Copper chelation reduces early collagen deposition and preserves saliva secretion in irradiated salivary glands

Abstract

Radiation therapy is a first-line treatment for head and neck cancer; however, it typically leads to hyposalivation stemming from fibrosis of the salivary gland. Current strategies to restore glandular function are dependent on the presence of residual functional salivary gland tissue, a condition commonly not met in patients with extensive fibrotic coverage of the salivary gland resulting from radiation therapy. Fibrosis is defined by the pathological accumulation of connective tissue (i.e., extracellular matrix) and excessive deposition of crosslinked (fibrillar) collagen that can impact a range of tissues and given that collagen crosslinking is necessary for fibrosis formation, inhibiting this process is a reasonable focus for developing anti-fibrotic therapies. Collagen crosslinking is catalyzed by the lysyl oxidase family of secreted copper-dependent metalloenzymes, and since that copper is an essential cofactor in all lysyl oxidase family members, we tested whether localized delivery of a copper chelator into the submandibular gland of irradiated mice could suppress collagen deposition and preserve the structure and function of this organ. Our results demonstrate that transdermal injection of tetrathiomolybdate into salivary glands significantly reduced the early deposition of fibrillar collagen in irradiated mice and preserved the integrity and function of submandibular gland epithelial tissue. Together, these studies identify copper metabolism as a novel therapeutic target to control radiation induced damage to the salivary gland and the current findings further indicate the therapeutic potential of repurposing clinically approved copper chelators as neoadjuvant treatments for radiation therapy.

1. Introduction

Radiation therapy is commonly used to treat head and neck cancer [1]; however, ionizing radiation typically leads to chronic oral complications such as fibrosis of the salivary gland and subsequent hyposalivation [2]. Standard care for radiation-induced hyposalivation includes the use of saliva substitutes or secretory agonists (e.g., pilocarpine) which only provide temporary relief and often cause significant side effects [3]. Current experimental strategies to restore functionality to damaged salivary glands include aquaporin-1 gene therapy [4], stem cell transplantation [5] and use of scaffolds [6,7]; however, they are all dependent on the presence of residual functional salivary gland tissue, a condition commonly not met in patients with extensive fibrotic coverage of the salivary gland resulting from radiation therapy [2]. Fibrosis is defined by the pathological accumulation of connective tissue (i.e., extracellular matrix) and excessive deposition of crosslinked (fibrillar) collagen [8,9] that can impact a range of tissues, and given that collagen crosslinking is necessary for fibrosis formation [10], inhibiting this process is a reasonable focus for developing anti-fibrotic therapies [[11], [12], [13]]. Specifically, collagen crosslinking is the process by which aldehyde-containing amino acids join other amino acids between collagen molecules to create junctions that limit movement and functionality of the tissue. The formation of crosslinks between collagen molecules is catalyzed by members of the lysyl oxidase (LOX) family of secreted copper-dependent metalloenzymes, which in mammals comprises five members (i.e., LOX, LOXL1-LOXL4) [[13], [14], [15]]. Although a role for LOX enzymes in radiation-induced fibrosis has not previously been investigated in salivary glands, it is noteworthy that numerous LOX family members have been studied in association with fibrotic disorders affecting a range of organs including lung [11,16], liver [17,18], eye [19], kidney [13], heart [20,21], and skin [22]. This has prompted efforts to develop monoclonal antibodies targeting specific LOX enzymes; nevertheless, recent Phase II clinical trials of a monoclonal antibody specifically raised against LOXL2 (Simtuzumab) failed to show efficacy against primary sclerosing cholangitis (a type of hepatic fibrosis) [23] and idiopathic pulmonary fibrosis [24]. These findings underscore the importance of considering functional redundancy when targeting individual LOX enzymes, as other LOX enzymes may be upregulated to compensate for LOX2 inhibition in the above-mentioned study. Furthermore, because copper is an essential cofactor in all LOX family members, blocking its delivery to these enzymes through the use of copper-specific chelators offers what would appear to be a viable antifibrotic strategy [25,26]. In light of the above, the goal of the current study was to evaluate the therapeutic potential of a locally administered copper chelator in preventing radiation-induced damage to the submandibular gland.

