Special AT‐rich sequence‐binding protein 2 (Satb2) synergizes with Bmp9 and is essential for osteo/odontogenic differentiation of mouse incisor mesenchymal stem cells

Abstract Objectives Mouse incisor mesenchymal stem cells (MSCs) have self‐renewal ability and osteo/odontogenic differentiation potential. However, the mechanism controlling the continuous self‐renewal and osteo/odontogenic differentiation of mouse incisor MSCs remains unclear. Special AT‐rich sequence‐binding protein 2 (SATB2) positively regulates craniofacial patterning, bone development and regeneration, whereas SATB2 deletion or mutation leads to craniomaxillofacial dysplasia and delayed tooth and root development, similar to bone morphogenetic protein (BMP) loss‐of‐function phenotypes. However, the detailed mechanism underlying the SATB2 role in odontogenic MSCs is poorly understood. The aim of this study was to investigate whether SATB2 can regulate self‐renewal and osteo/odontogenic differentiation of odontogenic MSCs. Materials and methods Satb2 expression was detected in the rapidly renewing mouse incisor mesenchyme by immunofluorescence staining, quantitative RT‐PCR and Western blot analysis. Ad‐Satb2 and Ad‐siSatb2 were constructed to evaluate the effect of Satb2 on odontogenic MSCs self‐renewal and osteo/odontogenic differentiation properties and the potential role of Satb2 with the osteogenic factor bone morphogenetic protein 9 (Bmp 9) in vitro and in vivo. Results Satb2 was found to be expressed in mesenchymal cells and pre‐odontoblasts/odontoblasts. We further discovered that Satb2 effectively enhances mouse incisor MSCs self‐renewal. Satb2 acted synergistically with the potent osteogenic factor Bmp9 in inducing osteo/odontogenic differentiation of mouse incisor MSCs in vitro and in vivo. Conclusions Satb2 promotes self‐renewal and osteo/odontogenic differentiation of mouse incisor MSCs. Thus, Satb2 can cooperate with Bmp9 as a new efficacious bio‐factor for osteogenic regeneration and tooth engineering.


| INTRODUC TI ON
Mesenchymal stem cells (MSCs) are subjected to complex and tight regulation by diverse growth factors and cytokines in osteogenic regeneration and tooth engineering. 1,2 The continuously growing mouse incisor provides a superior model for understanding the mechanisms of odontogenic MSCs self-renewal and osteo/odontogenic differentiation. 3 In the mouse incisor apical papilla, the pool of continuously growing MSCs (named mouse incisor MSCs) provides precursor cells to maintain renewal growth and mineralization. 4,5 Therefore, the identification and characterization of important regulatory factors that govern the stem cell properties and differentiation potential of mouse incisor MSCs should significantly expand our understanding of osteogenic regeneration and thus facilitate tooth engineering. Our previous studies indicated that mouse incisor MSCs, which are recognized as one of the most promising odontogenic stem cells in bone and tooth regeneration engineering, show the osteo/odontogenic differentiation potential would induced by effective biological factors. 6,7 As a member of a special family of AT sequence-binding proteins, special AT-rich sequence-binding protein 2 (SATB2), which binds to nuclear matrix-associated regions and activates gene transcription programs, is a multifunctional regulator involved in development, particularly in craniofacial patterning, palate formation, osteoblast differentiation and bone regeneration. 8 Human SATB2 is a highly evolutionarily conserved chromatin remodelling gene located on chromosome 2q33.1, 9 and mutation of SATB2 induces not only severe bone-associated conditions, including cleft palate, facial cleft, micrognathia and alveolar bone dysplasia [10][11][12][13] but also odontogenic abnormalities, including missing teeth, delayed tooth and root development. [14][15][16] Satb2 is responsible for osteoblast function to promote osteoblastogenesis and enhance bone regeneration by upregulating the expression of bone matrix proteins and osteogenic transcription factors in bone marrow mesenchymal stem cells (BMSCs). 10,17,18 These reports highlighted that SATB2 serves as a candidate biofactor for bone regeneration engineering. In order to further gain insights into the molecular mechanism through which SATB2 regulates osteo/odontogenic differentiation in odontogenic MSCs, we have recently conducted a comprehensive transcriptomic analysis of SATB2-regulated expression and demonstrated the SATB2 effectively regulates numerous osteogenic regulators and marker genes in a panel of human dental stem cells, including periodontal ligament stem cells, dental pulp stem cells and stem cells from human exfoliated deciduous teeth. 19 While these findings provided important insights into possible underlying mechanism of SATB2-regulated osteo/odontogenic differentiation, potential upstream regulators of SATB2 it remains to be fully investigated.
Bone morphogenetic protein 9 (BMP9), also called growth differentiation factor 2, has been reported to play a pivotal role in skeletal development, bone formation and stem cell differentiation. 20 Many transcription factors and cytokines, such as Runt-related transcription factor 2 (Runx2) 21 and Osterix (Osx), 22 mediate Bmp9-induced osteogenic differentiation of MSCs. We and others have recently demonstrated that BMP9 regulates dentinogenesis, 23 tooth root development and alveolar ridge height. 24,25 Interestingly, mice with Satb2 deficiency exhibit phenotypes similar to Bmp loss-of-function phenotypes. 26,27 Furthermore, Satb2 is considered a downstream effector of Bmp signalling and directly binds to Smad1/5 during osteoblast differentiation in facial skeletal development. 8 Nonetheless, the detailed mechanism through which SATB2 may mediate BMP9induced osteo/odontogenic differentiation of odontogenic MSCs remains to be fully elucidated.
In this study, we sought to investigate the function and possible mechanism of Satb2 inducing osteo/odontogenic differentiation of mouse incisor MSCs, as well as its potential synergy with the osteo/ odontogenic signalling factor, Bmp9. We found that Satb2 is expressed in mesenchymal cells in the apical papilla of mouse incisors and effectively enhances the self-renewal capacity of mouse incisor MSCs. We further demonstrated that Satb2 acts synergistically with Bmp9 in inducing osteo/odontogenic differentiation of mouse incisor MSCs in vitro and in vivo. Therefore, our findings strongly suggest that a thorough understanding of Satb2 functions should significantly facilitate our efforts to develop efficacious bone and tooth engineering approaches.

