KR-12

Polyetheretherketone (PEEK) is considered to be a promising bone implant material owing to its biocompatibility and elastic modulus, which is similar to that of natural bone. However, the clinical potential of PEEK is severely limited by its bioinertness, which leads to poor osseointegration, and the lack of antibacterial properties. In this study, the antimicrobial peptide, KR-12, was immobilized on the surface of PEEK implants with the assistance of polydopamine (PDA) to inhibit bacterial infection as well as to promote osteogenesis and osseointegration. Compared to unmodified PEEK, the PEEK with immobilized KR-12 showed significantly improved antibacterial activity against Staphylococcus aureus (ATCC 25923), both in vitro and in vivo. For the in vitro and in vivo evaluation of the osteogenic properties of modified PEEK, rat bone mesenchymal stem cells (rBMSCs) and a rat femoral defect model were used, respectively. The in vitro studies showed that compared to rBMSCs treated with unmodified PEEK, those treated with KR-12-coated PEEK exhibited improved adhesion, proliferation, and osteogenic differentiation. Moreover, micro-computed tomography and histological analysis suggested that the KR-12 coating promoted osteointegration in vivo in rat femurs. Taken together, these results suggest that the KR-12 coating could improve the antibacterial ability of pure or PDA-coated PEEK against Staphylococcus aureus (ATCC 25923), both in vitro and in vivo. Overall, KR-12 combined with the PDA film coating synergistically induced osteogenic effects both in vitro and in vivo. Thus, the surface-modified material, which exhibits both anti-bacterial and osteointegration properties, shows considerable potential for use as an orthopedic implant.

KR-12-a5 is an analogue of the antimicrobial peptide KR-12. Both of these two agents can play key effects in the treatment of infections such as osteomyelitis. Our previous work demonstrated that the osteogenic differentiation of human bone marrow mesenchymal stem cells (HBMSCs) can be enhanced by KR-12. The present study investigated if KR-12-a5 could reverse the adverse effects of lipopolysaccharides (LPS) on HBMSC osteogenesis and the involved molecular mechanisms. We observed the proliferation, cell cycle, and apoptosis of HBMSCs in the presence of KR-12-a5 by a cell counting kit-8 assay and flow cytometry. The osteogenic differentiation of HBMSCs was studied by alkaline phosphatase, Alizarin Red staining, and quantitative assays. Osteogenic differentiation marker levels were detected using real-time quantitative PCR analysis, which demonstrated that KR-12-a5 treatment reversed the inhibition of osteogenesis. Western blot analysis indicated that LPS-activated P38 mitogen-activated protein kinase (MAPK) signaling was inhibited and BMP/Smad pathway was reactivated after KR-12-a5 treatment under induced osteogenic conditions. Furthermore, flow cytometry results demonstrated that KR-12-a5 relieved LPS-induced oxidative stress. Combining the LPS-treated mouse model results, we proved that KR-12-a5 reversed the adverse effects of LPS on HBMSC osteogenic differentiation by influencing the BMP/Smad and P38 MAPK signaling pathways.

Considering the increased resistance to antibiotics in the clinic and the ideal antibacterial properties of KR‑12, the effects of KR‑12‑a6, an important analogue of KR‑12, on the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) were investigated. Osteogenic differentiation‑associated experiments were conducted in hBMSCs, and KR‑12‑a6 was used as an additional stimulating factor during osteogenic induction. Quantitative analysis of alkaline phosphatase (ALP) and alizarin red staining, and reverse transcription‑quantitative PCR analysis of the expression of osteogenesis‑associated genes were performed to determine the effects of KR‑12‑a6 on the osteogenic differentiation of hBMSCs. LDN‑212854 was selected to selectively suppress BMP/SMAD signaling. Western blotting was performed to investigate the underlying mechanisms. The intensity of ALP and alizarin red staining gradually increased with increasing KR‑12‑a6 concentrations. KR‑12‑a6 induced the strongest staining at 40 µg/ml, whereas 60 µg/ml and 80 µg/ml concentrations did not further increase the intensity of staining. The mRNA expression levels of RUNX2 and ALP increased in a dose‑dependent manner as early as 3 days post‑KR‑12‑a6 treatment. The mRNA expression of COL1A1, BSP and BMP2 exhibited significant upregulation from day 7 post‑KR‑12‑a6 treatment. In contrast, the mRNA levels of OSX, OCN and OPN were enhanced dramatically at day 14 following KR‑12‑a6 stimulation. Additionally, KR‑12‑a6 significantly promoted the phosphorylation of Smad1/5. Furthermore, LDN‑212854 suppressed the activation of Smad1/5 and inhibited the upregulation of several osteogenic differentiation‑associated genes in KR‑12‑a6‑treated hBMSCs. KR‑12‑a6 promoted the osteogenic differentiation of hBMSCs via BMP/SMAD signaling.