BACKGROUND:Conventjonally,the osteoinduction of bone substitute materials is detected in vivo,which is unsatisfactory regarding the reliability,chronergy and precision,especially for a large amount of substitute materials.OBJECTIVE:To search an in vitro assay for determining in vitro osteogenic activities of bone graft substitutes.DESIGN:Controlled cytological trials.TIME AND SETTING:The experiments were carried out in the Institute of Orthopaedics.Chinese PLA General Hospital(Beijing,China)from August 2006 to May 2007.MATERIALS:C2C 12 cells was offered by the Cell Center of Peking Union Medical College;Human decalcified bone matrix and bone protein were offered by the Institute of Orthopaedics in Chinese PLA General Hospital;Type Ⅰ collagen extracted from bovine tendon was purchased from Beijing Yierkang Bioengineering Development Center;Recombinant human bone morphogenetic protein 2 was purchased from Hangzhou Gene Technology of Huadong Medicine Group.METHODS:By means of dialysis,a composite material was prepared with the bone protein extracted from human,human decalcified bone matrix and type Ⅰ collagen of bovine tendon.The samples of decalcified bone matrix.composite material and recombinant human bone morphogenetic protein 2 were respectively co-incubated with C2C12cells for 72 hours.Negative control group comprised pure cells without materials.Then C2C 1 2 cells were lysed and the lysate were assayed for the absorbance of alkaline phosphatase(ALP)and total protein by chromatometry.ALP is (the specific mark of osteoblastic phenotype.The relative ratio of absorbance value between ALP and total protein could represent ALP activity in the unit quantitative C2C12 cells.MAIN OUTCOME MEASURES:The ratio of absorbance value between ALP and total protein.RESULTS:The ALP activity was the highest in the recombinant human bone morphogenetic protein 2 group,then in the decalcified bone matrix group and composite material group,and the lowest in the negative control group.There were significant differences in the ALP activity between the three trial groups and the negative control group(P＜0.05).CONCLUSION:The assay in vitro is effective to detect the ALP activity and it can be used to determine the osteoinduction of bone graft substitutes.

Objective The tissue-engineered composite graft was formed with induced marrow-derived stromal cells (MSCs)and PLGA double-layer scaffold. The effectiveness of this graft for the repair of osteochondral defects in the knee of rabbits was investigated. Methods MSCs were isolated from 20 adult rabbits with density gradient centrifugation and was divided into two groups. In group A, the MSCs were cultivated with regular medium. In group B they were cultivated with chondrogenic differentiation medium. The mRNA of MSCs and articular cartilage cells were extracted, and the expression of mRNA for type Ⅰ and Ⅱ collagen was tested by RT-PCR. The distribution and compound of MSCs with PLGA double-layer scaffold was examined with scanning electron microscopy. 28 adult rabbits were divided into 3 groups, osteochondral defect of 3.5 mm in diameter and 3 to 4 mm in depth were created in the patellar groove. Group A (10 rabbits), the MSCs cultivated with regular medium was grafted into the defects. In group B (10 rabbits), the MSCs cultivated with chondrogenic differentiation medium was grafted into the defects. In group C (8 rabbits), the defects were repaired with autologous osteochondral grafts as control. Specimens were harvested at 4th, 8th, 16th and 24th week post operation respectively, histological examination was performed and graded. Results For the MSCs cultivated with regular medium, the expression of mRNA for type Ⅰ collagen was found with RT-PCR, but no expression for Ⅱ collagen was found. For the induced MSCs, the expression of mRNA both for type Ⅰ and type Ⅱ collagen were found. The adhesion and growth of MSCs on the PLGA double-layer scaffold were well visualized with scanning electron microscopy, and some cells were found in the deep porotic area. For the specimens of group B, no significant difference was found comparing with normal cartilage at 24th week, and the specimens were defined as matured hyaline-like cartilage(4/6)with histological examination, superior to those specimens of group A (1/4). Conclusion The MSCs have osteogenic and chondrogenic potentiality. Combined with PLGA double-layer scaffold, it can be served as seeded cell to form tissue-engineered composite grafts, which can be used to repair osteochondral defects in rabbit models.

