Adipose-derived stem cells prevent the onset of bisphosphonate-related osteonecrosis of the jaw through transforming growth factor β-1-mediated gingival wound healing
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Due to its complex pathogenesis and low clinical cure rate, bisphosphonate-related osteonecrosis of the jaw (BRONJ) poses a substantial challenge for oral and maxillofacial surgeons. Therefore, the treatment of BRONJ should focus on prevention. In clinical studies, primary wound closure can significantly reduce the incidence of BRONJ. Whether local stem cell transplantation can promote primary gingival healing in patients with a medication history and prevent BRONJ has not been reported.
In this study, animals were divided into a healthy group (non-drug treatment), a BP group, a hydroxyapatite (HA) group, and an adipose-derived stem cell (ADSC) group. All groups except the healthy group were treated with BPs and immunosuppressive drugs once per week for 8 weeks, simulating clinical use for the treatment of cancer patients with bone metastasis, to induce BRONJ-like animals. After the sixth drug treatment, the bilateral premolars were extracted in all groups. In contrast to the healthy and BP groups, the extraction sockets in the HA and ADSC groups were filled with HA or HA + ADSCs simultaneously post extraction to observe the preventive effect of ADSCs on the occurrence of BRONJ. At 2 and 8 weeks post extraction, animals from all groups were sacrificed.
At 8 weeks post transplantation, ADSCs prevented the occurrence of BRONJ, mainly through accelerating healing of the gingival epithelium at 2 weeks post extraction. We also found that ADSCs could upregulate the expression of transforming growth factor β1 (TGF-β1) and fibronectin in tissue from animals with a medication history by accelerating gingival healing of the extraction socket. A rescue assay further demonstrated that TGF-β1 and fibronectin expression decreased in TGF-β1-deficient ADSC-treated animals, which partially abolished the preventive effect of ADSCs on the onset of BRONJ.
ADSCs prevent the onset of BRONJ, mainly by upregulating the expression of TGF-β1 and fibronectin to promote primary gingival healing, ultimately leading to bone regeneration in the tooth extraction socket. Our new findings provide a novel stem cell treatment for the prevention of BRONJ.
KeywordsBisphosphonate-related osteonecrosis of the jaw Wound healing Zoledronic acid Fibroblast Fibronectin Adipose-derived mesenchymal stem cells
Adipose-derived stem cells
Basic fibroblast growth factor
Bone morphogenic protein
Bisphosphonate-related osteonecrosis of the jaw
Collagen type 1a1
Epidermal growth factor
Insulin-like growth factor
Medication-related osteonecrosis of the jaw
Mesenchymal stem cells
Peroxisome proliferator-activated receptors
Runt-related transcription factor 2
Transforming growth factor
Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is defined as a type of disease in which jaw bone is exposed in the maxillofacial region and persists for no less than 8 weeks in patients who are undergoing bisphosphonate treatment and have no oral radiation history . BRONJ is a type of medication-related osteonecrosis of the jaw (MRONJ) and has been correlated with high doses of bisphosphonates (BPs) . In addition, BRONJ was frequently observed in bone metastatic tumor patients who were treated in conjunction with immunosuppressive agents, and its incidence increased to 18% . Dexamethasone, as the most common immunosuppressive drug, was used to treat multiple myeloma and bone metastasis and was also required for the development of a BRONJ-like disease model [4, 5]. The main clinical manifestations are impaired gingival wound healing and bone exposure, with necrotic bone formation accompanied by pain or sinus formation . Among the risk factors, jaw trauma, especially tooth extraction and apical surgery, is a high-risk factor for BRONJ . To date, this disease still lacks effective treatments, and only 59% of cases can be partially or completely healed . In view of the low clinical cure rate, the treatment of BRONJ should focus on prevention.
Recent studies have indicated that primary closure after tooth extraction in BP-treated patients can effectively reduce the occurrence of BRONJ [8, 9, 10]. Notably, no MRONJ was observed when the tooth extraction wound was completely closed with a relaxation incision and/or sharp bone edges were removed, whereas an open wound increased the incidence to 56.1% . Therefore, a soft tissue covering would reduce the occurrence of BRONJ.
