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Tissue-resident stem cells are essential for development and repair, and in the skeleton, this function is fulfilled by recently identified skeletal stem cells (SSCs). However, recent work has identified that SSCs are not monolithic, with long bones, craniofacial sites, and the spine being formed by distinct stem cells. Recent studies have utilized techniques such as fluorescence-activated cell sorting, lineage tracing, and single-cell sequencing to investigate the involvement of SSCs in bone development, homeostasis, and disease. These investigations have allowed researchers to map the lineage commitment trajectory of SSCs in different parts of the body and at different time points. Furthermore, recent studies have shed light on the characteristics of SSCs in both physiological and pathological conditions. This review focuses on discussing the spatiotemporal distribution of SSCs and enhancing our understanding of the diversity and plasticity of SSCs by summarizing recent discoveries.

The formation of the epiphyseal bone plate, the flat bony structure that provides strength and firmness to the growth plate cartilage, was studied in the present study by using light, confocal, and scanning electron microscopy. Results obtained evidenced that this bone tissue is generated by the replacement of the lower portion of the epiphyseal cartilage. However, this process differs considerably from the usual bone tissue formation through endochondral ossification. Osteoblasts deposit bone matrix on remnants of mineralized cartilage matrix that serve as a scaffold, but also on non-mineralized cartilage surfaces and as well as within the perivascular space. These processes occur simultaneously at sites located close to each other, so that, a core of the sheet of bone is established very quickly. Subsequently, thickening and reshaping occurs by appositional growth to generate a dense parallel-fibered bone structurally intermediate between woven and lamellar bone. All these processes occur in close relationship with a cartilage but most of the bone tissue is generated in a manner that may be considered as intramembranous-like. Overall, the findings here reported provide for the first time an accurate description of the tissues and events involved in the formation of the epiphyseal bone plate and gives insight into the complex cellular events underlying bone formation at different sites on the skeleton.

The craniofacial area is prone to trauma or pathologies often resulting in large bone damages. One potential treatment option is the grafting of a tissue‐engineered construct seeded with adult mesenchymal stem cells (MSCs). The dental pulp appears as a relevant source of MSCs, as dental pulp stem cells display strong osteogenic properties and are efficient at bone formation and repair. Fibroblast growth factor‐2 (FGF‐2) and/or hypoxia primings were shown to boost the angiogenesis potential of dental pulp stem cells from human exfoliated deciduous teeth (SHED). Based on these findings, we hypothesized here that these primings would also improve bone formation in the context of craniofacial bone repair. We found that both hypoxic and FGF‐2 primings enhanced SHED proliferation and osteogenic differentiation into plastically compressed collagen hydrogels, with a much stronger effect observed with the FGF‐2 priming. After implantation in immunodeficient mice, the tissue‐engineered constructs seeded with FGF‐2 primed SHED mediated faster intramembranous bone formation into critical size calvarial defects than the other groups (no priming and hypoxia priming). The results of this study highlight the interest of FGF‐2 priming in tissue engineering for craniofacial bone repair. Stem Cells Translational Medicine 2019;8:844&857 Priming stem cells from human exfoliated deciduous teeth with fibroblast growth factor‐2 within plastically compressed collagen hydrogels improves osteoformation in vivo in the context of craniofacial bone repair with a faster intramembranous bone formation into calvarial bone critical size defects when compared with either of the 2 other groups (no priming and hypoxia priming) that produced smaller amounts of new bone.

The cranial endo and dermal skeletons, which comprise the vertebrate skull, evolved independently over 470 million years ago and form separately during embryogenesis. In mammals, much of the cartilaginous chondrocranium is transient, undergoing endochondral ossification or disappearing, so its role in skull morphogenesis is not well studied and it remains an enigmatic structure. We provide complete 3D reconstructions of the laboratory mouse chondrocranium from embryonic day (E) 13.5 through E17.5 using a novel methodology of uncertainty-guided segmentation of phosphotungstic enhanced 3D micro-computed tomography images with sparse annotation. We evaluate the embryonic mouse chondrocranium and dermatocranium in 3D, and delineate the effects of a Fgfr2 variant on embryonic chondrocranial cartilages and on their association with forming dermal bones using the Fgfr2c C342Y/+ Crouzon syndrome mouse. We show that the dermatocranium develops outside of and in shapes that conform to the chondrocranium. Results reveal direct effects of the Fgfr2 variant on embryonic cartilage, on chondrocranium morphology, and on the association between chondrocranium and dermatocranium development. Histologically, we observe a trend of relatively more chondrocytes, larger chondrocytes, and/or more matrix in the Fgfr2c C342Y/+ embryos at all timepoints before the chondrocranium begins to disintegrate at E16.5. The chondrocrania and forming dermatocrania of Fgfr2c C342Y/+ embryos are relatively large, but a contrasting trend begins at E16.5 and continues into early postnatal (P0 and P2) timepoints, with the skulls of older Fgfr2c C342Y/+ mice reduced in most dimensions compared to Fgfr2c +/+ littermates. Our findings have implications for the study and treatment of human craniofacial disease, for understanding the impact of chondrocranial morphology on skull growth, and potentially on the evolution of skull morphology.

