| The repair of large segmental defects in diaphyseal bone is a significant problem faced by orthopaedic surgeons during treating trauma, diseases and tumors of bone. Alternative solutions include implantation of the defect site with autografts or allografts of bone tissue, or other resorbable biomaterials. Autografts of bone are limited by the amount of tissue can be harvested; allografts spark an immune reaction, and its resorption and creeping substitution is a long and time-consuming process; and implanted biomaterials, not bioactive materials, generally act as a matrix for new osseous ingrowth and creeping substitution. However, current cell based tissue engineered approaches open up a novel and promising prospect for repair of bone defects, for it provides viable osteoblastic cells, which are essential to bone regeneration especially for patients who have a diminished pool of osteoprogenitors.Repair of bone defect using tissue engineered bone, which is constructed with culture-expanded seeding cells and resorbable biomaterials, will not be a remote dream. Of late years, construction of tissue engineered bones using autologous cells is developing rapidly, and making the grade in clinical trial. But it involves a complex process in which individualized cell preparations must be harvested, expanded, and transplanted. So it takes several weeks to achieve. Not only does this rule out emergency repairs, but it also makes the procedure expensive. An alternative approach would be the use of an allogeneic cell source, but the immunogenecity of allogeneic seeding cells needs be solved. In previous studies, we showed, by implanting in lower-class animals tissue engineered tendon, cartilage and bone which constructed using allogeneic seeding cells, that no visible immune reaction was detected, so allogeneic cells may be potentially used.Marrow stromal cells (MSCs) can be isolated from other cells in marrow by their tendency to adhere to tissue culture plastic.The cells have many of the characteristics of stem cells and can be differentiated in culture into osteoblast. The present study was designed to construct tissue engineeredbone with allogeneic MSCs and bio-derived bone materials, and to explore the immunogenicity of tissue engineered bone as well as the capacity to repair segmental bone defects in rhesus monkey.Objects (1) To construct tissue engineered bone with allogeneic bone marrow stromal cells (MSCs) loading onto bio-derived materials, and to observe the cellular compatibility with bio-derived materials. ( 2 ) To investigate the osteogenic capacity of the tissue engineered bone to repair critical-sized segmental bone defects in rhesus monkey. (3) To explore the intensity of immune reaction sparked by allogeneic osteoblastic cells, after being implanted in bone defect as seeding cells, and its influence on osteogenic capacity. (4) To investigate the osteogenic mechanism on repair of the bone defect implanted with tissue engineered bone which was constructed using allogeneic MSCs.Methods (1) 2~5 ml bone marrow harvested aseptically from tibias tuberosities of young rhesus monkeys (macaca mulattta) were applied to density gradient centrifugation to isolate MSCs. Then the cells were culture-expanded in Dulbecco's modified Eagle's medium plus 10% fetal bovine serum. When the cells were 90% confluency, they were passaged. At the second passage, the MSCs were cultivated for 2 days in the condition of 1 X 10"7mol/L dexamethasone, 1 X 10'2mol/L P -glycerophosphate, and 50mg/L ascorbic acid, to be induced into osteoblastic cells, which were then labeled for 1 hour with 5-bromo-2-deoxyuridine (BrdU) by delivering them into serum-free DMEM containing 0.2% BrdU. Eventually, these labeled cells were transferred at a sum of 1 X 106 cells onto bio-derived bone material. They were incubated for 3 days at 37 degrees Celsiusin in a humidified 5 per cent carbon-dioxide environment. Thus, tissued engineered bone were constructed. (2) 2.5cm segmental osteoperiosteal defects of bilateral radius were created surgically in 17 rhesus monkeys, of 15 monkeys the defects in one lateral were bridged with tissue engineered bones and in the other lateral, were bridged with bio-derived materials only; of 2 remaining monkeys the defects left unfilled were blank group. At the host-graft overlapping portion, a hole was bored and a steel wire was passed through to fasten the graft on host bone. After rinsed, the wound was sutured meticulously in layers. (3 ) The monkeys were observed generally for several weeks after the operation. Radiographs were made at 3, 6. 