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. Author manuscript; available in PMC: 2020 Nov 24.
Published in final edited form as: Acta Biomater. 2019 Apr 25;91:209–219. doi: 10.1016/j.actbio.2019.04.052

Improving Hard Palate Wound Healing Using Immune Modulatory Autotherapies

Samir A Ballestas 1, Thomas Turner 3,4, Archana Kamalakar 1, Yvonne Stephenson 1, Nick Willett 2,4, Steven L Goudy 1, Edward A Botchwey 3,4
PMCID: PMC7685787  NIHMSID: NIHMS1640574  PMID: 31029828

Abstract

Oral cavity wound healing occurs in an environment that sustains ongoing physical trauma and is rich in bacteria. Despite this, injuries to the mucosal surface often heal faster than cutaneous wounds and leave less noticeable scars. Patients undergoing cleft palate repair have a high degree of wound healing complications with up to 60% experiencing oronasal fistula (ONF) formation. In this study, we developed a mouse model of hard palate mucosal injury, to study the endogenous injury response during oral cavity wound healing and ONF formation. Immunophenotyping of the inflammatory infiltrate following hard palate injury showed delayed recruitment of non-classical LY6Clo monocytes and failure to resolve inflammation. To induce a pro-regenerative inflammatory response, delivery of FTY720 nanofiber scaffolds following hard palate mucosal injury promoted complete ONF healing and was associated with increased LY6Clo monocytes and pro-regenerative M2 macrophages. Alteration in gene expression with FTY720 delivery included increased Sox2 expression, reduction in pro-inflammatory IL-1, IL-4 and IL-6 and increased pro-regenerative IL-10 expression. Increased keratinocyte proliferation during ONF healing was observed at day 5 following FTY720 delivery. Our results show that local delivery of FTY720 from nanofiber scaffolds in the oral cavity enhances healing of ONF, occurring through multiple immunomodulatory mechanisms.

Keywords: Cleft Palate, Wound Healing, Oral Cavity, Oronasal Fistula, FTY720

1. Introduction

Oral cavity wound healing occurs in a bacteria-laden environment that undergoes constant trauma and is exposed to saliva [1]. Despite the frequency of injury to the oral cavity and its clinical significance, relatively little is known about the healing of oral mucosa. One particularly challenging problem is healing in the oral cavity following cleft palate repair, wherein all of the palate tissues are rotated to the midline to separate the nasal and oral cavities. Poor wound healing following cleft palate repair can occur in up to 60% of patients, leading to a persistent oronasal fistula (ONF) and reflux of liquids from the nose and air escape during speech [2]. The morbidity of ONF is significant as the children require additional anesthesia, overnight stay in the hospital, and revision surgeries, having an efficacy of 50%. The only available regenerative strategies to reduce ONF formation involve the use of donated human material to be implanted as a barrier, carrying the risk of transmissible disease [3].

Efforts to better understand host inflammation following injury have drawn interest for the development of novel autotherapies, which are based on the body’s natural ability to heal and protect itself. The role of pro-regenerative inflammatory signals in oral cavity wound healing is less well characterized compared to cutaneous wound healing but is known to have less scarring and happens more quickly. Importantly, mononuclear cell infiltration, including monocytes and macrophages, is inherent to the post-injury microenvironment [4, 5]. Two distinct populations of monocytes exist in both mouse and human blood. Specifically, Ly6ChiCX3CR1lo “classical” monocytes in mice have been identified as a class of pro-inflammatory monocytes, whereas Ly6CloCX3CR1hi non-classical, “anti-inflammatory” monocytes patrol the resting endothelium and exhibit pro-regenerative functions after injury [6]. Monocyte-derived macrophages also respond to cues within the injury microenvironment that determine their role in wound healing responses and the polarization state ranging from pro-inflammatory M1 macrophages to alternatively activated pro-regenerative M2a/c macrophages [7].

Innovative biomaterial technologies are needed to harness pro-regenerative potential of host inflammation. We have previously reported on the development of “immunoregenerative” materials in which endogenous tissue repair programs are unlocked by small molecules that re-engineering the temporal recruitment profile, spatial distribution, and niche signaling of monocyte / macrophage subsets [7]. It is essential to thoroughly understand the properties and actions of these cells in their tissue context to implement immune autotherapies. In order to tailor this powerful autotherapy for applications in dentistry and oral surgery, we investigated the recruitment of mononuclear phagocyte subsets after oral mucosal injury [7]. We specifically investigate whether recruitment of pro-regenerative non-classical monocytes using degradable nanofiber scaffolds loaded with immune modulatory drug FTY720 will improve oral cavity wound healing and reduce ONF.

