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. 2020 Jun 25;17(5):705–715. doi: 10.1007/s13770-020-00277-1

Promoting Osseointegration of Dental Implants in Dog Maxillary Sinus Floor Augmentation Using Dentin Matrix Protein 1-Transduced Bone Marrow Stem Cells

Dong Ma 1, Yuanyin Wang 1, Yongxiang Chen 1, Gang Yang 1, Xin Liu 1,2,✉
PMCID: PMC7524955  PMID: 32588341

Abstract

Background:

Beta-tricalcium phosphate (β-TCP) has been employed successfully as a synthetic graft material in maxillary sinus floor augmentation (MSFA) for placing dental implants. However, the lack of osteogenic and osteoinductive properties of this substitute invariably results in bone regeneration of low quality and quantity. The purpose of this study was to determine whether loading dentin matrix protein-1 (DMP1) gene-modified bone marrow mesenchymal stem cells (BMSCs) onto β-TCP promoted bone regeneration and osteointegration of dental implants in MSFA of dogs.

Methods:

BMSCs were transduced with a lentiviral vector overexpressing the DMP1 gene (Lenti-DMP1) and with a lentiviral vector overexpressing enhanced green fluorescent protein (Lenti-EGFP) in vitro and were loaded into β-TCP scaffolds for autologous sinus grafting. Beagles received bilateral MSFA with four biomaterials (① Lenti-DMP1-transduced BMSCs/β-TCP, ② Lenti-EGFP-transduced BMSCs/β-TCP, ③ BMSCs/β-TCP, ④ β-TCP) and simultaneous implant placement at each sinus. Twelve weeks post operation, the maxillae were explanted, and every sinus was evaluated by radiographic observation, micro-CT and histological analysis. The osteogenic outcomes of bone regeneration and osseointegration were compared between the four groups.

Results:

The sinuses grafted with Lenti-DMP1-transduced BMSCs/β-TCP constructs presented a significantly higher increase in compact radiopaque area, higher local bone mineral densities, greater bone-implant contact and greater bone density when compared to other three groups.

Conclusion:

These results demonstrated that combinations of β-TCP and DMP1 gene-modified BMSCs could be used to construct tissue-engineered bone to enhance mineralization of the regenerated bone and osseointegration of dental implants in MSFA.

Keywords: Bone marrow mesenchymal stem cells, Dentin matrix protein-1, Oral implant

Introduction

In recent decades, titanium dental implants have been demonstrated to be an effective tool to support prostheses and have been widely used to restore missing teeth. Alveolar resorption and continued pneumatization of the sinus after loss of teeth in the posterior maxilla invariably result in an inadequate amount of bone for a successful implant placement [1]. Maxillary sinus floor augmentation (MSFA) using the lateral window technique has been demonstrated to be a reliable procedure to increase the alveolar bone height to permit placement of longer implants with high success rates [2, 3]. Standard MSFA procedures necessitate elevating the sinus membrane and inserting bone grafts into the augmented cavity. Currently, an increasing number of bone substitutes are used to avoid the complications accompanying autogenous bone grafting [1, 2, 4, 5]. However, most of these biomaterials, such as β-TCP, are characterized by poor osteogenic or osteoinductive properties in contrast to autogenous bone grafts [1, 6, 7]. To improve the osteogenic capability of these biomaterials, stem cells and growth factors are always used in combination with the biomaterials [5, 8–10]. However, direct application of osteoinductive proteins results in temporal and spatial expression of the functional protein [8, 11]. Comparatively, the gene therapy approach could be a promising alternative strategy because it allows targeted delivery of the protein of interest at the desired site and prolonged expression of growth factors [12, 13].

Dentin matrix protein-1 (DMP1) is a highly phosphorylated protein that has a crucial role in bone mineralization. In vitro and in vivo experiments confirmed enhanced osteogenesis and mineralization upon overexpression of DMP1 [12, 14–16]. In our latest animal study, the combination of DMP1-mediated bone marrow stromal cells (BMSCs) and Bio-Oss was used to enhance bone regeneration during MSFA and yielded encouraging results [12]. However, that study did not explore the impact of transduction of the DMP1 gene on the biological characteristics of BMSCs in vitro. Furthermore, the growth and differentiation of mesenchymal stem cells (MSCs) was greatly influenced by the composition of the scaffold materials. Bio-Oss is an inorganic bone graft obtained from bovine bone after removal of organic components that is a long-lasting material that will not be completely resorbed over time [2, 4]. Several studies have reported that Bio-Oss may hamper the interactions between osteogenic cells and native bone tissue and delay the replacement of new bone formation in less-vascularized environments [17, 18]. Thus, the success of our stem cell-based study was limited and was associated with poor clinical predictability within a short time frame because the resistance to resorption of Bio-Oss may influence the reliability and efficiency of biological processes, such as cell growth, vascularization and bone regeneration in MSFA.

