| ObjectiveMassive bone defects constitute a major challenge to reconstructive surgery. Currently, clinically established therapeutic approaches for critical-size bone defects mainly include autografts,allografts and artificial materials alone or in combination with bone grafts. However, autogenous bone grafting has a limited source of graft material and causes surgical morbidity of the donor site. Allografts have a risk of immunogenic reactions,disease transmission and inflammation.There have been study on treating bone defects by tissue-engineering technology recent years,which attracted increasing interest.There are three key elements in tissue engineering technology:(1) cells; (2) growth factors; (3) scaffolds. At the same time,the culture environment of the tissue engineering bone also applys a significant impact.BMSCs are multipotential cells that can differentiate into into osteoblasts,adipocytes,tenocytes and marrow stroma. BMSCs can be extracted from bone marrow and rapidly expanded in vitro,so it has been widely used as seed cells.Platelet-rich plasma(PRP) is rich in growth factors that influence bone regeneration.When activated by agonist such as thrombin,the PRR releases those factors. Scaffolds play a critical role in the practical realization of bone tissue engineering. We chose β-TCP for it’s advantages of high biocompatibility,good biological absorbability and ability to induce the osteoblastic differentiation and amplification spontaneous. A perfusion bioreactor was used to simulate in vivo conditions and create a 3D environment, promoting cell adhesion, proliferation and differentiation. The activity of ALP is an important index for the evaluation of osteogenesis differentiation. In addition, ALP is an iconic enzyme of mature osteoblasts; thus, plays a critical role in the in vitro calcification process. Quantitative detection of the ALP concentration is frequently used for in vitro osteogenesis experiments as a conventional symbol of early osteoblast differentiation. BGLAP2 mainly appears in a mineralized formation of the cells as a sign of osteoblast maturation.In this study, we utilized perfusion bioreactor, BMSCs and β-TCP to build tissue engineering bone in vitro. In addition, we used PRP as the source of growth factors. By detecting the expression level of ALP and Bglap2 of the different combination of the above factors, we assessedthe influencing factors of tissue engineering bone formation.Materials and Methods1.Ten adult male New Zealand white rabbits (2-3 months of age), weighing between 1.7 and 2.3 kg, were used. With the animal under general anesthesia, Bone marrow aspirate (5 ml) was aspirated from the tuberosity of the tibia using a sterile bone marrow aspiration needle containing 1 ml heparin. The bone marrow was mixed with lml 5%sodium citrate before placed in a ice tray. The BMSCs were isolated using the Percoll separation method. After mixed with equal volume D-hanks and homogenized, the aspirate was centrifuged at 1000g for 6 min. The supernatant was abandoned and the remaining was mixed with equal volume D-hanks and homogenized. Then equal volume Percoll separating medium was added to the sample. The cloudy coat in the middle of the centrifuge tube was harvested after centrifuged at 2500g for 20 min. After mixed with 5ml D-Hanks in the centrifuge tube, the sample was centrifuged at 1000g for 6 min. Then BMSCs were harvested at the bottom of the centrifuge tube. The BMSCs were cultured in DMEM medium containing 10% fetal bovine serum and identified by flow cytometry. Cell growth was observed under a inverted phase-contrast microscope. After two passages the BMSCs were used to build cell scaffold composites.2.PRP was prepared according to the two-step centrifugation method. With the animal under general anesthesia,5ml blood was drawn from rabbit’s central ear artery with a 10-ml sterilized syringe containing 1 ml of sodium citrate used as anticoagulant.After homogenized, the mixture was centrifuged for 6 min at 3500g to separate the cells from the serum components. The plasma and buffy coat were collected and subjected to another centrifugation at 3000g for 6 min. After removing the top layer, the lower part was resuspended and designated as PRP. Then the PRP was mixed with lml coagulant (mixed of 1ml 10% CaCl2 and 1000 U thrombin) and homogenized. Platelet rich plasma gel (PRG), a jelly-like substance, was harvested.3.The third generation BMSCs was prepared into suspension before added to the (3-TCP scaffold. When adding the suspension, the bracket was flipped up and down to make sure that each part of the bracket surface had cell adhesion. Then 3ml cell culture media was added to each cultivation orifice plate. The scaffold was then placed in a 37℃,5% CO2 incubator overnight. The constructed bracket composites were then placed in the bioreactor perfusion column and cultured 3 weeks using culture media containing PRP or not. The entire system was placed in a 37℃,5% CO2 incubator and cultured for 21 days. The flowrate was maintained at 3.5 ml/min. The culture medium was changed every 2-3 days, when the glucose content in the medium was depleted. Adhesion, proliferation and growth of the BMSCs in the β-TCP scaffold were examined under a scanning electron microscope. The bracket composites were divided into five groups. Group A:composite cultured with PRP in bioreactor; Group B:composite cultured with PRP without bioreactor; Group C:composite cultured in bioreactor without PRP; Group D:composite cultured without PRP nor bioreactor; Group E:β-TCP scaffold only (negative control group).4.One from each five groups of composites was implanted between the shallow and deep fascia under the right side of the waist skin of each rabbit, with the animal under general anesthesia. The composites were fixed with sterile sutures and their positions were recorded. The rabbits were humanly killed 3 months after surgery and the composites with tissue cells on were taken out to perform Quantitative RT-PCR.5.The Quantitative RT-PCR was carried out 3 times. Data were represented as mean ±standard deviation (SD) and were analyzed using the SPSS 18.0 statistical software package (SPSS, USA). One-way analysis of variance or t-tests were used for comparisons between groups and P<0.05 was considered statistically significant.Results1.Observation of the BMSCs using an inverted phase-contrast microscope revealed a small amount of cell adhesion at day three following seeding. The cells were found to have a spindle-shape at this time. Cell colonies were formed after the primary cells had been cultured for seven days, and the cells were gathered into swirl-shaped colonies at day seven following passage.2. Flow cytometric analysis of BMSCs was used to determine the gene expression profiles of cell passage 3. The results indicated that 2% of the cells were CD34-positive and 99.8% were CD44-positive.3. Scanning electron microscopy showedgood adhesion and distribution of BMSCs on the p-TCP scaffold.4. The expression of the markers in experimental group were higher compared with the negative control group. Comparisons between the experimental groups also revealed statistical significance.Conclusion:l.Bone marrow mesenchymal stem cells is an ideal seed cells for tissue engineering.2. The platelet-rich plasma is rich in a variety of cytokines which can induce bone formation.3. β-TCP scaffold is suitable for bone marrow mesenchymal stem cells to adhere and proliferate.4. The bioreactor can provide a 3D culture mode which promoted BMSCs adhesion, growth and differentiation at the same time and,hence,promoted bone formation. |
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