2. Material and Methods

2.1. Oronasal Fistula Creation

All animal experiments were performed in accordance with Emory IACUC procedures. To create an ONF, the mice were anesthetized with Isoflurane 3% and Ketamine/Xylazine 100mg/kg/10mg/kg, and a retractor was used to open their mouth (Supplemental Figure 1). Following this, a 1.5 mm full thickness hard palate mucosal injury was created in the midline using an ophthalmologic cautery. Nanofiber scaffold implantation following the injury was secured using TA-5 veterinary tissue glue (Oasis Medical Mettawa, IL.), as explained in the timeline graph of Supplemental figure 2. No major injury is produced to the septal bone as it can be seen in Supplemental Figure 3.

2.2. Nanofiber scaffold fabrication

Electrospun nanofiber scaffolds were fabricated using a 1:1 weight/weight ratio of Polycaprolactone (PCL, Sigma) and poly(lactic-co-glycolic-acid) (PLAGA, Lakeshore Biomaterials, Birmingham, AL). These polymers were then dissolved in a 1:3 volume ratio solution of methanol to chloroform to achieve an 18% polymer concentration for blank fibers and 20% polymer concentration for FTY720-loaded fibers. For FTY720 loaded fibers, drug was added into the solution at a 1:200 drug:polymer weight ratio. The solution was then agitated for 2+ hours until the polymer was fully dissolved and then 2mL of either polymer solution was loaded into a 3mL syringe with a 10mm diameter. Electrospinning was performed at a flow rate of 1mL/hr and at an applied voltage of 19kV for both blank and FTY720 fibers [8, 9]. The working distance was 12 cm for FTY720 fibers and 10 cm for blank fibers. After 2mL of polymer was spun, fibers were wrapped in low-binding plastic folders and stored at −20°C. These polymer fiber sheets were then made into 1.5mm diameter scaffolds using a biopsy punch. A release kinetics study of FTY720 from the nanofiber scaffold was performed as shown in Supplemental Figure 4. In 100% of cases the scaffolds were present 24 hours after the surgery (N=120). At Day 3 and 5 following injury, the majority of scaffolds were dislodged from the oronasal fistula. 47/60 (78.3%) mice had no scaffold by day 3, and 55/60 (91.6%) mice had no scaffold by day 5.

2.3. Flow Cytometry

For analysis of immune infiltrate, oral mucosal tissues were harvested and digested in 1mg/ml collagenase I (Sigma) for 45 minutes at 37°C. The digested palate mucosa was filtered through a cell strainer to obtain a single cell suspension. Single-cell suspensions from palatal samples were stained for live cells using either Zombie Green or Zombie NIR (Biolegend) dyes in cell-culture grade PBS per manufacturer instructions. Cells were then stained with cell phenotyping antibodies in a 1:1 volume ratio of 3% FBS and Brilliant Stain Buffer (BD Biosciences) according to standard procedures and analyzed on a FACS AriaIIIu flow cytometer (BD Biosciences). The following antibodies were used for cell phenotyping: Zombie NIR Fixable Viability Kit (BioLegend), BV421-conjugated anti-CD11b (BioLegend), BV510-conjugated anti-Ly6C (BioLegend), BV711-conjugated anti-CD64 (BioLegend), APC anti-mouse/PE-conjugated anti-MerTK (Biolegend), FITC-conjugated anti-CD206 (BioLegend), 30μL of Accucheck Counting Beads (Invitrogen) were added per sample for absolute quantification of cell populations.

2.4. Histology and Immunohistochemistry

Samples used for histology sectioning were isolated palates. They were placed in 10% NBF (Neutral buffered formalin), then into a histology cassette and labeled, followed by 10% formic acid decalcification reagent. Models were embedded in paraffin and cut using coronal planes in 4 μm sections, which were then stained in hematoxylin and eosin (H&E) and Phosphohistone 3 (PH3) (Cell Signaling Technology, Denver, MA) (#9701S). PH3 positive cells were counted per 20x high powered field adjacent to the wound bed in triplicate from 3 separate specimens and compared using Chi-squared analysis.

2.5. Slide scans and analysis

After having our H&E and PH3 slides, Hamamatsu NanoZoomer 2.0 HT processor was used to scan slides and they were then analyzed in Hamamatsu NDP.view2 Viewing Software.

