Mitigation of BMP-induced Inflammation in Craniofacial Bone Regeneration and Improvement of Bone Parameters by Dietary Hesperidin

Based on anti-inflammatory and osteogenic properties of hesperidin (HE), we hypothesized its systemic administration could be a cost-effective method of improving BMP-induced bone regeneration. Sprague-Dawley rats were allocated into 4 groups (n=10/group): a 5-mm critical-sized mandible defect + collagen scaffold or, scaffold + 1 μg of BMP2 with and without dietary HE at 100 mg/kg. HE was administered by oral gavage 4 weeks prior to surgeries until euthanasia at day 7 or 14. The healing tissue within the defect collected at day 7 was subjected to gene expression analysis. Mandibles harvested at day 14 were subjected to microcomputed tomography and histology. HE+BMP2-treated rats had a statistically significant decrease in expression of inflammatory genes compared to BMP2 alone. The high-dose BMP2 caused cystic-like regeneration with incomplete defect closure. HE+BMP2 showed virtually complete bone fusion. Red collagen fibrils were significantly higher in BMP2-induced newly formed bone (NFB) in HE-supplemented group (p<0.05) indicating high organization. Clear changes in osteocyte lacunae as well as a statistically significant increase in osteoclasts were found around NFB in HE rats. A significant increase in trabecular volume and thickness, and trabecular and cortical density was found in femurs of HE-supplemented rats (p<0.05). Our findings show, for the first time, that dietary HE has a remarkable modulatory role in locally delivered high-dose BMP2-induced bone possibly via control of inflammation, osteogenesis, changes in osteocyte and osteoclast function and collagen maturation in regenerated and native bone. In conclusion, HE has a significant skeletal bone sparing effect and the ability to provide a more effective BMP-induced craniofacial regeneration.


Introduction
Recombinant human bone morphogenetic protein 2 (BMP-2) is a potent osteoinductive cytokine that plays a critical role in bone regeneration and has been studied extensively as a biologic for bone repair in dental and orthopedic elds [1][2][3][4] . The growth factor BMP-2 is currently used as a bone-inducing agent for alveolar bone reconstruction, and several biomaterials and scaffolds have been introduced and evaluated as BMP-2 carriers, e.g., absorbable collagen sponges, allogenic bone, bovine bone, hydroxyapatite and biphasic calcium phosphate 5 .
Although the usefulness of BMP-2 for bone induction has not been questioned, the exponential rise in the clinical use of BMP-2 (Infuse TM Bone Graft, Medtronic) use has been described to be associated with serious side effects 6 , such as in ammatory complications that may cause signi cant swelling and compromise a patient's airway, cause ectopic bone, and tumor-like formation 7 . Other orthopedicassociated conditions like radiculitis, vertebral osteolysis, and increased microfracture occurrence has also been reported 8 . Therefore, it is important to nd ways to better utilize BMP2 as a biologic either by controlling its in ammatory effects and/or potentiating its osteogenic function allowing for smaller quantities to be used [9][10][11][12] . Therapeutics that act in synergy with BMP-2, limiting or avoiding side effects thus improving e cacy in many clinical applications, would be a paradigm shift in the eld of regenerative dentistry and medicine. Hesperidin (HE) is a natural compound found in citrus fruits that has been associated with various functions such as anti-in ammatory, anti-oxidative, anti-clastogenic and osteogenic [13][14][15] . Brie y, avonoids present in citrus fruits, such as HE, have been shown to stimulate osteoblast differentiation through activation of BMP signaling 16 . Pre-clinical studies have demonstrated that HE intake results in bone density protection in senescent and ovariectomized rats as well as reduction in oxidative stress and overall lipid content [17][18][19][20] . In a human clinical trial, our group investigated the use of grape seed extract and grapefruit extract (the latter rich in HE) in bone healing of extraction sockets and found that grapefruit extract led to downregulation of in ammatory genes such as interleukin (IL)-1 β, IL-6 and CXCL2 in the healing tissue 21 . More recently, we reported the positive effects of this promising phenolic compound on pre-osteoblastic cell differentiation, on the quantity and quality of mineralization, and on the quality of the organic type I collagen-rich matrix in vitro. Such characteristics are critical for optimal bone propertiesas well as a bone-inducing role in vivo 12 . Our ndings showed, for the rst time, that HE has a modulatory role in mineralized tissue formation via promotion of osteoblast cell differentiation and improvement of matrix organization and mineral-to-matrix ratio. Moreover, the in vivo rat mandible regenerative bone model showed that HE combined with a suboptimal dose of BMP2 (a dose not able to promote fusion of the bone defect) in a collagenous scaffold promoted a well-controlled (not ectopic) pattern of bone formation as compared to a larger dose of BMP2 12 . Therefore, we have shown that HE delivered locally can promote BMP2 function suggesting that it could be a potential adjunct to the use of BMP2 in clinic.
