Faculty Bibliography

Using a physiologic model of mouse cranial suture fusion, the authors' laboratory has previously demonstrated that transforming growth factor (TGF)-betas appear to be more abundantly expressed in the suture complex of the fusing posterior frontal compared with the patent sagittal suture. Furthermore, the authors have shown that by blocking TGF-beta signaling with a replication-deficient adenovirus encoding a defective, dominant negative type II TGF-beta receptor (AdDN-TbetaRII), posterior frontal suture fusion was inhibited. In this study, the authors attempt to further elucidate the role of TGF-beta in cranial suture fusion by investigating possible mechanisms of AdDN-TbetaRII-mediated cranial suture patency using both an established organ culture model and a novel in vitro co-culture system that recapitulates the in vivo anatomic dura mater/cranial suture relationship. In this article, the authors demonstrate that blocking TGF-beta signaling with the AdDN-TbetaRII construct led to inhibition of cellular proliferation in the suture mesenchyme and subjacent dura mater during the early period of predicted posterior frontal suture fusion. Interestingly, co-culture experiments revealed that transfecting osteoblasts with AdDN-TbetaRII led to alterations in the gene expression levels of two important bone-related molecules (Msx2 and osteopontin). Inhibiting TGF-beta signaling prevented time-dependent suppression of Msx2 and prevented induction of osteopontin, thereby retarding osteoblast differentiation. Furthermore, the authors demonstrated that the AdDN-TbetaRII construct was capable of blocking TGF-beta -mediated up-regulation of collagen IalphaI, an extracellular matrix molecule important for bone formation. Collectively, these data strongly suggest that AdDN-TbetaRII maintains posterior frontal patency, in part by altering early events in de novo bone formation, including cellular proliferation and early extracellular matrix production

In CD-1 mice, the posterior frontal suture (analogous to the human metopic suture) fuses while all other cranial sutures remain patent. In an in vitro organ culture model, the authors previously demonstrated that posterior frontal sutures explanted immediately before the onset of suture fusion (at 25 days old) mimic in vivo physiologic fusion. In the first portion of this study, the authors defined how early in development the posterior frontal suture fuses in their tension-free, serum-free organ culture system by serially analyzing posterior frontal suture fusion from calvariae explanted at different stages of postnatal development. Their results revealed a divergence of suture fate leading to abnormal patency or physiologic fusion between the first and second weeks of life, respectively, despite viability and continued growth of the calvarial explants in vitro. From these data, the authors postulated that the gene expression patterns present in the suture complex at the time of explant may determine whether the posterior frontal suture fuses or remains patent in organ culture. Therefore, to elucidate potentially important differences in gene expression within this 'window of opportunity,' they performed a cDNA microarray analysis on 5-day-old and 15-day-old posterior frontal and sagittal whole suture complexes corresponding to the age ranges for unsuccessful (1 to 7 days old) and successful (14 to 21 days old) in vitro posterior frontal suture fusion. Overall, their microarray results reveal interesting differential expression patterns of candidate genes in different categories, including angiogenic cytokines and mechanosensitive genes potentially important in cranial suture biology

We analyzed mechanobiological influences on successful distraction osteogenesis (DO). Mandibular distraction surgeries were performed on 15 adult male Sprague-Dawley rats. Animals underwent gradual distraction (GD), progressive lengthening by small increments (5-day latency followed by 0.25 mm distractions twice daily for 8 days followed by 28-day maturation period). Distracted hemimandibles were harvested on postoperative days (POD) 5, 7, 10, 13, and 41. Load-displacement curves were then recorded for ex vivo distractions of 0.25 mm and stresses determined. Histologically, new bone formation appeared in GD specimens on distraction day 2 (POD 7), filling 50-60% of the gap by distraction day 8 (POD 13), with nearly complete bony bridging at end maturation (POD 41). Average tensile strains imposed by each incremental distraction ranged from approximately 10% to 12.5% during distraction days 2-8 and were associated with bone apposition rates of about 260 microm/day. Because this GD protocol was previously determined to be optimal for DO, we conclude that strains within this range provide an excellent environment for de novo bone apposition. Distraction caused tissue damage in distraction day 2, 5, and 8 specimens as evidenced by distinct drops in the load/displacement curves. Taken together, our interpretation of these data is that daily distractions cause daily tissue damage which triggers new mesenchymal tissue formation

