| Experiment OneThe use of augmented demineralized bone matrix with caninebone-derived protein in the repair of segmental bone defectsDemineralized bone matrix (DBM) has osteoinductive and osteoconductive properties The osteoinductive principle of DBM is a kind of bone -derived protein(BP), it has been termed bone morphogenetic protein(BMP) The effects of DBM augmented with BP(BP/DBM) on bone marrow stem cells(BMSCs) biologic and the repair of segmental defects in a canine model were evaluated an vitro and an vavo The DBM was prepared from the long bones that had been harvested from freshly killed canines All soft tissue was removed and the bones were washed in sterile deionized water The cleaned bones were extracted for 2h in a 1 1 mixture of chloroform methanol(fifty milliliters per gram of bone),dried overnight,milled in a mill, sieved to between 2OO1Lm7OOl.Jm in size, and decalcified in 0 6M hydrochloric acid for 24h at 2C The resulting powder was rinsed with serial changes of sterile deionized water to bring the pH to 5 o or more and then was lyophulized The BP was extracted from DBM The particles of DBM were extracted with a solution of 4M guanidine hydrochloride buffered with 1 N Tris(pH 7 6) for 24h at 4C The extracted7protein was purified with use of a 8000-molecular-weight cutoff ultrafilterThe residuals of extracted DBM was inactive DBM(IDBM) The BP that0was extracted from 10 grams was redissolved in 50 milliliters 4M guanidinehydrochloride buffered with 1 N Tris(pH 7 6) The supernatant was then combined with 10 grams of DBM, prepared as already described, by placing the two together in dialysis tubing and dialyzed against deionized water for 24h After dialysis, the DBM was separated from the supernatant by centrifugation at 40,000g and was lyophilized This material, consisting of 10 grams of DBM and the BP that had been extracted by 4M guanidine hydrochloride from another 10 grams of DBM, was called augmentedDBM(BP/DBM)Lixivium of BP/DBM, DBM, and IDBM were prepared by placing them in the solution of Dulbecco s modified eagle s medium(DMEM) for 120h at 370C (1:100) Bone marrow stem cells were obtained from the canine iliacBone marrow cells were aspirated from the iliac bone marrow and cultured in DMEM with 10% fetal bovine serum(FBS) Cells were continually passaged until passage 5 and were harvested for the bioassay Bone marrow stem cells , passage 5, were collected from the flasks and cultured in the hxivium of BP/DBM, DBM, IDBM and DMEM with 10%FBS Cell growth curves were protracted and the toxicity of these lixivium to BMSCs were mensurated using MTT test The stages of cell cycle were analyzed with flow cytometry(FCM)The masses of BP/DBM and DBM were prepared by placing these materials into 5% solution of poly-l-lactic acid(PLA) with chloroform and being dried by airing These materials were sterilized with ethylene oxide gas A8standard procedure was used to make a 1 Scm long defect bilaterally in theradius of ten skeletally mature male canines All of the 20 defects were0randomly divided into four subgroups group I ,five defects were graftedwith mass of BP/DBM, group II, five defects were grafted with mass of DBM, grouplll, autologous cancellous bone from the iliac crest was grafted into the third five-defects, group IV, as a control group, the five defects were empty All defects were internally fixed with stainless plates and screws The grafts were periodically evaluated by radiographs, and four months after surgery the grafts were harvested and tested the biomechanical strength, The mechanical parameters were calculated, and histological examination of major fragments of the grafts was performedThe cell biologic results showed that the counts of cell treated with lixivium of BP/DBM was higher than the counts of cell treated with the lixivium of0DBM, IDBM and DMEM from 2 to 6 days In the presence of the lixivium of BP/DBM, the relative growth rate(RGR) of cultured BM...
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