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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Experimental research of repairing large segment of dogs bone defect with massive bioactive bone substitutes / 中国矫形外科杂志

[Objective]To evaluate the effect of massive bioactive bone substitute in repairing large animals bone defect and to know its degrading rate.[Method]The massive Polylevolactic acid?collage calcium phosphate(PLLA?cTCP) carrieres by rapid forming technology was making,and then compounding rhBMP-2 and carrieres in a ratio of 3mg rhBMP-2 to one carrier was compounded to prepare the massive bioactive bone substitutes for dogs bone defect.Then the massive bone substitutes were implanted into 2.0cm dogss radius defects in the experiment group,and the massive carriers were implanted into in the control group.The repairing effect was evaluated by radiography,histology and biomechanics,and the degrading rate of the substitues was calculated in an image analysis apparatus.[Result]Radiographically,in the experiment group,the defects were connected by callus in all dogs in 12 weeks postoperatively;in 24 weeks,the callus rebuilt well.But in the control group,there was no callus formed in 24 weeks postoperatively,and the defects were not repaired.Histologically,in 12 weeks postoperatively,the outer layer of the callus in the experiment groups was lamellar bone and the center were trabecular bone,myeloid tissue and partial degrading carrier;in 24 weeks,the lamellar bone was more compact,trabecular bone decreased,myeloid tissue increased,and the carrier degraded more.In the control group,in 12 weeks postoperatively,the fibrous tissue wrapped and infiltrated into carrier,at the same time,part of the carrier degraded;in 24 weeks,the carrier was divided up by fibrous tissue and degraded more.The degrading rate of the carder in 12 weeks in the experiment group was 43.2%,in the control group was 35.7%,in 24 weeks 58.4% and 45.4%.Biomechanics,in 24 weeks after postoperation,the radius strength in the experiment group was superior to that in the normal bone.[Conclusion]The massive bioactive bone substitutes have satisfactory repairing effect on the radius bone defects of the large animal,but its degrading rate needs improving.

Comparison of two osteoarthritis models in rabbit / 中国矫形外科杂志

[Objective]To establish osteoarthritis model in rabbit knees by two kinds of surgery methods,and to explore their applicable conditions.[Method]Seventeen rabbits were divided into three groups(Hulth group,ligament-excised group and control group),they were anesthetized and operated differently according to their group.The rabbits were sacrificed at 1,3 and 6 weeks after surgery.The femoral condylars were harvested and studied in both gross morphological and pathohistological aspect.[Result]The degeneration of articular cartilage of the two surgery groups got worse by time,and their Mankin's scores were significantly higher than those in the control group(P

Osteogenic differentiation of adipose-derived stem cells from rabbits in vitro / 中国矫形外科杂志

[Objective]To observe the differentiation of adipose derived stem cells(ADSCs)into osteogenic cells in rabbit in vitro.[Method]ADSCs were prepared by collagenase Ⅰ digestion of subcutaneous fat from the nape of Japanese white rabbit after being excised and finely minced.Cells were identified by stro-1 immunocy to chemistry,and collagen Ⅰ immunocytochemistry,alkaline phosphatase assay and Von Kossa stain after osteogenic differentiation was performed.[Result](1)ADSCs were stro-1 positive.(2)The expression of collagen Ⅰ,alkaline phosphatase and calcium deposit of ADSCs were all positive under the influence of osteogenic differentiation medium.[Conclusion]ADSCs have similar characteristics to bone marrow stromal cells,but are much more easier to have high number upon harvest and bring less pain will occur during taking ADSCs than taking BMDCs.

Influence of continuous pressure on the function of rabbit bone marrow stromal cells proliferation / 中国矫形外科杂志

[Objective]To investigate the influence of pressure on the proliferation of rabbit bone marrow stromal cells(MSCs) and explain the relationship of the hip prosthesis submerge and loosening with continuous pressure after revision of total hip replacement.[Method]A unit of MSCs pressure model was set up to load different pressures on MSCs cultured in vitro.[Result]MSCs showed more proliferation capacity under discontinuous pressure.MTT assay was used to determine the cell proliferation of primarily cultured MSCs under discontinuous pressure.The MSCs OD value of pressured groups was much smaller than the control group at different times.Various magnitudes and durations of the discontinuous pressure could significantly suppress MSCs proliferation with the magnitude-dependent.[Conclusion]At the early period after revision of total hip replacement,patients should lie and exercise on the bed until 6 weeks.Otherwise,the weight-bearing pressure may restrain MSCs proliferation which is harmful to union and may cause prosthesis submerge and loose.

