Click chemistry extracellular vesicle/peptide/chemokine nanocarriers for treating central nervous system injuries

Central nervous system (CNS) injuries, including stroke, traumatic brain injury, and spinal cord injury, are essential causes of death and long-term disability and are difficult to cure, mainly due to the limited neuron regeneration and the glial scar formation. Herein, we apply extracellular vesicles (EVs) secreted by M2 microglia to improve the differentiation of neural stem cells (NSCs) at the injured site, and simultaneously modify them with the injured vascular targeting peptide (DA7R) and the stem cell recruiting factor (SDF-1) on their surface via copper-free click chemistry to recruit NSCs, inducing their neuronal differentiation, and serving as the nanocarriers at the injured site (Dual-EV). Results prove that the Dual-EV could target human umbilical vascular endothelial cells (HUVECs), recruit NSCs, and promote the neuronal differentiation of NSCs in vitro. Furthermore, 10 miRNAs are found to be upregulated in Dual-M2-EVs compared to Dual-M0-EVs via bioinformatic analysis, and further NSC differentiation experiment by flow cytometry reveals that among these miRNAs, miR30b-3p, miR-222-3p, miR-129-5p, and miR-155-5p may exert effect of inducing NSC to differentiate into neurons. In vivo experiments show that Dual-EV nanocarriers achieve improved accumulation in the ischemic area of stroke model mice, potentiate NSCs recruitment, and increase neurogenesis. This work provides new insights for the treatment of neuronal regeneration after CNS injuries as well as endogenous stem cells, and the click chemistry EV/peptide/chemokine and related nanocarriers for improving human health.

Research progress in the prevention and treatment of developmental dysplasia of the hip / 中华骨科杂志

Developmental dysplasia of the hip (DDH) is one of the most common orthopaedic diseases in children. Due to the complexity of risk factors, the molecular regulatory mechanism of its occurrence and development is still not clear. Understanding the molecular regulatory mechanism and morphological changes of DDH is of great significance for the exploration of the mechanism, the formulation of early screening, diagnosis and treatment strategies. Recently, with the development of development biology, molecular biology, preclinical medicine and clinical medicine, the risk factors and potential mechanism of DDH have been investigated deeply. Here, we reviewed the formation of anatomical structure during hip joint development, genes expression and signal pathways involved in endochondral ossification. Further, we analyzed the possible development stages, which might lead to the developmental instability. Previous studies have shown that the interaction between the femoral head and the acetabulum in the embryonic development of the hip joint determines the morphogenesis of the hip joint. The embryonic cartilage of the hip joint begins to develop at 5 to 12 weeks after fertilization, followed by the development of the primary and secondary ossification processes to form the hip joint with a complete structure. Transcription factors of SOX9 and RUNX2, which regulate chondrogenesis and osteogenesis during bone development, are mediated by HIF, WNT, FGF and PTHRP signal pathways. In addition, there are 28 potential pathogenic genes of DDH identified by clinical DDH case gene detection techniques, including whole genome sequencing and whole exon sequencing, which are of great significance for revealing the molecular mechanism of DDH occurrence. In addition, we summarized the current clinical screening methods, risk factors, diagnosis and treatment of DDH. Finally, we discussed the remaining challenges and possible future directions for DDH research and interventions, which may provide new ideas for the mechanism research, and clinical diagnosis and treatment strategies for DDH.

Experimental study on the prevention of bone loss by deferoxamine in type 1 diabetic mice with osteoporosis / 中华内分泌代谢杂志

