Nanofibrous scaffolds incorporating PDGF-BB microspheres induce chemokine expression and tissue neogenesis in vivo

Abstract

Platelet-derived growth factor (PDGF) exerts multiple cellular effects that stimulate wound repair in multiple tissues. However, a major obstacle for its successful clinical application is the delivery system, which ultimately controls the in vivo release rate of PDGF. Polylactic-co-glycolic acid (PLGA) microspheres (MS) in nanofibrous scaffolds (NFS) have been shown to control the release of rhPDGF-BB in vitro. In order to investigate the effects of rhPDGF-BB release from MS in NFS on gene expression and enhancement of soft tissue engineering, rhPDGF-BB was incorporated into differing molecular weight (MW) polymeric MS. By controlling the MW of the MS over a range of 6.5 KDa-64 KDa, release rates of PDGF can be regulated over periods of weeks to months in vitro. The NFS-MS scaffolds were divided into multiple groups based on MS release characteristics and PDGF concentration ranging from 2.5-25.0 microg and evaluated in vivo in a soft tissue wound repair model in the dorsa of rats. At 3, 7, 14 and 21 days post-implantation, the scaffold implants were harvested followed by assessments of cell penetration, vasculogenesis and tissue neogenesis. Gene expression profiles using cDNA microarrays were performed on the PDGF-releasing NFS. The percentage of tissue invasion into MS-containing NFS at 7 days was higher in the PDGF groups when compared to controls. Blood vessel number in the HMW groups containing either 2.5 or 25 microg PDGF was increased above those of other groups at 7d (p<0.01). Results from cDNA array showed that PDGF strongly enhanced in vivo gene expression of the CXC chemokine family members such as CXCL1, CXCL2 and CXCL5. Thus, sustained release of rhPDGF-BB, controlled by slow-releasing MS associated with the NFS delivery system, enhanced cell migration and angiogenesis in vivo, and may be related to an induced expression of chemokine-related genes. This approach offers a technology to accurately control growth factor release to promote soft tissue engineering in vivo.

Figure 1. PDGF-containing microspheres (MS) in nanofibrous scaffolds (NFS) stimulates tissue invasion in vivo.

Left panel at low magnification (2X) shows the tissue completely penetrated the entire NFS for the groups with PDGF encapsulated in PLGA microspheres, while other groups show a portion of scaffold occupied by ingrown tissue. In addition, the scaffolds were enlarged by the penetrated tissues in the groups with high dose PDGF encapsulated in PLGA microspheres. Right panel at high magnification (10X) demonstrated that most of the cells in the penetrated tissues were fibroblast-like cells and lymphocyte-like cells. On the scaffold pore surfaces multiple macrophage-like cells were seen. Bars indicate 0.5 mm.

Figure 2. Histomorphometric analysis results of PDGF-inducing tissue penetration and neogenesis.

A is percentage of tissue penetration. The percentage of tissue penetration within the groups of PDGF encapsulated in PLGA MS was statistically greater than that for the other groups with the exception of the 25 µg PDGF coating group. The percentage of tissue penetration in the 25 µg PDGF coating group is significantly greater than for the NFS only group. B demonstrates the areas for all groups. The areas in the groups for PDGF encapsulated in PLGA MS are statistically greater than those in the groups for NFS only, NFS containing empty, slow, and fast release PLGA microspheres, and 2.5 µg PDGF coating group. The groups with 2.5 µg PDGF encapsulated in slow release PLGA microspheres and 25 µg PDGF encapsulated in slow and fast release PLGA microspheres have larger areas than the groups with 25 µg PDGF coating and 2.5 µg PDGF encapsulated in fast release PLGA microspheres. The largest area was found in the group of 25 µg PDGF encapsulated in fast release PLGA microspheres. * indicates p<0.01, ** indicates p<0.05

Figure 3. Nanofibrous scaffolds with PDGF microspheres promote vasculogenesis in vivo.

Left panel is low magnification (10×), right panel is high magnification (40×). Positive Factor VIII stained blood vessels were located in the central regions of the pores within penetrated tissues. The blood vessels also permeated through the inter-openings between each pore. The group with 25 µg PDGF encapsulated in slow release PLGA microspheres had measurably more vascularization.

Figure 4. PDGF-containing microspheres in nanofibrous scaffolds increases angiogenesis in vivo.

The blood vessel number was measured within each group using sections immunostained with Factor VIII antibody. Blood vessel number within both PDGF encapsulated, slow release groups was significantly greater that that in other groups. The blood vessel number of PDGF-encapsulated in the fast release PLGA group showed no difference from that in the NFS-only group. * indicates p<0.01

Figure 5. Chemokine gene induction in PDGF encapsulated microspheres in vivo.

A: CXCL1 gene expression, B: CXCL2 gene expression, C: CXCL5 gene expression, D: CCL21b gene expression. * indicates p<0.01, ** indicates p<0.05.