Kelly A. Lenton

Applications of a mouse model of calvarial healing: Differences in regenerative abilities of juveniles and adults

Young children are capable of healing large calvarial defects, whereas adults lack this endogenous osseous tissue-engineering capacity. Despite the important clinical implications, little is known about the molecular and cell biology underlying this differential ability. Traditionally, guinea pig, rabbit, and rat models have been used to study the orchestration of calvarial healing. To harness the research potential of knockout and transgenic mice, the authors developed a mouse model for calvarial healing. Nonsuture-associated parietal defects 3, 4, and 5 mm in diameter were made in both juvenile (6-day-old, n = 15) and adult (60-day-old, n = 15) mice. Calvariae were harvested after 8 weeks and analyzed radiographically and histologically. Percentage of healing was quantified using Scion Image software analysis of calvarial radiographs. A significant difference in the ability to heal calvarial defects was seen between 6-day-old and 60-day-old mice when 3-, 4-, or 5-mm defects were created. The authors’ analysis revealed that juvenile mice healed a significantly greater percentage of their calvarial defects than adult mice (juvenile mean percentage of healing: 3-mm defects, 59 percent; 4-mm defects, 65 percent; 5-mm defects, 44 percent; adult mean percentage of healing: <5 percent in all groups; p < 0.05). All three defect sizes were found to be critical in the adult, whereas significant healing was seen regardless of the size of the defect in juvenile mice. The establishment of this model will facilitate further, detailed evaluation of the molecular biology underlying the different regenerative abilities of juvenile versus adult mice and enhance research into membranous bone induction by making available powerful tools such as knockout and transgenic animals.

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.

Cranial Suture Biology

Publisher Summary This chapter focuses on the cranial suture biology. The term “craniosynostosis” was first used in 1830 by Otto to describe the premature fusion of cranial sutures. Since this first identification of craniosynostosis as a distinct clinical entity, several theories have been proposed to explain both the pathogenesis of premature suture fusion and the resultant aberrations in calvarial growth that result in a dysmorphic skull. Recent advances in clinical genetics have resulted in the identification of genetic mutations in the major craniosynostostic syndromes. Despite these insights into the rudimentary disturbances leading to craniosynostosis, the processes by which mutations in these genes trigger premature suture fusion remain largely unknown. Rodents are proving to be extremely valuable in unraveling the cellular and molecular mechanisms of cranial suture morphogenesis and pathology. The cranial sutures include the metopic or interfrontal suture (between the frontal bones), the sagittal suture (between the parietal bones), the coronal suture (between the frontal and parietal bones), and the lambdoid sutures (between the parietal and interparietal bones). The sutures can be thought of as a complex consisting of four principal components: (1) the osteogenic fronts of the approximating bone plates; (2) the suture mesenchyme spanning the osteogenic fronts; (3) the overlying pericranium or cranial periosteum; and (4) the underlying dura mater, a tough, fibrous membrane that constitutes the outer meningeal layer that envelops the brain and forms the inner lining of cranial bones and sutures. The main objective is to obtain a thorough understanding of normal and pathological suture morphogenesis and development. Armed with this knowledge, researchers will be prepared to devise biologically based therapeutic strategies that could be used both in utero or postnatally to prevent craniosynostosis, potentially alleviating any adverse sequelae and avoiding the morbidity of current surgical approaches.

Indian hedgehog positively regulates calvarial ossification and modulates bone morphogenetic protein signaling

Much is known regarding the role of Indian hedgehog (Ihh) in endochondral ossification, where Ihh regulates multiple steps of chondrocyte differentiation. The Ihh−/− phenotype is most notable for severely foreshortened limbs and a complete absence of mature osteoblasts. A far less explored phenotype in the Ihh−/− mutant is found in the calvaria, where bones form predominately through intramembranous ossification. We investigated the role of Ihh in calvarial bone ossification, finding that proliferation was largely unaffected. Instead, our results indicate that Ihh is a pro‐osteogenic factor that positively regulates intramembranous ossification. We confirmed through histologic and quantitative gene analysis that loss of Ihh results in reduction of cranial bone size and all markers of osteodifferentiation. Moreover, in vitro studies suggest that Ihh loss reduces Bmp expression within the calvaria, an observation that may underlie the Ihh−/− calvarial phenotype. In conjunction with the newly recognized roles of Hedgehog deregulation in craniosynostosis, our study defines Ihh as an important positive regulator of cranial bone ossification. genesis 49:784–796, 2011.

