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Mesenchymal stem cells (MSCs) have been repeatedly shown to be a valuable source for cell-based therapy in regenerative medicine, including bony tissue repair. However, engraftment at the injury site is poor. Recently, it has been suggested that MSCs and other cells act through a paracrine signaling mechanism. Exosomes are nanostructures that have been implicated in this process. They carry DNA, RNA, proteins, and lipids and play an important role in cell-to-cell communication directly modulating their target cell at a transcriptional level. In a bone microenvironment, they have been shown to increase osteogenesis and osteogenic differentiation in vivo and in vitro. In the following review, we will discuss the most advanced and significant knowledge of biological functions of exosomes in bone regeneration and their clinical applications in osseous diseases. IMPACT STATEMENT: Mesenchymal stem cells have been shown to be a promising tool in bone tissue engineering. Recently, it has been suggested that they secrete exosomes containing messenger RNA, proteins, and lipids, thus acting through paracrine signaling mechanisms. Considering that exosomes are nonteratogenic and have low immunogenic potential, they could potentially replace stem-cell based therapy and thus eradicate the risk of neoplastic transformation associated with cell transplantations in bone regeneration.

Skin fibrosis is the excessive deposition of extracellular matrix in the dermis. Cutaneous fibrosis can occur following tissue injury, including burns, trauma, and surgery, resulting in scars that are disfiguring, limit movement and cause significant psychological distress for patients. Many molecular pathways have been implicated in the development of skin fibrosis, yet effective treatments to prevent or reverse scarring are unknown. The Wnt signaling pathways are known to play an important role in skin homeostasis, skin injury, and in the development of fibrotic skin diseases. This review provides a detailed overview of the role of the canonical Wnt signaling pathways in regulating skin scarring. We also discuss how Wnt signaling interacts with other known fibrotic molecular pathways to cause skin fibrosis. We further provide a summary of the different Wnt inhibitor types available for treating skin scarring. Understanding the role of the Wnt pathway in cutaneous fibrosis will accelerate the development of effective Wnt modulators for the treatment of skin fibrosis.

Osteoarthritis (OA) is a degenerative disease resulting in irreversible, progressive destruction of articular cartilage1. The etiology of OA is complex and involves a variety of factors, including genetic predisposition, acute injury and chronic inflammation2–4. Here we investigate the ability of resident skeletal stem-cell (SSC) populations to regenerate cartilage in relation to age, a possible contributor to the development of osteoarthritis5–7. We demonstrate that aging is associated with progressive loss of SSCs and diminished chondrogenesis in the joints of both mice and humans. However, a local expansion of SSCs could still be triggered in the chondral surface of adult limb joints in mice by stimulating a regenerative response using microfracture (MF) surgery. Although MF-activated SSCs tended to form fibrous tissues, localized co-delivery of BMP2 and soluble VEGFR1 (sVEGFR1), a VEGF receptor antagonist, in a hydrogel skewed differentiation of MF-activated SSCs toward articular cartilage. These data indicate that following MF, a resident stem-cell population can be induced to generate cartilage for treatment of localized chondral disease in OA. Endogenous skeletal stem cells are recruited to form cartilage in mice when stimulated by microfracture surgery together with localized delivery of growth factors, pointing to a new approach for treating cartilage defects.

Treacher Collins syndrome (TCS: OMIM 154500) is an autosomal dominant craniofacial disorder belonging to the heterogeneous group of mandibulofacial dysostoses. Objective To investigate four Treacher Collins syndrome patients of the Sgaw Karen family living in Thailand. Method Clinical examination, hearing tests, lateral cephalometric analyses, Computed tomography, whole exome sequencing and Sanger direct sequencing were performed. Results All of the patients affected with Treacher Collins syndrome carried a novel TCOF1 mutation (c.4138_4142del; p.Lys1380GlufsTer12), but clinically they did not have the typical facial gestalt of Treacher Collins syndrome, which includes downward-slanting palpebral fissures, colobomas of the lower eyelids, absence of eyelashes medial to the colobomas, malformed pinnae, hypoplastic zygomatic bones and mandibular hypoplasia. Lateral cephalometric analyses identified short anterior and posterior cranial bases, and hypoplastic maxilla and mandible. Computed tomography showed fusion of malleus and incus, sclerotic mastoid, hypoplastic middle ear space with a soft tissue remnant, dehiscence of facial nerve and monopodial stapes. Conclusion Treacher Collins syndrome in Sgaw Karen patients has not been previously documented. This is the first report of monopodial stapes in a TCS patient who had a TCOF1 mutation. The absence of a common facial phenotype and/or the presence of monopodial stapes may be the effects of this novel TCOF1 mutation.

Trichorhinophalangeal syndrome type 1 (TRPS1; Online Mendelian Inheritance in Man #190350) is an autosomal dominant disorder caused by mutations in TRPS1. We report a Thai male with TRPS1 who carried a c.1842C>T (p.Arg615Ter) mutation. He had 15 supernumerary teeth, double mental foramina, hypoplastic mandibular condyles with slender condylar necks and unique ultrastructural hair findings. Body hair was absent. The hair in the area of a congenital melanocytic nevus had a greater number of hair cuticles than normal. Occipital hair had abnormal hair follicles and cuticles. The scale edges of the hair cuticles were detached and rolled up. Hypoplastic mandibular condyles with slender condylar necks, double mental foramina and the rolled up edges of hair cuticles have not been reported in patients with TRPS1.