Currently, there isn't a way to effectively and consistently deliver HE concomitantly with BMP2 locally to a bone defect. Further, the aqueous insoluble nature of hesperidin without a delivery system may pose standardization of treatment di cult at this time. We proposed to evaluate the effect of systemic administration of HE as a dietary supplement to BMP2/collagen scaffold-induced (locally delivered) bone regeneration to circumventing these limitations. The use of HE for local delivery warrants further investigation of a release system such as nanoparticles, branched polymers, or hydrogels with predictable rates of degradation to facilitate its solubility and ensure cell and matrix uptake as well as concomitant BMP2 delivery 12 . Since HE is already available as an over-the-counter (OTC) supplement, dietary administration of HE could, in the meantime, be a practical and immediate alternative to modulation of BMP2-based regeneration while local delivery system(s) are being developed. OTC supplementation can be utilized as adjunctive and concomitant treatment during bone graft procedures in dentistry and orthopedics.
Thus, taking into consideration the role of HE in in ammation and in osteogenesiss/osteoclastogenesis, our study aimed to evaluate bone healing in a critical-sized mandible defect of rats under HE supplementation by evaluating early in ammatory/osteogenic gene expression and histopathological and tomographic bone parameters in bone healing sites treated with or without BMP2. Our hypothesis was that systemic HE supplementation would positively modulate in ammation associated with use of high-dose BMP2, promote osteogenesis, in uence osteoclastogenesis and favor mandible bone quality, ultimately leading to a more desirable regenerative therapy free of abnormal and poor-quality bone formation. Since the rats would be on dietary HE for 6 weeks, off target effect on skeletal bones were anticipated, thus, femurs were imaged for effect on skeletal bone mass. This report is the rst in the literature showing a bene t in consumption of a dietary compound to in uence bone healing.

Animals and groups
The Institutional Animal Care and Use Committee (IACUC) at the University of North Carolina at Chapel Hill (IACUC ID: 18-115, 04/30/2021) approved the animal experiment protocol and the procedures were conducted in compliance with ethical standards that fully comply with Animal Research: Reporting of in vivo Experiments (ARRIVE) guidelines 22 .
Forty male Sprague-Dawley rats (350-400 grams) were randomly allocated into 4 groups (n=10/group): mandible defect + scaffold with (1) or without (2) HE 100mg/kg dietary supplementation and, mandible defect + scaffold with 1µg of BMP2 with (3) or without (4) supplementation with HE 100mg/kg. The animals were kept in an environment with controlled temperature, humidity, and light cycles, and fed with water and feed ad libitum.
Hesperidin treatment HE (97% purity, ACROS Organics, Belgium) concentration at 100 mg/kg of rat weight was diluted in 0.9% sodium chloride mixed fresh at time of ingestion and administered systemically once daily via oral gavage by experienced personnel as reported 23 . HE treatment started 4 weeks before mandible surgery and was maintained until euthanasia at day 7 or 14 post-surgery.