Expression and possible mechanisms of regulation of bmp3 in rat cranial sutures

Background: Clinical genetics data and investigative studies have contributed greatly to our understanding of the role of numerous genes in craniosynostosis. Recent studies have introduced antagonists of osteogenesis as potential key regulators of suture fusion and patency. The authors investigated the expression pattern of the bone morphogenetic protein antagonist BMP3 in rat cranial sutures and the factors regulating its expression in vitro. Methods: Microarray analysis was performed on rat posterior frontal and sagittal cranial sutures at 5, 10, 15, 20, and 30 days of life (n = 30 per group). Gene expression was confirmed using quantitative real-time reverse transcriptase polymerase chain reaction. Regulation of BMP3 expression was determined using primary rat calvarial osteoblasts stimulated with recombinant human fibroblast growth factor 2 or recombinant human transforming growth factor &bgr;1, or cultured with primary rat nonsuture dura mater. Gene expression was quantified with quantitative real-time reverse transcriptase polymerase chain reaction. Results: BMP3 expression in the posterior frontal suture decreased over the time course analyzed, whereas it increased in the sagittal suture. Notably, BMP3 expression was higher in the patent sagittal suture during the window of posterior frontal suture fusion. Stimulation of osteoblasts with recombinant human fibroblast growth factor 2 led to a rapid and sustained suppression of BMP3 expression (85 percent, p < 0.01) when compared with controls. Co-culture with dural cells decreased BMP3 mRNA by 50 percent compared with controls (p < 0.01). Conclusions: BMP3 is expressed in rat cranial sutures in a temporal pattern suggesting involvement in cranial suture patency and fusion. Furthermore, BMP3 is regulated in calvarial osteoblasts by recombinant human fibroblast growth factor 2 and by paracrine signaling from dura mater. These data add to our knowledge of the role of osteogenic antagonists in cranial suture biology.

Primary cilia act as mechanosensors during bone healing around an implant

The primary cilium is an organelle that senses cues in a cells local environment. Some of these cues constitute molecular signals; here, we investigate the extent to which primary cilia can also sense mechanical stimuli. We used a conditional approach to delete Kif3a in pre-osteoblasts and then employed a motion device that generated a spatial distribution of strain around an intra-osseous implant positioned in the mouse tibia. We correlated interfacial strain fields with cell behaviors ranging from proliferation through all stages of osteogenic differentiation. We found that peri-implant cells in the Col1Cre;Kif3a(fl/fl) mice were unable to proliferate in response to a mechanical stimulus, failed to deposit and then orient collagen fibers to the strain fields caused by implant displacement, and failed to differentiate into bone-forming osteoblasts. Collectively, these data demonstrate that the lack of a functioning primary cilium blunts the normal response of a cell to a defined mechanical stimulus. The ability to manipulate the genetic background of peri-implant cells within the context of a whole, living tissue provides a rare opportunity to explore mechanotransduction from a multi-scale perspective.

Ex vivo Model of Cranial Suture Morphogenesis and Fate

Background/Aims: Craniosynostosis, the premature fusion of cranial sutures, is a common congenital defect. In vivo models for studying cranial suture biology impose inherent restrictions on tissue accessibility and manipulation. The present study was performed to investigate the utility of the renal capsule assay in overcoming these limitations and providing a reproducible model system for studying cranial suture morphogenesis and fate. Materials and Methods: The posterior frontal suture, which fuses physiologically, and the coronal and sagittal sutures, which remain patent, were dissected from postnatal and embryonic mouse calvaria and placed under the renal capsule of syngeneic recipient mice (n = 72 in total). Sutures were harvested from 1–14 days after transplantation for histological and morphometric analysis. Suture transplants were compared with nonmanipulated sutures at equivalent developmental stages. The derivation of cells associated with the growing transplants was analyzed using β-actin-GFP (green fluorescent protein) transgenic mice. Results: Sutures transplanted under the renal capsule maintained normal suture morphology and fate with the posterior frontal suture fusing and the coronal and sagittal sutures remaining patent. In posterior frontal suture transplants, the fusion process mimicked in vivo suture fusion with a delay of 1–2 days. In comparison to in vivo suture complexes, transplant thickness and trabeculation were significantly increased. In addition, we found that osteoblasts within the growing transplant were derived from the transplant itself rather than the host. Conclusion: The renal capsule supports the growth of cranial sutures. In this system transplanted sutures recapitulate the anatomical development and fate (fusion or patency) of cranial sutures in vivo. This model system will facilitate controlled ex vivo manipulations of both embryonic and postnatal sutures.

Contents Vol. 190, 2009

F. Beck, Leicester A.L. Boskey, New York, N.Y. R.C. Burghardt, College Station, Tex. G. Burnstock, London F. Eckstein, Salzburg A.C. Enders, Davis, Calif. C. Farnum, Ithaca, N.Y. R.H.W. Funk, Dresden N.E. Fusenig, Heidelberg A. Gibson, Phoenix, Ariz. M. Glickstein, London J.W. Hermanson, Ithaca, N.Y. C.J. Kirkpatrick, Mainz P. Köpf-Maier, Berlin W. Kummer, Giessen J.W. Lichtman, Cambridge, Mass. K.G. Marra, Pittsburgh, Pa. O. Ohtani, Toyama P.J. Reier, Gainesville, Fla. R. Roy, Los Angeles, Calif. R. Segal, Chapel Hill, N.C. F. Sinowatz, Munich M. Sittinger, Berlin T. Skutella, Tübingen G.B. Stark, Freiburg i.Br. E. Th ompson, Melbourne C.G. Widmer, Gainesville, Fla. in vivo, in vitro