2.6. VEGF Serum ELISA

Mouse VEGF Quantikine ELISA kit (R&D Systems, Inc. Minneapolis, MN) (#MMV00) was used. This assay uses a quantitative sandwich enzyme immunoassay technique, with specific mouse VEGF polyclonal antibodies pre-coated in the microplate. 50 μL of Assay Diluent RD1N were added to each well, then 50 μL of Standard, control and samples were added with posterior incubation of 2 hours at room temperature. Wells were washed with Wash Buffer five times, and 100 μL of Mouse VEGF Conjugate were added to each well, letting the samples incubate for another 2 hours. Wells were washed again five times and 100 μL of Substrate Solution were added to each well. Samples were incubated for another 30 minutes protected from light, and 100 μL of Stop Solution were finally added. Enzyme reactions produced a blue product, which turned yellow after stop solution was added. Color intensity was measured with microplate reader set at 450 nm. (dilution factor 1:5)

2.7. Serum Sodium Analysis

Mice serum was extracted and analyzed with Colorimetric Sodium Assay Kit (Abcam, ab211096) (dilution factor 1:250). 40 μL of standards, samples and background sample control were added into a 96-well plate. Β-Gal was diluted in DTT/Assay Buffer (1:200) and 20 μL of the resultant solution were added into the wells. The plate was incubated at 37°C for 10 minutes protected from light, and 40 μL of Substrate were added into each well. Plate was incubated for 30 minutes at 37°C protected from light. 100 μL of Na Developer were then added. The generated color signal was observed at OD = 405 nm.

2.8. Quantitative PCR

For gene expression we used qPCR technique with TRIzol reagent (Ambion, 15596018) for total RNA isolation as described before [10]. Primer pairs are shown in Supplemental Table 1.

2.9. Statistical Analysis

Data were analyzed using GraphPad Prism 7 software, and all statistical tests were conducted with α=0.05. We used Shapiro-Wilk test in order to determine that continuous variables were normally distributed. Results were analyzed with repeated measures one-way ANOVA with Bonferroni’s multiple comparisons test. Data are plotted as mean with standard deviation.

2.10. Murine Model

Wild-type C57BL/6 were obtained from The Jackson Laboratories. All mice were 8–12-week females and had a weigh of 20–25 g. All mice had 1.5 mm oronasal fistulas verified using a 1.5mm biopsy punch as a measuring tool. In the event of creation of an oronasal fistula bigger than 1.5 mm, animals were immediately eliminated from the study. Mortality rates for the 174 animals included in this study were as follows. After oronasal fistula creation and no implantation (n=54), 15 mice died or were euthanized (27.77%) and 39 survived (72.77%). Mortality after Blank nanofiber scaffold implantation (n=60) was of 15 mice who died or were euthanized (25%) with 45 who survived (75%). Mortality after FTY720 nanofiber scaffold implantation (n=60) was of 10 mice who died or were euthanized (16.66%), in this group 50 mouse survived (83.33%).

3. Results

An oronasal fistula (ONF) was created in the hard palate mucosa, in mice, connecting it with the nasal mucosa, similar to that seen following fistula formation in cleft palate patients (supplemental Figure 1). Following injury, the ONF failed to close, creating a phenocopy of human ONF following cleft palate repair (Figure 1). At day 7, the mice had difficulty feeding and became weak despite being given soft chow, and ultimately more than 25% died due to their inability to eat and dehydration (supplemental Figure 5). When comparing the mortality rate in mice where nanofiber scaffolds were implanted, the mortality rate decreased from 25% with blank scaffolds to 16.66% with FTY720 scaffolds. Histologic analysis of the ONF reveals loss of hard palate mucosa, denuded bone and ONF formation (Figure 2).

Figure 1. Postoperative hard palate injuries in hard palate.

Figure 1.

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(A) Day 3 postoperative ONF. (B) Day 5 postoperative ONF. (C) Day 7 postoperative ONF.

Figure 2. Oronasal fistula in hard palate after critical size defect.

Figure 2.

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(Day 3) (A) No-injury control, (B) No-injury control 20X, (C) Oronasal fistula, (D) Oronasal fistula 20X. (OC = Oral Cavity, NC= Nasal Cavity)

To examine the monocyte and macrophage environment during ONF wound healing, we performed flowcytometric analysis of the hard palate mucosal tissue surrounding the ONF. We compared the accumulation of pro-regenerative population of LY6Clo non-classical monocytes and M2 macrophage relative to (uninjured) hard palate mucosa. There were no significant changes in the LY6Clo or LY6Chi classical monocyte populations (Figure 3). In addition, while the total number of M2 macrophages to the total macrophage population was not increased, the percentage of M2 macrophages at day 5 of injured tissue did increase compared to uninjured controls.

Figure 3. Monocytes and Macrophages migration into the injured hard palate.

