Basic Fibroblast Growth Factor Fused with Tandem Collagen-Binding Domains from Clostridium histolyticum Collagenase ColG Increases Bone Formation

Abstract

Basic fibroblast growth factor 2 (bFGF) accelerates bone formation during fracture healing. Because the efficacy of bFGF decreases rapidly following its diffusion from fracture sites, however, repeated dosing is required to ensure a sustained therapeutic effect. We previously developed a fusion protein comprising bFGF, a polycystic kidney disease domain (PKD; s2b), and collagen-binding domain (CBD; s3) sourced from the Clostridium histolyticum class II collagenase, ColH, and reported that the combination of this fusion protein with a collagen-like peptide, poly(Pro-Hyp-Gly)10, induced mesenchymal cell proliferation and callus formation at fracture sites. In addition, C. histolyticum produces class I collagenase (ColG) with tandem CBDs (s3a and s3b) at the C-terminus. We therefore hypothesized that a bFGF fusion protein containing ColG-derived tandem CBDs (s3a and s3b) would show enhanced collagen-binding activity, leading to improved bone formation. Here, we examined the binding affinity of four collagen anchors derived from the two clostridial collagenases to H-Gly-Pro-Arg-Gly-(Pro-Hyp-Gly)12-NH2, a collagenous peptide, by surface plasmon resonance and found that tandem CBDs (s3a-s3b) have the highest affinity for the collagenous peptide. We also constructed four fusion proteins consisting of bFGF and s3 (bFGF-s3), s2b-s3b (bFGF-s2b-s3), s3b (bFGF-s3b), and s3a-s3b (bFGF-s3a-s3b) and compared their biological activities to those of a previous fusion construct (bFGF-s2b-s3) using a cell proliferation assay in vitro and a mouse femoral fracture model in vivo. Among these CB-bFGFs, bFGF-s3a-s3b showed the highest capacity to induce mesenchymal cell proliferation and callus formation in the mice fracture model. The poly(Pro-Hyp-Gly)10/bFGF-s3a-s3b construct may therefore have the potential to promote bone formation in clinical settings.

Effect of Single Injection of Recombinant Human Bone Morphogenetic Protein-2-Loaded Artificial Collagen-Like Peptide in a Mouse Segmental Bone Transport Model

“…Collagen materials have been used for the sustained release of rhBMP-2 in clinical settings [9][10][11]. We previously reported that collagen-like polypeptide poly(POG)n is superior to animalderived collagen with regard to thermal stability and lack of pathogenicity and is a useful carrier which retains bFGF well [17,18]. Our preliminary study showed that 2.0 μg of rhBMP-2 without poly(POG)n failed to promote bone formation in a mice bone defect model.…”

“…We previously reported that the artificial collagen-like peptide poly(POG)n was a useful carrier for growth factors. Injection of bFGF-loaded poly(POG)n into fracture sites stimulated periosteal bone formation in mouse fracture models [17,18]. In the present study, we investigated whether a single injection of rhBMP-2-loaded artificial collagen-like peptide gel (rhBMP-2/ACG) accelerates consolidation at the bone defect site and bone union at the docking site in a mouse SBT model.…”

Acceleration of bone union by in situ-formed hydrogel containing bone morphogenetic protein-2 in a mouse refractory fracture model

“…The femur fracture model was generated using 9-week-old C57BL/6 J mice [ 19 ]. The mice were maintained at Nippon Charles River Laboratories (Kanagawa, Japan) in a semi-barrier system under controlled temperature (23 ± 28 °C), humidity (55 ± 10%), and lighting (12-h light/dark cycle), and received standard rodent chow (CRF-1; Oriental Yeast, Tokyo, Japan).…”

Growth Factor Engineering Strategies for Regenerative Medicine Applications

“…For example, a bacterial collagen-binding domain (CBD), was fused to fibroblast growth factor-2 (FGF-2), allowing improved bone formation in a spinal fusion model (Inoue et al, 2017). In another study, CBD-fused FGF-2 showed the ability to induce significantly higher mesenchymal cell proliferation and callus formation in a mice fracture model compared to wild-type FGF-2 (Sekiguchi et al, 2018). Similarly, a CBD-fused hepatocyte growth factor (HGF) delivered via hydrogel improved recovery after spinal cord injury in mice compared to wild-type HGF (Yamane et al, 2018).…”