Abstract
Current clinically used delivery methods for bone morphogenetic proteins (BMPs) are collagen based and require large concentrations that can lead to dangerous side effects. Fibrin hydrogels can serve as osteoinductive bone substitute materials in non-load bearing bone defects in combination with BMPs. Two strategies to even further optimize such a fibrin based system include employing more potent BMP heterodimers and engineering growth factors that can be covalently tethered to and slowly released from a fibrin matrix. Here we present an engineered BMP-2/BMP-7 heterodimer where an N-terminal transglutaminase substrate domain in the BMP-2 portion provides covalent attachment to fibrin together with a central plasmin substrate domain, a cleavage site for local release of the attached BMP-2/BMP-7 heterodimer under the influence of cell-activated plasmin. In vitro and in vivo results revealed that the engineered BMP-2/BMP-7 heterodimer induces significantly more alkaline phosphatase activity in pluripotent cells and bone formation in a rat calvarial model than the engineered BMP-2 homodimer. Therefore, the engineered BMP-2/BMP-7 heterodimer could be used to reduce the amount of BMP needed for clinical effect.
Highlights
While bone autograft is the gold standard in healing critical-sized bone defects, there are significant drawbacks, including lack of supply and morbidity at the harvest site
Through the use of infrared secondary antibodies in a Western blot detecting bone morphogenetic proteins (BMPs)-2 and BMP-7, the heterodimer was confirmed by the overlap of the BMP-2 and BMP-7 signal (Figure 1a–c)
An SDS-PAGE verified that the TG-BMP-2/BMP-7 heterodimer is pure, showing only a single band (Figure 1d)
Summary
While bone autograft is the gold standard in healing critical-sized bone defects, there are significant drawbacks, including lack of supply and morbidity at the harvest site. Since the growth factors are tethered to the material, immobilization further limits side effects due to diffusion to undesired areas This strategy enables spatial growth factor delivery and replicates physiological matrix interactions [11,12]. Successful bone healing in these fibrin delivery systems with BMP-2 non-covalently incorporated, required high amounts of BMP-2 [22]. To address this issue with the delivery and retention of BMPs, we developed a covalent BMP binding strategy that utilizes an engineered BMP with an amino acid sequence that can be enzymatically attached to fibrin by using transglutaminase Factor XIII, an enzyme naturally found in blood clots [22]. We characterize these engineered TG-BMP-2 and TG-BMP-2/BMP-7 growth factors in vitro and study their in vivo efficacy with a rat calvarial defect model in conjunction with a fibrin matrix
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