Craniofacial Skeletal Defects Research Articles

Loss of Neogenin alters branchial arch development and leads to craniofacial skeletal defects.

The formation of complex structures, such as the craniofacial skeleton, requires precise and intricate two-way signalling between populations of cells of different embryonic origins. For example, the lower jaw, or mandible, arises from cranial neural crest cells (CNCCs) in the mandibular portion of the first branchial arch (mdBA1) of the embryo, and its development is regulated by signals from the ectoderm and cranial mesoderm (CM) within this structure. The molecular mechanisms underlying CM cell influence on CNCC development in the mdBA1 remain poorly defined. Herein we identified the receptor Neogenin as a key regulator of craniofacial development. We found that ablation of Neogenin expression via gene-targeting resulted in several craniofacial skeletal defects, including reduced size of the CNCC-derived mandible. Loss of Neogenin did not affect the formation of the mdBA1 CM core but resulted in altered Bmp4 and Fgf8 expression, increased apoptosis, and reduced osteoblast differentiation in the mdBA1 mesenchyme. Reduced BMP signalling in the mdBA1 of Neogenin mutant embryos was associated with alterations in the gene regulatory network, including decreased expression of transcription factors of the Hand, Msx, and Alx families, which play key roles in the patterning and outgrowth of the mdBA1. Tissue-specific Neogenin loss-of-function studies revealed that Neogenin expression in mesodermal cells contributes to mandible formation. Thus, our results identify Neogenin as a novel regulator of craniofacial skeletal formation and demonstrates it impinges on CNCC development via a non-cell autonomous mechanism.

The non-canonical Wnt receptor Ror2 is required for cartilage cell polarity and morphogenesis of the craniofacial skeleton in zebrafish.

Non-canonical/β-catenin-independent Wnt signaling plays crucial roles in tissue/cell polarity in epithelia, but its functions have been less well studied in mesenchymal tissues, such as the skeleton. Mutations in non-canonical Wnt signaling pathway genes cause human skeletal diseases such as Robinow syndrome and Brachydactyly Type B1, which disrupt bone growth throughout the endochondral skeleton. Ror2 is one of several non-canonical Wnt receptor/co-receptors. Here, we show that ror2-/- mutant zebrafish have craniofacial skeletal defects, including disruptions of chondrocyte polarity. ror1-/- mutants appear to be phenotypically wild type, but loss of both ror1 and ror2 leads to more severe cartilage defects, indicating partial redundancy. Skeletal defects in ror1/2 double mutants resemble those of wnt5b-/- mutants, suggesting that Wnt5b is the primary Ror ligand in zebrafish. Surprisingly, the proline-rich domain of Ror2, but not its kinase domain, is required to rescue its function in mosaic transgenic experiments in ror2-/- mutants. These results suggest that endochondral bone defects in ROR-related human syndromes reflect defects in cartilage polarity and morphogenesis.

Loss of KDM4B impairs osteogenic differentiation of OMSCs and promotes oral bone aging

Aging of craniofacial skeleton significantly impairs the repair and regeneration of trauma-induced bony defects, and complicates dental treatment outcomes. Age-related alveolar bone loss could be attributed to decreased progenitor pool through senescence, imbalance in bone metabolism and bone-fat ratio. Mesenchymal stem cells isolated from oral bones (OMSCs) have distinct lineage propensities and characteristics compared to MSCs from long bones, and are more suited for craniofacial regeneration. However, the effect of epigenetic modifications regulating OMSC differentiation and senescence in aging has not yet been investigated. In this study, we found that the histone demethylase KDM4B plays an essential role in regulating the osteogenesis of OMSCs and oral bone aging. Loss of KDM4B in OMSCs leads to inhibition of osteogenesis. Moreover, KDM4B loss promoted adipogenesis and OMSC senescence which further impairs bone-fat balance in the mandible. Together, our data suggest that KDM4B may underpin the molecular mechanisms of OMSC fate determination and alveolar bone homeostasis in skeletal aging, and present as a promising therapeutic target for addressing craniofacial skeletal defects associated with age-related deteriorations.

Are ERK and Wnt Fate d to Reinforce Calvarial Osteoblast Identity?

