| In the preparation of reparation materials for bone regeneration, the ideal materials should mimic the chemical and topographical structure of native bone tissues, in which, nanoscale hydroxyapatite crystals grow along the orientation of collagen nanofibers to form organic-inorganic composite material. This kind of special structure provides bone tissues with excellent mechanical and biological properties. Biomineralization in vitro is usually considered as a good method to prepare bone reparation materials. In this study, several simulated body fluids (SBF) were designed and used to perform the biomineralization study on nanofibers, with the purpose to extend the understanding of the in vivo biomineralization and to provide guide for the preparation of organic-inorganic composite materials for bone regeneration.At first, composite nanofibers containing poly(L-lactide) (PLLA) and gelatin were prepared via blend electrospinning to mimic the natural nanofibrous structure in native bone extracecullar matrix. PLLA/gelatin nanofibers were then crosslinked using chemicals of EDC and NHS. A proper mechanical stability and hydrophilicity, thus, they could be used in the following biomineralizaiton study. After a series of preliminary experiments, 2.5 times SBF was chosen for the study for its relatively fast apatite formation within one week. Three amino acids with different charge characterics, i.e. glycine (neutral), aspartic acid (acidic) and arginine (basic), were added into the 2.5SBF separately. The pre-nucleation behaviors in these systems were investigated using methods of freeze-drying and HRTEM observation after 8 hrs standing. The control 2.5SBF and 2.5SBF-Gly demonstrated the formed aggregations in an ordered pattern of nano-scale clusters. While the other two displayed random net-like structure of aggregated clusters. The sizes of the clusters in the four SBF systems were found 4.7nm (2.5SBF-blank),4.4nm (2.5SBF-Gly),8.8nm (2.5SBF-Alu) and 9.3nm (2.5SBF-Arg), respectively. These facts revealed that amino acids were able to participate in the formation of prenucleation clusters and thus might influence the following crystal growth. Subsequently, PLLA/gelatin nanofibers were immersed in the four aforementioned SBF solutions. Macroscopically, the deposited minerals demonstrated minor difference in appearance, chemical composition and crystalline structure. The depositons were identified calcium-deficient (Ca/P=1.4-1.5) HA by characterizations of SEM、XRD、FTIR andEDX. However, subtle difference in crystal growth was found via further HRTEM observation and SAED evaluation. In the presence of amino acids, the transformation from amorphous calcium phosphate to crystalline HA was enhanced, leading the orientated growth of needle-like HA along.Secondly, several cell culture media were used for the biomineralization study. By soaking the PLLA/gelatin nanofibers in a-MEM, a layer of apatite was detected on fiber surface, which displayed different morphology to those in the previous SBF study. The apatite layer attached well to the PLLA/gelatin fiber. The Ca/P ratio obtained in culture media was found a little higher (Ca/P ～1.8) that those from SBF study, which revealed the difference in inducing mineralization from different solutions. The addition of fetal bovine serum (10%) into a-MEM, the biomineralization was found being accelerated that the growth rate of crystalline HA was increased.In summary, the incorporation of compounds as amino acids or protein into SBF, might be helpful in preparing biomimetic organic-inorganic materials for bone regeneration via in vitro biomineralization. |
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