Figure 3.

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(A) Ly6Clo (AM) monocyte % of cells. (B). Ly6Clo (AM) monocyte percentage of CD11B+ cells. (C) Ly6Chi (IM) monocyte % of cells. (D). Ly6Chi (IM) monocyte percentage of CD11B+ cells. (E) M2 Macrophages % of cells show an increase when reaching day 5 (F) CD64+MerTK+ Macrophage % of cells show an increase when arriving to day 5. (G) M2 Macrophages % of CD64+MerTK+ Macrophages. (Similar symbols = no difference, different symbols = significant difference) (S.D. p<0.05).

To determine if a targeted recruitment of pro-regenerative LY6Clo monocytes and macrophages would lead to improved ONF healing, we tested the delivery of FTY720, a sphingosine analog that increases pro-regenerative monocytes and macrophages, in a nanofiber scaffold [11]. Following injury to the hard palate mucosa, we implanted blank and FTY720 impregnated nanofiber scaffolds in the ONF site immediately following injury (supplemental Figure 6). Following scaffold placement, there was significant improvement in wound healing of the ONF with the FTY720 scaffold at day 3 and day 5 compared to unmodified scaffolds (Figure 4). Histologic examination of the injured site at day 3 showed persistence of the ONF in the blank scaffold group (Figure 5) whereas the FTY720 scaffold group showed re-epithelialization and ongoing repair of the submucosal tissues. Histologic evaluation at day 5 confirmed persistence of the ONF in the blank scaffold group whereas there was re-epithelialization in the FTY720 scaffold group (Figure 6). Histologic evaluation of cellular proliferation demonstrated increased keratinocyte proliferation at Day 5 in the FTY720 scaffold group compared to blank (Figure 7). Examination of the pro-regenerative population of LY6Clo monocytes demonstrated that the insertion of the FTY720 scaffold showed increased LY6Clo monocytes at day 3 compared to blank scaffolds group, however, there was no difference noted at day 5 (Figure 8). Examination of the pro-regenerative M2 macrophages revealed an increase in the total number of M2 Macrophages and the percentage of M2 macrophages to all macrophages at day 3, whereas there were no differences observed at day 5 (Figure 9).

Figure 4. Oral injury creation followed by nanofiber scaffold implantation models (Blank and FTY720 scaffolds).

Figure 4.

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(A) Day 3 oral injury where blank scaffold was implanted with ONF. (B) Day 3 oral injury where FTY scaffold was implanted and shows re-epithelization. (C) Day 5 oral injury where blank scaffold was implanted with ONF. (D) Day 5 oral injury where and FTY scaffold was implanted and shows re-epithelization. N=3 for all experiments.

Figure 5. Day 3 ONF Healing with Blank and FTY720 Nanofiber.

Figure 5.

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(A) Oronasal fistula found in critical size defect in the palate with implanted blank scaffold. DAY 3 (B) H&E histology of ONF DAY 3 5X, (C) H&E histology of ONF DAY 3 20X, (D) Critical size defect in the palate with implanted FTY720 scaffold with proper healing by DAY 3. (E) H&E histology of healing palate DAY 3 5X, (F) H&E histology of healing palate DAY 3 20X

Figure 6. Day 5 ONF Healing with Blank and FTY720 Nanofiber.

Figure 6.

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(A) Oronasal fistula found in critical size defect in the palate with implanted blank scaffold. DAY 5 (B) H&E histology of ONF DAY 5 5X, (C) H&E histology of ONF DAY 5 20X, (D) Critical size defect in the palate with implanted FTY720 scaffold with proper healing by DAY 5. (E) H&E histology of healing palate DAY 5 5X, (F) H&E histology of healing palate DAY 5 20X

Figure 7. PH3 + cells staining in the wound edge of the hard palates.

Figure 7.

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(A) DAY 3 Blank scaffold, (B) Day 3 FTY scaffold (C) DAY 5 Blank scaffold, (D) DAY 5 FTY scaffold, (E) cells per high powerfield show no significant difference between these groups by day 3. (F) cells per high powerfield show statistically significant difference between blank and FTY group, showing higher migration and proliferation of keratinocytes when FTY720 is used. *=P<0.05.

Figure 8. LyC6lo (Anti-inflammatory) monocytes (AM) percentages.

Figure 8.

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(A) AMs % of total amount of cells day 3. (B) AMs % of CD11B+ cells day 3. (C) AMs % of cells day 5. (D) AMs % of CD11B+ cells day 5. N=3 for all experiments *=P<0.05.

Figure 9. M2 Macrophages migration in nanofiber scaffold groups.

