Sustained-released anti-tuberculosis drugs: a choice of carrier materials / 中国组织工程研究

BACKGROUND:Local administration of anti-tuberculosis drugs is a commonly used therapy. Due to the rapid absorption, the drugs cannot have the durable therapy effect; therefore, it is necessary to seek an optimal carrier material for the agents. OBJECTIVE:To review the new development for the carrier materials of anti-tuberculosis drugs. METHODS:A computer-based search of PubMed and VIP databases was performed by the first author to search articles related to sustained-released anti-tuberculosis drugs published from January 1990 to December 2014. The key words were osteoarticular tuberculosis; anti-tuberculosis; sustained-released drugs in English and Chinese, respectively. RESULTS AND CONCLUSION:Inorganic materials (calcium phosphate, calcium sulfate), polymer materials (polylactic acid, polyglycolic acid, polylactic-co-glycolic acid) and biomaterials (protein, glutin, alginates, chitin, demineralized bone matrix) are the main three kinds of carrier materials for anti-tuberculosis drugs. These carrier materials have their own advantages and disadvantages, which cannot be the optimal carrier materials. However, the complex of these materials is a promising technology for the optimal carrier materials in the future.

Tendon-to-bone healing using an injectable calcium phosphate cement combined with bone xenograft/BMP composite.

Injectable calcium phosphate cement (ICPC) has been applied to enhance the tendon-to-bone healing. However, its slow degradation delays the osteointegration of grafted tendon in bone tunnels. We therefore constructed a synthetic biomaterial of ICPC combined with recombined bone xenograft granules (RBX). In this study, the first stage study demonstrated that the ICPCB contained 3 mg BMPs (ICPCB-3) obtained a porous structure. More importantly, the values of ICPCB-3 were highest in cell proliferation, alkaline phosphatase (ALP) activity, expression of osteogenic genes, and newly ectopic bone-forming area (P < 0.05). Then, ICPCB-3 was used in an anterior cruciate ligament (ACL) reconstruction model. Ninety skeletal mature rabbits underwent bilateral ACL reconstructions and were assigned to 3 groups: control group, ICPC alone group, and ICPCB-3 group. Animals were sacrificed at 6, 12 and 24 weeks. The results showed compared with ICPC, ICPCB-3 composite markedly accelerated tendon-to-bone healing. In addition, little remnants were observed in ICPCB-3 group. Moreover, the maximum loads to failure of ICPCB-3 group was significantly higher than ICPC group at 24 weeks (P < 0.01). We conclude that the ICPCB composite, with a porous structure and better osteointegration effect, has direct clinical instruction to arthroscopic techniques of the ACL reconstruction.

Biphasic Ceramic Biologic Bone, Bone Morphogenetic Protein, and Basic Fibroblast Growth Factor Complex Transplanted for Femoral Head Necrosis / 中国康复理论与实践

@#Objective To observe the efficacy of a kind of complex composed of biphasic ceramic biologic bone (BCBB), bone morphogenetic protein (BMP) and basic fibroblast growth factor (bFGF) on the repair of necrotic areas of the femoral head. Methods The femoral head necrosis model of 64 femoral heads in 32 rabbits induced with microwave heating were randomly divided into four groups, which implanted with nothing (group A), BCBB/BMP (group B), BCBB/BMP/bFGF (group C) and with cancellous bone autograft (group D). The specimens were harvested separately at the end of 2, 4, 8 and 12 weeks after operation. 4 femoral heads were taken off at each interval in every group. A series of examinations were carried out including of naked eyes and gross anatomic observation, X-ray, histology, and blood vessel immunohistochemical staining. Results In group A, 1 femoral head collapsed by the end of 12 weeks, and there was only a little osteoid tissue formed. At the same time, a lot of new bone formed in group B and group C, and the boundary between the bone grafting area and the post bone still existed, but the boundary was unclear in group D, with the density consistent to the post bone. Under X-ray, the defect could be found and one femoral head collapsed in group A by the end of 12 weeks. The density of bone grafting area was high and the boundary to the post bone was unclear in group B and in group C. The density of bone grafting area was the same as the post bone and the boundary between them was unclear in group D. There was only a little osteoid tissue formed in group A by the end of 4 weeks. At the same time, there was a little new bone formed in group B, and BCBB was partly degraded. There was a lot of new bone formed in group C and group D, and BCBB was partly degraded in group C, but cancellous bone autograft was almost absorbed in group D. The new bone area by the end of 4, 8 and 12 weeks from more to less were: group C and group D (P>0.05), group B, and group A (P<0.05). At the end of 2, 4 and 8 weeks, the blood vessel area of group C was more than that of group A, group B, and group D (P<0.05). Conclusion The BCBB/BMP/bFGF complex can induced osteoinduction and revascularization, to repair rabbit femoral head necrosis as effective as cancellous bone autograft.

