Animals and experimental groups

A total of 66 age-matched (10-week-old) male rats from Wistar and SHR strains (Rattus norvegicus albinus) from the Central Animal Facility of School of Dentistry of Araçatuba (Unesp) were used. Animals were housed in a controlled light, temperature, and humidity environment (12h light/dark cycle, 22±2°C, and 55±5%), and offered a standard pellet diet and drinking water ad libitum. Animals from each strain (Wistar, W; and SHR, S) were randomly divided into the following experimental groups: Control (C), animals with PD (PD), and mast cell-depleted animals with PD (48/80+PD). Additionally, for a mast-cell depleted control group (without PD; W+48/80 and S+48/80) six male Wistar and six male SHR without PD were used. Experimental protocols were in accordance with Brazil’s National Council for Animal Experiments Control and were approved by the Ethics Committee on Animal Use from the School of Dentistry of Araçatuba (Unesp) (Process FOA-00686-2016).

Mast cells depletion

Mast cells depletion was conducted by the mast cell degranulation-inducing drug, compound 48/80 (c48/80; Sigma-Aldrich; St. Louis, Missouri, USA) [21, 22]. Briefly, c48/80 was administered intraperitoneally twice a day for four days, with increasing doses (day 1: 0.6; day 2 and 3: 1.2; and day 4: 2.4 mg/Kg), so that on day 5, periodontal disease was induced in the absence of filled-vesicles mast cells. Non-treated animals only received the vehicle (sterile phosphate-buffered saline, PBS).

Periodontal disease (PD) induction

PD was induced by bilateral ligature inserted around the lower first molars and kept for 15 days, as previously described [23]. Briefly, animals were anesthetized (ketamine and xylazine hydrochloride association, 80 and 10 mg/Kg) and placed in ventral decubitus on a dental table for rodents, with oral retractors supported on incisive teeth. A 4–0 silk thread (Shalon; Goiânia, Goiás, Brazil) was wrapped around the first inferior molars, carefully pushed into the gingival sulcus, and knotted medially.

Non-invasive blood pressure measurement

To confirm the hypertensive phenotype systolic blood pressure (SBP) was verified by tail plethysmography (NIBP and PowerLab System; ADInstruments; Sydney, Australia) after the 15 days of PD, pre-treated or not with c48/80, and animals were considered hypertensive when SBP ≥150 mmHg [24].

Euthanasia and sample harvest

On day 15 after PD induction, animals were euthanized by inhalational anesthetic overdose (Isoflurane, Cristália, Itapina, SP, Brazil), the presence of bilateral ligature was evaluated, and animals in which it was absent were excluded from the study. The hemi-mandibles and gingival tissue were surgically collected, and the right-sided bones were stored in PBS at -20°C for microtomography, or fixed in 10% buffered formaldehyde for histological processing, and left-sided bones were immediately frozen in liquid nitrogen and stored at -80°C, for gene expression analysis and cytokines quantitation.

Gingival mast cell count

Formaldehyde fixed gingiva was paraffin-embedded and 3-μm thick sagittal sections were obtained and used for toluidine blue staining. For mast cell counting, representative slides were photographed, the connective gingival tissue area was determined, and the mast cell number (purpled-stained cells) were counted (n = 5/group), using the ImageJ software (v1.47, National Institutes of Health).

Micro-computed tomography (microCT) analysis

Tomographic images from left hemi-mandibles (n = 5/group) were acquired using a SkyScan 1272 system (Bruker microCT; Kontich, Belgium) [70 kVp and 142 μA; 0.5 mm aluminum filter; 9 μm isotropic voxel; 1100 ms exposure time, 2 frames averaging, and 180° rotation (0.5° rotation step)], and three-dimensionally reconstructed (NRecon software; v1.6; Bruker microCT). Alveolar bone loss was evaluated in the first molar region, a region of interest (ROI) was standardized from defined anatomical points (upper limit: furcation roof; lower limit: proximal root apex; distal limit: 2nd molar proximal root; proximal limit: 1st molar proximal root, and vestibular and lingual limits: limits of the alveolar bone). A volume of interest (VOI) was automatically delimited by the bone edges and the tooth volume exclusion. The bone percentage (BV/TV), trabecular number (Tb.N), thickness (Tb.Th), and separation (Tb.Sp) were analyzed [25].

