Protease-Triggered Release of Stabilized CXCL12 from Coated Biomaterials for Improved Implant Integration and Wound Healing

Abstract

Biomaterials are designed to improve impaired healing of injured tissue. To accomplish better cell integration and to accelerate revascularization, it is suggested to coat implant surfaces with bio-functional proteins. Here, a mussel-derived surface-binding peptide was coupled to the chemokine CXCL12 (stromal cell-derived factor 1α), a chemokine that activates the CXCR4 and consequently recruits tissue specific stem and progenitor cells. CXCL12 variants with either non-releasable or protease-mediated release properties were designed and compared. Whereas CXCL12 was stabilized at the N-terminus for protease resistance, a C-terminal linker was designed that allowed specific cleavage-mediated release by matrix metalloproteinase 9 and 2, as both enzymes are frequently found in wound fluid. These surface adhesive CXCL12 derivatives were produced by expressed protein ligation and subsequent refolding. Functionality of the modified chemokines was assessed by inositol phosphate accumulation assay and cell migration assays. Increased migration of keratinocytes as well as the activation of the downstream signaling cascade by the PI3K-Akt and Ras-Raf-Erk1/2 pathway have been demonstrated. Immobilization to biomaterials was studied for titanium surfaces and PCL-co-LC scaffolds. Osteoblast-like cells spread significantly more on titanium, coated with the modified chemokine, indicating increased cell adhesion. Accelerated wound closure in an ex vivo wound healing assay has been shown with coated PCL- co -LC scaffolds on porcine skin grafts. After 24 h, a significantly improved CXCL12-specific growth stimulation of the epithelial tips has been observed. The presented data emphasizes a successful translation of protein-coated biomaterials for bone as well as for skin regeneration.