2. Materials and methods

2.1. Materials

Tetrathiomolybdate, avertin, pilocarpine, goat serum, xylene, Tween® 20, fluorescein sodium salt, hematoxylin and eosin Y solution were purchased from MilliporeSigma (Burlington, MA). Ethanol was purchased from Decon Laboratories (King of Prussia, PA). Tris-EDTA buffer, Picro Sirius Red Stain Kit and 6-diamidino-2-phenylindole (DAPI) were purchased from Abcam (Cambridge, UK). Triton X-100, phosphate buffered saline (PBS), xylene-based mounting solution and rabbit anti-zona occludens 1 (ZO-1) were purchased from Thermo Fisher Scientific (Newington, NH). Mouse anti-E-cadherin was purchased from BD Biosciences (Franklin Lakes, NJ). Alexa Fluor 488 conjugated anti-rabbit IgG secondary antibody and Alexa Fluor 568 conjugated anti-mouse IgG secondary antibody were purchased from Invitrogen (Carlsbad, CA).

2.2. Animals

Female C57BL/6J mice (8-week-old, 18–20 g) were purchased from Jackson Laboratory and allowed to acclimate for two weeks. Mice were kept under regular light/dark cycles (12 h light, 12 h dark). The animals’ behavior, food intake, signs of pain, and overall health were evaluated daily by a veterinarian who was not directly involved in the experiment. This independent care and monitoring ensured that the experimental results were not influenced by the animal management process.

2.3. Salivary gland irradiation in mice

A total of 42 mice were randomly assigned to three treatment groups (14 mice per group): radiation therapy (RT) injected with tetrathiomolybdate, RT injected with PBS, and a control group that did not undergo radiation. The sample size per group for saliva flow measurement (6 mice per group) was determined using G*Power software (v 3.1.9.7) with a power of 0.95, a significance level of 0.05, and an effect size (f) of 1.1. Additionally, 8 mice per group (4 mice per time point) were used for histological studies and 10 μL volume of tetrathiomolybdate (1 mg/mL) or PBS solution was injected transdermally into both submandibular glands of each mouse using an insulin syringe (28G) one day before radiation therapy (D-1). The day after tetrathiomolybdate and/or PBS injections (D0), mice were anesthetized and fitted with a protective lead shield with customized ventral opening of 1 cm × 4 cm above the submandibular gland region. Mice were exposed to a single 15 Gy (X-Ray) radiation dose (Fig. 1A). Two days after radiation (D2), mice received a second dose of tetrathiomolybdate or PBS injections (as above) and cohorts from both treatment groups were analyzed on days 8, 14 and 27 (Fig. 1B–F). All animal management, anesthesia and euthanasia (CO₂ administration followed by cervical dislocation) were performed during the morning and followed a protocol approved by the Animal Care and Use Committee (ACUC protocol # 43008) at the University of Missouri. Also, this study conforms to the Animal Research: Reporting In Vivo Experiments (ARRIVE) guidelines. Finally, no adverse effects were observed during our experiments.

Fig. 1.

Radiation treatment and local tetrathiomolybdate delivery used in this study. (A) Mice received a single 15 Gy radiation dose with a customized lead shield having a 1 cm × 4 cm slit opening above the neck region. (B) Both 1 day prior to and 2 days after radiation treatment, the submandibular gland of each mouse was injected with tetrathiomolybdate (TTM) or PBS control via transdermal injection. (C) Images of the neck area were taken to show progressive penetration of the fluorescent dye (D) or tetrathiomolybdate (E) into the submandibular gland, and fluorescent signal was confirmed using a ChemiDoc system. (F) Finally, tissues were harvested at 8 and 14 days and saliva was collected at 27 days Black arrows indicate the injection points of the drugs.

2.4. Histological studies

Four mice per group were randomly selected and euthanized at day 8 and day 14 post-radiation therapy. Next, submandibular glands were dissected and fixed in 10 % formalin at room temperature for 1 day and dehydrated in 70 % ethanol solution at 4 °C for 1 day. Then, specimens were dehydrated in 100 % ethanol, embedded in paraffin wax and cut into 5 μm sections. The sections were deparaffinized with xylene and rehydrated with serial ethanol solutions (100 %, 95 % 80 %, 70 % and 50 %, v/v) followed by a rinse with distilled water. For hematoxylin and eosin staining, the rehydrated sections were stained with Mayer's hematoxylin solution for 5 min and washed twice with distilled water. Next, slides were destained with acid alcohol (0.3 % HCl in ethanol) for 10 s and washed twice with distilled water. Following this step, slides were stained with Eosin Y Solution for 2 min, rinsed with absolute ethanol, cleared in xylene and mounted with a xylene-based mounting solution. Finally, the samples were imaged using the Leica DMI6000B microscope. For collagen staining, the rehydrated sections were stained with Picro Sirius Red for 1 h and washed twice with acetic acid solution (1 %, v/v). Then, slides were rinsed with absolute ethanol, cleared in xylene and mounted with a xylene-based mounting solution. Finally, the samples were captured using the Leica DMI6000B microscope under both bright field and fluorescence conditions, with 3 slides per mouse used (e.g., top, middle and bottom from each salivary gland, as previously described [27]) and a total of 4 mice per group. To identify regions of interest of collagen, fluorescence imaging was applied to artificial intelligence-guided image analysis (AIVIA) and the difference in stained area was calculated using the following equation (1) [28,29].

