Laminin-1 Peptides Conjugated to Fibrin Hydrogels Promote Salivary Gland Regeneration in Irradiated Mouse Submandibular Glands

Previous studies demonstrated that salivary gland morphogenesis and differentiation are enhanced by modification of fibrin hydrogels chemically conjugated to Laminin-1 peptides. Specifically, Laminin-1 peptides (A99: CGGALRGDN-amide and YIGSR: CGGADPGYIGSRGAA-amide) chemically conjugated to fibrin promoted formation of newly organized salivary epithelium both in vitro (e.g., using organoids) and in vivo (e.g., in a wounded mouse model). While these studies were successful, the model’s usefulness for inducing regenerative patterns after radiation therapy remains unknown. Therefore, the goal of the current study was to determine whether transdermal injection with the Laminin-1 peptides A99 and YIGSR chemically conjugated to fibrin hydrogels promotes tissue regeneration in irradiated salivary glands. Results indicate that A99 and YIGSR chemically conjugated to fibrin hydrogels promote formation of functional salivary tissue when transdermally injected to irradiated salivary glands. In contrast, when left untreated, irradiated salivary glands display a loss in structure and functionality. Together, these studies indicate that fibrin hydrogel-based implantable scaffolds containing Laminin-1 peptides promote secretory function of irradiated salivary glands.

Regarding stem cells/progenitors, previous studies showed that c-Kit + cells, which normally are found in very low numbers within salivary gland specimens (Nanduri et al., 2011;Nanduri et al., 2013) can be expanded ex vivo for restoring salivary gland function; however, further characterization (e.g., how they incorporate into host tissue as well as long term secondary effects such as tumorigenesis and survival rates) must be determined before translating this approach into humans. Another technology involves the use of embryonic organ culture transplantation, where embryonic salivary cells grown in culture can be transplanted in vivo (Ogawa et al., 2013); nonetheless, a diminished gland size and an absence of studies showing long-term outcomes following treatment significantly decrease the utility of this model for translational applications. Bioprinting strategies have shown the possibility of assembling glandular compartments (e.g., acinar/ductal epithelial, myoepithelial, endothelial, and neuronal) into salivary gland organotypic cultures; however, this technology does not mimic the salivary gland native architecture (e.g., cell polarity and organization (Ferreira et al., 2016;Adine et al., 2018)). Cell sheets made of salivary gland cells have demonstrated positive results, as they promote cell differentiation and tissue integrity in wounded mouse submandibular gland (SMG) models, yet the main challenge facing this technology is the need to standardize cell composition within the sheets and thereby achieve greater reproducibility (Nam et al., 2019a;dos Santos et al., 2020). Regarding scaffolds other than the Fibrin Hydrogels (FH), various biomaterials (Aframian et al., 2000;Sun et al., 2006;Cantara et al., 2012;Soscia et al., 2013;Hsiao and Yang, 2015;Yang and Hsiao, 2015) have been shown to promote cell growth and attachment but the degree of structural organization, as demonstrated by hollow multi-lumen formation, cell polarity and functionality, has been modest. Likewise, studies have shown that human cells grown on a hyaluronic acid-based scaffold and transplanted into a wounded mouse parotid gland lead to improved secretory function (Pradhan-Bhatt et al., 2014); nevertheless, these results included neither monitoring for degradation of the scaffold nor evidence of new tissue formation, thus raising concerns with the stability of the biomaterial and capacity for regeneration, respectively. Together, these technologies offer the potential for more advanced solutions to hyposalivation due to head and neck radiation therapy but have yet to truly deliver.
In response to these needs and challenges, we developed FH with conjugated Laminin-1 peptides (L 1p ) A99 and YIGSR that were used successfully to repair salivary gland tissue in a wounded SMG mouse model (Nam et al., 2017a;Nam et al., 2017b;Nam et al., 2019b). To apply these results to a more translational setting, the goal of the current study is to determine whether transdermal injection with the L 1p A99 and YIGSR chemically conjugated to FH can promote secretory function in irradiated salivary glands.

