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

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.

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 ASCs^GFP+ 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. STEM CELLS 2011;29:286–296

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.

The utilization of growth factors for bone regeneration is a widely studied field. Since the approval of bone morphogenetic protein-2 (BMP-2) for therapeutic use in humans, the concept of utilizing growth factors for bone regeneration in translational medicine has become even more attractive. Despite many studies published on individual growth factors in various bone models, comparative analysis is largely lacking. The aim of our study was to compare three different proosteogenic factors under identical in vivo conditions. Thus, we tested the bone regeneration capacity of the three different growth factors BMP-2, fibroblast growth factor-2 (FGF-2), and vascular endothelial growth factor A (VEGFA) in a calvarial defect model. We demonstrated that BMP-2 and VEGFA had similar bone healing capacities, resulting in complete calvarial healing as early as week 3. FGF-2 also showed a significantly higher bone regeneration capacity; however, the healing rate was lower than with BMP-2 and VEGFA. Interestingly, these findings were paralleled by an increased angiogenic response upon healing in BMP-2– and VEGFA-treated calvarial defects as compared with FGF-2. Immunohistochemistry for proliferating and osteoprogenitor cells revealed activity at different points after surgery among the groups. In conclusion, we demonstrated an efficient bone regeneration capacity of both BMP-2 and VEGFA, which was superior to FGF-2. Moreover, this study highlights the efficient bone regeneration of VEGFA, which was comparable with BMP-2. These data provide a valuable comparative analysis, which can be used to further optimize growth factor–based strategies in skeletal tissue engineering.

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.