Wnt signaling regulates cell fate decisions in diverse contexts during development and disease. In the mouse cranial mesenchyme (CM) Wnt signaling pathway components and reporters are spatially distributed during calvarial osteoblast fate selection. Within 24 hours, loss of Wnt signaling in the mouse embryonic CM results in a robust and binary cell fate switch, from calvarial bone to ectopic cartilage. The mechanism by which Wnt signaling regulates this cell fate switch is not clear. Extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) activation is required for bone-cartilage cell fate decisions in long bone perichondrium, and we have demonstrated that ERK1/2 activation is present in calvarial osteoblasts. Here, we test the hypothesis that ERK signaling is a mediator of binary Wnt-dependent bone-cartilage cell fate decisions. First, we used three distinct Wnt signaling loss-of-function mouse models to demonstrate that Wnt signaling is required for activation of ERK signaling in calvarial bone progenitors. Second, we showed that loss of ERK signaling precedes formation of ectopic cartilage in CM-Wnt signaling mutants and ERK activation is highly sensitive to levels of Wnt signaling within the cranial mesenchyme. Third, loss of Erk1/2 in the CM results in elevated levels of the master cartilage determinant, SOX9, by E13.5 and loss of calvarial bone by E16.5. These results demonstrate a link between the Wnt and ERK signaling pathways in regulating calvarial bone cell fate decisions in vivo. We propose a new model whereby reciprocal regulation of both canonical and non-canonical Wnt pathways in the CM generates a gradient of Wnt signaling and utilizes ERKs to reinforce binary cell fate decisions in vivo. This offers a new opportunity for therapeutic targeting of Wnt signaling in craniofacial skeletal defects and disease.

BRCA1 and BRCA2 tumor suppressors in neural crest cells are essential for craniofacial bone development.

Craniofacial abnormalities, including facial skeletal defects, comprise approximately one-third of all birth defects in humans. Since most bones in the face derive from cranial neural crest cells (CNCCs), which are multipotent stem cells, craniofacial bone disorders are largely attributed to defects in CNCCs. However, it remains unclear how the niche of CNCCs is coordinated by multiple gene regulatory networks essential for craniofacial bone development. Here we report that tumor suppressors breast cancer 1 (BRCA1) and breast cancer 2 (BRCA2) are required for craniofacial bone development in mice. Disruption of Brca1 in CNCC-derived mesenchymal cells, but not in epithelial-derived cells, resulted in craniofacial skeletal defects. Whereas osteogenic differentiation was normal, both osteogenic proliferation and survival were severely attenuated in Brca1 mutants. Brca1-deficient craniofacial skeletogenic precursors displayed increased DNA damage and enhanced cell apoptosis. Importantly, the craniofacial skeletal defects were sufficiently rescued by superimposing p53 null alleles in a neural crest-specific manner in vivo, indicating that BRCA1 deficiency induced DNA damage, cell apoptosis, and that the pathogenesis of craniofacial bone defects can be compensated by inactivation of p53. Mice lacking Brca2 in CNCCs, but not in epithelial-derived cells, also displayed abnormalities resembling the craniofacial skeletal malformations observed in Brca1 mutants. Our data shed light on the importance of BRCA1/BRCA2 function in CNCCs during craniofacial skeletal formation.

Seckel Syndrome in a 9 Year Old Child

Seckel Syndrome first defined by Seckel in 1959, is a rare (incidence 1:10000) genetically heterogeneous, autosomal recessive disorder presenting at birth. This syndrome is characterised by a proportionate dwarfism of prenatal onset, severe microcephaly with a bird headed appearance (beaked nose, receding forehead, prominent eyes and micrognathia) and mental retardation in addition to the characteristics craniofacial dysmorphism and skeletal defects, abnormalities have been described in the cardiovascular hematopoietic, endocrine, gastrointestinal and central nervous system. Usually such patients have poor psychomotor development.

