**4. Tissue engineering and reparative/regenerative medicine: current regimens and strategies**

Several tissues and organs are highly sensitive to irradiation, such as the skin, esophagus and bone marrow. However, the salivary glands are intricately radiosensitive, given their highly-differentiated cell content marked with a very low or slow proliferative rate [50]. This can help explain why the salivary glands, in specific, are somewhat unique in their early- and delayed-effects post-RT, when compared to other tissues and organs. Nonetheless, salivary gland dysfunction and/or hypofunction has been shown, in some cases, to be reversible. Such treatment intervention is multi-factorial and highly-dependent on original causality, for example, in cases of alcohol abuse and dehydration or hypothyroidism. RT-induced salivary gland damage and dysfunction is a far more challenging scenario. Auto-immune/chronic inflammatory diseases, such as SS or systemic lupus erythematosus also result in irreversible damage to the salivary glands [26].

Today, as mentioned earlier, only palliative and efficacy-limited regimens are commercially-available [47]. **Tables 1–4** highlight a selection of various radioprotection strategies, at different stages of development, pre-clinically (*in vitro* and in *in vivo* testing) and clinical (human clinical trials). Briefly, database search was performed in PubMed-indexed articles using a multi-search of the following keywords: "Salivary Glands AND Radioprotection [Title/Abstract]", "Salivary Glands AND Radioprotection [MeSH]", "Salivary AND Glands AND Radioprotection [Title/Abstract]", "Salivary Gland AND Radioprotection [Title/Abstract]", "Salivary AND Gland AND Radioprotection [ALL FIELDS]", "Salivary Glands AND Radioprotection [ALL FIELDS] and "Salivary Gland AND Radioprotection [ALL FIELDS]". Eligibility and inclusion criteria included English articles reporting radio-protection data from *in vitro*, *in vivo* and/or clinical setting/trials. Articles dated back to 1978 up to the search end-date of December 31st of 2019 were analyzed. Reviews, communications or articles with preliminary results were not included in our analysis (**Figure 3**). Herein, our purpose is to screen the available literature and assess the level of development of new strategies, regimens and/or innovative solutions, to provide a usable prior-Art formatted report. Hence, not all included articles, which are tabulated for the reader, were aimed to be presented and dissected to be discussed in detail. This review attempts to provide an overview of the current understanding, status and prospect of salivary gland radioprotection

*Salivary Gland Radio-Protection, Regeneration and Repair: Innovative Strategies DOI: http://dx.doi.org/10.5772/intechopen.94898*


#### **Table 1.**

*Radioprotection of salivary glands,* in vitro*.*





#### **Table 2.**

*Radioprotection of salivary glands,* in vivo *using murine models.*


#### **Table 3.**

*Radioprotection of salivary glands,* in vivo *using non-murine models.*

systems, with a look onto potential reparative and regenerative keys, where we, amongst other clinicians and researchers, do aspire for a superior, safe, efficacious and long-term innovative solution that reverses RT-induced damage to the salivary glands of our HNC patients. Moreover, we opted to avoid concluding our *overview* with calls for additional research or validation, given that vital tissue engineering strategies employing the design, characterization and optimization of novel biomaterials (and 3D printing), that can also be housing/incorporating release-controlled nanoparticles or nanocapsules that also are designed to encapsulate distinct mesenchymal stem cells, induced pluripotent stem cells (iPSCs), growth factors or cytokines and/or pharmaceutical agents or drugs, currently investigated at different levels of development are limitless in distinctions and details.