In the present study, the impact of transduction of the DMP1 gene on the biological characteristics of BMSCs in vitro was explored. Furthermore, we investigated the osteogenic outcome of the transplantation of a tissue-engineered complex of β-TCP combined with BMSCs transduced with a lentiviral vector overexpressing the DMP1 gene (Lenti-DMP1) in MSFA of beagles. A series of clinical, radiographic and histologic evaluations by dental computed tomography, microcomputed tomography and histomorphological analysis were conducted to test the effectiveness of this gene therapy.

Materials and methods

Animal models

Twelve adult female beagles within 24 sinuses in a weight of 19.80 ± 2.15 kg were used for the experiments under a protocol approved by the Animal Research Committee of Anhui Medical University, China (No. 20160130). Beagles received bilateral MSFA within four biomaterials (① Lenti-DMP1 transduced BMSCs/β-TCP constructs, ② Lenti-EGFP transduced BMSCs/β-TCP, ③ BMSCs/β-TCP, ④ β-TCP) at each sinus respectively, and each group involved six samples.

BMSC isolation and culture

Six milliliters of autologous bone marrow was aspirated from the iliac crest of each beagle. BMSCs were isolated from the marrow by Percoll density gradient centrifugation and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY, USA) with 10% fetal bovine serum (FBS) (HyClone, Logan, UT, USA) and 1% penicillin/streptomycin (Invitrogen, Shanghai, China) at 37 °C in 5% CO2. The medium was renewed 2 times a week to remove non-adherent cells. BMSCs at passage 3 were used in the study.

Gene transduction of BMSCs

BMSCs were transduced with lentivirus overexpressing green fluorescent protein (EGFP) or lentivirus overexpressing DMP1 and EGFP (DMP1/EGFP) at a multiplicity of infection (MOI) of 4 pfu/cell. Cell morphology was evaluated under a light microscope and fluorescence microscope (Leica DM 1 RB, Wetzlar, Germany).

Gene expression of osteogenic markers

All procedures were carried out according to the instructions of the manufacturers. Total cellular RNA was extracted on days 3, 6, 9 and 12 after gene transduction using TRIzol Reagent (Invitrogen Life Technologies, Carlsbad, CA, USA). The quality and quantity of the RNA were obtained using a RNeasy Mini kit (Qiagen, Hilden, Germany). Reverse transcription was performed using 2 μg of RNA in a 20 μl final volume using a RevertAid First Stand cDNA Synthesis Kit (Thermo Scientific™, Waltham, MA, USA). Quantitative PCR was carried out with the ABI 7300 Real Time PCR System (Applied Biosystems, Foster City, CA, USA) under the following cycling conditions: 120 s at 95 °C for predenaturation, denaturation at 95 °C for 5 s, 40 cycles of annealing at 60 °C for 15 s and a final dissociation step at 95 °C for 30 s, followed by 55 °C for 30 s, and 95 °C for 30 s. Gene-specific primers for collagen 1 (Col1), runx2 (Runx2), osteocalcin (OCN), osteopontin (OPN) and the reference gene actin were synthesized (Invitrogen Biotechnology Co., LTD, Shanghai, China). Threshold cycle values were calculated by normalizing to the actin values. The relative expression levels of these osteogenic genes were compared to those in the Lenti-EGFP-transduced controls on days 3, 6, 9, 12 and 15.

Seeding BMSCs onto β-TCP

OSTEON™ Sinus, a commercial β-tricalcium coating on an HA scaffold with a porosity of 77%, was selected as the cell scaffold. Twenty-four hours after infection, Lenti-DMP1/EGFP- and Lenti-EGFP-transduced BMSCs and control BMSCs were collected and concentrated to 1 × 105 cells/500 µl in sterile saline solution separately. Each cell suspension was gently pipetted onto the β-TCP scaffolds until homogeneous drop saturation. The BMSC/Bio-Oss constructs were incubated for an additional 1 h to allow for cell attachment and were cultured in 1 ml complete medium for 24 h.