Sf3b4-depleted Xenopus embryos: A model to study the pathogenesis of craniofacial defects in Nager syndrome

Mandibulofacial dysostosis (MFD) is a human developmental disorder characterized by defects of the facial bones. It is the second most frequent craniofacial malformation after cleft lip and palate. Nager syndrome combines many features of MFD with a variety of limb defects. Mutations in SF3B4 (splicing factor 3b, subunit 4) gene, which encodes a component of the pre-mRNA spliceosomal complex, were recently identified as a cause of Nager syndrome, accounting for 60% of affected individuals. Nothing is known about the cellular pathogenesis underlying Nager type MFD. Here we describe the first animal model for Nager syndrome, generated by knocking down Sf3b4 function in Xenopus laevis embryos, using morpholino antisense oligonucleotides. Our results indicate that Sf3b4-depleted embryos show reduced expression of the neural crest genes sox10, snail2 and twist at the neural plate border, associated with a broadening of the neural plate. This phenotype can be rescued by injection of wild-type human SF3B4 mRNA but not by mRNAs carrying mutations that cause Nager syndrome. At the tailbud stage, morphant embryos had decreased sox10 and tfap2a expression in the pharyngeal arches, indicative of a reduced number of neural crest cells. Later in development, Sf3b4-depleted tadpoles exhibited hypoplasia of neural crest-derived craniofacial cartilages, phenocopying aspects of the craniofacial skeletal defects seen in Nager syndrome patients. With this animal model we are now poised to gain important insights into the etiology and pathogenesis of Nager type MFD, and to identify the molecular targets of Sf3b4.

Newborn male presented with congenital diaphragmatic hernia and ileal atresia: A case report

Infants with congenital diaphragmatic hernia (CDH) have an increased incidence of associated malformations. The malformations often include craniofacial abnormalities, skeletal defects, and cardiac defects. Ileal atresia is an anomaly that has not yet been described in association with CDH. We describe a patient with a left congenital diaphragmatic hernia who was later diagnosed with ileal atresia. It is our belief that this is the first report of its kind in the literature.

Enzyme replacement for craniofacial skeletal defects and craniosynostosis in murine hypophosphatasia

Hypophosphatasia (HPP) is an inborn-error-of-metabolism disorder characterized by deficient bone and tooth mineralization due to loss-of function mutations in the gene (Alpl) encoding tissue-nonspecific alkaline phosphatase (TNAP). Alpl−/− mice exhibit many characteristics seen in infantile HPP including long bone and tooth defects, vitamin B6 responsive seizures and craniosynostosis. Previous reports demonstrated that a mineral-targeted form of TNAP rescues long bone, vertebral and tooth mineralization defects in Alpl−/− mice. Here we report that enzyme replacement with mineral-targeted TNAP (asfotase-alfa) also prevents craniosynostosis (the premature fusion of cranial bones) and additional craniofacial skeletal abnormalities in Alpl−/− mice. Craniosynostosis, cranial bone volume and density, and craniofacial shape abnormalities were assessed by microscopy, histology, digital caliper measurements and micro CT. We found that craniofacial shape defects, cranial bone mineralization and craniosynostosis were corrected in Alpl−/− mice injected daily subcutaneously starting at birth with recombinant enzyme. Analysis of Alpl−/− calvarial cells indicates that TNAP deficiency leads to aberrant osteoblastic gene expression and diminished proliferation. Some but not all of these cellular abnormalities were rescued by treatment with inorganic phosphate. These results confirm an essential role for TNAP in craniofacial skeletal development and demonstrate the efficacy of early postnatal mineral-targeted enzyme replacement for preventing craniofacial abnormalities including craniosynostosis in murine infantile HPP.

Isolation and enrichment of human adipose-derived stromal cells for enhanced osteogenesis.

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the process by which they must be harvested can be associated with significant donor site morbidity. In contrast, adipose-derived stromal cells (ASCs) are more readily abundant and more easily harvested, making them an appealing alternative to BM-MSCs. Like BM-MSCs, ASCs can differentiate into osteogenic lineage cells and can be used in tissue engineering applications, such as seeding onto scaffolds for use in craniofacial skeletal defects. ASCs are obtained from the stromal vascular fraction (SVF) of digested adipose tissue, which is a heterogeneous mixture of ASCs, vascular endothelial and mural cells, smooth muscle cells, pericytes, fibroblasts, and circulating cells. Flow cytometric analysis has shown that the surface marker profile for ASCs is similar to that for BM-MSCs. Despite several published reports establishing markers for the ASC phenotype, there is still a lack of consensus over profiles identifying osteoprogenitor cells in this heterogeneous population. This protocol describes how to isolate and use a subpopulation of ASCs with enhanced osteogenic capacity to repair critical-sized calvarial defects.