**Palliative care for RT-induced salivary gland dysfunction- c**urrent and commercially-available palliative options for HNC patients undergoing RT include chewing gum (sugar-free), saliva substitutes, oral and topical lubricants, malic and ascorbic acid, saliva stimulants and sialogogue such as pilocarpine (Salagen, for

*Salivary Gland Radio-Protection, Regeneration and Repair: Innovative Strategies DOI: http://dx.doi.org/10.5772/intechopen.94898*


#### **Table 4.**

*Radioprotection of salivary glands, clinically in human subjects.*

#### **Figure 3.**

*PRISMA flow diagram for the bibliographic electronic search on PubMed central.*

example) and cevimeline (Evoxac, for example). As mentioned above, none have proved to restore normal QoL and patient satisfaction, mainly due to their limited efficacy and effectiveness [30, 42]. On top, adverse side effects are common, and such options are often costly to patients, requiring multiple daily use over long

periods of time. In parallel, patients, especially the elderly, institutionalized and frail, need to go through education and training to acquire new eating and life-style habits, learn to prevent or avoid impaired swallowing and potential choking, and improve their oral and dental hygiene practices and tools to prevent (or halt the progression of) dental and oral mucosal diseases, infections and tooth loss. Other palliative care options including acupuncture and electro-stimulation (enhancement of salivary reflexes) are currently undergoing investigation [30, 93].

The only Food and Drug Administration–approved radioprotective and anti-xerostomia drug for clinical use (adjuvant setting) is Amifostine, an organic thiophosphate, cryoprotective agent and free radical scavenger administered subcutaneously or most often intravenously upon reconstitution with normal saline prior to or simultaneously with RT to then accumulate within the salivary glands, has been extensively-studied since its development, initially under the nuclear warfare program [14, 94]. Today, while it continues to benefit some patients, prophylactically, via minimizing the effects of xerostomia and taste loss, it is often associated with severe side effects including a rapid decrease in blood pressure (hypotension), nausea and emesis or vomiting. Recent analysis of several clinical trials associated Amifostine to low-quality and mixed evidence in preventing dry mouth complaints in patients receiving RT to the head and neck region, in the short- to medium-terms (up to three months post-RT) and have questioned its potential in tumor cell protection, thereby further narrowing its clinical safety and efficacy window, especially in light of its high cost [94, 95]. Essentially, its use in radiation-induced xerostomia has already been cautioned in the year 2008 by the American Society of Clinical Oncology [96], and so, its controversial and debatable safety and use in all cancer cases lingers.

**Preventive and interventional care for RT-induced salivary gland dysfunction-** the main objective of any planned and/or prescribed option should be the relief of symptoms and complications associated with hypo-salivation and xerostomia in HNC patients scheduled to receive RT, in order to prevent deteriorations in their QoL thereby enhancing their battle with cancer, its treatment and consequences [97]. As discussed earlier, despite advancements in irradiation techniques and regimens including IMRT, only palliative and prophylactic options are available, all of which do suffer substantial short-comings [98, 99]. One might even consider IMPT or intensity modulated proton therapy, used to deliver a muchreduced irradiation dose and subsequently less toxic than IMRT, thereby alleviating much of the typical side effects of RT, however, IMPT is known to be more expensive and lacks accessibility and availability [43, 98, 99].

1.**Surgical Intervention alternative-** to prevent RT-induced hyposalivation, sub-mandibular gland preservation and protection from irradiation via surgical relocation to the sub-mental space, thereby away or out of irradiation zone, has been explored, with positive results. It is perhaps worth mentioning herein that sub-mandibular salivary gland supplies up to 90% of the un-stimulated saliva formation/secretion. However, such highly-invasive interventional procedures are peculiar and require exquisite surgical manipulation skills and settings. Further, surgical transfer of salivary glands is not indicated or possible for cancers of the oral cavity or patients undergoing (systemic) chemotherapy. In addition, for the gland to either retain or restore functionality, the connection of the gland to the main duct must be maintained or restored, respectively [100], altogether render it a very limit-ed/−ing option.

In terms of innovative approaches, Rao *et al.* [101] recently described the use of a synthetic hydrogel (TraceIT, composed of water and iodinated crosslinked polyethylene glycol), injected via an 18-gauge needle, to serve as a

*Salivary Gland Radio-Protection, Regeneration and Repair: Innovative Strategies DOI: http://dx.doi.org/10.5772/intechopen.94898*

minimally-invasive "spacer" (previously demonstrated in the treatment of prostate cancer), and displace or relocate the sub-mandibular gland in order to protect it from irradiation toxicity and be able to deliver a reduced irradiation dose, however the experimental model used comprised of four refrigerated cadaveric specimens and no further *in vivo* or clinical studies evaluated usability, malleability, safety and efficacy, amongst other factors, in clinical organ spacing.