Evaluation of a novel reconstituted bone xenograft using processed bovine cancellous bone in combination with purified bovine bone morphogenetic protein.

BACKGROUND: Xenogeneic grafting represents an alternative to autogenous grafting in osseous reconstruction and exhibits many beneficial properties. However, the usefulness of xenogeneic bone relies on necessary processing procedures for removing antigens and viruses, and preserving biological activities simultaneously. By chemical treatment of bovine cancellous bone to make it an antigen-free scaffold, and extraction of bone morphogenetic protein (BMP) from bovine cortical bone, followed by recombination of the scaffold with the BMP, we developed a new grafting material, reconstituted bone xenograft (RBX). METHODS: In this study, scanning electron microscope and energy dispersive X-ray were first employed to observe the structure and components of RBX. Then the biomechanical property was evaluated by applying compression in a materials testing machine. Subsequently, the immunologic evaluation was performed by measuring galactose-alpha-1,3-galactose (α-gal) epitope in vivo and proinflammatory cytokine (TNF-α) secreted by human monocytic cell line (THP-1) in vitro. Finally, this RBX was implanted into segmental radial defects in a rabbit model, and its ability to treat large bone defects was specifically evaluated. RESULTS: Although the compressive strength of RBX was 10% lower than that of unprocessed bovine cancellous bone (UBCB), the basic porous structure and natural components were still kept in this composite. The α-gal xenoantigen level was significantly lower in RBX (P < 0.05) compared with UBCB. Moreover, the TNF-α level was significantly (P < 0.05) reduced compared with UBCB when THP-1 was exposed to RBX. On the other hand, RBX appeared to induce cartilage formation from immature cell populations and resulted in osteogenesis through endochondral-like ossification from 4 to 12 weeks in repairing segmental bone defects. CONCLUSIONS: These results demonstrate that RBX, with its natural microstructure and components, certain mechanical strength and strong osteoinductivity without evoking immune rejection, has significant potential for the treatment of bone defects.

Preparation of tissue engineered bone and in vivo osteogenesis using alginate and xenograft bone composite technology / 中国组织工程研究

BACKGROUND: Alginic acid has a relatively mild gel condition and good biocompatibility, and it has been widely used in bio-tissue engineering.OBJECTIVE: To construct bone tissue engineering scaffolds using alginate gel composite bone xenograft approach, and to observe the cell biological properties and in vivo osteogenic potential in scaffolds.METHODS: The bone marrow was harvested from two 2-week-old New Zealand rabbits, 1 ×10~(-8)mol/L recombinant human bone morphogenetic protein-2 was used to induce bone marrow mesenchymal stem cells. The induced bone marrow mesenchymal stem cells at the second generation were incubated into 1% sodium alginate gel, after cultured for 4 days, the cell morphology in gel was observed by hematoxylin-eosin staining. Bone marrow mesenchymal stem cells at the second generation were divided into simple DMEM gel group and DMEM containing 1% sodium alginate gel group, followed by a culture of 7 days. Then bone morphogenic protein-2 immunohistochemical staining was performed. A total of 24 nude mice were randomly divided into two groups, both sides of the thigh muscle pockets were implanted with bone marrow-derived mesenchymal stem cells/alginate gel/bovine cancellous bone complex as an experimental group, with bone marrow-derived mesenchymal stem cells/bovine cancellous bone as a control group. At 2 and 4 weeks post-operation, the osteogenesis in the composite was observed by histological examination, the percentage area of new bone or cartilage was determined using image analysis system.RESULTS AND CONCLUSION: The bone marrow-derived mesenchymal stern cells in the sodium alginate gel exhibited a well-stacked morphology, they suspended in a gel, showing cell division and mitosis phase. In the simple DMEM gel group and DMEM gel containing 1% sodium alginate group, the immunohistochemical results showed that, cell division and proliferation were normal, with prominence at a variety of forms, large nucleus, and clear nucleolus. The bone morphogenetic protein-2 expression had no significant difference between the simple DMEM gel group and DMEM gel containing 1% sodium alginate group (P>0.05).Scanning electron microscopy revealed that, the alginate gel evenly composited in bovine cancellous bone micropores, cell grew at different planes. Animal experiments showed that there were significant differences regarding the percentage of new bone or cartilage area between the experimental group and control group at 2 and 4 weeks postoperation (P< 0.05). It is indicated that constructing bone tissue engineering scaffolds by using alginate gel/bovine cancellous bone, complies with the ultra-structural principle of tissue engineering scaffolds, can maximize the cell loads, achieve good bio-performance, without adverse affects on the proliferation, osteogenic phenotype and related biological properties of bone marrow-derived mesenchymal stem calls, the in vivo osteogenic efficiency was high.

