Bolander, Mark E.

OBJECTIVE A bileaflet aortic valve (BAV) occurs in 1–2% of the population, making it the most common cardiovascular malformation. Patients with BAV have increased risk of thoracic ascending aortic aneurysm (TAA) and dissection compared to those with trileaflet aortic valves (TAV). We have evaluated the gene expression profile of the aorta of patients with BAV and TAV with TAA. METHODS Segment of aneurysmal tissue was collected from 12 patients with BAV (age 43–68) and 19 patients with TAV (age 37–79). RNA was extracted using the Invitrogen RNA extraction Kit. RNA that was of adequate quality by the Agilent Bioanalyzer was used in the Affymetrix protocol for the U133A gene chips probing for over 16,000 genes on each chip. Real‐time RT‐PCR was used to confirm the microarray results for a subset of genes. RESULTS Data filtering (using two different statistical methods) identified significant differences in gene expression between the BAV and TAV groups. The real time RT‐PCR independently confirmed that the expression levels of osteopontin and tenascin were consistently increased in TAA with TAV. CONCLUSIONS The results indicate that many genes are involved in a complicated manner in the pathogenesis of TAA. This study also demonstrates two basic gene expression profiles in the TAA, one characteristic of patients with BAV and the other of those with TAV. Variations in the molecular mechanisms underlying different types of TAA warrant further investigation. (This work was supported by an SS‐50 award to Dr. Thoralf Sundt from the Department of Surgery, Mayo Clinic)

Low-intensity pulsed ultrasound is a commonly prescribed therapy for delayed unions and nonunions after fractures. Several prospective double-blind clinical trials have shown that a 1.5-MHz ultrasound signal at a 200-s tone burst repeating at 1 kHz shortens the time to clinically evaluated healing by 30%. We have shown in vitro that pulsed ultrasound increases aggrecan gene expression and aggrecan production in chondrocytes, a key cell in the fracture healing process. Because the square wave pulsed 1.5-MHz signal produces radiation force vibration at 1 kHz, we tested the hypothesis that dynamic radiation force, not ultrasound, is responsible for the biological effect of the signal. Experiments showed that a 1-kHz acoustic square wave, simulating the radiation force caused by the clinically used signal, induced chondrogenesis similarly to pulsed ultrasound treatment in ATDC5 cells. These results may have implications for remote ultrasound or acoustic stimulation of different types of strain-sensitive cells in addition to those responsible for fracture healing.

The fact that modulated ultrasound at a pulse intensity of 160 mW/cm2 can induce fracture healing implies that cells that are sensitive to strain can feel and respond to this signal. A hypothesis that uses Nyborg inspired methods to investigate this effect will be presented along with data from a wide variety of simulations and experimental data. It appears that the response is mediated through the cytoskeleton and may be associated with interaction of the sound with focal adhesion complexes at one end of the skeletal fibers for high frequency responses and with the nuclear membrane at the other end of the fibers for low frequency responses. Although this hypothesis is nascent it is supported with experimental data from the literature.

In vitro studies have shown that treatment with 1.5 MHz ultrasound signal (160 mW/cm2) at a 200 μs tone burst repeating at 1 kHz increases proteoglycan synthesis in chondrocytes [J. Parvisi et al., J. Orthop. Res. 17, 488–494 (1999)]. It was hypothesized that a continuous 1 kHz signal would be similar to the pulsed 1.5 MHz signal in stimulating chondrocytes to produce proteoglycan, which may cause accelerated fracture healing. In vitro experiments were performed with ATDC5 cells, a chondrogenic clonal cell line, plated in 6-well plates for 3 to 7 days before receiving ultrasound treatments. Cells were treated with either 1.5 MHz pulsed signal or 1 kHz signal for 20 minutes per day for 9 to 11 days. The signals were calibrated so that the bottom of the 6-well plate moved 10 nm for each condition. After the final treatment, cell layers were stained with Alcian blue, which stains cartilage nodules providing a measure of chondrogenesis. Both 1.5 MHz and 1 kHz led to a highly significant increase in chondrogenesis compared to control. Quantitative image analysis of stained wells showed that treatments with either signal increased number of nodules 2.3-fold (p<0.02) and total area of nodules 3-fold (p<0.02) compared to controls.

