Kenton Fong

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.

Increased transcriptional response to mechanical strain in keloid fibroblasts due to increased focal adhesion complex formation

Clinicians have observed that keloids preferentially form in body areas subject to increased skin tension. We hypothesized a difference exists in the transcriptional response of keloid fibroblasts to mechanical strain compared with normal fibroblasts. Normal and keloid fibroblasts were seeded in a device calibrated to deliver a known level of equibiaxial strain. We examined the transcriptional response of TGF‐β isoforms and collagen Iα, genes differentially expressed in keloids. Keloid fibroblasts produced more mRNA for TGF‐β1, TGF‐β2, and collagen Iα after mechanical strain compared to normals, and this was correlated with protein production. Inhibiting the major mechanical signal transduction pathway with the ERK inhibitor, U0126, blocked upregulation of gene expression. In addition, keloid fibroblasts formed more focal adhesion complexes as measured by immunofluorescence for focal adhesion kinase, integrin β1, and vinculin. Finally, there is increased activation of focal adhesion kinase when we detected the phosphorylated form of focal adhesion kinase with immunofluorescence and immunoblotting. In summary, keloid fibroblasts have an exaggerated response to mechanical strain compared to normal fibroblasts leading to increased production of pro‐fibrotic growth factors. This may be one molecular mechanism for the development of keloids. J. Cell. Physiol. 206: 510–517, 2006.

Equibiaxial tensile strain affects calvarial osteoblast biology.

Mechanical tensile strain is believed to play an important role in regulating calvarial morphogenesis. To better understand the effects of mechanical strain on pathologic calvarial growth, we applied 10% constant equibiaxial tensile strain to neonatal rat calvarial osteoblast cultures and examined cellular proliferation, cytokine production, and extracellular matrix molecule expression. Mechanical strain markedly increased osteoblast proliferation as demonstrated by increased proliferating cell nuclear antigen (PCNA) protein. In addition, both transforming growth factor-&bgr;1 (TGF-&bgr;1) mRNA expression and fibroblast growth factor-2 (FGF-2) protein production were increased with exposure to strain. Moreover, mechanical strain induced expression of the extracellular matrix molecule collagen I&agr;I. To further explore the relationship between mechanotransduction, osteogenesis, and angiogenesis, we examined the effect of mechanical strain on calvarial osteoblast expression of vascular endothelial growth factor (VEGF). Interestingly, we found that mechanical strain induced a rapid (within 3 hrs) increase in osteoblast VEGF expression. These data suggest that constant equibiaxial tensile strain-induced mechanotransduction can influence osteoblasts to assume an “osteogenic” and “angiogenic” phenotype, and these findings may have important implications for understanding the mechanisms of pathologic strain-induced calvarial growth.

Tools and Techniques for Craniofacial Tissue Engineering

Craniofacial surgery is an important conduit for tissue-engineering applications. As interdisciplinary collaborations improve, we can expect to see remarkable progress in de novo tissue synthesis, replacement, and repair. Ultimately, we may one day find that gene-modified cell-based tissue-engineering strategies will succeed todays reconstructive strategies. In this review, we highlight the major gene- and cell-based preclinical tools and techniques that are currently being developed to solve common craniofacial problems.

Mechanical strain affects dura mater biological processes: implications for immature calvarial healing.

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-&bgr;1 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.

Regional dura mater differentially regulates osteoblast gene expression.

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.

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.

Frustrative nonreward and pituitary–adrenal activity in squirrel monkeys

Little is known about frustration-induced changes in stress physiology in humans and nonhuman primates. Here we assess in two experiments with squirrel monkeys plasma levels of pituitary-adrenal stress hormones in conditions designed to provoke frustrative nonreward. In the first experiment 18 prepubertal monkeys were trained to feed from one of eight sites, and then tested without food at any of the sites. These monkeys responded with significant increases in cortisol and adrenocorticotropic hormone (ACTH). In the second experiment 18 adult monkeys were trained to feed from one of eight sites, and then tested after food was moved to a different foraging site. Nine monkeys found food at the relocated site, discontinued foraging at the previously baited site, and responded with decreases in cortisol. The other nine monkeys failed to find the relocated site, initially increased their visits to the previously baited site, and responded with elevations in cortisol and ACTH. In keeping with comparable findings in rats, our observations indicate that frustrative nonreward elicits ACTH-stimulated secretion of cortisol in primates.

New strategies for craniofacial repair and replacement: a brief review.

Craniofacial anomalies can severely affect the appearance, function, and psychosocial well being of patients; thus, tissue engineers are developing new techniques to functionally and aesthetically rebuild craniofacial structures. In the past decade, there have been tremendous advances in the field of tissue engineering that will substantially alter how surgeons approach craniofacial reconstruction. In this brief review, we highlight some of the preclinical recombinant protein, gene transfer, and cell-based strategies currently being developed to augment endogenous tissue repair or create structures for replacement. In addition, we discuss the importance of studying endogenous models of tissue induction and present some of the current in vitro and in vivo approaches to growing complex tissues/organs for craniofacial reconstruction.

The SNaP System: Biomechanical and Animal Model Testing of a Novel Ultraportable Negative-Pressure Wound Therapy System

BACKGROUND Negative-pressure wound therapy is traditionally achieved by attaching an electrically powered pump to a sealed wound bed and applying subatmospheric pressure by means of gauze or foam. The Smart Negative Pressure (SNaP) System (Spiracur, Inc., Sunnyvale, Calif.) is a novel ultraportable negative-pressure wound therapy system that does not require an electrically powered pump. METHODS Negative pressure produced by the SNaP System, and a powered pump, the wound vacuum-assisted closure advanced-therapy system (Kinetic Concepts, Inc., San Antonio, Texas), were compared in vitro using bench-top pressure sensor testing and microstrain and stress testing with pressure-sensitive film and micro-computed tomographic scan analysis. In addition, to test in vivo efficacy, 10 rats underwent miniaturized SNaP (mSNaP) device placement on open wounds. Subject rats were randomized to a system activation group (approximately -125 mmHg) or a control group (atmospheric pressure). Wound measurements and histologic data were collected for analysis. RESULTS Bench measurement revealed nearly identical negative-pressure delivery and mechanical strain deformation patterns between both systems. Wounds treated with the mSNaP System healed faster, with decreased wound size by postoperative day 7 (51 percent versus 12 percent reduction; p < 0.05) and had more rapid complete reepithelialization (21 days versus 32 days; p < 0.05). The mSNaP device also induced robust granulation tissue formation. CONCLUSIONS The SNaP System and an existing electrically powered negative-pressure wound therapy system have similar biomechanical properties and functional wound-healing benefits. The potential clinical efficacy of the SNaP device for the treatment of wounds is supported.