Cranial sutures are major growth centers for the calvarial vault, and their premature fusion leads to a pathologic condition called craniosynostosis. This study investigates whether skeletal stem/progenitor cells are resident in the cranial sutures. Prospective isolation by FACS identifies this population with a significant difference in spatio-temporal representation between fusing versus patent sutures. Transcriptomic analysis highlights a distinct signature in cells derived from the physiological closing PF suture, and scRNA sequencing identifies transcriptional heterogeneity among sutures. Wnt-signaling activation increases skeletal stem/progenitor cells in sutures, whereas its inhibition decreases. Crossing Axin2LacZ/+ mouse, endowing enhanced Wnt activation, to a Twist1+/− mouse model of coronal craniosynostosis enriches skeletal stem/progenitor cells in sutures restoring patency. Co-transplantation of these cells with Wnt3a prevents resynostosis following suturectomy in Twist1+/− mice. Our study reveals that decrease and/or imbalance of skeletal stem/progenitor cells representation within sutures may underlie craniosynostosis. These findings have translational implications toward therapeutic approaches for craniosynostosis., Cranial sutures are major growth centers for the skull vault and premature fusion leads to pathological fusion, craniosynostosis. Here the authors isolate Wnt responsive skeletal stem and progenitor cells from sutures, that can be transplanted together with Wnt3a protein to repair craniosynostosis in a mouse model.

Adult-onset immunodeficiency syndrome (AOID) with anti-interferon (IFN)-γ autoantibodies is characterized by an AIDS-like illness with disruptive IFN-γ signaling. Patients generally present with recurrent and disseminated opportunistic infections along with neutrophilic dermatoses. Generalized pustular psoriasis (GPP; Online Mendelian Inheritance in Man #614204) is characterized by acute generalized erythema and scaling with numerous aseptic pustules. Mutations in SERPINA3 have been reported as predisposing risk factors for both AOID and GPP. Here, we report two unrelated patients, one with AOID and a pustular skin reaction and the other with GPP, who both carried the same heterozygous variant c.718G>A (p.Val240Met) in SERPINA1. Our observation of a shared mutation in SERPINA1 in AOID and GPP indicate possible pathobiological and disease mechanism similarities in these two disorders. Thus, variants in both SERPINA1, SERPINA3, and potentially other SERPIN family members may be associated with the etiology of GPP and AOID.

Summary: Regenerative paradigms exhibit nerve dependency, including regeneration of the mouse digit tip and salamander limb. Denervation impairs regeneration and produces morphological aberrancy in these contexts, but the direct effect of innervation on the stem and progenitor cells enacting these processes is unknown. We devised a model to examine nerve dependency of the mouse skeletal stem cell (mSSC), the progenitor responsible for skeletal development and repair. We show that after inferior alveolar denervation, mandibular bone repair is compromised because of functional defects in mSSCs. We present mSSC reliance on paracrine factors secreted by Schwann cells as the underlying mechanism, with partial rescue of the denervated phenotype by Schwann cell transplantation and by Schwann-derived growth factors. This work sheds light on the nerve dependency of mSSCs and has implications for clinical treatment of mandibular defects. : Jones et al. demonstrate the nerve dependency of mandibular bone repair after degeneration of the inferior alveolar nerve. Schwann cell paracrine signaling is required for skeletal stem cell enactment of bone healing. Rescue of healing by Schwann cell transplantation highlights skeletal stem cell-Schwann cell circuitry during mandibular repair. Keywords: skeletal stem cells, Schwann cells, mandibular repair, nerve-dependent mandibular regeneration

Craniofacial development is a program exquisitely orchestrated by tissue contributions and regulation of genes expression. The basic helix-loop-helix (bHLH) transcription factor Twist1 expressed in the skeletal mesenchyme is a key regulator of craniofacial development playing an important role during osteoskeletogenesis. This study investigates the postnatal impact of Twist1 haploinsufficiency on the osteoskeletal ability and regeneration on two calvarial bones arising from tissues of different embryonic origin: the neural crest-derived frontal and the mesoderm-derived parietal bones. We show that Twist1 haplonsufficiency as well Twist1-sh-mediated silencing selectively enhanced osteogenic and tissue regeneration ability of mesoderm-derived bones. Transcriptomic profiling, gain-and loss-of-function experiments revealed that Twist1 haplonsufficiency triggers its selective activity on mesoderm-derived bone through a sharp downregulation of the bone-derived hormone Fgf23 that is upregulated exclusively in wild-type parietal bone.

Marfan syndrome (MFS) is a hereditary disease caused by mutations in the gene encoding Fibrillin-1 (FBN1) and characterized by a number of skeletal abnormalities, aortic root dilatation, and sometimes ectopia lentis. Although the molecular pathogenesis of MFS was attributed initially to a structural weakness of the fibrillin-rich microfibrils within the extracellular matrix, more recent results have documented that many of the pathogenic abnormalities in MFS are the result of alterations in TGFβ signaling. Mutations in FBN1 are therefore associated with increased activity and bioavailability of TGF-β1, which is suspected to be the basis for phenotypical similarities of FBN1 mutations in MFS and mutations in the receptors for TGFβ in Marfan syndrome-related diseases. We have previously demonstrated that unique skeletal phenotypes observed in human embryonic stem cells carrying the monogenic FBN1 mutation (MFS cells) are faithfully phenocopied by cells differentiated from induced pluripotent-stem cells (MFSiPS) derived independently from MFS patient fibroblasts. In this study, we aimed to determine further the biochemical features of transducing signaling(s) in MFS stem cells and MFSiPS cells highlighting a crosstalk between TGFβ and BMP signaling. Our results revealed that enhanced activation of TGFβ signaling observed in MFS cells decreased their endogenous BMP signaling. Moreover, exogenous BMP antagonized the enhanced TGFβ signaling in both MFS stem cells and MFSiPS cells therefore, rescuing their ability to undergo osteogenic differentiation. This study advances our understanding of molecular mechanisms underlying the pathogenesis of bone loss/abnormal skeletogenesis in human diseases caused by mutations in FBN1.

