Bethany J. Slater

Cranial sutures: a brief review.

Summary: Craniosynostosis, or the premature fusion of one or more cranial sutures, is a relatively common congenital defect that causes a number of morphologic and functional abnormalities. With advances in genetics and molecular biology, research of craniosynostosis has progressed from describing gross abnormalities to understanding the molecular interactions that underlie these cranial deformities. Animal models have been extremely valuable in improving our comprehension of human craniofacial morphogenesis, primarily by human genetic linkage analysis and the development of knock-out animals. This article provides a brief review of perisutural tissue interactions, embryonic origins, signaling molecules and their receptors, and transcription factors in maintaining the delicate balance between proliferation and differentiation of cells within the suture complex that determines suture fate. Finally, this article discusses the potential implications for developing novel therapies for craniosynostosis.

Cell-based therapies for skeletal regenerative medicine

Skeletal deficits represent a substantial biomedical burden on the US healthcare system. Current strategies for reconstructing bony defects are fraught with inadequacies. Cell-based therapies for skeletal regeneration offer a paradigm shift that may provide alternative solutions. Substantial work has identified a host of cellular sources that possess the potential for osteogenic differentiation. Significant efforts have been devoted toward characterizing the role of postnatal cellular sources that are relatively abundant and easily accessible. Among these, the potential of using adipose-derived stromal cells for skeletal regeneration has garnered much interest. Integral to these efforts directed at characterizing cellular sources are studies that seek to understand the factors that initiate and regulate osteogenic differentiation of progenitor cells. Specifically, focus has been directed on elucidating the role of bone morphogenetic protein and fibroblast growth factor signaling in regulating osteogenic differentiation of osteoprogenitor cells. Concurrent studies in the field of scaffold design have also helped to advance the potential for cell-based therapies.

Tissue Engineering in Cleft Palate and Other Congenital Malformations

Contributions from multidisciplinary investigations have focused attention on the potential of tissue engineering to yield novel therapeutics. Congenital malformations, including cleft palate, craniosynostosis, and craniofacial skeletal hypoplasias represent excellent targets for the implementation of tissue engineering applications secondary to the technically challenging nature and inherent inadequacies of current reconstructive interventions. Apropos to the search for answers to these clinical conundrums, studies have focused on elucidating the molecular signals driving the biologic activity of the aforementioned maladies. These investigations have highlighted multiple signaling pathways, including Wnt, fibroblast growth factor, transforming growth factor-β, and bone morphogenetic proteins, that have been found to play critical roles in guided tissue development. Furthermore, a comprehensive knowledge of these pathways will be of utmost importance to the optimization of future cell-based tissue engineering strategies. The scope of this review encompasses a discussion of the molecular biology involved in the development of cleft palate and craniosynostosis. In addition, we include a discussion of craniofacial distraction osteogenesis and how its applied forces influence cell signaling to guide endogenous bone regeneration. Finally, this review discusses the future role of cell-based tissue engineering in the treatment of congenital malformations.

Applications of an athymic nude mouse model of nonhealing critical-sized calvarial defects.

Calvarial bone defects are a common clinical scenario in craniofacial surgery. Numerous approaches are used to reconstruct skull defects, and each possesses its own inherent disadvantages. This fact underscores the opportunity to develop a novel method to repair osseous defects in craniofacial surgery. Recent literature strongly suggests that cell-based therapies in the form of regenerative medicine may be a developing paradigm in reconstructive surgery. Although numerous studies have probed osteoprogenitor cells from mice, few have explored the biology of human cells in the setting of osteogenesis in an equally rigorous manner. This study proposes a nude mouse model of critical-sized calvarial defects to study the in vivo biology of human osteoprogenitor cells. Critical-sized 4.0-mm calvarial defects were created in nude mice (n = 15) with a custom trephine drill bit outfitted to a dental drill handpiece. During the craniotomy, the dura mater was spared from injury. Gross inspection, routine histology, and micro-computed tomographic scanning were performed at 2, 4, 8, and 16 weeks postoperatively. There was no calvarial healing in any of the animals by 16 weeks. The dura mater remained intact in all subjects. Gross, histologic, and radiographic assays confirmed these findings. Although several studies have implanted human osteoprogenitor cells in vivo in various animal models, few have documented the appropriate controls or conditions necessary to support the potential to translate benchtop findings into clinical applications. We propose in this study that the nude mouse critical-sized calvarial defect model will be valuable with increasing investigations with human osteoprogenitor cells.

Mesenchymal cells for skeletal tissue engineering.

Background: Skeletal defects represent a significant socioeconomic burden to the US healthcare system. Current options for reconstructing osseous deficits have shortcomings. Objective: To review the use of mesenchymal stem cells for skeletal tissue engineering. Methods: We focused on the application of mesenchymal cells in skeletal regeneration, optimization of this technique, tropic effects of multipotent mesenchymal cells, and future directions. Results/conclusion: A number of cell-based modalities have been investigated. We have been interested in the role of adipose-derived stromal cells in bone regeneration and understanding the mechanisms behind osteogenic differentiation of progenitor cells and acceleration of this process. Future clinical applications of multipotent mesenchymal cells will depend on better understanding of the molecular signaling involved in osteogenic differentiation and maintaining pluripotency.

