Jian-Quan Feng

Fibroblast growth factor 23 (FGF23) is a phosphate-regulating (Pi-regulating) hormone produced by bone. Hereditary hypophosphatemic disorders are associated with FGF23 excess, impaired skeletal growth, and osteomalacia. Blocking FGF23 became an effective therapeutic strategy in X-linked hypophosphatemia, but testing remains limited in autosomal recessive hypophosphatemic rickets (ARHR). This study investigates the effects of Pi repletion and bone-specific deletion of Fgf23 on bone and mineral metabolism in the dentin matrix protein 1–knockout ( Dmp1 ^KO ) mouse model of ARHR. At 12 weeks, Dmp1 ^KO mice showed increased serum FGF23 and parathyroid hormone levels, hypophosphatemia, impaired growth, rickets, and osteomalacia. Six weeks of dietary Pi supplementation exacerbated FGF23 production, hyperparathyroidism, renal Pi excretion, and osteomalacia. In contrast, osteocyte-specific deletion of Fgf23 resulted in a partial correction of FGF23 excess, which was sufficient to fully restore serum Pi levels but only partially corrected the bone phenotype. In vitro, we show that FGF23 directly impaired osteoprogenitors’ differentiation and that DMP1 deficiency contributed to impaired mineralization independent of FGF23 or Pi levels. In conclusion, FGF23-induced hypophosphatemia is only partially responsible for the bone defects observed in Dmp1 ^KO mice. Our data suggest that combined DMP1 repletion and FGF23 blockade could effectively correct ARHR-associated mineral and bone disorders.

Wnt–β-catenin signaling plays a key role in orthodontic tooth movement (OTM), a common clinical practice for malocclusion correction. However, its targeted periodontal ligament (PDL) progenitor cells remain largely unclear. In this study, we first showed a synchronized increase in Wnt–β-catenin levels and Axin2⁺ PDL progenitor cell numbers during OTM using immunostaining of β-catenin in wild-type mice and X-gal staining in the Axin2-LacZ knock-in line. Next, we demonstrated time-dependent increases in Axin2⁺ PDL progenitors and their progeny cell numbers within PDL and alveolar bones during OTM using a one-time tamoxifen-induced Axin2 tracing line ( Axin2^CreERT2/+; R26R^tdTomato/+). Coimmunostaining images displayed both early and late bone markers (such as RUNX2 and DMP1) in the Axin2^Lin PDL cells. Conversely, ablation of Axin2⁺ PDL cells via one-time tamoxifen-induced diphtheria toxin subunit A (DTA) led to a drastic decrease in osteogenic activity (as reflected by alkaline phosphatase) in PDL and alveolar bone. There was also a decrease in new bone mass and a significant reduction in the mineral apposition rate on both the control side (to a moderate degree) and the OTM side (to a severe degree). Thus, we conclude that the Axin2⁺ PDL cells (the Wnt-targeted key cells) are highly sensitive to orthodontic tension force and play a critical role in OTM-induced PDL expansion and alveolar bone formation. Future drug development targeting the Axin2⁺ PDL progenitor cells may accelerate alveolar bone formation during orthodontic treatment.

Transforming growth factor beta (TGFβ) signaling plays an important role during osteogenesis. However, most research in this area focuses on cortical and trabecular bone, whereas alveolar bone is largely overlooked. To address the role of TGFβR2 (the key receptor for TGFβ signaling) during postnatal alveolar bone development, we conditionally deleted Tgfβr2 in early mesenchymal progenitors by crossing Gli1-Cre^ERT2; Tgfβr2^flox/flox; R26R^tdTomato mice (named early cKO) or in osteoblasts by crossing 3.2kb Col1-Cre^ERT2; Tgfβr2^flox/flox; R26R^tdTomato mice (named late cKO). Both cKO lines were induced at postnatal day 5 (P5) and mice were harvested at P28. Compared to the control littermates, early cKO mice exhibited significant reduction in alveolar bone mass and bone mineral density, with drastic defects in the periodontal ligament (PDL); conversely, the late cKO mice displayed very minor changes in alveolar bone. Mechanism studies showed a significant reduction in PCNA+ PDL cell numbers and OSX+ alveolar bone cell numbers, as well as disorganized PDL fibers with a great reduction in periostin (the most abundant extracellular matrix protein) on both mRNA and protein levels. We also showed a drastic reduction in β-catenin in the early cKO PDL and a great increase in SOST (a potent inhibitor of Wnt signaling). Based on these findings, we conclude that TGFβ signaling plays critical roles during early alveolar bone formation via the promotion of PDL mesenchymal progenitor proliferation and differentiation mechanisms.