Figure 9.

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(A) M2 Macrophages % of cells day 3. (B) CD64+MerTK+ Macrophages % of cells day 3. (C) M2 Macrophages % of CD64+MerTK+ macrophages day 3. (D) M2 Macrophages % of cells day 5, (E) CD64+MerTK+ Macrophages % of cells day 5. (F) Macrophages % of cells day 5. (G) M2 Macrophages % of CD64+MerTK+ macrophages day 5. N=3 for all experiments *=P<0.05.

To examine the effects of the FTY720 on the host inflammatory response, we performed quantitative PCR on the mucosal tissue surrounding the injury and found that IL-1, IL-4 and IL-6 were decreased in the presence of FTY720 whereas IL-10 was increased (Figure 10). Analysis of Sox2, Pax9 and Pitx2, transcription factors important in cutaneous wound healing, demonstrated increased expression associated with FTY720 scaffold delivery at day 3 (Figure 11).

Figure 10. Gene expression of hard palate mucosa following injury and implantation with blank or FTY720 nanofiber scaffolds at day 3 (N=3) and 5 (N=3) following injury.

Figure 10.

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(A) IL-1 gene expression, (B) IL-4 gene expression, (C) IL-6 gene expression, (D) IL-10 gene expression (Similar symbols = no difference, different symbols = significant difference) (S.D. p<0.05).

Figure 11. SOX 2, PAX9 and PITX2 gene expression of hard palate mucosa following injury and implantation with blank or FTY720 nanofiber scaffolds at day 3 (N=3) and 5 (N=3) following injury.

Figure 11.

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(A) Sox2 gene expression day 3, (B) Sox2 gene expression day 5, (C) Pax9 gene expression day 3, (D) Pax9 gene expression day 5, (E) Pitx2 gene expression day 3, (F) Pitx2 gene expression day 5. *=P<0.05.

4. Discussion

Oral cavity wounds are characterized by reduced inflammation, less angiogenesis, quick healing and less scar formation [12]. Unfortunately patients undergoing cleft palate repair suffer wound healing complications in up to 60% of patients who develop persistent oronasal fistula (ONF) [13]. Oral cavity wound healing occurs in a bacteria-laden environment that sustains constant force as the tongue masticates food against the healing tissues [14]. Occurrence of an ONF results in ongoing nasal reflux of liquid from the nose during eating and nasal air escape from the nose during speech, greatly affecting the well-being of the child. Currently, the only available regenerative approach to preventing ONF formation is the use of donor-derived tissue implantation which does not harness a reparative response and exposes the patient to potential infectious transmission [3]. Understanding the normative oral cavity wound healing response and developing targeted therapies to improve oral cavity wound healing will potentially reduce the occurrence of ONF in children.

There are important differences in the oral cavities in mice and human with regards to dentition structure and organization. Basic components such as lips, oral mucosa, tongue, salivary glands and teeth are the same and they all begin the digestion process. In mice and humans, the hard palate is lined by orthokeratinized squamous epithelium, that increases in keratin thickness in fasted or anorexic rodents [15]. The squamous epithelium of the soft palate may be variably keratinized in mice and nonkeratinized to parakeratinized in humans. They both have three pairs of major salivary glands: Parotid, Sublingual and Submandibular, as well as several minor salivary glands over the oral cavity [15]. The intermolar diameter of our mice was measured to be 4.7 +/− 0.2 mm as shown in Supplemental figure 7 and a 1.5 mm injury would represent 31.91% of that diameter being compromised. A 1-year-old child, the typical age of cleft palate repair, has an intermolar width of 38.51 +/− 4.12 mm [16]. Thus, a 1.5 mm injury in mice represents an initial oronasal fistula size of 12.2mm (32% of the diameter of the palate) in a 1-year-old child.

Oral cavity mucosal wound healing has previously been studied by creating superficial palate mucosal wounds that were found to completely re-epithelialize over a 7 day period [17]. Healing of ONF in this study occurred over a 5 day period following a 1.5 mm full-thickness injury. However, unlike the mucosal-only injuries, there is persistence of the ONF causing ongoing difficulty eating, a phenocopy of ONF formation in humans (Figure 1). The presence of an ONF was associated with 25% mortality rate in this study following injury due to dehydration and malnutrition Supplemental Figure 5. A similar model of ONF was created in a mini-pig model, however these animals had no physiologic consequences, like dehydration or malnutrition, due to the very small size of the porcine ONF in relationship to the palate size compared to the murine ONF model reported here [18]. Without intervention, the mini-pigs had persistence of their ONF, however, several donor-derived implantable materials were used to repair the small ONF with various degrees of success. Histologic evaluation of the mini-pigs demonstrated complete re-epithelialization of the palate and closure of the ONF. Using partial thickness palate mucosal injury models, investigators have evaluated multiple therapeutic targets to aid in wound healing including VEGFa and Thymosin B4 [19]. These prior studies have highlighted the inflammatory, histologic, and transcriptional differences between cutaneous and oral cavity wound healing and suggest that these differences can be targeted using regenerative approaches to improve oral cavity wound healing.

