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/mm2 on scaffold at 1 day; cells proliferated to (1808 ± 317) cells/mm2 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)/(μg 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.

Angiogenesis and bone regeneration of porous nano-hydroxyapatite/coralline blocks coated with rhVEGF165 in critical-size alveolar bone defects in vivo

“…28 Some studies have reported that the aggregated space between the pores acts as the passageway for the metabolism of cells and the transportation of nutrition that promote the ingrowth of blood vessels. [29][30][31] Yoshikawa et al 32 suggested that the pore size in grafts used for bone healing should be at least 50 µm. Larger pore sizes result in greater quantities of nutrition and oxygen being transported into the inside of the scaffold.…”

“…31,33,34 However, Grynpas et al 33 noted that oversized pores not only reduce mechanical strength but also influence cell function and new bone formation. High levels of interconnectivity between the pores enhance the porosity and enlarge the superficial area and consequently increase the numbers of endothelial cells and active osteoblasts.…”

Cell-material interaction

“…These coatings are described to induce an increased bone-to-implant contact and are regarded as osteoinductive [65]. In vitro, several studies have shown that a calcium phosphate coated implant surface promotes osteogenic differentiation of mesenchymal stem cells, which implies a stimulating effect of calcium phosphate for the formation of new bone [66][67][68].…”

Development of magnesium calcium phosphate biocement for bone regeneration

“…The ALP activity of MG 63 cells cultured on the MCPB was significantly higher than that of CPB and the control (TCP) at 7 and 14 days ( p , 0.05). A previous study showed that the ALP for mesenchymal stem cells on CPC -chitosan was higher than on CPC at 14 days (Moreau & Xu 2009). There were no significant differences for ALP at 7 and 14 days for the same sample.…”