| Cell culture and chemicals
The 293pTP cell line was used for adenovirus packaging and amplification as previously described. 6 Both primary mouse incisor MSCs (see below) and 293pTP cells were maintained in complete Dulbecco's modified Eagle's medium containing 10% foetal bovine serum (FBS, HyClone, New York, USA), 100 units of penicillin and 100 µg of streptomycin at 37°C in 5% CO 2 . Unless mentioned otherwise, all chemicals were purchased from Beyotime (Shanghai, China) or HyClone(New York, USA).

| Immunofluorescence staining of Satb2 and Bmp9 expression in mouse incisors
The use and care of C57BL/6 mice and Sprague-Dawley (SD) rats in this study followed an animal protocol approved by the Research Ethics Committee of College of Stomatology, Chongqing Medical University, Chongqing, China (CQHS-REC-2020(LSNo.55) ).
Mandibular specimens from C57BL/6 mice (4-week-old males and females) were extracted for isolation of the incisor apical papilla.
Control IgGs and an absence of primary antibody were used as negative controls.

| Isolation, culture and characterization of mouse incisor MSCs
Primary mouse incisor MSCs were isolated from mouse incisor apical papilla tissues obtained from C57BL/6 mice (4 weeks old, both male and female) as previously reported. 28,29 See Supporting Information for details.

| Construction and amplification of recombinant adenoviruses expressing Bmp9, Satb2, siSatb2 and G/Rfp
Ad-Bmp9, Ad-Satb2, Ad-Gfp and Ad-Rfp recombinant adenoviruses were constructed using AdEasy technology as previously described. 6,19 Briefly, the coding region of the mouse Satb2 gene was PCR-amplified and subcloned into the adenoviral shuttle vector pAdTrack-TOX, which was subsequently used for homologous recombination with the adenoviral backbone vector in BJ5183 bacterial cells. The resulting recombinant plasmid was verified and used for adenovirus packaging in 293pTP cells, leading to production of the adenoviral vector Ad-Satb2, which also co-expressed the marker Gfp for tracking infection efficiency (Figure 3Aa).
To generate siRNA adenovirus targeting mouse/rat Satb2, we employed the siDESIGN program (Dharmacon, a Horizon Discovery Group company, Lafayette, Colorado, USA) and designed three siR-NAs that target the coding region of mouse Satb2, while two of these three siRNAs also target the coding region of rat Satb2 (Table S1;

Subconfluent cells were seeded in 35-mm dishes and infected
with Ad-Satb2, Ad-siSatb2 or Ad-G/Rfp. The infected cells were stained with crystal violet at the indicated time points.
Macrographic staining images were recorded. The stained cells were dissolved in 10% acetic acid at room temperature, and the optical density was measured at 570-590 nm for quantitative measurement.

