Schematic representation of BMP-2 adsorption to ceramic scaffolds with the properties that generally have increased the mass of BMP-2 adsorbed to its surface.

Schematic representation of BMP-2 incorporation into a dried polymer network that swells to form a hydrogel and properties that generally increase the mass of BMP-2 incorporated into the hydrogel network.

Results of studies that compared rhBMP-2 release from acidic and basic gelatin hydrogels in vitro. A) rhBMP-2 release was faster from acidic gelatin hydrogel disks compared to basic gelatin hydrogel disks in PBS with permission. rhBMP-2 release was also faster from acidic gelatin hydrogel microparticles (B) crosslinked with 10 or 40mM glutaraldehyde at low or high rhBMP-2 doses compared to basic gelatin hydrogel microparticles (C) with permission.

Influence of convective fluid flow on growth factor release in vitro. A) Mechanical strain (top) triggered the release of VEGF from alginate hydrogels over the course of 6 loading cycles (bottom) with permission. B) Schematic representation of dynamic hydrogels in which specific biochemical ligands induced protein-conformational changes, hydrogel volume decreases, and growth factor release with permission. C) Temporal control over BMP-2 release from dynamic hydrogel networks was controlled by varying the timing that hydrogels underwent trifluoperazine (TFP)-induced volume decreases with permission.

The effect of rhBMP-2 on the expression of desmin and alkaline phosphatase by the mc13 cells. Desmin staining of freshly isolated mc13 clones (A) showed that all of these cells express desmin. The phase-contrast view (B) is shown to demonstrate that a high percentage of the cells were desmin positive. Mc13 cells were incubated in growth media containing 200 ng/ml rhBMP-2 for 6 d. Cells were then stained for desmin expression, and percent desmin positive cells calculated by visualization with immunofluorescence. As a control, mc13 cells were grown in parallel without addition of rhBMP-2. When grown without rhBMP-2, mc13 cells remained uniformly (90每100%) desmin positive (C). With exposure to rhBMP-2, there is a significant decrease (*P < 0.05) in relative number of desmin positive cells (30每40%) within 6 d (C). The npmc and mc13 cells were also analyzed for expression of alkaline phosphatase after 6 d of growth with and without exposure to 200 ng/ml rhBMP-2. With rhBMP-2 stimulation, mc13 cells show a >600-fold increase in alkaline phosphatase activity than nonstimulated cells (D). The npmc show only minimal alkaline phosphatase activity with or without rhBMP-2 (D). *, Indicates a significant difference using the t test (P < 0.05). Bar, 100 µm.

Morphologic change and expression of osteocalcin by mc13 cells with exposure to rhBMP-2. Mc13 cells were incubated in growth media without rhBMP-2 for 6 d. When cells became >50% confluent, they began to fuse and form multinucleated myotubes (A). When mc13 cells were incubated in growth media containing 200 ng/ml rhBMP-2, cells remained mononucleated and did not fuse (B). When cells reached >90% confluency without rhBMP-2, almost all the cells fused to form myotubes (C). With rhBMP-2, when cells reached >90% confluency, round, hypertrophic cells began to appear in the culture (D, arrows). These round, hypertrophic cells were highly positive for osteocalcin expression (E, arrows). Bar, 50 µm.

Distribution of BMP-2 in human sclera fibroblasts in vitro using indirect immunofluorescence. FITC marked the secondary antibody (green; A), and PI dyed the nucleus (red; B). The first (A) and second images (B) are combined to form the third image shown (C). BMP-2 is localized in the cytoplasm and weakly in the nucleus of HSF (A-C). Magnification: 400x.

The changed pattern of human sclera fibroblasts incubated with rhBMP-2. The HSFs showed only a sparse and vortex pattern before incubation (A) whereas incubation with 100 ng/ml rhBMP-2 changed the HSFs into the intensive and polygonal cells (B). Inverted phase contrast microscope, original magnification 100 x.

Time-dependent effects of rhBMP-2 on the cell proliferation of primary cultured human sclera fibroblasts. Cell proliferation was determined by MTT assay. The optical density value at A490 and the mean㊣SD is provided for each time point (n=8). The cells were incubated with no rhBMP-2 or 100ng/ml rhBMP-2 for seven days. A significant difference was seen in cell proliferation between 100 ng/ml rhBMP-2 treatment and control after four days. The asterisk indicates that P<0.05 when compared with the control without rhBMP-2.

Distribution of BMP-2 in human sclera by indirect immunofluorescence. FITC marked the secondary antibody (green; A) and Hoechst33342 dyed the nucleus (blue; B). The first (A) and second images (B) are combined to form the third image shown (C). BMP-2 is localized in the cytoplasm of HSF and extracellular matrices(A-C). Magnification: 400x.

Effect of rhBMP-2 on mRNA expression levels of MMP-2 and TIMP-2 in human sclera fibroblasts. Ethidium-bromide agarose gels indicated the level of 汕-actin message relative toMMP-2 (A) and TIMP-2 (B) levels from total RNA. Bar graphs revealed changes in mRNA expression where values were normalized to beta-actin values and expression was state as a ratio of optical dencity. The double asterisk means that the semi-quantitative RT-PCR revealed significant changes for MMP-2 and TIMP-2 with varying concentration of rhBMP-2.

Distribution of BMP-2 in human sclera by indirect immunofluorescence. FITC marked the secondary antibody (green; A) and Hoechst33342 dyed the nucleus (blue; B). The first (A) and second images (B) are combined to form the third image shown (C). BMP-2 is localized in the cytoplasm of HSF and extracellular matrices(A-C). Magnification: 400x.