Objective To observe the effect of a hypoxia mimicking agent deferoxamine (DFO) on the mineral density,volume,architecture,strength,and metabolism of the bones in type 1 diabetic mice withosteoporosis.Methods Type 1 diabetic mice model was established by intraperitoneal injections of streptozotocin.The mice were divided into control (normal mice),diabetes mellitus,and DFO groups.Micro-CT was used to analyze the bone mineral density,volume,architecture,and strength of the trabecule in the distal part of femurs.Three point bending test was carried out to evaluate the bone strength.Hematoxylin and eosin (HE) staining was performed to observe the alteration in the number of osteoblasts.Real-time PCR was used to detect the mRNA expressions of Runt-related gene 2 (Runx-2),osteoclacin,and tartrate resistant acid phosphatase (TRAP) in tibias.Western blot was used to detect the protein expressions of Hypoxia-inducible factor-1α(HIF-1α) and vascular endothelial growth factor (VEGF) in tibias.Results There was a decrease in mineral density,volume,strength of bones as well as deteriorated trabecular microarchitecture in diabetic mice as compared to control mice,which were partially improved by DFO treatment.Moreover,DFO treatment increased the number of osteoblasts and mRNA expression levels of Runx-2,osteoclacin,TRAP,as well as protein expression levels of HIF-1 α and VEGF(P＜0.05).Conclusion Bone loss could be partially prevented by DFO treatment in type 1 diabetic osteoporosis mice,which might be ascribed to increased bone formation via stimulating hypoxia inducible factor singnaling pathway.

Research status and progress of biomaterials for bone repair and reconstruction / 中国修复重建外科杂志

Artificial bone repair material is the best substitute for autologous bone transplantation. Bone repair materials are constantly being replaced and upgraded, which can be roughly divided into three generations: bioinert materials, bioactive materials, and smart materials. Research and development of bone repair materials with multiple biological activities, in vivo degradation property that perfectly fit for new bone formation, and ability of complete reconstruction of bone tissue in physiological state are the focus of future research.

Histomorphometryof the vertebra and its correlations with MRI signal intensity in rabbits with fluoride treatment / 中华骨科杂志

Objective To analyze histomorphometrical characteristics of the bone and bone marrow tissues of the lumbar vertebrae in rabbits with fluoride treatment,and its correlation with signal intensity of MRI.Methods Forty New Zealand albino rabbits aged three months old were randomly divided into fluoride exposure of 30 cases and control of 10 cases,male and female,half each.One hundred milligrams of sodium fluoride were added to the municipal water each liter (fluoride content 100 mg/L) as drinking waterto fluorine for 180 days.Twenty-four of 30 cases with fluoride exposure had complete data (male10 casesand female14 cases).The same municipal water was used as control drinking water (fluoride content ＜ 0.9 mg/L).Eight of 10 cases with control had complete data (male andfemale in half).Twenty-four cases with fluoride treatment and complete data were classified into sensitive and resistant type according to the MRI signal intensity of the lumbar vertebra.Histomorphometrics of the vertebra and its correlation with the MRI signal intensity,and sensitivity in early diagnosis of osteofluorosis and feasibility of susceptibility to osteofluorosis detected with MRI were analyzed.Results Theratios of trabecular bone volume (BV),hematopoietic cell volume (HV) and fluid volume (FV) in bone marrow tissue to total cavernous tissue volume (TT) in group with fluoride treatment were 18.3％±2.6％,45.2％±6.0％ and 10.4％±5.7％ respectively.These were 14.5％±2.8％,36.3％±7.3％ and 6.2％±2.1％ in control group respectively.These parameters in fluoride group were significantly increased compared to control group.The ratio 26.0％± 8.0％ of adipocyte volume (AV) to TV in fluoride group was significantly lower than that 43.3％±5.6％ in control group.Two of 24 cases with fluoride exposure (8.3％,2/24) were sensitive and the remaining 22 (91.7％,22/24) were in resistance.The valuesof BV/TT,HV/TV and FV/TV were considered to be sensitive,resistant and control from large to small,while AV/TV value were opposite.A comparison resuhs of signal intensity in MRI showed that vertebra T1WI contrast to noise ratio (CNR) in the sensitive was the minimum (3.0±0.8),followed by resistance (21.3±3.8) andmaximum in the control (28.3±3.1),but CNR of FsT2WIwas opposite.There were positive associations between T1WI and AV/TV,FV/TV and BV/TV,and between FsT2WI and FV/TV and BV/ TV.There were inverse associationsbetween FsT2WI and AV/TV.Theoptimal threshold value of the vertebra T1WI CNR was 23.2 or lessin early diagnosis of skeletal fluorosis,with sensitivity of 83.3％ and specificity of 100％.FsT2WI was 5.7 or more,with sensitivity of 45.8％ and specificity of 100％.Conclusion The pathogenesis of osteofluorosis is relative to changes in bone marrow microenvironment and cells number in bone marrow tissue,and is correlated to MRI signal intensity.