F I G U R E 2 Isolation and characterization of stem cells of the incisor apical papilla. A, Isolation of mouse incisor mesenchymal stem cells (MSCs)
from mouse lower incisors. The incisors of adult C57BL/6 mice were separated (a) and carefully extracted (b). The apical papilla regions (b, dotted circles) were resected and retrieved for cell isolation. B, Mouse incisor MSCs were cultured in dishes for 48 h (a) and 96 h (b). C, Endogenous expression of SATB2, the MSCs markers CD90 and CD29 and the proliferation marker KI67 in mouse incisor MSCs

MSCs encapsulated in GelMA hydrogel
The proliferation and viability of mouse incisor MSCs encapsulated in GelMA hydrogel were evaluated via CCK-8 assays and live/dead staining. See Supporting Information for details.

| Rat calvarial defect repair model and micro-CT analysis
SD rats (8 weeks old) were anaesthetized, and a 5-mm-diameter critical-sized defect was created on each side of the rat calvarial bone using a dental bur (Figure 6Fa). See Supporting Information for details.

| Histologic evaluation
The retrieved rat cranial and mouse mandibular specimens were fixed, decalcified, dehydrated and paraffin embedded. Three randomly selected cross sections from each implant were stained with H&E. Trichrome staining was also carried out on rat cranial specimens.

| Statistical analysis
All quantitative data are presented as the means ± SDs. The continuous data were normally distributed, and the statistical significance was determined using Student's t test or one-way analysis of variance. P < .05 was accepted as statistically significant. All statistical analyses were conducted with the SPSS 26.0 statistical software (SPSS Inc, Chicago, USA).

| Satb2 and Bmp9 expression pattern in mouse incisors
Micro-CT (μCT) image showed a schematic diagram of a lower jaw incisor in a 4-week-old mouse ( Figure 1A). Magnification of the H&E-stained image shows the apical papilla region of the incisors characterized by continuous growth. The apical mesenchyme was located between the lingual cervical loop (LiCL) and labial cervical loop (LaCL) in the mouse incisor ( Figure 1B). To determine whether Satb2 and Bmp9 are expressed in the incisor growth region, immunofluorescence staining was conducted. Satb2 was found to be coexpressed with Bmp9 in pre-odontoblasts/odontoblasts. In addition, Satb2 was found in the mesenchyme, where Bmp9 expression was not detectable ( Figure 1C). Furthermore, qPCR analysis and Western blot revealed the Satb2 and Bmp9 mRNA and protein expression levels in the incisor growth region ( Figure 1D,E).

| Isolation and characterization of stem cells in the incisor apical papilla
To obtain insights into the role of Satb2 in the apical mesenchyme, mouse incisor MSCs were isolated from the lower incisors of mice ( Figure 2A). The primary mouse incisor MSCs grew well to at least 5 passages (Figure 2Ba-b). Mouse incisor MSCs from passages 3-5 were used in this study. MSCs markers (CD90, CD29) and a proliferation marker (KI67) 4,5 were expressed in the mouse incisor MSCs, while CD34 and CD45 were negative, as revealed by immunofluorescence staining. Moreover, mouse incisor MSCs were positive for SATB2 staining, which was observed in the nucleus ( Figure 2C).
Collectively, these results demonstrate that mouse incisor MSCs express MSCs markers and SATB2, suggesting that these cells may exhibit MSC-like characteristics.

| Validation of adenovirus-mediated Satb2 overexpression and silencing
To investigate the function of Satb2 in mouse incisor MSCs, we con-  BMSCs. 31 Moreover, ARS staining demonstrated that Satb2 significantly enhanced mineralized node formation ( Figure 4G).

| Satb2 is essential for the self-renewal and osteo/odontogenic differentiation capabilities of mouse incisor MSCs in vitro
As important markers of odontoblastic differentiation, the expression of dentin matrix protein 1 (Dmp1) and dentin sialophosphoprotein (Dspp) in mouse incisor MSCs was also assessed.
We found Satb2 overexpression infected with Ad-Satb2 slightly promoted Dmp1 and Dspp expression on day 3 and markedly upregulated on day 7, which was inhibited by knockdown of Satb2 expression with Ad-siSatb2 infection ( Figure 4E,F). Thus, these results indicate that Satb2 can induce the odontoblast-like differentiation process in mouse incisor MSCs.

