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. 2026 Jan 4;114(4):2214–2225. doi: 10.1007/s10266-025-01288-x

Effect of vitamins A/C primed human gingival mesenchymal stem/progenitor cells in preventing medication-related osteonecrosis of extraction socket: an animal study

Fatma I Elfaiedi 1, Nahed S Korany 1, Karim Fawzy El-Sayed 2,3,4, Nermeen AbuBakr 1,✉
PMCID: PMC13630899  PMID: 41484660

Abstract

Human gingival mesenchymal stem/progenitor cells (hGMSCs) exert remarkable tissue regeneration properties. A combination of vitamins C and A at specified concentrations enhanced cellular dedifferentiation and multipotency of hGMSCs. To our knowledge, vitamins A/C-primed hGMSCs in the prevention of medication-related osteonecrosis of the jaw (MRONJ) have not been investigated. Thus, this study evaluated the impact of vitamins A/C in pluripotency-inductive concentrations on the regenerative/reparative potential of hGMSCs in preventing MRONJ development in rats. Thirty albino rats (n = 10/group) were administered zoledronic acid and dexamethasone three times a week for 4 weeks. The upper first molar was extracted 2 weeks following the first administration. Immediately after extraction, rats received normal saline (group Ι), hGMSCs (group ΙΙ), and hGMSCs primed with vitamins A/C (group ΙΙΙ). Rats were euthanized 2 and 4 weeks after extraction. The maxillae were analyzed using dual-energy X-ray absorptiometry, histologically, histomorphometrically, and by quantitative real-time polymerase chain reaction analysis. Groups II and III displayed elevated bone mineral density, with a significant rise in group ΙΙΙ relative to group ΙΙ at 4 weeks. Histomorphometrically, groups II and III revealed an elevation in area percentage of newly generated bone, with a significant rise in group ΙΙΙ at both time intervals. Gene expression of transforming growth factor-β1 (TGF-β1) and runt-related transcription factor 2 (Runx2) presented a significant elevation in groups II and III, with a significant rise in group ΙΙΙ at both time intervals. In conclusion, vitamins A/C-primed hGMSCs at the tested pluripotency-inductive concentrations prevented MRONJ development, primarily through encouraging wound healing and bone repair in the extraction sockets of rats.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1007/s10266-025-01288-x.

Keywords: Ascorbic acid, Vitamin A, Wound healing, Osteonecrosis, Stem cells

Introduction

Medication-related osteonecrosis of the jaw (MRONJ) is a multifactorial condition, in which an infection, following bone injury (e.g., tooth extraction), combined with repressed bone remodeling caused by antiresorptive medications (e.g., bisphosphonates (BPs)), can result in persistently exposed bone without epithelial coverage accompanied by pain, infection, and sinus formation. The resultant clinically challenging situation could markedly reduce the patient’s life quality [1].

To date, although different therapeutic approaches were advocated, no consensus exists for a widely accepted effective strategy for the prevention/treatment of MRONJ. Among the strategies examined, mesenchymal stem/progenitor cells (MSCs)-based tissue engineering was reported to hold a promising potential in this context [2]. Human gingival mesenchymal stem/progenitor cells (hGMSCs), adult stem/progenitor cells, which can be easily separated from the gingiva, are marked by multipotent differentiation abilities, remarkable proliferation and regeneration capacities [3, 4], as well as immunomodulatory properties [5–7]. Previous studies have shown that hGMSCs can be effectively combined with bioactive regenerative biomaterials and dentinal derivatives. These combinations supported cell viability, matrix deposition, and osteogenic differentiation, highlighting their promising role in bone and soft tissue regeneration [8, 9].

Ascorbic acid (vitamin C) is a strong antioxidant, necessary for collagen generation and cellular growth and activity [10, 11]. It exerts anti-inflammatory activities, can promote immune defense as well as wound healing events [12], and encourages the growth of mesenchymal cells into osteoblasts [13]. An in vitro investigation demonstrated that ascorbic acid at a certain concentration enhances hGMSCs’ stemness, proliferation, and differentiation properties [14].

