| Background:Bone defect caused by trauma, infection, bone tumor and other diseases needs bone repair and reconstruction. Clinically, bone graft can provide mechanical support for the body and promote bone regeneration. At present, the number of cases that need bone transplantation each year in the world is huge, and it is next to blood transfusion. Among treatments of bone defect, Autologous bone transplantation is still considered to be the gold standard. However, there exists some shortcomings of autologous transplantation, such as limited supply of bone, lengthened operation time, donor bone defect, pain, bleeding and some related complications. Allograft bone transplantation is also common used in clinical, but it also has disadvantages like limited supply, risk of rejection and spread of disease. With the gradual increase of the population, incidence of traffic accident is gradually rising. incidence of bone disease, trauma and infection caused by trauma is also increasing. Meanwhile, massive bone defect can be caused by these diseases, therefore, clinical demand for bone graft is increasing. We need an ideal material to act as alternative of autograft or allograft bone in reconstructive surgery. With the rapid development of tissue engineering, breakthroughs have been made in biomaterials research and fabrication technology. The researchers are trying to fabricate appropriate scaffold materials for bone tissue engineering through tissue engineering related techniques, in order to replace clinical used autograft bone and allograft bone. Researches of tissue engineering mainly focus on scaffolds, seed cells and bioactive factors.The ideal bone repair materials should have good biocompatibility, considerable mechanical strength, biodegradability and non-toxic degradation products. At the same time, bone substitute materials should also have good osteogenic properties. At present, there are several kinds of materials most commonly used:1.inorganic materials like beta tricalcium phosphate, hydroxyapatite, calcium phosphate cement and bioactive glass etc..2. Natural polymeric materials like collagen, chitosan, fibrin, alginate and hyaluronic acid etc..3. Synthetic polymeric materials like polylactic acid, polyglycolic acid, copolymer of polylactic acid and polyglycolic acid, polycaprolactone, polyhydroxybutyrate and poly phosphazenes etc..4. Composite material consisted of two or more than two kinds of single material according to a certain proportion. It has the advantages of each single material, and conforms to the requirements of bone tissue engineering scaffolds.At present, electrospinning technique is now attracting more and more researchers’ attention. The polymer solution is fabricated into nano-fiber mat with special morphology in a high voltage electric field. The diameter of fibers in fiber mat is small, and reaches the level of nano-scale. And the structure of the fiber mat is similar to the structure of the body’s extracellular matrix, which can simulate the living environment of the cells in physiological conditions. So the electrospinning technique has been applied in the bone tissue engineering as a technique to fabricate extracellular matrix or scaffold.PCL is a kind of polymers, which not only has good biocompatibility, but also has good mechanical properties. Gelatin is a derivative of collagen, which has good biocompatibility and hydrophilicity, and it is good for cell adhesion. The researchers usually combine these two materials together to fabricate composite scaffold with good mechanical properties and good adhesion properties for cells in tissue engineering.Because of its strong self-renewal, proliferation and differentiation ability, Adipose derived stem cells (ADSCs) has become the hotspot of the study of seed cells in tissue engineering. It has the potential to differentiate in multiple directions. Under different conditions, it may be differentiate into osteoblasts, adipocytes, fibroblasts, nerve cells, vascular endothelial cells respectively. ADSCs have many advantages, such as easy to isolate, rich in sources and convenient to cultivate, moreover it does not involve the problem of the ethics and morals. So ADSCs has great application prospect in tissue engineering.Hydroxyapatite is an important part of bone tissue, which can provide strength support for bone. Previous studies have shown that it can promote the proliferation and osteogenic differentiation of ADSCs, and Hydroxyapatite has been widely used in bone tissue engineering. The natural bone contains not only inorganic part like hydroxyapatite, but also contains collagen and various bioactive factors. At present, bone tissue, as a whole, has not yet been applied in bone tissue engineering. And there is no research on combining bone powder with nanofibers to form bone tissue engineering scaffolds by electrospinning.Therefore, in this study, we are trying to obtain bone powder and combine bone powders in gelatin/PCL fiber by electrospinning to form a bone tissue engineering scaffold. This scaffold not only has good biocompatibility, but also has a variety of bioactive factor to promote adhesion, proliferation and osteogenic differentiation of ADSCs. Based on the above background, we put forward the following hypothesis: bone powder can be united into gelatin/PCL nanofiber by by electrospinning technology; Gelatin/PCL/bone powder fiber mat has good compatibility and for ADSCs grow, adhere and proliferate. If the hypothesis is correct, we will obtain an ideal scaffold for bone tissue engineering. And this research can provide new ideas for the preparation of scaffold in bone tissue engineering; it has great significance for the further study of natural bone in fundamental and clinical orthopedics.Objective:To prepare porous, biomimetic bone tissue engineering scaffold by electrospinning technology. To detect of the surface characteristics of prepared scaffold by transmission electron microscopy(TEM) and scanning electron microscopy (SEM); To isolate adipose-derived stem cells from SD rat inguinal fat by enzyme digestion, and