| Objective To explore the in vivo osteogenic ability and feasibility of repairation of large segmental long bone defects with cultivated the BMSCs derived osteoblast combined with porcine SIS to fabricate the tissue engineered periosteum in New Zealand rabbit. The purpose of this study was to supply experimental therapeutic data for the future clinical use.Methods The BMSCs was harvested from2week old rabbit marrow aspirates. In vitro,BMSCs were expanded and selected to be induced to osteoblasts.The cell was observed the morphological characteristics and draw the growth curve and identified by ALP,s Gomori staining and Alizarin red S staining. Osteocalcin immunocytochemistry was used to detect MSCs and osteoblasts quality. According Abraham method, SIS was prepared by the procedure performed mechanical disassociation, degrease, enzyme digestion, detergent treatment, lyophilization and sterilization as an artificial scaffold to comfine with osteoblasts to fabricate the tissue engineered periosteum. The natural bone, based on paper technique, was taken from the limbs of healthy home-raised rabbit at2-3months after killing. The fresh specimens were pierced pores compactly with pore diameter of500mm. Then,the sample was incubated in the30%hydrogen peroxide and99%ether for24h and three times, rinsed with the deionized water continuously to remove the organic solvents.After lyophilization and hermetic packages, all the products were sterilized by gamma irradiation before use.The large segmental2.5-3.0cm long bone defects at radius of72New Zealand rabbit were established as the animal model and divided randomly into six groups. Ⅰ group only the tissue engineered periosteum (BMSCs derived osteoblast+SIS scaffold)was at right radius as experimental group. Ⅱ group bone defect was treated w ith internal fixation with periosteum.Ⅲ group only the deproteinised allograft bone (DPB) was implanted into the animal. Ⅳ group the deproteinised bone (DPB) coupled with fixation interne was used to establish the model. Ⅴ group the DPB with the artifical periosteum was transplanted into the bone defect area.Ⅵ group the DPB accompanied with the artifical periosteum and internal fixation was grafted into the right upper limb.Animals were sacrificed at4,8,16weeks postoperation respectively to harvest the specimen. The healing outcome was evaluated by radiograph, three-dimensional CT scan and histological examination methods.Results and Conclusion1. MSCs isolated from skeletal tissues can be cultured and purified in vitro. The progenitor cells are a typical morphological fibroblast and an important member of stem cell family which proliferate rapidly and can be induced to differentiate into osteoblasts under suitable condition. It revealed that adherence screening method is relatively simple and reliable.2. Several structural characteristics such as physical and biological nature are similar to periosteum. Thus,SIS is regarded as an ideally natural biomaterial scaffold for tissue engineering applications of bone. The BMSCs and osteoblasts are seeded onto the scaffold and grow well and foster cellular differentiation.3. All animals tolerated the surgical procedures well and had a normal diet, behavior and ambulation;the incision healed normally:Inspection of imageology revealed bone formation much more followed with time, and weave bone and marrow cavity shape after12weeks incompletely at experimental side, but there was not any bone formed at control side.In histological examination,no lymphocytes infiltrated and a lot of irregular newly formed vessels horned at experimental group,while the defect space of control group were occupied by some connective tissue. The tissue engineered periosteum has osteogenic ability and feasibility of repairation of large segmental long bone defectsin allogenic rabbit. The results provide novel and useful information on the clinical potentials of artifical periosteum in orthopedic tissue engineeering. |
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