The vertical reduction mammaplasty is an evolving technique. Its proponents report significantly decreased scarring, better breast shape, and more stable results compared with the standard inverted-T method, but the learning curve is long and cosmetic outcomes can be inconsistent. Many surgeons have experimented with the vertical closure before returning to methods more familiar to them. The authors present their modifications to the vertical reduction mammaplasty. Their changes simplify the preoperative markings and the intraoperative technique to shorten the learning curve while maintaining reliable aesthetic results. With the patient standing, only four preoperative marks are made: (1) the inframammary fold; (2) the breast axis; (3) the apex of the new nipple-areola complex; and (4) the medial and lateral limbs of the vertical incision. In the operating room, a medial or a superomedial pedicle is developed. Excess breast skin is resected with the inferior and lateral parenchyma as a C-shaped wedge. The lateral skin-adipose flap is redraped inferomedially and sutured to the chest wall. The inferior aspect of the breast is aggressively debulked and a gathering subcuticular stitch is started 2 cm below the nadir of the nipple-areola complex. Finally, a 38-mm to 42-mm nipple-areola complex marker is used to create a circular defect that is offset 0.5 cm medial to the vertical axis of the breast. In their series, 56 patients were treated and no major complications were noted. The median follow-up period was 17 months. The average reduction was 554.5 g per breast; however, the reduction was greater than 1000 g per breast in eight patients. The authors found that (1) chest wall anchoring improves lateral contour and minimizes axillary fullness; (2) aggressive debulking inferiorly avoids the persistent inferior bulge; and (3) starting the subcuticular gathering suture 2 cm below the nipple-areola complex followed by placement of a nipple-areola complex marker at the conclusion of the case prevents lateral deviation and corrects the nipple-areola complex teardrop deformity. These innovations accelerate the learning curve by simplifying the preoperative markings and lead to more consistent postoperative results and an improved cosmetic outcome. In conclusion, these modifications yield a simple, easily learned vertical reduction mammaplasty with aesthetically reliable results

Accumulating clinical genetic data support the hypothesis that alterations in osteoblast differentiation are closely associated with craniosynostoses. Gain-of-function mutations in FGFR1, FGFR2, FGFR3, and Msx2 and loss-of-function mutations in Twist are examples of such alterations. Several studies have examined how these mutations alter the expression patterns for transcription factors such as Runx2 and noncollagenous extracellular matrix molecules such as osteopontin and osteocalcin. One limitation of such studies is that they examine samples derived from craniosynostotic patients with sutures that have already fused, thus missing the dynamic osteogenic process of suture fusion. In this study, in situ hybridization was used to localize Runx2, osteopontin, and osteocalcin expression in the sagittal and posterior frontal sutures in mice (n = 20), before (day 13), during (days 23, 33, and 43), and after (day 53) the period of physiological posterior frontal suture fusion. The data demonstrated similar patterns of expression in fusing (posterior frontal) and nonfusing (sagittal) sutures. The expression of all three genes was primarily concentrated in the osteogenic fronts of both sutures and decreased with time. Notably, none of the three genes was expressed in the mesenchyme of either fusing or nonfusing sutures. The data suggest that the molecular signals leading to bone formation along the osteogenic fronts in fusing and nonfusing sutures are similar, raising the possibility that other factors, such as antagonists of osteogenesis, might have a role in maintaining suture patency

The human brain grows rapidly during the first 2 years of life. This growth generates tensile strain in the overlying dura mater and neurocranium. Interestingly, it is largely during this 2-year growth period that infants are able to reossify calvarial defects. This clinical observation is important because it suggests that calvarial healing is most robust during the period of active intracranial volume expansion. With a rat model, it was previously demonstrated that immature dura mater proliferates more rapidly and produces more osteogenic cytokines and markers of osteoblast differentiation than does mature dura mater. It was therefore hypothesized that mechanical strain generated by the growing brain induces immature dura mater proliferation and increases osteogenic cytokine expression necessary for growth and healing of the overlying calvaria. Human and rat (n = 40) intracranial volume expansion was calculated as a function of age. These calculations demonstrated that 83 percent of human intracranial volume expansion is complete by 2 years of age and 90 percent of Sprague-Dawley rat intracranial volume expansion is achieved by 2 months of age. Next, the maximal daily circumferential tensile strains that could be generated in immature rat dura mater were calculated, and the corresponding daily biaxial tensile strains in the dura mater during this 2-month period were determined. With the use of a three-parameter monomolecular growth curve, it was calculated that rat dura mater experiences daily equibiaxial strains of at most 9.7 percent and 0.1 percent at birth (day 0) and 60 days of age, respectively. Because it was noted that immature dural cells may experience tensile strains as high as approximately 10 percent, neonatal rat dural cells were subjected to 10 percent equibiaxial strain in vitro, and dural cell proliferation and gene expression profiles were analyzed. When exposed to mechanical strain, immature dural cells rapidly proliferated (5.8-fold increase in proliferating cell nuclear antigen expression at 24 hours). Moreover, mechanical strain induced marked up-regulation of dural cell osteogenic cytokine production; transforming growth factor-beta1 messenger RNA levels increased 3.4-fold at 3 hours and fibroblast growth factor-2 protein levels increased 4.5-fold at 24 hours and 5.6-fold at 48 hours. Finally, mechanical strain increased dural cell expression of markers of osteoblast differentiation (2.8-fold increase in osteopontin levels at 3 hours). These findings suggest that mechanical strain can induce changes in dura mater biological processes and gene expression that may play important roles in coordinating the growth and healing of the neonatal calvaria