Osteogenesis and angiogenesis are two closely correlated processes during bone growth, development, remodelling and repair. Vascular endothelial growth factor （VEGF） is an essential mediator during the process of angiogenesis. Based on an extensive literature search, which was carried out using the PubMed database and the keywords of osteogenesis, VEGF, endochondral ossification and intramembranous ossification, this manuscript reviews the role of VEGF in ossification, with emphasis on its effect in endochondral and intramembranous ossification. Osteogenesis and angiogenesis are closely correlated processes. VEGF acts as an essential mediator durin~ these processes. It not only functions in bone an~io~enesis but also in various aspects of bone develooment.

Intermuscular bones (IBs) are slender linear bones embedded in muscle, which ossify from tendons through a process of intramembranous ossification, and only exist in basal teleosts. IBs are essential for fish swimming, but they present a choking risk during human consumption, especially in children, which can lead to commercial risks that have a negative impact on the aquaculture of these fish. In this review, we discuss the morphogenesis and functions of IBs, including their underlying molecular mechanisms, as well as the advantages and disadvantages of different methods for IB studies and techniques for breeding and generating IB-free fish lines. This review reveals that the many key genes involved in tendon development, osteoblast differentiation, and bone formation, e.g., scxa, msxC, sost, twist, bmps, and osterix, also play roles in IB development. Thus, this paper provides useful information for the breeding of new fish strains without IBs via genome editing and artificial selection.

Epigenetic and chromatin regulation of craniofacial development remains poorly understood. Ankyrin Repeat Domain 11 ( ANKRD11 ) is a chromatin regulator that has previously been shown to control neural stem cell fates via modulation of histone acetylation. ANKRD11 gene variants, or microdeletions of the 16q24.3 chromosomal region encompassing the ANKRD11 gene, cause KBG syndrome, a rare autosomal dominant congenital disorder with variable neurodevelopmental and craniofacial involvement. Craniofacial abnormalities include a distinct facial gestalt, delayed bone age, tooth abnormalities, delayed fontanelle closure, and frequently cleft or submucosal palate. Despite this, the dramatic phenotype and precise role of ANKRD11 in embryonic craniofacial development remain unexplored. Quantitative analysis of 3D images of KBG syndromic subjects shows an overall reduction in the size of the middle and lower face. Here, we report that mice with heterozygous deletion of Ankrd11 in neural crest cells (Ankrd11 nchet ) display a mild midfacial hypoplasia including reduced midfacial width and a persistent open fontanelle, both of which mirror KBG syndrome patient facial phenotypes. Mice with a homozygous Ankrd11 deletion in neural crest cells (Ankrd11 ncko ) die at birth. They show increased severity of several clinical manifestations described for KBG syndrome, such as cleft palate, retrognathia, midfacial hypoplasia, and reduced calvarial growth. At E14.5, Ankrd11 expression in the craniofacial complex is closely associated with developing bony structures, while expression at birth is markedly decreased. Conditional deletion of Ankrd11 leads to a reduction in ossification of midfacial bones, with several ossification centers failing to expand and/or fuse. Intramembranous bones show features of delayed maturation, with bone remodeling severely curtailed at birth. Palatal shelves remain hypoplastic at all developmental stages, with a local reduction in proliferation at E13.5. Our study identifies Ankrd11 as a critical regulator of intramembranous ossification and palate development and suggests that Ankrd11 nchet and Ankrd11 ncko mice may serve as pre-clinical models for KBG syndrome in humans.

