Animal experiments were approved by the Animal Care and Use Committee of Chulalongkorn University (approval no. 012; March 03/2019).

A total of 45 8-week-old male Wistar rats (150-180 g) were obtained from Namura Animal Laboratory Center (Bangkok, Thailand). The rats were acclimated for 7 days under a controlled 12-h light/dark cycle, fed standard mice chow, and provided ad libitum access to water. To create second-degree burns, the rats were anesthetized with 2% isoflurane (Sigma-Aldrich; Merck KGaA) (45), and the dorsal skin was shaved. Two burn wounds were created on the back of each rat using a 10-mm-diameter aluminum rod, which was heated to 100˚C and placed on the skin for 10 sec (46). The rats were divided into three groups with 15 rats/group. Normal saline solution (NSS; 100 µl), calcium phosphate (Ca-P) nanoparticles, or 150 nM B1 siRNA in 100 µl of a solution containing Ca-P nanoparticles was applied daily to each wound on the rats in the control and treatment groups.

The wounds of three animals in each group (a total of six) were imaged on days 0, 7, 14, 21 and 28 after injury, and the wound areas were measured using ImageJ version 1.52t (National Institutes of Health) with a freehand selection tool. Wound areas are reported as a percentage of the initial wounded area. The rats were euthanized by exposing them to 5% isoflurane for 5-10 min until respiration ceased and they were confirmed dead on day 28(45). For 12 animals in each group (a total of 24 wounds), three rats (a total of six wounds) were euthanized with 5% isoflurane on days 3, 7, 14 and 21. On each of the specified euthanasia days, wound tissues were excised, and three of these samples (from the initial six wounds) were immediately subjected to DNA extraction and analyzed to determine the methylation levels. The remaining three wound tissues were fixed in 10% neutral-buffered formalin at room temperature (28-30˚C) for at least 48 h and embedded in paraffin for pathological examination (Fig. 1).

Rat epidermal keratinocytes (REK) (Cell Applications) were cultured in DMEM supplemented with 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin (Thermo Fisher Scientific, Inc.). Cells were incubated at 37˚C in a humidified incubator supplied with 95% air and 5% CO₂. The cells were cultured in T-75 Corning^™ U-shaped cell culture flasks and harvested at a confluency of ~80% using 0.05% Trypsin-EDTA with phenol red (Thermo Fisher Scientific, Inc.).

The B1 siRNA sequences were 5'-CGCACGCCUUUAAUCCCAGCACUCGUU-3' (sense) and 5'-CGAGUGCUGGGAUUAAAGGCGUGCGUU-3' (antisense). The scrambled siRNA sequences were 5'-GGCCAUUUCCCCGACUGCACACCUAUU-3' (sense) and 5'-UAGGUGUGCAGUCGGGGAAAUGGCCUU-3' (antisense). The B1 siRNAs and scrambled siRNA were purchased from Bioneer, Inc. The siRNA sequences were designed using siRNA wizard software version 3.1 (InvivoGen). The day before transfection, 5x10⁴ REK cells/well were seeded in a 24-well plate in 0.5 ml DMEM supplemented with 10% fetal bovine serum. After 24 h, the medium in each well in the control groups was replaced with normal saline solution (NSS; 100 µl) and experiment groups was replaced with 100 µl Ca-P nanoparticles, 150 nM B1 siRNA in 100 µl Ca-P nanoparticles and 150 nM scramble siRNA in 100 µl Ca-P nanoparticles. The cells were incubated at 37˚C in a CO₂ incubator for 48 h, following which, the cells were collected through trypsinization.

To deliver siRNAs to target cells, the siRNAs were coated with a nanoparticle solution before topical administration on the burn wounds. The most effective siRNA:nanoparticle solution ratio was 150 nM B1 siRNA in 100 µl CA-P nanoparticle solution, based on the results of previous studies (15,47,48).