Maxillofacial sinus floor augmentation and simultaneous implant placement

The bilateral maxillary first molars (M1) of each beagle were extracted. After 3 months of healing, anesthesia was performed by intravenous injection of sodium pentobarbital (0.5 mg/kg). The sidewalls of the frontal sinuses were exposed by elevation of a full thick mucoperiosteal flap, and rectangular bone windows measuring approximately 10 mm × 15 mm were fenestrated to access the sinus. In the case of the perforation, the sinus membrane was peeled carefully and elevated inward to a height of approximately 6 mm from the bony floor (Fig. 1A). An implant measuring 4.1 mm in diameter and 10 mm in length (Straumann Standard SLA implants, RN Plus; Institut Straumann AG, Basel, Switzerland) was placed parallel to the midpoint line of the residual ridge and penetrated the floor of each sinus by 3–4 mm (Fig. 1B). Every implant protruded more than 4 mm into the right and left sinuses of each animal, and 4 different grafts of approximately 0.5 cc (DMP1 group: Lenti-DMP1/EGFP-transduced BMSCs + β-TCP; Control groups: Lenti-EGFP-transduced BMSCs + β-TCP; BMSCs + β-TCP; β-TCP) were consolidated into the sinus cavity underneath the membrane (Fig. 1C), and the wound was closed using interrupted sutures.

Fig. 1.

Fig. 1

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MSFA model in beagles: A elevation of the sinus membrane through lateral approach; B simultaneous implant placement; and C filling of the bone substitutes

Sample preparation

The animals were sacrificed 12 weeks after surgery. They were exsanguinated and perfused through the jugular veins with 10% buffered paraformaldehyde solution. After CT scan, the bone blocks with implants for the augmented site were removed from the maxillae, fixed in the same 10% buffered paraformaldehyde solution and embedded in polymethylmethacrylate.

Computed tomography (CT) scan analysis

After removing the maxillae of the animal, a maxillae three-dimensional (3D) CT scan for each beagle was acquired through a dental CT scanner (Meyer Digital 3D Oral Imaging System SS-X9010DPro-3D, Meyer, Hefei, China). The Meyer Digital 3D Oral Imaging System DCTViewer v.1.08 was used to analyze the CT data by reconstructing coronal tomograms vertical to the occlusal plane and the nasal septum plane of each sinus. The mean bone density values in Hounsfield units (HU) were evaluated for the sinus area excluding the implant site. According to the classification by Lekholm and Zarb [19], bone mineral density values > 400 HU were designated as bone type I between 200 HU to 400 HU as bone types 2 and 3, and < 200 HU as bone types IV and V. The percentages of bone type I and type IV were recorded and compared between groups. All measurements were taken 3 times by 2 independent researchers.

Micro-CT measurement

The radiographic morphology of each block sample was scanned and assessed using an animal micro-CT scanner system (SkyScan 1176, Bruker AXS Inc., Kontich, Belgium) with the following parameter settings: Header length (bytes) = 1134, pixel matrix = 692 × 692; pixel size = 35.034 um; and threshold value from 40 to 100. In addition, the bone from a distance of 1 mm to the implant surface was segmented and evaluated by a three-dimensional histomorphometric analysis. The ratio of bone volume to total volume (BV/TV) and bone mineral density (BMD) were recorded and compared between groups.

Histological analysis

The fixed specimens were cut into 100-µm sections longitudinally through the mid-plane of the implants and were further cut into 30 µm thick sections by grinding. The sections were stained with hematoxylin and eosin, observed and imaged using a microscope (Carl Zeiss, Inc., Oberkochen, Germany). Then, a series of quantitative analyses, including osseointegration assessment, bone area fraction and residual bone substitute material volume (BSMV), were performed using Image Pro 6.0 software (Media Cybernetics, Silver Springs, MD, USA). To calculate these parameters in terms of the grafting area, the osseointegration assessment was evaluated by the bone implant contact (BIC) excluding the native bone surrounding the neck of implants. Bone area fraction and BSMV were calculated at four randomly selected areas of no less than 4 mm apical from the native bone and 1 mm from the implant surface. BIC was defined as the direct contact length of the bone surface and the implant surface divided by the total length of the implant surface. The bone area fraction was reported as the percentage of red fluorescence band area of the total area at this site, and the BSMV was defined as the corresponding remaining scaffold area divided by the total area.