Enhancing Calvarial Regeneration through Inhibition of TGF-β1 Signaling

INTRODUCTION: Given the significant biomedical burden posed by calvarial defects and the limitations of contemporary craniofacial skeletal reconstructive approaches, a need exists to better understand the biology of calvarial osteoblasts and how to direct them towards endogenous bone regeneration. The mammalian skull vault is the result of a tightly regulated evolutionary process which components of disparate embryonic origin are integrated with frontal bones arising from neural crest cells and parietal bones arising from paraxial mesoderm. Frontal neural crest-derived osteoblasts possess greater osteogenic potential relative to parietal bone derived cells. This is, in part, due to increased TGF-β1 signaling in parietal cells, which is known to promote apoptosis in a number of different cell types. We therefore investigated the potential of enhancing calvarial regeneration through modulation of TGF-β1 signaling.1–5 METHODS: 2mm calvarial defects were created in both the frontal and parietal bones of wild-type CD-1 mice. A collagen sponge was then used to deliver either human recombinant TGF-β1 (800ng) or the TGF-β signaling pathway inhibitor SB431542 (26 mM) into defects. Micro-Computed Tomography was employed to evaluate bone regeneration and percent osseous healing was calculated using the GE Microview program. Immunohistochemistry was performed on coronal sections through treated calvarial defects for Phospho-Smad 2 and 3, the downstream effectors for TGFβ signaling to assess endogenous activation of this pathway and whether our treatment with SB431542 was effectively inhibiting this pathway in vivo. RESULTS: Inhibition of TGF-β1 signaling using SB431542, a specific small molecule inhibitor of TGF-β1 mediated signaling, resulted in significantly enhanced bone regeneration in parietal defects (*p<0.05) and engendered a more ‘frontal like’ healing capacity (Figure 1). In contrast, delivery of TGF-β1 significantly reduced healing relative to non-treated defects (*p<0.05). Immunohistochemistry for downstream effectors of TGF-β1 signaling, Phospho-Smad 2 and 3, established that this pathway was being effectively inhibited by SB431542 in vivo.Figure 1: Calvarial Healing with SB431542 or DMSO control treatment.CONCLUSION: Increased TGF-β1 signaling impairs calvarial healing whereas inhibition of this pathway promotes bone regeneration, potentially through inhibition of apoptotic activity. This study provides an insight into the potential use of specific small molecule inhibitors of TGF-β signaling as a novel approach that could be used in conjunction with currently available treatments and may allow treatment of larger, more complex craniofacial skeletal defects.

Pax6 regulates craniofacial form through its control of an essential cephalic ectodermal patterning center

Normal patterning and morphogenesis of the complex skeletal structures of the skull requires an exquisite, reciprocal cross-talk between the embryonic cephalic epithelia and mesenchyme. The mesenchyme associated with the jaws and the optic and olfactory capsules is derived from a Hox-negative cranial neural crest (CNC) population that acts much as an equivalence group in its interactions with specific local cephalic epithelial signals. Craniofacial pattern and morphogenesis is therefore controlled in large part through the regulation of these local cephalic epithelial signals. Here, we demonstrate that Pax6 is essential to the formation and maturation of the complex cephalic ectodermal patterning centers that govern the development and morphogenesis of the upper jaws and associated nasal capsules. Previous examinations of the craniofacial skeletal defects associated with Pax6 mutations have suggested that they arise from an optic-associated blockage in the migration of a specific subpopulation of midbrain CNC to the lateral frontonasal processes. We have addressed an alternative explanation for the craniofacial skeletal defects. We show that in Pax6(SeyN/SeyN) mutants regional CNC is present by E9.25 while there is already specific disruption in the early ontogenetic elaboration of cephalic ectodermal expression, associated with the nascent lambdoidal junction, of secreted signaling factors (including Fgf8 and Bmp4) and transcription factors (including Six1 and Dlx5) essential for upper jaw and/or nasal capsular development. Pax6 therefore regulates craniofacial form, at stages when CNC has just arrived in the frontonasal region, through its control of surface cephalic ectodermal competence to form an essential craniofacial patterning center.