Clinical translation of cell-based strategies for tissue regeneration remains challenging because survival of implanted cells within hostile, hypoxic wound environments is uncertain. Overexpression of B-cell lymphoma 2 (Bcl-2) has been shown to inhibit apoptosis in implanted cells. The present study describes an “off the shelf” prefabricated scaffold integrated with magnetic nanoparticles (MNPs) used to upregulate Bcl-2 expression in implanted adipose-derived stromal cells for bone regeneration. Iron oxide cores were sequentially coated with branched polyethyleneimine, minicircle plasmid encoding green fluorescent protein and Bcl-2, and poly-β-amino ester. Through in vitro assays, increased osteogenic potential and biological resilience were demonstrated in the magnetofected group over control and nucleofected groups. Similarly, our in vivo calvarial defect study showed that magnetofection had an efficiency rate of 30%, which in turn resulted in significantly more healing compared with control group and nucleofected group. Our novel, prefabricated MNP-integrated scaffold allows for in situ postimplant temporospatial control of cell transfection to augment bone regeneration.

Craniosynostosis describes the premature fusion of one or more cranial sutures and can lead to dramatic manifestations in terms of appearance and functional impairment. Contemporary approaches for this condition are primarily surgical and are associated with considerable morbidity and mortality. The additional post-operative problems of suture refusion and bony relapse may also necessitate repeated surgeries with their own attendant risks. Therefore, a need exists to not only optimize current strategies but also to develop novel biological therapies which could obviate the need for surgery and potentially treat or even prevent premature suture fusion. Clinical studies of patients with syndromic craniosynostosis have provided some useful insights into the important signaling pathways and molecular events guiding suture fate. Furthermore, the highly conserved nature of craniofacial development between humans and other species have permitted more focused and step-wise elucidation of the molecular underpinnings of craniosynostosis. This review will describe the clinical manifestations of craniosynostosis, reflect on our understanding of syndromic and non-syndromic craniosynostoses and outline the different approaches that have been adopted in our laboratory and elsewhere to better understand the pathogenesis of premature suture fusion. Finally, we will assess to what extent our improved understanding of the pathogenesis of craniosynostosis, achieved through laboratory-based and clinical studies, have made the possibility of a non-surgical pharmacological approach both realistic and tangible.

Human adipose-derived stem cells (hASCs) are known for their capability to promote bone healing when applied to bone defects. For bone tissue regeneration, both sufficient angiogenesis and osteogenesis is desirable. Vascular endothelial growth factor A (VEGFA) has the potential to promote differentiation of common progenitor cells to both lineages. To test this hypothesis, the effects of VEGFA on hASCs during osteogenic differentiation were tested in vitro. In addition, hASCs were seeded in murine critical-sized calvarial defects locally treated with VEGFA. Our results suggest that VEGFA improves osteogenic differentiation in vitro as indicated by alkaline phosphatase activity, alizarin red staining, and quantitative real-time polymerase chain reaction analysis. Moreover, local application of VEGFA to hASCs significantly improved healing of critical-sized calvarial defects in vivo. This repair was accompanied by a striking enhancement of angiogenesis. Both paracrine and, to a lesser degree, cell-autonomous effects of VEGFA-treated hASCs were accountable for angiogenesis. These data were confirmed by using CD31−/CD45− mouse ASCsGFP+ cells. In summary, we demonstrated that VEGFA increased osteogenic differentiation of hASCS in vitro and in vivo, which was accompanied by an enhancement of angiogenesis. Additionally, we showed that during bone regeneration, the increase in angiogenesis of hASCs on treatment with VEGFA was attributable to both paracrine and cell-autonomous effects. Thus, locally applied VEGFA might prove to be a valuable growth factor that can mediate both osteogenesis and angiogenesis of multipotent hASCs in the context of bone regeneration.

Premature closure of cranial sutures, which serve as growth centers for the skull vault, result in craniosynostosis. In the mouse posterior frontal (PF) suture closes by endochondral ossification, whereas sagittal (SAG) remain patent life time, although both are neural crest tissue derived. We therefore, investigated why cranial sutures of same tissue origin adopt a different fate. We demonstrated that closure of the PF suture is tightly regulated by canonical Wnt signaling, whereas patency of the SAG suture is achieved by constantly activated canonical Wnt signaling. Importantly, the fate of PF and SAG sutures can be reversed by manipulating Wnt signaling. Continuous activation of canonical Wnt signaling in the PF suture inhibits endochondral ossification and therefore, suture closure, In contrast, inhibition of canonical Wnt signaling in the SAG suture, upon treatment with Wnt antagonists results in endochondral ossification and suture closure. Thus, inhibition of canonical Wnt signaling in the SAG suture phenocopies craniosynostosis. Moreover, mice haploinsufficient for Twist1, a target gene of canonical Wnt signaling which inhibits chondrogenesis, have sagittal craniosynostosis. We propose that regulation of canonical Wnt signaling is of crucial importance during the physiological patterning of PF and SAG sutures. Importantly, dysregulation of this pathway may lead to craniosynostosis.

