The osteogenesis of bone marrow stem cells on mPEG-PCL-mPEG/hydroxyapatite composite scaffold via solid freeform fabrication

Abstract

The study described a novel bone tissue scaffold fabricated by computer-aided, air pressure-aided deposition system to control the macro- and microstructure precisely. The porcine bone marrow stem cells (PBMSCs) seeded on either mPEG-PCL-mPEG (PCL) or mPEG-PCL-mPEG/hydroxyapatite (PCL/HA) composite scaffold were cultured under osteogenic medium to test the ability of osteogenesis in vitro. The experimental outcomes indicated that both scaffolds possessed adequate pore size, porosity, and hydrophilicity for the attachment and proliferation of PBMSCs and the PBMSCs expressed upregulated genes of osteogensis and angiogenesis in similar manner on both scaffolds. The major differences between these two types of the scaffolds were the addition of HA leading to higher hardness of PCL/HA scaffold, cell proliferation, and VEGF gene expression in PCL/HA scaffold. However, the in vivo bone forming efficacy between PBMSCs seeded PCL and PCL/HA scaffold was different from the in vitro results. The outcome indicated that the PCL/HA scaffold which had bone-mimetic environment due to the addition of HA resulted in better bone regeneration and mechanical strength than those of PCL scaffold. Therefore, providing a bone-mimetic scaffold is another crucial factor for bone tissue engineering in addition to the biocompatibility, 3D architecture with high porosity, and interpored connection.

Figure 1

The surgery procedure for bone regeneration with a pig temporal bone defect model. (a) A full-thickness 2 cm × 2 cm bone defect was created at temporal bone area of a pig, which was reconstructed by (b) a tissue-engineered PCL/HA/PBMSCs construct.

Figure 2

Illustration of scaffold design: (a) frontal view, (c) cross-sectional view; microcomputed tomography (μ-CT) analysis of PCL/HA scaffolds prepared by APAD. (b) Frontal view, (d) cross-sectional view. The lines in 3D image (b) mark the planes of cross-sections where 2D cross-sectional views are taken ((e), (f), and (g)).

Figure 3

Comparison of cell proliferation of porcine bone marrow stem cells (PBMSCs) in PCL and PCL/HA scaffolds at different time points.

Figure 4

The viability of PBMSCs at PCL and PCL/HA group from day 0 to day 21 (green means live cells and red means dead cells); scale bar: 100 μm.

Figure 5

Relative (a) Runx II, (b) alkaline phosphatase (ALP), (c) osteocalcin (OCN), and (d) VEGF mRNA expression of PBMSCs in PCL and PCL/HA scaffolds at different time points. The relative qRT-PCR values were corrected using the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression levels and normalized with respect to the values on day 0 of culture. The values are the mean values ± SD of three independent experiments. *P < 0.05.

Figure 6

The SEM showed the cell-scaffold interaction at PCL and PCL/HAP group from day 0 to day 21.

Figure 7

The coronal view of two-dimensional view of bilateral pig bone defects which were reconstructed by PCL/PBMSCs at left side and PCL/HA/PBMSCs at right side. (a) Post-op 1 w, (b) post-op 2 months, (c) post-op 4 months, and (d) post-op 6 months.

Figure 8

Comparison of Hounsfield units (HU) among PCL/PBMSCs, PCL/HA/PBMSCs groups at post-op 1 w, 2 m, 4 m, and 6 m and normal bone.

Figure 9

Comparison of the hardness of harvested specimens between PCL/PBMSCs and PCL/HA/PBMSCs after 6-month implantation in pig temporal bone.

Figure 10

The histological examination of specimens post-op 6 months for PCL/PBMSCs: (a) H&E (40X), (c) H&E 100X, (e) Masson's trichrome stain (40X), and (g) Masson's trichrome stain (100X) and PCL/HA/PBMSCs group: (b) H&E (40X), (d) H&E 100X, (f) Masson's trichrome stain (40X), and (h) Masson's trichrome stain (100X). S: scaffold; B: bone formation area (deep blue area); F: fibrotic tissue area (light blue area). Black scale bar at 40X is 200 μm and black scale bar at 100X is 100 μm.