First, B1 siRNA was mixed with an appropriate proportion and concentration of calcium solution (MilliporeSigma), a B1 siRNA-binding reagent. Next, the B1 siRNA-calcium complex was added to a mixture of sodium carbonate (Na₂CO₃; MilliporeSigma) and sodium dihydrogen phosphate monohydrate (NaH₂PO₄·H₂O; MilliporeSigma). After the B1 siRNA-coating process, the nanoparticle-coated B1 siRNAs were stored at room temperature (28-30˚C) and prepared on the day of use. The nanoparticle solution was composed 50 µl of a mixture containing 0.5 M calcium chloride (CaCl₂) solution (MilliporeSigma) and 150 nM B1 siRNA + 50 µl of a mixture of 0.01 M Na₂CO₃ solution (MilliporeSigma) and 0.01 M NaH₂PO₄·H₂O solution (MilliporeSigma). A 31:1 molar ratio of CO₃^2-:PO₄^3- was used. The B1 siRNA was mixed with 16 µl 0.5 M CaCl₂ solution and adjusted to a final volume of 50 µl using sterile dH₂O. Thereafter, the B1 siRNA-calcium complex was added to 50 µl of a mixture of Na₂CO₃ solution and NaH₂PO₄·H₂O solution (16 µl) and 34 µl sterile dH₂O (47). All steps in the preparation of the nanoparticle solution were performed using sterile techniques. Each nanoparticle solution preparation was used to validate the transfection efficiency with rat dermal keratinocytes (Cell Applications) before each experiment (48-52).

Formalin-fixed wound tissues were dehydrated and embedded in paraffin, after which, 3-µm-thick sections were prepared using a microtome. Hematoxylin and eosin (H&E) staining was performed for scoring, using hematoxylin solution for 6 h and eosin Y ethanol solution for 48 h at a temperature of 60˚C. Immunohistochemical staining of the tissue sections was performed using monoclonal antibodies against γH2AX (1:100; cat. no. Ab2893; Abcam) and 8-OHdG (1:100; cat. no. Ab48508; Abcam) (53). The sections and antibodies were incubated at 37˚C for 32 min using the Ventana^® Benchmark XT (Ventana Medical Systems, Inc; Roche Diagnostics) automated slide strainer in combination with the Ventana ultraView DAB v3 IHC Detection Kit before mounting, according to the manufacturer's protocol. Thereafter, the slides were counterstained at 25˚C with Hematoxylin II for 8 min and Bluing Reagent for 4 min (53).

The histological sections of the wounds were graded using a histopathological scoring system, following a previously published method with some modifications (54-58) Three dermatopathologists who were blinded to the treatment regimen independently performed H&E scoring, based on five criteria: Epithelialization, polymorphonuclear leukocyte (PMNL) infiltration, collagen formation, number of fibroblasts and presence of new blood vessels. A score of 0 was assigned when there was no evident epithelialization and no increase in the number of fibroblasts, PMNLs or newly formed blood vessels. A score of 1 indicated an increased thickness of the edges of the cut epithelial tissue sections, or the presence of a few fibroblasts, PMNLs and newly formed blood vessels. A score of 2 indicated migration of epithelial cells, or the presence of a moderate number of fibroblasts, PMNLs and newly formed blood vessels. A score of 3 indicated epithelial bridging of the incision, or the presence of several fibroblasts, PMNLs and newly formed blood vessels. A score of 4 was assigned for sections where there was complete regeneration of the epithelium, or presence of excessive fibroblasts, PMNLs and newly formed blood vessels. The mean scores of the three scorers were determined and combined to obtain an overall pathology score. Sections positive for anti-cytokeratin immunohistochemical staining were scored in a similar manner; three dermatopathologists scoring the sections on a scale of 0-4, starting from no staining to strong staining.

DNA extraction from rat skin was performed using the phenol-chloroform method, which is the most commonly used method to purify and concentrate DNA (59), followed by the addition of phenol-chloroform to a solution of lysed cells, and mixing and separation of the two phases by centrifugation for 5 min at 16,000 x g. The resulting solution was separated into two phases: A lower organic phase and an upper aqueous phase. The top aqueous phase containing DNA was then carefully removed. A total of 1 µl 20 mg/ml Glycogen solution, 0.1X 3 M sodium acetate and 2.5X volume of absolute ethanol (100%) were added to the precipitated DNA, and the mixture was centrifuged for 1 min at 8,000 x g at 25˚C. Finally, the DNA was washed with 70% ethanol, air-dried and dissolved in dH₂O (59).