Application of anti-infective reconstituted bone xenograft in one-stage treatment of adult posttrau matic osteomyelitis / 中华创伤杂志

Objective To validate the effect of anti-infective reconstituted bone xenograft (ARBX) in treating posttraumatic osteomyelitis by one-stage grafting in the adults.Methods With clinical application approval of Medical Command,Logistics Ministry of PLA,ARBX was used to treat 27 adult patients (29 lesions) with posttraumatic osteomyelitis by one-stage grafting after debridement since September 2001.The study analyzed 27 patients (29 grafts) who were followed up for average 26 months (12-63 months).Results The follow-up for average 26 months (12-63 months) in 27 patients showed that infection of 22 patients (24 lesions) was controlled and cured,except for three with failure to control the infection or with recurrence of infection,two with controlled infection but with postoperative nonunion.The infection control rate was 89.7% (26/29) and the cure rate was 82.8% (24/29) ,which were better than the results of traditional therapy.Conclusions ARBX has high osteoinductive activity and enhanced anti-infective capability,which enables it to be used as one-stage grafting to treat posttraumatic osteomyelitis in the adults.

Pathological study on peripheral vessel injury induced by explosion shock wave / 中华创伤杂志

Objective To observe pathological features of peripheral vessel injury caused by ex-plosion shock wave so as to provide theoretical basis for emergency treatment, prevention and complication reduction of war extremity injuries. Methods A total of 18 rabbits were randomly divided into three groups (six rabbits in each group) and placed respectively at 1, 2 and 3 m away from the explosion cen-ter. The animal model with blast injury was made by using fluid dynamite that electrically exploded at 60 cm above the ground. The physical parameters of blast wave were recorded using pressure transducers (PCB, UAS). After explosion, the femoral arteries were examined grossly, histologically and immunohis-tochemically. Results The results showed that vascular endothelial cells were denudated, the spaces of contractile fiber cells increased and appeared puff, the vassular elastic fibers ruptured, flexed and de-formed visibly. Some parts of the vessel wall ruptured completely or partly, leading to bleeding. TUNEL staining and fluorescence microscope found large number of apoptotic cells in endothelium layer, smooth muscle layer and membrana adventitia layer of the blood vessels. Conclusion Explosion shock wave can result in severe large blood vessel injury, which should be paid much attention during treatment of ex-plosion shock injury.

Repairing articular cartilage defects in rabbits using bone marrow stromal cell-derived chondrocytes compounded with poly(lactic-co-glycolic acid) / 中国组织工程研究

