Efficacy of soluble lansoprazole-impregnated beta-tricalcium phosphate for bone regeneration

Abstract

The proton pump inhibitor lansoprazole has been previously identified to upregulate the expression and transcriptional activity of runt-related transcription factor 2 (Runx2) that promotes lineage commitment and differentiation of osteoprogenitor cells. We could not elicit the expected efficacy of insoluble lansoprazole in enhancing osteogenesis when combined with beta-tricalcium phosphate (β-TCP) bone substitutes. This study aimed to evaluate the effects of soluble lansoprazole on in vitro osteoblastogenesis and new bone formation in vivo. Commercially available human mesenchymal stem cells or patient-derived bone marrow-derived stromal cells were treated with 20 µM of soluble lansoprazole at the beginning of osteogenic induction. Soluble lansoprazole-impregnated β-TCP materials were embedded in the cortical bone defect model of rabbits. Rabbits were sacrificed four weeks postoperatively and undecalcified bone specimens were prepared for evaluation of intra-material new bone formation. Only a 1-day treatment with soluble lansoprazole facilitated osteoblastic differentiation and matrix calcium deposition when added to undifferentiated human mesenchymal stromal cells at the beginning of the osteogenic differentiation. Soluble lansoprazole dose-dependently accelerated intra-material new bone formation when being impregnated with porous β-TCP artificial bones. Local use of soluble lansoprazole can be applicable for fracture and bone defect repair when combined with porous β-TCP scaffolds.

Figure 1

Radiographic, µ-CT, and histological images of insoluble lansoprazole-impregnated artificial bones embedded for four weeks in a rabbit cortical bone defect model. Note that radiolucent areas around lansoprazole-impregnated materials correspond to dense fibrogranulomatous tissues on histology (Villanueva-Goldner staining). In contrast to the control one, the insides of the experimental materials surrounded with the fibrogranulomatous tissues are devoid of newly-formed bones (green). Scale bars, 1 mm.

Figure 2

Short-term treatment with soluble lansoprazole facilitates osteoblastic differentiation of human mesenchymal stem cells. (A) Expression levels of endogenous Runx2 mRNA in soluble lansoprazole-treated commercially available human MSCs. Cells were treated with soluble lansoprazole for 1 day at the beginning of osteogenic induction. The relative expression levels were normalized to the mean of the control. (B) ALP activity of cell lysates of soluble lansoprazole-treated commercially available human MSCs. Cells were cultured in the osteogenic medium for 14 days and treated with soluble lansoprazole for one, three, or five days at the beginning of osteogenic induction (day 0). The ALP activities were determined on day 5. The relative activity was normalized to the mean of the control. (C) Osteocalcin concentration of cell lysates of soluble lansoprazole-treated commercially available human MSCs. Cells were cultured in the osteogenic medium for 14 days and treated with soluble lansoprazole for one, three, or five days at the beginning of osteogenic induction (day 0). The osteocalcin concentrations were determined on day 14. The relative concentration was normalized to the mean of the control. In (A–C), the mean and SD (A, n = 3; B and C, n = 6) are indicated. (A) *p < 0.05 by unpaired t-test. (B, C) *p < 0.05 by one-way ANOVA with post hoc Tukey analysis. ns, not significant.

Figure 3

Short-term treatment with soluble lansoprazole enhances matrix calcium deposition of patient-derived bone marrow stromal cells (BMSCs). (A) Verification of stemness characteristics of plastic-adherent cells obtained from primary culture of patient-derived bone marrow aspirates in the growth medium. A pan-leukocyte marker CD45-negative adherent cells were evaluated for expression levels of typical MSC markers CD90, CD105, and CD73 by FACS analysis. Blue histograms show the fluorescent intensity for the indicated cell surface markers. Shadow histograms represent the fluorescent intensity obtained with the control antibodies. X-axis and Y-axis indicate fluorescent intensity and cell count, respectively. FSC, forward scatter. (B) Matrix calcium deposition detected by alizarin red staining in patient-derived BMSCs. Cells were treated with 20 µM of soluble lansoprazole for 1 day at the beginning of osteogenic induction and cultured for 21 days in the osteogenic medium without lansoprazole. Scale bar, 1 mm (upper panel) and 200 µm (lower panel). (C) Osteocalcin concentration of cell lysates of patient-derived BMSCs. Cells were treated with soluble lansoprazole for 1 day at the beginning of osteogenic induction and cultured for 14 days in the osteogenic medium without lansoprazole. The relative concentration was normalized to the mean of the control. The mean and SD (n = 3) are indicated. *p < 0.05 by unpaired t-test.

Figure 4

Radiographic and µ-CT images of soluble lansoprazole-impregnated artificial bones embedded for four weeks in a rabbit cortical bone defect model. In contrast to insoluble-lansoprazole ones (Fig. 1), experimental materials appeared to fuse with the host bones.

Figure 5

Soluble lansoprazole-impregnated artificial bones demonstrate the acceleration of intra-material bone regeneration. (A) Undecalcified bone histology of soluble lansoprazole-impregnated artificial bones embedded for four weeks in a rabbit cortical bone defect model (Villanueva-Goldner staining; upper panel). The surface area of newly formed bones within the materials was automatically measured using an image analyzer (WinROOF). The area of newly formed bones within the entire material (total bone area, TBA; middle panel) and that within the specific area of the material embedded in the cortical bone area (partial bone area, PBA; lower panel) are colored green. TMA, total material area; PMA, partial material area. Each of the area percentage ratios is indicated in the inset of the middle panel (TBA/TMA ratio) and the lower panel (PBA/PMA ratio). (B) The percentage of new bone formation within the entire material (TBA/TMA ratio, left) and the specific area of the material embedded in the cortical bone area (PBA/PMA ratio, right). (C) The percentage of new bone formation within the entire material (TBA/TMA ratio) of the lower concentration group (control and 20 µM) and the higher concentration group (200 µM and 2 mM). In (B, C), the mean and SD (n = 2 per each of three experimental subgroups and n = 6 for the control group) are indicated. *p < 0.05 by the Jonckheere-Terpstra tread test over the indicated concentration range of soluble lansoprazole solution (B) and by unpaired t-test (C). ns, not significant. Scale bars, 1 mm.