| Bone defects are a common clinical disease. Small bone defects can be regenerated by bone tissue around the defect, but when the defect exceeds bone regeneration ability, non-union will result, which seriously affects limb function. However, treating bone defects is sometimes problematic. The traditional treatments for bone defects are autologous bone graft transplant and allograft transplant, but because of the limited availability of bone, the immune response, and athic issues, autologous bone graft transplant and allograft transplant methods cannot be widely used for bone defect treatment, especially for large bone defects.Today, scientists are greatly interested in the field of bone tissue engineering as a method for the treatment of bone defects. Currently, bone tissue engineering research is focused on biological scaffolds, seed cells and growth factors.Layer-by-layer paper-stacking is a novel and simple way to transform a scaffold from a two-dimensional structure to a three-dimensional scaffold. This three-dimensional structure can change the relationship between the scaffold and the cells, making more space and providing proper biological microenvironments.Another technique used to form stable nanofiber scaffolds using a high voltage electric field with polymers are known as electrospinning. It has rapidly proliferated in recent years in the tissue engineering field. The current electrospinning technique is commonly used in constructing the extracellular matrix or tissue engineering scaffold because of the small diameter of the resulting fiber and similar nano-level structures of the fibers of the extracellular matrix, which is typically measured in nanometers (less than the diameter of a cell).Polycaprolactone (PCL) has good mechanical properties and it is biocompatible. Gelatin has good biocompatibility and cell adhesion ability. By combining both of these materials, we can build a scaffold which has certain mechanical properties and good cell adhesion, making it appropriate for tissue engineering scaffold applications.Adipose-derived mesenchymal stem cells (ADSCs) are currently promoting stem cells for tissue engineeringand attract the attention of much scientific attention and research. The ADSCs have the ability of self-renewal and proliferation, as well as the ability to differentiate multi-directional pluripotent cells under certain conditions. They can proliferate and differentiate into adipocytes, osteoblasts, fibroblasts, neurons, and vascular endothelial cells. Also, the adipose has a rich source and the ADSCs can be simply isolated, easily cultured and their usage does not result in ethical conflict. ADSCs are currently the most promising stem cells in the tissue engineering field.In this study, we use PCL and gelatin as raw materials, and apply the electrospinning technique to construct the electrospinning nanofibrous membrane. The resultant electrospinning membrane is highly porous and has fiber uniformity. The diameters of the fibers are almost in nanoscale. We seeded ADSCs on a PCL/gelatin electrospun nanofibrous membrane (NFM), and investigated the compatibility of the cell proliferation on the NFM. The results showed that the NFM has good biocompatibility. The ADSCs grew well on the NFM; they had good adhesion and growth, ample proliferation, and were normally shaped. We constructed the three-dimensional scaffold with ADSC-laden NFM in a paper-stacking layer by layer way, and detected the viability of the ADSCs within the scaffold using live/dead staining. The result showed that most of the ADSCs can survive within the 3D scaffold. The cell proliferation test was carried out using the CCK-8 kit, and the ADSCs showed a significantly higher proliferation rate after 24 hours than the control group.In the first 12 hours, the ADSCs laden NFM group had a lower proliferation rate than the control group, but after 24 hours, the proliferation rate of the ADSCs- laden NFM group was significantly increased, and was higher than the control group. The ADSCs-laden NFM were cultured to detect the osteogenic differentiation ability of the ADSCs, and the results showed that the ADSCs on the NFM secreted OCN, OPN and OPG proteins. The ADSCs-laden NFM paper-stacking layer-by-layer three-dimensional scaffold, which cultured in the osteogenic medium bone, had the highest osteogenic gene expression and best osteogenic differentiation effect. We implanted the ADSCs-laden NFM paper-stacking layer-by-layer three-dimensional scaffold to treat the skull defect in order to detect the osteogenic ability of the scaffold. The gross specimens, micro-CT results, and the results of the histological sections showed that the ADSCs-laden, NFM paper-stacking layer-by-layer three-dimensional scaffold has good osteogenic ability and bone repair capabilities.Part I:The identification and osteogenic effect investigation of mesenchymal stem cells from rat inguinal subcutaneous fatObjectives:To identify ADSCs from rat inguinal subcutaneous fat to ensure its purity and to investigate ADSCs osteogenic ability by culturing them in an osteogenic mediumMethods:We used