Adipose-derived stem cells (ADSCs) are harvested from subcutaneous fat, are a rich source of cellular factors and promote angiogenesis, which makes them suitable for soft tissue repair [12, 13, 14, 15]. ADSCs promote wound healing and antioxidation by paracrine cytokine activation [16, 17]. Stem cell-based treatment strategies are currently an effective method for the treatment of BRONJ. Notably, BRONJ-like lesions in mice and minipig models were successfully induced by BPs combined with dexamethasone and cured by systemic infusion of mesenchymal stem cells (MSCs) [5, 18]. Nevertheless, systemic infusion of MSCs may induce disseminated intravascular thrombosis and prolonged prothrombin time . Most importantly, in cancer patients, systemic infusion of MSCs increased the risk of cancer metastasis and recurrence [20, 21]. To avoid the side effects mentioned above, in this study, we attempted to elucidate whether local application of ADSCs can also prevent the occurrence of BRONJ.
Overall, the present study evaluated the effect of local ADSC transplantation on tooth extraction in BP-treated animals to prevent the occurrence of BRONJ. We hypothesized that local ADSC transplantation would induce soft tissue repair and prevent the occurrence of BRONJ after tooth extraction.
New England rabbits (n = 36) weighing 2.0 kg were used in this study. The animals were housed in a conventional room with a regular dark-light cycle (12 h) at room temperature (20–25 °C). Experimental procedures involving animals followed institutionally approved protocols for animal research at Peking University (LA2018017).
Zoledronic acid was purchased from Sigma (SML0223, St. Louis, MO, USA); dexamethasone was purchased from Sinopharm Group (Shanghai Chemical Reagent Co. Ltd., China); the human TGF-β1 ELISA kit (DB-100B) and anti-TGF-β1 (MAB-240) primary antibody were purchased from R&D Systems (R&D Systems, Inc., Minneapolis, MN, USA); and the anti-fibronectin primary antibody was purchased from Santa Cruz (sc-59826, Santa Cruz Biotech, Santa Cruz, CA, USA).
Induction of a BRONJ-like rabbit model and experimental design
Rabbits were maintained in a conventional room for 1 week to adapt to the environment. The BP, HA, and ADSC groups then received zoledronic acid (800 μg/kg, sml-0223, Sigma, USA) and dexamethasone (Dex, 10 mg/kg, Sinopharm Group, China) intravenously (I.V.) once per week for 8 weeks. The bilateral premolars were extracted under deep anesthesia by I.V. injection of pentobarbital (20 mg/kg, P3761, Sigma, USA) and xylazine (50 μl/kg, Jilin Huamu Animal Health Product Co., Ltd., China) after the sixth dose. For the ADSC group, ADSCs (passage 3–5) incubated on coral hydroxyapatite (HA) were immediately filled into the extraction sockets of animals with a medication history (ADSC group). Non-medication-treated animals and nonfilled animals (healthy group), medication-treated and nonfilled animals (BP group), and medication-treated and non-ADSC hydroxyapatite-filled animals (HA group) were used as controls. Then, at 2 and 8 weeks post extraction, the tooth extraction socket and the adjacent first molar were harvested at each indicated time. The unhealed area, necrotic bone, and new bone formation were measured based on clinical appearance and histology. All parameters mentioned above were quantified by ImageJ software (Version 1.51 s, US National Institutes of Health, Bethesda, Maryland).
Formation of the ADSC-hydroxyapatite complex
The complex of ADSCs and hydroxyapatite was made based on the previously described method . In brief, 5 × 106 ADSCs in 300 μl of serum-free α-MEM medium per 40 mg of spheroidal hydroxyapatite/tricalcium phosphate (HA, Beijing YHJ Science and Trade Co. Ltd.) were used and preincubated at 37 °C for 30 min and then kept on ice before filling the socket.