Calvarial bones arise from two embryonic tissues, namely, the neural crest and the mesoderm. In this study we have addressed the important question of whether disparate embryonic tissue origins impart variable osteogenic potential and regenerative capacity to calvarial bones, as well as what the underlying molecular mechanism(s). Thus, by performing in vitro and in vivo studies, we have investigated whether differences exist between neural crest–derived frontal and paraxial mesodermal–derived parietal bone. Of interest, our data indicate that calvarial bone osteoblasts of neural crest origin have superior potential for osteogenic differentiation. Furthermore, neural crest–derived frontal bone displays a superior capacity to undergo osseous healing compared with calvarial bone of paraxial mesoderm origin. Our study identified both in vitro and in vivo enhanced endogenous canonical Wnt signaling in frontal bone compared with parietal bone. In addition, we demonstrate that constitutive activation of canonical Wnt signaling in paraxial mesodermal–derived parietal osteoblasts mimics the osteogenic potential of frontal osteoblasts, whereas knockdown of canonical Wnt signaling dramatically impairs the greater osteogenic potential of neural crest–derived frontal osteoblasts. Moreover, fibroblast growth factor 2 (FGF-2) treatment induces phosphorylation of GSK-3β and increases the nuclear levels of β-catenin in osteoblasts, suggesting that enhanced activation of Wnt signaling might be mediated by FGF. Taken together, our data provide compelling evidence that indeed embryonic tissue origin makes a difference and that active canonical Wnt signaling plays a major role in contributing to the superior intrinsic osteogenic potential and tissue regeneration observed in neural crest–derived frontal bone. © 2010 American Society for Bone and Mineral Research.

Adipose-derived adult stem cells (ASCs), like their bone-marrow derived counterparts, possess the ability to differentiate down osteogenic, chondrogenic, adipogenic, and myogenic pathways. For bone differentiation of mouse ASCs (mASCs), retinoic-acid mediated upregulation of BMPR-IB has been found to be necessary. Interestingly, our previous work has also shown Fibroblast Growth Factor-2 (FGF-2) to strongly inhibit this osteogenic differentiation, even in the presence of retinoic acid. In this report, we investigated the molecular mechanisms underlying FGF-2 mediated osteogenic inhibition, demonstrating that addition of exogenous FGF-2 to mASCs antagonizes upregulation of BMPR-IB gene expression in response to retinoic acid. In addition, constitutive expression of BMPR-IB, but not BMPR-IA or BMPR-II, was found to counteract the inhibitory effects of FGF-2. Finally, p53(-/-) mASCs and human ASCs, both of which express high levels of endogenous BMPR-IB, underwent normal osteogenic differentiation even in the presence of FGF-2. Collectively, our data therefore indicate that FGF-2 antagonizes the response of mASCs to retinoic acid and also suggest that threshold levels of BMPR-IB may play a crucial role both in counteracting the inhibitory role of FGF-2 and in promoting osteogenic differentiation of ASCs in the absence of retinoic acid. Moreover, the present study also indicates that differences exist between mouse and human ASCs in relationship to FGF-2 activity in the osteogenic context.

Although dura mater tissue is believed to have an important role in calvarial reossification in many in vivo studies, few studies have shown the direct effect of dura mater cells on osteoblasts. In addition, no reports have yet identified the potential factor(s) responsible for various biological activities exerted by dura mater on calvarial reossification (e.g., cell proliferation). In this study, we tested the effect of dura mater on calvarial-derived osteoblasts by performing both heterotypic coculture and by culturing osteoblast cells with conditioned media harvested from dura mater cells of juvenile (3-day-old) and adult (30-day-old) mice. The results presented here demonstrate that cellular proliferation of juvenile osteoblast cells was significantly increased by juvenile dura mater either in the coculture system or when dura mater cell-conditioned medium was applied to the osteoblast cells. Moreover, high levels of FGF-2 protein were detected in juvenile dura mater cells and their conditioned medium. In contrast, low levels of FGF-2 protein were detected in adult dura mater cells, whereas FGF-2 protein was not detectable in their conditioned medium. Abrogation of the mitogenic effect induced by juvenile dura mater cell-conditioned medium was achieved by introducing a neutralizing anti-FGF-2 antibody, thus indicating that FGF-2 may be responsible for the mitogenic effect of the juvenile dura mater. Moreover, data obtained by exploring the three major FGF-2 signaling pathways further reinforced the idea that FGF-2 might be an important paracrine signaling factor in vivo supplied by the underlying dura mater to the overlying calvarial osteoblasts.

In addition to adult bone marrow-derived mesenchymal stem cells, more recently, a second large stromal compartment found in adipose tissue has received attention and is believed to contain multipotent cells. In vitro, adipose tissue-derived stromal cells (ADS) can differentiate down osteogenic, chondrogenic, myogenic, adipose, and even neuronal pathways. In this article, we explore the effect of fibroblast growth factors 2 (FGF-2) on ADS cells. Our results demonstrated that FGF-2 inhibits osteogenesis in ADS cells. The osteogenic inhibitory effects is dose-dependent and reversible, thus suggesting that the lack of osteogenesis observed in ADS cells exposed to FGF-2 is not due to a negative selection triggered by this factor on a subpopulation of osteoblast progenitors. Furthermore, either overexpression of FGF-2, or continuous FGF-2 treatment sustain the proliferative and osteogenic potential state of ADS cells. Therefore, FGF-2 appears to be a positive regulator of osteoprogenitor cells and a negative regulator of osteoblast differentiation in ADS cells. These FGF-2 functional characteristics may assist with cell selection and enrichment for the purpose of bone tissue engineering.