Monocytes are a key component of wound healing and are present during cutaneous and oral cavity wound healing [7]. Prior studies have revealed that oral cavity wounds have fewer monocytes present compared to cutaneous wounds. Monocytes in mice can take on a pro-inflammatory (Ly6C hi) and a pro-reparative (Ly6C lo) phenotype [20]. Once in the tissue, the monocytes can differentiate into macrophages that also play an important role in wound repair and they assume two phenotypes M1 (inflammatory) or M2 (pro-reparative) [21]. In this study, the presence of Ly6C hi and Ly6C lo monocytes did not change following hard palate mucosal injury or over the course of ONF healing (Figure 3). However, evaluation of the total macrophage population revealed an increase of total macrophages and M2 macrophages as a % of total cells at day 5, when the ONF wound had matured and there was little evidence of scar formation. Evaluation of cutaneous wounds demonstrated that inhibition of M2 macrophage production was associated with prolonged wound healing and more inflammation, suggesting that M2 macrophages are critical in reducing scarring and inflammation [22].

Multiple approaches have been used to improve the recruitment of the pro-regenerative monocytes and macrophages to cutaneous wounds. A recent report demonstrated the efficacy of FTY720, a sphingosine analog capable of attracting pro-regenerative monocytes to a cutaneous wound [11]. The delivery of FTY720 on a nanofiber scaffold increased the frequency of Ly6Clo pro-regenerative monocytes at 3 days and was associated with improved wound healing. FTY720 is also known to increase the migration of M2 macrophages into a wound bed. [20] In our data, we demonstrate that FTY720 delivery on a nanofiber scaffold following hard palate mucosal injury leads to wound contracture at day 3 and healing at day 5 compared to the blank scaffold where there was persistence of the ONF (Figure 4, 5, 6). Evaluation of the monocyte and macrophage population during the healing process following hard palate mucosal injury revealed an increase in Ly6Clo monocytes in the wound bed compared to blank scaffolds, similar to the results found in the cutaneous wound healing studies (Figure 8). Similarly, we found that the FTY720 nanofiber delivery increased the proportion of M2 macrophages in the wound bed at day 3 (Figure 9), instead of day 5 as seen in the control mice (Figure 3), as previously reported above.

The secretion of interleukins during oral wound healing is critical, as inhibition of their function is associated with reduced macrophage infiltration, angiogenesis and collagen deposition [1]. During wound healing monocytes and macrophages produce multiple cytokines, particularly IL-1, during the immediate inflammatory response [23]. Prior reports have demonstrated that high levels of IL-1 are associated with poor wound healing, however complete loss of IL-1 leads to delayed wound healing particularly in the oral cavity [24]. In our results, we identified that delivery of the FTY720 nanofiber reduced the expression of multiple pro-inflammatory interleukins including IL-1, IL-4, and IL-6 compared to blank scaffolds (Figure 10). Evaluation of the pro-regenerative interleukins demonstrated increased expression of IL-10 associated with FTY720 nanofiber delivery. Previous reports described improved wound healing with IL-10 delivery [25]. VEGF concentration in serum showed no significant difference among blank and FTY720 groups, while sodium concentration was higher in mice who received a blank scaffold compared to FTY720 at day 3, meaning a higher level of dehydration. (Supplemental Figure 8)

Evaluation of reported transcriptional regulators necessary for wound healing revealed increased Sox2, Pax9 and Pitx2 expression at day 3 following FTY720 nanofiber delivery (Figure 11). Sox2 (Sex determining region Y-box 2) is present at a basal state in oral mucosa, and is theorized to potentially reprogram skin keratinocytes to increase their migration and improve wound resolution working together with other transcription factors present in the process. [26] When a wound is produced in the skin, there is an upregulated transcriptional activity, but minimal or no differential transcription regulation in oral mucosa, suggesting that there are transcriptional regulatory networks present in the oral mucosa at a basal state that enhances wound healing process making it faster. Sox2 functions to increase migration and proliferation of keratinocytes, and leads to rapid wound healing, while Pax9 and Pitx2 have been described to be overexpressed transcripts in human and mice oral mucosa and keratinocytes. In our data we also observed increased keratinocyte proliferation (Figure 7) which may be a consequence of increased Sox2 expression. Never the less, expression of other transcription factors related to keratinocyte activation weren’t upregulated with our treatment. (Supplemental Figure 9) Further studies are needed to determine the mechanisms by which FTY720 nanofiber delivery enhances ONF healing.