A total of 1 µl bisulfited DNA taken from an EZ DNA methylation-Gold^TM kit (Zymo Research Corp.) was subjected to 45 cycles of PCR, using the PCR mastermix^TM (Thermo Scientific, Inc.). The following thermocycling conditions were used: 95˚C for 15 min; followed by 40 cycles at 95˚C for 45 sec, 53˚C for 45 sec and 72˚C for 45 sec; with a final extension step of 72˚C for 10 min using the B1 COBRA forward primer, 5'-YGYAYGYYTTTAATYYYAGYAAT-3' and reverse primer, 5'-CCCTRRCTRTCCTRRAACTCAC-3', which were designed using Primer 3 version 4.1.0 (2,60), and had an annealing temperature of 70˚C. Primer sequences with ‘Y’ represent pyrimidine bases (C or T) that have the ability to bind to G or A bases, and ‘R’ represents purine bases (A or G) that have the ability to bind to T or C bases in DNA sequences following bisulfite conversion (Fig. 2C). The amplified PCR products were digested with two units of Taq1 and Tas1 endonuclease in NE buffer II (MBI Fermentas; Thermo Fisher Scientific, Inc.) at 37˚C overnight. The digested PCR products were identified using 8% non-denaturing polyacrylamide gel electrophoresis and stained with SYBR Green JumpStart Taq ReadyMix (Sigma-Aldrich; Merck KGaA). All samples were assessed in duplicate. After enzyme digestion, six products of different lengths were detected after COBRA B1 (98, 78, 54, 44, 34 and 20 bp). The band intensities of the COBRA B1 products were determined using Image Quant version 8.2 (Molecular Dynamics; GE Healthcare).

According to the B1 analysis and calculations, two types of COBRA B1 products could be classified based on the methylation status of the CpG dinucleotides, namely methylated loci (mC) and unmethylated loci (uC). The percentage of B1 methylation was calculated as follows. The letters demonstrated the normalized scores of each band, and the percentage of intensity of each band was divided by the amplicon length in terms of bp: %98/98=A, %78/74=B, %54/54=C, %44/42=D, %34/34=E, and %20/20=F. Subsequently, the percentage of methylation was calculated by bring the normalized scores of the digested methylation fragment divided by the sum of the normalized scores of the undigested and digested products as following formula: % total methylation (%mC)=100 x (A + C + D)/2A + 2B + 2D. As an internal control, 25% methylated rat genomic DNA (EpigenDX) was used for the experiments and inter-assay adjustment.

Distribution of data were determined using a Shapiro-Wilk test. For normally distributed data, a one-way ANOVA followed by Bonferroni post-hoc corrections were used to compare the groups. For non-normally distributed data, a Kruskal-Wallis analysis followed by a Dunn's post-hoc test was used to test the differences between groups. Data are reported as the mean ± standard deviation. GraphPad Prism version 9.0.0 (GraphPad Software, Inc.) was used for all statistical analyses. P<0.05 was considered to indicate a statistically significant difference.

Second-degree burn wounds were treated each day with 150 nM B1 siRNA and the wound areas were measured using photographs taken on days 0, 7, 14, 21 and 28 post-injury (Fig. 2A). The improvements in wound healing were greater in the B1 siRNA group. The respective percentage wound healing ± standard deviation in the groups treated with saline or Ca-P nanoparticles, relative to that in the B1 siRNA-treated group, on different days was as follows: i) Day 7, 15.81±3.59 and 15.66±2.36 vs. 20.01±1.97%, both P<0.01; ii) day 14, 56.58±1.24 and 56.73±1.56 vs. 60.38±1.39%, both P<0.001; iii) day 21, 73.14±2.27 and 73.39±2.62 vs. 77.15±1.32%, both P<0.01; and iv) day 28, 90.63±1.39 and 90.81±0.86 vs. 97.21±0.84%, both P<0.001. There were no differences in wound areas between the NSS-treated control and Ca-P nanoparticle groups. Furthermore, significant differences were observed amongst the control and Ca-P nanoparticle compared with the B1 siRNA treatment group, particularly on days 14 and 28 post-injury (Fig. 2B).