FGF2 promotes Msx2 stimulated PC‐1 expression via Frs2/MAPK signaling

PC-1 is an enzymatic generator of pyrophosphate and a critical regulator of tissue mineralization. We previously showed that fibroblast growth factor-2 (FGF2) specifically induces PC-1 expression in calvarial pre-osteoblasts and that this occurs via a transcriptional mechanism involving Runx2. Because aberrant FGF signaling and Msx2 activity result in similar craniofacial skeletal defects and because Msx2 is an established regulator of osteoblastic gene expression, here we investigate Msx2 as an additional mediator of PC-1 gene expression. mRNA analysis and experiments utilizing PC-1 gene promoter/luciferase reporter constructs demonstrate that Msx2 promotes transcription of the PC-1 gene downstream of FGF2. Results indicate that both Msx2 and Runx2 are recruited to a conserved core Msx2 binding site within the PC-1 gene promoter upon FGF2 stimulation, and that Msx2 and Runx2 function together to induce PC-1 gene expression in osteoblastic cells. Here we show that FGF signaling promotes Msx2 transcriptional activity on the PC-1 gene promoter via the Frs2/MAPK signaling pathway. To our knowledge, this is the first report of Msx2 functioning as a transcriptional enhancer downstream of FGF2 in calvarial pre-osteoblasts. As activating mutations in FGF receptors and Msx2 result in similar craniofacial skeletal disorders, our findings support the idea that FGF signaling and Msx2 activity influence cranial osteogenesis via the same molecular mechanism.

Face, Upper Extremity, and Concomitant Transplantation: Potential Concerns and Challenges Ahead

From its origination involving successful rat hind-limb allograft studies using cyclosporine, face and upper extremity composite tissue allotransplantation has since developed into an exciting and promising subset of reconstructive transplant surgery. Current surgical technique involving composite tissue allotransplantation has allowed optimal outcomes in patients with massive facial and/or upper extremity defects; however, with its coexisting immunologic barrier, obligatory lifelong immunosuppression commits each patient to a daily risk of transplant-related complications with many unanswered questions. Since 1998, nearly 50 hand transplantations in 40 patients have been performed around the world at various levels ranging from wrist level to shoulder level. However, the risk-to-benefit ratio remains controversial in bilateral versus unilateral transplantation and has yet to be determined. From recent experience, the two most important determinants of the success of each patient's upper extremity transplant are patient compliance and intense rehabilitation. A total of nine face transplants have been performed since 2005. Multiple aesthetic subunits (i.e., nose, lips, eyelids) with or without underlying craniofacial skeletal defects (i.e., maxilla, mandible) have been successfully restored, thereby providing restoration of vital facial functions (i.e., smiling) in an unprecedented manner. As of today, face transplantation carries an estimated 2-year mortality of 20 percent. Concomitant composite tissue allotransplantation, which involves a variable combination of allograft subtypes, has been performed in two of the nine face transplant patients. These have included simultaneous bilateral hand transplants and tongue with mandible. Future study is warranted to investigate the potential advantages and disadvantages of using this approach versus a staged approach for reconstruction.

Periosteum-Guided Prefabrication of Vascularized Bone of Clinical Shape and Volume

Large craniofacial skeletal defects require complex reconstruction. Vascularized tissue transfer is the current standard in treatment, but these operations are technically difficult and associated with donor-site morbidity. Guided flap prefabrication offers a technique for endogenously engineering vascularized composite tissues with complex three-dimensional structure. This study evaluates the relationship between implantation time and tissue structure for generating tissues of clinically relevant volume and structure. Twenty skeletally mature domestic sheep were implanted with poly(methyl methacrylate) chambers designed to mimic the size and shape of the mental protuberance of the mandible. Each chamber was filled with morcellized bone graft and implanted with the open face apposed to the cambium layer of the rib periosteum. Chambers were harvested at 3, 6, 9, 12, and 24 weeks, and the tissue inside the chambers was analyzed for shape conformation to chamber geometry, gross tissue volume, and bone histomorphometric parameters. Histologically, active endochondral, direct, and appositional bone formation was observed. Calcified tissue area and new bone formation increased for each time point up to 12 weeks of implantation. The tissues formed maintained volumetric and geometrical structure consistent with the chamber up to 9 weeks after implantation. Significant decreases in total volume and agreement with chamber geometry were observed at 12 and 24 weeks. Periosteum-guided tissue prefabrication was found to be an effective means of engineering three-dimensional vascularized bone of clinical size and shape. The optimal duration of incubation before significant volume loss occurs is 9 weeks in this large-animal model.

