Adult subcutaneous fat tissue is an abundant source of multipotent cells. Previous studies from our laboratory have shown that, in vitro, adipose-derived adult stem (ADAS) cells express bone marker proteins including alkaline phosphatase, type I collagen, osteopontin, and osteocalcin and produce a mineralized matrix as shown by alizarin red staining. In the current study, the ADAS cell ability to form osteoid in vivo was determined. ADAS cells were isolated from liposuction waste of three individual donors and expanded in vitro before implantation. Equal numbers of cells (3 x 10(6)) were loaded onto either hydroxyapatite/tricalcium phosphate (HA-TCP) cubes or the collagen/HA-TCP composite matrix, Collagraft, and then implanted subcutaneously into SCID mice. After 6 weeks, implants were removed, fixed, and demineralized and sectioned for hematoxylin and eosin staining. Osteoid formation was observed in 80% of HA-TCP implants loaded with ADAS cells. Only 20% of Collagraft implants were positive for the presence of osteoid matrix. Whereas 100% of HA-TCP implants loaded with hFOB 1.19 cells formed osteoid, Collagraft loaded with hFOB 1.19 cells displayed a high degree of adipose tissue within the matrix. Immunostaining of serial sections for human nuclear antigen demonstrated that the osteoid contained human cells. Osteoid formation was not observed in control HA-TCP or Collagraft matrices implanted without cells. In summary, the data demonstrate the ability of ADAS cells to form osteoid matrix in vivo. Because of their abundance and accessibility, ADAS cells may prove to be a novel cell therapeutic for bone repair and regeneration.

Exogenous administration of vascular endothelial growth factor (VEGF) improves long-term viability of myocutaneous flaps. However, endogenous expression of this substance in flaps following ischemia-reperfusion injury has not been reported previously. Endogenous production of VEGF was measured in myocutaneous pig latissimus dorsi flaps after ischemia-reperfusion injury. Latissimus dorsi myocutaneous flaps (15 x 10 cm) were simultaneously elevated bilaterally in six Yorkshire-type male pigs (25 kg). Before elevation, three flap zones (5 x 10 cm) were marked according to their distance from the vascular pedicle. After isolation of the vascular pedicle, ischemia-reperfusion injury was induced in one flap by occlusion of the thoracodorsal artery and vein for 4 hours, followed by 2 hours of reperfusion. The contralateral flap served as a control. Perfusion in each zone was monitored by laser Doppler flowmetry at baseline, during ischemia, and during reperfusion. At the end of the protocol, skin and muscle biopsies of each flap zone and adjacent tissues were obtained for later determination of VEGF protein levels. VEGF concentrations were quantified using the Quantikine human VEGF immunoassay. Skin perfusion was similar among all flap zones before surgery. Flow fell in all flaps immediately after flap elevation. After 4 hours of ischemia, blood flow in the ischemic flaps was significantly decreased (p< 0.05) compared with nonischemic control flaps. After 2 hours of reperfusion, flow in ischemic flap skin recovered to levels similar to those in control flaps. VEGF protein concentrations in muscle tissue exceeded concentrations in skin and decreased from zones 2 to 3 in control and ischemic flaps. No significant differences in VEGF concentrations between ischemic and control muscle zones were observed. However, the concentration of VEGF in all muscle zones was significantly higher (p < 0.05) than muscle adjacent to the flap. Concentrations in skin zones 1 and 2 were significantly higher (p < 0.05) in ischemic flaps than in control flaps, but levels in zone 3 (most ischemic flaps) showed no significant difference.

Causative molecular mechanisms accounting for the potential link between Chlamydia pneumoniae and atherosclerosis are unknown. Formalin and heat-inactivated C. pneumoniae activated the transcription factor nuclear factor (NF)-kappaB in cultured porcine endothelium and up-regulated the expression of E-selectin messenger RNA and protein. This up-regulation was abolished by an IkappaB super-repressor, an NF-kappaB-specific inhibitor. Live bacteria are not necessary for the activation of endothelial NF-kappaB, and C. pneumoniae may contribute to atherogenesis without active infection.

The mitochondria and actin fibers of 3T3 fibroblasts cultured on microcarriers in spinner flasks were visualized using fluorescent stains. In contrast to cells grown on planar surfaces under static or steady laminar flow conditions, cells exposed to higher levels of turbulent agitation do not form actin stress fibers. Greater agitation also leads to a more diffuse appearance of the mitochondria and a wider distribution of them throughout the cytoplasm. This response may indicate damaged mitochondria, as similar results have been reported for chemical toxins.

This study assessed three in vitro techniques designed to measure the thrombogenicity of vascular grafts. All techniques immersed vascular grafts in rotating blood. In the gravimetric analysis, the weight of adherent thrombi was recorded at 2 min intervals for 20 min. In the torque analysis, a microviscometer continuously recorded the amount of torque developed as the graft rotated for 20 min. In the thrombin analysis, the blood sample was analyzed for fibrinopeptide A production indicating fibrinogen cleavage. Expanded polytetrafluoroethylene grafts were treated by removal of air nuclei (denucleation), binding of heparin, or binding of polyethylene oxide (PEO). The gravimetric analysis determined that the time at which each group experienced clot initiation was as follows: control after 6 min, denucleation after 14 min, heparin after 18 min, and PEO after 10 min. Similarly, in the torque analysis all treatment groups significantly delayed the initial increase in torque from 8.0 min for control to 12.5 min for denucleation (P < .01), > 20 min for heparin (P < .01), and 12 min for PEO (p < .05). The thrombin analysis determined that coagulation activity was reduced relative to control at 12 min with the denucleation group (P < .05) and heparin group (P< .01) and at 18 min with all treatment groups (P < .01). The similarity of results among the techniques increases confidence that each measurement accurately predicts in vitro thrombogenicity.