Human embryonic stem cell-derived mesenchymal stem cell seeding on calcium phosphate cement-chitosan-RGD scaffold for bone repair

Abstract

Calcium phosphate cement (CPC) has in situ-setting ability and excellent osteoconductivity. Human embryonic stem cells (hESCs) are exciting for regenerative medicine due to their strong proliferative ability and multilineage differentiation capability. However, there has been no report on hESC seeding with CPC. The objectives of this study were to obtain hESC-derived mesenchymal stem cells (hESCd-MSCs), and to investigate hESCd-MSC proliferation and osteogenic differentiation on novel CPC with chitosan immobilized with RGD (CPC-chitosan-RGD). RGD was covalently bonded with chitosan, which was then incorporated into CPC. The CPC-chitosan-RGD scaffold had higher strength and toughness than CPC-chitosan control without RGD (p<0.05). hESCs were cultured to form embryoid bodies (EBs), and the MSCs were then migrated out of the EBs. Flow cytometry indicated that the hESCd-MSCs expressed typical surface antigen profile of MSCs. hESCd-MSCs had good viability when seeded on CPC scaffolds. The percentage of live cells and the cell density were significantly higher on CPC-chitosan-RGD than CPC-chitosan control. Scanning electron microscope examination showed hESCd-MSCs with a healthy spreading morphology adherent to CPC. hESCd-MSCs expressed high levels of osteogenic markers, including alkaline phosphatase, osteocalcin, collagen I, and Runx2. The mineral synthesis by the hESCd-MSCs on the CPC-chitosan-RGD scaffold was twice that for CPC-chitosan control. In conclusion, hESCs were successfully seeded on CPC scaffolds for bone tissue engineering. The hESCd-MSCs had good viability and osteogenic differentiation on the novel CPC-chitosan-RGD scaffold. RGD incorporation improved the strength and toughness of CPC, and greatly enhanced the hESCd-MSC attachment, proliferation, and bone mineral synthesis. Therefore, the hESCd-MSC-seeded CPC-chitosan-RGD construct is promising to improve bone regeneration in orthopedic and craniofacial applications.

FIG. 1.

Effect of RGD immobilization in calcium phosphate cement (CPC) on physical properties: (A) Setting time, (B) flexural strength, (C) elastic modulus, (D) work-of-fracture (toughness), (E, F) scanning electron micrographs of fracture surfaces of CPC-chitosan-RGD scaffold at a low and high magnification, respectively. Each value is mean±standard deviation (sd); n=5. In each plot, bars with dissimilar letters are significantly different (p<0.05). RGD immobilization in CPC significantly increased the strength and toughness of CPC. The microstructures of CPC on fractured cross sections are shown in scanning electron microscope (SEM) images in (E, F) for CPC-chitosan control, and (G, H) for CPC-chitosan-RGD, at low and high magnifications, respectively. “P” indicates the intrinsic pores in CPC resulting from the powder–liquid mixing of the cement. Arrows indicate the fine crystallites that make up the CPC matrix. Color images available online at www.liebertpub.com/tea

FIG. 2.

Phase-contrast photos of human embryonic stem cells (hESC) culture. (A) hESC colony cultured on mouse embryonic fibroblast (MEF) feeder layer (example shown at 4 days). (B) Embryoid bodies (EBs) formed after 4 days of suspension culture. (C) MSCs that migrated out of the EBs were harvested and passaged. The example in (C) shows hESC-derived MSCs after passage 4. MSCs, mesenchymal stem cells. Color images available online at www.liebertpub.com/tea

FIG. 3.

Flow cytometry analysis of surface markers of hESC-derived MSCs (passage 4 MSCs). The names of the antigens are listed inside each plot. The black histogram represents isotype controls and the red histogram represents the conjugated antibody of each antigen. The number in each plot represents the percentage of positive cells. hESC-derived mesenchymal stem cells (hESCd-MSCs) expressed typical surface antigen profile of MSCs. For example, MSC surface markers CD29, CD44, CD73, and CD166 were expressed to levels greater than 99.4%, while expressions of hematopoietic markers (CD31, CD34, and CD45) were less than 1.5%. Color images available online at www.liebertpub.com/tea

FIG. 4.

Viability of hESCd-MSCs cultured on CPC-chitosan control and CPC-chitosan-RGD. (A–D) Representative photos of hESCd-MSCs. Live cells were stained green and were numerous on both scaffolds. Dead cells (stained red, not shown here) were relatively few. (E) The percentage of live cells. (F) Live cell density (number of live cells per mm²), which increased with time due to cell proliferation. Each value is mean±sd; n=5. Incorporation of RGD into CPC significantly improved the hESCd-MSCs attachment. Color images available online at www.liebertpub.com/tea

FIG. 5.

SEM micrographs of hESCd-MSCs attachment on: (A) CPC-chitosan control, and (B, C) CPC-chitosan-RGD at 4 days. Cells are designated as C. The cells had developed a healthy spreading and polygonal morphology. There were noticeably more cells attaching to CPC-chitosan-RGD than to CPC-chitosan control. The cells had developed long cytoplasmic extensions E, which is shown in (C) at a higher magnification. Color images available online at www.liebertpub.com/tea

FIG. 6.

RT-PCR results for osteogenic differentiation of hESCd-MSCs on CPC-chitosan control and CPC-chitosan-RGD: (A) Alkaline phosphatase (ALP), (B) Osteocalcin (OC), (C) Collagen type I (Coll I), and (D) Runx2 gene expressions. Each value is mean±sd; n=5. hESCd-MSCs attaching to both scaffolds showed osteogenic differentiation. The ALP peaked at 7 days. The OC, Coll I, and Runx2 peak at 14 days. In each plot, values with dissimilar letters are significantly different (p<0.05). Color images available online at www.liebertpub.com/tea

FIG. 7.

Mineral synthesis by hESCd-MSCs on CPC-chitosan-RGD and CPC-chitosan control: (A, B) 7 days, (C, D) 14 days, and (E, F) 21 days. (G) Results from the osteogenesis assay (mean±sd; n=5). Mineral synthesis by the cells increased with time for both scaffolds. Mineral synthesis by cells on CPC-chitosan-RGD was 2-fold of that on CPC-chitosan control at 21 days. Color images available online at www.liebertpub.com/tea