Cytokine quantification

The left hemi-mandibles (n = 6/group) were sectioned from the mandibular branch and incisor teeth, powdered in liquid nitrogen, and subsequently homogenized with a lysis buffer (100 mM Tris-HCl, 150 mM NaCl, 1% Tween 20, 0.5% sodium deoxycholate, 1 mM EDTA, pH 7.4). The samples were centrifuged and in the recovered supernatant, TNF-α, IL-6, IL-1β, CXCL3/CINC-2, and CCL20/MIP-3α were quantified by enzyme-linked immunosorbent assay (ELISA), with DuoSet® ELISA (in order: DY510, DY506, DY501, DY540, and DY516; R&D Systems, Minneapolis, MN, USA). Results were normalized by the total protein content, determined by the Lowry method [26].

Gene expression analysis

Left hemi-mandibles (n = 6/group) were sectioned, excluding the mandibular branch and the incisor tooth, powdered in liquid nitrogen, and total RNA was extracted using TRIzol reagent (Invitrogen, Thermo Fisher Scientific; Carlsbad, CA, USA) following the manufacturer’s instructions. RNA purity was assessed by 260/280 and 260/230 spectrophotometry ratio (satisfactory between 1.8–2.0, and 2.0–2.2, respectively). Samples were treated with DNAse I (Sigma-Aldrich), RNA was quantified (Quant-iT RiboGreen RNA Assay Kit, Invitrogen) and 2 μg of total RNA were reverse transcribed to complementary DNA (High Capacity RNA-to-cDNA™ Kit; Applied Biosystems, ThemoFisher Scientific; Foster City, California, USA), according to manufacturer’s instructions.

Gene expression analysis of bone markers and cytokines was performed by qRT-PCR, in StepOne PlusTM Real-Time PCR Systems, with TaqMan^TM Gene Expression Assays (FAM fluorophore reporter / non-fluorescent quencher MGB) (Applied Biosystems, Thermo Fisher Scientific). Targets expression was normalized by Actb expression, as the housekeeping gene, and relative transcripts abundance was determined by the 2^{(-delta Ct)} method [27]. Assay references are listed in Table 1.

Table 1

Taqman® assay reference list for qRT-PCR.

Transcription factors
Runx2	Runt-related transcription factor 2	Rn01512298_m1
Osx/Sp7	Osterix/Sp7 transcription factor	Rn02769744_s1
Ctnnb	β-catenin/Cadherin associated protein beta 1	Rn00584431_g1
Bone formation markers
Alp	Bone alkaline phosphatase	Rn01516028_m1
Col1a1	Collagen type I alpha 1	Rn01463848_m1
Opn/Spp1	Osteopontin/secreted phosphoprotein 1	Rn00681031_m1
Ocn/Bglap	Osteocalcin/Bone gamma-carboxyglutamate protein	Rn00566386_g1
Bsp/Ibsp	Bone sialoprotein/Integrin-binding sialoprotein	Rn00561414_m1
Bmp2	Bone morphogenetic protein 2	Rn00567818_m1
Bone formation/remodeling markers
Opg	Tumor necrosis factor receptor superfamily member 11b	Rn00563499_m1
Rankl	Tumor necrosis factor ligand superfamily member 11	Rn00589289_m1
Rank	Tumor necrosis factor receptor superfamily member 11a	Rn04340164_m1
Oscar	Osteoclast associated immunoglobulin-like receptor	Rn01530958_m1
Vtn	Vitronectin	Rn01466920_g1
Itgav	Integrin, alpha V	Rn01485633_m1
Itgb5	Integrin, beta 5	Rn01439348_m1
Mmp2	Matrix metalloproteinase 2	Rn01538170_m1
Mmp9	Matrix metalloproteinase 9	Rn00579162_m1
Trap/Acp5	Acid phosphatase 5, tartrate resistant	Rn00569608_m1
Ctsk	Cathepsin K	Rn00580723_m1
Cytokines
IL-1β	Interleukin-1β	Rn00580432_m1
TNF-α	Tumor necrosis factor	Rn01525859_g1
IL-6	Interleukin-6	Rn01410330_m1
Housekeeping gene
Actb	β-actin	Rn00667869_m1

Immunohistochemistry assays

Formaldehyde fixed mandibles (n = 5/group) were decalcified in 10% EDTA-buffered solution (Titriplex® III; Merck Millipore; Burlington, MA, USA), for paraffin histological processing. Mandibles 3-μm thick sagittal sections were obtained and used for hematoxylin and eosin staining, and immunostaining of bone markers. Briefly, tissue sections were deparaffinized, rehydrated, and submitted to endogenous peroxidase blocking, and hot citric acid buffer antigen retrieval. immunostaining was performed by primary antibody incubation (references listed in Table 2) and detected by Histofine® Simple Stain™ kit (Nichirei Biosciences Inc.; Tokyo, Japan), followed by the chromogenic substrate (3,3’-diaminobenzidine-tetrahydrochloride; Dako Corp., Carpinteria, CA, USA), according to manufactures instruction. Harry’s hematoxylin was used as counterstaining, and all assays were accompanied by a negative control slide which was submitted to the same procedures, except for the primary antibody incubation.