Statistical analysis

All the data obtained are reported as the mean ± standard deviation (SD). Statistical analyses were performed using the SAS 8.2 statistical software package (Cary, NC, USA). The statistical significance of the differences between two groups were determined by the Mann–Whitney test. For more than two groups, one-way ANOVA followed by Tukey’s post-test was utilized, and P < 0.05 was considered significant.

Results

Gene transduction and expression of DMP1

Approximately 95% of BMSCs showed green fluorescence without excessive cell death in vitro after transduction with Lenti-DMP1/EGFP and Lenti-EGFP for 3 days. Cells in the DMP1/EGFP group and EGFP group showed comparable normal cellular morphology compared to the untransduced control cells. Quantitative PCR showed significantly higher DMP1 gene expression in the DMP1 group than in the EGFP group and untransfected group (Fig. 2).

Fig. 2.

Fig. 2

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Lenti-DMP1/EGFP- and Lenti-EGFP-transduced BMSCs (A, C Lenti-DMP1/EGFP-transduced cells; B, D Lenti-EGFP-transduced cells; E DMP1 gene expression 3 days after transduction). *Statistically siginificant differences (p < 0.05) for the DMP1 group compared to the other groups

Detection of bone-associated gene expression in the Lenti-DMP1/EGFP group and Lenti-EGFP group by real-time quantitative PCR

The Lenti-DMP1/EGFP-transfected BMSC group had significantly higher expression of COL-1, RunX2, OCN and OPN gene transcripts than the Lenti-EGFP-transfected BMSC group at 9, 12, and 15 days. In the Lenti-DMP1/EGFP-transfected BMSC group, the expression of all four osteogenic genes showed a slight increase at day 9, dramatic upregulation at day 12 and moderate decrease to the basal level at day 15. In comparison, the transcripts of these osteogenic markers in the Lenti-EGFP-transfected BMSC group always stayed at the initial level (Fig. 3).

Fig. 3.

Fig. 3

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Gene expression analysis of 4 osteogenic markers in DMP1/EGFP-transduced BMSCs and EGFP-transduced BMSCs at specific time points: A COL-1; B RunX2; C OCN. *Statistically siginificant differences (p < 0.05) between the DMP1/EGFP group and EGFP group

Scanning electron microscope observation

The Lenti-DMP1/EGFP-transfected cells were flat and attached to the surface of the scaffold 24 h after seeding. After 7 days, BMSCs were found growing into the cores of the scaffold within fibrils spreading to other cells and colonized deposition of cellular confluence (Fig. 4).

Fig. 4.

Fig. 4

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Scanning electron microscopic view of Lenti-DMP1/EGFP-transfected BMSCs on the surface of β-TCP: A 24 h after seeding, B 7 days after seeding and C overall cellular morphology on the scafflold 24 h later

Computed tomography (CT) scan analysis results

An obvious compact radiopaque area could be observed in the sinuses grafted with Lenti-DMP1-transduced BMSCs/β-TCP constructs (Fig. 5A). A significantly larger proportion of type I bone with high radiographic density (48.92 ± 7.82%) and a lower percentage of type IV bone (19.98 ± 4.15%) were found in the Lenti-DMP1/EGFP group compared to the three control groups (Fig. 5B–D). No significant differences in the control groups were detected.

Fig. 5.

Fig. 5

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Computed tomography (CT) scan analysis of the newly formed bone classification: A coronal section of beagle maxilla; B Lenti-EGFP-transduced BMSCs/β-TCP in the right situ; C Lenti-DMP1/EGFP-transduced BMSCs/β-TCP in the left situ. Bone type I area of hounsfield units (HU) > 400 was recorded as red, and bone types IV area of HU < 200 was recorded as blue; D The percentages of bone type I and type IV were compared between groups. *Statistically siginificant differences (p < 0.05) for the DMP1 group when compared to the control groups

Micro-CT measurement results

The bone surrounding the implants as far as 2 mm from the surface of the implants was evaluated (Fig. 6A). The Lenti-DMP1/EGFP group presented a larger relative bone volume (BV/TV), greater thickness of the trabecular bone (Tb.Th) and more new bone trabeculae (Tb.N) than the three control groups based on the micro-CT scan (Fig. 6B–D). The difference of these parameters for three control groups were not statistically significant.

Fig. 6.