Animals
Female 6-week-old C57BL/6J mice weighing ∼17-20 g were purchased from Jackson Laboratory (Bar Harbor, ME). Power analysis was performed to determine mouse numbers using G*Power 3.1.9.7 software (Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany; http://www.gpower.hhu.de/). All calculations were conducted using a significance level of 0.05 with 95% power. Then, 105 mice were randomly distributed into three groups to receive the following treatments: non-irradiated (40 mice), irradiated without L 1p -FH injection (40 mice), and irradiated while also receiving the L 1p -FH injection (25 mice), comprising treatment groups 1-3, respectively. All animal usage, anesthesia and surgeries were conducted with the approval of the University of Utah Institutional Animal Care and Use Committee (IACUC) in compliance with the ARRIVE guidelines.

Radiation Treatment
Salivary gland tissue damage is a late degenerative response observed after radiation therapy (Wu and Leung, 2019;Jasmer et al., 2020). To confirm L 1p -FH regenerative effects in a more clinically relevant animal model, a widely accepted head and neck irradiated mouse model was used for this study (Deasy et al., 2010;Varghese et al., 2018). Briefly, mice were anesthetized with ketamine (100 mg/kg) and xylazine (5 mg/kg) solution administered intraperitoneally with the head and neck area positioned over the 1 cm slit of a customized lead shield, thereby protecting other areas of the body from radiation. SMGs then received a single 15 Gy radiation dose using a JL Shepherd 137 Cs irradiator ( Figure 1A). Animals were allowed to recover for 3 days and received hydrogel treatment soon after, as detailed below.

Transdermal Injection
C57BL/6J mice were anesthetized with 3% isoflurane using an oxygen flow rate set at 2.0 L/min, and 10 μL of freshly mixed L 1p -FH solution was transdermally injected using insulin syringe (G 28) to irradiated mouse SMGs at post-radiation day 3. L 1p -FH effects were studied at days 8 and 30. Using thrombin prior transdermal injection causes rapid polymerization of L 1p -FH which clogs the needle. To overcome this issue, the mixture was applied in a liquid form using endogenous thrombin for internal polymerization. To confirm scaffold implantation in Frontiers in Bioengineering and Biotechnology | www.frontiersin.org September 2021 | Volume 9 | Article 729180 vivo, FH was labeled with DyLight 680 and quantified within dissected glands using a Bio-Rad Chemi-Doc ™ MP imaging system ( Figure 1C).

Hematoxylin and Eosin and Masson's Trichrome Stain
SMGs were fixed in 10% formalin at room temperature overnight, dehydrated in 70% ethanol solution, embedded in paraffin wax and cut into 3 μm sections. Sections were then deparaffinized with xylene and rehydrated with serial ethanol solutions (100%, 95% 80, 70 and 50%, v/v) and distilled water. For hematoxylin and eosin staining, the rehydrated sections were stained with hematoxylin for 5 min, washed with distilled water for 5 min, tap water for 5 min and distilled water for 2 min. Next, slides were stained with eosin for 30 s, washed with tap water for 5 min and distilled water for 2 min. Finally, hematoxylin and eosin stained gland sections were dehydrated with 95 and 100% ethanol (v/v), cleared in xylene and mounted with a xylene-based mounting medium. As for Masson's trichrome staining, the rehydrated sections were re-fixed in Bouin's solution at 60°C for 1 h then washed with running tap water for 10 min and distilled water for 5 min. Next, sections were stained with Weigert's iron hematoxylin solution for 10 min then washed with running warm tap water for 10 min and distilled water for 5 min. For cytoplasm staining, sections were incubated with Biebrich scarlet acid fuchsine solution for 5 min and washed three times with distilled water for 2 min. Regarding collagen staining, sections were incubated in phosphotungstic/phosphomolybdic acid for 15 min, stained with aniline blue solution for 5 min and washed three times with distilled water for 2 min. Stained sections were then differentiated in 1% acetic acid solution for 1 min and washed two times with distilled water for 2 min. Finally, Masson's trichrome stained sections were dehydrated with serial ethanol solutions (95 and 100%), cleared in xylene and mounted with a xylene-based mounting medium. Finally, the samples were analyzed using a Leica DMI6000B (Leica Microsystems, Wetzlar, Germany) to determine tissue morphology.