0.05).There were better effect of removal of myelin(P2

[Objective]To evaluate the effect of post-operative rehabilitative nursing of patients repaired with acellular nerve allograft.[Method]From April 2003 to April 2006,39 inpatients with peripheral nerve defect were subjected to receive acellular nerve allograft in order to repair nerve defect.The patients were rehabilitated with special nursing after being operated and discharged.Among of them,21 patients were followed up over 6 months,the effect of treatment was analyzed.[Result]Among 21 patients,16 people had excellent and good effect of treatment and the efficient rate was 71.4%.[Conclusion]Post-operative rehabili tative nursing is important and effective for rehabilitation patients of peripheral nerve injuries repaired with acellular nerve allograft.

0.05), whereas the value of new bone grafted with pDBM was significantly lower than that of the normal group (P0.05); but the CUS of the pDBM grafted group was significantly lower than that of normal radius (P0.05). Histological analysis exhibited that most of the DBM was absorbed and substituted by matured new cortical bone in the treated defects of both groups 6 weeks postoperatively, whereas in the untreated group, the defects were only filled with fibrous connective tissue in their mid-portion. Conclusion The sDBM and pDBM are both effective in repairing segmental bone defects. The properties of new bone induced by grafts with sDBM are superior to that of pDBM in biomechanics. These materials can be used in clinical practice as bone graft extenders or enhancers.

[Objective]To study the effect of()~(60)Co-irridiated and ethylene oxide sterilization on acellular Achilles tendon-bone biomechanical properties.[Method]Twelve fresh Achilles tendon-bone were harvested from New-Zealand white rabbits.Those tendons were soaked in 1%TnBP[tri(n-butyl)phosphate] for 48h,then they were rinsed with deionized water and ethyl alcohol.They were conducted mechanics testing after lyophilization and received()~(60)Co-irridiated or ethylene oxide sterilization.Fresh Achilles tendon-bones were as positive control.[Result]There were no significant difference between fresh and sterilized by ethylene oxide Achilles tendon-bone on biomechanical properties(P

Objective To research the immunologic reaction and the potential of chemically extracted acellular nerve allograft(CEANA) to repair peripheral nerve defects in primates. Methods Adult SD rats were used as nerve donors and adult male Wistar rats used as nerve recipient hosts. 25 mm long nerve segments were excised from SD rats' sciatic nerves. The nerve segments were decellularized via an improved chemical decelluarization treatment as follows: 1) nerve segments were rinsed with cold sterile Ringer's solution; 2)stabilized by pinning the ends to a thin plastic support, and submerged in 4% Triton-100 solution 12 h; 3)soaked into 72 mM sodium deoxycholate for 12 h; 4)washed in distilled water for 6 h. The procedures were repeated once again. Median nerve segments were obtained from macaques and decellularized according to above procedures. The CEANA from SD rats were implanted into Wistar rats subcutanously. The control group was implantation of fresh nerve allografts from SD rats. The immunogenicity of acellular nerve allograft was tested by immunohistochemical examination of the intensity of CD3+, CD4+ and CD8+ cells that infiltrated the allografts. Median nerve defects for 5 cm were created in three macaques. CEANA were interposed across the gap. The CEANA were anastomosed microsurgically to the epineurium of proximal and distal stumps. Results The number of CD3, CD4 and CD8 positive lymphocytes infiltration in CEANA was far lower than that in the control group of fresh nerve allografts at 2 weeks and 4 weeks after implantation. There was no significant evidence of inflammatory in the CEANA grafted group. In the experiment of nerve regeneration of macaques, electromyographic activity was recorded across the allografts. The conduction velocity of regenerated nerve was (40.5?6.8) m/s. Regenerated axons sprouted from the proximal portion reached the distal portion of the grafts, and Schwann cells were also present in the central portion of the CEANA. Motor end-plates were observed in reinnervated muscles. Conclusion The immunogenicity that would have initiated cell-mediated immunological rejection of CEANA are removed. The implantation of CEANA could repair the defect of median nerve 5 cm long in the arm 5 months postoperatively. The CEANA as a type of substitute of nerve autografts has the potential to repair peripheral nerve defects in primates.