B1 siRNA promotes methylation through siRNA directed DNA methylation (21,23); it improves DNA stability and enhances cell proliferation (15). The methylation levels of the B1 siRNA-treated group were significantly higher than those of the saline-treated control and Ca-P nanoparticle groups, especially on days 7 and 14. The respective percentage methylation ± standard deviation in the NSS control, Ca-P nanoparticle and B1 siRNA-treated wounds on different days were: i) Day 3, 30.33±1.52 and 29.0±1.0 vs. 33.00±1.0%, both P<0.05; ii) day 7, 30.0±2.0 and 31.0±2.0 vs. 35.0±1.0%, both P<0.001; iii) day 14, 33.67±1.52 and 33.0±1.73 vs. 45.0±1.0%, both P<0.001; iv) day 21, 33.67±2.08 and 34.67±0.58 vs. 41.0±1.0%, both P<0.001; and v) day 28, 34.33±0.58 and 34.33±0.58 vs. 38.00±1.00%, both P<0.05 (Fig. 2D). Significant differences were observed in the B1 siRNA treated group compared with the control groups 3 days post-injury.

Individual histopathological scores were added together to provide an overall score for each wound. Blinded scoring of the wounds by the three dermatopathologists confirmed that the rats treated with B1 siRNA displayed considerably higher pathological scores than those treated with NSS or Ca-P nanoparticles, with the greatest improvement observed on days 14, 21 and 28 post injury. The pathological scores ± standard deviation in the NSS and Ca-P nanoparticle-treated groups (respectively), relative to that in the B1 siRNA-treated group, on different days were as follows: i) Day 3, 1.33±0.58 and 1.67±0.58 vs. 2.67±0.58%, both P>0.05; ii) day 7, 7.67±1.16 and 7.67±0.58 vs. 7.0±1.0%, both P>0.05; iii) day 14, 12.33±0.58 and 11.33±1.16 vs. 15.67±0.58%, both P<0.01; iv) day 21, 13.33±0.58 and 12.33±1.52 vs. 16.0±0.0%, P<0.05; and v) day 28, 15.0±1.0 and 14.33±0.58 vs. 16.67±1.53%, P<0.05 (Fig. 3A and B).

8-OHdG and γH2AX scores were determined to measure DNA damage response after introducing burn injuries. A low immunohistochemistry score for 8-OHdG and γH2AX reflected a low DNA damage response. The B1 siRNA-treated group exhibited lower levels of 8-OHdG than the NSS and Ca-P nanoparticle groups on days 14 and 21. In addition, the wounds in the B1 siRNA-treated group showed significantly lower γH2AX levels from days 7 to 21 than the NSS- and Ca-P nanoparticle-treated groups, with the highest level observed 14 days post-injury. The respective 8-OHdG immunohistochemistry score ± standard deviation in the NSS and Ca-P nanoparticle-treated groups (respectively), relative to that in the B1 siRNA-treated group was as follows: i) Day 3; 4.0±0.0 and 4.0±0.0 vs. 4.0±0.0%, both P>0.05; ii) day 7; 4.0±0.0 and 3.67±0.58 vs. 3.33±0.58%, both P>0.05; iii) day 14, 3.0±0.0 and 3.33±0.58 vs. 2.67±0.58%; both P<0.01; iv) day 21, 3.0±0.0 and 2.67±0.58 vs. 2.0%±0.0%, both P<0.05; and v) day 28, 2.0±0.0 and 2.0±0.0 vs. 1.67±0.58%, P<0.05 (Fig. 3C and D). Furthermore, the γH2AX immunohistochemistry scores ± standard deviation in the NSS and Ca-P nanoparticle groups, relative to that in the B1 siRNA-treated group, was as follows: i) day 3, 4.0±0.0 and 4.0±0.0 vs. 4.0±0.0%, both P>0.05, ii) day 7, 3.67±0.58 and 4.0±0.0 vs. 3.33±0.58%, both P>0.05, iii) day 14, 3.0±0.0 and 3.33±0.58 vs. 2.67±0.58%, both P<0.05, iv) day 21, 2.67±0.58 and 2.67±0.58 vs. 2.0±0.0%, both P<0.05; and v) day 28, 2.0±0.0 and 2.0±0.0 vs. 1.67±0.58%; both P>0.05 (Fig. 3E and F).