DiR fluorescence labeling of ADSCs
The commercially available lipophilic tracer 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide (DiR; Molecular Probes Inc.) was used to label live ADSCs. In brief, 1 × 106 ADSCs were stained with 1 ml (2.5 μg/ml) of DiR-labeling solution for 30 min at 37 °C, and the solution was then discarded after centrifugation (1500 rpm, 15 min) and resuspended in prewarmed (37 °C) phosphate-buffered saline (PBS). An extra centrifuge step was needed before incubation on HA.
The concentrations of TGF-β1 in human gingival fibroblasts (HGFs) and ADSCs were measured with culture supernatant in serum-free medium with a TGF-β1 ELISA kit (R&D, USA) following the manufacturer’s instructions.
Isolation and culture of ADSCs and HGFs
ADSCs were harvested from healthy donors. HGFs were harvested from healthy donors and BRONJ patients without bone metastases. All procedures were approved by the Ethics Committee of Peking University (IRB00001052-11002). The specific process is described in the Additional file 1: Supplemental methods.
The ADSCs were washed 3 times with PBS and harvested by digestion and centrifugation. The cells were then incubated with the following labeled antibodies: CD90-FITC, CD44-PE, CD73-APC, CD105-CY5.5, and negative cocktail-PE. Control-conjugated immunoglobulin G (IgG) was used as the isotype control. All antibodies were purchased from BD Biosciences (No.562245). Flow cytometry was performed with an Accuri C6 flow cytometer (BD Biosciences).
For osteogenic differentiation, third-passage ADSCs were cultured in osteogenic medium supplemented with 10 nm dexamethasone, 0.1 mm l-ascorbic acid phosphate, and 10 mm β-glycerophosphate (Sigma-Aldrich). The osteogenic medium was changed every 3 days. After 7 days, messenger RNA (mRNA) was isolated for osteogenic genes analysis. After 21 days, calcium nodes were stained with 2% Alizarin red (Sigma-Aldrich).
For adipogenic differentiation, third-passage BMSCs were cultured in adipogenic culture medium supplemented with 0.5 μm hydrocortisone, 0.5 mm 3-isobutyl-methylxanthine, 10 μg/ mL insulin, 60 μm indomethacin (Sigma-Aldrich), and 10% FBS. After 7 days, mRNA was isolated for adipogenic genes analysis. After 21 days, the cells were stained for cellular lipid droplets with Oil Red O (Sigma-Aldrich).
Immunochemistry-paraffin (IHC-P) staining
Immunochemistry was performed as previously reported . In brief, the tissues were fixed in 4% formalin for 72 h at 4 °C, decalcified in 10% EDTA for 3 weeks until easily pierced by a needle and then embedded in paraffin for the next procedure. Slides (4 μm) were made and deparaffinized, and endogenous peroxidase was eliminated. After antigen blocking and retrieval, the slides were incubated with primary TGF-β1 and fibronectin antibodies overnight at 4 °C, thoroughly rinsed with PBS, incubated for 20 min and then stained with a DAB kit. The area of tissue with positive staining was quantified by ImageJ software (US National Institutes of Health).
In vitro and in vivo siRNA interference assays
Double-stranded siRNA was used to silence TGF-β1 expression in ADSCs. The procedure and sequence used for the siRNA are available in the Additional file 1: Supplemental methods and Table S3. A satisfactory silencing efficiency in ADSCs was obtained after incubation on HA, and the cells were used in the animals with a medication history immediately post extraction (KD group).
Tissue and cell RNA isolation and real-time polymerase chain reaction (real-time PCR)
To lyse tissues into a paste, a mortar with liquid nitrogen and a high-throughput tissue crusher (Qiagen GmbH, Hilden, Germany) were used for bone and gingival tissues. Both tissue and cell messenger RNA (mRNA) were isolated using TRIzol reagent (15,596, Invitrogen Corp., Carlsbad, CA). Messenger RNA was reverse transcribed into complementary DNA (cDNA) with the GoScript Reverse Transcription System (Promega, Madison, WI, USA), and an ABI Prism 7500 (Applied Bioscience, PerkinElmer, Foster City, CA) was used to perform quantitative PCR. The relative mRNA abundance of target genes was determined by normalizing to the GAPDH threshold cycle and calculated using the △△Ct method. The primers are listed in Additional file 1: Tables S1 and S2.