Mandible model and femur phenotype
As previously described (Miguez et al., 2011(Miguez et al., , 2014(Miguez et al., , 2021, all animals were given a pre-operative dose of the antibiotic Cefazolin (10 mg/kg), and the anesthesia was achieved by using Ketamine (80 mg/kg) /Xylazine (10 mg/kg). Two-cm incisions were made along the inferior border of the hemi-mandibles and the masseter muscle, and the periosteum, were detached to expose the ramus (Fig 1A). Using a 5-mmdiameter trephine (Salvin Dental, Charlotte, NC, USA), through a mandible jig prototype (archetype jig: http://otc.unc.edu, Tech#20-0105) a critical-sized defect was placed at the ramus at a jig-oriented standardized location about 3mm above the lower border of the mandible and 2mm distal to the incisor root ( Fig 1B,C,D). A 5mm bone core was removed without rigged borders and a well-de ned critical-sized defect was obtained at a standardized and reproducible location (Fig 1E,F,G). The defects were lled with a UV-cross-linked type I collagen sponge (Nitta Gelatin, Japan) as a scaffold. Each scaffold was precut with a 5-mm-diameter tissue punch (Miltex Inc., York, PA, USA) and was soaked uniformly in 10 µl total solution of phosphate-buffered saline (PBS) with and without the growth factor BMP2 (rhBMP2, catalog #:355-BM, R&D Systems, Minneapolis, MN). Two groups received an empty scaffold pressed into the defect, while the other two groups, the bone defect was lled with the scaffold soaked with 10 µl PBS containing BMP2 at 1μg concentration ( Fig 1H). The muscle layer was passively, but tightly, sutured around the mandible with 5-0 chromic gut (Ethicon Inc., Cornelia, GA, USA) and the skin with 5-0 polypropylene suture (Ethicon). The rats were maintained on a diet of soft rat chow and water for 1 day and starting on day 2 had both soft and hard diet available. Rats received buprenorphine for pain management. Any complications such as seromas, signs of distress or change in behavior were recorded and managed immediately to avoid animal stress and discomfort. At day 7 post-surgery, animals (n=5/group) were anesthetized by ketamine/xylazine, the surgical access to the mandible was re-opened and the collagenous tissue present within the mandible bone defect was collected and immediately stored in an RNA stabilization solution (RNA later®, Sigma-Aldrige, Burlignton, MA) ( Fig 1I) until processed for gene arrays. Another cohort of animals (5/group) was euthanized at day 14 post-surgery and the mandibles were immediately xed in 10% neutral buffered formalin for 72 h, rinsed in PBS and stored in 70% v/v ethanol at 4 o C. Femurs of the groups treated with or without HE were also harvested 2 weeks post-surgery, to evaluate the impact of 6 weeks of systemic dietary HE on femoral phenotype. All animals were sacri ced according to the IACUC and ARRIVE guidelines with a lethal dose of pentobarbital followed by the physical method of thoracotomy. The formalin-xed mandibles with intact attached muscle and femurs were analyzed by microcomputed tomography (µCT). Mandibles were demineralized with 0.5M EDTA at pH 7.4 for 8 weeks under agitation, para n-embedded and processed for histology.

RNA isolation and PCR arrays
Seven-day samples stored in RNA later were processed for RNA extraction via Trizol (Invitrogen, Carlsbad, CA) as described previously (Miguez et al., 2021). RNA was further puri ed using the miRNeasy Mini Kit (Qiagen, Germantown, MD) and RNA integrity assessed using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). The cDNA synthesis and gDNA elimination were performed using the RT 2 Micro uidics qPCR Reagent System (Qiagen) with 100 ng ( rst experiment) /500 ng (second experiment) RNA used as input. Next, speci c target preampli cation was performed using the

Microcomputed tomography bone analysis
The mandibles were scanned using µCT system (Scanco µCT40 scanner -SCANCO Medical AG, Bruttisellen, Switzerland) 12 . The X-ray parameters were 70kVp at 114uA with a 200ms integration time. Image matrix size was 2048 × 2048 with acquired 2000 projections over a 360-degree rotation. A tube of 20.5mm diameter which allows a pixel size of 10 μm was used. Acquisitions were made using a conebeam geometry and a Feldkamp ltered backprojection reconstruction algorithm used to create the reconstructions. All samples were positioned and scanned in a standard manner using an airtight cylindrical sample holder lled with PBS. For the analyses of the acquired images, the CTAn analyser software (Skyscan) was used. The region of interest (ROI) was selected in all images through a standardized drawing of the area within the defect borders. The region was rst positioned in the middle of the surgical defect, and it was then extended to all slices of the data set. A new ROI was set every 20 images. Quantitative morphometric analysis of the mineralized tissue inside the defects was carried out on voxels that corresponded to bone. After tomographic acquisitions, 3D images were reconstructed through direct volume rendering from the series of 2D projections 9,10,12 . For the femurs, a 6 µm, 70 kVp, 142 µA using a 0.5 mm aluminum attenuation lter setting was used. The ROI included a speci c number of slices below the growth plate after a set anatomical marker for trabecular bone and cortical bone analysis (50 and 400 slices below growth plate, respectively). The bone microarchitecture parameters evaluated were trabecular and cortical bone volume fraction (BV/TV, %), trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular separation (Tb.S), and trabecular and cortical density (mgHA/ccm) as previously recommended 26 .