Fig. 6

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A The extent for micro-CT evaluation; B relative bone volume (BV/TV); C thickness of the trabecular bone (Tb.Th); D new bone trabeculae (Tb.N). *Statistically siginificant differences (p < 0.05) for the DMP1 group when compared to the control groups

Histological analysis results

Bright green fluorescence spots were observed distributing in the trabeculae like area in DMP1/EGFP and EGFP groups, and the BMSCs were supposed to actively participate in the new bone formation in both groups. More green fluorescent spots and greater green dyed area of connections between spots were found in the DMP1 group compared with the control groups, which indicated greater bone formation in the DMP1 group (Fig. 7A, B). New bone formation at the grafted area in 4 groups was close to that of the unabsorbed material. A compact bone with deep dyeing arranged trabecular was found in the DMP1 group (Fig. 8A–C). Comparatively, cancellous bone with less maturation was observed in the EGFP group (Fig. 8D–F). The BIC rate was 39.24 ± 6.72% in the DMP1 group and nearly 28% in the control groups. The bone area fraction was 58.75 ± 5.58% in the DMP1 group and nearly 35% in the control groups. The residual bone substitute material volume (BSMV) was 23.14 ± 6.74% in the DMP1 group and about 32% in the control groups (Fig. 9). A significant difference of above parameters was shown in DMP1 group when compared to the control groups, whereas the difference for three control groups was not statistically significant (p < 0.05).

Fig. 7.

Fig. 7

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Fluorescent evaluation for new bone formation in the DMP1 group (A) and the EGFP group (B) (A and B 4×). More green fluorescent spots and greater green dyed area of connections between spots were found in the DMP1 group compared with the EGFP group. NB new bone, RS residual scaffold

Fig. 8.

Fig. 8

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Histologically evaluated area of the BIC, bone area fraction and BSMV from square area (excluding the native bone) in the DMP1 group (A–C) and the EGFP group (D–F) (A and D: 1.25×; B and E: 4×; C and F: 20×). NB new bone, RS residual scaffold. Squared area in A and D corresponds to B and E respectively; Four randomly graft sites of more than 4 mm distance from sinus floor were selected for evaluation

Fig. 9.

Fig. 9

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Comparison results of DMP1 group and EGFP group in Bone-implant contact, Bone area fraction, Residual bone substitute material volume. *Statistically siginificant differences (p < 0.05) for the DMP1 group when compared to the control groups

Discussion

In dental implant surgery at posterior maxillary sites, MSFA using only bone substitutes has been documented as an effective approach to provide sufficient and qualified bone mass for successful oral implantation [1, 2, 4, 5]. Nonetheless, these bone substitutes are always associated with osteoconductive scaffolds lacking osteogenic and osteoinductive properties. The sole use of this material always results in poor bone regeneration and failed osseointegration with inadequate strength even after a prolonged healing time of no less than 9 months [1, 2, 4, 5]. Thus, numerous osteogenic cytokines have been tested to enhance the mineralization and maturation of regenerated bone in the presence or absence of stem cells [5, 8–10]. However, only adding stem cells to the bone substitute seemed to not contribute to the efficacy of bone regeneration in MSFA [5]. Based on the results of our previous study [12], DMP1 overexpression by transfected BMSCs can be used to improve the maturation of demineralized bovine bone mineral in MSFA. Functional cells from the genetically targeted group were observed to be distributed in the mineralized ECM, and a higher BIC and bone area fraction were documented in the DMP1 group compared with the EGFP group. However, a similar high BSMV in both groups was recorded because the scaffold used in that research showed great resistance to resorption, and the resorptive capacity of a graft is determined by the property of this biomaterial. The possible effects of DMP1 on bone formation and material resorption might not be evaluated correctly in a short healing time. Thus, β-TCP was used in this study since it resorbed faster than DBBM. The DMP1 group showed a higher BIC (39.24 ± 6.72%) and bone area fraction (58.75 ± 5.58%) than the control groups (EGFP group: BIC: 28.18 ± 7.81%, bone area fraction: 36.28 ± 7.81%). Correspondingly, the BSMV percentage in the DMP1 group (23.14 ± 6.74%) was lower than that in the control groups (EGFP group: 33.26 ± 8.86%). Compared to the values in our previous DMP1 study using the Bio-Oss scaffold, a more promising effect of DMP1 was observed when β-TCP was applied. The histological results are consistent with the CT scan analysis and radiographic micromorphology in this study. A significantly larger proportion of radiographic opaque area with higher density in the DMP1 group than in the control groups was revealed, especially within 2 mm from the surface of the implants. The bone area fraction in the control group was consistent with the value of 41.34% in Shayesteh et al.’s study with the addition of BMSCs after a healing period of 4 months [20]. Similarly, approximately 28–35% bone area fraction and 27–35% residual grafted material were reported in the absence of BMSCs at 6 months post surgery [6, 7].

