Mesenchymal stem cell proliferation and differentiation on an injectable calcium phosphate-chitosan composite scaffold

Abstract

Calcium phosphate cement (CPC) can be molded or injected to form a scaffold in situ, has excellent osteoconductivity, and can be resorbed and replaced by new bone. However, its low strength limits CPC to non-stress-bearing repairs. Chitosan could be used to reinforce CPC, but mesenchymal stem cell (MSC) interactions with CPC-chitosan scaffold have not been examined. The objective of this study was to investigate MSC proliferation and osteogenic differentiation on high-strength CPC-chitosan scaffold. MSCs were harvested from rat bone marrow. At CPC powder/liquid (P/L) mass ratio of 2, flexural strength (mean+/-sd; n=5) was (10.0+/-1.1) MPa for CPC-chitosan, higher than (3.7+/-0.6) MPa for CPC (p<0.05). At P/L of 3, strength was (15.7+/-1.7)MPa for CPC-chitosan, higher than (10.2+/-1.8)MPa for CPC (p<0.05). Percentage of live MSCs attaching to scaffolds increased from 85% at 1 day to 99% at 14 days. There were (180+/-37) cells/mm(2) on scaffold at 1 day; cells proliferated to (1808+/-317) cells/mm(2) at 14 days. SEM showed MSCs with healthy spreading and anchored on nano-apatite crystals via cytoplasmic processes. Alkaline phosphatase activity (ALP) was (557+/-171) (pNPP mM/min)/(microg DNA) for MSCs on CPC-chitosan, higher than (159+/-47) on CPC (p<0.05). Both were higher than (35+/-32) of baseline ALP for undifferentiated MSCs on tissue-culture plastic (p<0.05). In summary, CPC-chitosan scaffold had higher strength than CPC. MSC proliferation on CPC-chitosan matched that of the FDA-approved CPC control. MSCs on the scaffolds differentiated down the osteogenic lineage and expressed high levels of bone marker ALP. Hence, the stronger CPC-chitosan scaffold may be useful for stem cell-based bone regeneration in moderate load-bearing maxillofacial and orthopedic applications.

Figure 1

Flexural strength and elastic modulus of CPC control (using water as liquid) and CPC-chitosan scaffolds. Each value is the mean of five measurements, with the error bar showing one standard deviation (mean ± sd; n = 5).

Figure 2

Live/dead staining of rat bone-marrow-derived MSCs cultured for 1 day. (A) Live cells on CPC control without chitosan. (B) Live cells on CPC-chitosan disk. (C) Dead cells on CPC-chitosan disk. The live MSCs were stained green and appeared to have adhered and attained a normal polygonal morphology on both materials. Dead cells were stained red and were very few on both materials.

Figure 3

SEM micrographs of MSCs: (A) Lower magnification showing cells anchoring on a CPC-chitosan specimen; (B) Higher magnification showing a cell attaching to the nano-apatite crystals that make up CPC and CPC-chitosan specimens. C: Msenchymal stem cells. E: Cytoplasmic extensions of the cells. J: Cell-cell junctions. When the CPC powder is mixed with an aqueous solution, it goes through a dissolution-reprecipitation process to convert to nano-apatite. These crystals are consistent with those observed in previous studies, and the phase was verified with X-ray diffraction [30].

Figure 4

MSC proliferation on CPC and CPC-chitosan at 14 days. (A) Confluent monolayer of MSCs on CPC, typical on most disks. (B) Some areas of the disks had cell that were slightly less than completely confluent. (C) MSCs on CPC-chitosan were as confluent as cells on CPC control.

Figure 5

Percentage of live MSCs, P_Live. After 1 day, P_Live was 85–90%, and there was no significant difference between CPC and CPC-chitosan (p > 0.1). After 14 days, P_Live increased significantly to 99% (p < 0.05). Each value is mean ± sd, n = 5.

Figure 6

MSC attachment density, C_Attach, which is number of cells per specimen surface area. On both CPC and CPC-chitosan, C_Attach was increased by an order of magnitude from 1 day to 14 days. It should be noted that the C_Attach at 14 days represents a lower-end estimate, because the number of live cells at 14-day were only counted in areas of samples where the cells could be counted (such as Fig. 4B), while other areas had more cells that were too crowded to be counted. Each value is mean ± sd, n = 5.

Figure 7

Alkaline phosphatase activity (ALP), normalized to DNA concentration, with units of (pNPP mM/min)/(μg DNA). MSCs on both CPC-chitosan and CPC without chitosan had higher ALP than the baseline ALP for MSCs on tissue culture plastic in control media. Each value is mean ± sd, n = 5.