The ability of immature animals to orchestrate successful calvarial ossification has been well described. This capacity is markedly attenuated in mature animals and humans greater than 2 years of age. Few studies have investigated biological differences between juvenile and adult osteoblasts that mediate successful osteogenesis. To identify possible mechanisms for this clinical observation, we investigated cellular and molecular differences between primary osteoblasts derived from juvenile (2-day-old) and adult (60-day-old) rat calvaria. Data demonstrated that juvenile osteoblasts contain a subpopulation of less differentiated cells as observed by spindle-like morphology and decreased osteocalcin production. Juvenile, compared with adult, osteoblasts showed increased proliferation and adhesion. Furthermore, following rhFGF-2 stimulation juvenile osteoblasts increased expression of collagen I alpha 1 (5-fold), osteopontin (13-fold), and osteocalcin (16-fold), compared with relatively unchanged adult osteoblasts. Additionally, juvenile osteoblasts organized and produced more matrix proteins and formed 41-fold more bone nodules. Alternatively, adult osteoblasts produced more FGF-2 and preferentially translated the high molecular weight (22 kDa) form. Although adult osteoblasts transcribed more FGF-R1 and juvenile osteoblasts transcribed more FGF-R2 at baseline levels, juvenile osteoblasts translated more FGF-R1 and -R2 and showed increased phosphorylation. Collectively, these findings begin to explain why juvenile, but not adult, osteoblasts successfully heal calvarial defects

The vertical reduction mammaplasty can be challenging to learn. In addition, first attempts to perform the vertical reduction mammaplasty can lead to inconsistent aesthetic results. The authors describe their transition from a traditional inverted-T reduction mammaplasty to a modified vertical reduction mammaplasty based on a technique described by Elizabeth Hall-Findlay. In their early cases using the Hall-Findlay technique, they noted several aesthetic complications. These problems included a persistent vertical dog-ear deformity at the nadir of the incision, a teardrop deformity of the nipple-areola complex, lateral deviation of the nipple, and lateral axillary fullness. They developed several modifications to the Hall-Findlay technique to correct the aesthetic deficiencies and to simplify further the vertical reduction method. The authors think their innovations facilitate the transition from a traditional inverted-T breast reduction to a successful vertical reduction mammaplasty technique

Recent studies have suggested that regionally differentiated dura mater regulates murine cranial suture fate by providing growth factors to the osteoblasts in the overlying suture complex. To determine if regionally differentiated dura mater is capable of effecting changes in osteoblast gene expression, an in vitro coculture system was established in which osteoblast-enriched cell cultures derived from neonatal rat calvaria were grown in serum-free media in the presence of dural cells derived from posterior frontal (PF) or sagittal (SAG) dural tissues, recapitulating the in situ relation between the underlying dura mater and the osteoblasts in the overlying cranial suture. In this study, the changes in osteoblast gene expression induced by signaling from regional dura mater were examined by analyzing total cellular RNA isolated from osteoblasts cocultured with PF or SAG dural cells. The expression of extracellular matrix molecules (alkaline phosphatase, bone sialoprotein, osteopontin, and osteocalcin) and the transcription factor Msx2 was assessed. Consistent with previous data, the findings demonstrate that osteoblasts cocultured with dural cells undergo changes in gene expression indicative of a more differentiated osteoblast. Additionally, the data suggest that regionally differentiated dura mater isolated from the PF suture enhances the expression of osteogenic genes to a greater extent than SAG suture-derived dural cells. These data support an osteoinductive role for suture-derived dural cells in vitro that may have implications for suture biology in vivo