Histological analysis
After mandibles were demineralized and embedded, sections (6 µm) obtained from the mid-cross section of the mandible in the frontal plane were stained by Hematoxylin & Eosin (H&E) for a qualitative gross evaluation and picrosirius red (PSR) for quantitative collagen organization and maturation analysis. Osteoclast numbers were assessed by tartrate-resistant acid phosphatase (TRAP) staining as previously described 10,23 .
For PSR, slides were incubated in 0.1% (w/v) sirius red in saturated picric acid solution for 30 minutes at room temperature. This was followed by rinsing with distilled water, dehydration and mounting. The slides were imaged under bright eld and polarizing light using a Leica DMR microscope (Buffalo Grove, IL, USA). PSR images at 20x magni cation were analyzed using a custom generated algorithm in Matlab ® R2016a (Mathworks, Natick, MA, USA) as previously reported (Smith et al., Miguez et al). The percent area of red, yellow, and green color signals were normalized to total color signal for each sample 12,27 .
Non-serial histological sections (n=5/animal) were stained for TRAP-positive multinucleated osteoclasts, and slides were depara nized and rehydrated through graded ethanol to distilled water, and then immersed in TRAP staining solution mix (at 37ºC for 30 minutes). The sections were rinsed in distilled water and counterstained with 0.02% fast green for 30 seconds. For quanti cation, TRAP-positive cells stained in red containing three or more nuclei on the bone surface, were considered osteoclasts, and quanti ed in the region of interest comprising the whole area within the defect borders 23 .

Statistical analyses
A blinded evaluator from the bioinformatics core performing the statistical analyses was blinded to the identity of experimental groups for gene arrays. Gene arrays were evaluated by ANOVA and Tukey post hoc as described 25 . Data were submitted to the Shapiro-Wilk test to assess homogeneity and data distribution. Analysis of variance (ANOVA) with Tukey post hoc test or Kruskal-Wallis with Dunn post hoc test were performed to determine the differences among groups of animals for microcomputed tomography, osteoclast numbers as described 10,12 . PSR colors were quanti ed by MatLab software as described 12,27 and statistically evaluated by ANOVA with Tukey post test. GraphPad Prism 8 software (GraphPad Software Inc., San Diego, CA, USA) was utilized for analyses. All tests were applied with a 95% con dence level (p < 0.05). Data were expressed as mean ± standard deviation.

Adverse events
Systemic HE administration, surgical procedures and scaffolds did not promote any behavioral change, physical issues (e.g., skin lesions, hair loss), detrimental weight loss or feeding impairment. Three animals in the BMP2-treated group developed seromas in the submandibular space which were monitored as the animals were not stressed and were ambulating, behaving, and eating normally.
Hesperidin in uence in in ammatory and osteogenic genes the rat mandible model compared to scaffold alone after 7 days of healing, signi cantly decreased the expression of several genes including biglycan (bgn), bmp6, bmp5, bronectin (fn1), the main BMP receptor gene bmpr1a, coll3a1, anexin5, phex, ppc and csf2. One of the most signi cant downregulation in BMP2 treated rats vs. scaffold alone was tgfb3. BMP2 increased in ammation-associated genes such as ltb and il5rα.
When rats were on dietary HE and subjected to BMP2-induced bone regeneration (HE+BMP2) where compared to BMP2, the healing tissue showed a statistically signi cant decrease in expression of the in ammatory genes tnf, nf-κb and ccl12, respectively. Interestingly, expression of smad4 and spp1 (osteopontin) were signi cantly reduced potentially indicating some control of osteogenesis via HE. Coll3a1 was signi cantly upregulated as well as tgfb3, bmpr1a and bmp6 in HE-rats.