$$\text{Picro}\text{Sirius}\text{Red}\text{fluorescence}(\%)=\frac{\text{Fluorescence}\text{active}\text{area}\text{in}\text{submandibular}\text{gland}}{\text{Area}\text{of}\text{submandibular}\text{gland}}\times 100$$ (1)

2.5. Confocal immunofluorescence analysis

For antigen retrieval, the rehydrated submandibular gland sections were incubated in Tris-EDTA buffer [10 mM Tris, 1 mM EDTA, 0.05 % (v/v) Tween® 20, pH 9.0]. 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 overnight with the following primary antibodies: rabbit anti-ZO-1 and mouse anti-E-cadherin. Later, sections were incubated with Alexa Fluor 488 conjugated anti-rabbit IgG secondary antibody and Alexa Fluor 568 conjugated anti-mouse IgG secondary antibody at room temperature for 1 h and counterstained with 300 nM DAPI at room temperature for 5 min. Subsequently, specimens were analyzed using the STELLARIS 5 Confocal Microscope (Leica Microsystems, Wetzlar, Germany), and the disorganized duct was calculated using the following equation (2):

$$\text{Disorganized}\text{duct}=\frac{\text{Damaged}\text{duct}}{\text{Total}\text{number}\text{of}\text{ducts}}\times 100$$ (2)

2.6. Salivary secretion measurements

At day 27 post-radiation therapy, PBS- or tetrathiomolybdate-treated mice were anesthetized with an avertin solution followed by intraperitoneal injection with pilocarpine (2.5 mg/kg) to stimulate the secretion of saliva, which was collected over a 20-min period and measured using a 200 μL pipette. Next, the normalized saliva flow rate was calculated using the following equations (3), (4)):

$$\text{Saliva}\text{flow}\text{rate}=\frac{\text{Stimulated}\text{saliva}\text{flow}\text{rate}}{\text{Mouse}\text{body}\text{weight}(\mathrm{g})\times \text{collection}\text{time}(\min )}$$ (3)

$$\text{Normalized}\text{saliva}\text{flow}\text{rate}=\frac{\text{Saliva}\text{flow}\text{rate}\text{of}\text{irradiated}(\text{experimental})\text{group}}{\text{Saliva}\text{flow}\text{rate}\text{of}\text{non}-\text{irradiated}(\text{control})\text{group}}$$ (4)

Statistical significance was assessed using one-way ANOVA (P ≤ 0.05) followed by Dunnett's post-hoc test for multiple comparisons with irradiated submandibular gland treated with PBS. The data analysis was performed using GraphPad Prism (v 8) software.

2.7. Data analysis

To mitigate potential biases that may have emerged during the experimental plan, a comparison was made between the independently designed experiments and blind test results, the latter designation indicating that researchers performing the statistical and histological analyses could not identify groups for which analyses were being performed (i.e., labels indicating sample names were removed).

3. Results

3.1. Transdermal drug delivery into the submandibular glands of irradiated mice

We initially performed pilot studies to evaluate the extent to which transdermal injections can be used for drug delivery into the submandibular gland. Using anesthetized mice, the left submandibular gland was transdermally injected with fluorescein dye (10 μL per submandibular gland, 1 mg/mL, 376.27 g/mol) as a fluorescent drug proxy while the right submandibular gland remained untreated (Fig. 1C). Imaging of the submandibular gland over a 30-min period revealed a time-dependent diffusion of fluorescent signal in the left submandibular gland but not the untreated contralateral gland (Fig. 1C). Fluorescence imaging of the dissected submandibular glands using a ChemiDoc system indicated that the fluorescein dye was dispersed throughout much of the left submandibular gland, but importantly it did not extent beyond this organ (Fig. 1D). These results were consistent even when tetrathiomolybdate only was used (Fig. 1E).

