Apical expansion of calvarial osteoblasts and suture patency is dependent on fibronectin cues.

The skull roof, or calvaria, is comprised of interlocking plates of bones that encase the brain. Separating these bones are fibrous sutures that permit growth. Currently, we do not understand the instructions for directional growth of the calvaria, a process which is error-prone and can lead to skeletal deficiencies or premature suture fusion (craniosynostosis, CS). Here, we identify graded expression of fibronectin (FN1) in the mouse embryonic cranial mesenchyme (CM) that precedes the apical expansion of calvaria. Conditional deletion of Fn1 or Wasl leads to diminished frontal bone expansion by altering cell shape and focal actin enrichment, respectively, suggesting defective migration of calvarial progenitors. Interestingly, Fn1 mutants have premature fusion of coronal sutures. Consistently, syndromic forms of CS in humans exhibit dysregulated FN1 expression, and we also find FN1 expression altered in a mouse CS model of Apert syndrome. These data support a model of FN1 as a directional substrate for calvarial osteoblast migration that may be a common mechanism underlying many cranial disorders of disparate genetic etiologies.

The emergence of the calvarial hematopoietic niche in health and disease.

The diploë region of skull has recently been discovered to act as a myeloid cell reservoir to the underlying meninges. The presence of ossified vascular channels traversing the inner skull of cortex provides a passageway for the cells to traffic from the niche, and CNS-derived antigens traveling through cerebrospinal fluid in a perivascular manner reaches the niche to signal myeloid cell egress. This review will highlight the recent findings establishing this burgeoning field along with the known role this niche plays in CNS aging and disease. It will further highlight the anatomical routes and physiological properties of the vascular structures these cells use for trafficking, spanning from skull to brain parenchyma.

Craniolacunia and the Development of the Fetal Calvaria: An Autopsy Series With Clinical and Histological Analysis.

BACKGROUND: Craniolacunia, also known as lückenschädel, is a congenital abnormality of the calvaria featuring well-circumscribed areas of marked thinning, interspersed with more-normal bone. It is most commonly associated with myelomeningocele and/or Chiari 2 malformation. METHODS: Records, photographs, and histologic sections were reviewed from 13 autopsy cases with craniolacunia. To investigate normal calvarial development, 23 parietal bone samples from fetuses/infants of 16-42 weeks gestation were examined. RESULTS: Parietal bone development had reproducible morphologic stages. Bone thickness increased with gestational age, while osteoblast numbers decreased. Craniolacunia was mainly seen in neonates. Five patients had Chiari 2 malformation, 1 had hydrocephalus, and 2 had other structural CNS abnormalities. One had trisomy 18. Four had no congenital abnormalities. Two sustained intrapartum skull fractures. Histologic sections were available in 5 cases. Lacunae in term infants had architecture similar to normal calvaria at 16-20 weeks. Adjacent bone had age appropriate architecture but increased osteoblast numbers. CONCLUSIONS: This is the largest autopsy series of craniolacunia and first systematic histologic analysis of craniolacunia and the developing fetal calvaria. Decreased cerebrospinal fluid pressure, due to myelomeningocele or other structural abnormality, may promote craniolacunia development. The risk of intrapartum fracture through lacunae emphasizes the continued clinical relevance of this diagnosis.

Intraoperative extracorporeal irradiation for giant sarcoma-infiltrated calvarium: a case report.

Large bone defects that occur after resection of calvarial tumours are commonly remedied using titanium meshes or bone prostheses. However, these methods have several problems. While intraoperative extracorporeal radiotherapy for bone flaps could avoid these problems, there have been only a few reports wherein meningiomas were treated with 120 Gy irradiation. Moreover, no reports are available on calvarial metastasis of sarcoma, and the therapeutic radiation dose remains uncertain. Here, we report a case of giant calvarial metastasis of myxoid liposarcoma treated with intraoperative extracorporeal radiotherapy at a dose of 50 Gy. The treatment resulted in successful tumour control followed by favourable bone reconstruction.

Reconstruction and analysis of the DAN5/P1 and BSN12/P1 Gona Early Pleistocene Homo fossils.