BACKGROUND: Matrix material for cartilage tissue engineering exhibits too fast or too slow chondrocytes degradation in vivo, affecting tissue regeneration and shaping reconstruction, which has troubled scholars.OBJECTIVE: To amply bone marrow stromal cells (BMSCs) and induce them to chondrocytes in vitro, so as to study the feasibility of repairing articular cartilage defects in rabbits using poly(lactic-co-glycolic acid) (PLGA) loaded with BMSC-derived chondrocytes.DESIGN, TIME AND SETTING: Comparative experiment was performed at the Institute of Orthopaedics in the Fourth Military Medical University of Chinese PLA and the Center Laboratory of the Airforce General Hospital of Chinese PLA between June 2002 and June 2008.MATERIALS: A total of 36 two-month-old New Zealand white rabbits were used, and 4-6 mL bone marrow was aspirated from bilateral femoral trochanters in each animal. Primary culture and subcultures were done. In subcultures, the medium contained bone morphogenetic protein-2 (100 μg/L.), and high polymer hyaluronic acid was spread on bottom of the culture flasks in advance.In this way, the BMSCs were induced into chondrocytes and the third passage of cells at the adjusted density of 2.0×10~(10)/L wereco-cultured with PLGA for 24 hours, then PLGA-cell composites were prepared.METHODS: A defect of 4-mm in diameter and reaching medullary cavity were created in femoral condyles of 36 rabbits, and 36 right knees were treated with PLGA-cell composites, serving as experimental group, while 18 left knees with PLGA only as material group, and the other 18 knees remained untreated, as blank control group.MAIN OUTCOME MEASURES: At 4,8,12, 24 weeks after operation, the animals were euthanized and the newly formed tissues were observed macroscopically and microscopically, graded histologically, and analyzed statistically.RESULTS: Material group and blank control group shared identical outcomes of gross and histological observation, thus assigning into a control group. In the experimental group at 24 weeks, the defects were filled with white translucent cartilage tissue which appeared smooth and tenacious. The color and the luster were similar to that of normal cartilage, and was ill-demarcated from the surrounding normal cartilage. The cells on the surface paralleled to joint surface. Though the cells in the deep layer arranged disorderly, they tended to align vertically. The matrix was extensively stained. The subchondral bone formed.The tide mark basically recovered, and the new cartilage integrated with normal cartilage finely. In the control group, chondrocytes proliferated in the border, but in the bottom, there were mainly fibrous tissues. The histological grade of 12 and 24 weeks was different significantly from that of 4 and 8 weeks (P < 0.01). There were also significant differences between experimental group and control group at each time intervals after operation (P < 0.01).CONCLUSION: BMSCs were successfully induced into chondroncytes by use of bone morphogenetic protein-2 and high polymer hyaluronic acid. PLGA can be degraded and absorbed gradually while new cartilage tissues form. It can be used as a suitable scaffold material for repairing articular cartilage defects in tissue engineering.

Telomere,telomerase,cell of immortalization and aging / 国际生物医学工程杂志

Normal cells have limited proliferation ability.After certain cycles of proliferation,they will lose the response ability to growth factors and finally cease division and start the course of aging. In current opinion,lacking of the terminal end of a chromosome(telomere)is the cause for cells to loss the proliferation ability and leads cells to aging and death.The human telomerase catalytic subunit 1(hTERT)can activate telomemse which prolong DNAs of the terminal end of chmmosome and help cells gain genomic stabilization.The discoveries of telomere,telomerase and hTERT provide new idea for studying of cell aging and the findings are also applied in the establishment of immortal cell line. Also they may play an important role in the studv of biological feature of seed cell in tissue engineering and the establishment of cell bank.

Surface modification of biodegradable polymer/TCP scaffolds and related research / 生物医学工程学杂志

Under laboratory condition, the compound materials of Poly (DL-lactic-co-glycolic acid)/Tricalcium phosphate [PLGA/TCP(L), with component ratio of 7:3] were fabricated by combining the thermally induced phase separation (TIPS) with solvent-casting particulate-leaching (SCPL) approach. On the other hand, rapid prototyping (RP) technique manufactured PLGA/TCP scaffolds [PLGA/TCP(RP)] were obtained. These two kinds of carriers were coated with collagen type I (Col I). The extracted bovine bone morphogenetic protein (bBMP) was loaded into carriers to establish biomimetic synthetic bones. PLGA/TCP(L) scaffolds, demineralized bone matrices (DBM) of bovine cancellous bone, PLGA/TCP(L) scaffolds, biomimetic synthetic bones and OsteoSet bone graft substitutes were investigated. Scanning electron microscopy revealed that the microarchitecture of PLGA/TCP(RP) scaffolds was much better than that of PLGA/TCP(L) scaffolds. The diameter of macropore of PLGA/TCP(RP) scaffold was 350 microm. The porosities of PLGA/ TCP(L) scaffolds, DBM, PLGA/TCP(RP) scaffolds and OsteoSet bone graft substitutes were 21.5%, 70.4%, 58.6% and 0%, respectively (P<0.01). Modification of PLGA/TCP scaffolds with collagen type I [PLGA/TCP(L)-Col I and PLGA/TCP(RP)-Col I] essentially increased the affinity of the carriers to bBMP. Among these synthetic materials, PLGA/TCP(RP)-Col I-bBMP composite is promising as a novel bone graft substitute due to its advanced fabrication technique, good tri-dimensional microarchitecture and ideal components.