Histomorphometrical analysis of vertebral lamina with osteofluorosis and its correlation with signal intensity in MRI / 中华骨科杂志

Objective To analyze histomorphometrical characteristics of bone and bone marrow tissue in the vertebral lamina of patients with osteofluorosis, and to explore the influencing factors on signal intensity in MRI. Methods Spinal MRI of 109 patients (57 men, 52 women;age range 32-80 years;mean age 52 years) with osteofluorosis from December 2001 to May 2012 was analyzed retrospectively, including 48 patients in cervical segment, 31 in thoracic segment and 30 in lumbar segment. 36 pa?tients (16 men, 20 women;mean age 51 years;age range 34-68 years) had undergone laminectomy and the vertebral lamina speci?mens were collected. The cervical MRI of 48 patients with matching gender and age (26 men, 22 women;mean age 51 years, age range 34-71 years) was selected as control group, who were from areas where fluorosis is not endemic. All patients were divided in?to vertebra low, medium and high signal groups according to T1WI of MRI. The vertebra signal to noise ratio measure and stan?dardization of signal intensity were performed. Osteosclerosis, osteoporosis and normal bone were differentiated under spinal X?ray plain film. Combined with histomorphometric analysis of vertebra lamina in 36 patients, correlation between MRI signal intensity, histomorphometric parameters of the vertebra lamina and influencing factors on signal intensity were studied. Results 77 pa?tients (70.6%, 77/109) had osteosclerosis indicated by appearance of spine under X?ray, 29 (26.6%, 29/109) osteoporosis and 3 (2.8%, 3/109) normal bone. T1WI of MRI showed 25 cases had low signal vertebra, 52 medium signal and 32 high signal. The ver? tebra SNR in patients with osteofluorosis was lower on T1WI, T2WI and short time inversion recovery (STIR) sequences, compared with control group. Those with a low versus high signal on T1WI had 6.04 times the odds of osteosclerosis (OR=6.04, 95%CI 2.44-14.91, P<0.001). Histomorphometry of vertebral lamina in 36 patients with osteofluorosis was performed, revealing that not only the trabecular bone volume had changed, but also did the adipocyte volume and hemopoietic cell volume in the bone marrow tis?sues. Compared with normal reference values, trabecular bone volume was significantly increased (47.7%± 13.3% vs. 14.7%± 4.3%) (P<0.001);adipocyte volume was significantly decreased (12.3%±9.1%vs. 50.5%±8.7%);hematopoietic cell volume was decreased (40.0%±7.0%vs. 42.5%±8.5%) (P=0.038). There were inverse associations between trabecular bone volume and adipo?cyte volume (r=-0.869, P<0.001), and between trabecular bone volume and T1WI (r=-0.851, P<0.001) found by Pearson correla?tion test. In contrast, there were positive associations between T1WI and adipocyte volume (r=0.927, P<0.001). Conclusion The vertebra T1WI signal intensity is decreased in patients with osteofluorosis, resulting from increase of trabecular bone volume and re?duction of adipocyte volume. The vertebra STIR signal intensity is decreased, mainly caused by increase of trabecular bone volume.