Dental pulp stem cells (DPSCs) have the potential to polarize, differentiate, and form tubular dentin under certain conditions. However, the factors that initiate and regulate DPSC polarization and its underlying mechanism remain unclear. Identification of the factors that control DPSC polarization is a prerequisite for tubular dentin regeneration. We recently developed a unique bioinspired 3-dimensional platform that is capable of deciphering the factors that initiate and modulate cell polarization. The bioinspired platform has a simple background and confines a single cell on each microisland of the platform; therefore, it is an effective tool to study DPSC polarization at the single-cell level. In this work, we explored the effects of biophysical factors (surface topography, microisland area, geometry, tubular size, and gravity) on single DPSC polarization. Our results demonstrated that nanofibrous architecture, microisland area, tubular size, and gravity participated in regulating DPSC polarization by influencing the formation of the DPSC process and relocation of the Golgi apparatus. Among these factors, nanofibrous architecture, tubular size, and appropriate microisland area were indispensable for initiating DPSC polarization, whereas gravity served as an auxiliary factor to the process of DPSC polarization. Meanwhile, microisland geometry had a limited effect on DPSC polarization. Collectively, this work provides information on DPSC polarization and paves the way for the development of new biomaterials for tubular dentin regeneration.

Cementum, a specialized bony layer covering an entire molar root surface, anchors teeth into alveolar bone. Gli1, a key transcriptional activator in Hedgehog signaling, has been identified as a mesenchymal progenitor cell marker in various tissues, including the periodontal ligament (PDL). To address the mechanisms by which Gli1⁺ progenitor cells contribute to cementogenesis, we used the Gli1^lacZ/+ knock-in line to mark Gli1⁺ progenitors and the Gli1^CreERT2/+; R26R^tdTomato/+ line (named Gli1^Lin) to trace Gli1 progeny cells during cementogenesis. Our data unexpectedly displayed a biphasic feature of Gli1⁺ PDL progenitor cells and cementum growth: a negative relationship between Gli1⁺ progenitor cell number and cementogenesis but a positive correlation between Gli1-derived acellular and cellular cementoblast cell number and cementum growth. DTA-ablation of Gli1^Lin cells led to a cementum hypoplasia, including a significant reduction of both acellular and cellular cementoblast cells. Gain-of-function studies (by constitutive stabilization of β-catenin in Gli1^Lin cells) revealed a cementum hyperplasia. A loss of function (by conditional deletion of β-catenin in Gli1⁺ cells) resulted in a reduction of postnatal cementum growth. Together, our studies support a vital role of Gli1⁺ progenitor cells in contribution to both types of cementum, in which canonical Wnt/β-catenin signaling positively regulates the differentiation of Gli1⁺ progenitors to cementoblasts during cementogenesis.

Bone-forming osteoblasts have been a cornerstone of bone biology for more than a century. Most research toward bone biology and bone diseases center on osteoblasts. Overlooked are the 90% of bone cells, called osteocytes. This study aims to test the hypothesis that osteocytes but not osteoblasts directly build mineralized bone structures, and that defects in osteocytes lead to the onset of hypophosphatemia rickets. The hypothesis was tested by developing and modifying multiple imaging techniques, including both in vivo and in vitro models plus two types of hypophosphatemia rickets models (Dmp1-null and Hyp, Phex mutation mice), and Dmp1-Cre induced high level of β-catenin models. Our key findings were that osteocytes (not osteoblasts) build bone similar to the construction of a high-rise building, with a wire mesh frame (i.e., osteocyte dendrites) and cement (mineral matrices secreted from osteocytes), which is a lengthy and slow process whose mineralization direction is from the inside toward the outside. When osteoblasts fail to differentiate into osteocytes but remain highly active in Dmp-1-null or Hyp mice, aberrant and poor bone mineralization occurs, caused by a sharp increase in Wnt-β-catenin signaling. Further, the constitutive expression of β-catenin in osteocytes recaptures a similar osteomalacia phenotype as shown in Dmp1 null or Hyp mice. Thus, we conclude that osteocytes directly build bone, and osteoblasts with a short life span serve as a precursor to osteocytes, which challenges the existing dogma.

