted cells in the damaged tissue or the result of stimulation of the surviving endogenous cells by the transplanted cells—the paracrine effect. Some authors, while reporting the beneficial effects of stem cell transplantation, fail to demonstrate the integration of the transplanted donor cells in host tissue or demonstrate a very low level of engraftment that cannot justify the significant functional improvements as a result of transplantation. In the study of the effect of stem cell transplantation on amelioration of radiation-induced salivary gland damage by mobilisation of endogenous bone marrow stem cells [99], significant improvements were seen in the gland weight and salivary flow but transdifferentiation of stimulated bone marrow cells in the salivary glands were not observed. Stem cell transplantation showed therapeutic effects on irradiated lung tissue but the number of transplanted cells in irradiated lungs were so low that they could not justify the observed improvements [78]. Neural stem cells transplanted intradurally in spinal cord irradiated rats resulted in 30% reduction in the development of radiation myelopathy [60] but these authors failed to demonstrate the transdifferentiation of the transplanted cells in the irradiated spinal cords of engrafted rats. Similarly, despite improvements in irradiated liver tissue by exogenous cell transplantation, the transplanted cells were not found in the liver of the irradiated animals [76].

These findings suggest that the beneficial effects of stem cell transplantation are not necessarily due to the replacement of damaged cells by healthy transplanted cells or their trans differentiation into functional cells. It is probable that the paracrine effect also plays a role [112–114]. It is to say that the transplanted cells secrete some bioactive factors that stimulate endogenous stem cells. Bioactive factors secreted by MSCs are both immunomodulatory and trophic. Secretion of angiogenic and antiapoptotic factors by transplanted human ADSCs have been reported [115]. VEGF secretion were increased manyfold when the ADSCs were cultured under hypoxic conditions. In fact, paracrine effect was reported as early as 1971 by Little [116] who reported the repair of potentially lethal radiation damage by a conditioned medium of cultured mammalian cells. Later, it was shown that the growth of cultured endothelial cells was enhanced and endothelial apoptosis was reduced by the addition of conditioned media obtained from ADSCs grown under hypoxic conditions [115]. Regeneration of radiation damaged tissues by transplanted MSCs has been attributed to the indirect effect of stem cell transplantation due to the secretion of cytokines and growth factors [76]. Tissue regeneration, acceleration of angiogenesis, and growth of nerves have been reported after transplantation of ADSCs in mice [117]. The beneficial effects of ADSC transplantation were attributed to the secretion of neurotrophic genes and extracellular matrix proteins required for nerve growth and myelination. MSCs, besides trophic effects, exert immunomodulatory effects too [109] that inhibit the surveillance ability of lymphocytes. This prevents the immunogenicity and allows allogenic transplantation of MSCs. A total of 73 proteins secreted by human ADSCs have been reported that includes factors such as heat shock proteins, macrophage inflammatory proteins, proteases, protease inhibitors, cycloskelethal components, extracellular matrix components, metabolic enzymes, anti-inflammatory proteinsVEGF, IGF-1, EGF, EGF, and many others [118,119]. Besides, RNA-containing microparticles are also involved in the paracrine effect. Microparticles or microvesicles consist of extracellular vesicles (EVs) that are released by almost all bodily cells, including stem cells. Evs are referred to a heterogenous population of membrane-coated small vesicles with diameter of 30–1000 nm. Exosomes constitute the microvesicles of diameter less than 200 nanometer. EVs consist of a bilipid membrane and a cargo consisting of various proteins and miRNA. Intracellular communication of cells is facilitated by secreted microvesicles [120–123]. Microvesicles released by stimulation of MSCs show therapeutic characteristics against ischemia-repurfusion induced acute and

chronic kidney injury [124]. The same authors also demonstrated that inactivating RNA by pretreatment of microvesicles by RNase abrogated its therapeutic effect. This indicates the importance of the RNA component of microvesicles in exerting its therapeutic effect.

Evidence is mounting in support of paracrine effect of stem cells; in recent years particularly, EVs derived from stem cells have been the focus of extensive research efforts in the fields of regenerative medicine and radiation. The beneficial effects of MSCsecreted microvesicles have been demonstrated in vitro and in vivo treatment of many lesions [113,125–133]. Inhibition of tumour growth by MSC-derived microvesicles have also been demonstrated [68,128]. It has also been shown that platelet-derived microvesicles facilitate the homing of transplanted bone marrow stem cells in irradiated mice [134]. EVs extracted from human MSCs were injected into nude mice by three consecutive applications after a lethal whole body irradiation that resulted in 85% reduction in mortality [135]. Recently, the efficacy of MSC-derived EVs in amelioration of radiation-induced hematopoietic syndrome was reported [136]. Exosomes derived from mesenchymal stem cells have been used for conditioning macrophages to be used in the treatment of acute radiation syndrome [137]. It appears that, besides proteins and bioactive lipids, the RNA content of the cargo of EVs is the major component of the action of the beneficiary effects of EVs. This mode of action have been shown to be responsible for the amelioration of radiationinduced lung injury by mesenchymal cell-derived EVs [138]. The mode of action and potential of EVs in the treatment of radiation lesions are reviewed by Forsberg et al. [139]. EVs have also been indicated in mediating radiation-induced bystander effects [140].