Vascular endothelial growth factor promotes the proliferation and osteogenic differentiation of mouse bone marrow mesenchymal stem cells / 中国组织工程研究

BACKGROUND:Vascular endothelial growth factor (VEGF) is a crucial factor for regulating bone marrow mesenchymal stem cells in microenvironment, which can promote endothelial differentiation of bone marrow mesenchymal stem cells. But there is no report about VEGF effect on regulating the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells. OBJECTIVE:To investigate the regulatory role of VEGF in the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells. METHODS:The bone marrow mesenchymal stem cells were isolated and cultured from mouse long bones. cellCounting Kit-8 was used to test the proliferation of bone marrow mesenchymal stem cells cultured with different concentrations of recombinant VEGF proteins. The appropriate concentration of VEGF recombinant protein was selected to test their effects on the osteogenic differentiation of bone marrow mesenchymal stem cells. The mRNA and protein levels of Osterix, Runx2, alkaline phosphatase, osteocalcin and heme oxygenase-1 in bone marrow mesenchymal stem cells under induction of VEGF were detected by molecular biology methods. RESULTS AND CONCLUSION:(1) VEGF promoted the proliferation of bone marrow mesenchymal stem cells dose-dependently, and 100μg/L was the optimum concentration. (2) VEGF promoted the expression of Osterix, Runx2, alkaline phosphatase and osteocalcin in bone marrow mesenchymal stem cells under osteogenic induction. Similar results were obtained by alizarin red staining and quantification of numbers of calcium nodules. (3) VEGF induced the expression of heme oxygenase-1 in bone marrow mesenchymal stem cells at mRNA and protein levels. These findings indicate that VEGF can induce the expression of heme oxygenase-1 i in bone marrow mesenchymal stem cells, and promote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells.

Correlation between magnitude and duration of hydrostatic pressure and the differentiation of human bone marrow mesenchymal stem cells** / 中国组织工程研究

BACKGROUND:Mechanical signal has close correlation with the growth, development, repair and reconstruction of the skeletal system and the development of disease, the effect and the mechanism on bone marrow mesenchymal stem cel s is worthy to concern. OBJECTIVE:To explore the effect and mechanism of hydrostatic pressures on the differentiation of bone marrow mesenchymal stem cel s. METHODS:Short-term experiment:the human bone marrow mesenchymal stem cel s were incubated into the normal Dulbecco’s modified Eagle’s medium, osteogenic medium or the Dulbecco’s modified Eagle’s medium containing extracel ular signal-regulated kinase 1/2 inhibitor U0126, respectively. Homemade pressure loading system was used to impose 0, 40 and 80 kPa hydrostatic pressure for 1 and 4 hours. Long-term experiment:human bone marrow mesenchymal stem cel s were incubated into the normal Dulbecco’s modified Eagle’s medium or osteogenic medium respectively, and then 40 kPa hydrostatic pressures was loaded for 4 hours per day, and lasted for 14 days. The cel s without hydrostatic pressure were regarded as the control group. RESULTS AND CONCLUSION:Real-time quantitative reverse transcription PCR results showed that after osteogenic induction and simulated with 40 kPa hydrostatic pressure for 4 hours, the mRNA expressions of core binding factorα1 and osteocalcin in the bone marrow mesenchymal stem cel s were increased, while the mRNA expressions of peroxisome proliferator-activated receptorγ2 and adipsin were decreased, and the 80 kPa hydrostatic pressure did not cause such reactivity. The osteogenic induction effect of 40 kPa hydrostatic pressure could be partial antagonized with U0126. Histochemical staining showed that after simulated with 40 kPa hydrostatic pressure for 7 days, the expression and activity of alkaline phosphatase of bone marrow mesenchymal stem cel s were increased;after lasted for 14 days, the mRNA expressions of peroxisome proliferator-activated receptorγ2 and adipsin were increased. Certain intensity and duration of hydrostatic pressure stimulation can regulate the differentiation of bone marrow mesenchymal stem cel s, and the mechanism is only partly mediated by the extracel ular signal-regulated kinase 1/2 signaling pathway.

Effects of rat serum containing Chinese herbal medicine Sangen Decoction on osteoclastogenesis and bone resorption of osteoclasts induced by polymethylmethacrylate particles / 中西医结合学报

To investigate the effects of Sangen Decoction, a compound Chinese herbal medicine, on osteoclastogenesis and bone resorption function of osteoclasts induced by polymethylmethacrylate particles in vitro.

