Loss of KDM4B impairs osteogenic differentiation of OMSCs and promotes oral bone aging.

Aging of craniofacial skeleton significantly impairs the repair and regeneration of trauma-induced bony defects, and complicates dental treatment outcomes. Age-related alveolar bone loss could be attributed to decreased progenitor pool through senescence, imbalance in bone metabolism and bone-fat ratio. Mesenchymal stem cells isolated from oral bones (OMSCs) have distinct lineage propensities and characteristics compared to MSCs from long bones, and are more suited for craniofacial regeneration. However, the effect of epigenetic modifications regulating OMSC differentiation and senescence in aging has not yet been investigated. In this study, we found that the histone demethylase KDM4B plays an essential role in regulating the osteogenesis of OMSCs and oral bone aging. Loss of KDM4B in OMSCs leads to inhibition of osteogenesis. Moreover, KDM4B loss promoted adipogenesis and OMSC senescence which further impairs bone-fat balance in the mandible. Together, our data suggest that KDM4B may underpin the molecular mechanisms of OMSC fate determination and alveolar bone homeostasis in skeletal aging, and present as a promising therapeutic target for addressing craniofacial skeletal defects associated with age-related deteriorations.

Stem cells of the suture mesenchyme in craniofacial bone development, repair and regeneration.

Development of the skeleton is mediated through two distinct ossification mechanisms. Craniofacial bones are formed mainly through intramembranous ossification, a mechanism different from endochondral ossification required for development of the body skeleton. The skeletal structures are quite distinct between the two, thus they are likely to have their unique stem cell populations. The sutures serve as the growth center critical for healthy development of the craniofacial skeleton. Defects in suture morphogenesis cause its premature closure, resulting in development of craniosynostosis, a devastating disease affecting 1 in ~2,500 individuals. The suture mesenchyme has been postulated to act as the niche of skeletal stem cells essential for calvarial morphogenesis. However, very limited knowledge is available for suture biology and suture stem cells (SuSCs) have yet to be isolated. Here we report the first evidence for identification and isolation of a stem cell population residing in the suture midline. Genetic labeling of SuSCs shows their ability to self-renew and continually give rise to mature cell types over a 1-year monitoring period. They maintain their localization in the niches constantly produce skeletogenic descendants during calvarial development and homeostastic maintenance. Upon injury, SuSCs expand drastically surrounding the skeletogenic mesenchyme, migrate to the damaged site and contribute directly to skeletal repair in a cell autonomous fashion. The regeneration, pluripotency and frequency of SuSCs are also determined using limiting dilution transplantation. In vivo clonal expansion analysis demonstrates a single SuSC capable of generating bones. Furthermore, SuSC transplantation into injured calvaria facilitates the healing processes through direct engraftments. Our findings demonstrate SuSCs are bona fide skeletal stem cells ideally suited for cell-based craniofacial bone therapy as they possess abilities to engraft, differentiate.(Presented at the 1980th Meeting, April 16, 2019).

Signals from the brain and olfactory epithelium control shaping of the mammalian nasal capsule cartilage.

Facial shape is the basis for facial recognition and categorization. Facial features reflect the underlying geometry of the skeletal structures. Here, we reveal that cartilaginous nasal capsule (corresponding to upper jaw and face) is shaped by signals generated by neural structures: brain and olfactory epithelium. Brain-derived Sonic Hedgehog (SHH) enables the induction of nasal septum and posterior nasal capsule, whereas the formation of a capsule roof is controlled by signals from the olfactory epithelium. Unexpectedly, the cartilage of the nasal capsule turned out to be important for shaping membranous facial bones during development. This suggests that conserved neurosensory structures could benefit from protection and have evolved signals inducing cranial cartilages encasing them. Experiments with mutant mice revealed that the genomic regulatory regions controlling production of SHH in the nervous system contribute to facial cartilage morphogenesis, which might be a mechanism responsible for the adaptive evolution of animal faces and snouts.

Cell morphologic changes and PCNA expression within craniofacial sutures during monkey Class III treatment.