2.**Tissue Engineering and Regenerative Medicine alternative-** clearly, better approaches need to be explored and developed, driving the search elsewhere, into the multi-disciplinary areas of tissue engineering and regenerative medicine, in order to combine with and improve current options or to innovate and translate new alternative solutions, for wound healing. This is especially true, in light of accumulating knowledge and understanding of the underlying mechanisms governing radiation-induced salivary gland damage and dysfunction [47]. Indeed, from inducing DNA damage (via: a. the generation of ROS/ reactive oxygen species or b. the breakage of the DNA double strand), to mutations to cell death (by apoptosis or necrosis, depending on cell type, injury and cellular responses), to the loss of salivary progenitors, to the accruing evidence regarding the regenerative capacity (slow yet existent) of salivary glands following RT-induced injury, more evidently upon the administration of a stimuli (exogenous delivery of stem cells and/or growth factors, for example), altogether re-emphasize the potential of such complex yet innovative approaches in finding a better clinical alternative solution.

In a recent *clinical* study, Ho *et al.* [102] evaluated the effects of a commercially-available slowly-dissolving adhering disc/tablet formulation (OraCoat XyliMelts) on the oro-dental health, enamel remineralization, bio-film formation, saliva presence, pH and buffering in 5 patients diagnosed with xerostomia (criteria: un-stimulated whole saliva flow rate below 0.2 mL per minute and a stimulated saliva flow rate of less than 0.5 mL in 5 minutes). They also assessed patient self-reported comfort with the mint-flavored, xylitol-releasing tablets. Subjects were instructed to use the disc as often as needed for dry mouth symptoms relief. At the end, a mean of 4 + 1 discs each day and 2 discs each night, were used. Overall, desirable effects of the product on symptomatic alleviation and management of xerostomia were reported. The authors reported effective local palliation, reduced dental sensitivity, improved salivary production and buffering capacity, reduced plaque formation and alleviated xerostomia symptoms, without the need to use any systemic sialagogue medications throughout the 21 days of the study [102]. Yet, this is a pilot study, limited for involving a small of number of participants.

**Biomaterials and Cell Therapy-** one of the fundamental roles for the maintenance of the body of any living organism is regeneration, which enables the repair and restoration of lost or damaged tissue [47, 103]. Adult stem/stromal and progenitor cells have been identified in many tissues, and are known to have a key role in the regeneration and repair, initiated or activated either by the excessive loss of differentiated cells (pool) or via (niche) environmental cues. In the presence of functional biomaterials such as the previously-described injectable hydrogel spacer [101] and a feasible agent-delivery tablet or disc [102], *would loading, encapsulating or incorporating putative salivary progenitor or stem/stromal cells, for example, a distinct type of stimuli, yield better results?* Supplying salivary gland progenitor and stem/stromal cells, via a proper release-controlled dose-responsive carrier, might be able to re-establish the disrupted salivary stem/progenitor cell pool and niche,

restore glandular tissue homeostasis, reverse hypo-salivation, and perhaps control xerostomia, a hypothesis we are currently examining in our laboratory, employing natural and synthetic polymers, liposomes, solid lipid nanoparticles and core-shell nanocapsules, and further supplementing by other pharmaceutical agents.

Modern medicine and biomedical research aim to control and enhance radioprotective as well as regenerative and reparative capabilities through the utilization of cells (cell lineages or primary cells), growing surface control using bio-scaffolds and/or manipulating growth factor/cytokine concentrations [47, 104], strategies designed to stimulate residual cells to regenerate acini and other parenchymal elements (ductal ligation) and infiltrate growth factor doses to boost salivary gland repair post-RT [105].