HE administered systemically promotes a more robust and well-de ned BMP2-induced bone regeneration Three and two-dimensional analysis ( Fig. 2A, 2B, 2C) of newly formed bone within the defects showed a small percentage of bone formation in samples treated with scaffold only in both groups with and without HE supplementation. A signi cantly robust and cyst-like bone formation was observed in the defects treated with scaffolds infused with 1µg of BMP2 compared to no growth factor (p<0.05). Bone volume was also signi cantly increased compared to scaffold alone in rats under dietary HE regimen and treated with BMP2 (p<0.05) but was not different compared to BMP2 scaffold without HE. Remarkably, the bone formation in the HE-rats virtually closed the mandible defect without any evidence of abnormal bone formation such as cysts or ectopic sites in all rats. Histological evaluation (Fig. 2D) by H&E con rmed the pattern of bone regeneration seen on µCT and revealed pronounced in ammatory in ltrate associated with BMP2 groups without HE treatment including heme-lled cavities (Fig. 2F).
Dietary HE supplementation leads to a more mature extracellular matrix PSR staining showed a quanti able pattern of mature collagen brils of predominantly red birefringence with a lower percentage of green and yellow brils in BMP2-treated rats while supplemented with HE as compared to only scaffold or BMP2 alone (p<0.05) (Fig. 3).
Osteocytes lacunae were clearly dissimilar in rats under HE supplementation compared to no HE. The lacunae was marked by intensive sirus red staining (dense and of mostly unidirectional collagen matrix) revealing a spindle shape form of the osteocyte cavity compared to less de ned, poorly collagen-dense walls and round-shaped lacunae for non-HE rats (blue arrows, 3E, 3F).
Osteoclasts numbers are increased in NFB of HE-treated rats at 100 mg/kg TRAP staining (Fig 4) showed that there were no osteoclasts present at 2-weeks post-surgery in the NFB area indicating no detectable active remodeling in BMP2-treated or scaffold alone groups. Interestingly, only in the presence of dietary HE, it was possible to nd osteoclasts in the samples 14 days post-surgery with most being localized to the surrounding area of the NFB. BMP2+HE rats showed the highest number of osteoclasts (35.6+18.3) compared to HE (8.3+5.5) (p<0.05, n=5 slices/rat, n=3 rats).

Trabecular and cortical femoral bone parameters are improved by dietary hesperidin
Systemic administration of HE signi cantly affected femoral bone parameters 6 weeks-post introduction of the oral gavage with HE (Fig. 5A, B, C). Trabecular bone volume (BV), trabecular thickness (Tb.Th.) and density were signi cantly increased by HE when compared to no-HE (p<0.05). HE administration also promoted a slight but signi cant increase in cortical density compared to control group (no HE treatment).
Of note, rats under HE-regimen lost signi cant amount of weight after starting the oral gavage (although no measurable difference in food intake was noticed in the rst week, Fig. 5D). In the subsequent weeks, HE-rats maintained the statistically signi cant lower weight compared to no HE, were alert and had normal behavior and eating patterns. Both groups lost weight the week after surgery as expected due to the nature of the surgery with limited food intake in the rst days post intervention.

Discussion
The incorporation of BMP2 has been found to remarkably enhance the bone restorative effect of synthetic bone substitutes and greatly expand the horizon for the clinical application of bone grafts 28 . However, BMP2 tends to be rapidly degraded by proteases when injected directly into a defect site and, thus, a supraphysiological dose of the protein is typically required. This dose can cause undesired side effects 29 , e.g., excessive in ammation, occasional ectopic ossi cation, tumor-like bone growth, and edema 12,30-32 .
As previously demonstrated in our studies, HE when delivered locally to a critical-sized defect in a rat mandible model, combined with a suboptimal dose (not enough to regenerate the defect) of BMP2 in a collagenous scaffold, promoted a well-controlled pattern of bone formation as compared to a large dose of BMP2 12 . Complementary, the present study is the second evidence showing HE effects on BMP2induced regeneration and the rst evaluating dietary HE on BMP2-bone therapy. Defects lled with a high dose of BMP2 in a collagenous scaffold only formed bone within the boundary of the mandible defect when HE was administered systemically over the course of the healing period, which is in agreement with the results observed when HE was associated to BMP2 and directly delivered into bone defects 12 . HE effects observed in both models are paradigm changing and very promising in the eld of cost-effective regenerative medicine and dentistry. The remarkable effects of HE in controlling BMP function could be associated with its modulation of osteogenic, resorptive and in ammatory cell functions 12,20,33,34 .