Two Early Pleistocene fossils from Gona, Ethiopia, were originally assigned to Homo erectus, and their differences in size and robusticity were attributed to either sexual dimorphism or anagenetic evolution. In the current study, we both revisit the taxonomic affinities of these fossils and assess whether morphological differences between them reflect temporal evolution or sexual variation. We generated virtual reconstructions of the mostly complete ∼1.55 Ma DAN5/P1 calvaria and the less complete 1.26 Ma BSN12/P1 fossil, allowing us to directly compare their anterior vault shapes using landmark-based shape analysis. The two fossils are similar in calvaria shape to H. erectus and also to other Early Pleistocene Homo species based on a geometric morphometric analysis of calvaria landmarks and semilandmarks. The DAN5/P1 fossil bears a particularly close affinity to the Georgian H. erectus fossils and to KNM-ER 1813 (H. habilis), probably reflecting allometric influences on vault shape. Combined with species-specific traits of the neurocranium (e.g., midline keeling, angular torus), we confirm that these fossils are likely early African H. erectus. We calculated regression-based estimates of endocranial volume for BSN12/P1 of 882-910 cm3 based on three virtual reconstructions. Although BSN12/P1 is markedly larger than DAN5/P1 (598 cm3), both fossils represent the smallest adult H. erectus known from their respective time periods in Africa. Some of the difference in endocranial volume between the two Gona fossils reflects broader species-level brain expansion from 1.77 to 0.01 Ma, confirmed here using a large sample (n = 38) of H. erectus. However, shape differences between these fossils did not reflect species-level changes to calvaria shape. Moreover, the analysis failed to recover a clear pattern of sexually patterned size or shape differences within H. erectus based on our current assessments of sex for individual fossils.

Potential Use of 3D CORAGRAF-Loaded PDGF-BB in PLGA Microsphere Seeded Mesenchymal Stromal Cells in Enhancing the Repair of Calvaria Critical-Size Bone Defect in Rat Model.

Our previous study evidenced that the 3D CORAGRAF loaded with PLGA microsphere constitutes PDGF-BB can support cell attachment and proliferation and can induce an osteogenic commitment of mesenchymal stromal cells in the in vitro condition. However, how this construct can perform in pathophysiological conditions in terms of repairing critical bone defects is yet to be understood. A study was therefore conducted to investigate the regeneration potential of calvaria critical-size defects using CORAGRAF + PLGA with PDGF-BB + mesenchymal stromal cells (MSCs) in a rat model. A 5 mm critical bone defect was created on calvaria of 40 male Sprague-Dawley rats. CORAGRAF incorporated either with or without PDGF-BB and seeded with rat bone-marrow-derived MSCs was implanted at the defect region. The bone regeneration potential of implanted constructs was assessed using micro-CT imaging and histological staining in weeks 4 and 8. The micro-CT images indicated a significant closure of defects in the cranial bone of the rats treated with 3D CORAGRAF + PLGA with PDGF-BB + MSCs on week 4 and 8 post-implantation. This finding, further supported with the histology outcome where the rat cranial defect treated with CORAGRAF + PLGA with PDGF-BB + MSCs indicated neo-bony ingrowth with organized and mature bone-like morphology as compared with other groups. The previous in vitro results substantiated with our pre-clinical findings demonstrate that the combination of CORAGRAF + PLGA with PDGF-BB + MSCs could be an ideal construct to support bone regeneration in critical bone defects. Hence, this construct can be further investigated for its safety and efficacy in large animal models, or it can be skipped to human trial prior for commercialization.

Integra Dermal Regeneration Template in reconstructive surgery for cutaneous tumours: a two-year retrospective review.

OBJECTIVE: Integra Dermal Regeneration Template (IDRT) (Integra LifeSciences, US) is a bioengineered dermal matrix that has been widely used in burn reconstruction since its first description. However, little is reported on its use in oncologic dermatological defects. Our objective was to evaluate reconstruction using IDRT on cutaneous tumour defects. METHOD: We conducted a two-year retrospective review of patients with skin tumours who had an excision surgery, followed by reconstruction with IDRT, as a mid-step towards a final autograft procedure: a split-thickness skin graft. The records of all patients at a single academic institution were queried from the electronic medical record using data obtained from the operating surgeon. RESULTS: We identified 13 patients with different tumour types and locations. The mean defect size was 105.92cm². The matrix take rate was 92.3% and average postoperative day for definite autograft was 20 days. Patients were followed for a period of up to 12 months. Of the patients, one had exposed bone without periosteum; another patient showed recurrence six months after matrix placement, requiring a new second two-stage IDRT-autograft procedure before radiation therapy. Patients reported complete satisfaction with the cosmetic, functional and oncological results. No cases of infection were encountered. CONCLUSION: IDRT is a valid option for the reconstruction of oncologic surgical defects of the skin and can be used in different anatomical locations. Specifically, it is an alternative to the reconstructive ladder when grafts and local flaps are not possible in those patients, and an option for patients who will eventually need adjuvant radiotherapy.

Different Requirements of CBFB and RUNX2 in Skeletal Development among Calvaria, Limbs, Vertebrae and Ribs.

RUNX proteins, such as RUNX2, regulate the proliferation and differentiation of chondrocytes and osteoblasts. Haploinsufficiency of RUNX2 causes cleidocranial dysplasia, but a detailed analysis of Runx2+/- mice has not been reported. Furthermore, CBFB is required for the stability and DNA binding of RUNX family proteins. CBFB has two isoforms, and CBFB2 plays a major role in skeletal development. The calvaria, femurs, vertebrae and ribs in Cbfb2-/- mice were analyzed after birth, and compared with those in Runx2+/- mice. Calvarial development was impaired in Runx2+/- mice but mildly delayed in Cbfb2-/- mice. In femurs, the cortical bone but not trabecular bone was reduced in Cbfb2-/- mice, whereas both the trabecular and cortical bone were reduced in Runx2+/- mice. The trabecular bone in vertebrae increased in Cbfb2-/- mice but not in Runx2+/- mice. Rib development was impaired in Cbfb2-/- mice but not in Runx2+/- mice. These differences were likely caused by differences in the indispensability of CBFB and RUNX2, the balance of bone formation and resorption, or the number and maturation stage of osteoblasts. Thus, different amounts of CBFB and RUNX2 were required among the bone tissues for proper bone development and maintenance.

ANATOMICAL VARIATIONS OF THE PARIETAL FORAMEN AND ITS RELATIONS TO THE CALVARIAL LANDMARKS: A CROSS-SECTIONAL CADAVERIC STUDY.

OBJECTIVE: The aim: Estimate the prevalence of the parietal foramen in the adult human skulls of Ukrainian origin, and study its morphology and relationships to main anatomical landmarks of the skull. PATIENTS AND METHODS: Materials and methods: A cross-sectional observational study of PF was conducted with 42 random cadaveric adult human skull roofs (calvaria) collected from the laboratory and museum of Human Anatomy Department, Kharkiv National Medical University, Ukraine. The patency and the length of the PF canal were determined, and PF external/internal diameters and the distance to the calvarial landmarks from PF were measured using the caliper. Mean and standard deviation were calculated to compare with the existing data. RESULTS: Results: In the present study 85.7% (n = 36) of the calvaria had the PF, 54.8% (n = 23) had bilateral location of PF, 30.9% (n = 13) had unilateral presence of PF (right side: 23.8%, n=10 and left side: 7.1%, n=3), and 14.3 % (n = 6) demonstrated bilateral absence of PF. CONCLUSION: Conclusions: An anatomical variation in parietal foramen is not uncommon, and the differences can be based on multiple factors like geography and race. It is important to have detailed information on anatomical variations in different population groups to facilitate surgical and radiological interventions.

Aggressive growing of the infantile cavernous hemangioma of the calvaria: a case report and review of literature.

INTRODUCTION: Primary intraosseous cavernous hemangiomas of the skull are very rare in the pediatric age group and usually slow-growing tumors. CASE REPORT: We present a case of 5-month-old girl with a left occipital cavernous hemangioma that is rapidly growing. The subcutaneous occipital tiny mass was first noted at birth, and the lesion became rapidly enlarged in size and became soft for 3 months. The left occipital subcutaneous lesion was 4.0 × 4.0 × 2.0 cm (AP × LR × HT) in size. There was no history of trauma or bone tumor in her family. She underwent resection of the lesion, and a pathologic diagnosis of calvarial cavernous hemangioma was made. No recurrence was seen 1 year after surgery. CONCLUSION: The rapid growth of the infant cavernous hemangioma might be related to not only bleeding and/or congestion of the lesion but the immature thin skull of the infant.

Molecular patterning of the embryonic cranial mesenchyme revealed by genome-wide transcriptional profiling.

In the head of an embryo, a layer of mesenchyme surrounds the brain underneath the surface ectoderm. This cranial mesenchyme gives rise to the meninges, the calvaria (top part of the skull), and the dermis of the scalp. Abnormal development of these structures, especially the meninges and the calvaria, is linked to significant congenital defects in humans. It has been known that different areas of the cranial mesenchyme have different fates. For example, the calvarial bone develops from the cranial mesenchyme on the baso-lateral side of the head just above the eye (supraorbital mesenchyme, SOM), but not from the mesenchyme apical to SOM (early migrating mesenchyme, EMM). However, the molecular basis of this difference is not fully understood. To answer this question, we compared the transcriptomes of EMM and SOM using high-throughput sequencing (RNA-seq). This experiment identified a large number of genes that were differentially expressed in EMM and SOM, and gene ontology analyses found very different terms enriched in each region. We verified the expression of about 40 genes in the head by RNA in situ hybridization, and the expression patterns were annotated to make a map of molecular markers for 6 subdivisions of the cranial mesenchyme. Our data also provided insights into potential novel regulators of cranial mesenchyme development, including several axon guidance pathways, lectin complement pathway, cyclic-adenosine monophosphate (cAMP) signaling pathway, and ZIC family transcription factors. Together, information in this paper will serve as a unique resource to guide future research on cranial mesenchyme development.

DBBM shows no signs of resorption under inflammatory conditions. An experimental study in the mouse calvaria.

OBJECTIVES: Deproteinized bovine bone mineral (DBBM) is not resorbable. However, the behavior of DBBM under inflammatory conditions remains unclear. Aim of the study was therefore to evaluate the resorption of DBBM under local inflammatory conditions in vivo using the calvarial osteolysis model. METHODS: In thirty adult BALB/c mice, DBBM was implanted into the space between the elevated soft tissue and the calvarial bone. Inflammation was induced either by lipopolysaccharides (LPS) injection or by polyethylene particles (Ceridust) mixed with DBBM. Three modalities were randomly applied (n = 10 each): (a) DBBM alone (control), (b) DBBM + LPS, and (c) DBBM + polyethylene particles (Ceridust). Mice were euthanized on day fourteen, and each calvarium was subjected to histological and µCT analysis. Primary outcome was the size distribution of the DBBM particles. Secondary outcome was the surface erosion of the calvarial bone. RESULTS: Histological and µCT analysis revealed that the size distribution and the volume of DBBM particles in the augmented site were similar between DBBM alone and the combinations with LPS or polyethylene particles. Moreover, histological evaluation showed no signs of erosions of DBBM particles under inflammatory conditions. µCT analysis and histology further revealed that LPS and the polyethylene particles, but not the DBBM alone, caused severe erosions of the calvarial bone as indicated by large voids representing the massive compensatory new immature woven bone formation on the endosteal surface. CONCLUSIONS: Local calvarial bone but not the DBBM particles undergo severe resorption and subsequent new bone formation under inflammatory conditions in a mouse model.

Increase in the Number of Bone Marrow Osteoclast Precursors at Different Skeletal Sites, Particularly in Long Bone and Jaw Marrow in Mice Lacking IL-1RA.

Recently, it was shown that interleukin-1ß (IL-1ß) has diverse stimulatory effects on different murine long bone marrow osteoclast precursors (OCPs) in vitro. In this study, interleukin-1 receptor antagonist deficient (Il1rn-/-) and wild-type (WT) mice were compared to investigate the effects of enhanced IL-1 signaling on the composition of OCPs in long bone, calvaria, vertebra, and jaw. Bone marrow cells were isolated from these sites and the percentage of early blast (CD31hi Ly-6C-), myeloid blast (CD31+ Ly-6C+), and monocyte (CD31- Ly-6Chi) OCPs was assessed by flow cytometry. At the time-point of cell isolation, Il1rn-/- mice showed no inflammation or bone destruction yet as determined by histology and microcomputed tomography. However, Il1rn-/- mice had an approximately two-fold higher percentage of OCPs in long bone and jaw marrow compared to WT. Conversely, vertebrae and calvaria marrow contained a similar composition of OCPs in both strains. Bone marrow cells were cultured with macrophage colony stimulating factor (M-CSF) and receptor of NfκB ligand (RANKL) on bone slices to assess osteoclastogenesis and on calcium phosphate-coated plates to analyze mineral dissolution. Deletion of Il1rn increased osteoclastogenesis from long bone, calvaria, and jaw marrows, and all Il1rn-/- cultures showed increased mineral dissolution compared to WT. However, osteoclast markers increased exclusively in Il1rn-/- osteoclasts from long bone and jaw. Collectively, these findings indicate that a lack of IL-1RA increases the numbers of OCPs in vivo, particularly in long bone and jaw, where rheumatoid arthritis and periodontitis develop. Thus, increased bone loss at these sites may be triggered by a larger pool of OCPs due to the disruption of IL-1 inhibitors.

Developmental biology of the meninges.

The meninges are membranous layers surrounding the central nervous system. In the head, the meninges lie between the brain and the skull, and interact closely with both during development. The cranial meninges originate from a mesenchymal sheath on the surface of the developing brain, called primary meninx, and undergo differentiation into three layers with distinct histological characteristics: the dura mater, the arachnoid mater, and the pia mater. While genetic regulation of meningeal development is still poorly understood, mouse mutants and other models with meningeal defects have demonstrated the importance of the meninges to normal development of the calvaria and the brain. For the calvaria, the interactions with the meninges are necessary for the progression of calvarial osteogenesis during early development. In later stages, the meninges control the patterning of the skull and the fate of the sutures. For the brain, the meninges regulate diverse processes including cell survival, cell migration, generation of neurons from progenitors, and vascularization. Also, the meninges serve as a stem cell niche for the brain in the postnatal life. Given these important roles of the meninges, further investigation into the molecular mechanisms underlying meningeal development can provide novel insights into the coordinated development of the head.

Notch signaling inhibition protects against LPS mediated osteolysis.

Chronic inflammatory responses have profound effects on the differentiation and activity of both the bone-forming osteoblasts and bone-resorbing osteoclasts. Importantly, inflammatory bone diseases characterized by clinical osteolysis promote bone resorption and decrease bone formation by uncoupling the process in favor of excess resorption. Notch signaling regulates osteoclast development and thus its manipulation has the potential to suppress resorptive potential. Here, we have utilized a genetic model of Notch inhibition in osteoclasts by expression of dnMAML to prevent formation of transcriptional complex essential for downstream Notch signaling. Using this model and LPS as a tool for experimental inflammatory osteolysis, we have demonstrated that dnMAML-expressing osteoclasts exhibited significantly lower maturation and resorption/functional potential ex vivo using TRAP staining and calcium phosphate coated surfaces. Moreover, we observed that while LPS stimulated the formation of wildtype osteoclasts pre-treated with RANKL, dnMAML expression produced resistance to osteoclast maturation after LPS stimulation. Genetically, Notch-inhibited animals showed a significantly lower TRAP and CTX-1 levels in serum after LPS treatment compared to the control groups in addition to a marked reduction in osteoclast surfaces in calvaria sections. This report provides evidence for modulation of Notch signaling activity to protect against inflammatory osteolysis. Taken together, the findings of this study will help guide the development of Notch signaling-based therapeutic approaches to prevent bone loss.

Autophagy Regulates Craniofacial Bone Acquisition.

Increasing evidence has demonstrated the important role of autophagy in skeletal homeostasis; however, the role of autophagy in craniofacial bone development and acquisition is largely unknown. In this study, we investigated the effect of autophagy suppression on craniofacial bone acquisition by deleting Fip200 or Atg5, two essential autophagy genes, using Osterix-Cre (Osx-Cre). We found that the Osx-Cre transgene mildly decreased the bone mass of parietal bone but not frontal bone, and did not affect cranial base bone mass in adult mice. In the cranial vault, Fip200 or Atg5 deletion similarly decreased 50% bone mass of neural crest-derived frontal bone; Atg5 deletion decreased 50% and Fip200 deletion decreased 30% bone mass of mesoderm-derived parietal bone. In the cranial base, Fip200 or Atg5 deletion similarly decreased 30% bone mass of neural crest-derived presphenoid bone; Atg5 deletion decreased 30% and Fip200 deletion decreased 16% bone mass of mesoderm-derive basioccipital bone. Lastly, we used doxycycline treatment to inhibit the Osx-Cre expression until 2 months of age and showed that postnatal Fip200 deletion led to cranial vault bone mass decrease in association with a small increase in both bone volume/tissue volume and tissue mineral density. Altogether, this study demonstrated the important role of autophagy in craniofacial bone acquisition during development and postnatal growth.

A protocol for isolation and identification and comparative characterization of primary osteoblasts from mouse and rat calvaria.

Calvaria from neonatal mouse and rat is ideal resource for osteoblasts but can be easily contaminated by other cells such as fibroblasts. Here, we established a protocol for isolation and purification of primary osteoblast by enzyme sequential digestion and differential adhesion. In addition, we compared the phenotypic and functional traits of osteoblasts from mouse and rat which are commonly employed in studies. The method applied equally to rat and mouse in osteoblasts isolation and was corroborated its feasibility and validity. The results also provided us evidences for other experiments such as choosing a certain time point to give intervention and do the relevant tests.

[Occipital sinus pericranii: About an unusual situation and review of the literature]. / Sinus pericranii occipital : à propos d'une situation inhabituelle et revue de la littérature.

The sinus pericranii refers to a set of clinical presentations that share a pathological communication between the scalp veins and the underlying cranial venous sinus. The nature of this connection ranges from the simple dilatation of the emissary veins, to wide connections through a calvarial bone loss leaving almost the cranial venous sinus in direct contact with the subcutaneous tissue. The authors present the case of an occipital pericranii sinus of intraoperative discovery. Congenital, post-traumatic or spontaneous, this anomaly is most often frontal and located on or close to the midline. The usual clinical presentation is that of a soft mass that empties to the pressure while standing, while it fills in all situations that increase the intracranial pressure (Valsalva maneuver …). The diagnosis is not always easy to establish, and relies on a bundle of clinical and radiological arguments. Through a review of the literature we propose to focus on the clinical features, diagnostic means and therapeutic options of this rare entity.

The prevention of latanoprost on osteoclastgenesis in vitro and lipopolysaccharide-induced murine calvaria osteolysis in vivo.

Identification of agents that inhibit osteoclast formation and function is important for the treatment of osteolytic diseases which feature excessive osteoclast formation and bone resorption. Latanoprost (LTP), an analog of prostaglandin F2α, is a medication which works to lower pressure inside the eyes. Prostaglandin F2α was reported to regulate bone metabolism, however, the effect of LTP in osteoclastogenesis is still unknown. Here, we found that LTP suppressed RANKL-induced osteoclastogenesis in a dose-dependent manner as illustrated by TRAP activity and TRAP staining. In addition, the osteoclast function was also reduced by LTP treatment, as indicated in less osteoclastic resorption pit areas. Furthermore, LTP inhibited the mRNA expressions of osteoclast marker genes such as TRAP and cathepsin K. In order to illustrate its molecular mechanism, we examined the changing of mRNA and protein levels of NFATc1 and c-fos by LTP treatment, as well as the phosphorylation of ERK, AKT, JNK, and p38. The results suggested that LTP inhibited RANKL-induced osteoclastgenesis and function by inhibiting ERK, AKT, JNK, and p38 cascade, following by the c-fos/NFATc1 pathway. In agreement with in vitro results, using an in vivo lipopolysaccharide-induced murine calvaria osteolysis mouse model, we found that administration of LTP was able to reverse the lipopolysaccharide-induced bone loss. Together, these data demonstrated that LTP attenuated the bone loss in lipopolysaccharide-induced murine calvaria osteolysis mice through inhibiting osteoclast formation and function. Our study thus provided the evidences that LTP was a potential treatment option against osteolytic bone diseases.

VWC2 Increases Bone Formation Through Inhibiting Activin Signaling.

By a bioinformatics approach, we have identified a novel cysteine knot protein member, VWC2 (von Willebrand factor C domain containing 2) previously known as Brorin. Since Brorin has been proposed to function as a bone morphogenetic protein (BMP) antagonist, we investigated the binding of Brorin/VWC2 to several BMPs; however, none of the BMPs tested were bound to VWC2. Instead, the ßA subunit of activin was found as a binding partner among transforming growth factor (TGF)-ß superfamily members. Here, we show that Vwc2 gene expression is temporally upregulated early in osteoblast differentiation, VWC2 protein is present in bone matrix, and localized at osteoblasts/osteocytes. Activin A-induced Smad2 phosphorylation was inhibited in the presence of exogenous VWC2 in MC3T3-E1 osteoblast cell line and primary osteoblasts. The effect of VWC2 on ex vivo cranial bone organ cultures treated with activin A was investigated, and bone morphometric parameters decreased by activin A were restored with VWC2. When we further investigated the biological mechanism how VWC2 inhibited the effects of activin A on bone formation, we found that the effects of activin A on osteoblast cell growth, differentiation, and mineralization were reversed by VWC2. Taken together, a novel secretory protein, VWC2 promotes bone formation by inhibiting Activin-Smad2 signaling pathway.