A Pilot Study On The Experimental Research And Clinical Application Of ITEB Repairing Long Bone Defects

Background and objectiveThe Japanese Orthopaedic Association Committeeon Tissue Transplantation and Regenerative Medicinehas conducted a nationwide survey of the status of bone graftingin Japan every 5 years from 1985. In the most recently research in 2007 they found that with the improvement of the modern medical conditions, the employment of the bone grafts kept increasing by each year, a total of 163,564 tissue transplantations were performed, and 134 782 (82.4%) of them were bone grafts. Of the bone grafts, 76,015 (56.4%) were autografts, 53,735 (40%) used a synthetic bone substitute, and 4886 (3.6%) were banked bone allografts. The proportion of synthetic bone substitutes increased, and the proportion of autografts decreased compared to the past 5 years, with the number of autografts, synthetic bone substitutes, and banked bone allografts increased 1.32, 1.68, and 1.72 times, respectively, during this period. Most significantly, the results suggest that Japanese orthopedic surgeons are tending to use synthetic bone substitutes largely as a replacement for autografts especially in the bone transplantations on children. In spite of the predominant properties of osteoinduction and osteoconduction autografts may have, there are limitations to the volume, shape, and size of autografts that can be excised. Excision of autogenous bone is also an invasive procedure. Furthermore, the bone mass of the autografts used to be deficient in children, in addition, this kind of operations may cause the pain in the donor site, the increase of the operation time, cost and probability of infections, even lead to the impediment of the adolescents'bone development. Same to some allografts, bones from the patients'relatives are not the optimum selection due to the indeterminateness of their curative effects. Scientists all over the world have tried many artificial substitutes to repair the bone defects such as the artificial synthetic materials, allografts, demineralized bone matrices from allografts, but none of them could fix the problems when the amount of the bones required are too large or the conditions of the patients are not sound. As we have gained so many breakthroughs on the research of repairing large bone defect of big animals using tissue-engineered bone(TEB), it is necessary to carry out some clinical experiments restrainedly for the sake of the TEB's formal clinical applications and the accumulation of rudiment data in order to form an orthonormal therapy mode for long bone defect. But the application of TEB is limited for the harsh demands of its conservation and transportation. To meet the final desire of the ideal tissue-engineered bone grafts"reach on demand", literatures were reviewed by us and we found that in early research of William H's group, the tissue they froze-dried and sealed in vacuum may be stored at room temperature for periods of at least two years without detectable change. In 2007 Philip J. declared they got successful result on the treatment of periodontal intrabony defects using freeze-dried bone. In addition to the conclusion Moshe Salai drew in 2001 that prolonged cryopreservation on the biomechanical properties of bone allografts came out insignificant, all the information give us a hint that the preservation even the full scale operation of our TEB should go well in this way.This clinical research was approved by the Ethics Committee of Southwest Hospital, which made the department of orthopedics of southwest hospital come to be the first unit in PLA authorized to carry out such clinical applications of tissue-engineered bone by the ministry of health, PLA General Logistics Department.Methods1. The individualized TEB(iTEB) was constructed according to the SOP for the clinical application in the treatment of long bone defects 3-4 weeks before the admission. In this procedure, strict screening and determination based on SOP were used on the target cells or tissues in different phases of the construction. Commitments of the subject's physical security are sticked to from the beginning to the end in this research.2. The patients were informed to hospitalization after the construction of TEB was accomplished, all the perioperative check-ups, follow-up visits and the long-term developmental communications between the subjects and the clinicians during the restoration were executed according to the clinical path for these patients only. An identical resident is appointed to accomplish the above work, which is responsible to find out possible problems promptly and provide helpful recommendations for the subjects. The evaluation of the curative effect was mainly through the persistent monitoring of the radiology and hemotolgy results.3. Nude mice of 6 weeks old (male:female=1:1) were chosen to be the host of the implantation subcutaneously and were evaluated the osteogenesis or oncogenicity by periodic check through X-ray, CT scan and pathological slice and histochemical stain of the graft and the vital organs respectively.4. Key proteins like BMP-2, TGF-β1 and IGF-1 which should be produced in tissue-engineered bone and freeze-dried tissue-engineered bone were detected by western-blot method in order to explore the mechanism of the individualized tissue-engineered grafts'bone formation and the possible influence of freeze-drying on our TEB.Results1. Rapid proliferation, efficient induction and differentiation of the seedling cells for the construction of TEB was documented according to the SOP, productive secretion of the cell matrix was found in almost every pore of DBM under the inverted phase contrast microscope. Strict screening and determination of the cells and tissues all through the procedure were carried out and the physical security of the subjects is confirmed at least.2. Thirty cases of clinical probation were accomplished according to the SOP of TEB strictly and self-contained follow-up materials were obtained simultaneously. According to the materials, comparatively high-density calcification shadows were observed 3 months after the implantation when capsular bone defect cases were analyzed, then foci almost disappeared at about 12 months after operation, furthermore, cavum pulpi were found completely recanalized in the 1.5 years case. Bone callous was found across the gap 6 months after the implantation and the recanalization of cavum pulpi was observed by CT scan throughout the cases.3. Ectopia osteogenesis experiment on nude mice demonstrated gradually ossification along with the extension of the time post-operation. Both the internal and the external part of the tissue have formation signs of woven bone with major osteoblasts and small vessles infiltration. No obvious change of life habit was observed and no tumor-like tissue was discovered by HE staining of the slice of heart, lung, liver and kidney taken from the mice injected with cell suspensions. 4. The expressions of the three proteins are positive both in TEB and freeze-dried TEB, with significant difference to the pure DBM. The climax of their secretion lies in the period of 5-9 days after the cells were seeded into the scaffold.Conclusion1. No visible rejections were observed during the application of individualized TEB for the treatment of patients with long bone defects, which implies that the constructed TEB has good histocompatibility and extensive prospects for clinical application. Both the result of the clinical application and ectopia osteogenesis experiment in nude mice showed comparatively doughty bone induction and osteogenesis of TEB at the same time.2. The biological security and curative effect of TEB in the treatment of different kinds of long bone defects were tentatively confirmed by monitoring the serology and imageology results of the patients in differents periods of rehabilitation dynamically.3. Felicitous curative results were manifested in the treatments of children's long bone defects with individualized TEB, which provided a novel path for the theraphy of juvenile bone defects casued by different etiological factors.4. Much osteogenic key proteins such as BMP-2, TGF-β1 and IGF-1 in the individualized tissue-engineered bone constructed according to the standard operating procedure which we preferred were confirmed within the individualized tissue-engineered bone and freeze-dried TEB by WB method.