Emily L. Durham

Recombinant human bone morphogenetic protein-2-induced craniosynostosis and growth restriction in the immature skeleton.

Background: Recombinant human bone morphogenetic protein-2 (rhBMP-2) delivered on an absorbable collagen sponge is a U.S. Food and Drug Administration–approved therapy effective at generating bone formation. In pediatric patients for whom other therapeutic options have been exhausted, rhBMP-2 is used off-label to address problematic bony defects. In the skeletally immature patient, the safety of rhBMP-2 therapy remains uncertain. Experiments are needed that investigate the effect of rhBMP-2 on growth and development in clinically relevant models. Methods: Ten juvenile rabbits underwent creation of a parietal skull defect that was treated with either 0.2 mg/cc rhBMP-2/absorbable collagen sponge or a neutral buffer solution/absorbable collagen sponge. Amalgam markers were placed at suture confluences to track suture separation and skull growth. Cranial growth was assessed radiographically at 10, 25, 42, and 84 days of age. Means and standard deviations for the various craniofacial growth variables were calculated and compared. Mean differences were considered significant for values of p < 0.05. At 84 days, sutures were analyzed by means of micro–computed tomographic scanning and histologic staining. Results: Treatment with rhBMP-2 resulted in fusion of the coronal sutures bilaterally, with variable fusion of the sagittal suture by cephalometric, radiographic, and histologic analysis. There were statistically significant changes to coronal suture growth, sagittal suture growth, skull height, craniofacial length, and intracranial volume (p < 0.05). Conclusions: The use of rhBMP-2 in this juvenile animal model resulted in skeletal changes that may be undesirable in a clinical setting. The appearance of these fused sutures suggested a direct effect of rhBMP-2. Further work is required to limit the effect of rhBMP-2 to the target defect when used in the immature skeleton.

Novel Animal Model of Calvarial Defect in an Infected Unfavorable Wound: Reconstruction with rhbmp-2

Background: Recombinant human bone morphogenetic protein-2 (rhBMP-2) delivered on an absorbable collagen sponge has been shown to induce the healing of acute, primary, large-scale calvarial defects in rabbits. However, clinical circumstances often require the reconstruction of a previously infected and chronically scarred wound. This study was designed to evaluate the efficacy of rhBMP-2/absorbable collagen sponge to improve healing in the previously infected, unfavorable calvarial defect model. Methods: Subtotal defects were made in the calvariae of 15 adult New Zealand White rabbits. The bone flap was inoculated with Staphylococcus aureus and replaced in situ. After a 2-week infection period, animals underwent bone flap removal and a 10-day course of antibiotic therapy. On postoperative day 42, the defect was exposed and treated with (1) no intervention/control (group 1; n = 5), or (2) absorbable collagen sponge with 0.43 mg/ml of rhBMP-2 (group 2; n = 10). Bone growth was analyzed with serial computed tomographic imaging and postmortem histology. Percentage bone healing was compared between groups using the t test. Results: The treatment group (group 2) demonstrated statistically more healing (55.6 percent) compared with the control group (group 1) (29 percent; p < 0.01). However, rhBMP-2–induced bone was not histologically or radiographically similar to native bone, lacking both continuity and a well-defined diploic space. Conclusions: These data suggest that rhBMP-2-treated collagen sponges may be useful for the repair of calvarial defects following infection. However, the osseous healing observed in this study was significantly less than previous reports in acute, noninfected models and was dissimilar to native bone. Further work is needed to optimize treatment of the previously infected calvarial wound with rhBMP-2.