12 weeks postoperatively and radiographic assessments along with radiographic density measurement were performed. At 1. 2. 3. 6. 12 weeks the animals were sacrificed and the involved radius were removed. After macroscopic examination, the defects and adjacent bonewere prepared for undecalcified histological sections. Histological studies, immunohistochemical staining of BrdU, type I collagen and BMP-2, quantitatively assay for detecting alkaline phosphatase activity and osteocalcin, and real-time reverse transcription polymerase chain reaction (RT-PCR) assay for type I collagen and BMP-2 were carried out to examine the exist of labeled cells and the healing of the defects and the formation of novel bone in and around the implants. (4) At 1, 2, 3, 6, and 12 weeks timepoints, T lymphocyte subsets CD3+/CD4+/CD45+, CD3+/CD8+/CD45+ and CD28+ in implants and blood samples were assayed by flow cytometry, and contents of IL-2 and its receptor were detected quantitatively by enzyme-linked immuneosorbent assay.Results (1) MSCs flourished in Dulbecco's modified Eagle's medium plus 10% fetal bovine serum. At 2 weeks the MSCs were confluency and expanded in number for 20 folds. The induced cells were positive ALP staining and the positive rate reached 85%. Cells incubated for 3 days on bio-derived materials, observed through microscopy, were anchored, proliferated and secreted extracellular matrix on the surface and inner wall of the material. (2) After implantation of tissue engineered bone, the monkeys had no fever, and feeled no cold. Around all the implants, slight inflammatory reactions were visible at 1, 2 , 3 weeks postoperatively, but it weakened at 6 weeks and it disappeared at 12 weeks. Both intramembranous and endochondral ossification were involved in the osteogenic processes of the bone defect repair, but the latter process was the predominant. The bone defects implanted with tissue engineered bone were repaired ealier in multipoint way than that implanted with bio-derived materials which were repaired in 'creep substitution' way. Atrophic non-union occurred in blank group. (3) Histological and radiographic assessment, radiographic density measurement revealed that all the scores or values in tissue engineered bones group were significantly higher that that in bio-derived materials group at 3, 6, 12 weeks after operation (P<0.05) . (4) The labeled osteoblasts, by immunohistochemical staining of BrdU, were detected existing at 6 weeks but disappearing at 12 week postoperatively. By immunohistochemical staining of type I collagen and BMP-2, real-time RT-PCR assay for detecting type I collagen and BMP-2, the expression of type I collagen and BMP-2 in tissue engineered bone group was higher than that in bio-derived materials group at 1. 2. 3. 6 weeks after operation. (5) Higher alkaline phosphatase activity during 3 weeks and larger amounts of osteocalcin during 6 weeks were assayed in tissue engineered bone group than that in bio-derivedmaterials group. ( 6 ) T lymphocyte subsets CD3+/CD4+/CD45+, CD3+/CD8+/CD45+and CD28+ in tissue samples at above timepoints in tissue engineered bone group had no significant difference compared with bio-derived materials group (P > 0.05) ; and the contents of IL-2 and its receptor in tissue engineered bone group at above timepoints had no significant difference compared with that in bio-derived materials group (P > 0.05) .Conclusions ( 1 ) Engineered bones can be constructed with bio-derived materials and MSCs which were induced to differentiate into osteoblastic cells. (2) Engineered bones constructed with allogeneic seeding cells were capable of healing critical size segmental bone defects in rhesus monkeys. (3) Both intramembranous and endochondral ossification were involved in the osteogenic processes of the bone defect repair, but the latter process was the predominant, which sparked by inducing factors secreted by implanted osteoblastic cells. (4) Allogeneic osteoblastic cells could live and perform osteogenic function at least 6 weeks after implantion. (5) Slight immune reaction at early stage might accelerate the formation of novel callus and make the bone defect healed earier, for it recruited bone growth factors to local bone defect site.In conclusion, tissue engineered bones constructed with allogeneic MSCs and bio-derived materials could more rapidly repaired critical size segmental bone defects in monkeys than bio-derived materials only. Allogeneic MSCs as seeding cells co-cultured with bio-derived materials might be an alternative in construction of bone tissue engineering.
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