Effects of stress stimulation on repair process of rotator cuff tear / 中华创伤骨科杂志

Objective To explore the role and mechanism of stress stimulation in the repair process of the rotator cuff. Methods The supraspinatus tendon was cut off at the osteotendinous junction (OTJ) and then sutured in situ (Carpenter's model). The experimental group started treadmill running after one week immobility, and the control group were free in the cage. All animals were sacrificed at 4 time points (2, 4, 8 and 16 weeks after operation). Histological changes, immunohistology of yon Willebrand factor and tenascin-C at OTJ were compared between the 2 groups. Results There was no significant difference between the 2 groups in the early repair period. Significant differences between the 2 groups in histology and angiogenesis appeared 8 weeks after operation, and significant differences in expression Of Tenascin-C appeared 16 weeks after operation. Conclusion As stress stimulation can promote angiogenesis and expression of Tenascin-C at OTJ, facilitating the reconstruction of bone and tendon, it plays a critical role in the repair process of rotator cuff tear.

Role of hypoxia inducible factor-1α and VHL in murine endochondral ossification / 上海交通大学学报（医学版）

Objective To investigate the role of hypoxia inducible factor-1α (HIF-1α) and von Hippel-Lindau (VHL) in murine endochondral ossification. Methods The knockout of HIF-1α or VHL gene in murine osteoblasts was accomplished by conditional knockout technique at 4th, 8th and 12th week, and the differences between wild-type group and knock-out group in endochondral ossification were detected by HE staining, micro-CT scanning, trabecular bone area measurement, calcium content measurement, tetracycline fluorescence labeling, Real-time PCR and Western blotting. Results After knockout of HIF-1α gene in osteoblasts, the expression of vascular endothelial growth factor ( VEGF) reduced, the rate of new bone formation stepped down, the content of calcium became less, and the trabecular bone volume decreased (P <0.05) . After knockout of VHL gene in osteoblasts, the expression of VEGF increased, the rate of new bone formation stepped up, the content of calcium became more, and the trabecular bone volume was promoted (P < 0.001). Conclusion During murine endochondral ossification, VHL/HIF-1α signal pathway promotes angiogenesis through the stimulation of VEGF expression, which subsequently accelerates osteogenesis.

Osteogenic potential of fibrous ring tissue in cervical intervertebral disc / 中国组织工程研究

BACKGROUND: Ossification of intervertebral disc, and ossification of spinal ligament and fibrous tissues are still uncertain.OBJECTIVE: To observe osteogenic potential of fibrous ring in the cervical intervertebral disc during bone fusion.DESIGN, TIME AND SETTING: The comparative observation was performed at the Experimental Animal Center of Second Military Medical University and Shanghai Institute of Orthopedics in October 2006.MATERIALS: Ten healthy goats, including 6 males and 4 females; titanium alloy cervical hollow threaded columnar internal fixator (CHTF) for goat, simulated human used internal fixator by Kanghui Medical Innovation Co., Ltd., Changzhou.METHODS: Every goat underwent conventional anterior cervical decompression and internal fixation. Two adjacent intervertebral spaces among C2-6 were selected and implanted with 2 CHTFs for each space. Of the 4 CHTFs, 3 were filled with cancellous bone alone, cancellous bone plus fibrous ring, and fibrous ring alone, respectively; the other one filled with nothing served as blank control.MAIN OUTCOME MEASURES: Implant location and fusion condition on anteroposterior and lateral radiographs and CT plain scanning at 6 and 12 weeks postoperatively; bone graft fusion and regional tissue reaction by histology.RESULTS: Radiographs and CT showed that CHTF was in the position during the whole experimental procedure with no loosening, displacement or dislocation. At 6 weeks, bone tissue was found surrounding CHTF and the vertebral body, and bone bridge formed in the connection site of CHTF and the vertebral body. New cartilage and bone trabecula formation were found in the CHTF filled with cancellous bone alone, accompanied by necrotic original bone graft; in the CHTF filled with cancellous bone and fibrous ring, necrotic fibrous tissue and newly formed cartilage accumulation surrounding original bone trabecula and fibrous ring were found; 6 weeks after surgery, there were fibrocartilage in fibrous tissue of CHTF filled with fibrous ring alone, and at 12 weeks postoperatively, newly formed cartilage was observed. In blank control group, only few newly formed cartilages were found at 12 weeks postoperatively. CONCLUSION: Enchondral ossification of fibroblast may be the osteogenic pattern of fibrous ring of cervical intervertebral disc.