Likewise, fat-soluble vitamin A, an important element in the adaptive immune system, is a cofactor for the development and distinction of regulatory T cells [15] and is crucial for the osteogenic differentiation of hGMSCs [16] and regeneration [17]. Importantly, retinoic acid signaling pathways were suggested as therapeutic targets in bone diseases [18].

Strikingly, earlier studies demonstrated that a combination of vitamins C and A at specific doses impacts cellular epigenetics by demethylating nuclear bases, which causes adult cells to dedifferentiate into pluripotent ones [19, 20]. A combination of vitamin C and A at these pluripotency-inductive concentrations with hGMSCs could foster their therapeutic effect in the early control/prevention of MRONJ, and to our knowledge, it has not been investigated. Thus, this animal research intended to investigate the effect of vitamins A/C-primed hGMSCs in the early control/prevention of medication-related osteonecrosis in extraction sockets of albino rats.

Materials and methods

Isolation of hGMSCs

Gingival tissues were obtained from healthy partially impacted third molars that were surgically extracted with free gingival soft tissue collars. The gingival tissues were submerged in phosphate buffer solution complemented with 100 µg/ml streptomycin, 1% amphotericin, and 100 U/ml penicillin at 4 °C for a period not exceeding 24 h. Tissues were deepithelialized and divided into little pieces, washed with minimum essential medium (MEM), and antibiotics (100 µg/ml streptomycin, 100 U/ml penicillin). The specimens were put into dry tissue culture flasks for 30 min for adherence. The basic medium included MEM complemented with 400 mmol/ml L-glutamine, 15% fetal calf serum, 100 µg/ml streptomycin, and 100 U/ml penicillin. Cells were allowed to proliferate after the flasks were incubated at 37 °C with 5% CO2. Phase contrast inverted microscopy was utilized to inspect the culture flasks, and the basic medium was replaced three times every week. Cells were separated using 0.10% trypsin–EDTA and counted once they had reached 80–85% confluence. Trypan Blue was used to verify their vitality to be finally cultured at a density of 30 cells/cm2 in culture flasks. The flasks were then incubated at 37 °C with 5% CO2 [21].

Identification of hGMSCs

Flow cytometric analysis

Upon reaching confluence, third passage hGMSCs were characterized by flow cytometry. Primary antibodies against human CD34, CD44 (eBioscience, Waltham, USA), CD45, CD73, CD90, and CD105 (Miltenyi Biotec, Bergisch Gladbach, Germany) were utilized. Cells were stained with fluorescein isothiocyanate (FITC) or phycoerythrin (PE)-conjugated primary antibodies at room temperature. PE- or FITC-conjugated human IgG were employed as isotype controls. A CytoFLEX flow cytometer and CytExpert Software (Beckman Coulter, USA) were used for the analyses.

Multilineage differentiation assays

Osteogenic media was used to cultivate third passage hGMSCs. To identify calcified deposits, cell cultures were stained with Alizarin Red on day 14. hGMSCs were cultivated in adipogenic inductive media for 21 days to determine their capacity for adipogenic differentiation. Oil-Red-O staining was utilized to assess the existence of lipid drops. Chondrogenic inductive media was used to incubate hGMSCs to examine their capacity for chondrogenic differentiation. At day 35, chondrogenic differentiation was assessed by staining glycosaminoglycans with Alcian Blue. Basic medium was used for the same durations as the respective controls. Three times per week, all media were renewed [22].

Experimental study design

Sample size calculation

Using a 0.05 alpha threshold, a statistical power of 80%, and a 1.13 effect size (Cohen’s f) determined from a previous investigation [23], a total of 18 animals were required (n = 18). The sample size was raised to enhance the robustness of the statistical analysis to be 30 samples in total, with 10 samples in each group and 5 samples in each subgroup. R statistical analysis software version 4.5.0 for Windows was utilized to calculate the sample size.