OBJECTIVES: To evaluate and compare the cellular morphologic changes and proliferating cell nuclear antigen (PCNA) expression within craniofacial sutures in growing Rhesus monkeys treated with a Class III functional appliance. MATERIALS AND METHODS: Six Rhesus monkeys in the mixed dentition stage were divided into three groups: a 45-day experimental group, a 90-day experimental group, and a control group. Monkeys in the experimental groups were fitted with a Class III magnetic twin-block appliance. Cellular changes in six craniofacial sutures-the zygomaticomaxillary, zygomaticotemporal, transverse palatine, pterygopalatine, zygomaticofrontal, and frontomaxillary sutures were qualitatively and quantitatively evaluated by means of histomorphologic analysis, TEM, and immunohistochemical test of PCNA. RESULTS: Obvious and altered bone remodeling combined with bone deposition and resorption was present in craniofacial sutures in the experimental groups. Increased activity of enlarged fibroblasts with abundant organelles was revealed. PCNA expression increased in the 45-day group compared with the control group, followed by the 90-day group. The highest percentage of PCNA-positive cells was found in the pterygopalatine suture in the 45-day group and the zygomaticomaxillary suture in the 90-day group. CONCLUSIONS: The pterygopalatine and zygomaticomaxillary sutures are more active among the craniofacial sutures in the craniofacial complex remodeling during Class III treatment. The magnetic twin-block appliance effectively promoted suture remodeling by enhancing the activity and proliferation of osteoblasts, osteoclasts, and fibroblasts, especially in the early phase.

Tissue-engineered autologous grafts for facial bone reconstruction.

Facial deformities require precise reconstruction of the appearance and function of the original tissue. The current standard of care-the use of bone harvested from another region in the body-has major limitations, including pain and comorbidities associated with surgery. We have engineered one of the most geometrically complex facial bones by using autologous stromal/stem cells, native bovine bone matrix, and a perfusion bioreactor for the growth and transport of living grafts, without bone morphogenetic proteins. The ramus-condyle unit, the most eminent load-bearing bone in the skull, was reconstructed using an image-guided personalized approach in skeletally mature Yucatán minipigs (human-scale preclinical model). We used clinically approved decellularized bovine trabecular bone as a scaffolding material and crafted it into an anatomically correct shape using image-guided micromilling to fit the defect. Autologous adipose-derived stromal/stem cells were seeded into the scaffold and cultured in perfusion for 3 weeks in a specialized bioreactor to form immature bone tissue. Six months after implantation, the engineered grafts maintained their anatomical structure, integrated with native tissues, and generated greater volume of new bone and greater vascular infiltration than either nonseeded anatomical scaffolds or untreated defects. This translational study demonstrates feasibility of facial bone reconstruction using autologous, anatomically shaped, living grafts formed in vitro, and presents a platform for personalized bone tissue engineering.

Canonical and noncanonical intraflagellar transport regulates craniofacial skeletal development.

The primary cilium is a cellular organelle that coordinates signaling pathways critical for cell proliferation, differentiation, survival, and homeostasis. Intraflagellar transport (IFT) plays a pivotal role in assembling primary cilia. Disruption and/or dysfunction of IFT components can cause multiple diseases, including skeletal dysplasia. However, the mechanism by which IFT regulates skeletogenesis remains elusive. Here, we show that a neural crest-specific deletion of intraflagellar transport 20 (Ift20) in mice compromises ciliogenesis and intracellular transport of collagen, which leads to osteopenia in the facial region. Whereas platelet-derived growth factor receptor alpha (PDGFRα) was present on the surface of primary cilia in wild-type osteoblasts, disruption of Ift20 down-regulated PDGFRα production, which caused suppression of PDGF-Akt signaling, resulting in decreased osteogenic proliferation and increased cell death. Although osteogenic differentiation in cranial neural crest (CNC)-derived cells occurred normally in Ift20-mutant cells, the process of mineralization was severely attenuated due to delayed secretion of type I collagen. In control osteoblasts, procollagen was easily transported from the endoplasmic reticulum (ER) to the Golgi apparatus. By contrast, despite having similar levels of collagen type 1 alpha 1 (Col1a1) expression, Ift20 mutants did not secrete procollagen because of dysfunctional ER-to-Golgi trafficking. These data suggest that in the multipotent stem cells of CNCs, IFT20 is indispensable for regulating not only ciliogenesis but also collagen intracellular trafficking. Our study introduces a unique perspective on the canonical and noncanonical functions of IFT20 in craniofacial skeletal development.