Suitable concentration of alginate with xenograft for constructing bone tissue engineering carrier / 中国组织工程研究

BACKGROUND:The structure of tissue engineering carrier affects the bio-action of cells greatly.OBJECTIVE: To investigate the biological characteristics of bone marrow stem cells (MSCs) in different concentrations of alginate combined with de-antigen bone xenograft (DBX).DESIGN: Observational trial.SETTING: PLA Institute of Trauma and Orthopedics, the Fourth Military Medical University of Chinese PLA.MATERIALS: Alginate, calcium chloride, MSCs, bone xenograft.METHODS: Bovine cancellous bone was out into cubes, which were degreased, deproteinized and then lyophilized.Cubes in pore size within 300-500 μm were selected for use after ethylene oxide sterilization. The purified sodium alginate was dissolved in DMEM cell culture medium of concentrations as different as 0.5%, 2%, 8% and 16%; 1×1012 L-1 induced MSCs were blended with isopyknic alginate-DMEM and compounded with DBX at a status of 0.5 Mpa negative pressure for 5 minutes in order to make a cell suspension fully fill into the pores of the cancellous bone. Then alginate was crosslinked with 50 g/L calcium gluconic acid for 30 seconds. The complex was put into a CO2 incubator and cultured for 4 days. The gel compound and cell growth in the pores of the complex were grossly observed with an inverted microscope. Status of cell growth in the complex with different concentrations of alginate was observed with scanning electron microscopeMAIN OUTCOME MEASURES: Compound status of alginate and bone xenograft, cell growth status and matrix secretion in compound carries.RESULTS: When the concentration of alginate was 0.25% or 1%, alginate was equally combined in DBX, while that of 4% and 8% only combined on the surface of cancellous bone. After in vitro cultured for 4 days, alginate of 0.25% were broken off from DBX surface. But alginate of 1% was equally combined with DBX pores with cells secreting well in alginate. Development of cells in alginate of 4% was restricted and no cells were seen in alginate of 8%.CONCLUSION: Alginate of 1% is suitable for constructing the carrier of bone tissue engineering with bone xenograft.

Longitudinal stress-strain relation of human peripheral vessels ex vivo / 中国组织工程研究