Having successfully demonstrated transdermal injection as a method of drug delivery into the submandibular gland, we applied this approach to test the anti-fibrotic potential of the copper chelator tetrathiomolybdate in irradiated mice. As described in the Materials and methods section, irradiated mice were fitted with a customized lead shield with a 1 cm wide ventral opening positioned over the submandibular gland region. Female mice at 8 weeks of age were randomly divided into cohorts that received either a local transdermal injection of tetrathiomolybdate or PBS directly into both submandibular glands one day prior to and two days post-radiation therapy (Fig. 1). The therapeutic potential of tetrathiomolybdate was evaluated at 8 days and 14 days post irradiation as described below.

3.2. Tetrathiomolybdate diminishes epithelial tissue damage

3.2.1. Tetrathiomolybdate preserves salivary gland epithelial integrity

To examine the potential impact of tetrathiomolybdate on epithelial tissue damage in irradiated submandibular glands, tissue sections were stained with hematoxylin and eosin. Subsequently, irradiated samples treated with PBS were qualitatively analyzed and compared to a tetrathiomolybdate-treated group, as previously outlined [[30], [31], [32], [33], [34], [35]]. This analysis serves as a proof-of-concept demonstration indicating a reduction in radiation damage in the latter condition. As shown in Fig. 2, Fig. 3, Fig. 4 and Supplemental Fig. 1A, irradiated submandibular gland treated with PBS at day 8 post-radiation therapy displayed focal areas of nuclear atypia (red arrows), cytoplasmic vacuolization (blue arrows), cell degeneration (green arrows) and interstitial edema (yellow arrows). Such changes were also observed at day 14 post-radiation therapy (Fig. 2, Fig. 3, Fig. 4 and Supplemental Fig. 1B), along with an abundance of inflammatory cells. In contrast, irradiated submandibular gland treated with tetrathiomolybdate at day 8 post-radiation therapy (Fig. 2, Fig. 3, Fig. 4 and Supplemental Fig. 1C) displayed areas of nuclear atypia, vacuolization and cell degeneration, all of which were less evident at day 14 post-radiation therapy (Fig. 2, Fig. 3, Fig. 4 and Supplemental Fig. 1D) and were likewise comparable to non-irradiated submandibular gland controls (Fig. 2, Fig. 3, Fig. 4 and Supplemental Fig. 1E). Together, these results indicate that treatment with tetrathiomolybdate reduces epithelial tissue damage in irradiated submandibular gland.

Fig. 2.

Tetrathiomolybdate preserves epithelial integrity in submandibular gland from irradiated mice. Hematoxylin and eosin-stained submandibular gland tissue sections from irradiated mice that received either control or tetrathiomolybdate (TTM) treatments as follows: PBS-treated mice at day 8 (A1-4) and day 14 (B1-4); tetrathiomolybdate-treated mice at day 8 (C1-4) and day 14 (D1-4) and sham control at day 8 (E1-4). Black scale bars represent 50 μm. Tissue morphology was analyzed using the Leica DMI6000B and images are representative from a total of 4 mice per group.

Fig. 3.

Tetrathiomolybdate reduces early collagen deposition in submandibular gland from irradiated mice. Picro Sirius Red staining of collagen deposition in each treatment group at day 8 (A, C, E) and day 14 (B, D) post-radiation therapy. Images were analyzed using a Leica STELLARIS confocal microscope equipped with an artificial intelligence-guided image analysis software (AIVIA). (F) Quantification of Picro Sirius Red pixels is depicted in the lower right bar graph with data from each treatment expressed as mean ± SD of results from a total of 4 mice per group. Statistical significance was assessed by one-way ANOVA (***p < 0.001, ****p < 0.0001 and n.s. = not significant) and Dunnett's post-hoc test for multiple comparisons, with scale bars representing 1 mm.

Fig. 4.

Tetrathiomolybdate preserves ZO-1 apical localization in submandibular gland from irradiated mice. The localization of ZO-1 and E-cadherin are shown in submandibular gland tissue sections from each treatment group as follows: PBS-treated irradiated mice at day 8 (A) and day 14 (B); tetrathiomolybdate (TTM)-treated irradiated mice at day 8 (C) and day 14 (D); sham control mice (E). Submandibular gland sections were incubated with ZO-1 (green) and E-cadherin (red) antibodies, counterstained in blue with DAPI and analyzed using a Leica STELLARIS confocal microscope. Images are representative of a total of 4 mice per group, with scale bars set at 50 μm. White arrows indicate disorganized ZO-1. (F) Quantification of disorganized duct is depicted in the lower right bar graph with data from each treatment expressed as mean ± SD of results from a total of 4 mice per group (3 slides per mouse). Statistical significance was assessed by one-way ANOVA (*p < 0.05, ***p < 0.001, ****p < 0.0001 and n.s. = not significant) and Dunnett's post-hoc test for multiple comparisons.