Confocal Analysis
For antigen retrieval, the rehydrated and fixed tissue sections were incubated in Tris-EDTA buffer [10 mM Tris, 1 mM EDTA, 0.05% (v/v) Tween ® 20, pH 9.0] for ZO-1 and E-cadherin or with sodium citrate buffer [10 mM sodium citrate, 0.05% (v/v) Tween ® 20, pH 6.0] for TMEM16A, Na + /K + -ATPase, iNOS, Arg-1, VCAM-1 and ICAM-1 at 95°C for 30 min. Next, samples were permeabilized with 0.1% (v/v) triton X-100 in PBS at room temperature for 45 min. Specimens were then blocked in 5% (v/v) goat serum in PBS for 1 h at room temperature and incubated at 4°C with the following primary antibodies overnight: rabbit anti-ZO-1, mouse anti-E-cadherin, rabbit anti-TMEM16A, mouse anti-Na + /K + -ATPase, rabbit anti-VCAM-1 or mouse anti-ICAM-1. At that time, sections were incubated with antirabbit Alexa Fluor 488 and anti-mouse Alexa Fluor 568 secondary antibodies in 5% goat serum at room temperature for 1 h followed by 300 nM DAPI staining at room temperature for 5 min. For M1 and M2 marker staining, specimens were blocked in 3% (w/v) bovine serum albumin (BSA) in PBS for 1 h at room temperature and incubated with primary antibodies (rabbit anti-iNOS or rabbit anti-Arg-1) at 37°C for 1 h. Then, sections were incubated with anti-rabbit Alexa Fluor 568 in 3% BSA at room temperature for 1 h followed by 300 nM DAPI staining at room temperature for 5 min. Finally, specimens were analyzed using a STELLARIS Confocal Microscope (Leica Microsystems, Wetzlar, Germany).

Macrophage Ratio
M1 and M2 macrophage cells were determined using ImageJ. Specifically, the color threshold was set to isolate the colocalized signal of nuclei and M1 (Figure 4, white arrows)/M2 (Figure 4, red arrows) positive cells, which were counted and normalized by area. Statistical significance was assessed using one-way ANOVA (*p < 0.01) and Dunnett's post-hoc test for multiple comparisons to group 2 (irradiated with no L 1p -FH injection at day 30).

Saliva Flow Rate Measurements
Mice were anesthetized with ketamine (100 mg/kg) and xylazine (5 mg/kg) followed by intraperitoneal injection with pilocarpine (25 mg/kg) and isoproterenol (0.5 mg/kg). Then, whole saliva was collected using a micropipette for 5 min and flow rate was calculated using the following formula: Saliva flow rate Stimulated saliva µL Body weight of mouse g x collection time (5min)

Statistical Analysis
Experimental data were analyzed using one-way ANOVA and Dunnett's post hoc test for multiple comparisons to the nonirradiated group 1 at day 30. All values represent means ± SD (n 5), where p values <0.01 were considered statistically significant. Finally, these calculations were performed using GraphPad Prism 6.

A Head and Neck Irradiated Mouse Model was Achieved
To investigate whether L 1p -FH could restore irradiated SMG structure and function, C57BL/6J mice were subjected to a single radiation treatment as described in Materials and Methods ( Figure 1A). Mice treated with a single 15 Gy radiation dose displayed a significant reduction in saliva flow rates as compared to non-irradiated controls (i.e., from 1.43 to 0.80 μL/g/min, n 5, p < 0.01) in the first 8 days and remained steady thereafter until day 30 ( Figure 1B). These results demonstrated that the radiation dose utilized here caused significant loss of salivary secretory function and can thus be used as a head and neck irradiated preclinical model, consistent with previous studies (Lombaert et al., 2008;Varghese et al., 2018;Weng et al., 2018).

L 1p -FH was Successfully Implanted in Irradiated Mouse Submandibular Glands
Our previous studies showed the biocompatibility of L 1p -FH with host tissue when surgically implanted in a wounded mouse model (Nam et al., 2017a;Nam et al., 2017b).
To avoid an open wound surgery, we attempted to deliver the L 1p -FH to irradiated mouse SMG via transdermal injection as described in Materials and Methods. For these experiments, we used a fluorescently labeled hydrogel using DyLight 680 and successfully implanted L 1p -FH in irradiated mouse SMG via transdermal injection ( Figure 1C, white arrows).