Bone morphogenetic protein 4 promotes craniofacial neural crest induction from human pluripotent stem cells.

Neural crest (NC) cells are a group of cells located in the neural folds at the boundary between the neural and epidermal ectoderm. Cranial NC cells migrate to the branchial arches and give rise to the majority of the craniofacial region, whereas trunk and tail NC cells contribute to the heart, enteric ganglia of the gut, melanocytes, sympathetic ganglia, and adrenal chromaffin cells. Positional information is indispensable for the regulation of cranial or trunk and tail NC cells. However, the mechanisms underlying the regulation of positional information during human NC induction have yet to be fully elucidated. In the present study, supplementation of bone morphogenetic protein (BMP) 4 in defined serum-free culture conditions including fibroblast growth factor-2 and Wnt3a from day 8 after NC specification induced the expression of cranial NC markers, AP2alpha, MSX1, and DLX1, during NC cell differentiation from human pluripotent stem cells. On the other hand, the proportion of cells expressing p75(NTR) or HNK1 decreased compared with that of cells cultured without BMP4, whereas gene expression analysis demonstrated that the expression levels of cranial NC-associated genes increased in BMP4-treated NC cells. These BMP4-treated NC cells were capable of differentiation into osteocytes and chondrocytes. The results of the present study indicate that BMP4 regulates cranial positioning during NC development.

Stem cells of the suture mesenchyme in craniofacial bone development, repair and regeneration.

The suture mesenchyme serves as a growth centre for calvarial morphogenesis and has been postulated to act as the niche for skeletal stem cells. Aberrant gene regulation causes suture dysmorphogenesis resulting in craniosynostosis, one of the most common craniofacial deformities. Owing to various limitations, especially the lack of suture stem cell isolation, reconstruction of large craniofacial bone defects remains highly challenging. Here we provide the first evidence for an Axin2-expressing stem cell population with long-term self-renewing, clonal expanding and differentiating abilities during calvarial development and homeostastic maintenance. These cells, which reside in the suture midline, contribute directly to injury repair and skeletal regeneration in a cell autonomous fashion. Our findings demonstrate their true identity as skeletal stem cells with innate capacities to replace the damaged skeleton in cell-based therapy, and permit further elucidation of the stem cell-mediated craniofacial skeletogenesis, leading to revealing the complex nature of congenital disease and regenerative medicine.

Mesenchymal stem cells and alginate microcarriers for craniofacial bone tissue engineering: A review.

Craniofacial bone is a complex structure with an intricate anatomical and physiological architecture. The defects that exist in this region therefore require a precise control of osteogenesis in their reconstruction. Unlike traditional surgical intervention, tissue engineering techniques mediate bone development with limited postoperative risk and cost. Alginate stands as the premier polymer in bone repair because of its mild ionotropic gelation and excellent biocompatibility, biodegradability, and injectability. Alginate microcarriers are candidates of choice to mediate cells and accommodate into 3-D environment. Several studies reported the use of alginate microcarriers for delivering cells, drugs, and growth factors. This review will explore the potential use of alginate microcarrier for stem cell systems and its application in craniofacial bone tissue engineering.

Stem Cells in Teeth and Craniofacial Bones.

Stem cells are remarkable, and stem cell-based tissue engineering is an emerging field of biomedical science aiming to restore damaged tissue or organs. In dentistry and reconstructive facial surgery, it is of great interest to restore lost teeth or craniofacial bone defects using stem cell-mediated therapy. In the craniofacial region, various stem cell populations have been identified with regeneration potential. In this review, we provide an overview of the current knowledge concerning the various types of tooth- and craniofacial bone-related stem cells and discuss their in vivo identities and regulating mechanisms.