three two-month SD rats (purchased from Southern Medical University Experimental Animal Center), with an average weight of 227g±14.8g. After being anesthetized with the 10% chloral hydrate intraperitoneal injection (0.2ml/100g), they were sterilized with 70% alcohol. Following the routine surgical operation, we made incisions on both sides of the inguinal, exposing the subcutaneous adipose tissue, and isolated it. Fatty tissue was mashed into small pieces and digested. The ADSCs were isolated for the primary culture. All the ADSCs were undergone the following tests:(1) Proceding morphological observation under the microscope; (2) The ADSCs were identified by flow cytometry with the cell-surface-markers such as CD29、CD90、CD49d、CD45、CD11 and CD106; (3) All the ADSCs were proceded osteogenic induction (cultured in the osteogenic medium), ALP staining and Alizarin red staining were done in order to test the osteogenic ability of ADSCs.Results:(1) Under the microscope, we could see the ADSCs were about 50-100 μm in diameter. They had large cell sizes, large nuclei, and abundant cytoplasm. Also they were polygonal, spindle-shaped, and spirally arranged after cell fusion. (2) Flow cytometry tests on ADSCs:the ADSCs’ characteristic surface antibody positive rate of CD29 (99.65%), CD90 (68.38%) and CD49d (86.82%), and the ADSCs uncharacteristic positive rate of antibodies:CD45 (0.06%), CD11b (0.24%) and CD106 (0.84%). (3) ALP staining results were shown:ADSCs cultured in the osteogenic medium were dyed gray, and the result was positive. Alizarin red staining results were shown:most of the ADSCs were stained red, the calcium node could be seem in amount of the site view, the calcum nodes were scattering distribution, and the center of the nodes were stained deeply.Conclusions:(1) ADSCs provide a rich source, are easily isolated, easy to culture, and require no special culture conditions, making them favorable for applications; (2) We isolated the ADSCs from the rat inguinal subcutaneous fat, and found the morphology and phenotype characteristics of the cells to be similar to the ADSCs (3) ADSCs isolated from the rat inguinal subcutaneous fat are pure and they can be used in the tissue engineering field; (4) ADSCs from rat inguinal subcutaneous fat have good osteogenic differentiation ability.Part II Electrospun nanofibrous membrane scaffold construction biological propertiesinvestigation and their function in ADSCs’osteogenic ability in vitroObjectives:Using electrospinning technology to build electrospun nanofibrous membranes and to detect its biocompatibility and osteogenic differentiation ability. We applied the NFM to fabricate paper-stacking layer by layer scaffolds and detected their function on ADSCs’ osteogenic ability in vitro.Methods:In this study, the raw materials of PCL and gelatin were dissolved in trifluoroethanol at a concentration of 10% respectively. Then, the two solutions were mixed at a 1:1 ratio. We fabricated NFM using the electrospinning technique. (1) The NFM was observed and detected, and its morphology and porosity were also investigated. (2) The ADSCs were planted on the NFM and we investigated the biocompatibility of NFM and detected the ADSCs’ proliferation ability within the NFM paper-stacking layer by layer scaffolds. And evaluate the proliferation of ADSCs in NFM layers assembled on scaffolds using factorial ANOVA design information for statistical analysis and application of two independent samples t-test to compare two groups of four difference time points. (3) In order to see the fibrous structure, we tested the NFM using scanning electron microscopy. We measured its fiber diameter and observed the morphology of the cells on the NFM. (4) We applied live/dead staining technique to detect the viability of ADSCs on the NFM paper-stacking layer by layer scaffolds. (5) The ADSCs laden NFM papers were cultured in the osteogenic medium, the osteogenic proteins (OCN, OPN and OPG) were investigated with the fluorescence staining technique. (6) Also, the ADSCs laden NFM layer by layer paper stacking structure were incubated in the osteogenic medium and osteogenic gene expression (OCN, OPN, OPG, BSP, RUNX2 and OSX) were detected. The osteogenic ability of ADSCs on the NFM paper-stacking layer by layer scaffolds was evaluated. Univariate analysis of variance and independent samples t-test and other statistical methods were used to compare the value in each group, when a single sample compared with the control group, we used a single sample t test for analysis, P<0.05 was considered significantly different. Evaluate the effect of the three-dimensional scaffold for osteogenesis. Unless otherwise specified notification, each test were repeated in three biological replicates, repeated measurements for each sample were at least three times.Results:(1) Morphology:NFM’s thickness is about 50 μm and it has certain mechanical properties. (2) Scanning electron microscopy shows overall NFM uniform fiber diameter, as well as a few differences between the fiber diameters, about 500- 1500nm, with nanofibers intertwined. The pore sizes of the NFM are about 10-50μm, and the pores are interwoven. ADSCs can be seen on the NFM, and the cells were stretched and polygonal on the NFM. There were some links between cells and fibers; cells adhered on the fibers, and some grew into the pores. (3) ADSCs rapid proliferation on the NFM, after the 24-hour time point was significantly higher (t48h=-7.723, P48h=0.002;t72h=-14.912, P72h<0.001, Fgroup=1451.881,Pgroup<0.001, Ftime=69.878, Ptime<0.001。 