Animals

Thirty (n = 30) healthy adult 7-weeks-old male albino rats, weighing about 195 ± 20g were employed. The animals were kept in a pathogen-free environment with a humidity of 50 ± 1%, a temperature of 22 ± 1 °C, and a 12-h light/dark cycle and were fed normal chow and water.

Induction of MRONJ-like animal model

Three times a week for 4 weeks, zoledronic acid (ZA) (66 µg/kg) (Novartis, Basel, Switzerland) and dexamethasone (5 mg/kg) (Amriya Pharmaceuticals, Alexandria, Egypt) were injected subcutaneously to induce osteonecrosis. Two weeks following the first administration, all rats were subjected to maxillary left first molar atraumatic extraction [24]. The rats were anesthetized with an intraperitoneal injection of a mixture of ketamine (70 mg/kg; Ketamar® 5%, Amoun Pharmaceutical Co., Qalyubia, Egypt) and xylazine (10 mg/kg; Xyla-Ject® 2%, ADWIA Co., Cairo, Egypt). Each rat was then positioned with the head gently tilted backward, and the mouth was carefully opened while the tongue was depressed. The molar was grasped with a curved mosquito hemostat and gently luxated with slow bucco-palatal movements until loosening was achieved, followed by gradual buccal traction to remove the tooth with intact roots. Rats were fed a soft diet for 1 week after extraction to decrease trauma from mastication and hard food. ZA and dexamethasone injections were continued for 4 weeks. Subcutaneous injection of buprenorphine (0.05 mg/kg) was administered every 12 h for 48 h following extraction for pain relief [25].

Groups

Immediately after extraction, rats were split randomly into three groups (www.random.org, n = 10/group): (1) group I: was administered normal saline in the extraction site; (2) group II (hGMSCs group): received hGMSCs (1 × 106 cells/mL) [26] in the extraction site; (3) group III (vitamins A/C-primed hGMSCs group): received hGMSCs (1 × 106 cells/mL) with vitamin C 250 μmol/L [20] and vitamin A 20 μmol/L [18] in the extraction site. In each group, either saline solution or hGMSCs were loaded onto a sterile absorbable hemostatic gelatine sponge [27] (Cutanplast® Standard, Mascia Brunelli S.p.A, Milano, Italy), trimmed to approximately 2 × 3 × 2 mm. The sponge was gently compressed and placed into the extraction socket. It was left in situ and gradually resorbed within the socket.

Blinding

For each animal, four investigators were involved. The allocation to the experimental group was only known to the first investigator. The second investigator was the only one to evaluate dosage delivery for every animal during the trial. Without being aware of the groups, the third investigator evaluated the results in a masked manner. The experimental groups were unknown to the statistician who conducted the data analysis.

Animal euthanasia and tissue preparation

Rats from each group (n = 5/time point) were euthanized using anesthesia overdose 2 and 4 weeks post-extraction. All animals survived to the study end points, with no losses. The maxillae were collected and subjected to the following analyses.

Bone mineral density (BMD) analysis

BMD (primary outcome) of the collected maxillae was examined by dual-energy X-ray absorptiometry (DEXA) (MEDIX DR, Medilink, Mauguio, France) to determine hard tissue repair in teeth extraction sockets (region of interest). The scanning speed was 60 mm/s, and the resolution was 0.5 × 0.5 mm. Results were displayed automatically in g/cm2 [22].

Histopathologic examination

All maxillae, including the extraction sockets, were stained with hematoxylin and eosin (H&E) in accordance with standard protocols for histological interpretation by a light microscope [28].

Histomorphometric analysis

Leica QWin 500 software (Leica Microsystems, Heerbrugg, Switzerland) was used to evaluate the area percentage of the newly created bone in the extraction socket location. For every specimen, ten fields were measured at a total magnification of 200 × . A standard measuring frame with an area of 118,476.6 µm2 was used to compute the bone area percentage [22].