Indeed, an in ammatory pro le has been observed with BMP2 use in the rats in this and previous studies histologically. The literature has described an exaggerated in ammatory response after implantation of BMP2 in an absorbable collagen sponge in animal models or clinical settings [35][36][37][38][39][40] . A remarkable reduction in tnf, nfκb and ccl12 pro-in ammatory genes were found where HE was present systemically in rats treated with BMP2 compared to no HE. Corroborating our result, HE was described to attenuate alteration in lung NF-κB expression in rats 41 , while the topic application of 5% HE hydrogel decreased NF-κB expression in granulation tissue of skin wounds in mice 42 . In a mouse model of gout arthritis, HE inhibited NF-κB activation 43 . NF-κB signaling pathway is one of the most well-studied pathways related to in ammation, since this transcription factor regulates the expression of various genes involved in in ammation and the immune response (including TNF and CXCL2) [44][45][46] . The importance of NF-κB in both bone formation and bone resorption is well-known 47 , and genetic mutations in molecules involved in the NF-κB signaling pathway in mammals cause pathological bone phenotypes, including osteopetrosis 48 . Tnf, cxcl1 and ccl3 genes, were also signi cantly downregulated in HE+BMP2 rats compared to scaffold alone. Previous evidence has reported a regulatory effect by different classes of avonoids (apigenin, quercetin, setin, astragalin, HE, hesperitin) in the chemokine subfamilies CXC and CC, and TNF activity [49][50][51][52][53][54][55][56][57][58] . Our results share similarities with prior evidence demonstrating an antiin ammatory effect of HE during bone formation in our mandible model. In contrast, evaluating the systemic effects of HE in a model of periodontal in ammation 23 , this avonoid increased the in ammatory pro le, tissue damage and bone resorption in rats with periodontal disease, which can in part be attributed to the characteristics of the model studied. As previously described by de Paiva Gonçalves et al. 23 , the complexity of the diseased environment, local in ammation, as well as avonoid dosage, are crucial factors to be considered in the cell responses to HE. Nonetheless, the remarkable reduction in the in ammatory cell in ux, the evidence of organized, high quality and non-ectopic healing bone in rats treated with HE at 100mg/kg+BMP2 at 1µg are likely the direct result of a primarily nonhyperin ammatory healing site.
Our previous studies have demonstrated an osteogenic function of HE when used in combination with a low dose of BMP2 (both delivered locally) 12 . In this study, the use of systemically administered HE with high-dose BMP2 compared to rats not on HE and treated with the same dose of the growth factor, led to a substantial and signi cant decrease in smad4 and osteopontin expression, important in osteogenic signaling and mineralization. There was also an increase in coll3a1 (important in collagen maturation and angiogenesis), tgfb3 (known to control BMP function and matrix deposition and can control collagen crosslinking), bmpr1a (a known receptor for BMP2 signaling), and bmp6 (important in endochondral ossi cation) [59][60][61][62][63][64][65][66] .
The signi cant reduction observed in genes such as smad4 and spp1 (as well as bgn and bmp6 compared to scaffold alone) gene expression by HE-treated animals, is curious, as those are important in osteogenesis. Smad proteins mediate the signal transduction in TGF and BMP signaling pathways, affecting both osteoblast and osteoclast function, and therefore play a critical role in the regulation of bone remodeling 67 . BGN is detected at high levels in areas of endochondral and intramembranous bone formation 68 and has been extensively investigated in its role in promoting BMP function, vascularization and more recently, in ammation 9,68-70 . A previous study 71 showed that avopiridol, another avonoid, inhibits TGFβ-stimulated BGN synthesis by blocking linker region phosphorylation and nuclear translocation of smad2 in vascular smooth muscle cells. There seems to be a ne-tuned HE-mediated control of bone formation which is involving many key osteogenic players such as TGFβ3 and, in turn, bmpr1α, smad4, spp1 and bgn gene expression 60 .