Western blot assay
Proteins were extracted from ADSCs and fibroblasts, and Western blot assays were performed as previously described . Primary antibodies against GAPDH, transforming factor beta-1(TGF-β1), were purchased from R&D system (clone #9016).
Bone parameter acquisition and analysis in different groups
The bone parameters were analyzed as previously described . Computed tomography (CT) data were harvested by three-dimensional multi-image cone beam CT (60 kV, 2 mA, J. Morita Corp., Kyoto, Japan) and imported into the Inveon Research Workplace (SIEMENS, Munich, Germany) for further analysis. The region of interest was depicted, and the bone mineral density (BMD) and BV/TV were calculated.
Tartrate-resistant acid phosphatase (TRAP) staining
TRAP staining was performed as previously described . The specific process followed the instructions provided in the TRAP staining kit (Sigma-Aldrich, St. Louis, MO, USA), and the details are described in the Additional file 1: Supplemental methods.
All data are presented as the mean ± STD from at least 3 independent animals. The differences between experimental groups were analyzed by using one-way analysis of variance (ANOVA) followed by Tukey’s honestly significant difference (HSD) post hoc test by using GraphPad Prism 6 (GraphPad Software). A difference was considered significant if p < 0.05 (indicated by an asterisk).
Rabbit model of BRONJ-like lesions induced by BPs and DEX delivery
Local transplantation of ADSCs promotes gingival wound healing and prevents the occurrence of BRONJ-like lesions
Primary gingival healing by transplantation of ADSCs facilitate socket bone regeneration and upregulate osteogenic genes expression
Local ADSC transplantation improved suppressed TGF-β1 and fibronectin expression in BRONJ-like rabbits
TGF-β1 deficiency eliminated the ability of ADSCs to accelerate gingival wound healing in BP-treated animals
The highest incidence of BRONJ is observed in malignant tumor patients treated with a combination of BPs and DEX. However, based on the current treatment method, the cure rate of BRONJ is only 58.5% . It is particularly important to prevent the occurrence of BRONJ in patients with a medication history following a tooth extraction or other dentoalveolar surgery. In our study, we successfully induced a BRONJ-like rabbit model by simulating the clinical application of BPs and immunosuppressive drugs. BRONJ was successfully prevented when ADSCs were transplanted into the extraction wound. We first showed that ADSC transplantation elevated TGF-β1 and fibronectin expression under BRONJ conditions, contributing to the promotion of gingival wound healing and facilitating bone regeneration. Furthermore, when we knocked down TGF-β1 in ADSCs, these cells failed to promote primary gingival wound healing. In summary, local ADSC transplantation proved that ADSCs can mediate gingival wound healing through TGF-β1, and early soft tissue healing accelerated by ADSCs is important for preventing the onset of BRONJ.
In our study, we found that TGF-β1 and fibronectin expression was suppressed not only in BP-treated animal but also in patient gingival lesions, which may be connected to delayed gingival healing in BRONJ. During normal wound healing, fibronectin can induce the regeneration of epithelial tissue by recognizing the RGD sequence on the epithelial cell integrin receptor α5β1 [32, 33]. However, the absence of fibronectin expression in irradiated skin leads to skin wounds that do not heal properly . It was also reported that fibronectin is regulated by TGF-β1 in a c-Jun N-terminal kinase-dependent and Smad4-independent manner . Other studies also showed that BPs could inhibit the TGF-β1 pathway , leading to collagen deposition and restricted downstream signaling. The retarded gingival healing in BRONJ may be caused by suppressed fibronectin regulated by low levels of TGF-β1.