**Growth Factor Therapy-** somatomedin C is a hormone, similar to insulin in molecular structure, and actually is better known as IGF-1 or insulin-like growth factor 1 [106]. While a statement as "increased insulin-like growth factor signaling induces cell proliferation, survival and cancer progression" is true, it is traditional and partial, to a great extent. Today we understand that the issue is much more complex. For instance, IGF regulates cellular senescence which is known to halt proliferation of aged and stressed cells and do play a key role against cancer development. Actually, there is accruing evidence that, over time, IGF not only regulates but also induces pre-mature cellular senescence (tumor suppressor protein p53-dependant, in terms of acetylation, stabilization and activation) [107]. Hence, despite the *understandablyalarming, at first and for some, suggestion* to exogenously administer/supply cytokines and growth factors to sites of cancer, the recent years have indeed witnessed a noteworthy increase in the study of growth factors as cytoprotectants including their use as radioprotectors for salivary glands, and to reduce RT-induced symptoms, such as oral mucositis. To date, various growth factors have emerged as potential radioprotectors, including neurotrophic factors [108, 109], epidermal growth factor (EGF), fibroblast growth factor (FGF) [51, 110], keratinocyte growth factor (KGF) [111, 112] and the afore-mentioned insulin-like growth factor-1 or IGF-1 [55, 113, 114]. Meyer *et al.,* [113], for example, investigated and determined the radioprotectant and therapeutic effect of IGF-1, in a murine model. They found that IGF-1 is mediated by the activation and maintenance of a histone deacetylase, specifically the Sirtuin 1 (SirT-1). Pre-treatment with IGF-1 enabled the repair of double-stranded breaks in the DNA of parotid salivary gland cells within the first hours post-irradiation, thereby allowing for optimal DNA repair (*i.e.* IGF-1 promotes DNA repair in irradiated parotid salivary glands via the maintenance and activation of SirT-1) to fulfill the cell cycle checkpoints. However, hours later and as early as 8 h, RT-induced apoptotic cells were detected [113]. Such observations lead to further study the signaling cross-talk between IGF-1 and SirT-1, thereby identifying several activators, stabilizers and inhibitors, including the afore-mentioned inhibition of the p53-mediated apoptosis and the phosphoinositide 3-kinase (PI3K) – protein kinase B (Akt) pathway [107], indepth study-worthy topics, beyond the scope of this concise review. To date, studies, collectively indicate that cytokines can be radioprotective, anti-apoptotic and suggest/ promote that the exogenous and localized (via a release-controlled delivery system, preferably directly injectable) utilization of growth factors do stimulate endogenous stem cell populations/niche and will eventually contribute to the desired and/or pursued clinical solution suitable for preventing RT-induced damage, diminishing salivary hypofunction, as well as restoring salivary gland function in irradiated HNC cases.

**Gene Transfer Therapy-** the utilization of gene transfer, DNA transmission and cell transduction to produce high levels of transgenic protein in order to correct cellular dysfunction and/or induce a new cellular function, post-RT, is a wide area of investigation and development. Baum *et al.* [115], utilized an adenoviral technique to transfer the Aquaporin-1 (AQP1) gene into the sub-mandibular gland, reporting

*Salivary Gland Radio-Protection, Regeneration and Repair: Innovative Strategies DOI: http://dx.doi.org/10.5772/intechopen.94898*

an increase in salivary flow when compared to control viruses into rat or mini-pig models [115, 116]. Yet, key shortcomings continue to exist for non-viral as well as viral vectors [103], rendering translation for routine clinical use difficult. Likewise, the therapeutic potential of genetic modification and application of small-interfering RNAs or siRNA for the purpose of target gene silencing are intensively investigated, progressing from pre-clinical testing in animal models to ongoing clinical trials for cancer, lung disease and liver damage in human subjects. Thus far, highly limited in salivary gland tissues and accompanied with significant safety concerns [50]. For example, AQP-1 gene transfer into the salivary glands via adeno-viral vectors to treat disorders such as SS, yielded strong immune responses, mainly due to the limited or low efficiency of intra-cellular siRNA delivery [117, 118]. Herein, similar to growth factors, cell therapy and pharmaceutical agent administration, the availability of a reproducible, scalable, safe and effective, release-controlled carrier/ vehicle suitable for therapeutic siRNA delivery, directly into the salivary gland, ensuring sufficient residency/retention, is a challenge.