Table 2

Antibody reference list for immunohistochemistry assays.

Primary antibody	Source	Reference	Manufacturer
Anti-RUNX2 (F-2)	Mouse	sc-390351	Santa Cruz Biotechnology.
Anti-OSTERIX (Y-21)	Rabbit	sc-133871	Santa Cruz Biotechnology
Anti-CTNNB (12F7)	Mouse	sc-59737	Santa Cruz Biotechnology
Anti-COL1A1 (L-19)	Goat	sc-8783	Santa Cruz Biotechnology
Anti-OPN	Rabbit	ab8448	Abcam
Anti-OPG (N-20)	Goat	sc-8468	Santa Cruz Biotechnology
Anti-RANKL (N-19)	Goat	sc-7628	Santa Cruz Biotechnology
Anti-RANK (H-300)	Rabbit	sc-9072	Santa Cruz Biotechnology
Anti-VTN (EP873Y)	Rabbit	ab45139	Abcam
Anti-ITGαVβ3 (23C6)	Mouse	sc-7312	Santa Cruz Biotechnology
Anti-MMP2 (2C1)	Goat	sc-13594	Santa Cruz Biotechnology
Anti-MMP9 (C-28)	Goat	sc-6841	Santa Cruz Biotechnology
Anti-TRAP (N-17)	Goat	sc-30832	Santa Cruz Biotechnology
Anti-CTSK (N-20)	Goat	sc-6507	Santa Cruz Biotechnology
Anti-BMP2	Rabbit	ab1493	Abcam
Anti-OCN (FL-95)	Rabbit	sc-30045	Santa Cruz Biotechnology
Anti-BSP (M-154)	Rabbit	sc-292394	Santa Cruz Biotechnology

For IHC analysis, slides were blindly analyzed (n = 5/group) in 400X magnification bright-field microscopy in the region of interest (middle third of the first molar furcation region), and expression was based on the immunostaining pattern [negative (−); low staining (+); moderate staining (++); strong staining (+++)] [28]. Image boards present a photographed representative section, after digital correction for brightness and contrast, and black arrows indicate positive-stained cells.

Statistical analysis

Data are expressed as mean and standard error of the mean (SEM), and were analyzed by one-way ANOVA, followed by Šidak post hoc test for multiple comparisons, after being tested for normality distribution by Shapiro-Wilk test. Statistical difference is represented by brackets labelled by * p<0.05, ** p<0.01, *** p<0.001, and **** p<0.0001, comparing Control, PD and 48/80+PD, or Wistar and SHR in the same experimental condition. All analysis was performed on Graph Pad Prism v8.0 (GraphPad Software Inc.; San Diego, California, USA).

Previous mast cells depletion does not alter blood pressure after PD

To ensure the SHR phenotype, we evaluated the systolic blood pressure (SBP) after 15 days of PD. As expected, all SHR groups presented elevated SBP, compared to W groups (Table 3), but previous mast cell depletion alone or associated with PD did not alter this parameter.

Table 3

Systolic Blood Pressure (SBP) of Wistar and SHR with PD, depleted from mast cells (48/80+PD).

	SBP
	Mean (mmHg)	SEM	n
WC	102,40a	3,20	6
W+48/80 (without PD)	104,70 a	4,08	6
WPD	116,30 a	5,03	6
W48/80+PD	109,80 a	3,31	6
SC	162,30 b	2,58	6
S+48/80(without PD)	159,50 b	3,95	6
SPD	157,90 b	3,20	6
S48/80+PD	157,80b	1,18	6

SBP measured by tail plethysmography are shown as mean and SEM (n = 6). Significant statistical differences are represented by a different superscript letter (^a and ^b; p<0.001).

Mast cells are more abundant in SHR inflamed gingival tissue

The presence of MCs in the gingival tissue was verified by toluidine blue staining (Fig 1A and 1B). Few MCs were observed in the control groups (WC and SC). However, they were more abundant in groups with PD, especially in the SPD group, suggesting participation in PD inflammatory responses. As expected, MCs were less abundant in 48/80+PD groups, thus validating the previous MCs depletion in our model.