L 1p -FH Preserved Epithelial Integrity After Radiation Treatment
Our previous studies showed that L 1p -FH promoted tissue repair in a wounded SMG mouse model (Nam et al., 2017a;Nam et al., 2017b;Nam et al., 2019b). To determine whether these effects occur in the head and neck irradiated mouse model, we randomly distributed mice in three groups and applied this scaffold as follows: non-irradiated, irradiated without L 1p -FH injection and irradiated that received the L 1p -FH injection, comprising treatment groups 1-3, respectively (see Material and Methods section). As shown in Figure 2, group 1 (non-irradiated glands) displayed intact lobules where the parenchyma was separated by areas of thin connective tissue at days 8 (Figures 2A,B) and 30 ( Figures 2C,D). As for cytologic features, serous acini cells showed a typical pyramidal shape with basophilic cytoplasm and basal nuclei. In contrast, mucous cells showed a pale cytoplasm with flat basilar nuclei, intercalated ducts were lined by cuboidal and/or flat cells, striated ducts showed cuboidal to low columnar cells and granular convoluted ducts were lined by tall columnar cells containing intracytoplasmic eosinophilic granules. Together, these features indicate that the nonirradiated glands in group 1 showed the morphology of a healthy epithelium. In contrast, group 2 (irradiated with no L 1p -FH injection) demonstrated glandular parenchyma separated by thicker connective tissue strands, ductal areas with ectasia, intraluminal depositions and increased presence of fibrosis when compared to controls ( Figures 2E,F). Furthermore, tissue damage was even more severe at day 30 ( Figures 2G,H), where SMG showed an extensive disruption of the lobular architecture as indicated by the replacement of acini and ducts with sheets of vacuolated cells, adipocytes and fibrosis. Together, these results indicated that irradiated glands with no L 1p -FH injection (group 2) dramatically lost epithelial integrity.
Remarkably, mice in group 3 (irradiated with L 1p -FH injection) recovered many of the features of healthy glands. For instance, we observed the presence of serous acinar units with organized ductal structures surrounded by thin connective tissue strands  Figures 2K,L). These changes indicate that group 3 (irradiated glands treated with L 1p -FH) had a morphology consistent with a healthy salivary gland epithelium and results in this section indicate that L 1p -FH is a suitable scaffold for promoting epithelial integrity in irradiated SMG.

L 1p -FH Maintained Epithelial Polarity and Preserved Ion Transporter Expression
To determine whether L 1p -FH maintained epithelial polarity in an irradiated mouse model, we stained the SMG sections with the apical tight junction marker ZO-1 and basolateral marker E-cadherin. As shown in Figure 3A, group 1 (non-irradiated glands) displayed apical ZO-1 (green) and basolateral E-cadherin (red) after 30 days. However, in group 2 (irradiated glands with no L 1p -FH injection), a mild residual ZO-1 signal was detected at day 8 ( Figures 3B,F, blue solid line), and a weaker ZO-1 signal was expressed at day 30 ( Figure 3F, blue dotted line), together with ZO-1 disorganization ( Figure 3C), thereby indicating loss of epithelial polarity. In contrast, group 3 (irradiated glands treated with L 1p -FH) showed apical ZO-1 and basolateral E-cadherin signals both at days 8 ( Figure 3D) and 30 ( Figure 3E), indicating that the scaffold treatment helps to maintain epithelial polarity ( Figure 3F, red line and red dotted line). Regarding the presence of functional markers, group 1 (non-irradiated SMG) showed apical TMEM16A ( Figure 3G, green) and basolateral Na + /K + -ATPase localization ( Figure 3G, red) at day 30, consistent with a healthy salivary epithelium. In contrast, group 2 (irradiated glands with no L 1p -FH injection) showed a moderate TMEM16A signal ( Figure 3L, blue solid line) at day 8 ( Figure 3H, green) and weaker TMEM16A signal ( Figure 3L, blue dotted line) at day 30 ( Figure 3I, green). Interestingly, group 3 (irradiated glands treated with L 1p -FH) expressed strong apical TMEM16 (Figures 3J,K, green; Figure 3L, red line and red dotted line) and basolateral Na + /K + -ATPase similar to non-irradiated glands, thus suggesting that L 1P -FH treatment helps to maintain epithelial polarity and preserve ion transport expression, both of which are critical for saliva secretion.