59. Tissue-Harvest Procedure has no Effect on Osteogenic Differentiation of Adipose-Derived Stromal Cells in Vitro

Introduction: Bone tissue engineering mediated by adipose-derived stromal cells (ASCs) is a particularly interesting prospect due to the clinical burden of craniofacial skeletal defects and drawbacks of current techniques including limited supply and donor site morbidity of autogenous bone for grafting and demise of alloplastic materials. Current studies of ASCs largely employ the use of cells isolated from tissue harvested by suction-assisted lipoaspiration (SAL). However, ultrasound-assisted lipoaspiration (UAL) is gaining clinical popularity as the immediate benefits to the patient include reduced tissue trauma and bleeding and faster recovery. Therefore, there is a widening dichotomy between clinical principle and research focus. No current study has rigorously examined the potential for UAL-derived ASC-mediated osteogenesis. The current study investigates the in vitro osteogenic potential of UAL-derived ASCs with regard to their proliferative and differentiative capacities compared to more commonly used SAL-derived ASCs. Methods: UAL- and SAL-derived ASCs were harvested from lipoaspiration specimens and established in primary culture. After a brief period of expansion, cells were trypsinized and replated for proliferation and osteogenic assays. Cell proliferation was assessed by seeding equal numbers of first passage UAL- and SAL-derived ASCs in side-by-side culture dishes in triplicate. Cell counting was performed by trypan blue exclusion and hemacytometer 1, 3 and 7 days after seeding to obtain growth curves of these cells. Osteogenic differentiation was also assayed for UAL- and SAL-derived cells. First passage UAL- and SAL-derived ASCs were seeded in equal numbers in side-by-side culture dishes in triplicate. After a brief period of expansion to subconfluence, both cell types were induced with osteogenic differentiation media (ODM) for a period of 10 days. Cells were harvested 3, 7 and 10 days after induction for analysis of osteogenic gene expression analysis by RNA extraction and quantitative RT-PCR. Finally, UAL- and SAL-derived ASCs were assayed for their ability to form a mineralized extracellular matrix in vitro by staining for alkaline phosphatase activity (early) and Alizarin Red (terminal differentiation) which was normalized to protein content. Results: UAL- and SAL-derived ASCs exhibited similar proliferative capacities. There were no significant differences in cell numbers 1, 3 or 7 days after seeding. Furthermore, there was no significant difference in rate of cell death by trypan blue exclusion. Osteogenic differentiation by gene expression revealed similar numbers of RUNX2, osteopontin and osteocalcin transcripts that were not significantly different during the differentiation period. Finally, osteogenesis by staining revealed alkaline phosphatase activity (3 days of ODM) and Alizarin Red staining (7 and 10 days of ODM) that were not significantly different when normalized to protein content. Conclusions: With growing popularity and clinical benefits of UAL, research efforts should elect to further explore the biology of these cells in bone tissue engineering applications. Preliminary observations have revealed that UAL-derived ASCs may exhibit equal in vitro proliferative and osteogenic differentiative capacities compared to ASCs collected from conventional SAL. Future directions will include an in vivo assay of these cells to further investigate their potential in bone tissue engineering.

Loss of Gbx2 results in neural crest cell patterning and pharyngeal arch artery defects in the mouse embryo

Several syndromes characterized by defects in cardiovascular and craniofacial development are associated with a hemizygous deletion of chromosome 22q11 in humans and involve defects in pharyngeal arch and neural crest cell development. Recent efforts have focused on identifying 22q11 deletion syndrome modifying loci. In this study, we show that mouse embryos deficient for Gbx2 display aberrant neural crest cell patterning and defects in pharyngeal arch-derived structures. Gbx2 −/− embryos exhibit cardiovascular defects associated with aberrant development of the fourth pharyngeal arch arteries including interrupted aortic arch type B, right aortic arch, and retroesophageal right subclavian artery. Other developmental abnormalities include overriding aorta, ventricular septal defects, cranial nerve, and craniofacial skeletal patterning defects. Recently, Fgf8 has been proposed as a candidate modifier for 22q11 deletion syndromes. Here, we demonstrate that Fgf8 and Gbx2 expression overlaps in regions of the developing pharyngeal arches and that they interact genetically during pharyngeal arch and cardiovascular development.