Fig 1

Mast cell count in the gingival tissue of Wistar and SHR with PD, depleted from mast cells (48/80+PD).

(A) Representative images of toluidine blue stained gingival section. The black arrows indicate mast cells (purple-stained cells). (B) Graph shows mean ± SEM (n = 5) of mast cell count per mm², and statistical differences are represented by brackets labeled by *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. Microtomography analysis of mandible of Wistar and SHR with PD, depleted from mast cells (48/80+PD). (C) Mandible three-dimensional reconstruction in lingual view with sagittal cut detail, (D) Volume rendering of the analyzed region (first molar teeth in white and surrounding alveolar bone in brown), and graphs showing (E) %BV/TV, (F) Tb.Nb, (G) Tb.Th, and (H) Tb.Sp as mean ± SEM (n = 5). Statistical differences are represented by brackets labeled by * p<0.05, ** p<0.01, *** p<0.001, and **** p<0.0001.

Mast cells depletion prevented PD-induced alveolar bone loss in SHR

We analyzed the alveolar bone loss and bone architecture parameter by microCT (Fig 1C–1H). As expected, WPD and SPD groups showed significant bone loss with lower %BV/TV and Tb.Th, compared to their respective controls (Fig 1E and 1G), while Tb.Nb and Tb.Sp were not significantly altered (Fig 1F and 1H). Although WC and SC had similar alveolar bone structures (Fig 1C–1E), bone loss was more severe in SPD, as compared to WPD. Interestingly, MCs depletion was able to prevent this loss, more significantly in S48/80+PD, but the trabecular architecture was not significantly altered (Fig 1F–1H). We also evaluated if previous MC depletion alone would alternate the alveolar bone loss, which was not noticed (S1 Fig).

Mast cells depletion reduced PD-induced inflammation in SHR

To assess the inflammatory process, we evaluated the gene expression of Tnfa, Il6, and Il1b by qRT-PCR, and the production of TNF-α, IL-6, IL-1β, CXCL3/CINC-2, and CCL20/MIP-3α by ELISA in the rat mandibles (Fig 2A–2H). Tnfa, Il6, and Il1b expression were higher in PD groups, compared to their respective controls (Fig 2A–2C). MCs depletion only reduced the expression of Il6 and Il1b in S48/80+PD group, compared to SPD (Fig 2B and 2C). When quantified at the protein level, TNF-α, IL-6, and IL-1β were also increased in WPD and SPD compared to their controls, and were significantly higher in SPD, compared to WPD (Fig 2D–2F). In S48/80+PD, all mediators were reduced, compared to SPD, while in W48/80+PD only IL-6 was reduced, compared to WC (Fig 2D–2F).

Fig 2

Inflammatory status in mandibles of Wistar and SHR with PD 15d, depleted of mast cells (48/80+PD).

qRT-PCR for (A) Tnfa, (B) Il6, and (C) Il1b, and ELISA for (D) TNF-α, (E) IL-6, (F) IL-1β, (G) CXCL3 and (H) CCL20. Graphs shown mean ± SEM (n = 6), and statistical difference are represented by brackets labeled by * p<0.05, ** p<0.01, *** p<0.001, and **** p<0.0001. (I-N) H&E staining of the first mandibular molar, in the furcation region, image board shows representative images from each experimental group (n = 5).

The chemotactic proteins CXCL3/CINC-2 and CCL20/MIP-3α were also quantified, which are associated with neutrophil and macrophage infiltrate, respectively. CCL20 was not altered in PD, but had inherently higher levels in SHR groups, compared to Wistar groups (Fig 2H). CXCL3, however, was significantly increased in PD groups but was partially prevented in 48/80+PD groups.

H&E staining corroborated and further demonstrated the periodontal inflammation response (Fig 2I–2N). Histological observation showed increased leukocyte infiltration and periodontal ligament disorganization to be more severe in SPD (Fig 2M), compared to WPD (Fig 2J), which was prevented in 48/80+PD groups (Fig 2K and 2N).