Objective Osteosarcoma arises predominantly in the metaphyseal growth plate of children during the growth spurt years. These tumors develop during physiological growth from an expanding cell population, suggesting that the transformed cell is a bone-forming progenitor. An absence of the p53 oncogene has been implicated in the origin and progression of osteosarcoma, and because mesenchymal stem cells (MSCs) are the physiological osteogenic progenitor cell population, we hypothesized that a p53 −/− mutation would enhance bone differentiation of MSC in a mouse model of in vitro osteogenesis. Methods Clonal MSC populations were derived from p53 −/− mice. P53 −/− and wild-type cells were placed in osteogenic culture and assessed via Alizarin Red quantification and alkaline phosphatase staining. The osteogenic marker genes Cbfa1 , osteopontin, and osteocalcin were assessed by quantitative real time polymerase chain reaction during differentiation. Results Bone nodule formation and alkaline phosphatase staining was accelerated and enhanced in the p53 −/− cells. The early and intermediate osteogenic markers, Cbfa1 and osteopontin, were upregulated in p53 −/− MSCs compared with wild-type cells during osteogenesis. The terminal osteogenic marker gene osteocalcin was paradoxically lower in p53 −/− MSCs indicating impaired terminal differentiation. Conclusion The p53 −/− mutation enhances and accelerates early osteogenesis in MSCs, but prevents terminal differentiation toward a mature osteocyte phenotype. These findings may have important implications for the regulation of the MSC compartment during the derivation of osteosarcoma in children.

In adults and children over two years of age, large cranial defects do not reossify successfully, posing a substantial biomedical burden. The osteogenic potential of bone marrow stromal (BMS) cells has been documented. This study investigates the in vivo osteogenic capability of adipose-derived adult stromal (ADAS) cells, BMS cells, calvarial-derived osteoblasts and dura mater cells to heal critical-size mouse calvarial defects. Implanted, apatite-coated, PLGA scaffolds seeded with ADAS or BMS cells produced significant intramembranous bone formation by 2 weeks and areas of complete bony bridging by 12 weeks as shown by X-ray analysis, histology and live micromolecular imaging. The contribution of implanted cells to new bone formation was 84-99% by chromosomal detection. These data show that ADAS cells heal critical-size skeletal defects without genetic manipulation or the addition of exogenous growth factors.

Improvements in medical care, nutrition and social care are resulting in a commendable change in world population demographics with an ever increasing skew towards an aging population. As the proportion of the world’s population that is considered elderly increases, so does the incidence of osteodegenerative disease and the resultant burden on healthcare. The increasing demand coupled with the limitations of contemporary approaches, have provided the impetus to develop novel tissue regeneration therapies. The use of stem cells, with their potential for self-renewal and differentiation, is one potential solution. Adipose-derived stem cells (ASCs), which are relatively easy to harvest and readily available have emerged as an ideal candidate. In this review, we explore the potential for ASCs to provide tangible therapies for craniofacial and long bone skeletal defects, outline key signaling pathways that direct these cells and describe how the developmental signaling program may provide clues on how to guide these cells in vivo. This review also provides an overview of the importance of establishing an osteogenic microniche using appropriately customized scaffolds and delineates some of the key challenges that still need to be overcome for adult stem cell skeletal regenerative therapy to become a clinical reality.

The mammalian skull vault, a product of a unique and tightly regulated evolutionary process, in which components of disparate embryonic origin are integrated, is an elegant model with which to study osteoblast biology. Our laboratory has demonstrated that this distinct embryonic origin of frontal and parietal bones confer differences in embryonic and postnatal osteogenic potential and skeletal regenerative capacity, with frontal neural crest derived osteoblasts benefitting from greater osteogenic potential. We outline how this model has been used to elucidate some of the molecular mechanisms which underlie these differences and place these findings into the context of our current understanding of the key, highly conserved, pathways which govern the osteoblast lineage including FGF, BMP, Wnt and TGFβ signaling. Furthermore, we explore recent studies which have provided a tantalizing insight into way these pathways interact, with evidence accumulating for certain transcription factors, such as Runx2, acting as a nexus for cross-talk.

During the first month of life, the murine posterior-frontal suture (PF) of the cranial vault closes through endochondral ossification, while other sutures remain patent. These processes are tightly regulated by canonical Wnt signaling. Low levels of active canonical Wnt signaling enable endochondral ossification and therefore PF-suture closure, whereas constitutive activation of canonical Wnt causes PF-suture patency. We therefore sought to test this concept with a knockout mouse model. PF-sutures of Axin2(-/-) mice, which resemble a state of constantly activated canonical Wnt signaling, were investigated during the physiological time course of PF-suture closure and compared in detail with wild type littermates. Histological analysis revealed that the architecture in Axin2(-/-) PF-sutures was significantly altered in comparison to wild type. The distance between the endocranial layers was dramatically increased and suture closure was significantly delayed. Moreover, physiological endochondral ossification did not occur, rather an ectopic cartilage appeared between the endocranial and ectocranial bone layers at P7 which eventually involutes at P13. Quantitative PCR analysis showed the lack of Col10α1 upregulation in Axin2(-/-) PF-suture. Immunohistochemistry and gene expression analysis also revealed high levels of type II collagen as compared to type I collagen and absence of Mmp-9 in the cartilage of Axin2(-/-) PF-suture. Moreover, TUNEL staining showed a high percentage of apoptotic chondrocytes in Axin2(-/-) PF-sutures at P9 and P11 as compared to wild type. These data indicated that Axin2(-/-) PF-sutures lack physiological endochondral ossification, contain ectopic cartilage and display delayed suture closure.

Neural crest-derived (FOb) and mesoderm-derived (POb) calvarial osteoblasts are characterized by distinct differences in their osteogenic potential. We have previously demonstrated that enhanced activation of endogenous FGF and Wnt signaling confers greater osteogenic potential to FOb. Apoptosis, a key player in bone formation, is the main focus of this study. In the current work, we have investigated the apoptotic activity of FOb and POb cells during differentiation. We found that lower apoptosis, as measured by caspase-3 activity is a major feature of neural crest-derived osteoblast which also have higher osteogenic capacity. Further investigation indicated TGF-β signaling as main positive regulator of apoptosis in these two populations of calvarial osteoblasts, while BMP and canonical Wnt signaling negatively regulate the process. By either inducing or inhibiting these signaling pathways we could modulate apoptotic events and improve the osteogenic potential of POb. Taken together, our findings demonstrate that integration of multiple signaling pathways contribute to imparting greater osteogenic potential to FOb by decreasing apoptosis.