In vivo loss of function study reveals the short stature homeobox-containing (shox) gene plays indispensable roles in early embryonic growth and bone formation in zebrafish.

BACKGROUND: Congenital loss of the SHOX gene is considered to be a genetic cause of short stature phenotype in Turner syndrome and Leri-Weill dyschondrosteosis patients. Though SHOX expression initiates during early fetal development, little is known about the embryonic roles of SHOX. The evolutionary conservation of the zebrafish shox gene and the convenience of the early developmental stages for analyses make zebrafish a preferred model. Here, we characterized structure, expression, and developmental roles of zebrafish shox through a loss-of-function approach. RESULTS: We found a previously undiscovered Shox protein that has both a homeodomain and an OAR-domain in zebrafish. The shox transcript emerged during the segmentation period and it increased in later stages. The predominant domains of shox expression were mandibular arch, pectoral fin, anterior notochord, rhombencephalon, and mesencephalon, suggesting that Shox is involved in bone and neural development. Translational blockade of Shox mRNA by an antisense morpholino oligo delayed embryonic growth, which was restored by the co-overexpression of morpholino-resistant Shox mRNA. At later stages, impaired Shox expression markedly delayed the calcification process in the anterior vertebral column and craniofacial bones. CONCLUSIONS: Our data demonstrate evolutionarily conserved Shox plays roles in early embryonic growth and in later bone formation.

An assessment of the long-term effects of simulated microgravity on cranial neural crest cells in zebrafish embryos with a focus on the adult skeleton.

It is becoming increasingly important to address the long-term effects of exposure to simulated microgravity as the potential for space tourism and life in space become prominent topics amongst the World's governments. There are several studies examining the effects of exposure to simulated microgravity on various developmental systems and in various organisms; however, few examine the effects beyond the juvenile stages. In this study, we expose zebrafish embryos to simulated microgravity starting at key stages associated with cranial neural crest cell migration. We then analyzed the skeletons of adult fish. Gross observations and morphometric analyses show that exposure to simulated microgravity results in stunted growth, reduced ossification and severe distortion of some skeletal elements. Additionally, we investigated the effects on the juvenile skull and body pigmentation. This study determines for the first time the long-term effects of embryonic exposure to simulated microgravity on the developing skull and highlights the importance of studies investigating the effects of altered gravitational forces.

Contribution of SATB2 to the stronger osteogenic potential of bone marrow stromal cells from craniofacial bones.

Previous studies have shown that craniofacial bone marrow stromal cells (BMSCs) have a strong osteogenic potential. However, the mechanism by which BMSCs of various embryonic origins develop diverse osteogenic potentials remains unclear. To investigate the mechanisms regulating osteoblast differentiation in two different types of BMSCs, we compared the temporal and spatial mRNA and protein expression patterns of Satb2 and its downstream gene Hoxa2 by using real-time polymerase chain reaction, Western blotting and fluorescent immunostaining in mandible BMSCs (M-BMSCs) and tibia BMSCs (T-BMSCs) undergoing osteoblast differentiation. Higher levels of alkaline phosphatase, greater calcium accumulation and earlier expression of Runx2 were observed in osteogenic-induced M-BMSCs compared with T-BMSCs. Low levels of Satb2 were detected in both types of uninduced BMSCs but the majority of SATB2 was located in the nuclei of M-BMSCs. Notably, Satb2 was expressed earlier in M-BMSCs and Hoxa2, a downstream target of Satb2, was not expressed in uninduced M-BMSCs or during osteoblast differentiation, just as during embryonic mandible development. In contrast, Hoxa2 was reactivated in T-BMSCs during osteoblast differentiation. Based on these results, we conclude that SATB2 plays a different role during osteoblast differentiation of M-BMSCs and T-BMSCs. The earlier activation of Satb2 expression in M-BMSCs compared with T-BMSCs might explain the stronger osteogenic potential of M-BMSCs.

Presence of galanin-like immunoreactivity in mesenchymal and neural crest origin tissues during embryonic development in the mouse.