Effect of oxygen tension on chondrocytic phenotype in vitro / 中国矫形外科杂志

[Objective] To examine the specific gene and protein expression of primary and passaged chondrocytes in normal oxygen tension and hypoxia.[Methods]Articular chondrocytes were isolated from limb joint cartilage of C57BL/6 mice(3~5 days).Primary chondrocytes(P0)and passaled chondrocytes(P1,P2)were cultured in normal oxygen tension and hypoxia respectively for two days.The mRNA levels of collagen II,aggrecan,sox9,Indian hedgehog(ihh),parathyroid hormone-related protein(PTHrP),bone morphogenetic protein(BMP)-4 and wnt5a were examined with RT-PCR,and protein levels of collagen II,aggrecan were examined with immunohistochemistry and toluidine blue staining.[Results]During the culture of primary chondrocyte,the expression of collagen II and aggrecan increased under hypoxia,mRNA levels of collagen II,wnt5a increased and ihh decreased at the same time.The mRNA levels of special genes and differentiation related genes of passaged chondrocytes were not altered under hypoxia.[Conclusion]Protein and mRNA level of Collagen II of primary chondrocyte under hypoxia increased.It may be regulated by chondrocyte differentiation gene ihh,and wnt5a.The chondrocyte phenotype could not be resumed under hypoxia through short-term monolayer culture in vitro.

Study on estrogen receptor of adult human osteoblasts / 中西医结合学报

OBJECTIVE: To examine estrogen receptor (ER) in osteoblasts from adult human and to elucidate the mechanism of estrogen in modulating bone metabolism. METHODS: The cultured osteoblasts were harvested from bone chips by modified sequential digestive enzyme release and immunohistochemical assay of ER in osteoblasts were carried out in three groups of female adults: normal control (group 1), patients with moderate osteoporosis (group 2) and patients with serious osteoporosis (group 3). The percentages of ER-positive osteoblasts from the three groups were compared by t test. RESULTS: The brown marks that indicate ER were found in nuclei and plasma of the osteoblasts, and the percentages of ER-positive osteoblasts among three groups were significantly different. CONCLUSION: ERs exist in nuclei and plasma of the osteoblasts. Estrogen may modulate bone metabolism through binding ER in nuclei and plasma of the osteoblasts. The reduction of ER of osteoblasts may play an important role in the pathogenesis of postmenopausal osteoporosis.

Culture of human osteoclasts / 中西医结合学报

To establish a stable, useful culture system for human osteoclasts and to investigate the effect of osteoblasts on the differentiation, proliferation and activation of osteoclasts so as to provide a base for the studies on prevention and treatment of osteolysis and osteoporosis.

Expression of core-binding factor a1 by human skin fibroblasts induced in vitro / 中华外科杂志

<p><b>OBJECTIVE</b>To investigate the probabilities of core-biding factor a1 (Cbfa1) expression by human skin fibroblasts induced in vitro.</p><p><b>METHODS</b>The fibroblasts were isolated, purified from human skin, and were grown in incubation in the media of TNF-alpha, BMP-2, and combined TNF-alpha and BMP-2 at certain concentrations, respectively. The changes in biological features of these fibroblasts correlated with osteogenesis were detected by immunohistochemistry and RT-PCR assay.</p><p><b>RESULTS</b>TNF-alpha could switch phenotype of collagen in fibroblasts from Type I and III to Type I and induce fibroblasts to express Ras and BMP type I receptor (BMPR-IA). TNF-alpha in combination with BMP-2 could induce fibroblasts to express Cbfa1 and osteocalcin mRNA.</p><p><b>CONCLUSION</b>Human skin fibroblast could be induced into pro-osteoblast expressing Cbfa1, an osteoblast-specific transcription factor and a regulation of osteoblast differentiation, and combined use of TNF-alpha and BMP-2 was one of the regulating factors.</p>