F I G U R E 3 Constructs and validation of adenoviral vector-mediated
Satb2 overexpression and silencing. Aa,b, Molecular cloning and construction of Satb2 overexpression (a) and silencing (b) adenoviral vectors. Notably, the Ad-siSatb2 vector encodes three siRNAs targeting mouse Satb2, two of which (simSatb2-4 and simSatb2-5) also target rat Satb2 expression (NM_001109306). Satb2 target gene and protein expression levels in mouse incisor mesenchymal stem cells (MSCs) in the Ad-Satb2 and Ad-siSatb2 groups were determined by qPCR (B) and Western blot (C). Target gene and protein expression levels in all samples were normalized to the corresponding Gapdh levels. Each assay condition was analysed in triplicate. Quantitative analysis of Western blot results was performed with data from at least three independent experiments. All values are the means ± SDs; *P <.05 and **P <.01, # no statistical significance

| Satb2 participates in Bmp9-induced osteo/ odontogenic differentiation of mouse incisor MSCs in vitro
Satb2 was co-expressed with Bmp9 in pre-odontoblasts/odontoblasts. To assess whether Satb2 and Bmp9 exhibit crosstalk during osteo/odontogenic differentiation, we analysed the effect of Satb2 on Bmp9-induced osteoblast differentiation of mouse incisor MSCs in vitro. After mouse incisor MSCs were infected with Ad-Satb2 or Ad-siSatb2 separately, Bmp9-induced osteo/odontogenic was detected via ALP and ARS staining, followed by quantitative analysis ( Figure 5A,B). Compared with transduction with only Ad-Satb2 or Ad-Bmp9, co-transduction with Ad-Satb2 and Ad-Bmp9 strongly augmented ALP activity (Figure 5Aa,b). Interestingly, Satb2 mRNA expression was not induced after Ad-Bmp9 infection, suggesting that Satb2 may not act as a direct target of Bmp9 osteogenic signalling (Figure 5Ca). Nonetheless, we found that Runx2, Ocn and Opn expression was upregulated by Satb2 in Bmp9-induced osteo/ odontogenic differentiation (Figure 5Cb-d). ARS staining and quantitative analysis confirmed this tendency in the late stage of mineralization (Figure 5Ba,b).

| Enhancement of calvarial bone healing in rats synergistically induced by Satb2 and Bmp9
The concentration of GelMA hydrogel suitable for supporting in vitro mouse incisor MSCs survival and proliferation was assessed.
On day 14, 5% and 10% (w/v) GelMA hydrogels began to degrade ( Figure 6B). However, 20% (w/v) GelMA hydrogel retained a stiff, self-standing microporous environment and promoted cell adhesion, proliferation and rapid 3D seeding. Thus, 20% (w/v) GelMA hydrogel F I G U R E 5 Satb2 participates in Bmp9-induced osteo/odontogenic differentiation of mouse incisor MSCs in vitro. A, Cells were seeded in 24-well culture plates and infected with Ad-G/Rfp, Ad-Satb2, Ad-siSatb2, Ad-Bmp9, Ad-Satb2 + Ad-Bmp9 or Ad-siSatb2 + Ad-Bmp9 for 3 and 5 d. The effects of Satb2 modulation on ALP staining (a) and ALP activity (b) in Bmp9-induced mouse incisor MSCs were assessed. Scale bar = 400 µm. B, Alizarin Red S staining (a) and quantification (b). Infected mouse incisor MSCs were maintained in matrix mineralization culture medium for 14 d. Matrix mineralization nodules were stained with Alizarin Red S. Stained cells were imaged under a microscope. Scale bar = 400 µm. Ca-d, Satb2, Runx2, Opn and Ocn mRNA levels were determined in different groups after 3 d. All values are the means ± SDs; *P <.05 and **P <.01 was used in subsequent in vitro and in vivo studies. Mouse incisor MSCs (5 × 10 6 ) were suspended in 50 µL of 20% (w/v) GelMA hydrogel and allowed to form into a hemispheric 3D shape (Figure 6Ab).
Then, via confocal laser scanning microscopy, we observed that mouse incisor MSCs were uniformly distributed throughout the 3D GelMA hydrogel at 24 hours ( Figure 6C). CCK-8 assays and live/dead staining were applied to detect mouse incisor MSCs proliferation and viability in the GelMA hydrogel. As shown in Figure 6D,E, mouse incisor MSCs were maintained and exhibited high proliferation and viability up to day 7. These results demonstrated that hemispheric 3D GelMA hydrogel at a 20% (w/v) concentration was appropriate for subsequent in vivo studies.
To investigate the participation of Satb2 in bone healing, two critically sized defects with a non-healing full thickness diameter of 5 mm were made in both sides of rat calvaria (Figure 6Fa)