Quantitative real-time polymerase chain reaction (qRT-PCR) examination

qRT-PCR was employed to identify the mRNA expression of transforming growth factor-β1 (TGF-β1) and runt-related transcription factor 2 (Runx2) in bone and mucosal tissues of the extraction socket. β-Actin was employed as a housekeeping gene. Total RNA was isolated from homogenized tissue specimens using a Qiagen RNeasy mini kit. Complementary DNA (cDNA) was synthesized from the total RNA of each sample using a high-capacity cDNA reverse transcription kit. The resultant cDNA was then amplified on a 48-well plate utilizing a StepOne™ System with software version 2.3 (Applied Biosystems, Waltham, Massachusetts, USA). The thermocycling profile involved 15 min of enzyme activation at 95 °C, followed by 40 cycles of 60 s at 95 °C for denaturation, 60 s at 60 °C for annealing, and 60 s at 72 °C for extension [29]. Primer sequences of the genes are listed in Table 1.

Table 1.

Primer sequences of the studied genes

Gene Forward Reverse Accession number
TGF-β1 5′-CCTGGAAAGGGCTCAACAC-3′ 5′-CTGCCGTACACAGCAGTTCT-3′ X52498
Runx2 5′-TCCCAGTATGAGAGTAGGTGTCC-3′ 5′-GGCTCAGATAAGAGGGGTAAGAC-3′ NM_001278483
β-actin 5′-CTATGTTGCCCTAGACTTCG-3′ 5′-AGGTCTTTACGGATGTCAAC-3′ NM_031144.3
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Statistical analysis

Microsoft Excel 2016, GraphPadPrism, and the Statistical Package for Social Sciences computer program version 20 (SPSS 20) were used to conduct the statistical analysis. Shapiro–Wilk and Kolmogorov–Smirnov tests were used to explore data for normality, and the results revealed that all of the data were normally distributed. Mean and standard deviation were utilized to display all quantitative data. A paired t test was utilized to compare measures taken 2 and 4 weeks apart, and the one-way ANOVA test and Tukey post hoc test for multiple comparisons were utilized to compare groups. A significant threshold of P < 0.05 was established.

Results

Phenotypic identification of hGMSCs

Flow cytometric analysis of cell surface markers showed positivity for CD44 (84.28%), CD73 (93.50%), CD90 (84.67%), and CD105 (69.12%) and negativity for CD34 (1.29%) and CD45 (1.29%) (Fig. 1).

Fig. 1.

Fig. 1

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Flow cytometric analysis of cell surface marker expression showing that hGMSCs were positive for MSCs markers CD44, CD73, CD90, and CD105, while negative for hematopoietic markers CD34 and CD45

Morphological identification of hGMSCs

At the third passage, hGMSCs exhibited a fibroblast-like morphology (Fig. 2a). Alizarin red-positive calcified deposits formed as a sign of the osteogenic differentiation of hGMSCs (Fig. 2b). Intracellular presence of Oil-Red-O positive lipid drops indicated adipogenic differentiation (Fig. 2c). The synthesis of glycosaminoglycans that stained favorably with Alcian Blue indicated chondrogenic differentiation (Fig. 2d).

Fig. 2.

Fig. 2

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A photomicrograph showing a Phase contrast microscopic appearance of hGMSCs, b Alizarin Red staining of the osteogenically stimulated hGMSCs, c Oil-Red-O staining of the adipogenically stimulated hGMSCs, d Alcian Blue staining of the chondrogenically stimulated hGMSCs

Bone mineral density

Two and four weeks post-extraction, a significant difference in BMD was notable between the groups (p = 0.0001). Group Ι showed the lowest BMD, while groups II and III showed a significant increase in BMD compared to group Ι, with insignificant differences between them at 2 weeks. However, there was a significant variation between groups II and III, in favor of group III, at 4 weeks (Fig. 3a; Supplementary Table S1). In addition, quantified data of DEXA showed a significant rise in BMD from 2 to 4 weeks in all groups (Fig. 3b; Supplementary Table S2).

Fig. 3.