It is important to highlight how the high-dose BMP2 affected the healing bone compared to the collagen scaffold alone. It mostly decreased the expression of bgn (a BMP2 and 4 function promoter), bmp5 and bmp6 (which are associated with induction of chondrogenesis), fn1 (of key importance to cell adhesion, migration, growth and differentiation), bmpr1a (likely controlling osteogenic function due to over activation of BMP receptors), coll3a1 (involved in collagen organization and angiogenesis), and phex 59,64,66,69,72,73 . High dose BMP2 also signi cantly increased ltb (a chemoattractant of leukocytes), and decreased anexin5 (with function largely unknown but thought to modulate coagulation) and ccl10 (suggested to promote osteoclast differentiation) 74 . Both ltb and anexin5 could correlate with the high presence of heme in the cystic area and hematomas associated with some rats 75,76 .
PSR staining showed very de ned, active areas around osteocytes in the presence of systemic HE. However, without HE, BMP2-treated bone had non-distinct lacunae (characterized by poor collagen packing and mineralization). BMP samples showed a signi cant decrease in phex gene expression. Phex is important in cleavage of proteins such as osteopontin, is expressed in early and mature osteocytes and is involved in phosphate metabolism as well as growth of bone. Decrease in phex may mean poor phosphate clearance, altered mineralization and may be important in controlling remodeling 77 .
The clear increase in coll3a1 expression by systemic HE treatment, could be correlated and potentially in uenced by active osteocytes as well, considering that those cells synthesize several proteins involved in bone formation such as Type I collagen, osteocalcin, sclerostin and Dkk-1 78 . Through this mechanism, the suggested active osteocyte function may contribute to a well-de ned and mature bone healing pattern. Osteopontin (OPN) is a highly phosphorylated and glycosylated sialoprotein that is expressed by several cell types including osteoblasts, osteocytes, and odontoblasts 79 . This noncollagenous protein is encoded by the spp1 gene and is involved in biomineralization of bone tissue, bone remodeling, wound healing and apoptosis 79,80 . OPN was described to contribute to bone remodeling by promoting osteoclastogenesis and osteoclast activity through CD44-and αvβ3-mediated cell signaling. Further, it plays a de nitive role in bone remodeling by the formation of podosomes, osteoclast survival, and osteoclast motility 79 . Mirroring these considerations, the signi cant decrease of spp1 gene expression encoding OPN by HE, may constitute a mechanistic pathway to control remodeling (and phex increase also would decrease spp1 availability in BMP2 alone). Furthermore, the polarized imaged area around osteocytes (lacunae) in HE-treated groups, is marked by yellow and red matrix, suggesting active osteocyte function even in the native bone area. OPN is highly observed in the osteocytes during initial stages of orthodontic tooth movement 81 , and a model of mechanical stress shows that the number of OPN-mRNA expressing osteocytes on the pressure side after 48h of mechanical stress reach a maximum value at 72h, coinciding with bone resorption 82 . Therefore, the increased osteocyte activity could also be contributing to the marked increase in osteoclast presence in HE-treated rats.
Correlated with the mandibular bone regeneration results, HE presented signi cant effects on femoral bone parameters 6 weeks-post avonoid intake. HE improved trabecular and cortical bone microarchitecture, as previously observed in a similar study in rats performed by our group 23  In conclusion, our ndings show, for the rst time, that HE as a dietary supplement has a clear and promising modulatory role in BMP2-induced bone regeneration potentially via control of in ammation, effect on bone cells' function, and collagen maturation and can lead to a positive control of BMP2induced regeneration. This work supports the idea that HE may be a fast-track, implementable, costeffective method of improving BMP2 application in current regenerative bone therapies due to its multiple positive effects on controlling in ammation, improving bone matrix quality and ensuring su cient and adverse-event free bone healing. This study exempli es the bene ts of dietary supplementation in modulation of craniofacial and skeleton bone parameters improving oral and systemic health.  the in ammatory in ltrate and blood-lled cavity within bone is histologically evident n BMP2 alone samples (blue arrow). One-way analysis of variance, Tukey's post hoc test performed for imaging and gene expression parametric data at 95% con dence interval.