[Objective]The purpose of this paper is to demonstrate whether the nerve length could affect the quality of acellular nerve and investigate the properly repairable distance of nerve defects with acellular nerve allografts.[Method]Fresh sciatic nerves were obtained from adult dogs and divided into 12 cm long segments.The nerve segments were decellularized via an improved chemical decelluarization treatment as following: Nerve segments were rinsed with cold sterile Ringer's solution and submergedin 5% Triton-100 solution 12h,and then soaked the nerve segments into 5% sodium deoxycholate for 12h.The treated nerve segments were washed in distilled water for 3h.This procedures were repeated once again.In vitro,the degrees of decellularization,demyelination and integrity of nerve fiber tubal of chemically extracted acellular nerves were observed with microscope and assessed by a score system.In vivo,the sciatic nerve of dogs on the right was exposed.In 8 cm grafted group(n=6),a 7 cm segment of sciatic nerve was removed from the midthigh level.In 10 cm grafted group(n=6),a 9 cm segment of sciatic nerve was removed at the same level.The gaps were bridged with acellular nerve allografts by 8 cm and 10 cm long segments respectively.The follow-up period was 12 month postoperatively.Motor functional recovery of the right hind following allografting was examined by neurobehavioral,electrophysiological,histological and immunohistochemical assessment.[Result]There was no difference on the degrees of decellularization,demyelination and integrity of nerve fiber tubal among every fraction of the acellular nerve from the two ends to the central portion.In 8 cm grafted group,all survival dogs(n=5) were held upright with the affected hindlimb extended so that the body's weight was supported by the distal metatarsus and toes.In 10 cm grafted group,animals were failed to held upright with the affected hindlimb.Electrophysiological studies showed that elctromyographic activity was observed in both groups.After 12 month the conduction velocity was 32.1+5.1 m/s in 8 cm grafted animals and 18.3+6.0m/s in 10 cm grafted group.In normal animals, the conduction velocity was 106.6+16.4 m/s.The conduction velocity in 10 cm grafted group was lower than 8 cm grafted(P

0.05).[Conclusion]The values of three-dimensional parameters in old femoral neck fracture patient are different with that of normal persons in the loading region of the femoral head although there is no difference in BMD values between them.This may provide new evidence to predict osteoporotic femoral neck fracture.

Objective To observe the effect of based biomimetic biphasic calcium phosphate(BCP)tissue engineered bone masses to repair the bone defects and prevent the collapse of femoral head in dogs.Methods Biomimetic BCP ceramic scaffolds was fabricated using three-dimensional(3D)gel-lamination technology according the micro-computed tomography(Micro-CT)information of cancellous bone of dogs' femoral heads.Tissue engineered bone masses were constructed by the combination of biomimetic BCP scaffolds and induced osteogenic bone marrow stromal cells(BMSCs)in vitro.In 10 dogs,tissue engineered bone was implanted into the bone defect on the weight-bearing area of femoral head.In the control group of another 10 dogs,autogenous cancellous bone chips were impressed into the bone defect on the weight-bearing area of femoral head.The gross appearance and regeneration of new bone were observed in 30 weeks postoperatively.Results The fabricated biomimetic BCP scaffolds had better 3D structure.The trabecular system of the scaffolds had some orientation along the weight-bearing line of femoral head and represented plate-like model.BMSCs grow flourishing on the surface of the scaffolds.After 30 weeks operatively,in the tissue engineered bone implanted dogs,the gross appearances of the femoral head were integrity and a great lot of new bone tissue wraps the trabecula of the scaffolds under microscope.In the control group,the mostly femoral heads were collapse gross appearance.Conclusion Tissue engineered biomimetic BCP had better biocompatibility and could prevent the collapse of femoral head after it is implanted into the bone defects of femoral head.