BACKGROUND: Due to the difference of species, the data of vessel in human are particularly useful for the clinical practice.OBJECTIVE: To analyze the longitudinal residue strain and the relationship between stress and strain of human limb arteries and veins, and explore the influence of different biomechanical properties on the repairs of limb injury.DESIGN: Observational trials.SETTING: Institute of Orthopaedics, Xijing Hospital of the Fourth Military Medical University of Chinese PLA.PARTICIPANTS: The experiment was carried out in the Xijing Hospital Affiliated to the Fourth Military Medical University of Chinese PLA from September 2005 to September 2006. The specimens were taken from 13 male amputee donors(who treated for accident injury), aged 18 to 30 years. Those tissue samples were used with the approval from the donors and offered by Xijing Hospital Affiliated to the Fourth Military Medical University of Chinese PLA.METHODS: ①Harvest and preservation of samples: The samples were obtained within 2 hours after death. The vessels were calibrated and harvested without any large branch to avoid the influence on the mechanical property of vessel wall,and then token on major vessels of limbs with Methylene Blue. The distance between the points token on vessel was measured by vernier caliper. The token vessels were cut and taken into Kreb's liquid in ice casement, then were kept into freezer (0-5 ℃). ②Longitudinal stretch ratio measurement: The vessels were taken into Kreb's liquid and the distance between the points token on vessel was measured by vernier caliper. The longitudinal residue strain was expressed by longitudinal stretch ratio. Lab temperature was 20-25 ℃, experiment was finished in 2 hours after sampling.③Stretch test: The vessel cut 1.0 cm was set into the instrument with Kreb's liquid for uniaxial tension test. The change length of each vascular specimen with or without the load and each load was measured three times and was averaged, lab temperature was 20-25 ℃, and experiments were finished in 5 hours after sampling. The curve of stress-strain was fitted by the measured data.MAIN OUTCOME MEASURES: Longitudinal stretching ratio, residue strain and stress-strain relationship of normal limb arteries and veins.RESULTS: The longitudinal stretch ratio of each artery decreased along vascular branch from proximal heart part to distal heart part, and that of each vein was contrast; There were significant difference in the longitudinal stretch ratios of major artery compared with those of saphena megna vein and branchiocephalicae vein (P ＜ 0.001). The curve of artery shifting right showed the stiffness of vessels decreased along vascular branch from proximal heart part to distal heart part. That of vein shifting left showed the stiffness of vessels increased along vascular branch.CONCLUSION: With the major artery of human limbs from proximal end to distal end, both the longitudinal residue strain and the vascular stiffness gradually decreases, as for the vein, the condition is contrast. It suggests that the longitudinal biomechanical property should be involved into the consideration of repairing the artery and vein injuries of different sites.

Transforming growth factor beta as a regulator of the regional expression of the immune cytokines in xenogeneic bone transplantation / 中国组织工程研究

BACKGROUND: For xenogeneic bone transplantation, immune rejection is the major problem that affects the prognosis. However, the understanding about the expression and regulation of the immune factors in heterogenic bone transplantation is limited. Interleukin-1 (IL-1), interleukin-6 (IL-6)and tumor necrosis factor alpha (TNF-α) are important immune factors, and are closely related with post-transplantation rejection.OBJECTIVE: To observe the regional expressions of mRNA and protein of IL-1α, IL-6 and TNF-α in xenogeneic bone transplantation, and to in vestigate the effect of transforming growth factor (TGF)-β on these immune factors.DESIGN: A randomized controlled experiment. SETTING: The Orthopaedic Institute of Xijing Hospital of the Fourth Military Medical University of Chinese PLA MATERIALS: Totally 72 male Balb/c mice, with a body mass of 20 to 25 g, were randomly divided into 3 groups: combined bovine cancellous bone (bCB) granule group (Group A), simple bCB granule group (Group B) and blank control group (Group C) with 24 mice in each group.INTERVENTIONS: This experiment was conducted at the Institute of Orthopaedics, Xijing Hospital, Fourth Military Medical Univessity of Chinese PLA from June 2003 to June 2004. In Group A, one bCB combined TGF-β was implanted into the muscles of left thigh of each mouse. In Group B, one bCB alone was implanted, and in Group C, no bCB was implanted. The number of proliferation of cells in bone implantation area or adjacent tissues of the operated area was observed at postoperative 4, 7, 14 and 21 days; and the regional expressions of several immune factors in im plants were detected with in situ hybridization and immunocytochemistry methods.MAIN OUTCOME MEASURES: Histological observation and detection of regional cellular density of the implants of the mice in each group; the regional expressions of mRNA and protein of IL-1α,IL-6 and TNF-α in the implants RESULTS: ①) Histological observation and detection of regional cellular density of the implants of the mice in each group: on day 7, the cellular density of the proliferated tissues was significantly higher in the Group A than in the Group B [(470.63±132.89), (311.46±93.69)/field,P ＜ 0.01];But on days 14 and 21, there was no significant difference. ②The regional expressions of mRNA and protein of IL-lα, IL-6 and TNF-α of the im plants of the mice in each group. On days 7 and 14 after xenografts were implanted, the regional expressions of IL-1α, IL-6 was respectively lower the xenografts combined with TGF-βthan in the simple xenogeneic bone (day 7: IL-1α 42.55±9.65 vs 67.95±17.82,IL-6 48.26±11.17 vs 77.21±15,16;day 14: IL-1α mRNA 84.77 ±7.42 vs 112.94±7.02,78.1 ±17.22 vs 121.18±15.44,P ＜ 0.01) ,but for the TNF-α, there was no significant difference (P ＞ 0.05).CONCLUSION: In the region of bone xenograft, a variety of cells express mRNAs and proteins of IL-1α, IL-6 and TNF-α, and their expressions are regulated by TGF-β. It may imply a kind of regulation of the immunity of bone xenograft by TGF-β.

