| ObjectiveInoculation of seed cells into the three-dimensional porous scaffolds is the first and most critical step in construction of the tissue-engineering bone. However, several obstacles inhibits the development of bone tissue engineering technology, which including the limit number of seed cells located in the central areas of the scaffolds, the non-uniform distribution of seed cells and the delay complete of vascular ization of the cells/scaffold compound. In order to apply the bone tissue engineering technology to the clinical usage, it is urgent for us to modify the existing inoculation methods to improve the initial seeding efficiency, to promote the uniform distribution of seed cells and to enhance the mineralization and vascularization of cells/scaffold compounds.In the first part of my study, Chitosin/p-tricalcium phosphate scaffolds were prepared by freeze drying and the safety of the scaffolds were detected.Once impaired by trauma or disease, natural bone tissues initiate the regenerative process to retain their functional integrity by continuously transporting stem cells into the injured site to replace the lost cells through the blood circulation. The currently used inoculation method, in which seed cells are delivered into the scaffold one time in vitro, cannot mimic the bone regeneration in vivo. Consequently, we compared cells proliferation and distribution and vascularization of the cells/scaffold compounds treated by multi-time inoculation methods and one time inoculation method in the second part.It is hard to inoculate seed cells into the central pores of scaffolds by merely relying on the penetration of culture medium with seed cells. Consequently, we designed a new inoculation method-which improved the positive pressure-assisted inoculation method in the third part. Then, we inoculated the seed cells into the scaffolds using the above mentioned novel method and Two-side inoculation method, evaluated cells proliferation and distribution within the scaffold, and also compared the mineralization and vascularization of the cells/scaffold compounds.Methods1. Preparation of chitosin/?-tricalcium phosphate scaffolds and detection of biological compatibility for scaffolds(1). Preparation of chitosin/?-tricalcium phosphate scaffolds using freeze drying(2). Isolate, culture and identification of bone marrow stromal cells (MSCs)(3). Pore size of scaffolds was evaluated by scanning electron microscope(4). Porosity of scaffolds was analyzed by law of Archimedes(5). To analysis the biocompatibility of the chitosin/?-tricalcium phosphate scaffolds, we have cultured MSCs with 1×,5×,0.25× extraction medium of the chitosin/?-tricalcium phosphate scaffolds in vitro and evaluated the viability of MSCs anchored in the scaffolds at different time points(6). MSCs with number of 1×106cells were inoculated into the scaffolds for testing culture. After culture for 1 week, the cells/scaffold compounds were dyed with Acridine orange and observed by the fluorescence microscope to verify whether the scaffolds have a negative effect on MSCs.2. Comparing the cells proliferation, cells distribution, mineralization and vascularization of the cells/scaffold compounds treated by multi-time inoculation and one-time inoculation methods.(1).7.2×105MSCs were inoculated into the CS/?-TCP scaffolds by three, two and one time. After one hour of inoculation, the initial seeding efficiency of cells/scaffold in all groups was evaluated.(2). Cells viability in cells/scaffold compounds was measured by cell counting kit-8 after 0,7,14,21 and 28 days'osteogenic differentiation. Then, the curve of MSCs viability was draw.(3). After 0,7,14,21 and 28 days'osteogenic differentiation, the DNA and protein content of the samples in every group were extracted and measured. ALP activity of the cell-scaffolds compounds was detected and compared. Then, the curves of total DNA and ALP activity were draw.(4). To evaluate whether multi-time or one-time inoculation method has an influence on osteogenic differentiation of MSCs, RT-PCR was used to detect the genetic expression of osteogenic markers (OC, ON and OP).(5). After 0,14 and 28 days of osteogenic-induction, the cell/scaffold compounds were stained with propidium iodide, and observed by laser confocal microscope to evaluate the cells proliferation and distribution in the central area of the scaffolds.(6). The cell/scaffold compounds in all groups were transplanted into the defect area of rabbit's mandible. X ray film and Masson staining was used to evaluate the bone formation at 8 and 16 weeks of operation.3. Comparing the cells proliferation, cells distribution, mineralization and vascularization of the cells/scaffold compounds treated by positive pressure aided inoculation method and two-side inoculation method.(1).10,20,40, and 60 times of positive pressure produced by the plunger of the syringe was performed to make the MSC suspension (7.85×105) penetrate through the entire thickness of the scaffold. As to control group, the same number of MSCs was inoculated into the both surfaces of the scaffold. The initial seeding efficiency of all groups was evaluated at 1h of inoculation. In addition, the cell viability was evaluated by CCK-8 method at 0,3,5,8 and 12 days of inoculation to test whether the positive pressure produced by plunger of syringe has an influence on viability of MSCs.