FGF-2 (basic fibroblast growth factor) was recently detected in the nucleus of a variety of cell types. The large isoforms contain a functional nuclear localization signal that allows their nuclear accumulation in producing cells, while a small amount of FGF-2 added exogenously to target cells is translocated to the nucleus in phase G1 of the cell cycle according to an unknown process. We report here using Chinese hamster ovary cell mutants bearing deficiency in heparan sulfate proteoglycans (HSPGs) synthesis that HSPGs are required for transport of exogenous FGF-2 to the nucleus. Furthermore a co-transport was suggested since an active complex containing FGF-2 and HSPGs was isolated from nuclei of treated cells. Several FGF-2 nuclear targets were described. In vivo as in vitro, it activates rDNA transcription and it binds to a specific DNA sequence that is present in the non-transcribed spacer of ribosomal genes. In vitro, FGF-2 has a strong affinity for histone H1 and it activates the protein kinase CKII. In the nucleus FGF-2 could regulate gene expression through modulation of chromatin structure.

Clinically available sources of bone for repair and reconstruction are limited by the accessibility of autologous grafts, infectious risks of cadaveric materials, and durability of synthetic substitutes. Cell-based approaches for skeletal regeneration can potentially fill this need, and adipose tissue represents a promising source for development of such therapies. Here, we enriched for an osteogenic subpopulation of cells derived from human subcutaneous adipose tissue utilizing microfluidic-based single cell transcriptional analysis and fluorescence-activated cell sorting (FACS). Statistical analysis of single cell transcriptional profiles demonstrated that low expression of endoglin (CD105) correlated with a subgroup of adipose-derived cells with increased osteogenic gene expression. FACS-sorted CD105(low) cells demonstrated significantly enhanced in vitro osteogenic differentiation and in vivo bone regeneration when compared with either CD105(high) or unsorted cells. Evaluation of the endoglin pathway suggested that enhanced osteogenesis among CD105(low) adipose-derived cells is likely due to identification of a subpopulation with lower TGF-β1/Smad2 signaling. These findings thus highlight a potential avenue to promote osteogenesis in adipose-derived mesenchymal cells for skeletal regeneration.

Bone regeneration is a complex event that requires the interaction of numerous growth factors. Fibroblast growth factor (Fgf)-ligands have been previously described for their importance in osteogenesis during development. In the current study, we investigated the role of Fgf-18 during bone regeneration. By utilizing a unicortical tibial defect model, we revealed that mice haploinsufficient for Fgf-18 have a markedly reduced healing capacity as compared with wild-type mice. Reduced levels of Runx2 and Osteocalcin but not Vegfa accompanied the impaired bone regeneration. Interestingly, our data indicated that upon injury angiogenesis was not impaired in Fgf-18(+/-) mice. Moreover, other Fgf-ligands and Bmp-2 could not compensate for the loss of Fgf-18. Finally, application of FGF-18 protein was able to rescue the impaired healing in Fgf-18(+/-) mice. Thus, we identified Fgf-18 as an important mediator of bone regeneration, which is required during later stages of bone regeneration. This study provides hints on how to engineering efficiently programmed bony tissue for long bone repair.

An understanding of the molecular mechanisms controlling bone formation is central to skeletal tissue engineering efforts. The observation that immature animals are able to heal calvarial defects while adult animals are not has proven to be a useful tool for examining these mechanisms. Thus, the authors compared expression of sclerostin, a bone inhibitor, between the calvariae of juvenile and adult mice.Parietal bone was harvested from juvenile (6-day-old; n = 20) and adult (60-day-old; n = 20) mice. Sclerostin transcript and protein levels were compared between the parietal bone of juvenile and adult mice using polymerase chain reaction, Western blotting, and immunohistochemistry. Finally, osteoblasts from the parietal bone of juvenile and adult mice were harvested and cultured under osteogenic differentiation conditions with and without recombinant sclerostin (200 ng/ml). Terminal osteogenic differentiation was assessed at 21 days with alizarin red staining.Polymerase chain reaction, Western blot analysis, and immunohistochemistry all confirmed greater expression of sclerostin in the parietal bone of adult mice when compared with that of juvenile mice. Osteoblasts, whether from juvenile or adult parietal bones, demonstrated reduced capacity for osteogenic differentiation when exposed to recombinant sclerostin.Given the role of sclerostin in inhibiting bone formation, the authors' findings suggest that differences in expression levels of sclerostin may play a role in the differential regenerative capacity of calvariae from juvenile and adult animals. These findings suggest it as a potential target to abrogate in future tissue engineering studies.