5. Conclusion

Oral cavity wound healing occurs in a bacteria laden environment and there is constant physical trauma to the healing tissues. Clinically oronasal fistula formation occurs commonly after cleft palate repair and currently available therapies to reduce ONF formation do not harness the innate immune system. Delivery of FTY720 nanofiber scaffolds promote rapid oral cavity wound healing and prevent ONF formation by increasing pro-regenerative monocyte and macrophage infiltration, inducing favorable interleukin expression, increasing Sox2 expression and increasing keratinocyte migration and proliferation. Future studies will focus on the mechanisms by which FTY720 scaffolds improve oral cavity wound healing. These findings suggest that FTY720 nanofiber patches may be an effective treatment strategy to prevent ONF during cleft palate repair.

Supplementary Material

supplemental data

6. Acknowledgement

This paper was supported by the Pediatric Technology Center Therapeutic Child Impact Grand Award from Georgia Tech and Children’s Healthcare of Atlanta.

List of Abbreviations:

ONF

Oronasal Fistula

H&E

Hematoxylin and eosin

PH3

Phosphohistone 3

7. References

  • [1].Chen L, Arbieva ZH, Guo S, Marucha PT, Mustoe TA, DiPietro LA, Positional differences in the wound transcriptome of skin and oral mucosa, BMC Genomics 11 (2010) 471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [2].Phua YS, de Chalain T, Incidence of oronasal fistulae and velopharyngeal insufficiency after cleft palate repair: an audit of 211 children born between 1990 and 2004, Cleft Palate Craniofac J 45(2) (2008) 172–8. [DOI] [PubMed] [Google Scholar]
  • [3].Kirschner RE, Cabiling DS, Slemp AE, Siddiqi F, LaRossa DD, Losee JE, Repair of oronasal fistulae with acellular dermal matrices, Plast Reconstr Surg 118(6) (2006) 1431–40. [DOI] [PubMed] [Google Scholar]
  • [4].Mosser DM, Edwards JP, Exploring the full spectrum of macrophage activation, Nat Rev Immunol 8(12) (2008) 958–69. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [5].Nahrendorf M, Swirski FK, Aikawa E, Stangenberg L, Wurdinger T, Figueiredo JL, Libby P, Weissleder R, Pittet MJ, The healing myocardium sequentially mobilizes two monocyte subsets with divergent and complementary functions, J Exp Med 204(12) (2007) 3037–47. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [6].Avraham-Davidi I, Yona S, Grunewald M, Landsman L, Cochain C, Silvestre JS, Mizrahi H, Faroja M, Strauss-Ayali D, Mack M, Jung S, Keshet E, On-site education of VEGF-recruited monocytes improves their performance as angiogenic and arteriogenic accessory cells, J Exp Med 210(12) (2013) 2611–25. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [7].Ogle ME, Segar CE, Sridhar S, Botchwey EA, Monocytes and macrophages in tissue repair: Implications for immunoregenerative biomaterial design, Exp Biol Med (Maywood) 241(10) (2016) 1084–97. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [8].Chaurey V, Block F, Su YH, Chiang PC, Botchwey E, Chou CF, Swami NS, Nanofiber size-dependent sensitivity of fibroblast directionality to the methodology for scaffold alignment, Acta Biomater 8(11) (2012) 3982–90. [DOI] [PubMed] [Google Scholar]
  • [9].Petrie C, Tholpady S, Ogle R, Botchwey E, Proliferative capacity and osteogenic potential of novel dura mater stem cells on poly-lactic-co-glycolic acid, J Biomed Mater Res A 85(1) (2008) 61–71. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [10].Kamalakar A, Oh MS, Stephenson YC, Ballestas-Naissir SA, Davis ME, Willett NJ, Drissi HM, Goudy SL, A non-canonical JAGGED1 signal to JAK2 mediates osteoblast commitment in cranial neural crest cells, Cell Signal 54 (2019) 130–138. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [11].Olingy CE, San Emeterio CL, Ogle ME, Krieger JR, Bruce AC, Pfau DD, Jordan BT, Peirce SM, Botchwey EA, Non-classical monocytes are biased progenitors of wound healing macrophages during soft tissue injury, Sci Rep 7(1) (2017) 447. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [12].Glim JE, van Egmond M, Niessen FB, Everts V, Beelen RH, Detrimental dermal wound healing: what can we learn from the oral mucosa?, Wound Repair Regen 21(5) (2013) 648–60. [DOI] [PubMed] [Google Scholar]