Osteogenic potential of rabbit marrow stromal stem cells cultured in vitro: a histochemical and scanning electron microscopic study / 中华创伤杂志(英文版)

<p><b>OBJECTIVE</b>To further investigate the osteogenic potential of rabbit marrow stromal stem cells cultured in vitro.</p><p><b>METHODS</b>Rabbit marrow stromal stem cells were isolated by density gradient centrifugation method and amplified in the flasks, using the osteogenic inducing conditions (OGC) as the culture media. The osteogenic potential of marrow stromal stem cells were investigated by means of bone-seeking fluorescence (tetracycline) labelling, Alizarin red S (ARS) staining, Alcian blue-Sirius red (AS) staining, and scanning electron microscope.</p><p><b>RESULTS</b>After being passaged, the marrow stromal stem cells increased in number, became confluent and formed multi-layer structure. The stromal stem cells excreted innumerable tiny granules, heaping up on the cell body and merging gradually into foggy substances. These foggy substances kept on enlarging and formed round, oval, or flake-like nodules. These nodules revealed bright golden yellow fluorescence under fluorescence microscope when labelled with tetracycline. Histochemical study with specific new bone staining with ARS revealed positive calcium reaction, both denoting that they were newly formed bone tissues. After they were stained with AS, collagen and acid mucopolysaccharide were shown. Under scanning electron microscope, three types of cells with different configurations were found. They were globular cells, spindle-shaped cells and polygonal or polygonal cells. Granules were excreted from the cells and heaped up on the cell body. Needle-shaped and irregularly rectangular crystals also appeared and agglomerated with the granules to form nodules and trabecula-like or flake-like structures.</p><p><b>CONCLUSIONS</b>Sequence of events of bone formation by rabbit marrow stromal stem cells cultured in vitro is fully depicted and confirmed, which provides the foundation for further investigating the mechanisms of osteoblast differentiation from marrow stromal stem cells and the possible application in orthopaedics.</p>

Repair of articular cartilage by using induced autogeneic mesenchymal cells derived from bone marrow in rabbits / 中华骨科杂志

Objective To observe the tissus implanted into the damaged articular cartilage of rabbits with induced autogeneic mesenchymal cells. Methods The autologous mesenchymal cells derived from bone marrow of New Zealand rabbits were harvested. With basic fibroblast growth factor(bFGF， 25 ng/ml) and transforming growth factor beta 1(TGF-? 1， 2 ng/ml)， the cells were induced and expanded in cell culture. The induced cells with absorbable gelatin sponge as a carrier were then implanted into the damaged articular cartilage in rabbits as experimental group. The absorbable gelatin sponge without cells were served as control. Specimens were harvested at the end of 4, 12 and 24 week after implantation, and were stained with toluidine blue. Results By RT- PCR, it was confirmed that there was expression of typeⅡ procollagen mRNA in the induced mesenchymal cell. After implantation, it was difficult to macroscopically distinguish the repaired tissues from the normal cartilaginous tissue in the experimental group in 24 weeks. While the defect of articular cartilage was filled with white and swampy tissue in the control group at the same time. Histologically, the defective area of the articular cartilage was replaced by the formation of neo- cartilage which showed positive staining of toluidine blue in 4 weeks in the experimental group. The neo- cartilage was modeled to normal cartilage tissues in 12 weeks and was similar to the surrounding cartilage in 24 weeks. But in the control group, the defect of articular cartilage was not repaired by cartilage tissue at every stage and were replaced by fibrocartilage which was shown weakly positive staining of toluidine blue in 24 weeks. Conclusion The transplant of the induced autogeneic mesenchymal cells derived from bone marrow might promote repair of articular cartilage, and restore its structure and function.