Antimicrobial Peptide Combined with BMP2-Modified Mesenchymal Stem Cells Promotes Calvarial Repair in an Osteolytic Model

Abstract

Repair and regeneration of inflammation-induced bone loss remains a clinical challenge. LL37, an antimicrobial peptide, plays critical roles in cell migration, cytokine production, apoptosis, and angiogenesis. Migration of stem cells to the affected site and promotion of vascularization are essential for tissue engineering therapy, including bone regeneration. However, it is largely unknown whether LL37 affects mesenchymal stem cell (MSC) behavior and bone morphogenetic protein 2 (BMP2)-mediated bone repair during the bone pathologic remodeling process. By performing in vitro and in vivo studies with MSCs and a lipopolysaccharide (LPS)-induced mouse calvarial osteolytic bone defect model, we found that LL37 significantly promotes cell differentiation, migration, and proliferation in both unmodified MSCs and BMP2 gene-modified MSCs. Additionally, LL37 inhibited LPS-induced osteoclast formation and bacterial activity in vitro. Furthermore, the combination of LL37 and BMP2 markedly promoted MSC-mediated angiogenesis and bone repair and regeneration in LPS-induced osteolytic defects in mouse calvaria. These findings demonstrate for the first time that LL37 can be a potential candidate drug for promoting osteogenesis and for inhibiting bacterial growth and osteoclastogenesis, and that the combination of BMP2 and LL37 is ideal for MSC-mediated bone regeneration, especially for inflammation-induced bone loss.

Keywords: LL37; antimicrobial peptide; bone loss; bone regeneration; bone repair; inflammation; mesenchymal stem cells; osteolysis.

Figure 1

LL37 Promotes Proliferation, Osteogenic Differentiation, and Mineralization of MSCs (A) Cell proliferation activity of MSCs with LL37 (5 or 10 μg/mL) at a cell seeding density of 5 × 10³ per well in a 96-well plate (n = 6). **p < 0.01. (B) Cell proliferation activity of mMSC, mMSC/B2, mMSC+LL37, and mMSC/B2+LL37 at day 7 (n = 6). **p < 0.01. (C) ALP activity of MSCs with LL37 (5 or 10 μg/mL) treatment (n = 3). The cells were induced with osteogenic media for 7 days. *p < 0.05; **p < 0.01. (D) Alizarin red staining of mMSC, mMSC/B2, mMSC+LL37, and mMSC/B2+LL37. Cells were cultured in osteogenic medium for 14 days. (E) Quantitative analysis of cell mineralization shown in (D) (n = 3). *p < 0.05.

Figure 2

LL37 Promotes MSC Migration (A) Giemsa staining of cells (purple) that migrated through a transwell membrane pore (8 μm, black circle) in response to a different concentration of LL37. (B) The calculated migration cell number is shown in (A). **p < 0.01; ***p < 0.001.

Figure 3

LL37 Shows Anti-bacterial Activity and Inhibits LPS-Induced Osteoclast Formation (A) TRAP staining of the osteoclast formation by M-CSF/RANKL (RANKL) or M-CSF/LPS (LPS), as indicated. LL37 was added to the culture at 5 μg/mL (5) or 10 μg/mL (10). (B) Quantification of TRAP+ MNCs shown in (A) (n = 3). **p < 0.01. (C) Photographs of disk diffusion assay showing the inhibition zones of E. coli treated with LL37. Positive control is penicillin-streptomycin solution, and negative control is PBS. (D) Measurement of the diameter of inhibition zone in (C). n = 3. **p < 0.01.

Figure 4

LL37 with BMP2-Modified MSCs Protects against LPS-Induced Osteolysis (A) Schematic of LPS-induced osteolysis and treatment for each group. (B) Representative images showing calvarial bone harvested from each group. Red dot box is the ROI for Micro-CT quantitative analysis. (C) Representative coronal MicroCT image of each group. (D) Quantitative analysis of the ratio of newly formed bone volume (BV) to total volume (TV). Newly formed bone volume = total bone volume from each group at 3 weeks − bone volume from the LPS-injected group at day 5. ***p < 0.001 for the mMSC/B2+LL37 group versus each of the other groups.

Figure 5

Histological Evaluation of Newly Formed Bone in LPS-Induced Osteolysis (A) H&E staining of calvarial bone for the analysis of new bone formation. The sagittal section through the midline of defects is shown. Lower magnification, scale bar (black), 150 μm; higher magnification, scale bar (blue), 30 μm. (B) Quantitative analysis of relative bone thickness shown in (A) (n = 3). *p < 0.05; **p < 0.01; ***p < 0.0001; ns, not statistically significant. (C) Quantitative analysis of relative bone surface shown in (A). **p < 0.01; ***p < 0.0001; ns, not statistically significant.

Figure 6

Section of Calvarial Bone Stained with Antibody against VWF (A) Representative images from each group. Scale bar, 100 μm. (B) Quantitative analysis of relative staining area per bone area (%) in (A) (n = 3). *p < 0.05; ns, not statistically significant.