3.3. Tetrathiomolybdate reduces early collage deposition

To determine the effect of tetrathiomolybdate on early collagen deposition in irradiated submandibular gland, tissue sections were stained with Picro Sirius Red. The collagen deposition in irradiated samples treated with PBS was compared to tetrathiomolybdate-treated samples and non-irradiated controls to allow for a comprehensive evaluation of the impact of tetrathiomolybdate on collagen levels in the irradiated submandibular gland. As shown in Fig. 3A, irradiated submandibular gland treated with PBS at day 8 post-radiation therapy exhibited moderate levels of early collagen deposition that were more pronounced at day 14 post-radiation therapy (Fig. 3B). In contrast, irradiated submandibular gland treated with tetrathiomolybdate at day 8 post-radiation therapy (Fig. 3C) displayed fewer areas with fibrosis that were less evident at day 14 post-radiation therapy (Fig. 3D) comparable to non-irradiated controls (Fig. 3E). Quantification of Picro Sirius Red staining in irradiated submandibular gland using the AVIA software and ImageJ showed that tetrathiomolybdate significantly reduced early collagen deposition at day 14 post-radiation therapy relative to irradiated submandibular gland treated with PBS and was comparable to non-irradiated submandibular gland controls (Fig. 3F). Together, these results indicate that treatment of the submandibular gland with tetrathiomolybdate significantly reduces radiation-induced early collagen deposition.

3.4. Tetrathiomolybdate preserves polarized saliva secretion

To validate the reported loss of epithelial polarity caused by radiation therapy [33], we conducted experiments to analyze apicobasal polarity using confocal microscopy, as described in the Materials and methods section. As shown in Fig. 4A, irradiated submandibular gland treated with PBS at day 8 post-radiation therapy displayed areas with disorganized ZO-1; however, both E-cadherin basolateral expression and acinar/ductal lumens were conserved. These changes were also observed at day 14 post-radiation therapy though more abundant (Fig. 4B). Compared to the PBS control, the irradiated submandibular gland treated with tetrathiomolybdate at day 8 post-radiation therapy exhibited fewer areas of disorganized nuclei and ZO-1 in the submandibular gland (Fig. 4C). Notably, by 14 days post-radiation therapy, there was significant tissue recovery relative to day 8, as indicated by reduced disorganization of nuclei and ZO-1 staining, along with the presence of intact luminal structures (Fig. 4D); this staining pattern was comparable to that of non-irradiated controls (Fig. 4E). Further quantitative analysis to confirm these results is presented in Fig. 4F, indicating a significant increase in disorganized ducts in irradiated submandibular glands treated with PBS at both days 8 and 14 post-radiation therapy compared to non-irradiated sham controls. In contrast, irradiated submandibular glands treated with tetrathiomolybdate at both days 8 and 14 exhibited an organized ZO-1 fluorescent signal comparable to non-irradiated sham controls.

3.5. Tetrathiomolybdate preserves saliva secretion in irradiated mice

Next, we investigated whether tetrathiomolybdate might also protect the submandibular gland against functional defects arising from radiation damage. Previous studies showed that the time point where more than 50 % reduction of saliva secretion occurs after radiation treatment is 27 days (Supplemental Fig. 1A) [35], and to this end, we compared saliva secretion in tetrathiomolybdate-treated mice with control mice at day 27 post-radiation therapy. As shown in Fig. 5, irradiated mice treated with PBS (red bar) exhibited 65.24 % reduction in average saliva flow rate compared to non-irradiated controls [0.35 μL/g/min, n = 6, p < 0.05, blue bar]. By contrast, the mean reduction in saliva flow rate was only 32.15 % in irradiated submandibular gland treated with tetrathiomolybdate [0.62 μL/g/min, n = 6, p < 0.05, yellow bar] at day 27. Finally, no significant differences in the tetrathiomolybdate -treated groups at other time points were observed (Supplementary Fig. 2B), and when taken together, these findings suggest that transdermal injection of the submandibular gland with tetrathiomolybdate significantly protects against radiation-induced hyposalivation.

Fig. 5.