Effect of concentrations of serum on differentiation of adipose-derived adult stem cells into chondrocytes / 中国矫形外科杂志

[Objective]To study the differentiation of adipose-derived adult stem cells(ADASCs)into chondrocytes induced by TGF-?1 in the presence of different concentrations of serum in vitro.[Method]Adipose tissue-derived adult stem cells were isolated from rabbits subcutaneous adipose tissue using enzymatic digestion.The adipose tissue was minced and digested with collagenase type I,and the released cells were collected by density centrifugation and then placed in culture.After being passaged three times,ADASCs were induced to differentiate into chondrocytes by chondrogenic culture media containing 1% or 10% of FBS for 2 weeks.MTT,alkaline phosphatase toluidine blue staining and type Ⅱ collagen immunohistochemistry staining were then tested in both groups.Gray value was analyzed by Leica patho-image analysis system to observe if there was any difference in differentiative status between the two groups.[Result]MTT showed that the cells of 10% FBS group had higher proliferation activity than those of 1% FBS group(P

Study of hTERT activating telomerase in human bone marrow mesenchyme stem cells / 中国矫形外科杂志

[Objective]To explicate whether the telomerase activity is regulated by human telomerase reverse transcriptase (hTERT) in human bone marrow mesenchyme stem cells(hBMSC).[Method]hBMSC were cultured and transfected with eukaryotic expressing plasmid pCIneo-hTERT encoding hTERT. After selection with G418 to stabilize the transfection,expression of hTERT mRNA was detected with TR-PCR, detecting the expression of hTERT protein was detected with Western Bolt, and the telomerase activity in untransfected and transfected cells were detected by RT-PCR.[Result]The hMSCs grew well after transfecting plasmid pCIneo-hTERT.The cells began to suspend and die after the day of the G418 selection. At the tenth day,all the untransfected cells were dead, but the transfected cells began to clone proliferation. So the density of G418 subdued to 100 ?g/ml for maintaining selecting, at the twentieth day,there were obvious anti-G418 cell clones. After stable transfection, hTERT was expressed at mRNA and protein level in these anti-418 cell clones, and meanwhile telomerase activity was positive and obviously raise up in these transfected cells.[Conclusion]In human' bone marrow mesenchyme cells,telomerase could be activated by exogenous hTERT. This is a foundation to establish immortalized human bone marrow mesenchyme stem cell line.

Research on a novel bilayered scaffold being used in tissue engineering (TE) of osteochondral repair / 中国矫形外科杂志

[Objective]To explore the potential of a novel collagen I /chitosan /Nano?-tricalcium phosphate(?-TCP)bilayered scaffold for being used in tissue engineering(TE)of osteochondral repair.[Method]Bilayered scaffolds were produced with collagen Ⅰ,chitosan and?-TCP,using a special cross-linking and freeze drying method.The pore size,porosity and interpores of the scaffold were observed by scanning electron microscopy(SEM).Rabbit bone mesenchymal stem cells(BMSC)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.The cell-scaffold composite were then induced to differentiate towards cartilage by 3-D culturing,and then implanted into muscle pouches 2 weeks later.The result was observed 6 weeks later by HE staining,toluidine blue staining and type Ⅱ collagen immunohistochemistry.[Result]The scaffold possessed high porosity and proper pore size,the porosity was above 95%.BMSC could adhere onto the scaffold well,and the proliferation rate of the cells on the scaffolds was perfectly good.After in vitro induction,BMSC-scaffold composite can differentiate toward cartilage ectopicly.[Conclusion]The novel collagen I /chitosan /?-TCP bilayered scaffold possesses good pore structure and biocompatibility,and will possibly become a new biomaterial of TE used for osteochondral repair.

Comparison of the preparation of high purity collagen type I from four biomaterials / 中国矫形外科杂志

[Objective]To compare the purity and extraction rate of four purecollagen type I products prepared from various biomaterials by a limited enzyme digestion method for the use in tissue engineering.[Method]Bovine cortical bone, bovine achilles tendon, porcine achilles tendon and porcine skin were splitted into pieces of 0.2-0.5 mm. After being immersed in glacial acetic acid, they were extracted with pepsin. Then the crude products were dissolved, centrifuged, dialyzed and freeze drying to prepare pure collagen type I. The final products were confirmed by absorbance, amino acid analysis and'SDS-PAGE electrophoresis comparing them with the products of Sigma Company.[Result]The wave length of maximum absorbance of the final products was 230 nm, and the amino acid analysis and SDS-PAGE electrophoresis confirmed that the final products were collagen type I. The purity of product extracted from bovine cortical bone was the highest (96.12%) and higher than that from Sigma Company. The extraction rate of bovine achilles tendon collagen was the highest (75.34%).[Conclusion]Collagen type I of higher purity and higher extraction rate can be prepared using a limited enzyme digestion method.And the product from bovine cortical bone is better than the others,which has a promising prospect.