Osteoblastic induction on calcium phosphate cement-chitosan constructs for bone tissue engineering

Abstract

Calcium phosphate cement (CPC) is osteoconductive and moldable, and it can conform to complex cavity shapes and set in situ to form hydroxyapatite. Chitosan could increase the strength and toughness of CPC, but there has been no investigation on recombinant human bone morphogenic protein-2 (rhBMP-2) delivery via CPC-chitosan composite and its effect on osteogenic induction of cells. The objective of this research was to investigate the mechanical properties and osteoblastic induction of MC3T3-E1 cells cultured on CPC-containing chitosan and rhBMP-2. Cell viability for CPC with chitosan and rhBMP-2 was comparable with that of control CPC, whereas the CPC-chitosan composite was stronger and tougher than CPC control. After 14 days, osteoblastic induction was quantified by measuring alkaline phosphatase (ALP) activity. ALP (mean +/- SD; n = 6) of cells seeded on conventional CPC without rhBMP-2 was (143 +/- 19) (mM pNpp/min)/(mug DNA). The addition of chitosan resulted in an ALP of 161 +/- 27. Further addition of rhBMP-2 to the CPC-chitosan composite increased the ALP to 305 +/- 111 (p < 0.05). All ALP activity on CPC composites was significantly higher when compared with the 10.0 +/- 3.3 of tissue culture polystyrene (p < 0.05). Flexural strength of CPC containing 15% (mass fraction) chitosan was 19.8 +/- 1.4 MPa, which is more than double the 8.0 +/- 1.4 MPa of conventional CPC (p < 0.05). The addition of chitosan to CPC increased the fracture toughness from 0.18 +/- 0.01 MPa.m(1/2) to 0.23 +/- 0.02 MPa.m(1/2) (p < 0.05). The relatively high strength, self-hardening CPC-chitosan composite scaffold is promising as a moderate load-bearing matrix for bone repair, with potential to serve as an injectable delivery vehicle for osteoinductive growth factors to promote osteoblastic induction and bone regeneration. (c) 2010 Wiley Periodicals, Inc. J Biomed Mater Res, 2010.

Figure 1

(A) Flexural strength and (B) Elastic modulus of CPC as a function of mass fraction of chitosan in liquid. Each value is the mean of six measurements with the error bar showing one standard deviation (mean ± sd; n = 6). Dissimilar letters in the plot indicate values that are significantly different (Tukey’s multiple comparison test; family confidence coefficient = 0.95).

Figure 2

Fracture toughness of CPC + 0% chitosan and CPC + 15% chitosan. Each value is the mean of six measurements with the error bar showing one standard deviation (mean ± sd; n = 6). Dissimilar letters in the plot indicate values that are significantly different (Tukey’s multiple comparison test; family confidence coefficient = 0.95).

Figure 3

Cells cultured for 1 d on: (A) live cells on CPC+0% chitosan, (B) live cells on CPC+0% chitosan + 5 μg rh-BMP-2, (C) live cells on CPC+15% chitosan (D) live cells on CPC+15% chitosan + 5 μg rh-BMP-2 (E) live cells on TCPS control (F) dead cells on TCPS control.

Figure 4

(A) Cell viability (or percentage of live cells) = number of live cells / (number of live cells + number of dead cells) at 1 d cell culture. (B) Live cell density (or cell attachment per specimen surface area) = number of live cells / mm² (mean ± sd; n = 6) at 1 d cell culture. Dissimilar letters in the plot indicate values that are significantly different (Tukey’s multiple comparison test; family confidence coefficient = 0.95).

Figure 5

Cells cultured for 14 d. Live cells are stained green in panels (A) to (E). Dead cells are stained red in panel (F). (A) CPC with 0% chitosan, (B) CPC specimens with 0% chitosan + rhBMP-2, (C) CPC specimens with 15% chitosan, (D) CPC specimens with 15% chitosan + rhBMP-2, (E) TCPS without rhBMP-2 and (F) dead cells on CPC specimens with 0% chitosan + rhBMP-2.

Figure 6

(A) Cell viability (or percentage of live cells) = number of live cells / (number of live cells + number of dead cells) at 14 d cell culture. (B) Live cell density (or cell attachment per specimen surface area) = number of live cells / mm² (mean ± sd; n = 6) at 14 d cell culture. Dissimilar letters in the plot indicate values that are significantly different (Tukey’s multiple comparison test; family confidence coefficient = 0.95).

Figure 7

Relative alkaline phosphatase activity of MC3T3-E1 cells at 14 d cultured on TCPS, CPC, CPC+15% chitosan and CPC+15% chitosan+5 μg rhBMP-2. ALP activity is normalized with respect to the DNA concentration in each sample to give units of [(mM pNpp/min)/(μg DNA)]. Dissimilar letters in the plot indicate values that are significantly different (Tukey’s multiple comparison test; family confidence coefficient = 0.95). Each value is the mean of six measurements with the error bar showing one standard deviation (mean ± sd; n = 6).