Tetrathiomolybdate restores saliva secretion in irradiated mice. At day 27 post-radiation therapy, mice were anesthetized and stimulated with pilocarpine and saliva collected for 20 min. Data represent the mean ± SD of n = 6 mice per condition. Statistical significance was assessed by one-way ANOVA (*p < 0.05, ***p < 0.001 and ****p < 0.0001) and Dunnett's post-hoc test for multiple comparisons.

4. Discussion

Previous studies demonstrated that radiation therapy triggers collagen crosslinking in salivary glands that results in a loss of function [33,36], with collagen crosslinking catalyzed by the LOX family of secreted copper-dependent metalloenzymes [[13], [14], [15]]. Building on these findings, the current study demonstrates for the first time that copper chelation using tetrathiomolybdate reduces early collagen deposition in irradiated salivary glands. Mechanisms of this process may best be understood in relation to Wilson's disease, a condition characterized by copper overload [37], which has been managed by reducing bioavailable copper using tetrathiomolybdate, which binds with high affinity and specificity to copper ions [[38], [39], [40]]. In addition to its established use in Wilson's disease, tetrathiomolybdate has also shown promise in both preclinical and clinical studies as a potential therapeutic agent for other conditions involving copper dysregulation, including certain types of cancer and fibrosis [41,42]. To this end, tetrathiomolybdate has proven efficacy in preclinical models for fibrosis in multiple tissues [[43], [44], [45], [46]]; however, its application to radiation-induced submandibular gland damage was previously untested and perhaps even had not been posited, such that the current study represents an entirely new approach to the as-yet untreatable problem of radiation-induced salivary gland damage. In light of the above, transdermal administration of tetrathiomolybdate into mouse submandibular gland was attempted and found to mitigate several pathological hallmarks of radiation-induced damage including abnormal histopathology, increased collagen deposition, loss of cell polarity and hyposalivation. Limitations of this study include the following: a) A lack of mechanistic studies to understand the downstream effects of copper chelation on salivary gland fibrosis, and to this end, future studies will investigate whether copper chelation-mediated decrease of collagen deposition occurs via LOX enzymes; b) Absence of sex-mediated biological differences in the study, and to this end, experiments involving male mice will be necessary to investigate whether sexual dimorphism influences the effects of tetrathiomolybdate of radiation-induced early collagen deposition; c) The study did not show long-term effects of tetrathiomolybdate on collagen deposition, and to this end, future studies demonstrating the same are warranted; d) Finally, although tetrathiomolybdate is a newer copper chelator that has yet to receive FDA approval for Wilson's disease, other copper chelators such as penicillamine and trientine have a long history of oral use in patients with this disorder [47], and to this end, monitoring of ongoing clinical trial results in relation to potential secondary effects is advisable. By way of summary and based on the indicated treatment effect as well as perceived likelihood of tolerance in a clinical setting, it would appear that our results open the door for further studies that may ultimately lead to clinical trials of locally administered neoadjuvant copper chelators for the preservation of salivary gland function in patients undergoing radiotherapy for head and neck cancers.

Ethics statement

The study was conducted with the approval of the University of Missouri's Animal Care and Use Committee (ACUC) protocol number: 43008. Also, this research adhered to the Animal Research: Reporting In Vivo Experiments (ARRIVE) guidelines, ensuring ethical animal treatment and research reporting standards.

Data availability statement

The study data are available upon request from the corresponding author for the purpose of scientific inquiry and validation.

Funding statement

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study is supported by the National Institutes of Health-National Institute of Dental and Craniofacial Research (grants R01DE022971 and R01DE027884 to O.J.B) and Start-up funds from the University of Missouri.

Ethics declarations

This study was reviewed and approved by the University of Missouri Animal Care and Use Committee (ACUC), with the Approval Number: 42833 and Animal Welfare Assurance Number: D16-00249.

CRediT authorship contribution statement

Kihoon Nam: Writing – review & editing, Writing – original draft, Validation, Supervision, Methodology, Investigation, Data curation, Conceptualization. Harim Tavares Dos Santos: Writing – review & editing, Investigation, Data curation. Frank M. Maslow: Writing – review & editing, Methodology, Investigation. Travis Small: Writing – review & editing, Methodology, Investigation. Vinit Shanbhag: Writing – review & editing, Methodology, Investigation. Michael J. Petris: Writing – review & editing, Writing – original draft, Supervision, Investigation, Formal analysis, Conceptualization. Olga J. Baker: Writing – review & editing, Writing – original draft, Supervision, Resources, Project administration, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Olga J. Baker reports financial support was provided by National Institute of Dental and Craniofacial Research, National Institutes of Health. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following is the Supplementary data to this article: