Expression of high-affinity receptors for TGF-beta during rat cranial suture fusion.

The etiology of craniosynostosis is unknown. The elucidation of the biological pathways responsible for this disorder has been hampered by an inability to evaluate cranial sutures before, during, and after cranial suture fusion. The programmed fusion of the rat posterofrontal (PF) suture postnatally provides an excellent model to study the molecular events that occur during cranial suture fusion. Previous experiments have implicated transforming growth factor beta (TGF-beta) growth factors in the regulation of PF suture fusion. The purpose of these experiments was to localize the expression of high-affinity receptors for these growth factors during cranial suture fusion. Four rats were sacrificed on postnatal days 8, 12, 17, and 40 (N = 16). The PF and sagittal sutures were harvested and prepared for immunohistochemical localization of TGF-beta receptor 1 and receptor 2 (Tbeta-RI, Tbeta-RII) protein. Results indicate that immunostaining for Tbeta-RI and Tbeta-RII is markedly increased in the dura mater and osteoblasts of the sutural margin of the PF suture during active suture fusion (on postnatal days 12, 17, and 40) compared with the osteoblasts and dura mater underlying the patent sagittal suture. These results, in combination with the authors' previous findings as well as studies supporting a role for TGF-beta molecules in the regulation of osteogenesis, implicate TGF-beta signaling in the regulation of suture fusion. The possible mechanisms of ligand-receptor interaction are discussed.     

Analysis of TGF-beta production by fusing and nonfusing mouse cranial sutures in vitro.

The role of transforming growth factor beta (TGF-beta) in the regulation of cranial suture fusion has been studied by various qualitative techniques such as in situ hybridization and immunohistochemistry. Although the relative expression of TGF-beta isoforms has been assessed in these studies, increased expression of TGF-beta has not been demonstrated in a quantitative fashion. Therefore, the purpose of this study was to quantify TGF-beta production by fusing (posterofrontal [PF]) and nonfusing (sagittal) mouse sutures using two different quantitative TGF-beta assays. The PF and sagittal sutures of 25-day-old mice were harvested and cultured separately in vitro. Culture media conditioned for 48 hours were collected after 3, 6, 9, 12, 15, 18, 21, 24, 27, and 30 days of culture, and total TGF-beta production was assessed using a TGF-beta bioassay. For a quantitative TGF-beta1 immunoassay, media conditioned for 48 hours were collected after 3, 5, 7, 9, 14, 22, and 28 days of culture. The TGF-beta bioassay revealed large amounts of total TGF-beta activity in both PF and sagittal sutures during the first week of culture, with decreasing amounts thereafter. Absolute TGF-beta activity in conditioned media collected from PF sutures at several early time points was higher than those obtained from sagittal sutures; however, these differences were not statistically significant. The results of the TGF-beta1 immunoassay (enzyme-linked immunosorbent assay) were similar to the bioassay in that the highest TGF-beta1 levels were noted during the first week of culture period and decreased thereafter. Analysis of variance of these samples, however, revealed significantly more TGF-beta1 protein in samples collected from the PF suture compared with the sagittal suture on days 3 and 5 of culture (p < 0.05). TGF-beta1 levels in the conditioned media obtained from PF sutures remained elevated compared with the sagittal suture on days 7 and 9; however, these differences were not statistically significant. Increased production of TGF-beta in the conditioned media of fusing PF sutures is the first such quantitative demonstration of growth factor upregulation during suture fusion and supports the hypothesis that TGF-beta expression may be important in cranial suture fusion.     

Toward an understanding of nonsyndromic craniosynostosis: altered patterns of TGF-beta receptor and FGF receptor expression induced by intrauterine head constraint

Although the etiology of nonsyndromic forms of craniosynostosis remains uncertain, recent experiments from our laboratory have demonstrated that fetal head constraint induces cranial suture fusion in mice through a process associated with altered patterns of transforming growth factor beta (TGF-beta) isoform expression. Other recent studies have highlighted the role of secreted signaling molecules, including members of the TGF-beta superfamily and the fibroblast growth factors (FGFs), as well as their receptors, in regulating suture development and fusion. The purpose of these experiments was to examine the potential role of TGF-beta receptors and FGF receptor 2 (FGFR2) in nonsyndromic craniosynostosis by determining their temporospatial patterns of expression during development complicated by intrauterine head constraint. This study consisted of two groups of C57BI/6J mice: an experimental group subjected to intrauterine constraint and a control unconstrained group. Fetal head constraint was induced by performing uterine cerclage on day 17.5 of gestation and allowing intrauterine fetal growth to continue 24 and 48 hours beyond the normal gestational period. Control animals underwent hysterotomy on day 17.5 and the nonconstrained pups were allowed to continue intra-abdominal fetal growth 48 hours beyond normal gestation. Expression of TGF-beta receptor types I and II, and FGFR2 in the calvarial tissue was determined by immunohistochemical analysis. In the unconstrained control animals, there was minimal immunoreactivity for both TGF-beta receptors and FGFR2 within the coronal suture. After 24 hours of constraint, however, there was a marked increase in immunoreactivity of TGF-beta receptors and FGFR2 in the osteoblasts along the osteogenic fronts and in the dural cells. After 48 hours, there was continued expression of both type I and type II receptors and FGFR2 within the midsutural mesenchyme of the coronal suture, in the osteoblasts, and in the dura. The authors demonstrated substantial upregulation of TGF-beta receptor types I and II and FGFR2 in coronal sutures subjected to in utero constraint. These results suggest an important role for TGF-beta/TGF-beta receptor, and FGF/FGFR signaling in the pathogenesis of constraint-induced craniosynostosis.     

Microfocal CT: a method for evaluating murine cranial sutures in situ

INTRODUCTION: The murine model is a well-established surrogate for studying human cranial suture biology. In mice, all sutures with the exception of the posterior frontal (PF) suture remain patent throughout life. Histology is regarded as the gold standard for analyzing sutures. On this basis, PF suture fusion begins on day of life 25 and is complete by day 45. Cranial suture histology, however, requires sacrifice of the animal to obtain tissue for analysis. As a result, knowledge of the kinetics of cranial suture fusion is based on a patchwork analysis of many sutures from many different animals. The behavior of a single suture through time is unknown. Our goal is to develop a noninvasive means to repeatedly image mouse cranial sutures in vivo. As a first step, the present study was performed to evaluate microfocal computer tomography (micro-CT) technology for the use of capturing images of a mouse cranium in situ. METHODS: The micro-CT system consists of a microfocal X-ray source and a large format CCD camera optically coupled to a high-resolution X-ray image intensifier, digitally linked to a computer. The PF and sagittal sutures lie in continuity along the midline of the skull. Holes were drilled in the calvaria on both sides of the PF and sagittal sutures of a 45-day-old euthanized mouse. A micro-CT scan of this animal was performed and hundreds of cross-sectional images were generated for the cranium. These images were used to reconstruct three-dimensional volumetric images of the entire cranium. Comparisons were made between (1). the gross specimen and the three dimensional reconstructions; (2). two-dimensional coronal images obtained by micro-CT and those obtained by histology. RESULTS: Analysis of PF and sagittal sutures demonstrated the following: (1). The drilled holes were accurately rendered by micro-CT, when compared to both the gross specimen and the histology. (2). The sagittal suture was found to be patent by both micro-CT and histology. (3). The PF suture is fused by histology, but unexpectedly, the PF suture appears incompletely fused by micro-CT. By micro-CT, however, the anterior and endocranial regions appear more extensively fused than the remainder of the PF suture, a finding consistent with published histologic analysis. CONCLUSIONS: We successfully imaged 45-day-old mouse cranial sutures in situ using micro-CT technology. Precise correlation between histologic sections and radiologic images is difficult, but convincing similarities exist between the gross specimen and images from micro-CT and histology. PF suture fusion in a 45-day-old animal appears different by micro-CT than by histology. One possible explanation for this apparent discrepancy is that suture fusion in histology is determined based on the appearance of bone morphology and not tissue density, as the specimens are necessarily decalcified to section the bone. Micro-CT, on the other hand, distinguishes tissues on the basis of density. Newly forming bone may require bone matrix formation prior to complete calcification; PF suture in 45-day-old mice may be morphologically complete but incompletely ossified. Studies correlating histologic and micro-CT assessment of suture development are underway. Micro-CT appears to be a promising method for noninvasive imaging of mouse cranial suture.     

Fibroblast growth factors lead to increased Msx2 expression and fusion in calvarial sutures.

Craniosynostosis, the premature fusion of the skull bones at the sutures, represents a disruption to the coordinated growth and development of the expanding brain and calvarial vault and is the second most common birth defect that affects the craniofacial complex. Mutations in the human homeobox-containing gene, Msx2, have been shown to cause Boston type craniosynostosis, and we have shown that overexpression of Msx2 leads to craniosynostosis in mice. Activating mutations in fibroblast growth factor (FGF) receptors are thought to cause craniosynostosis in Crouzon, Apert, Jackson-Weiss, Beare-Stevenson, and Muenke syndromes. To mimic activated signaling by mutated FGF receptors, we used heparin acrylic beads to deliver FGF ligands to mouse calvaria and demonstrated increased Msx2, Runx2, Bsp, and Osteocalcin gene expression, decreased cell proliferation, and suture obliteration and fusion. FGF2 elicited the greatest increase in Msx2 expression, and FGF1 was most likely to cause suture obliteration and fusion. Of the three sutures studied, the coronal suture exhibited the greatest increase in Msx2 expression and was the most likely to undergo obliteration and fusion. These results are intriguing because the coronal suture is the most commonly affected suture in syndromic craniosynostosis. These results suggest that Msx2 is a downstream target of FGF receptor signaling and that increased FGF signaling leads to osteogenic differentiation by sutural mesenchyme in mouse calvaria. These results are consistent with the hypotheses that increased Msx2 expression and activated signaling by mutated FGF receptors lead to craniosynostosis.     

SD大鼠颅缝细胞不同时期GPC3基因表达变化的研究

目的 探索GPC3基因在颅缝组织和细胞中不同时期的表达情况,为后续的疾病模型研究提供参照。方法研究并观察1 d、3 d、7 d的SD大鼠颅缝细胞组织,应用组织切片免疫荧光染色、RT-q PCR、Western Blot等方法,检测其在不同年龄SD大鼠中的表达水平,判断其与颅骨成骨及颅缝闭合的关系。结果 免疫荧光显示,GPC3在大鼠未闭合颅缝组织不同阶段胞内、细胞表面及胞外均有表达;RT-q PCR、Western Blotting结果显示,GPC3基因表达随着大鼠年龄增长呈现下降趋势。结论 SD大鼠中,GPC3与颅缝闭合密切相关。     

Responses of intramembranous bone and sutures upon in vivo cyclic tensile and compressive loading

Cranial vault and facial sutures interpose between mineralized bones of the skull, and may function analogously to appendicular and cranial base growth plates. However, unlike growth plates that are composed of chondrocyte lineage, cranial and facial sutures possess heterogeneous cell lineages such as mesenchymal cells, fibroblasts, and osteoblasts, in addition to vascular-derived cells. Despite recently intensified effort, the biological responses of intramembranous bone and sutures to mechanical loading are not well understood. This study was designed to investigate whether brief doses of tensile or compressive forces induce modeling and growth responses of intramembranous bone and sutures. In different groups of growing rabbits in vivo, cyclic tensile or compressive forces at 1 N and 8 Hz were applied to the maxilla for 20 min/day over 12 consecutive days. Computerized histomorphometric analyses revealed that the average sutural widths of both the premaxillomaxillary suture (PMS) and nasofrontal suture (NFS) loaded in either tension or compression were significantly higher than age- and sex-matched sham controls (P<0.01). The average cell densities of tension- or compression-loaded PMS and NFS were significantly higher than sham controls (P<0.01). The average osteoblast occupied sutural bone surface loaded under tension was significantly higher than that of sham control (P<0.05). Interestingly, tensile loading significantly reduced the average osteoclast surface, in comparison to sham control (P<0.05). For the NFS, tensile loading significantly increased the average osteoblast occupied sutural bone surface, in comparison with that of sham control (P<0.05). Also for the NFS suture, compression significantly reduced the average sutural osteoclast surface in comparison with sham control (P<0.05). Taken together, the present data suggest that high-frequency cyclic forces in either tension or compression induce modeling and growth changes in cranial sutures. Due to the structural complexity of cranial vault and facial sutures, either tensile or compressive forces likely are transmitted as shear stresses and upregulate genes and gene products responsible for sutural growth.     

Latent TGF-beta binding proteins (LTBPs)-1 and -3 coordinate proliferation and osteogenic differentiation of human mesenchymal stem cells

Mesenchymal stem cells (MSCs) possess the capability to differentiate into bone forming cells, osteoblasts, and thus represent a new therapeutic tool in regenerative medicine. Transforming growth factor (TGF)-beta is abundantly present in bone tissue where it regulates osteoblast and osteoclast functions in a complex manner. Latent TGF-beta binding protein (LTBP)-1 mediates the extracellular matrix (ECM) targeting and accumulation of most TGF-beta in the bone. We describe here an important regulatory role for LTBP-3 in TGF-beta activation and autocrine growth control in MSCs. LTBP-3 knockdown via siRNA mediated silencing resulted in reduced cell proliferation and reduced osteogenic differentiation. When MSCs were induced to undergo differentiation, LTBP-3 levels became downregulated in parallel with reduced TGF-beta activation. These changes coincided with the matrix maturation phase of osteogenic differentiation. The mechanism of LTBP-3 is most likely via TGF-beta activation in the early proliferative phase of the differentiation process. Later, when TGF-beta activity would inhibit further maturation and mineralization, LTBP-3 expression becomes downregulated and LTBP-1 containing large latent TGF-beta1 complexes accumulate into the ECM. These complexes represent readily available targets for osteoclast mediated release and activation of TGF-beta in bone tissue. Our results provide evidence that LTBP isoforms can differentially regulate TGF-beta activation and ECM accumulation during osteogenic differentiation.     

A model of the cranial vault as a tensegrity structure,and its significance to normal and abnormal cranial development

BackgroundTraditional views of the human cranial vault are facing challenges as researchers find that the complex details of its development do not always match previous opinions that it is a relatively passive structure. In particular, that stability of the vault is dependant on an underlying brain; and sutural patency merely facilitates cranial expansion. The influence of mechanical forces on the development and maintenance of cranial sutures is well-established, but the details of how they regulate the balance between sutural patency and fusion remain unclear. Previous research shows that mechanical tensional forces can influence intracellular chemical signalling cascades and switch cell function; and that tensional forces within the dura mater affect cell populations within the suture and cause fusion.Understanding the developmental mechanisms is considered important to the prevention and treatment of premature sutural fusion – synostosis – which causes skull deformity in approximately 0.05% of live births. In addition, the physiological processes underlying deformational plagiocephaly and the maintenance of sutural patency beyond early childhood require further elucidation.MethodUsing a disarticulated plastic replica of an adult human skull, a model of the cranial vault as a tensegrity structure which could address some of these issues is presented.ConclusionsThe tensegrity model is a novel approach for understanding how the cranial vault could retain its stability without relying on an expansive force from an underlying brain, a position currently unresolved. Tensional forces in the dura mater have the effect of pushing the bones apart, whilst at the same time integrating them into a single functional unit. Sutural patency depends on the separation of cranial bones throughout normal development, and the model describes how tension in the dura mater achieves this, and influences sutural phenotype. Cells of the dura mater respond to brain expansion and influence bone growth, allowing the cranium to match the spatial requirements of the developing brain, whilst remaining one step ahead and retaining a certain amount of autonomy. The model is compatible with current understandings of normal and abnormal cranial physiology, and has a contribution to make to a hierarchical systems approach to whole body biomechanics.     

Regulation of bone morphogenetic protein signalling and cranial osteogenesis by Gpc1 and Gpc3

From birth, the vault of the skull grows at a prodigious rate, driven by the activity of osteoblastic cells at the fibrous joints (sutures) that separate the bony calvarial plates. One in 2500 children is born with a medical condition known as craniosynostosis because of premature bony fusion of the calvarial plates and a cessation of bone growth at the sutures. Bone morphogenetic proteins (BMPs) are potent growth factors that promote bone formation. Previously, we found that Glypican-1 (GPC1) and Glypican-3 (GPC3) are expressed in cranial sutures and are decreased during premature suture fusion in children. Although glypicans are known to regulate BMP signalling, a mechanistic link between GPC1, GPC3 and BMPs and osteogenesis has not yet been investigated. We now report that human primary suture mesenchymal cells coexpress GPC1 and GPC3 on the cell surface and release them into the media. We show that they inhibit BMP2, BMP4 and BMP7 activities, which both physically interact with BMP2 and that immunoblockade of endogenous GPC1 and GPC3 potentiates BMP2 activity. In contrast, increased levels of GPC1 and GPC3 as a result of overexpression or the addition of recombinant protein, inhibit BMP2 signalling and BMP2-mediated osteogenesis. We demonstrate that BMP signalling in suture mesenchymal cells is mediated by both SMAD-dependent and SMAD-independent pathways and that GPC1 and GPC3 inhibit both pathways. GPC3 inhibition of BMP2 activity is independent of attachment of the glypican on the cell surface and post-translational glycanation, and thus appears to be mediated by the core glypican protein. The discovery that GPC1 and GPC3 regulate BMP2-mediated osteogenesis, and that inhibition of endogenous GPC1 and GPC3 potentiates BMP2 responsiveness of human suture mesenchymal cells, indicates how downregulation of glypican expression could lead to the bony suture fusion that characterizes craniosynostosis.     

Overexpression of Nell-1, a craniosynostosis-associated gene, induces apoptosis in osteoblasts during craniofacial development.

We studied the cellular function of Nell-1, a craniosynostosis-related gene, in craniofacial development. Nell-1 modulates calvarial osteoblast differentiation and apoptosis pathways. Nell-1 overexpression disrupts these pathways resulting in craniofacial anomalies such as premature suture closure. INTRODUCTION: Craniosynostosis (CS), one of the most common congenital craniofacial deformities, is the premature closure of cranial sutures. Previously, we reported NELL-1 as a novel molecule overexpressed during premature cranial suture closure in patients with CS. Nell-1 overexpression induced calvarial overgrowth and resulted in premature suture closure in a rodent model. On a cellular level, Nell-1 is suggested to promote osteoblast differentiation. MATERIALS AND METHODS: Different levels of Nell-1 were introduced into osteoblastic cells by viral infection and recombinant protein. Apoptosis and gene expression assays were performed. Mice overexpressing Nell-1 were examined for apoptosis. RESULTS: In this report, we further showed that overexpression of Nell-1 induced apoptosis along with modulation of apoptosis-related genes. The induction of apoptosis by Nell-1 was observed only in osteoblastic cells and not in NIH3T3 or primary fibroblasts. The CS mouse model overexpressing Nell-1 showed increased levels of apoptosis in the calvaria. CONCLUSION: We show that Nell-1 expression modulates calvarial osteoblast differentiation and apoptosis pathways. Nell-1 overexpression disrupts these pathways resulting in craniofacial anomalies such as premature suture closure.     

Impaired posterior frontal sutural fusion in the biglycan/decorin double deficient mice

Biglycan (Bgn) and decorin (Dcn) are highly expressed in numerous tissues in the craniofacial complex. However, their expression and function in the cranial sutures are unknown. In order to study this, we first examined the expression of biglycan and decorin in the posterior frontal suture (PFS), which predictably fuses between 21 and 45 days post-natal and in the non-fusing sagittal (S) suture from wild-type (Wt) mice. Our data showed that Bgn and Dcn were expressed in both cranial sutures. We then characterized the cranial suture phenotype in Bgn deficient, Dcn deficient, Bgn/Dcn double deficient, and Wt mice. At embryonic day 18.5, alizarin red/alcian blue staining showed that the Bgn/Dcn double deficient mice had hypomineralization of the frontal and parietal craniofacial bones. Histological analysis of adult mice (45-60 days post-natal) showed that the Bgn or Dcn deficient mice had no cranial suture abnormalities and immunohistochemistry staining showed increased production of Dcn in the PFS from Bgn deficient mice. To test possible compensation of Dcn in the Bgn deficient sutures, we examined the Bgn/Dcn double deficient mice and found that they had impaired fusion of the PFS. Semi-quantitative RT-PCR analysis of RNA from 35 day-old mice revealed increased expression of Bmp-4 and Dlx-5 in the PFS compared to their non-fusing S suture in Wt tissues and decreased expression of Dlx-5 in both PF and S sutures in the Bgn/Dcn double deficient mice compared to the Wt mice. Failure of PFS fusion and hypomineralization of the calvaria in the Bgn/Dcn double deficient mice demonstrates that these extracellular matrix proteoglycans could have a role in controlling the formation and growth of the cranial vault.     

Surgical resection of calvarial metastases overlying dural sinuses

OBJECTIVE: Few reports have addressed the surgical management of cranial metastases that overlie or invade the dural venous sinuses. To examine the role of surgery in the treatment of dural sinus calvarial metastases, we reviewed retrospectively 13 patients who were treated with surgery at the University of Texas M.D. Anderson Cancer Center between 1993 and 1999. We compared them with 14 patients who had calvarial metastases that did not involve a venous sinus. METHODS: Clinical charts, radiological studies, pathological findings, and operative reports were analyzed retrospectively. RESULTS: The median age of patients with dural sinus calvarial metastases was 54 years. Nine patients were men and four were women. Renal cell carcinoma and sarcoma were the most common primary tumors. Similar features were noted in the 14 patients with nonsinus calvarial metastases. Of the 13 dural sinus calvarial metastases, 11 involved the superior sagittal sinus, and 2 involved the transverse sinus. In nine patients, the involved sinus was resected, and in four patients, the sinus was reconstituted after tumor removal. Nine patients in the dural sinus calvarial metastases group received en bloc resection, and four received piecemeal resection. No operative deaths occurred. The overall median actuarial survival was 16.5 months. The survival times of the two groups were comparable. In the group with dural sinus calvarial metastases, transient postoperative neurological deficits occurred in two patients (15%), and a permanent deficit occurred in one patient (8%). No patients in the group with nonsinus calvarial metastases experienced deficits after resection. Compared with piecemeal resection, en bloc resection was associated with significantly less blood loss. CONCLUSION: Complete extirpation of calvarial metastases that overlie or invade a dural sinus can be achieved with only slightly more morbidity than complete removal of calvarial metastases that are located away from the sinuses. En bloc resection is as safe as piecemeal resection and is more effective in limiting operative blood loss. The overall recurrence and survival rates of patients with dural sinus calvarial metastases are similar to those of patients with calvarial metastases that do not involve the sinuses. Therefore, involvement of a dural venous sinus should not discourage resection of calvarial metastases. In carefully selected cancer patients, surgery provides effective palliation of symptomatic calvarial metastases that overlie or invade the venous sinuses.     

Roentgen stereophotogrammetric analysis of neurocranial suturectomy in rabbits

This investigation was conducted to further elucidate both the significance of a calvarial suture and the compensatory ability of the cranial vault. Four-week-old male New Zealand White rabbits were subjected to unilateral or bilateral extirpation of the coronal suture after insertion of metallic markers, and were then followed regularly by roentgen stereophotogrammetry until age 21 weeks. Bilateral extirpation of the normal coronal suture resulted in a dramatically increased initial rate of bone separation, which tended to remain supranormal for the rest of the investigation. Unilateral suturectomy showed differences in growth between the sides, the operated side initially separating significantly more than the other. Volumetric calvarial growth in rabbits with unilateral extirpation terminated similar to that in control animals, while volumes in rabbits with bilateral extirpations constantly exceeded control volumes, finally exceeding these by 65%. Responses at intact sutures confirmed the compensatory capacity of cranial vaults. The results indicate that the passive longitudinal and volumetric cranial vault bone growth responds quickly to growth disturbances, thereby demonstrating its plasticity, and that the neurocranial suture is a restraining and modulating component in cranial growth.     

Cell Surface Expression of Stem Cell Antigen-1 (Sca-1) Distinguishes Osteo-, Chondro-, and Adipoprogenitors in Fetal Mouse Calvaria

The flat bones of the skull (calvaria) develop by balanced cell proliferation and differentiation in the calvarial sutures and the bone tips. As the brain grows and the calvaria expand, cells within the sutures must remain undifferentiated to maintain suture patency, but osteoprogenitors also need to be recruited into the osteogenic fronts. The exact identity of calvarial osteoprogenitors is currently not known. We used immunomagnetic cell sorting to isolate Sca-1⁺ and Sca-1⁻ cells from fetal mouse calvaria and determined their differentiation potential in in vitro differentiation asssays and in vivo subcutaneous transplantations. Cells within the Sca-1⁺ cell fraction have a higher adipogenic potential, whereas cells within the Sca-1⁻ cell fraction have a higher osteogenic and chondrogenic potential. The Sca-1⁻ fraction retains its chondrogenic potential after in vitro expansion but not its osteogenic potential. The Sca-1⁺ fraction does not retain its adipogenic potential after in vitro expansion. Subcutaneous transplantation resulted in islands of bone and cartilage in implants that had been seeded with Sca-1⁻ cells. In conclusion, immunomagnetic cell sorting with Sca-1 antibodies can be used to separate a Sca-1⁺ cell fraction with adipogenic potential from a Sca-1⁻ cell fraction with osteogenic and chondrogenic potential. Isolation of pure populations of calvarial adipoprogenitors, osteoprogenitors, and chondroprogenitors will be beneficial for cellular studies of calvarial development, adipogenesis, osteogenesis, and chondrogenesis. Calvaria-derived osteogenic cell populations may be useful in craniofacial tissue regeneration and repair.