culture ADSCs with prepared porous, biomimetic scaffold to study the biocompatibility of these scaffolds. To obtain an ideal bone tissue engineering scaffold with good biocompatibility, high porosity and good properties for ADSCs to grow, adhere and proliferate through this study and to give insight into the fabrication of scaffold in future fundamental and clinical study.Methods:1. Preparing materials and scaffold:Prepare natural bone powder by using an automatic freeze grinding instrument. Gelatin and PCL were dissolved in TFE to obtain a 10 wt% solution separately. We make a 1:1 mixture of the gelatin and PCL solutions, we get solution A, then solution B was prepared on the basis of solution A when 10 wt% of nHA was added in, and C was prepared on the basis of solution A when 10 wt% of bone powder was added in. Three types of scaffolds were fabricated by electrospinning and sterilized for later use.2. Detection of materials and scaffolds:We prepared a small amount of nHA and bone powder for SEM detection, also three kinds of fiber mats were collected and sent out for SEM detection. For TEM detection, fibers of each group were collected by Copper omentum.3. Isolation and culture of ADSCs:ADSCs were isolated from inguinal adipose tissue of SD rats by enzymatic digestion, and the following experiments were carried out by using cells of passage three.4. Scanning electron microscopy detection:After 2 days of co culture scaffolds with cells on them were taken out of culture medium in each group. Treated with gradient ethanol dehydration, and the samples were lyophilized and detect by scanning electron microscopy (SEM).5. Cell proliferation assay:For the cell proliferation assay, the cells were seeded on scaffold (6mm) in 96-well plates. Cell proliferation was assayed by using Cell Counting kit-8 according to the manufacturer’s protocol at time point 12h,24h,48h, and 72h. OD values were measured at 490 nm.6. Cell viability analysis:Firstly, agarose solution was processed by autoclave sterilization, before solidification, agarose solution was added into 24-pore plate to prevent cell adhesion. Then, scaffolds with diameter of 8mm of each group were placed on the bottom of the 24-pore plate. ADSCs were seeded on scaffolds, after co-culturing for 48, scaffolds of each group were taken out and washed with PBS. Then cells were stained using live/dead staining kit according to instructions. Finally, the processed samples were observed under inverted fluorescence microscope, and photos were taken randomly. Living cells on scaffold were counted by using Image-Pro Plus 6.0 software.7. Statistical analysis:All assays were repeated with a minimum of three times independently, determinations for each data point and data were presented as mean±standard deviation (SD). Data were analyzed by SPSS 20.0 software and statistical differences were assessed by one-way analysis of variance (ANOVA), followed by a post hoc Tukey’s Test. Value of P< 0.05 was considered as statistically significant.Results:1. Bone powders we use in this study were ground to micro-scale and nano-scale. The diameter of nHA bought from Sigma (USA) is smaller than 200nm and they are in round shape. Three types of scaffolds were successfully fabricated by electrospinning method, namely gelatin/PCL fiber mat, gelatin/PCL/nHA fiber mat and gelatin/PCL/bone powder fiber mat. According to TEM images, nHA and bone powder were successfully united in fibers of gelatin/PCL/nHA fiber mat and gelatin/PCL/bone powder fiber mat.2. Diameter of fibers in scaffolds became smaller when nHA and bone powder are added in, and diameter of fibers in gelatin/PCL/nHA fiber mat was the smallest. The diameters of nanofibers in the three groups were normally distributed. There is a significant difference between the diameter of fibers in gelatin/PCL fiber mat, gelatin/PCL/nHA fiber mat and gelatin/PCL/bone powder fiber mat (P<0.001). Diameter of fibers in gelatin/PCL fiber mat was the biggest. Diameter of fibers in gelatin/PCL/bone powder fiber mat was between gelatin/PCL/nHA fiber mat and gelatin/PCL fiber mat.3. ADSCs were successfully isolated from inguinal fat tissue of SD rat by means of enzyme digestion. ADSCs were in good condition.4. ADSCs covered the surface of the porous nanocomposites and spread with their pseudopodia into the inner part of each scaffold. More cells can be seen on scaffold gelatin/PCL/nHA fiber mat and gelatin/PCL/bone powder fiber mat, and cells on them are close to each other.5. There is no significant difference of proliferation rate between cells on scaffold gelatin/PCL fiber mat, gelatin/PCL/nHA fiber mat and gelatin/PCL/bone powder fiber mat in 24 hours of co-culture, and OD values are all lower than control group. But after time point 24h, cells proliferated to a high degree, cells on gelatin/PCL/bone powder fiber mat proliferate faster than control group, and at time point of 48h, OD value is higher than control group.6. Cells on fiber mats showed a homogeneous distribution. Cells on scaffolds were mainly stained green and just a few red cells can be seen. Number of living cells seems to be different between the three groups, and the difference is statistically significant (P<0.05). Number of cells on gelatin/PCL fiber mat is the least, number of cells on gelatin/PCL/bone powder fiber mat is the most, and cells on gelatin/PCL/nHA fiber mat is between gelatin/PCL fiber mat and gelatin/PCL/bone powder fiber mat.Conclusion:1. Bone powders can be successfully united into gelatin/PCL fibers by electrospinning.2. Gelatin/PCL/bone powder fiber mat has good biocompatibility and can be an ideal scaffold for bone tissue engineering.3. The fabrication of gelatin/PCL/bone powder fiber mat and study of its biocompatibility can lay the foundation for latter study of its osteogenic conducting property in vivo and in vitro. This project-may provide new ideas for bone tissue engineering scaffold preparation; it is of great significance in the further research of natural bone in clinical and fundamental orthopedics. |
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