Preparation, Characterization, And Function Study Of Hepairnized CNTs/Collagen/HA Composite Scaffold

Bone tissue culture scaffold is one of the hot spots in the field of tissue engineering.Collagen/Hydroxyapatite (Col/HA) composite scaffolds attracted many attentions due tosimilarity of their structure and composition to natural bone tissue, good biocompatibility aswell as bioactivity. However, the low mechanical strength and easily degradation naturegreatly limited their clinic applications. Carbon nanotubes (CNTs) are well known to be ableto increase the mechanical properties of many polymeric materials, but they are highlyhydrophobic and trend to aggregate, hard to be dispersed in aqueous solution and cytotoxic,which hinder their applications in tissue engineering. This work aimed to prepare heparinizedCNTs and use it to improve the mechanical property and biological function ofCollagen/Hydroxyapatite (Col/HA) composite scaffolds.Firstly, the carboxylic groups on single-walled carbon nanotubes (SWCNTs) surfacewere acylated, aminated stepwise and finally reacted with heparin using EDC as the catalystto immobilize heparin on SWCNTs surface via covalent bond. FTIR, Carbazole assay andZeta potential measurements indicated that heparin was successfully immobilized. Theamount of heparin on SWCNTs surface was measured to be257.53μg/mg, and heparinized–SWCNTs (SWCNTs-Hep) were negatively charged. TEM results found that SWCNTs-Hepwere well dispersed in aqueous solution and stable in a long period without any aggregation.The anti-Xa activity and APTT assay found that SWCNTs-Hep had significant anticoagulantactivity. MTT assay revealed that SWCNTs-Hep have no cytotoxicity. Taken together,immobilization of heparin on SWCNTs will not only improve its solubility and stability inwater, but also endow its excellent biocompatibility.Next, SWCNTS-Hep with a mass concentration ranges from0.4%to1.6%was mixedwith Col to prepare SWCNTs/Col composite scaffold. Adding SWCNTs into Col solutioninduced gelation, suggesting the form of physical hydrogel due to the electrostatic interactionbetween SWCNTs and Col. SEM observation showed that the Col/SWCNTs-Hep spongespossess homogeneous and orderly porous microscopic structure, which had lager pore sizethan that of SWCNTs-Col sponge, and tendency to form gel membrane. After the treatment ofSBF solution, more HA crystal formed on the Col/SWCNTs-Hep sponges, suggesting a bettermineralization. Mechanical property test found that the scaffold’s tensile strength, breakingelongation and compressive modulus were all improved significantly by SWCNTs-Hep, betterthan SWCNTs-COOH. Physic-chemical characterization showed that sponges have greatswelling properties. Col/SWCNTs-Hep sponges did not cause hemolysis in vitro，and werenon cytotoxic. Further study indicated that SWCNTs-Hep and Col/SWCNTs-Hep scaffoldcould not only promote BMSCs cell proliferation but also induce it differentiation to bonecells.