Endothelial progenitor cells with stem cells enhance osteogenic efficacy

Abstract

Background: Mesenchymal stem cell (MSC)-based bone tissue engineering is a promising treatment option for maxillary sinus augmentation. Rapid vascularization is necessary to enhance the osteoinductive efficacy and prevent necrosis of the tissue-engineered bone. This study investigated whether the co-autotransplantation of endothelial progenitor cells (EPCs) could significantly enhance the in vivo osteogenic efficacy of MSCs and prevent necrosis of the tissue-engineered bone in a maxillary sinus augmentation model in dogs.

Methods: We evaluated the in vitro osteogenic activities of a clinically-used scaffold-deproteinized bovine bone (Bio-Oss) by examining cell adhesion and alkaline phosphatase (ALP) activity. In vivo, sinus augmentations were performed identically on both sides of dogs (n = 3 per group) using three treatment groups: (A) Bio-Oss with MSCs and EPCs; (B) Bio-Oss with MSCs; and (C) Bio-Oss with EPCs. The tissue implants were evaluated 24 weeks post-implantation.

Results: In vitro, co-application of EPCs and MSCs on Bio-Oss significantly enhanced adhesion and ALP activity. In vivo, co-autotransplantation of MSCs and EPCs resulted in a significantly higher height, compressive strength, bone volume density, trabecular thickness, and trabecular number and a significantly lower trabecular separation compared with the other groups. The fluorescent test showed co-autotransplantation caused a significantly higher mineral apposition rate than the other groups. Histomorphometric analysis showed co-application resulted in the highest rate of new bone formation. Newly formed bone was frequently in the center of the implants with EPCs and MSCs, but not the other implants.

Conclusions: Co-autotransplantation of EPCs and MSCs significantly enhanced the in vivo osteogenic efficacy, suggesting promising potential for sinus augmentation.

Keywords: Endothelial progenitor cells; angiogenesis; bone marrow stromal cells; mesenchymal stem cells; osteogenesis; tissue-engineered bone.

Figure 1

Characterization of mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) used in this study. (A) Canine MSCs at P2 displayed spindle morphology. (B) ALP staining and (C) Alizarin Red S staining verify osteogenic differentiation of MSCs after three weeks of induction. (D) The adipogenic differentiation of MSCs was verified by positive Oil Red O staining after 4 weeks of mass culture and induction. (E) MSCs also had the capacity to undergo chondrogenic differentiation. This was evident through Alcian blue staining after induction for 4 weeks. (F) Flow cytometry analysis of the expression of indicated cell surface markers related to MSCs. (G) Morphology of EPCs at 12 days of culture. EPCs could take up (H) FITC-UEA-1 and bind to (I) Dil-Ac-LDL as shown by the green and red fluorescence. (J) Merged images show that most cells were dual-positive. Dual-positive cells were defined as EPCs. (K) Flow cytometry analysis of the expression of indicated cell surface markers related to EPCs. Scale bar = 100 μm.

Figure 2

MSCs-derived osteogenic cells alone, EPCs alone, and the co-cultured cells at the same density were dripped on the Bio-Oss granules layer and incubated for 3, 6, and 12 h at 37°C. (A) The percentage of adherent cells was calculated. Results are presented as the mean ± SD. *: P ≤ 0.05 vs MSC-derived osteogenic cells, #: P ≤ 0.05 vs EPCs. The SEM shows the morphological appearance of (B) MSCs-derived osteogenic cells and (C) EPCs on the Bio-Oss granules after 7 days of culture.

Figure 3

ALP activity in the Bio-Oss granules with MSC-derived osteogenic cells, EPCs and co-cultured cells for 24 h, 3 days, 7 days, and 14 days. Results are presented as mean ± SD. *: P ≤ 0.05. ×: No expression.

Figure 4

The augmented maxillary sinus of each group at 24 weeks post-operation shown by sagittal maxillofacial CT images (A1: group A, B1: group B, C1: group C). 3-D-micro-CT images of the total mineralized volume in groups A, B, and C were also displayed (A2: group A, B2: group B, C2: group C). Three-dimensional reconstructed images of the volume of interest (VOI) for the three groups represent remnants of Bio-Oss granules (pink) and newly formed bone (white) (A3: group A, B3: group B, C3: group C). (D) The mean height value and (E) the microhardness of each group was measured. Each value was expressed as mean ± SD. *: P ≤ 0.05 vs group C, #: P ≤ 0.05 vs group B. Group A contained scaffold Bio-Oss/MSCs-derived osteogenic cells and EPCs (n = 3); Group B contained scaffold Bio-Oss/MSCs-derived osteogenic cells (n = 3) as a control group; Group C contained scaffold Bio-Oss/EPCs (n = 3) as a control group.

Figure 5

The 3-D microarchitectural indices analyzed using micro-CT: total volume ratio (BV/TV), connectivity density (Conn.Dn), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and trabecular number (Tb.N). Data are expressed as mean ± SD. *: P ≤ 0.05 vs group C, #: P ≤ 0.05 vs group B.

Figure 6

New bone formation and mineralization were determined by hydrochloride tetracycline (TE), calcein (CA), and Alizarin Red S (AL) fluorescent observation at 4, 12, and 20 weeks after the operation (A: group A, B: group B; C: group C). (D) Mineral apposition rate (MAR) was analyzed for the three groups. The result is presented in a bar form as mean ± SD. *: P ≤ 0.05 vs group C, #: P ≤ 0.05 vs group B. Scale bar = 100 μm.

Figure 7

Representative histological sections of bone formation in augmented maxillary sinus in three groups. (A1) Group A contained scaffold Bio-Oss/MSCs-derived osteogenic cells and EPCs; (B1) Group B contained scaffold Bio-Oss/MSCs-derived osteogenic cells; (C1) Group C contained scaffold Bio-Oss and EPCs. A2 (group A), B2 (group B), and C2 (group C) show higher magnification images. Bone marrow was found in group A (A2). ×: residual Bio-Oss particles; Δ: the newly formed bone; black arrow: osteoblasts; red arrow: multinucleated giant cells. Scale bar = 100 μm. Osteocalcin (OCN) immunohistochemistry detection (A3: group A, B3: group B, C3: group C). (D, E) The new bone formation and remnant particles are shown. Each value is expressed as mean ± SD. *: P ≤ 0.05 vs group C, #: P ≤ 0.05 vs group B.

Figure 8

Representative histological sections in group A. (A) A compact bone area in an active phase with osteoblasts blood vessels near the (B) newly formed bone. black arrow: osteoblasts; white arrow: blood vessels. Scale bar = 100 μm.