To delineate the competence window in which canonical wingless (Wnt)-signaling can either inhibit or promote osteogenic differentiation, we have analyzed cells with different status, specifically undifferentiated mesenchymal cells, such as adipose-derived stem cells and embryonic calvarial mesenchymal cells, and differentiated mesenchymal cells such as juvenile immature calvarial osteoblasts and adult calvarial osteoblasts. Our analysis indicated that undifferentiated mesenchymal cells and juvenile calvarial osteoblasts are endowed with higher levels of endogenous canonical Wnt signaling compared to fully differentiated adult calvarial osteoblasts, and that different levels of activation inversely correlated with expression levels of several Wnt antagonists. We have observed that activation of canonical Wnt signaling may elicit opposite biological activity in the context of osteogenic differentiation depending on the status of cell, the threshold levels of its activation, and Wnt ligands concentration. The results presented in this study indicate that treatment with Wnt3 and/or expression of constitutively activated#x03B2;-catenin inhibits osteogenic differentiation of undifferentiated mesenchymal cells, whereas expression of dominant negative transcription factor 4 (Tcf4) and/or secreted frizzled related protein 1 treatment enhances their osteogenic differentiation. Wnt3a treatment also inhibits osteogenesis in juvenile calvarial osteoblasts in a dose-dependent fashion. Conversely, Wnt3a treatment strongly induces osteogenesis in mature calvarial osteoblasts in a dose-dependent manner. Importantly, in vitro data correlated with in vivo results showing that Wnt3a treatment of calvarial defects, created in juvenile mice, promotes calvarial healing and bone regeneration only at low doses, whereas high doses of Wnt3a impairs tissue regeneration. In contrast, high doses of Wnt3a enhance bony tissue regeneration and calvarial healing in adult mice. Therefore, the knowledge of both endogenous activity of canonical Wnt signaling and appropriate concentrations of Wnt3a treatment may lead to significant improvement for bony tissue engineering, as well as for the efficient implement of adipose-derived stem cells in bone regeneration. Indeed, this study has important potential implications for tissue engineering, specifically for repair of juvenile bone defects.

Bone healing requires a complex interaction of growth factors that establishes an environment for efficient bone regeneration. Among these, FGFs have been considered important for intrinsic bone-healing capacity. In this study, we analyzed the role of Fgf-9 in long bone repair. One-millimeter unicortical defects were created in tibias of Fgf-9 +/− and wild-type mice. Histomorphometry revealed that half-dose gene of Fgf-9 markedly reduced bone regeneration as compared with wild-type. Both immunohistochemistry and RT-PCR analysis revealed markedly decreased levels of proliferating cell nuclear antigen (PCNA), Runt-related transcription factor 2 (Runx2), osteocalcin, Vega-a, and platelet endothelial cell adhesion molecule 1 (PECAM-1) in Fgf-9 +/− defects. μCT angiography indicated dramatic impairment of neovascularization in Fgf-9 +/− mice as compared with controls. Treatment with FGF-9 protein promoted angiogenesis and successfully rescued the healing capacity of Fgf-9 +/− mice. Importantly, although other pro-osteogenic factors [ Fgf-2 , Fgf-18 , and bone morphogenic protein 2 ( Bmp-2 )] still were present in Fgf-9 +/− mice, they could not compensate for the haploinsufficiency of the Fgf-9 gene. Therefore, endogenous Fgf-9 seems to play an important role in long bone repair. Taken together our data suggest a unique role for Fgf-9 in bone healing, presumably by initiating angiogenesis through Vegf-a. Moreover, this study further supports the embryonic phenotype previously observed in the developing limb, thus promoting the concept that healing processes in adult organisms may recapitulate embryonic skeletal development.

In the skull vault, neural crest derived frontal bones have an increased healing capacity and higher expression levels of Fibroblast Growth Factor-ligands as compared to mesoderm-derived parietal bones. Thus, we asked whether Fibroblast Growth Factor-ligands are responsible for the superior healing potential of frontal bones. Parietal defects in juvenile and adult mice treated with Fibroblast Growth Factor-2, -9 and -18 showed increased bone regeneration, comparable to frontal defects. Immunohistochemistry revealed increased recruitment of osteoprogenitors and activation of FGF-signaling pathways in FGF-treated parietal defects. Conversely, calvarial defects in Fgf-9 + /− and Fgf-18 + /− mice showed impaired calvarial healing which could be rescued by exogenous Fibroblast Growth Factor-ligands. Moreover, by utilizing Wnt1Cre/R26R mice, the migration and contribution of dura mater and pericranium cells to calvarial healing could be demonstrated. Taken together our results demonstrated that different endogenous threshold levels of Fibroblast Growth Factor-ligands in frontal and parietal bones have a profound impact on calvarial regeneration. The present study thereby opens new avenues for translational medicine.

Cranial suture development involves coordinated expression of multiple genes and tissue contribution from neural crest cells and paraxial mesoderm for timely sutural morphogenesis. Transcription factors, growth factors, and neural crest determinant genes play critical roles in calvarial growth ensuring normal development of the underlying brain. In vitro studies have implicated cell-cell adhesion molecules as a driving force behind suture closure. We performed cDNA microarray to study differential expression of adhesion molecules during the timing of suture closure in a mouse model where only the posterior frontal (PF) suture closes. Our results indicate increased expression of E-cadherin during the period of PF suture closure. Quantitative RT-PCR analysis of E- and N-cadherin in PF closing suture revealed a biphasic expression of N-cadherin, the first phase coinciding with cellular condensation preceding chondrogenesis followed by a second phase coinciding with E-cadherin co-expression and suture closure. Furthermore, expression analysis of the N-cadherin and E-cadherin transcriptional repressors Wnt7a and Snail indicate a specific temporal regulation of these genes, suggesting their potential role as regulators of both E- and N-cadherin during the PF suture development and closure. Finally, given the in vitro evidence of fibroblast growth factor (FGF)-2 as a potential regulator of E- and N-cadherin we investigated the expression of E-cadherin during PF suture closure in Fgf-2 deficient mice. In contrast to in vitrodata previously reported, E-cadherin expression is normal in these animals, and PF suture closure occurs properly, probably due to potential redundancy of FGF ligands ensuring normal temporal expression of E-cadherin and PF suture closure.