Tissue interactions between craniosynostotic dura mater and bone

Background Cells within the dura mater have been implicated in the determination of suture patency and fusion. Craniosynostosis (CS), the premature fusion of 1 or more of the cranial sutures, could result from abnormal control over the differentiation of osteoprogenitor cells from the dura mater. This study tested whether dura mater cells derived from rabbits with congenital CS were different from cells derived from normal rabbits and investigated the effects that CS dura mater had on osteogenic differentiation in vitro and in vivo. Methods Cells were derived from the dura mater from wild-type rabbits (WT; n = 23) or CS rabbits (n = 16). Cells were stimulated with bone morphogenetic protein 4, and alkaline phosphatase (ALP) expression and cell proliferation were assessed. Dura mater–derived cells were also cocultured with primary rabbit bone-derived cells, and ALP was assessed. Finally, interactions between the dura mater and overlying tissues were manipulated in vivo. Results Craniosynostotic dura mater–derived cells proliferated faster than did WT cells but were not more ALP positive. Coculture experiments showed that CS dura mater cells induced increased ALP activity in CS bone-derived cells, but not in WT bone-derived cells. In vivo experiments showed that a physical barrier successfully inhibited dura mater–derived osteogenesis. Conclusions Coculture of CS bone- and CS dura mater–derived cells evoked an abnormal phenotype in vitro. Covering the CS dura mater led to decreased bone formation in vivo. Further investigations will focus on the signaling molecules involved in the communication between these 2 CS tissue types in vitro and in vivo.

Molecular Analysis of Coronal Perisutural Tissues in a Craniosynostotic Rabbit Model Using Polymerase Chain Reaction Suppression Subtractive Hybridization

Background: In the United States, the incidence of craniosynostosis (premature fusion of the sutures of the cranial vault) is one in 2000 to 3000 live births. The condition can cause increased intracranial pressure, severely altered head shape, and mental retardation. The authors have previously described a colony of rabbits with heritable coronal suture synostosis. This model has been instrumental in describing the postsurgical craniofacial growth associated with craniosynostosis. The molecular analysis of this model has been limited by the lack of molecular tools for use in rabbits. To understand the pathogenesis of craniosynostosis, the authors compared gene expression in perisutural tissues between wild-type and craniosynostotic rabbits using polymerase chain reaction suppression subtractive hybridization. Methods: Suppression subtractive hybridization polymerase chain reaction was performed on RNA derived from pooled samples of calvariae from 10-day-old wild-type (n = 3) and craniosynostotic (n = 3) rabbits to obtain cDNA clones enriched in either wild-type tissues (underexpressed in craniosynostotic tissue) or craniosynostotic tissues (overexpressed in craniosynostotic compared with wild-type). Results: Differential expression was identified for approximately 140 recovered cDNA clones up-regulated in craniosynostotic tissues and 130 recovered clones for wild-type tissues. Of these, four genes were confirmed by quantitative reverse-transcriptase polymerase chain reaction as being overexpressed in craniosynostotic sutural tissue: &bgr;-globin (HBB), osteopontin (SPP1), osteonectin (SPARC), and cathepsin K (CTSK). Two genes were confirmed to be underexpressed in the craniosynostotic samples: collagen 3, alpha 1 (COL3A1) and ring finger protein 12 (RNF12). Conclusion: The differential expression of these gene products in our naturally occurring craniosynostotic model appears to be the result of differences in the normal bone formation/resorption pathway.

The Vomeronasal Complex of Nocturnal Strepsirhines and Implications for the Ancestral Condition in Primates

This study investigates the vomeronasal organ in extant nocturnal strepsirhines as a model for ancestral primates. Cadaveric samples from 10 strepsirhine species, ranging from fetal to adult ages, were studied histologically. Dimensions of structures in the vomeronasal complex, such as the vomeronasal neuroepithelium (VNNE) and vomeronasal cartilage (VNC) were measured in serial sections and selected specimens were studied immunohistochemically to determine physiological aspects of the vomeronasal sensory neurons (VSNs). Osteological features corresponding to vomeronasal structures were studied histologically and related to 3‐D CT reconstructions. The VNC consistently rests in a depression on the palatal portion of the maxilla, which we refer to as the vomeronasal groove (VNG). Most age comparisons indicate that in adults VNNE is about twice the length compared with perinatal animals. In VNNE volume, adults are 2‐ to 3‐fold larger compared with perinatal specimens. Across ages, a strong linear relationship exists between VNNE dimensions and body length, mass, and midfacial length. Results indicate that the VNNE of nocturnal strepsirhines is neurogenic postnatally based on GAP43 expression. In addition, based on Olfactory Marker Protein expression, terminally differentiated VSNs are present in the VNNE. Therefore, nocturnal strepsirhines have basic similarities to rodents in growth and maturational characteristics of VSNs. These results indicate that a functional vomeronasal system is likely present in all nocturnal strepsirhines. Finally, given that osteological features such as the VNG are visible on midfacial bones, primate fossils can be assessed to determine whether primate ancestors possessed a vomeronasal complex morphologically similar to that of modern nocturnal strepsirhines. Anat Rec, 296:1881–1894, 2013.