Repair of craniotomy defects using bone marrow stromal cells.

Techniques used to repair craniofacial skeletal defects parallel the accepted surgical therapies for bone loss elsewhere in the skeleton and include the use of autogenous bone and alloplastic materials. Transplantation of a bone marrow stromal cell population that contains osteogenic progenitor cells may be an additional modality for the generation of new bone. Full thickness osseous defects (5 mm) were prepared in the cranium of immunocompromised mice and were treated with gelatin sponges containing murine alloplastic bone marrow stromal cells derived from transgenic mice carrying a type I collagen-chloramphenicol acetyltransferase reporter gene to follow the fate of the transplanted cells. Control surgical sites were treated with spleen stromal cells or gelatin sponges alone, or were left untreated. The surgical defects were analyzed histologically for percent closure of the defect at 2, 3, 4, 6, and 12 weeks. Cultured bone marrow stromal cells transplanted within gelatin sponges resulted in osteogenesis that repaired greater than 99.0+/-2.20% of the original surgical defect within 2 weeks. In contrast, cranial defects treated with splenic fibroblasts, vehicle alone, or sham-operated controls resulted in minimal repair that was limited to the surgical margins. Bone marrow stromal cells carrying the collagen transgene were immunodetected only in the newly formed bone and thus confirmed the donor origin of the transplanted cells. These studies demonstrate that mitotically expanded bone marrow cells can serve as an abundant source of osteoprogenitor cells that are capable of repairing craniofacial skeletal defects in mice without the addition of growth or morphogenetic factors.

Bone Repair by Regeneration

Bone repair by regeneration as we know it continues to undergo changes, with advances approaching that may change our treatment of patients with craniofacial deformities and skeletal defects. Perhaps by the turn of the century, patients born with asymmetric deformities due to lack of growth will be treated early in life by skeletal stretching, and then later in life by skeletal distraction that is followed by use of accelerating factors to assist the healing processes. All of these available modalities are part of the regeneration of new bone formation. The future of such changes is very interesting, and our ability to help our patients will be maximized. We may even look back 25 years from now at bone grafting and find it to be obsolete and crude. It is hoped that with the new modalities being developed, we will not deviate from the use of a bone grafting procedure, which is the workhorse of the craniofacial surgeon. Bone grafting is used by all surgeons working on the craniofacial skeleton despite the problems of unpredictability of healing and an inability to calculate what percentage of the original graft will survive. The transplantation issue will be solved. The problems with donor site morbidity will continue. The use of inorganic bone substitutes will continue to have its limitation, particularly in type II wounds, which we as plastic surgeons see in the craniofacial region. As we redefine our approach to skeletal repair, we may look back and find solutions to some of the major problems we have had. The rapid stretch of soft tissue after facial advancement or structural alteration that is accompanied by a relapse due to the elastic recoil of the soft tissue could be eliminated by gradual distraction. The bone will undergo better functional adaptation when it has a gradual change in structure based on adjustment and molding in a gradual fashion. The problem of donor site morbidity and a prediction formula for bone could be resolved with new bone formation in situ by mineralization of the area under repair. Bone healing enhancers are here to stay and their clinical application will produce a far-reaching better final outcome (Fig. 11).

Pedicled Osseous Flaps

Craniofacial skeletal defects are most optimally reconstructed with vascularized autogenous bone. There are two methods of achieving such a vascularized osseous reconstruction: microvascular bone transfers and pedicled osseous flaps. Although microvascular composite grafts allow a greater quantity and variety of bone to be moved to the reconstructive site, pedicled osseous flaps remain an excellent reconstructive option. Pedicled osseous flaps are not technically complex, result in minimal donor site morbidity, and demonstrate acceptable reliability in selected cases. This article reviews the realm of pedicled osseous flaps that can be used in craniofacial reconstruction. Although infrequently employed, these useful flaps should remain in the armamentarium of the head and neck surgeon.