Expression and purification of rat brain NT-3 fusion protein and its antibody preparation / 基础医学与临床

Objective To clone NT-3 gene from normal rat brain and to purify its fusion protein and to prepare specific high titer antibody so that to provide a foundation for further study for peripheral nerve injury.MethodsWe amplified target gene by RT-PCR and cloned it into the vector of pMD-18T,then analyzed its sequence and compared it with the sequence from GenBank.We subcloned it into pRSET-A vector and introduced it into Escherichia coli BL21.The expression was induced by IPTG,and identified by SDS-PAGE.The fusion protein was purified by niccolum purify kit.We immuned rabbits with immunological adjuvant for specificity antibody preparation.Results We got a 777 bp gene segment by RT-PCR.The DNA sequence was identical to rat NT-3 gene sequence in GenBank.It proved that the target gene was correctly inserted into the vector.A new protein band of about 34 ku appeared on SDS-PAGE after induction of IPTG.A specific high titer antibody of 1∶64000 was gained by immunizing rabbits with adjuvant.

Preparation of a three-dimentional acellular cartilage matrix scaffold and its characteristics / 中国矫形外科杂志

[Objective]To prepare a cartilage acellular matrix scaffold and to explore its feasibility in cartilage tissue engineering. [Methods]Microparticles about 100 ?m~154 ?m were prepared after calf cartilage physically shattered and experienced gradient centrifugation,and then treated by a modified Courtman's four-step method which was improved to produce acellular cartilage matrix.After this treatment the microparticles were made into 3% suspension which was placed into moulds.With the freeze-drying method,3-D cartilage acellular matrix (CACM) was prepared.The scaffolds were cross-linked by a neotype crosslinking agent genepin for 48h,and then placed into glycine solution server times for removing redundant genepin.The freeze-drying method was used to prepare CACM.The scaffolds were investigated by gross observation,histological staining (haematoxylin-eosin,toluidine blue) ,scanning electron microscope (SEM) observation and porosity measurement,water absorption rate and degradation rate analysis.After being cultivated for ten days,bone marrow stranal cells (BMSCs) of rabbit were seeded into the scaffold.MTT test and SEM were done to assess the growth and proliferation of BMSCs.[Results]Gross observation showed the scaffolds had a loosely porous and dark blue appearance after being cross-linked by genepin.The histological staining (haematoxylin-eosin,toluidine blue staining) showed that there were no chondrocyte fragments in the scaffold.The CACM scaffold had 90% porosity,(1314?337) % water absorption rate,and (13.69?7.3)% or (25.99?8.9) % degradation rate at 2 or 4 weeks.MTT test showed that BMSCs grew well in the 3-D CACM scaffolds of logarithmic trend,supporting that the scaffolds had no cytotoxic effect on BMSCs.SEM micrographs indicated that the scaffolds were porous and the cells covered the scaffolds firmly with cell processes.[Conclusion]The improved Courtman's four-step method makes a more thoroughly acellular scaffold.The 3-D CACM scaffold retains most of extracelluar matrix.After being cross-linked by genepin,the 3-D CACM scaffold has good biocompatibility and degradation rate of the scaffolds is decreased,which makes it a suitable carrier for cartilage tissue engineering.

Preparation of a collagenⅠ-sodium hyaluronate-fibrin glue tri-copolymer scaffold bonding with antigen-extracted bovine cancellous bone and its characteristics / 中国矫形外科杂志

0.05).[Conclusion]The osteochondral scaffold of the collagenⅠ-sodium hyaluronate-fbrin glue tri-copolymer scaffold bonding with antigen-extracted bovine cancellous bone has an appropriate structure and a good biocompatibility,which makes it a useful scaffold in the osteochondral tissue engineering.

