Study Of Calcium Phosphate Cement Reinforced By Nanocrystalline Cellulose

Calcium phosphate cement (CPC) has been widely used as bone substitutes in clinic, due to its abilities of excellent biocompatibility and self-setting. However, its poor mechanical performance has limited its applicability to non-stress-bearing bone. Nanocrystalline Cellulose (NCC) has been widely used as reinforcing component in composites because of its good mechanical strength, high crystallinity, high surface area, large aspect ratio and low price. Meanwhile, NCC is biodegradable and biocompatible, which can be used in biomaterials. Therefore, in this study, NCC was incorporated as reinforcing component with CPC and the preparation process of NCC/CPC composites were optimized to carry out the homogeneous dispersion of NCC in CPC. The influence of NCC on the compressive strength, setting time, phase composition and other properties of CPC were studied. In addition, the response of osteoblasts (OB) was evaluated by co-culturing with NCC/CPC composite.Firstly, different weight percentages of NCC(0,2 wt.% and 4 wt.%) were dispersed in the liquid phase of CPC (Phosphate Buffer Solution, PBS) to study the influence of different NCC contents on the physical and chemical properties of CPC. CPC with different NCC contents were prepared by mixing the liquid phase with powder phase of CPC according to Biocement D, and a liquid/powder ratio of 0.3 mL/g was applied. As a result, both the contents of 2% and 4% of NCC could significantly increase the compressive strength and fracture toughness of CPC. The highest compressive strength was about 27 MPa when the content of NCC was 2%, which had a 50% increase compared that of the control CPC. Setting times of CPC were prolonged with the increased content of NCC, but it still met the clinical requirements when the content of NCC was 2%. XRD and XPS indicated that the combination of NCC with Ca2+ could form the unstable coordination compound and NCC promoted the dissolution and conversion of dicalcium phosphate dehydrate (DCPD) and CaCO3. SEM showed that CPC became denser with fewer pores and cracks by addition of NCC. Meanwhile, the high aspect ratio of NCC might bridge cracks in CPC and play a role of reinforcing and tonghening. In conclusion, the above reasons would lead the significant increasing of compressive strength of NCC/CPC composites. Fluorescence microscope demonstrated that the NCC labeled by 7-amino-4-methylcoumarin (AMC) was not homogeneous dispersed in CPC.The TEMPO-mediated oxidation was applied to modify NCC, which was expected to improve its dispersion in solution. The carboxylated NCC (named CNCC) with maxmium carboxyl content was 0.89 mmol/g under the condition of 0～5℃, pH=10.5 and 14 mg TEMPO per one gram NCC, and then the powder of CNCC was gained by freeze drying. After that,2% NCC and 2% CNCC powders were dispersed in PBS, and then mixed with CPC to prepare NCC/CPC and CNCC/CPC, respectively. As a result, TEM results showed that NCC was about 200nm long and 15nm wide, while CNCC had a length of 180 nm and a width of 10 run. XRD showed that CNCC had the higher crystallinity and a smaller crystallite dimension compared to those of NCC due to the degradation of amorphous components of NCC during the oxidation progress during the oxidation progress. However, CNCC formed the aggregation after freeze-dried and hardly dispersed in PBS solution, which would form some defects in CNCC/CPC. It would result in the poor compressive strength of CNCC/CPC compared to that of NCC/CPC.The in-situ synthesis method was introduced to prepare hydroxyapatite (HA) powder containing 2% NCC and 2% CNCC (to the mass percentage of CPC powders), respectively. They were mixed with other CPC powders to prepare NCC-CPC and CNCC-CPC, respectively. The results showed that HA synthesized in situ with NCC and CNCC were presented as needle-like and petaloid with nanometer size, and the latter had a higher crystallinity. The compressive strength of NCC-CPC and CNCC-CPC was 38 MPa and 45 MPa, respectively, which the compressive strength of the later had a 80% increase compared to that of control CPC, and had a 20% increase compared to that of the former. XRD showed that the dissolution and conversion of DCPD were promoted by adding NCC and CNCC via in-situ synthesis. SEM showed that CPC became denser with fewer pores and cracks by addition of NCC and CNCC in situ. The high aspect ratio of NCC and CNCC might bridge cracks in CPC and play a role of reinforcing and tonghening. Meanwhile, NCC and CNCC could bind well with CPC matrix via chemical bonding. In addition, CNCC might have stronger binding of Ca2+ because of more hydroxyl and carboxyl in its surface compared to that of NCC, and CNCC had better dispersibility and can block the crack propagation more effectively. It would give rise to the increased compressive strength of CNCC-CPC compared that of the NCC-CPC.The response of OB to CNCC-CPC, NCC-CPC and control CPC were evaluated by in vitro co-culture experiment. The results of live cell fluorescence staining and SEM indicated that mounts of OB adhered to the surface of all CPC samples and spread with filopodia and formed cell-cell junctions, presenting variety forms and a strong sense of three-dimension. There were many tiny filopodia closely connected with the material surface, indicating a good cell activity. The results of co-culture experiment demonstrated that there was no significant difference among the Alamar Blue proliferation activity of different CPC samples, so did the Alkaline phosphate (ALP) differentiation activity, which were in time independence. It was indicated that the addition of 2 wt.% NCC and CNCC had no significant influence on proliferation and differentiation of OB.In this study, NCC was incorporated into CPC and it increased the compressive strength of CPC significantly by optimizing the preparation process of NCC/CPC composites, which was proved to have good biocompatibility. This study provides a new method for toughening and reinforcing CPC, which has potential applications.