Mast cells depletion reduced Ctnnb expression

To study the mechanisms modulated by MCs in the PD-associated alveolar bone loss, we analyzed the expression of the main bone markers in the rat mandibles by qRT-PCR and immunostaining. The transcription factors Runx2, Osx, and Ctnnb were initially analyzed (Fig 3A–3C). SC had a lower Runx2 expression compared to WC (Fig 3A), while Osx and Ctnnb expression were similar in both groups (Fig 3B and 3C). PD led to an increased Ctnnb expression in SPD only. However, MCs depletion was able to significantly reduce its expression in WPD and S48/80+PD (Fig 3C). Immunostaining confirmed this response (Fig 3F) as CTNNB showed no alteration in WPD compared to WC, but weaker staining in the W48/80+PD, while it was increased in SPD as compared to SC, which was partially prevented in S48/80+PD (Fig 3D). RUNX2 and OSX labeling did not show significant differences between any groups (Fig 3D and 3E).

Fig 3

Transcription factors expression in mandibles of Wistar and SHR with PD, depleted of mast cells (48/80+PD).

qRT-PCR for (A) Runx2, (B) Osterix, (C) Catnb, (D) immunostaining for (D) RUNX2, (E) OSTERIX, and (F) CTNNB. Graphs shown mean ± SEM (n = 6), and statistical difference are represented by brackets labeled by * p<0.05, ** p<0.01, *** p<0.001, and **** p<0.0001. The image boards show representative images from each experimental group (n = 5), and tables describe the average immunostained pattern of each target.

Mast cells modulate Opn expression in the alveolar bone

We then analyzed the expression of bone formation markers Alp, Bmp2, Col1a1, Opn, Ocn, and Bsp (Fig 4A–4F). We observed a significant decrease in Alp expression in PD groups compared to control groups, but MCs depletion did not modulate this response (Fig 4A). Bmp2, Col1a1, Ocn, and Bsp were not altered in PD (Fig 4B, 4C and 4F). However, the S48/80+PD group showed a decreased expression of Bmp2 and Col1a1 (Fig 4B and 4C). IHC analysis did not show significant differences in the BMP2 staining (S2 Fig), however, COL1A1 staining was increased in PD groups, but only reduced in S48/80+PD, compared to SPD (Fig 4G).

Fig 4

Bone formation markers expression in mandibles of Wistar and SHR with PD, depleted of mast cells (48/80+PD).

qRT-PCR for (A) Alp, (B) Bmp2, (C) Col1a1, (D) Opn, (E) Ocn, and (F) Bsp, and immunostaining for (G) COL1A1, and (H) OPN. Graphs shown mean ± SEM (n = 6), and statistical difference are represented by brackets labeled by * p<0.05, ** p<0.01, *** p<0.001, and **** p<0.0001. The image boards show representative images from each experimental group (n = 5), and tables describe the average immunostained pattern of each target.

SC showed an inherently lower Opn expression compared to WC, but PD led to an increased expression in WPD and SPD, compared to the respective controls (Fig 4D). Interestingly, MCs depletion significantly reduced Opn expression in WPD and S48/80+PD (Fig 4D). IHC analysis confirmed the OPN response, by and increased staining in WPD compared to WC, mostly seen in the bone adjacent connective tissue, while it was reduced in W48/80+PD in both the connective tissue and bone cells (Fig 4H). SC presented a constitutive higher OPN staining, which was further increased in bone cells in the SPD group, but reduced in S48/80+PD (Fig 4H).

Mast cells depletion prevented altered Opg/Rankl/Rank axis expression

We also evaluated the Opg/Rankl/Rank axis expression in rat mandibles (Fig 5), which plays a major role in bone remodeling and resorption. We noticed a constitutive lower Opg expression in SC compared to WC, but Rankl and Rank expression was similar (Fig 5A–5C). In Wistar rats, WPD showed a reduced Opg and increased Rankl and Rank expression. MCs depletion further decreased Opg expression and prevented increases in Rankl and Rank expression (Fig 5A–5C). Conversely in SHR, SPD groups had increased expression of all three axis components, which were reduced in the MCs-depleted group (Fig 5A–5C).

Fig 5

Opg, Rankl and Rank axis in mandibles of Wistar and SHR with PD, depleted of mast cells (48/80+PD).

qRT-PCR for (A) Opg, (B) Rankl, (C) Rank, and immunostaining for (D) OPG, (E) RANKL, and (F) RANK. Graphs shown mean ± SEM (n = 6), and statistical difference are represented by brackets labeled by * p<0.05, ** p<0.01, *** p<0.001, and **** p<0.0001. The image boards show representative images from each experimental group (n = 5), and tables describe the average immunostained pattern of each target.