Abstract We characterized gene expression in the reparative callus that formed after fracture of the rat femur. The callus was divided into regions of bone formation (hard callus) and cartilage formation (soft callus), and gene expression was examined separately in each region. Expression of extracellular matrix protein genes varied with the progression of repair and differed between hard and soft calluses. Messenger ribonucleic acids (mRNAs) for osteonectin, alkaline phosphatase, and type I procollagen were detected in the hard callus at maximal levels during endochondral ossification and bone remodeling (day 15) and at 50% maximal levels during intramembranous bone formation (day 7). Messenger RNAs for these proteins in the soft callus were detected at low levels during chondrogenesis (day 9) but increased to 80% of maximal levels with chondrocyte hypertrophy and mineralization of the cartilage matrix (day 13). Messenger RNAs for type II procollagen and proteoglycan core protein were detected at maximal levels in the soft callus during chondrogenesis (day 9). Osteocalcin gene expression was detected in the hard callus during endochondral ossification and remodeling but not during intramembranous bone formation or at any time in the soft callus. Osteonectin mRNA was detected in both the hard and soft callus throughout the entire course of fracture repair. Expression of cartilage and bone-related genes correlated with the temporal sequence of histologic changes, suggesting transcriptional regulation of gene expression during repair. Differences in gene expression between hard and soft callus and in each of these regions as repair progressed suggest local regulation of gene expression during cell differentiation and matrix synthesis.

Abstract Chondrogenesis can occur during a bone repair process, which is related to several growth factors. Transforming growth factor β1, (TGF-β1,) downregulates the expression of type II collagen by chondrocytes in vitro, but injection of TGF-β1, into the periosteum in vivo increases type II collagen mRNA levels and initiates chondrogenesis.(1) We examined the effect of TGF-β1, on collagen gene expression in a bovine periosteum-derived cell culture system to evaluate its direct effect on the periosteum. Cultured cells expressed alkaline phosphatase and collagen proα1,(I) and proα1(II) mRNAs. A low level of type II collagen synthesis was demonstrated by immunoprecipitation. TGF-β1, had no effect on periosteal cell proliferation. Expression of collagen proα1(I) mRNA did not change with TGF-β1, treatment, but alkaline phosphatase mRNA showed a dose-dependent decrease. Expression of collagen proα1(II) mRNA was stimulated 2.7-fold by TGF-β1. TGF-β1, also caused a 2.6-fold increase in type II collagen synthesis by immunoprecipitation. These findings indicate that TGF-β1 is an enhancer of the expression of the chondrocyte phenotype of the periosteal cells and suggest that TGF-β1, is important in initiating and promoting cartilage formation in vivo.

The number of osteoporotic hip fractures is projected to increase two to three fold in the next 30years. This will lead to increased mortality and nursing home placement with significant costs to our society. Although in clinical trials drug and hormone therapies have shown an important decrease in fracture risk there is a significant role of surgical implantation treatments to further reduce the risk in specific subpopulations. Strength testing was completed on four pairs of osteoporotic, two pairs of osteopenic, and two pairs of normal cadaver femurs. The fracturing was recorded with a video camera at 4000 frames per second. The specially designed test fixture was instrumented with three load cells to measure the femoral forces. Force and moment data were recorded simultaneously and correlated with the frames. The fracture force at the greater trochanter was used to characterize the strength of implanted∕nonimplanted osteoporotic femurs. All femurs began fracturing within about 60ms at a test speed of 100mm∕s. During the first 20ms, the relationship between force and displacement was nonlinear, but linear thereafter until the fracture occurs. The fracture events resolved completely within 10–20ms. The average strength increase of the implanted femurs was 79% compared to the nonimplanted controls. The simultaneous acquisition of fracture images and load data is a powerful tool to understand and characterize numerically the femoral fracture. This methodology allows the researcher to define the best metrics to compare competing reinforcement materials, implant designs and surgical procedures.

Abstract Pregnancy-associated plasma protein A (PAPP-A), a metalloproteinase that regulates IGF bioavailability in vitro through cleavage of inhibitory IGF-binding protein-4 (IGFBP-4), has been implicated in skeletal development and injury repair responses. However, direct in vivo data are lacking. In this study, we used PAPP-A knock-out (KO) mice to determine the role of PAPP-A in fracture repair. Stabilized mid-shaft fractures were produced in femurs of 3-month-old mice. At 14 days post-fracture, complete bony bridging of the fracture callus was seen radiographically in wild-type but not in PAPP-A KO mice. Histological examination 5 to 28 days post-fracture showed reductions in the amount of intramembranous bone formation, cartilage production, endochondral ossification and remodeling in PAPP-A KO compared with wild-type mice. However, fracture healing appeared similar in both groups at 42 days post-fracture when analyzed by histology. A similar degree of healing strength in wild-type and PAPP-A KO femurs was demonstrated by mechanical testing at 28 and 42 days post-fracture. Untreated cultures of day 5 fracture calluses from wild-type mice showed robust IGFBP-4 protease activity and IGF receptor phosphorylation, whereas fracture calluses from PAPP-A KO mice had no IGFBP-4 protease activity and reduced IGF receptor phosphorylation. These data demonstrate a marked delay in fracture healing in PAPP-A KO compared with wild-type mice, and suggest that PAPP-A is necessary in the early phases of the process for expeditious fracture repair. The ability of PAPP-A to enhance local IGF action may be an important mechanism for optimizing the fracture repair response.