Engineering vascularized bone grafts by integrating a biomimetic periosteum and β-TCP scaffold

Abstract

Treatment of large bone defects using synthetic scaffolds remain a challenge mainly due to insufficient vascularization. This study is to engineer a vascularized bone graft by integrating a vascularized biomimetic cell-sheet-engineered periosteum (CSEP) and a biodegradable macroporous beta-tricalcium phosphate (β-TCP) scaffold. We first cultured human mesenchymal stem cells (hMSCs) to form cell sheet and human umbilical vascular endothelial cells (HUVECs) were then seeded on the undifferentiated hMSCs sheet to form vascularized cell sheet for mimicking the fibrous layer of native periosteum. A mineralized hMSCs sheet was cultured to mimic the cambium layer of native periosteum. This mineralized hMSCs sheet was first wrapped onto a cylindrical β-TCP scaffold followed by wrapping the vascularized HUVEC/hMSC sheet, thus generating a biomimetic CSEP on the β-TCP scaffold. A nonperiosteum structural cell sheets-covered β-TCP and plain β-TCP were used as controls. In vitro studies indicate that the undifferentiated hMSCs sheet facilitated HUVECs to form rich capillary-like networks. In vivo studies indicate that the biomimetic CSEP enhanced angiogenesis and functional anastomosis between the in vitro preformed human capillary networks and the mouse host vasculature. MicroCT analysis and osteocalcin staining show that the biomimetic CSEP/β-TCP graft formed more bone matrix compared to the other groups. These results suggest that the CSEP that mimics the cellular components and spatial configuration of periosteum plays a critical role in vascularization and osteogenesis. Our studies suggest that a biomimetic periosteum-covered β-TCP graft is a promising approach for bone regeneration.

Figure 1

Step-by-step procedures for preparing cell sheet/β-TCP composite grafts. Preparing three cell sheet/β-TCP grafts including OM/UM/β-TCP, OM/HUVEC-UM/β-TCP, and HUVEC-UM/OM/β-TCP (A). (B) Macroscopic view of an hMSCs sheet on a dish (I) and a porous β-TCP scaffold (II). SEM image demonstrates the morphology of β-TCP pores (III). Point forceps were used to wrap the cell sheet onto a β-TCP scaffold (IV), thus generating a HUVEC-UM/OM/β-TCP graft (V). SEM images show a very dense extracellular matrix of cell sheet on a β-TCP scaffold (VI, VII).

Figure 2

HUVECs on an undifferentiated hMSCs sheet formed numerous networks. Networks started at day 3 (A), and elongated to form many lumens at day 5 (B) and day 7 (C). Arrows indicate lumens. Immunofluorescent staining images of CD31 show several networks on the hMSCs sheet at day 7 (D, 10× magnification; E, 20× magnification); 3D-reconstructed confocal images display lumen formation (F). Asterisks show the lumens. Alizarin red staining (G) and vov Kossa staining (H) show the mineralized matrix of osteogenic hMSC cell sheet.

Figure 3

Fluorescent images of DAPI staining, GFP-HUVECs, and SEM images show cell migration. Cells migrated into the periphery of the scaffold but did not reach its center at day 3. With time, cells started to migrate further from the periphery toward the center at 7 days and 14 days (A); SEM images show a very dense cell sheet wrapped on the β-TCP scaffold and the morphology of ECM. Cells migrated from the peripheral cell sheet into the β-TCP scaffold at day 3 and day 14 (B).

Figure 4

H&E staining results reveal that cells grew into the β-TCP scaffold and OM/UM/β-TCP groups at 2, 4, and 8 weeks, but few blood vessels were observed. However, many blood vessels containing red blood cells were seen in prevascularized groups, HUVEC-UM/OM/β-TCP and OM/HUVEC-UM/β-TCP (A). Magnified image from HUVEC-UM/OM/β-TCP group at 2 weeks shows murine blood cells in a blood vessel lumen (B). A quantitative assay shows the microvessel densities of fours groups at 2, 4, and 8 weeks (* p < 0.05, n = 4) (C).

Figure 5

Immunohistochemistry staining of human CD31 shows that many antihuman CD31 positive-expressing lumens were seen in HUVEC-UM/OM/β-TCP and OM/HUVEC-UM/β-TCP (black arrows), but there was no expression in plain β-TCP scaffolds and the nonprevascularized OM/UM/β-TCP group (A). A magnified image from HUVEC-UM/OM/β-TCP group at 2 weeks shows that the preformed human blood vessel lumen (brown color, red arrow) carried murine blood cells (semitransparent round ball, blue arrow) (B). The density of the CD31 positive-expressing lumens was higher in HUVEC-UM/OM/β-TCP than that in OM/HUVEC-UM/β-TCP at 2, 4, and 8 weeks (* p < 0.05, n = 4) (C). Immunofluorescent double staining shows the expressions of antihuman CD31 (green) and antimouse CD31 (magenta) in nonprevascularized and prevascularized scaffold group. The overlap or partial overlap of the two colors implies the anastomosis of the preformed human capillaries with the host vasculature (yellow, white arrow) (D).

Figure 6

Immunohistochemistry staining of antihuman vimentin shows that many positive-expressing human cells were seen in cell sheet/β-TCP groups from 2 weeks, 4 weeks, to 8 weeks. Human cells migrated from cell sheet to the inner core of β-TCP scaffolds. Host mouse cells (negative expression) also migrated into the plain β-TCP scaffolds.

Figure 7

Quantitative assay shows that HUVEC-UM/OM/β-TCP induced higher bone volume than all other groups at 4 and 8 weeks (* p < 0.05, n = 4).

Figure 8

Immunohistochemistry staining of osteocalcin shows a denser osteocalcin matrix in HUVEC-UM/OM/β-TCP compared to those in other three groups at 2, 4, and 8 weeks (A); TRAP staining shows osteoclastic activity in the HUVEC-UM/OM/β-TCP and OM/HUVEC-UM/β-TCP at 2 weeks (B).