There is a distinct clinical need for new therapies that provide an effective treatment for large bone defect repair. Herein we describe a developmental approach, whereby constructs are primed to mimic certain aspects of bone formation that occur during embryogenesis. Specifically, we directly compared the bone healing potential of unprimed, intramembranous, and endochondral primed MSC-laden polycaprolactone (PCL) scaffolds. To generate intramembranous constructs, MSC-seeded PCL scaffolds were exposed to osteogenic growth factors, while endochondral constructs were exposed to chondrogenic growth factors to generate a cartilage template. Eight weeks after implantation into a cranial critical sized defect in mice, there were significantly more vessels present throughout defects treated with endochondral constructs compared to intramembranous constructs. Furthermore, 33 and 50% of the animals treated with the intramembranous and endochondral constructs respectively, had full bone union along the sagittal suture line, with significantly higher levels of bone healing than the unprimed group. Having demonstrated the potential of endochondral priming but recognizing that only 50% of animals completely healed after 8 weeks, we next sought to examine if we could further accelerate the bone healing capacity of the constructs by pre-vascularizing them prior to implantation. The addition of endothelial cells alone significantly reduced the healing capacity of the constructs. The addition of a co-culture of endothelial cells and MSCs had no benefit to either the vascularization or mineralization potential of the scaffolds. Together, these results demonstrate that endochondral priming alone is enough to induce vascularization and subsequent mineralization in a critical-size defect.

Successfully regenerating damaged or diseased bone and other joint tissues will require a detailed understanding of how joint specific environmental cues regulate the fate of progenitor cells that are recruited or delivered to the site of injury. The goal of this study was to explore the role of cyclic tensile strain (CTS) in regulating the initiation of mesenchymal stem cell/multipotent stromal cell (MSC) differentiation, and specifically their progression along the endochondral pathway. To this end, we first explored the influence of CTS on the differentiation of MSCs in the absence of any specific growth factor, and secondly, we examined the influence of the long-term application of this mechanical stimulus on markers of endochondral ossification in MSCs maintained in chondrogenic culture conditions. A custom bioreactor was developed to apply uniaxial tensile deformation to bone marrow-derived MSCs encapsulated within physiological relevant 3D fibrin hydrogels. Mechanical loading, applied in the absence of soluble differentiation factors, was found to enhance the expression of both tenogenic (COL1A1) and osteogenic markers (BMP2, RUNX2, and ALPL), while suppressing markers of adipogenesis. No evidence of chondrogenesis was observed, suggesting that CTS can play a role in initiating direct intramembranous ossification. During long-term culture in the presence of a chondrogenic growth factor, CTS was shown to induce MSC re-organization and alignment, increase proteoglycan and collagen production, and to enhance the expression of markers associated with endochondral ossification (BMP2, RUNX2, ALPL, OPN, and COL10A1) in a strain magnitude-dependent manner. Taken together, these findings indicate that tensile loading may play a key role in promoting both intramembranous and endochondral ossification of MSCs in a context-dependent manner. In both cases, this loading-induced promotion of osteogenesis was correlated with an increase in the expression of the osteogenic growth factor BMP2. The results of this study demonstrate the potent role that extrinsic mechanical loading plays in guiding stem cell fate, which must be carefully considered when designing cell and tissue-engineering therapies if they are to realize their clinical potential.

In contrary to what has traditionally been believed, bone formation can occur through two different types of osteogenesis: static (SO) and dynamic (DO) osteogenesis, which are thus named because the former is characterized by pluristratified cords of unexpectedly stationary osteoblasts which differentiate at a fairly constant distance from the blood capillaries and transform into osteocytes without moving from the onset site, while the latter is distinguished by the well-known typical monostratified laminae of movable osteoblasts. The two types of osteogenesis differ in multiple aspects from both structural and functional viewpoints. Besides osteoblast arrangement, polarization, and motion, SO and DO differ in terms of time of occurrence (first SO and later DO), conditioning factors to which they are sensitive (endothelial-derived cytokines or mechanical loading, respectively), distribution of osteocytes to which they give rise (haphazard or ordered in planes, respectively), the collagen texture resulting from the different deposition types (woven or lamellar, respectively), the mechanical properties of the bone they form (poor for SO due to the high cellularity and woven texture and good for DO since osteocytes are located in more suitable conditions to perceive loading), and finally the functions of each, i.e., SO provides a preliminary rigid scaffold on which DO can take place, while DO produces bone tissue according to mechanical/metabolic needs.