(2). After 0,14 and 28 days of osteogenic-induction, the cell/scaffold compounds were stained with propidium iodide, and observed by laser confocal microscope to evaluate the cells proliferation and distribution in the central area of the scaffolds.(3). The cell/scaffold compounds in all groups were transplanted into the dorsal skin of the nake mice. After 2,4 and 6 weeks of implantation, the samples in all groups were taken out, embedded in OCT.5?m-thick consecutive sections cut from the center area of the cell/scaffold compounds were incubated with the DAPI solution and observed by fluorescence microscope to evaluate the cells proliferation and distribution in the central area of the scaffolds.(4). Voncossa staining was performed to evaluate the mineralization of cell/scaffold compounds.(5). To evaluate the influence of the two inoculation methods mentioned above on the host's vascularization, immunofluorescence of CD31 was undertaken.Results(1).We have finally fabricated the Chitosan/?-tricalcium phosphate scaffolds (Chitosan:?-tricalcium phosphate=4:2.7) with porosity of 87.5%±4.1% and pore size ranges from 80?m to 130?m. The extracted medium with different concentration has been proved nontoxic to MSCs according to the results of CCK-8; there are a large amount of cells with yellow fluorescence located in the scaffolds which observed by fluorescence microscope. The cells we isolated from rabbits can be induced into osteoblasts, chondrocytes and adipocytes, which proved that the cells we extracted are MSCs.(2). Initial adhesion ratio of three-time inoculation group is 81.9%±0.03%, which is higher than that of two (71.5%±0.02%) and one (60.3%±0.06%) time inoculation method. MSCs in the three-time group expressed a lower viability than those in the two-time and one-time groups at 3 and 5 days of osteogenic induction, and a higher viability than those in the two-time and one-time groups at 8 and 12 days of osteogenic induction. At 0 day of osteogenic induction, the number of MSCs in the one-time group was larger than that in the two-time and three-time groups. From days 21 to 28 after osteoinduction, a higher number of MSCs was found in three-time group than that of two-time or one-time groups. Furthermore, two-time group also showed higher cell numbers than the three-time group during the entire culture period, with the exception of day 28, when the three-time group exhibited higher DNA content. At all-time points, the cells/scaffold compounds in three-time and two-time groups were all showed a more uniform cells distribution than those in one-time group. During the entire culture period, ALP activity and the gene expression of OC, ON and OP in the three-time and two-time groups were all higher than that of the one-time group. The new bone density of the treated defects in three-time and two-time groups was higher than that of one-time group according to X ray images. Furthermore, new bone formation and reconstruction of the defect area in three-time group was fastest among all groups, two-time group ranked second and one-time group was the last.(3). Initial adhesion ratio of two-side inoculation group is 79.42%±9.87%, and 10 times of positive pressure accounts for the lowest seeding efficiency: 53.74%±9.96%. With the increase times of positive aspiration, the ratio of initial seeding efficiency is also increased.20,40 and 60 times of positive pressure reached the ratio of 92.63%±3.17%,95.26%±4.78% ?97.14%±1.30%. However, CCK-8 results showed that 40 and 60 times of positive pressure severely impaired the viability of MSCs.20-time positive aspiration of MSCs resulted in a relatively lower viability and initial seeding efficiency as compared with that of other groups. In the following experiments,20-time positive aspiration was chosen by us. During all time points, the cells viability, cells distribution, mineralization and vascularization in cells/scaffold compounds in the positive pressue-aided inoculation group were all better than those in two-side inoculation group according to the results of Voncossa staining and CD31 immnofluorescence.Conclusion(1). The Chitosan/?-tricalcium phosphate scaffolds we fabricted in this study have good biological compatibility, its pore diameter and porosity meets the requirements of bone tissue engineering scaffolds.(2). Multi-time inoculation methods have the following advantages over one-time inoculation method:(1) improve the seeding efficiency; (2) promote the proliferation of cells; (3) resulted in an even distribution of MSCs within the scaffold; (4) enhance the resorption of the scaffold; (5) promote the formation and reconstruction of bone.(3). The positive pressue-aided inoculation method used in this study resulted in a higher number and seeding efficiency of seed cells, a more uniform distribution of cells within the scaffold, enhanced graft vascularization and mineralization of the scaffolds when compared with two side inoculation method. To the authors' knowledge, it is a simple and high efficiency inoculation method for bone tissue engineering. |
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