Biological properties of a collagen/hydroxyapatite integrated composite scaffold used in osteochondral repair / 中国矫形外科杂志

[Objective]To observe the biological properties of a novel collagen/hydroxyapatite composite scaffold in vitro and to evaluate the possibility of application being used in tissue engineering for osteochondral repair.[Method]The scaffolds were constructed of collagen I and hydroxyapatite.The pore size and interpores of the scaffold were observed by scanning electronmicroscopy(SEM).The porosity was measured by liquid displacement method.Rabbit bone marrow stromal cells(BMSCs) were isolated and amplified,then inoculated onto the scaffold.By SEM scanning,the condition of the cells adhering onto the scaffold was observed.The proliferation of the cells on the scaffolds was examined using MTT method,and the growth curve was drawn.[Result]The scaffold possessed high porosity and proper pore size.The pore diameter of the collagen layer was about 90?m,the pore diameter of the HA layer was about 120?m,and the overall porosity of the composite scaffold was 75%.The proliferation of the cells on the scaffold was good.[Conclusion]The novel collagen/hydroxyapatite composite scaffold possesses desirable pore structure and good biocompatibility,and it can be used in tissue engineering for osteochondral repair.

Effect of reconstituted bone xenografts on tendon-to-bone healing after anterior cruciate ligament reconstruction / 中国矫形外科杂志

[Objective]To investigate the effect of reconstituted bone xenografts(RBX) on tendon-to-bone healing by means of imaging,histological and biomechanical studies.[Method]Anterior cruciate ligament(ACL) reconstruction was performed bilaterally in 25 skeletally mature rabbits using long digital extensor tendon grafts.RBX were implanted into the treated knee of each rabbit,with the contralateral knees as controls.Every three rabbits were killed at 2,6 and 12 weeks postoperatively for routine histological studies.The samples were processed through Micro CT and subsequent HE and toludine blue staining.The remaining 16 rabbits were sacrificed at 6 and 12 weeks.Their femur-tendon graft-tibia complexes were harvested for subsequent mechanical testing.[Result]At 6 and 12 weeks after operation,the values of BMD in the RBX group were higher than those in the control group(P

Spinal fusion of lumbar intertransverse process in rabbits using two different densities of autologous marrow mesenchymal stem cells combined with ?-TCP / 中国矫形外科杂志

[Objective]To investigate the effects of two different densities of bone marrow mesenchymal stem cells (BMSCs) combined with ?-TCP on spinal fusion in rabbits.[Method]Spinal fusion surgery of lumber intertransverse process were performed using MSCs/?-TCP as a graft in two groups of rabbits (low density group versus high density group),and rate of lumbar fusion,image characteristics,bone mineralization content,bone mineralization density,bone mineralization tissue volume and rate of new bone formation were observed.[Result]Compared with the low intensity group,the rate of lumbar fusion was greatly improved in the high density group(P

Effect of temperature on crosslinking of collagen/chitosan scaffolds with genipin / 中国矫形外科杂志

[Objective]To investigate the changes of pore sizes,crosslinking index,swelling ration,degradation rate,cytotoxicity and biocompatibility of genipin crosslinked collagen/chitosan scaffolds affected by the different crosslinking temperature points.[Method]The freeze-dried collgaen/chitosan scaffolds crosslinked with 0.5% genipin within 24 h were divided into 3 groups by crosslinking temperature:4℃ group,20℃ group and 36℃ group.The characteristics of pore sizes,crosslinking index,swelling ratio,degradation rate,cytotoxicity and biocompatibility were evaluated.Collagen/chitosan scaffolds without crosslinking were chosen as control group.[Result]With the increase of temperature,crosslinking index was increased,but swelling ratio and degradation rate were decreased.In 4℃ group,the crosslinking index was 47.88%?6.4%,the swelling ratio was 721%?46%,and the degradation rate was decreased by 3.95%?6.4% at 4 weeks.In 20℃ group,the crosslinking index was 67.69%?3.6%,the swelling ration was 662%?72%,and the degradation rate was 0.91%?5.9% in 4 weeks.In 36℃ group,the crosslinking index was 70.32%?5.7%,the swelling ration was 635%?27%,and the degradation rate was 0.66%?7.3% at 4 weeks.The above indexes of the three groups were much better than those of control group(P