| D ISCUSS I ON
In this study, we first found that Satb2 was broadly expressed in the Dentin derived from odontoblasts comprises >70% of the entire tooth structure and functions as a protective barrier for dental pulp. 33,34 Dentin formation requires MSCs to differentiate into odontoblast-like cells. 5,32,35 Loss of dentin causes several tooth problems, such as pulpitis. External molecules that can stimulate odontoblast differentiation are urgently needed to accelerate dentin repair. 36  Osteo/odontogenic differentiation is a well-orchestrated process and requires interactions among many factors. 44 BMP9 is the BMP with the greatest potential to induce osteo/odontogenic differentiation in odontogenic stem cells and has been found to be useful as an efficacious bio-factor in tooth engineering in many published studies. 6,24,45 We found that Satb2 was coactivated with Bmp9 in pre-odontoblasts/odontoblasts, while Satb2 but not Bmp9 was activated in the apical mesenchyme. Odontoblast precursors migrate from the apical mesenchyme to the odontogenic region, where they differentiate into dentin-forming odontoblasts. To better understand the role of Satb2 in osteo/odontogenic differentiation, Bmp9 was introduced in this study. Our results showed that Satb2 elevated Bmp9-induced osteo/odontogenic differentiation of mouse incisor MSCs in vitro and in vivo. Additionally, Bmp9 reversed the inhibition of osteo/odontogenic differentiation after transduction with Ad-siSatb2. In this study, we found that Runx2 and Opn are interactive genes in the process by which Satb2 participates in Bmp9-induced osteo/odontogenic differentiation.
Satb2 has been reported to repair bone defects in mice 46 ; thus, scaffolds seeded with Ad-Satb2-infected mouse incisor MSCs were implanted in a calvarial bone defect model. We found that Satb2 promoted bone regeneration, accompanied by increases in BV/TV and Tb.N. Importantly, we demonstrated that compared with other groups, mouse incisor MSCs infected with Ad-Satb2 and Ad-Bmp9 significantly enhanced the healing of bone defects. Our study provides the first confirmation that combined expression of Satb2 and Bmp9 in stem cells is a promising gene therapy approach in bone regeneration.
Previous studies have revealed that SATB2 deletion or mutation results in phenotypes similar to those of BMP deficiency in humans and mice, such as cleft palate and calvarial defects. 26  Whether Satb2 indirectly interacts with Bmp9 to induce osteo/odontogenic differentiation requires further study for verification.
Our previously demonstrated that SATB2 was expressed in multiple types of human odontogenic stem cells. 19 Moreover, SATB2 was shown to regulate the expression of MSC markers and osteogenic genes. In this report, we further confirm the important osteo-

ACK N OWLED G EM ENTS
The reported work was supported in part by research grants from

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

AUTH O R CO NTR I B UTI O N S
Qiuman Chen contributed to the study design, data acquisition, analysis, and interpretation, and drafting of the manuscript; Liwen Zheng and Yuxin Zhang contributed to the data acquisition and analysis and critical revision of the manuscript; Xia Huang and Feilong Wang contributed to the data acquisition and analysis and critically revised the manuscript; Shuang Li, Zhuohui Yang, Fang Liang, Jing Hu, Yucan Jiang, Yeming Li and Pengfei Zhou contributed to the data acquisition and analysis and drafted the manuscript; Wenping Luo and Hongmei Zhang contributed to the study conception and design and data interpretation, as well as drafted and critically revised the manuscript. All authors provided final approval and agree to be accountable for all aspects of the work.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.