Fig. 3

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Bar chart showing bone mineral density in all groups after 2 and 4 weeks (a) intergroup comparison, * indicates a significant difference between groups using ANOVA. Bars with different letters indicate a significant difference using Tukey post hoc test, and (b) intragroup comparison, * indicates a significant difference between groups using the paired t test. Significance at P < 0.05. n = 5/time point

Histological results

Histologically, group Ι showed necrotic bone with empty osteocytic lacunae, empty marrow spaces, and uneven resorption of the bone surface. The extraction socket was filled with dense inflammatory cell infiltrates and numerous blood vessels (Fig. 4a, d). After 4 weeks, a higher amount of necrotic bone, more empty lacunae, and bone sequestrate with scalloped borders were visible in the socket. The socket cavity revealed intense inflammatory cell infiltration with a decreased number of blood vessels (Fig. 4g, j).

Fig. 4.

Fig. 4

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Representative H&E-stained photomicrograph of tooth extraction sockets of groups I, II, and III at 2 weeks (a–f) and 4 weeks (g–l). Group I (a, d, g, j) showing granulation tissue (GT), blood vessels (BV), inflammatory cell infiltrate (curved arrows), scalloped borders (arrows), empty lacunae (black arrowheads), empty marrow spaces (BM), sequestrate (Sq), and osteoclasts (Oc). Group II (b, e, h, k) showing granulation tissue (GT), blood vessels (BV), marrow spaces (BM), bone trabeculae (dashed arrows), fibroblasts (dotted arrows), fibrous tissues (FT), osteocytes (black arrowheads), and osteoblasts (white arrowheads). Group III (c, f, i, l) showing marrow spaces (BM), osteocytes (black arrow heads), bone trabeculae (dashed arrows), woven bone (WB), oral epithelium (OE), and fibrous tissues (FT)

Group II at 2 weeks revealed that the newly formed bone was sparse and dispersed, enclosing randomly distributed large-sized viable osteocytes and fibrocellular marrow spaces. The new bone was restricted mainly to the internal surfaces of the alveolar socket with predominant osteoblastic activity. The alveolus was filled with granulation tissue, which contained more dilated blood vessels, more fibroblasts, and fewer inflammatory cells (Fig. 4b, e). At 4 weeks, the socket cavity contained larger, more interconnected bone trabeculae that were lined with osteoblasts and enclosing plumbed osteocytes. Fibrous tissue enclosing an increased number of dilated blood vessels and mature collagen fiber bundles was observed filling most of the socket (Fig. 4h, k).

In group III, at 2 weeks, there was newly created woven bone lining the socket basally and laterally, enclosing plumbed osteocytes and fibrocellular marrow cavities. The entire core region of the socket was filled with fibrous tissue as mature collagen fibers intermingled with immature ones (Fig. 4c, f). At 4 weeks, there was clear evidence of bone healing, and the socket was completely filled with more organized tissue. At the coronal end of the socket, there was fibrous tissue that surrounded mature collagen fiber bundles that were covered with an oral epithelial layer. Large, interconnected mature bone trabeculae enclosing plumbed osteocytes filled the socket (Fig. 4i, l).

Histomorphometric results

After 2 and 4 weeks of extraction, there was a significant variation in the area percentage of newly formed bone among all groups (p = 0.0001). Group Ι showed the least area percentage of newly created bone, while groups II and III showed a rise in the area percentage of newly formed bone compared to group Ι. Additionally, there was a significant variation between groups II and III, in favor of group III, at both time points (Fig. 5a; Supplementary Table S3). In the same context, there was a significant rise in the quantity of newly created bone in the extraction socket of all groups from 2 to 4 weeks (Fig. 5b; Supplementary Table S4).

Fig. 5.