L 1p -FH Promoted Macrophage Polarization
Our previous studies indicated that treatment with L 1p -FH promoted macrophage polarization in a wounded SMG female mouse model (Brown et al., 2020). To determine whether similar effects occur in an irradiated mouse model, we identified the presence of M1 and M2 subtypes within the SMG using macrophage-specific antibodies (i.e., iNOS and Arg-1, corresponding to M1 and M2, respectively). As shown in Figures 4A,F, group 1 (non-irradiated glands) expressed iNOSpositive cells with approximately 0.94 macrophages per 100,000 µm (Sroussi et al., 2017). In contrast, group 2 (irradiated glands with no L 1p -FH injection) showed a significant increase in M1 macrophages (approximately 28.65 iNOS-positive cells) at day 30 ( Figures 4C,F). Notably, group 3 (irradiated glands treated with L 1p -FH) showed a significant decrease of M1 macrophages (approximately 5.92 iNOS-positive cells) at day 30 ( Figures 4E,F) compared to group 2. Regarding the presence of M2 markers, group 2 (irradiated glands with no L 1p -FH injection) expressed Arg-1-positive cells with approximately 5.92 macrophages at day 30 ( Figures 2I,L), which is not a significant difference from group 1 (Figures 4G,L, 2.60 macrophages). Interestingly, group 3 (irradiated glands treated with L 1p -FH) expressed a significant increase of Arg-1-positive cells at day 30 (approximately 11.37 macrophages, Figure 4K,L). Together, these results indicate that L 1p -FH causes a decrease in M1 macrophages together with an increase in M2 macrophages in SMG following radiation treatment.

L 1p -FH Increased Saliva Secretion After Radiation Treatment
Our previous studies indicate that treatment with L 1p -FH enhances saliva secretion in a wounded SMG mouse model Frontiers in Bioengineering and Biotechnology | www.frontiersin.org September 2021 | Volume 9 | Article 729180 6 (Nam et al., 2017a;Nam et al., 2017b;Nam et al., 2019b). To determine whether similar effects occur in an irradiated mouse model, we treated irradiated SMG with a transdermal injection of L 1p -FH as described in Materials and Methods. As shown in Figure 5, group 1 (non-irradiated glands) showed intact saliva flow rates (i.e., 1.43 μL/g/min), as expected. In contrast, group 2 (irradiated untreated glands) exhibited a significant reduction in saliva flow rates (i.e., 0.80 μL/g/min, n 5, p < 0.01). Notably, group 3 (irradiated glands treated with L 1p -FH) showed a significant increase of saliva flow rates (1.32 μL/g/min, n 5, p < 0.01) at day 30, thereby demonstrating that L 1p -FH restores saliva secretion after radiation treatment.