Objectives In this study, we attempted to define the precise window of time for molar root elongation using a gain‐of‐function mutation of β‐catenin model. Materials and Methods Both the control and constitutively activated β‐catenin (CA‐β‐cat) mice received a one‐time tamoxifen administration (for activation of β‐catenin at newborn, postnatal day‐3, or ‐5, or ‐7, or ‐9), and were harvested at the same stage of P21. Multiple approaches were used to define the window of time of postnatal tooth root formation. Results In the early activation groups (tamoxifen induction at newborn, or P3 or P5) there was a lack of molar root elongation in the CA‐β‐cat mice. When induced at P7, the root length was slightly reduced at P21. However, the root length was essentially the same as that in the control when β‐cat activated at P9. This study indicates that root elongation occurs in a narrow time of window, which is highly sensitive to a change of β‐catenin levels. Molecular studies showed a drastic decrease in the levels of nuclear factor I‐C (NFIC) and osterix (OSX), plus sharp reductions of odontoblast differentiation markers, including Nestin, dentin sialoprotein (DSP) and dentin matrix protein 1 (DMP1) at both mRNA and protein levels. Conclusions Murine molar root elongation is precisely regulated by the Wnt/β‐catenin signaling within a narrow window of time (newborn to day 5).

Cell lineage tracing, an old technique which originated in the nineteenth century, regains popularity and relevance due to introduction of a more sensitive tomato fluorescent protein under the control of a ubiquitous promoter (Rosa 26 gene). In addition, various tissue specific CreERT2 mouse lines are widely available, making cell lineage tracing studies more specific and powerful. In this protocol, we provide a practical guide for researchers to map progeny of specific cells such as chondrocytes during development using a fluorescent reporter (tomato, red) and multiple chondrocyte Cre lines. Further, we provide valuable examples in which these tracing lines, combined with a bone reporter mouse line (2.3 Col 1a1-GFP) or costained with different immunofluorescent proteins, revealed how a chondrocyte transdifferentiates into a bone cell in vivo.

Background: Periodontitis is caused by multiple factors involving a bacterial challenge and a susceptible host, although there is no report on gene mutation directly linked to this common disease. Mutations in the proteinase bone morphogenetic protein 1 (BMP1) were identified in patients with osteogenesis imperfecta, who display some dentin defects and alveolar bone loss. We previously reported essential roles of BMP1 and tolloid‐like 1 (TLL1), two closely related extracellular proteinases with overlapping functions, in mouse periodontium growth by simultaneous knockout (KO) of both genes, although the separate roles of BMP1 and TLL1 have remained unclear. Here, we have investigated whether and how BMP1 and TLL1 separately maintain periodontal homeostasis by comparing single Bmp1 KO and Tll1 KO with double KO (dKO) phenotypes.Methods: Floxed Bmp1 and/or Tll1 alleles were deleted in transgenic mice via ubiquitously expressed Cre^ERT2 induced by tamoxifen treatment starting at 4‐weeks of age (harvested at 18‐weeks of age). Multiple approaches, including X‐ray, micro‐CT, calcein and alizarin red double‐labeling, scanning electron microscopy, and histological and immunostaining assays, were used to analyze periodontal phenotypes and molecular mechanisms.Results: Both Bmp1 KO and double KO mice exhibited severe periodontal defects, characterized by periodontal ligament (PDL) fiber loss and ectopic ossification in the expanded PDL area, and drastic reductions in alveolar bone and cementum volumes, whereas Tll1 KO mice displayed very mild phenotypes. Mechanistic studies revealed a sharp increase in the uncleaved precursor of type I collagen (procollagen I), leading to defective extracellular matrices.Conclusions: BMP1, but not TLL1, is essential for maintaining periodontal homeostasis. This occurs at least partly via biosynthetic processing of procollagen I, thereby maintaining appropriate levels of procollagen I and its activated products such as mature collagen I.