Fig. 5

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Bar chart showing the area percentage of newly formed bone in the extraction socket of all groups after 2 and 4 weeks (a) intergroup comparison, * indicates a significant difference between groups using ANOVA. Bars with different letters indicate a significant difference using Tukey post hoc test, and (b) intragroup comparison, * indicates a significant difference between groups using the paired t test. Significance at P < 0.05. n = 5/time point

Quantitative real-time polymerase chain reaction (qRT-PCR) results

There was a significant variation in TGF-β1 and Runx2 expressions in groups II and III relative to group I (p = 0.0001), with a further significant difference between groups II and III, in favor of group III, at 2 and 4 weeks post-extraction. Intragroup comparisons revealed a significant elevation in TGF-β1 gene expression from 2 to 4 weeks in groups ΙΙ and ΙΙΙ. On the contrary, a significant decline in Runx2 gene expression was noticed from 2 to 4 weeks in groups ΙΙ and ΙΙΙ (Figs. 6 and 7; Supplementary Tables S5–S8).

Fig. 6.

Fig. 6

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Bar chart showing mRNA gene expression of TGFβ-1 in all groups after 2 and 4 weeks (a) intergroup comparison, * indicates a significant difference between groups using ANOVA. Bars with different letters indicate a significant difference using Tukey post hoc test, and (b) intragroup comparison, * indicates a significant difference between groups using the paired t test. Significance at P < 0.05. n = 5/time point

Fig. 7.

Fig. 7

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Bar chart showing mRNA gene expression of Runx2 in all groups after 2 and 4 weeks (a) intergroup comparison, * indicates a significant difference between groups using ANOVA. Bars with different letters indicate a significant difference using Tukey post hoc test, and (b) intragroup comparison, * indicates a significant difference between groups using the paired t test. Significance at P < 0.05. n = 5/time point

Discussion

Since the first report on MRONJ was released [30], various modalities aimed at preventing or treating this life-threatening morbidity have been investigated [31, 32]. The objective of the present study was to evaluate the reparative/regenerative ability of vitamins A/C-primed hGMSCs at pluripotency-inductive concentrations in the prevention of MRONJ in an osteonecrotic rat model.

In line with earlier research [33], ZA was used in combination with dexamethasone to induce osteonecrosis. The maxilla was selected as the evaluation site in this investigation, as it presents a lower risk of complications compared to the mandible. Extractions in the mandible are often associated with fractures and feeding difficulties, whereas the maxilla presents a safer alternative. Its thinner cortical bone and greater vascularity favor better healing outcomes and provide a greater consistency in the subsequent assessment. This methodological choice was also supported by previous studies that have successfully induced MRONJ in the maxilla [34, 35].

hGMSCs were chosen, being readily available and possessing superior biological characteristics [3], including fostered bone repair [5] and a notable capacity for repair/regeneration [7, 36]. A combination of vitamins A and C at particular concentrations would affect cellular epigenetics, via demethylation of nuclear bases, with a subsequent induction of hGMSCs’ stemness and reparative/regenerative capacity [19, 20]. Although this combined application demonstrated remarkable effects on hGMSCs in vitro [37], a combination of both vitamins was not utilized before in an animal model for the prevention of MRONJ.

In the herein study, all interventions were administered immediately after extraction and delivered locally since local administration, as opposed to a systemic one, permits the agent to act on the target without its bioavailability being impacted by digestion and gastrointestinal absorption. Moreover, as it can be administered when the tooth is being extracted, a subsequent translational clinical practice implementation would be straightforward [35].

No graft rejection was observed in the present work, likely due to the combined effects of hGMSCs and dexamethasone. hGMSCs possess low immunogenicity and actively release anti-inflammatory factors that reduce host immune responses [38, 39], while dexamethasone provides additional immunosuppression [40]. Together, these factors could have created a favorable environment that prevented rejection of the transplanted cells during the 2–4 weeks experimental period. This was in line with previous studies that also reported the successful use of hGMSCs in animal models for regenerative approaches [36, 41].

In this investigation, rats were euthanized at 2 and 4 weeks following extraction. This 2–4 weeks observation period was chosen because rodents exhibit faster bone turnover and healing than humans, so this time frame would analogously represent the early and intermediate phases of healing, during which MRONJ-like lesions can be reliably induced in rodents, and is consistent with previous preclinical MRONJ studies [24, 33, 34].

