Culture human mesenchymal stem cells with calcium phosphate cement scaffolds for bone repair

Abstract

Because of its moldability and excellent osteoconductivity, calcium phosphate cement (CPC) is highly promising for craniofacial and orthopedic applications. The objectives of this study were to investigate the response of human mesenchymal stem cells (hMSCs) to a high-strength CPC-chitosan scaffold and to examine cell proliferation and osteogenic differentiation. hMSCs were seeded onto CPC-chitosan composite, CPC control, and tissue culture polystyrene (TCPS). Alkaline phosphatase activity (ALP) and mineralization of hMSCs were measured. CPC-chitosan had a flexural strength (mean + or - SD; n = 5) of (19.5 + or - 1.4) MPa, higher than (8.0 + or - 1.4) MPa of CPC control (p < 0.05). The percentage of live hMSCs on CPC-chitosan was (90.5 + or - 1.3)% at 8 days, matching (90.7 + or - 3.8)% of CPC control (p > 0.1). The CPC-chitosan surface area covered by the attached hMSCs increased from (51 + or - 11)% at 1 day to (90 + or - 4)% at 8 days (p < 0.05), matching those of CPC control (p > 0.1). Hence, the CPC strength was significantly increased via chitosan without compromising the hMSC response. At 8 days, there was a significant increase in ALP of cells in osteogenic media (10.99 + or - 0.93) [(mM pNpp/min)/(microg DNA)] versus control media (3.62 + or - 0.40) (p < 0.05). hMSCs in osteogenic media exhibited greater mineralization area of (47.5 + or - 19.7)% compared with (6.1 + or - 2.3)% in control medium on TCPS (p < 0.05). In conclusion, hMSCs showed excellent attachment and viability on the strong and tough CPC-chitosan scaffold, matching the hMSC response on CPC control. hMSCs were successfully differentiated down the osteogenic lineage. Hence, the strong, in situ hardening CPC-chitosan scaffold may be useful as a moderate load-bearing vehicle to deliver hMSCs for maxillofacial and orthopedic bone tissue engineering.

Figure 1

(A) SEM micrograph of typical surface of CPC showing pores (arrows). (B) High magnification showing the nanosized hydroxyapatite crystals in CPC. (C) Smaller crystals in CPC-chitosan. Arrows indicate hydroxyapatite nanocrystals. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 2

hMSCs cultured for 1 day in osteogenic media. (A) Live cells (stained green) on CPC-chitosan; (B) live cells on CPC; (C) live cells on TCPS; (D) dead cells (stained red) on CPC-chitosan; (E) percent of live cells. Horizontal line indicates values that are not significantly different (p > 0.1). Each value is the mean of five measurements with the error bar showing one standard deviation (mean ± SD; n = 5). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 3

hMSCs cultured for at 8 days in osteogenic media. (A) Live cells (stained green) on CPC-chitosan; (B) live cells on CPC; (C) live cells on TCPS; (D) dead cells (stained red) on CPC-chitosan; (E) percent of live cells at days 4 and 8. Horizontal line indicates values that are not significantly different (p >0.1). Each value is mean ± SD; n = 5. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 4

Specimen area covered by the attached live hMSCs, C_ATTACH. Cells were cultured in osteogenic media. Horizontal line indicates values that are not significantly different (p > 0.1). Each value is mean ± SD; n = 5. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 5

Alkaline phosphatase activity (ALP) of hMSCs at 1, 4, and 8 days cultured on TCPS in control and osteogenic medium. ALP was normalized by the DNA concentration, with units of (mM pNpp/min)/(μg DNA). Horizontal line indicates values that are not significantly different (p > 0.1). Each value is mean ± SD; n = 5. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 6

Mineralization by hMSCs cultured for 21 days on TCPS. Live cell culture was stained with xylenol orange in (A) control media, and (B) osteogenic media. (C) Mineralization area stained with xylenol orange. Live cell culture stained with calcein blue in (D) control media and (E) osteo-genic media. (F) Mineralization of cells stained with calcein blue. Each value is mean ± SD; n = 5. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 7

SEM of hMSCs on CPC-chitosan specimen: (A,B) 4 days; (C,D) 8 days. Arrows in (A) indicate globular mineral formation and collagen bundles. At higher magnification in (B), mineral formation is more clearly seen as individual mineral granules. The cell body in (C) is indicated by the letter C, and the cytoplasmic extensions of the cell are indicated by E. (D) Mineralization on cell surface at 8 days, similar to those at 4 days in (B). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]