Direct comparison of progenitor cells derived from adipose, muscle, and bone marrow from wild-type or craniosynostotic rabbits

Background: Reports have identified cells capable of osteogenic differentiation in bone marrow, muscle, and adipose tissues, but there are few direct comparisons of these different cell types. Also, few have investigated the potential connection between a tissue-specific abnormality and cells derived from seemingly unrelated tissues. In this article, the authors compare cells isolated from wild-type rabbits or rabbits with nonsyndromic craniosynostosis, defined as the premature fusion of one or more of the cranial sutures. Methods: Cells were derived from bone marrow, adipose, and muscle of 10-day-old wild-type rabbits (n = 17) or from age-matched rabbits with familial nonsyndromic craniosynostosis (n = 18). Cells were stimulated with bone morphogenetic protein-4 (BMP4), and alkaline phosphatase expression and cell proliferation were assessed. Results: In wild-type rabbits, cells derived from muscle had more alkaline phosphatase activity than cells derived from either adipose or bone marrow. The cells derived from craniosynostotic rabbit bone marrow and muscle were significantly more osteogenic than those derived from wild-type rabbits. Adipose-derived cells demonstrated no significant differences. Although muscle-derived cells were most osteogenic in wild-type rabbits, bone marrow-derived cells were most osteogenic in craniosynostotic rabbits. Conclusions: These results suggest that cells from different tissues have different potentials for differentiation. Furthermore, cells derived from rabbits with craniosynostosis were different from cells from wild-type rabbits. Interestingly, cells derived from the craniosynostotic rabbits were not uniformly more responsive compared with wild-type cells, suggesting that specific tissue-derived cells may react differently in individuals with craniosynostosis.

The vomeronasal organ of Lemur catta

The vomeronasal organ (VNO), also known as the Jacobsons organ, is a bilateral chemosensory organ found at the base of the nasal cavity specialized for the detection of higher‐molecular weight (non‐volatile) chemostimuli. It has been linked to pheromone detection. The VNO has been well studied in nocturnal lemurs and lorises, but poorly studied in diurnal/cathemeral species despite the large repertoire of olfactory behaviors noted in species such as Lemur catta. Here, the VNO and associated structures were studied microanatomically in one adult female and one adult male L. catta. Traditional and immunohistochemical procedures demonstrate the VNO epithelium consists of multiple rows of sensory neurons. Immunoreactivity to Growth‐associated protein 43 (GAP43) indicates the VNO is postnatally neurogenic. In volume, the VNO neuroepithelium scales similarly to palatal length compared to nocturnal strepsirrhines. Numerous taste buds present at the oral opening to the nasopalatine duct, with which the VNO communicates, provide an additional (or alternative) explanation for the flehmen behavior that has been observed in this species. The VNO of L. catta is shown to be microanatomically comparable to that of nocturnal strepsirrhines. Like nocturnal strepsirrhines, the VNO of L. catta may be functional in the reception of high‐molecular weight secretions. Am. J. Primatol. 77:229–238, 2015.