Immunostaining was performed to better observe this response (Fig 5D–5F). WPD showed weaker OPG staining in bone cells compared to WC, which was partially prevented in the W48/80+PD (Fig 5D). Interestingly, SC had constitutive higher OPG staining in the bone adjacent connective tissue, also observed in the SPD, but with weaker OPG staining in the bone cells (Fig 5D), and average weaker staining was observed in S48/80+PD (Fig 5D). RANKL, in turn, had a weak immunostaining pattern. However, increased staining was noticed in the PD group bone cells and adjacent connective tissues, which was slightly reduced in 48/80+PD groups (Fig 5E). Interestingly, RANK had an average intenser staining in SHR groups. In the PD groups, an increased staining was observed compared to the respective control groups, which was partially prevented in the 48/80+PD groups (Fig 5F).

Mast cells depletion prevented an increase in resorption markers

Finally, we evaluated the markers associated with bone resorption, including markers of osteoclast recruitment and activation, namely Oscar, Vtn, Itga5, and Itgb5 (Fig 6A–6F); organic matrix degradation, Mmp2 and Mmp9 (Fig 7A and 7D); and osteoclast function Trap and Ctsk (Fig 8A–8D).

Fig 6

Osteoclast recruitment and activation markers expression in mandibles of Wistar and SHR with PD, depleted of mast cells (48/80+PD).

qRT-PCR for (A) Oscar, (B) Vtn, (C) Itga5, (D) Itgb5, and immunostaining for (E) VTN, and (F) ITGαVβ3. Graphs shown mean ± SEM (n = 6), and statistical difference are represented by brackets labeled by * p<0.05, ** p<0.01, *** p<0.001, and **** p<0.0001. The image boards show representative images from each experimental group (n = 5), and tables describe the average immunostained pattern of each target.

Fig 7

Organic matrix degradation markers expression in mandibles of Wistar and SHR with PD, depleted of mast cells (48/80+PD).

qRT-PCR for (A) Mmp2 and (B) Mmp9, and immunostaining for (C) MMP2, and (D) MMP9. Graphs shown mean ± SEM (n = 6), and statistical difference are represented by brackets labeled by * p<0.05, ** p<0.01, *** p<0.001, and **** p<0.0001. The image boards show representative images from each experimental group (n = 5), and tables describe the average immunostained pattern of each target.

Fig 8

Osteoclast function markers expression in mandibles of Wistar and SHR with PD, depleted of mast cells (48/80+PD).

qRT-PCR for (A) Trap and (B) Ctsk, and immunostaining for (C) TRAP, and (D) CTSK. Graphs shown mean ± SEM (n = 6), and statistical difference are represented by brackets labeled by * p<0.05, ** p<0.01, *** p<0.001, and **** p<0.0001. The image boards show representative images from each experimental group (n = 5), and tables describe the average immunostained pattern of each target.

The results showed SC to have a higher constitutive expression of Oscar, Mmp9, Trap, and Ctsk, compared to WC (Figs 6A, 7A, 8A and 8B). In WPD, there was an increased expression of all analyzed markers, compared to WC, except for Vtn (Fig 6B). We observed a reduced Vtn, Itga5, Itgb5, Mmp2, and Trap expression in W48/80+PD, compared to WPD (Figs 6B–6D and 7A). SHR showed a slightly different response, where PD increased the expression of all analyzed targets, except for Mmp2 (Fig 7A), while in S48/80+PD there was a reduced expression of Vtn, Itga5, Itgb5, Trap, and Ctsk (Figs 6B–6D, 8A and 8B).

To further confirm these tissue responses in the previous MCs-depleted animals, we evaluated the immunostaining of the main altered markers. Immunostaining confirmed the response patter of VTN and ITG, showing an increased staining in the PD groups, while reduced in 48/80+PD. However, ITG staining alterations were concentrated in the bone cells, while VTN was equally distributed in bone cells and the adjacent connective tissue (Fig 6E and 6F).

MMP2 staining patterns alteration were more evident in the bone cells, while for MMP9 it was in the adjacent connective tissue. Different from gene expression analysis, MMP2 showed to be slightly higher in SC, compared to WC, while MMP9 showed a similar staining intensity between the control groups. Interestingly, IHC confirmed a MMP2 reduction in W48/80+PD, but MMP9 showed no significant changes in the 48/80+DP (Fig 7C and 7D).

Regarding the osteoclast functional markers, constitutive differences between WC and SC were not as apparent as in the gene expression of those markers. However, as expected, PD groups had more abundant TRAP- and CTSK-positive cells, especially in SPD, compared to SC and WPD. The response was prevented in W48/80+PD and S48/80+PD, which showed less stained cells compared to PD groups (Fig 8C and 8D).