In the present study, DEXA results exhibited a significant elevation in BMD in groups II and III with a significant rise in group III at 4 weeks. These findings were similar to earlier findings where after 4 and 8 weeks of MSC injection into the tail vein of ovariectomy mice (in a cell homing approach), a remarkable rise in BMD was observed [42]. Comparably, the tibias of ovariectomized rats receiving vitamin C therapy recovered their BMD [43]. In the same context, it was reported that a greater BMD was linked to increased vitamin A consumption through diet [44].

In the current work, the histopathological examination of group I at 2 weeks displayed clear osteonecrotic signs, which were aggravated at 4 weeks, verifying the osteonecrosis model employed. The resultant condition imitated the pathology of MRONJ‐like lesions 2 and 4 weeks post-extraction [24] and could be attributed to the fact that administering ZA may hinder tissue healing and regeneration through preventing stem/progenitor cells that are located around the jawbone from migrating to the necrotic area [45]. Moreover, BPs cause blockage in the signaling pathway of bone marrow cells and inhibit the deposition of bone mineral of osteoblasts and bone remodeling in the extraction socket [46]. Additionally, BPs are directly harmful to the oral epithelium, preventing the overlying soft tissue from healing [47].

In group II, many of the histopathologic events linked to osteonecrosis were significantly reversed by hGMSCs at 2 weeks, with more healing signs observed at 4 weeks. The results were concomitant with previous findings, in which MSCs injected into the osteonecrotic femoral head of the rabbit model achieved greater repair, with large quantities of fibrous tissue and cartilage covering the necrotic femoral head [48]. In group III, the onset of MRONJ was suppressed at 2 weeks, with new mature bone formation at 4 weeks, in accordance with earlier findings [49], demonstrating that the local use of an osteogenic inducer improved extraction socket healing in rabbits, enhancing the quality of the created bone, with substantial production of lamellar bone in the sockets. Moreover, all-trans retinoic acid (ATRA) was shown to potentiate bone morphogenic protein (BMP) 9-induced osteogenic differentiation of the mesenchymal progenitor cell line by inducing alkaline phosphatase (ALP) activity in vitro [50]. The histomorphometric analysis of new bone formation further supported these histopathological results. In line with earlier results [51, 52], the area percentage of newly created bone exhibited a significant increase in groups II and III compared to group Ι, with a significant rise in group III at both time intervals.

In accordance with earlier findings observed with adipose-derived stem cells injected in animals receiving BPs [53], in the current work, a significant elevation in TGF-β1 in groups II and III relative to group I, with a significant increase in group III at both time intervals, was further evident. Similarly, mice supplemented with ascorbic acid had significantly elevated TGF-β expression compared to deficient mice after 7 days of wound healing [54]. Moreover, a study on necrotizing enterocolitis found that vitamin A administration activated TGF-β1, which contributed to accelerated healing [55].

In the herein study, the expression of Runx2 in both groups II and III showed a significant rise relative to group I. These findings were consistent with a previous study where ascorbic acid supplementation in hGMSCs cultures was linked to osteogenesis, leading to enhanced mineralization and elevation of osteogenic genes Runx2, BMP-2/4, and osteopontin (OPN) [56]. Furthermore, it was demonstrated that osteoblast differentiation was greatly accelerated by low dosages of vitamin C, as was evident by the increase of Runx2 gene expression [57]. Treatment with retinoic acid was additionally shown to significantly increase Runx2 expression and osteoblastic differentiation [58]. ATRA encouraged bone marrow MSCs to differentiate into osteoblasts and increased Runx2 expression, bone formation, and mineralization density [59]. Yet, being an early osteogenic marker and concomitant with earlier findings [48], Runx2 expression in both hGMSCs groups significantly decreased at 4 weeks compared to 2 weeks.