Galanin is a highly conserved neuropeptide with a wide range of biological effects. Recently, through transcriptome analysis, galanin was identified in undifferentiated mouse embryonic stem cells as one of the most abundant transcripts. We have examined the developmental expression of galanin-like immunoreactivity in mice from embryonic day 10 (E10) to embryonic day 15 (E15). At E10, galanin was readily detected in the undifferentiated head and trunk mesenchyme of both mesodermal and neural crest origin. There was also strong immunoreactivity in the mesenchymal spiral ridges of the outflow tract of the heart and the endocardial cushions. The highest level of galanin detected was at E13 in the craniofacial mesenchyme and proliferating chondrocytes in bones of both neural crest and mesoderm origin. Dorsal root ganglia and trigeminal ganglia contained galanin immunoreactive cells as well. These data indicate the presence of galanin peptide during periods of morphogenesis and thus a developmental role for the peptide in mesenchymal and neural crest origin tissues in the mouse embryo. Whether galanin has a growth and/or differentiating role, still remains to be demonstrated.

Cell passage and composition of culture medium effects proliferation and differentiation of human osteoblast-like cells from facial bone.

Cells loose their capability to multiply and to differentiate when they are serial subcultivated. However, both, multiplication and differentiation are of utmost importance to obtain sufficient amounts of cells for the translation of tissue regeneration into cell based therapeutic approaches. Thus, for the clinical application more information about ideal culture conditions are necessary. Therefore, aim of this study was to assess culture conditions of human osteoblast-like cells during long-term culture focusing on effects of different culture media and ascorbic acid. Biopsies of maxilla and mandible were obtained from 17 patients to test different cell culture media and from 10 patients to analyse differentiation and proliferation related to number of subcultures and ascorbic acid content. Histochemical and immunhistochemical tests (EZ4U assay, ALP histochemistry, type I collagen immunohistochemistry, osteocalcin Elisa) were performed to determine cell proliferation and differentiation. Opti-MEM with 10% FCS produced statistically significant the highest increase in cell counts. The highest proliferation rate in long-term cultivation was seen in the 4th cell passage. A reciprocal relationship between cell proliferation and differentiation over 5 passages with a turning point in the 4(th) passage was found. An ascorbic acid content of 50 microg/ml triggered an optimal increase in differentiation. For osteoblast-like cells, Opti-MEM with 10% FCS proved to be the best culture medium. After 3 passages there is the highest amount of cells with osteogenic differentiation which is enhanced by the addition of ascorbic acid. This approach is suitable for tissue engineering of bone grafts.

Maxillofacial-derived stem cells regenerate critical mandibular bone defect.

Stem cell-based bone tissue regeneration in the maxillofacial complex is a clinical necessity. Genetic engineering of mesenchymal stem cells (MSCs) to follow specific differentiation pathways may enhance the ability of these cells to regenerate and increase their clinical relevance. MSCs isolated from maxillofacial bone marrow (BM) are good candidates for tissue regeneration at sites of damage to the maxillofacial complex. In this study, we hypothesized that MSCs isolated from the maxillofacial complex can be engineered to overexpress the bone morphogenetic protein-2 gene and induce bone tissue regeneration in vivo. To demonstrate that the cells isolated from the maxillofacial complex were indeed MSCs, we performed a flow cytometry analysis, which revealed a high expression of mesenchyme-related markers and an absence of non-mesenchyme-related markers. In vitro, the MSCs were able to differentiate into osteogenic, chondrogenic, and adipogenic lineages. Gene delivery of the osteogenic gene BMP2 via an adenoviral vector revealed high expression levels of BMP2 protein that induced osteogenic differentiation of these cells in vitro and induced bone formation in an ectopic site in vivo. In addition, implantation of genetically engineered maxillofacial BM-derived MSCs into a mandibular defect led to regeneration of tissue at the site of the defect; this was confirmed by performing micro-computed tomography analysis. Histological analysis of the mandibles revealed osteogenic differentiation of implanted cells as well as bone tissue regeneration. We conclude that maxillofacial BM-derived MSCs can be genetically engineered to induce bone tissue regeneration in the maxillofacial complex and that this finding may be clinically relevant.