Dentin matrix protein 1 (DMP 1) is a highly phosphorylated protein that is essential for the normal development of hard tissues such as teeth and bones [21–23]. As a member of the SIBLING (Small integrin-Binding Ligand, N-linked Glycoprotein) family, it contains a large number of acidic clusters for calcium binding. Numerous in vitro and in vivo studies have demonstrated that DMP1 plays an important role in the biomineralization of dentin and bone, from initiating nucleation of hydroxyapatite to further regulating mineral deposition [24–26]. The expression of the DMP1 gene has been found in multiple new bone formation regions, and experimental studies have also proven that the deletion of the DMP1 gene can cause bone formation defects [21–23]. High expression of osteogenesis-related genes, including Col-1, RunX2, OCN and OPN, in DMP1-transduced cells was observed in our study at 12 days, and a similar upregulation of RunX2 activity was found in studies of the DMP1 functional mechanism. DMP1 was found to bind to a 78-kDa glucose-regulated protein called GRP78 and type 1 collagen in vitro to increase calcium deposition and upregulate bone-related genes by promoting Runx2 activity. [27–30]. The results of our research are also in line with Narayanan and Lu’s studies. Enhanced osteogenic differentiation and an increased size of mineralization nodules through overexpression of DMP1 in pluripotent embryonic undifferentiated mesenchymal cells or rat osteoblastic cells were documented in their studies [14, 31]. Full-length DMP1 is proteolytically cleaved into NH2-terminal 37-kDa and COOH-terminal 57-kDa fragments in the extracellular matrix [32]. Although the function of the full-length precursor and its cleavage products remains controversial, the proteolytic processing of DMP1 was speculated to be responsible for its function, and the C-terminal 57-kDa fragment was the critical product for mineralization [32, 33]. Gaijeraman’s research proved that posttranslationally modified full-length DMP1 and its C-terminal fragment had a positive effect on hydroxyapatite nucleation in the presence of type 1 collagen [24]. Conversely, Tartaix’s research indicated that phosphorylated recombinant full-length DMP1 inhibited mineralization [26]. In our research, quantitative PCR showed higher DMP1 gene expression in the Lenti-DMP1/EGFP groups using primers for full-length DMP1. An increased proportion of the COOH-terminal domain was assumed to be secreted in the presence of BMSCs, due to mechanisms such as BMP1/Tolloid-like proteinases present in the cells that are capable of processing DMP1 into discrete cleavage fragments [34]. However, simply coating the implant surface with DMP1 protein might result in a delay rather than an enhancement of osseointegration [35]. This may be explained by the lack of the appropriate circumstances for proteolytic processing of DMP1. Additionally, a previous study has proven that the molecular mass of posttranslationally modified DMP1 from tissues is larger than that of full DMP1 because DMP1 in mammalian tissues is always heavily phosphorylated [36]. Transgenic mice with inactivated phosphorylating SIBLING proteins always presented several bony defects, and the functional C-terminal fragment was much more highly phosphorylated than the N-terminal fragment, indicating that phosphorylation seemed to be another necessary process for the functional activation of this protein [37]. The phosphorylation status of DMP1 generally has a critical impact on the apatite crystal size. Tartaix reported that a highly phosphorylated C-terminal fragment in murine bone induced mineralization, whereas phosphorylated recombinant full-length DMP1 inhibited mineralization [26]. Thus, our future research should focus on the effect of proteolytic processes and phosphorylation on bone regeneration and maturation.

Acknowledgements

Thanks to the lab for technical support and the support of the leaders. The study was supported by the National Natural Science Foundation of China (No. 81271162) and the Provincial Natural Science Foundation (No. 1708085MH194).

Compliance with ethical standards

Conflict of interest

The authors have no financial conflicts of interest.

Ethical statement

The experiment was carried out after being approved by the Ethics Review Committee of Anhui Medical University (No. 20160130). All authors gave their informed consent to all matters related to the publication of this paper.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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