interact effect:F=48.482, P<0.001). (4) ADSCs were cultured on the NFM at two time points (two weeks and four weeks). Immunofluorescence staining was conducted, and the result showed there were OCN, OPN and OPG proteins expressed. (5) At 1,2,3 and 4 week time points, the osteogenic gene expressions (OCN, OPN, OPG, BSP, RUNX2 and OSX) of ADSCs laden NFM paper-stacking layer by layer scaffold cultured in the osteogenic medium group were significantly higher than the other groups OCN(F3w=54.049, P3w<0.001; F4w=60.811, P4w,<0.001)、OPN(F3w=47.975, P3w<0.001; F4w=67.588, P4w<0.001)、 OPG(F3w=28.306, P3w<0.001; F4w=151.551,P4w<0.001,)、BSP(F5W=84.607, P3w<0.001; F4w=216.219, P4w<0.001)、RUNX2(F3w=10.950,P3w=0.003 F4w=4655.158, P4w<0.001) 及 OSX(F3w=2314.216, P3w<0.001;F4w=1405.869, P4w<0.001), significant difference.Conclusion:(1) The NFM fabricated with PCL/gelatin had good compatibility, the cells attached well to the NFM, and grew well; (2) The NFM has extensive porosity, which contributes to the diffusion of the nutrition and the production of the cells. Its fibrous structure is similar to the extracellular matrix and it can be used as an extracellular matrix to promote cell growth. (3) ADSCs can grow well on NFM and there were proteins secretion (OCN, OPN and OPG) when the ADSCs laden NFM were cultured in the osteogenic medium. (4) The ADSCs laden NFM paper-stacking layer by layer scaffold can promote ADSCs differentiated into osteoblasts, and promote the expression of osteogenic genes that are vital for bone repair.Part Ⅲ:ADSCs laden NFM paper-stacking layer by layer 3D scaffold for parietal defects repair in ratsObjective:To implant the ADSCs laden NFM paper-stacking layer by layer scaffold in the parietal defects in rats, and investigate the osteogenic effect of the scaffoldMethods:(1) We fabricated the ADSCs laden NFM paper-stacking layer by layer scaffold as shown in Part II. (2) The above 3D scaffold was planted into a rat parietal defect.12 weeks later, the parietal gross specimen was observed. (3) All the samples were scanned with micro-CT to get the bone mineral density data of regenerated bone in all the samples. All the scanning section images of the samples were reconstructed into a three-dimensional image for detecting. (4) HE staining and Masson staining were done for bone repair investigation in each group. (5) RT-qPCR experiments were carried out for clarifying the osteogenic gene expression in each group, use the independent-sample t test to do the statistical analysis and investigated the differences between the groups.Results:(1) The gross images of samples in each group were compared: samples from the ADSCs laden NFM paper-stacking layer by layer scaffold repair group have good results, and their surface is flat, with no residual defects in the original defect place. This group best repaired the defect. (2) As the micro-CT results showed, the mineral density of the regenerated bone in the ADSCs laden NFM paper-stacking layer by layer scaffold group was 823.047mg/cm3, but it is higher when compared with the other two groups(in the defect group, the BMD is 58.348±4.435, in the NFM group, the BMD is 610.666±51.733, in the ADSC-NFM group, the BMD is 823.047±47.010, compared with each group F=857.565, P<0.001, the difference is significant). In the ADSCs laden NFM paper-stacking layer by layer scaffold group, the three-dimensional reconstruction of the skull defect area has been largely repaired, and the repaired tissue was without obvious sense steps. (3) HE staining and Masson staining results showed ADSCs loads generated the structure most similar to normal bone and which repaired most effectively. (4) The RT-qPCR experiments were carried out for osteogenic gene detection. As shown in the images, osteogenic gene expression in the ADSCs laden NFM paper-stacking layer by layer group was higher than NFM group OCN (t=13.355, P=0.006), OPN (t=22.535, P=0.002)% OPG (t=22.67, P=0.002);BSP (t=24.268, PBSP=0.002)^ RUNX2 (t=8.510, P=0.014), OSX (t=12.722, P=0.006) when compared with NFM group OCN (t=-7.815, P=0.001). OPN (t=-10.288, P=0.002), OPG (t=-5.528, P=0.05)、BSP (t=-22.721,P<0.001)、RUNX2 (t=-7.053, P=0.001) and OSX (t=-9.441,P=0.001)Conclusions:(1) ADSCs can differentiate into osteoblasts when they are cultured in the paper-stacking layer by layer 3D scaffold, and this scaffold can promote bone formation. (2) ADSCs laden NFM paper-stacking layer by layer scaffolds can be used in bone tissue engineering for bone defects repair. (3) ADSCs laden NFM paper-stacking layer by layer scaffolds can improve the ADSCs ability to differentiate into osteoblasts. |
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