The pronounced preventive potential observed in vitamins A/C-primed hGMSCs group against MRONJ could be attributed to the complementary roles of retinol and ascorbate in regulating the epigenetic and osteogenic pathways. Retinol and ascorbate act in combination to promote DNA demethylation and enhance reprogramming toward naïve pluripotency by targeting ten–eleven translocation (TET) enzymes. Retinol upregulates TET2 and TET3 expression, while ascorbate sustains their catalytic activity by maintaining iron in its reduced Fe2⁺ state. Through these mechanisms, both vitamins facilitate the removal of epigenetic memory and support stem cell renewal [60]. In addition, vitamin C has been shown to potentiate osteoblastic function via enhancing osteoblast proliferation, increasing trabecular bone formation, and inducing bone matrix genes such as ALP, OPN, and Runx2 [61, 62]. Analogously, vitamin A contributes to bone physiology regulation through retinoic acid receptor-mediated signaling [63], with ATRA markedly elevating osteoblastogenesis and osteogenesis [59].

Limitations

Yet, the current findings should be carefully interpreted in light of the present animal study’s limitations. First, the 2- to 4-week observation period, while appropriate for capturing early and intermediate phases of post-extraction healing in rodents, does not fully replicate the chronic progression of MRONJ in humans, which typically develops over several months. Second, although our findings demonstrate the beneficial effects of primed vitamins A and C in combination with hGMSCs, the exact underlying molecular mechanisms remain unclear. Third, only male rats were included in this study, so sex-specific differences were not considered, which may limit the generalizability of the findings. Future studies with longer follow-up periods and deeper mechanistic investigations at cellular and molecular levels are warranted to better understand how biomolecular priming enhances stem cell-mediated regeneration and to more closely imitate the clinical progression of MRONJ in experimental models.

Conclusions

The current findings reveal that priming hGMSCs with vitamins A and C at the defined pluripotency-inductive concentrations may enhance soft tissue integrity and promote osteogenic homeostasis, thereby contributing to the prevention and early treatment of MRONJ. This preventive strategy appears to sustain bone formation and mineral density, facilitating balanced bone remodeling through the upregulation of Runx2-mediated osteogenic differentiation. The present study’s results highlight the potential of combined vitamins A/C-based preconditioning as a prophylactic approach against MRONJ and offer new insights and strategies for reducing the risk of other bone-related complications.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 30 KB) (30.3KB, docx)

Acknowledgements

The authors would like to thank Dr. Bassam Ahmed for calculating the sample size and Dr. Sherihan Eissa for performing the statistical analysis of the current work.

Abbreviations

ALP

Alkaline phosphatase

ATRA

All-trans retinoic acid

BPs

Bisphosphonates

BMD

Bone mineral density

BMP

Bone morphogenic protein

DEXA

Dual-energy X-ray absorptiometry

hGMSCs

Human gingival mesenchymal stem/progenitor cells

MRONJ

Medication-related osteonecrosis of the jaw

MSCs

Mesenchymal stem/progenitor cells

OPN

Osteopontin

Runx2

Runt-related transcription factor 2

TGF-β1

Transforming growth factor- β1

ZA

Zoledronic acid

Author contributions

NSK, KFE, and NA contributed to the study’s conception and design; data acquisition, analysis, and interpretation; drafting and revising of the manuscript. FIE contributed to data acquisition, analysis, and interpretation; drafting and revising of the manuscript. All authors provided their final approval and agreed to be accountable for all aspects of this work.

Funding

Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB). No funding was received for conducting this study.

Data availability

All data generated or analyzed during this study are included in this published article.

Declarations

Conflict of interest

The authors have no relevant financial or non-financial interests to disclose.

Ethical approval

This animal experiment was accepted by the Institutional Animal Care and Use Committee (IACUC) of Cairo University with approval number (CU|III|F|58|21). All animal procedures were performed according to the ARRIVE guidelines. Gingival sample collection was carried out in compliance with Cairo University’s Faculty of Dentistry’s Research Ethics Committee guidelines with approval number (1|1|23). This work was carried out in accordance with the Declaration of Helsinki.

Informed consent

Donors provided written informed consent for the use of free gingival soft tissue collars that were surrounding their extracted third molars for research purposes.

Footnotes

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