Retarded gingival healing with jaw bone exposure is the key clinical manifestation of BRONJ. Many studies have proven that soft tissue covering is the key factor in preventing BRONJ by mitigating secondary contamination and re-establishing a vascularized tissue bed that may nourish the diseased bone tissue [8, 37, 38]. Soft tissue is superior in terms of promoting inferior bone regeneration, as it prevents wound infection, serves as a local source of growth factors, and upregulates bone remodeling [39, 40, 41, 42]. Soft tissue also provides a bone anabolic environment through the expression of members of the TGF-β superfamily of growth and differentiation factors, including the bone morphogenetic proteins (BMPs), osteocalcin (OCN), and receptor activator of nuclear factor kappa-B ligand (RANKL) [42, 43, 44]. However, delayed gingival healing led to the invasion of bacteria into the tooth extraction wound, contributing to inflammatory infiltration and the development of BRONJ . In our study, local transplantation of ADSCs accelerated gingival healing and formed a sealed internal environment. We also found that early gingival healing significantly reduced inflammatory infiltration in subgingival connective tissue and provided a stable internal environment for bone regeneration. In addition, with coverage of the gingival tissue, we found that activated osteoclasts were rescued and that the expression of BMP-2, OCN, and other osteogenesis-related genes in the bone tissue was elevated. Therefore, the prevention of BRONJ by ADSCs is mainly achieved by the ability of the cells to promote early gingival healing and isolate the wound environment from the oral cavity, thus facilitating bone remodeling.
An increasing number of studies have proved that secreted cytokines perform the major therapeutic functions of transplanted stem cells [45, 46]. TGF-β1 is an important cytokine, particularly in ADSC-mediated wound healing. This cytokine can promote epithelial healing by mediating extracellular matrix (ECM) synthesis through upregulation of fibroblast collagen type I and III and fibronectin expression and facilitating fibroblast migration and proliferation to promote granulation tissue formation and reepithelization [27, 47, 48]. Even under hypoxic conditions, stem cells enhance TGF-β1 expression, accelerating dermal fibroblast migration, further elevating fibronectin expression and ultimately, promoting skin wound healing . In addition, due to low expression of TGF-β1 in diabetic ulcers, ADSCs can promote epithelial regeneration and granulation tissue formation by supplementing exogenous TGF-β1, thus promoting wound healing [50, 51]. Consistent with these studies, we also found that impaired gingival healing is accompanied by less TGF-β1 expression in BRONJ. In addition, local ADSC transplantation increased TGF-β1 expression and accelerated gingival healing. To further clarify the role of TGF-β1 in ADSC-mediated gingival healing, we knocked down TGF-β1, which eliminated the healing effects of ADSCs in BP-treated animals. Therefore, we believe that ADSCs induce gingival healing mainly mediated by TGF-β1 and thus prevent the onset of BRONJ.
In summary, we discovered that local ADSC transplantation provides a safe and effective therapeutic modality for preventing the onset of BRONJ by promoting primary gingival healing and represents a promising approach to clinical stem cell treatment.
We have revealed that local transplantation of ADSCs in animals with a medication history could prevent the onset of BRONJ, mainly by upregulating TGF-β1 and fibronectin expression and promoting gingival healing. Furthermore, TGF-β1 secreted from ADSCs is essential in the effects of primary gingival wound healing. This study provides a new perspective concerning how local stem cells act to prevent the occurrence of BRONJ.
Authors thank the center laboratory of Peking University School and Hospital of Stomatology for providing the research facilities. We appreciate the assistance from the Department of Imaging for data collection.
XLZ made substantial contributions to the conception and design and acquisition and analysis of data and contributed to drafting the manuscript. LHH contributed to the data acquisition and analysis and revised it critically for important intellectual content. LZ contributed to the animal surgery. YH contributed to the conception and design and has given final approval of the version to be published. EX made substantial contributions to the conception and design and interpretation of data. YZ has given final approval of the version to be published and revised the manuscript critically for important intellectual content. All authors read and approved the final manuscript.
This work was supported by a grant from the National Natural Science Foundation of China (No. 81371117) and the Beijing Natural Science Foundation (No. 7152155 & No. 7192230). The funding agencies had no role in the project design, experimental execution, analysis of the results, or preparation of the manuscript.
Ethics approval and consent to participate
The experimental procedures in this experiment to animals were under an institutionally approved protocols of animal research at Peking University (LA2018017).
Consent for publication
The authors declare that they have no competing interests.
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