Early Postoperative Outcomes and Medication Cost Savings after Laparoscopic Sleeve Gastrectomy in Morbidly Obese Patients with Type 2 Diabetes

Background. We investigated the effect of laparoscopic sleeve gastrectomy (LSG) on morbidly obese diabetics and examined the short-term impact of LSG on diabetic medication cost. Methods. A prospective database of consecutive bariatric patients was reviewed. Morbidly obese patients with type 2 diabetes who underwent LSG were included in the study. Age, gender, body mass index (BMI), diabetic medication use, glucose, insulin, and HbA1c levels were documented preoperatively, and at 2 weeks, 2 months, 6 months, and 12 months postoperatively. Insulin resistance was estimated using the homeostatic model assessment (HOMA). Use and cost of diabetic medications were followed. Results. Of 178 patients, 22 were diabetics who underwent LSG. Diabetes remission was observed in 62% of patients within 2 months and in 75% of patients within 12 months. HOMA-IR improved after only two weeks following surgery (16.5 versus 6.6, P < 0.001). Average number of diabetic medications decreased from 2.2 to <1, within 2 weeks after surgery; corresponding to a diabetes medication cost savings of 80%, 91%, 99%, and 99.7% after 2 weeks, 2 months, 6 months, and 12 months, respectively. Conclusion. Morbidly obese patients with diabetes who undergo LSG have high rates of diabetes remission early after surgery. This translates to a significant medication cost savings.

Cranial Osteogenesis and Suture Morphology in Xenopus laevis: A Unique Model System for Studying Craniofacial Development

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

Global age-dependent differences in gene expression in response to calvarial injury.

Children less than 2 years of age are capable of healing large calvarial defects, whereas adults have been found to lack this endogenous ability. In this study, we used microarray analysis to compare genomewide expression patterns during active regeneration after injury with calvaria in skeletally immature and mature mice. Parietal bone defects were created in 6-day-old (juvenile) and 60-day-old (adult) mice using a 4-mm trephine bit (n = 20 mice per age group). The calvarial disc was removed, leaving the underlying dura mater intact. Two weeks after injury, the region of regeneration with the underlying dura mater was harvested, and RNA was extracted for microarray analysis. The 25 most differentially upregulated genes in juvenile regenerates compared with adults were listed, as well as selected bone-related genes. In addition, QRT-PCR confirmation of specific genes was performed for validation. Juvenile regenerates expressed significantly greater amounts of BMP-2, -4, -7, as well as FGF-2 and its receptor FGFR-1. Various other growth factors were also noted to be upregulated, including IGF-2 and Ptn. This corresponded with the increased expression of markers for osteogenic differentiation of Sparc and Oc. Markers of osteoclast activity, Acp5, Ctsk, and Mmp2, were noted to be greater in juvenile regenerates compared with adults. The observation of Mmp14 upregulation, however, highlights the importance of balanced osteoclast-mediated bone resorption for ultimate healing. The 2 most differentially regulated genes, transthyretin (Ttr) and prostaglandin D2 synthase (Ptgds), highlight the potential role of retinoic acid signaling and the prostaglandin axis on skeletal regeneration. These findings underscore the multitude of biomolecular mechanisms at play, allowing juvenile calvaria to heal after injury. The identification of various growth factors and cytokines involved also suggests novel therapeutic strategies for tissue-engineering purposes.

The role of regional posterior frontal dura mater in the overlying suture morphology.

Background: Craniosynostosis, the premature fusion of one or more cranial sutures, is a common developmental disorder resulting in morphologic and functional consequences. The rat model is useful for studying pathologic and normal suture fusion because the posterior frontal suture undergoes fusion but the remaining sutures remain patent. The authors investigated the influence of regional posterior frontal dura mater on the overlying suture morphology and fate. Methods: In 8-day-old Sprague-Dawley rats, an 8-mm calvarial disk was excised without disrupting the underlying dura mater (n = 22) and flipped so that the previously ectocranial aspect was adjacent to the dura mater. The animals were humanely killed after 5, 7, 9, 11, and 28 days, and the posterior frontal sutures were analyzed histologically. A comparison was made to control animals in which the disk was excised and then placed back into its anatomical position (n = 5). Immunohistochemistry of the transforming growth factor (TGF)-β isoforms was performed to investigate their differential, temporal, and spatial expression. Results: Posterior frontal suture fusion occurred on the side adjacent to the dura mater (previously patent ectocranial aspect) in an anterior-to-posterior direction, similar to that in the control group. There was specific expression of the TGF-β isoforms in the dura mater and suture mesenchyme adjacent to the dura mater. Conclusions: Regional dura mater plays an important role in suture morphology, and the posterior frontal–associated dura mater possesses potent, pro-osteogenic signals that influence the overlying suture fate. The differential expression pattern of TGF-β signaling from the dura mater further supports the regional paracrine effect of the dura mater.

Microarray Analysis of the Role of Regional Dura Mater in Cranial Suture Fate

Background: Craniosynostosis, the premature fusion of cranial sutures, results in serious neurologic and morphologic abnormalities when left untreated. Surgical excision of the fused sutures and remodeling of the skull remains the standard therapy. Development of novel, minimally invasive therapies for craniosynostosis will undoubtedly be dependent on a more thorough understanding of the molecular mechanisms underlying this abnormality. Significant evidence suggests the influence of regional dura mater on the behavior of the overlying suture complex. The mouse model has been instrumental in investigating this observation because of the natural juxtaposition of the posterior frontal suture, which fuses early in life, with the other cranial sutures, which remain patent. Methods: The authors used microarray analysis to compare genomic changes in the dura mater underlying the posterior frontal and sagittal sutures of mice. Suture-associated dura mater was harvested from mice before (postnatal day 5), during (postnatal day 10), and after (postnatal day 20) posterior frontal suture fusion (n = 20 mice for each of the three time points). Results: Microarray results confirmed differential regulation of genes involved in paracrine signaling, extracellular matrix, and bone remodeling between the dura mater underlying the fusing posterior frontal suture and the patent sagittal suture. Conclusions: These data confirm global differences in gene expression between regional dura mater underlying fusing and patent sutures. These results provide further insight into potential molecular mechanisms that may play a role in cranial suture biology.