Human Faces Are Slower than Chimpanzee Faces

Background While humans (like other primates) communicate with facial expressions, the evolution of speech added a new function to the facial muscles (facial expression muscles). The evolution of speech required the development of a coordinated action between visual (movement of the lips) and auditory signals in a rhythmic fashion to produce “visemes” (visual movements of the lips that correspond to specific sounds). Visemes depend upon facial muscles to regulate shape of the lips, which themselves act as speech articulators. This movement necessitates a more controlled, sustained muscle contraction than that produced during spontaneous facial expressions which occur rapidly and last only a short period of time. Recently, it was found that human tongue musculature contains a higher proportion of slow-twitch myosin fibers than in rhesus macaques, which is related to the slower, more controlled movements of the human tongue in the production of speech. Are there similar unique, evolutionary physiologic biases found in human facial musculature related to the evolution of speech? Methodology/Prinicipal Findings Using myosin immunohistochemistry, we tested the hypothesis that human facial musculature has a higher percentage of slow-twitch myosin fibers relative to chimpanzees (Pan troglodytes) and rhesus macaques (Macaca mulatta). We sampled the orbicularis oris and zygomaticus major muscles from three cadavers of each species and compared proportions of fiber-types. Results confirmed our hypothesis: humans had the highest proportion of slow-twitch myosin fibers while chimpanzees had the highest proportion of fast-twitch fibers. Conclusions/significance These findings demonstrate that the human face is slower than that of rhesus macaques and our closest living relative, the chimpanzee. They also support the assertion that human facial musculature and speech co-evolved. Further, these results suggest a unique set of evolutionary selective pressures on human facial musculature to slow down while the function of this muscle group diverged from that of other primates.

Of mice, monkeys, and men: Physiological and morphological evidence for evolutionary divergence of function in mimetic musculature

Facial expression is a universal means of visual communication in humans and many other primates. Humans have the most complex facial display repertoire among primates; however, gross morphological studies have not found greater complexity in human mimetic musculature. This study examines the microanatomical aspects of mimetic musculature to test the hypotheses related to human mimetic musculature physiology, function, and evolutionary morphology. Samples from the orbicularis oris muscle (OOM) and the zygomaticus major (ZM) muscle in laboratory mice (N = 3), rhesus macaques (N = 3), and humans (N = 3) were collected. Fiber type proportions (slow‐twitch and fast‐twitch), fiber cross‐sectional area, diameter, and length were calculated, and means were statistically compared among groups. Results showed that macaques had the greatest percentage of fast fibers in both muscles (followed by humans) and that humans had the greatest percentage of slow fibers in both muscles. Macaques and humans typically did not differ from one another in morphometrics except for fiber length where humans had longer fibers. Although sample sizes are low, results from this study may indicate that the rhesus macaque OOM and ZM muscle are specialized primarily to assist with maintenance of the rigid dominance hierarchy via rapid facial displays of submission and aggression, whereas human musculature may have evolved not only under pressure to work in facial expressions but also in development of speech. Anat Rec, 297:1250–1261, 2014.

Molecular Analysis of Twist1 and FGF Receptors in a Rabbit Model of Craniosynostosis: Likely Exclusion as the Loci of Origin

Craniosynostosis is the premature fusion of the cranial vault sutures. We have previously described a colony of rabbits with a heritable pattern of nonsyndromic, coronal suture synostosis; however, the underlying genetic defect remains unknown. We now report a molecular analysis to determine if four genes implicated in human craniosynostosis, TWIST1 and fibroblast growth factor receptors 1–3 (FGFR1–3), could be the loci of the causative mutation in this unique rabbit model. Single nucleotide polymorphisms (SNPs) were identified within the Twist1, FGFR1, and FGFR2 genes, and the allelic patterns of these silent mutations were examined in 22 craniosynostotic rabbits. SNP analysis of the Twist1, FGFR1, and FGFR2 genes indicated that none were the locus of origin of the craniosynostotic phenotype. In addition, no structural mutations were identified by direct sequence analysis of Twist1 and FGFR3 cDNAs. These data indicate that the causative locus for heritable craniosynostosis in this rabbit model is not within the Twist1, FGFR1, and FGFR2 genes. Although a locus in intronic or flanking sequences of FGFR3 remains possible, no direct structural mutation was identified for FGFR3.