  • [13].Hardwicke JT, Landini G, Richard BM, Fistula incidence after primary cleft palate repair: a systematic review of the literature, Plast Reconstr Surg 134(4) (2014) 618e–27e. [DOI] [PubMed] [Google Scholar]
  • [14].Mak K, Manji A, Gallant-Behm C, Wiebe C, Hart DA, Larjava H, Hakkinen L, Scarless healing of oral mucosa is characterized by faster resolution of inflammation and control of myofibroblast action compared to skin wounds in the red Duroc pig model, J Dermatol Sci 56(3) (2009) 168–80. [DOI] [PubMed] [Google Scholar]
  • [15].Treuting PM DS, Montine KS, Comparative Anatomy and Histology: A Mouse, Rat, and Human Atlas, 2nd ed., Elsevier; 2018. [Google Scholar]
  • [16].Pradel W, Senf D, Mai R, Ludicke G, Eckelt U, Lauer G, One-stage palate repair improves speech outcome and early maxillary growth in patients with cleft lip and palate, J Physiol Pharmacol 60 Suppl 8 (2009) 37–41. [PubMed] [Google Scholar]
  • [17].Keswani SG, Balaji S, Le LD, Leung A, Parvadia JK, Frischer J, Yamano S, Taichman N, Crombleholme TM, Role of salivary vascular endothelial growth factor (VEGF) in palatal mucosal wound healing, Wound Repair Regen 21(4) (2013) 554–62. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [18].Kesting MR, Loeffelbein DJ, Classen M, Slotta-Huspenina J, Hasler RJ, Jacobsen F, Kreutzer K, Al-Benna S, Wolff KD, Steinstraesser L, Repair of oronasal fistulas with human amniotic membrane in minipigs, Br J Oral Maxillofac Surg 48(2) (2010) 131–5. [DOI] [PubMed] [Google Scholar]
  • [19].Lee SI, Yi JK, Bae WJ, Lee S, Cha HJ, Kim EC, Thymosin Beta-4 Suppresses Osteoclastic Differentiation and Inflammatory Responses in Human Periodontal Ligament Cells, PLoS One 11(1) (2016) e0146708. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [20].Awojoodu AO, Ogle ME, Sefcik LS, Bowers DT, Martin K, Brayman KL, Lynch KR, Peirce-Cottler SM, Botchwey E, Sphingosine 1-phosphate receptor 3 regulates recruitment of anti-inflammatory monocytes to microvessels during implant arteriogenesis, Proc Natl Acad Sci U S A 110(34) (2013) 13785–90. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [21].Krieger JR, Ogle ME, McFaline-Figueroa J, Segar CE, Temenoff JS, Botchwey EA, Spatially localized recruitment of anti-inflammatory monocytes by SDF-1alpha-releasing hydrogels enhances microvascular network remodeling, Biomaterials 77 (2016) 280–90. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [22].Klinkert K, Whelan D, Clover AJP, Leblond AL, Kumar AHS, Caplice NM, Selective M2 Macrophage Depletion Leads to Prolonged Inflammation in Surgical Wounds, Eur Surg Res 58(3–4) (2017) 109–120. [DOI] [PubMed] [Google Scholar]
  • [23].Graves DT, Nooh N, Gillen T, Davey M, Patel S, Cottrell D, Amar S, IL-1 plays a critical role in oral, but not dermal, wound healing, J Immunol 167(9) (2001) 5316–20. [DOI] [PubMed] [Google Scholar]
  • [24].Thomay AA, Daley JM, Sabo E, Worth PJ, Shelton LJ, Harty MW, Reichner JS, Albina JE, Disruption of interleukin-1 signaling improves the quality of wound healing, Am J Pathol 174(6) (2009) 2129–36. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [25].King A, Balaji S, Le LD, Crombleholme TM, Keswani SG, Regenerative Wound Healing: The Role of Interleukin-10, Adv Wound Care (New Rochelle) 3(4) (2014) 315–323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [26].Iglesias-Bartolome R, Uchiyama A, Molinolo AA, Abusleme L, Brooks SR, Callejas-Valera JL, Edwards D, Doci C, Asselin-Labat ML, Onaitis MW, Moutsopoulos NM, Gutkind JS, Morasso MI, Transcriptional signature primes human oral mucosa for rapid wound healing, Sci Transl Med 10(451) (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]

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