The mammalian skull vault consists mainly of 5 flat bones, the paired frontals and parietals, and the unpaired interparietal. All of these bones are formed by intramembranous ossification within a layer of mesenchyme, the skeletogenic membrane, located between the dermal mesenchyme and the meninges surrounding the brain. While the frontal bones are of neural crest in origin, the parietal bones arise from mesoderm. The present study is a characterization of frontal and parietal bones at their molecular level, aiming to highlight distinct differences between the neural crest-derived frontal and mesodermal-derived parietal bone. We performed a detailed comparative gene expression profile of FGF ligands and their receptors known to play crucial role in skeletogenesis. This analysis revealed that a differential expression pattern of the major FGF osteogenic molecules and their receptors exists between the neural crest-derived frontal bone and the paraxial mesoderm-derived parietal bone. Particularly, the expression of ligands such as Fgf-2, Fgf-9 and Fgf-18 was upregulated in frontal bone on embryonic day 17.5, postnatal day 1 and postnatal day 60 mice. Frontal bone also elaborated higher levels of Fgf receptor 1, 2 and 3 transcripts versus parietal bone. Taken together, these data suggest that the frontal bone is a domain with higher FGF-signaling competence than parietal bone.

Bone defects remain a substantial biomedical burden. The skull is an interesting model to study these defects, since it contains bones of different embryonic origins. The parietal bone is mesodermderived, whereas the frontal bone’s origin is the neural crest. In the latter one, we have previously demonstrated, that higher expression levels of Fibroblast Growth Factor (FGF) ligands and receptors exist. In our current work, we demonstrate a higher osteogenic healing potential of the frontal bone compared to the parietal bone in vivo and in vitro. In order to further investigate this interesting observation, we tried to mimic the healing potential of the frontal bone by adding exogenous FGF-2 in parietal defects.

Background The tremendous diversity in vertebrate skull formation illustrates the range of forms and functions generated by varying genetic programs. Understanding the molecular basis for this variety may provide us with insights into mechanisms underlying human craniofacial anomalies. In this study, we provide evidence that the anuran Xenopus laevis can be developed as a simplified model system for the study of cranial ossification and suture patterning. The head structures of Xenopus undergo dramatic remodelling during metamorphosis; as a result, tadpole morphology differs greatly from the adult bony skull. Because of the extended larval period in Xenopus, the molecular basis of these alterations has not been well studied. Methodology/Principal Findings We examined late larval, metamorphosing, and post-metamorphosis froglet stages in intact and sectioned animals. Using micro-computed tomography (μCT) and tissue staining of the frontoparietal bone and surrounding cartilage, we observed that bone formation initiates from lateral ossification centers, proceeding from posterior-to-anterior. Histological analyses revealed midline abutting and posterior overlapping sutures. To determine the mechanisms underlying the large-scale cranial changes, we examined proliferation, apoptosis, and proteinase activity during remodelling of the skull roof. We found that tissue turnover during metamorphosis could be accounted for by abundant matrix metalloproteinase (MMP) activity, at least in part by MMP-1 and -13. Conclusion A better understanding of the dramatic transformation from cartilaginous head structures to bony skull during Xenopus metamorphosis may provide insights into tissue remodelling and regeneration in other systems. Our studies provide some new molecular insights into this process.

The ability of Adipose-derived Stem Cells (ASCs) to differentiate into various tissues in vitro and in vivo, a function known as "stem cell plasticity", makes them an appealing cell source for tissue engineering. Our laboratory is particularly focused on the potential role of adipose tissue as a readily available postnatal source of osteoprogenitor. Fibroblast growth factors (FGF) and their receptors (FGFR) are important regulators of osteogenesis. The goal of this study was to elucidate how changes in temporal expression patterns of individual components of the fibroblast growth factor (FGF) signaling axis correlate with osteogenic differentiation of mASCs. Our results indicate that FGF ligand genes, such as Fgf-2, -4, -8, and -18, displayed a differential and dynamic profile during mouse ASC (mASC) osteogenesis. Fgf-2 transcript was down-regulated, while Fgf-18 transcript level was strongly up-regulated. Interestingly, a drift in the ratio of different FGF-2 protein forms, with translation favoring the HMWFGF-2 forms, occurred during osteogenic differentiation, whereas, the expression of LMWFGF-2 form was down-regulated. This finding shares similarity with a previous study suggesting that preferential expression of the HMWFGF-2 forms is associated with a more osteogenic differentiated state of calvarial osteoblast. Moreover, a differential expression of Fgf Receptor 1 and 2 resembling that previously found in in vivo osteogenic study was observed. Thus, mASCs undergoing osteogenesis recapitulate the in vivo osteogenic differentiation expression pattern of FGF ligands and receptors of calvarial mesenchymal cells during their own osteogenic differentiation. Indeed, this observation further validates ASCs as a suitable resource for skeletal tissue engineering.

Although the multilineage potential of human adipose-derived adult stromal cells (ADAS) has been well described, few published studies have investigated the biological and molecular mechanisms underlying osteogenic differentiation of mouse ADAS. We report here that significant osteogenesis, as determined by gene expression and histological analysis, is induced only when mouse ADAS are cultured in the presence of retinoic acid with or without recombinant human bone morphogenetic protein (BMP)-2 supplementation. Furthermore, a dynamic expression profile for the BMP receptor (BMPR) isoform IB was observed, with dramatic up-regulation during osteogenesis. Western blot analysis revealed that retinoic acid enhanced levels of BMPR-IB protein during the first 7 days of osteogenic differentiation and that RNAi-mediated suppression of BMPR-IB dramatically impaired the ability of ADAS to form bone in vitro . In contrast, absence of BMPR-IA did not significantly diminish ADAS osteogenesis. Our data therefore demonstrate that the osteogenic commitment of multipotent mouse ADAS requires retinoic acid, which enhances expression of the critical BMPR-IB isoform.