Effects of tensile stress on the alpha1 nicotinic acetylcholine receptor expression in maxillofacial skeletal myocytes.

This study was to investigate the alterations of the alpha1 nicotinic acetylcholine receptor (nAChR) levels under tensile stress stimulation in maxillofacial skeletal myocytes. The skeletal muscle satellite cells from two to three days post-natal BALB/C mice's maxillofacial muscle were collected for primary cell culture. The second passage cells in the loaded groups were subjected to cyclic tensile stress (0.5 Hz, 2000 micro strain) produced by a four point bending system for 2, 4, 6, 8, or 12 h. In the control groups, cells were cultured on similar plates and kept in the same incubator without mechanical stress loading. The examination of nAChR alpha1 receptor expression was performed by receptor binding of [125I] a-bungarotoxin. The nAChR alpha1 mRNA transcript level was analyzed by semi-quantitative RT-PCR. The result showed that the nAChR alpha1 receptor expression was elevated significantly in stress-stimulated group (P < 0.05). An increase of nAChR alpha1 in mRNA transcript level was also observed in stress groups as compared with controls (P < 0.05). It is concluded that nAChR was a possible molecular mechanism which might play an important role in mechanotransduction of tensile stress loading on maxillofacial skeletal myocytes.

Vitamin D3, vitamin K2, and warfarin regulate bone metabolism in human paranasal sinus bones.

Several recent studies have indicated that the paranasal sinus bones undergo pathophysiological changes in patients with chronic sinusitis. We examined the mineralization activity of osteoblasts and the production of osteocalcin and cytokines in cultured human osteoblasts derived from ethmoidal bones treated with vitamin D3, vitamin K2, and warfarin to investigate the metabolic effects of these treatments on paranasal sinus bones. In the bones treated with vitamin D3 plus vitamin K2, osteocalcin production and the ratio of the mineralization of osteoblasts were increased. Warfarin inhibited the promotive effects of vitamin K2 in the presence of vitamin D3. With regard to TGF-beta production, there was quite a difference in response depending on the isoforms. In conclusion, we have demonstrated that these vitamins and warfarin may be useful in improving bone metabolism in paranasal sinus bones, and may additionally improve the pathogenesis of chronic sinusitis.

Secondary pneumatization of the maxillary sinus in callitrichid primates: insights from immunohistochemistry and bone cell distribution.

The paranasal sinuses remain elusive both in terms of function and in the proximate mechanism of their development. The present study sought to describe the maxillary sinuses (MSs) in three species of callitrichid primates at birth, a time when secondary pneumatization occurs rapidly in humans. The MSs were examined in serially sectioned and stained slides from the heads of two Callithrix jacchus, one Leontopithecus rosalia, and two Saguinus geoffroyi. Specimens were examined microscopically regarding the distribution of osteoclasts and osteoblasts along the osseous boundaries of the MS and other parts of the maxillary bone. Selected sections were immunohistochemically evaluated for the distribution of osteopontin (OPN), which facilitates osteoclast binding. Taken together, OPN immunoreactivity and bone cell distribution suggested trends of bone resorption/deposition that were consistent among species for the superior (roof) and inferior (floor) boundaries of the MS. Expansion at the roof and floor of the MS appeared to correspond to overall vertical midfacial growth in callitrichids. Much more variability was noted for the lateral (alveolar) and medial (nasal walls) of the MS. Unlike the other species, the nasal wall of Saguinus was static and mostly composed of inferior portions of the nasal capsule that were undergoing endochondral ossification. The variation seen in the alveolar walls may relate to the presence or absence of adjacent structures, although it was noted that adjacency of deciduous molars influenced medial drift of the alveolar wall in Saguinus but not Leontopithecus. The results of this study are largely consistent with the "structural" or "architectural" hypothesis of sinus formation with respect to vertical MS enlargement, and the variable cellular/OPN distribution found along the nasal and alveolar walls was evocative of Witmer's (J Vert Paleontol 1997;17:1-73) epithelial hypothesis in revealing that most expansion occurred in regions unopposed by adjacent structures.