DISCUSSION
Our previous studies indicated that treatment with FH alone promotes neither cell polarity nor differentiation in salivary gland epithelium, both in vitro or in vivo (Nam et al., 2016;Nam et al., 2017a;Nam et al., 2017b;Nam et al., 2019b;Dos Santos et al., 2021). However, specific L 1p sequences (A99: CGGALRGDN-amide, YIGSR: CGGADPGYIGSRGAAamide) proved to be useful for improving salivary gland regeneration (Hoffman et al., 1998). Specifically, freshly isolated SMG cells grown on L 1p chemically attached to FH induced lumen formation and secretory function (Nam et al., 2016). Moreover, L 1p -FH promoted salivary gland regeneration in an in vivo wound-healing mouse model (Nam et al., 2017a;Nam et al., 2017b), thus leading to increased saliva secretion. Such functional recovery indicates that FH-based scaffolds can be used to promote salivary gland function in radiation-induced hyposalivation. Additionally, we developed a transdermal delivery system specifically for this study with the aim of using the patient's own blood for polymerization to increase biocompatibility (Froelich et al., 2010;Dietrich et al., 2013) and having the ancillary benefits of displaying optimal rheological properties (i.e., softness) and being less invasive than other delivery methods (i.e., retro-ductal delivery (Nair et al., 2016) and surgical application (Ogawa et al., 2013)), all of which indicates a greater degree of clinical applicability for our newly designed mouse model.
Regarding results of the current study, salivary gland morphology was significantly improved by L 1p -FH FIGURE 4 | L 1p -FH promotes macrophage polarization. Macrophage marker expression was analyzed using confocal microscopy with specific antibodies against iNOS (A-F), Arg-1 (G-L), and DAPI (blue; everywhere). Scale bars represent 100 µm. White and red arrows indicate iNOS or Arg-1 positive cells, respectively. Representative image from a total of five mice per group. iNOS (F) and Arg-1 (L) positive cells were analyzed using ImageJ and GraphPad Prism 6. Data represent the means ± SD of n 5 mice per condition with statistical significance assessed using one-way ANOVA (*p < 0.01) and Dunnett's post-hoc test for multiple comparisons to group 2 (irradiated with no L 1p -FH injection at day 30).
FIGURE 5 | L 1p -FH increases saliva secretion after radiation treatment. Mice were anesthetized and stimulated with pilocarpine and isoproterenol at days 8 and 30 with saliva collected for 5 min. Data represent the means ± SD of n 5 mice per condition with statistical significance assessed using one-way ANOVA (*p < 0.01) and Dunnett's post-hoc test for multiple comparisons to group 1 (non-irradiated mice at day 30). The symbol (+) indicates L 1p -FH injection, while the symbol (−) indicates no L 1p -FH injection, and n. s indicates no significant differences from group 1 (non-irradiated mice at day 30).
Frontiers in Bioengineering and Biotechnology | www.frontiersin.org September 2021 | Volume 9 | Article 729180 (Figures 2I-L and Figure 3D,E) and saliva secretion ( Figure 5) was likewise restored by day 30 post-radiation; however, such treatment gains cannot be counted on to persist, given the residual fibrosis noted ( Figure 2L). Additionally, future studies will use growth factors specifically targeted for angiogenesis (i.e., VEGF and FGF9) (Nam et al., 2019b) in response to current results demonstrating L 1p -FH promoted macrophage polarization (Figure 4) but gave rise to no blood vessel formation (Supplementary Figure S1). Moreover, should such gains in fact prove persistent (e.g., maintained over long periods of time), we as yet have limited knowledge of the mechanisms responsible for this recovery. These issues notwithstanding, the results to date are important because they are the first time that L 1p -FH has been used in irradiated glands to restore their form and function. It is noteworthy to mention three major differences between our previous studies and the current work. First, our previous studies used L 1p in trimeric form (Dos Santos et al., 2021) and in combination with growth factors (Nam et al., 2019b), while the current work employs only monomeric forms and no growth factors. Next, our previous studies used a more invasive SMG surgical punch model (Nam et al., 2017a;Nam et al., 2017b;Nam et al., 2019b) as compared to currently used transdermal injection implantation method. Finally, we replaced the SMG wounded mouse model of our prior studies with a radiation model for greater specificity in terms of clinical features and increased translational application.
To expand on this work, future studies will perform extended saliva secretion studies and track the appearance of fibrosis at multiple time points via histological studies and investigate how L 1p used here (i.e., A99 (Mochizuki, 2003;Rebustini et al., 2007;David et al.,2008) and YIGSR (Caiado and Dias, 2012;Frith et al., 2012;Huettner et al., 2018;Motta et al., 2019)) bind to specific integrins, thus addressing the questions noted above in relation to treatment duration and mechanisms. Finally, should this treatment near the stage of clinical trials, it would be important to replace the current single dose of radiation used for proof of concept and early exploration with more clinically appropriate fractionated doses.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

ETHICS STATEMENT
The animal study was reviewed and approved by University of Utah IACUC.