Osteoinductive Properties Of The Ultrafine Silk Fibroin Fibers Loaded With A Small Molecule Purmorphamine

Silk fibroin(SF) is a kind of well- liked natural biological mater ial due to its outstanding comprehensive performance. Electrospinning SF into ultrafine f ibers to mimic the native extra cellular matrix(ECM) has been extensively studied for applications in bone tissue engineer ing(BTE). In combination with the regenerative stem cells, electrospun-SF fibers are anticipated to improve the regeneration efficacy of BTE methods for repairing bone defects. It is thus imperative to improve the osteoinductive potent of the electrospun-SF scaffolds. Purmorphamine(PM) belonging to the type of purine derivatives, is a new small molecule drug with excellent osteoinductive property. Based on the electrospun-SF fibers, the main objective of this thesis research is to explore the osteoinductive potential of the electrospun-SF fibers after the introduction of PM.Firstly, in order to examine the osteoinductive attribute of SF itself, SF was prepared and characterized from silkworm cocoon by the classical Na2CO3 degumming method, and bone marrow mesenchymal stem cells(BMSCs) were isolated and characterized from the rat tibia by the well-documented whole bone marrow adherent method. Then, we prepared culture media containing different concentrations of SF(0.01%, 0.05% and 0.1%) to culture BMSCs and tested the growth, proliferation and osteogenic differentiation of BMSCs in vitro. The results obtained are stated as follows: 1) As FTIR spectra of the sample extracted from silkworm cocoon showed distinct absorption peaks at 1653(amide I), 1530.5(amide II) and 1212.3 cm-1(amide III), the extracted substance could be confirmed to be SF. For the generated cells, the markers of MSCs, e.g., CD29, CD44 and CD90 reached up to high levels of 97.65%、99.01% and 99.52%%, respectively. Whereas the marker of hematopoietic stem cell, CD45, was as low as 0.07%. Meanwhile, the cells could differentiate into osteocytes, chondrocytes and adipocytes under specific induction conditions, which further confirmed the extracted cells being BMSCs; 2) 0.05% SF was not only in favor of cell adhes ion, migration and proliferation, but also promoted the ALP expression in BMSCs. This helps to draw a conclusion that SF itself has the osteoinductive capability with its effects dependent upon the mass concentration of SF. These results allow SF to be used as a kind of osteoinductive materials for constructing functional scaffolds.Secondly, based on the parallel structure of collagen fibers in the natural bone(cortical bone), we prepared ultrafine electrospun-SF fibers(containing a little bit of polyethylene oxide, PEO, for the goodness of electrospinnability) with high degree of orientation by the stable jet electrospinning(SJES). Then the SF fibers were sequentially treated in aqueous methanol solution(90%) to realize transformation in secondary structure of SF for stabilizing the fibrous structure in aqueous environment, and in water to leach PEO out of the fiber matrix. Results showed that mechanical properties of the SF/PEO fibers were improved tremendously after soaking in aqueous methanol. However, PEO-leaching resulted in weakened mechanical performance but increased hydrophilicity. Cellular compatibility evaluation results showed that compared to methanol-treated SF/PEO fiber scaffold, the PEO-leached SF fibers supported much better BMSC adhes ion, spread, and growth along the fiber orientation direction. In terms of osteoinductive effect, it was found that although the aligned SF fibers had the same parallel structure as that of collagen fib ers in bone, and promoted cell proliferation, it was inferior in stimulating secretory expression of ALP in BMSCs. In contrast, the non-woven SF fibers were proved to be advantageous in promoting the osteogenic differentiation of BMSCs.Based on the above results, hydroxyapatite(HAp) was introduced to enhance the mechanical and biological properties of the non-woven SF fibers because of its excellent biocompatibility, osteoconductive and osteoinductive nature. The optimum amount of HAp to generate HAp/SF composite fibers was determined from considering the effects on mechanical and osteoinductive properties of the HAp/SF fibers. Results showed that: 1) HAp could disperse well in the SF fibers and there existed chemical interactions between the carboxyl groups of SF and the HAp. Compared with the ?plain‘ SF fibers, addition of 10% HAp gave rise to signif icant improvement in the Young’s modulus of the HAp/SF fibers, reaching the best increasement of up to 5.58 times, but its hydrophilicity was declined; 2) The addition of 10% HAp could not only improve the adhesion, migration, proliferation and spread-out of BMSCs, but also promoted secreting ALP and COL proteins and mRNA expression of ALP, COL, OCN and ON in BMSCs, indicative of its excellent osteoinductive capability.Finally, PM was introduced to further enhance the osteoinductive capacity of the HAp/SF composite fibers by performing osteoinductive assays, from which the underlying mechanism was verified as well. In order to detect the best concentration of PM in inducing the osteogenesis of BMSCs, the culture media containing different concentrations of PM(0.1, 3 and 10 μM) were prepared to culture BMSCs and its osteoinductive ability was compared with the conventional osteogenic supplements including dexamethasone, ascorbic acid and β-glycerin sodium phosphate. Results showed that 3 μM was the optimum PM concentration to induce osteogenisis of BMSCs and comparable to that of the conventional induc ing system. This means that PM could replace the conventional osteogenic supplements in BTE. Afterwards, we explored the best loading amount of PM in prepar ing PM/HAp/SF fibers, and evaluated the effect of PM concentration on the physical properties(including morphology, mechanical properties and hydrophilic ity), PM release behavior and cytocompatibility. The obtained results are: 1) Introduction of PM benefited the reduction in average diameter of fibers, but led to a slight decline in mechanical and hydrophilic properties; 2) HAp component in the fiber matrix played an important role in realizing sustained release of PM from the PM/HAp/SF fibers; 3) Addition of PM was conducive to cell adhes ion, spread-out and proliferation. Finally, we tested the osteogenic differentiation and working mechanism of BMSCs on two types of fibers, i.e., 10-PM/HAp/SF and 20-PM/HAp/SF. It was found that PM and HAp could synergistically promote the expression of ALP, COL, OCN and ON genes, and it was the Hedgehog signaling pathway that could mainly activate the osteogenic differentiation of BMSCs.In conclusion, this thesis confirmed the intrinsic osteoinductive capability in SF per se. Aligned ultraf ine SF fibers resembling the parallel fiber structure of natural bone(compact bone) were successfully prepared via SJES. However, its osteoinductive ability was not good as compared to non-woven SF fibers. Introduction of 10% HAp was found to be beneficial to improving the mechanical and osteoinductive properties of the SF fibers. The PM concentration of 3 μM was optimal to induce the osteogenic differentiation of BMSCs. After introducing PM into the HAp/SF fibers(i.e., PM/HAp/SF), osteoinductive capacity of the HAp/SF composite fibers was signif icantly improved by activating the Hedgehog signaling pathway. All the results show that the fibrous PM/HAp/SF scaffolds could significantly induce the osteogenic differentiation of BMSCs. This study laid a solid foundation for moving forward to in vivo animal test with the new biomimetic fibers of PM/HAp/SF, showing great potential in BTE applications.