Preparation of human meniscus acellular matrix / 中国骨伤

<p><b>OBJECTIVE</b>To investigate development of a cell extraction process for preparing human meniscus acellular matrix, and morphology and biomechanical properties.</p><p><b>METHODS</b>Human meniscus were subjected to modified eight-step detergent, then, the specimens were assessed by staining with haematoxylin-eosin, toluidine blue, sirius red, saffron O, alcain blue and hoechst-33258, et al. The ultrastructure of the specimens was observed with scanning electron microscope. Transient recovery rate of deformation, maximal recovery rate of deformation and maximal compressive strength were tested to determine the biomechanical properties of the scaffold.</p><p><b>RESULTS</b>Every stain confirmed that the celluar constituents of the specimens were removed. The specimens stained positively by staining with sirius red. Lacuna were found irregularly not only on the surface of the meniscus,but also in the meniscus with scanning electron microscope. Pores in the specinmens were large, the diameter of pores was 80 to 760 microm, porosity was over 67%. The transient recovery rate of deformation was (89.62 +/- 1.04)%, the maximal recovery rate of deformation was 100% and the maximal compressive strength was (3.04 +/- 0.13)N, when the specimens were compressed 30%.</p><p><b>CONCLUSION</b>The modified eight-step detergent can remove the immunogenic cell components from human meniscus, in addition, 3D extracellular matrix can be retained. The scaffold has good biomechanical properties. This scaffold stands a good chance to be an implant for future tissue engineering of the human meniscus.</p>

Mechanisms of autologous chondrocytes mass transplantation in the repair of cartilage defects of rabbits' knee / 中国骨伤

<p><b>OBJECTIVE</b>To trace the pathological changes of the cultured autologous chondrocytes mass after implanted in cartilage defects and investigate the pathophysiological mechanisms of the antologous chondrocytes mass transplantation in the repair of cartilage defects.</p><p><b>METHODS</b>Twenty-four New Zealand white rabbits of 4 to 6 month-old and weighing more than 3.0 kg (female and male was unrestricted) were randomly divided into experiment group and the control group. For 12 rabbits of experiment group, the cartilage defects were repaired with the autologous chondrocytes mass and sealed with one piece of periosteum. Firstly, cartilage tissue of 10 to 30 mg was obtained from the shoulder of the rabbits after anaesthetized by 1 mg/kg 20% sumianxin. Then, chondrocytes were isolated from the cartilage tissue with 0.2% type II collagenase digestion and were cultured in DMEM/F-12 supplemented with 20% fetal bovine serum (FBS), 50 microg/ml ascorbic acid-2-phosphate, 0.4 mM proline, 5 microg/ml insulin and 1 mM non-essential amino acids (NEAA) in flasks in vitro. The cells were harvested until a thin film of the cells covered the bottom of the flask could be seen with naked eyes. Then the film was collected with a curled glass stick and formed a solid mass. On this time, the animal was anaesthetized again and the full-thickness cartilage square defect of 4.0 mm x 6.0 mm was fabricated in the patellar grove of distal femur, and then the cellular mass was transplanted into the defect covered by one piece of periosteum which obtained from the upper anterior of tibia and sealed with the femoral condyles. For 12 rabbits of the control group, the defects were sealed with one piece of periosteum only. The animals were sacrificed in the 1st, 3rd, 6th and 12th weeks after the operation respectively. The histologic sections were stained with safranin O-fast green, hematoxylin-eosin (H&E) and picric acid-Sirius red and immunostained for type II collagen and aggrecan.</p><p><b>RESULTS</b>In the 1st week, the transplanted cells oriented to articular surface differentiated to matured hyaline chondrocytes and excrete large amount cartilage matrix. In the 3rd week, the trend was more obvious and the periosteum was union to the cell mass. In the 12th week, the defects were repaired with hyaline-like cartilage tissue, and in the 24th week, the repair tissue turned to matured hyaline cartilage. In the control group, the defects were repaired with fibrocartilage tissues.</p><p><b>CONCLUSION</b>It was evidenced that the defects were repaired by the autologous chondrocytes mass transplantation. The procedure was gradual and initialed from up toward joint to down to the deep of the defect.</p>

Hydroxyapatite modified titanium promotes superior adhension and proliferation of corneal fibroblast in comparison with pure titanium / 国际眼科杂志(Guoji Yanke Zazhi)

AIM: To determine whether hydroxyapatite modified titanium promotes superior adhension and proliferation of rabbit corneal fibroblast in comparison with pure titanium.METHODS: We used bioactive hydroxyapatite to modify titanium surfaces. Fourth passage fibroblasts of rabbit cornea were seeded on hydroxyapatite modified titanium surfaces, pure titanium and glass surfaces. Cell adhension, proliferation and morphology were detected at 24 hours, 48 hours, and 72hours using a acridine orange stain. Further studies of cell morphology were performed using scanning electron microscopy.RESULT: Ceil counts were significantly greater on hydroxyapatite modified titanium surfaces at each time point(P＜0.05).At 24 hours, cell spreading was greater on hydroxyapatite-coated titanium and glass than on the pure titanium. At 72 hours, compared with pure titanium and glass surfaces, the cells on hydroxyapatite modified titanium surfaces had greater spreading area and longer stress fibers.CONCLUSIONS: Hydroxyapatite modified titanium promotes superior adhension and proliferation of rabbit corneal fibroblast in comparison with pure titanium.

Biological characterization of rabbit's articular chondrocytes by confluent culture in vitro / 中华外科杂志

<p><b>OBJECTIVE</b>To obtain large amount of differentiated chondrocytes in vitro, examine and compare the biological characterization of rabbits' articular chondrocyte cultured in different density in vitvo.</p><p><b>METHODS</b>From November 2001 to June 2004, articulate tissues were obtained from the joints of the adult rabbits. Chondrocytes were isolated from the cartilage tissue with type II collagenase digestion and cultured in DMEM/F-12 supplemented with 20% fetal bovine serum (FBS). The chondrocytes were cultured with low density of monolayer culture and high density of confluent culture respectively. The differentiated phenotype was evaluated by histochemistry or immunohistochemistry.</p><p><b>RESULTS</b>When chondrocytes cultured in monolayer and in low density, it proliferated rapidly during the three generations, but with the same time, dedifferentiation was also rapid. After the third passage, most of the passage cells lost the phenotype, and the proliferation also stagnated. While chondrocytes cultured in high density, dedifferentiation slowed down. And even the phenotypes of the dedifferentiated chondrocyte which were cultured in low density could reduced partly by followed high density culture.</p><p><b>CONCLUSIONS</b>Culture chondrocytes by high density in vitro can effectively maintain the differentiated phenotype of chondrocyte. It also keeps the proliferation character as monolayer culture. The dedifferentiated chondrocyte caused by many passages could redifferentiate partly. So it is indicated that confluent culture of original or expanded chondrocytes in high density is a better culture methods than culture in low density.</p>

The preparation, structure evaluation and preliminary application of biomimetic biphasic calcium phosphate scaffold / 中华外科杂志

<p><b>OBJECTIVE</b>To fabricate biomimetic biphasic calcium phosphate BCP ceramic scaffolds using three-dimensional (3D) gel-lamination technology and evaluated their structure with 3D parameters and related method.</p><p><b>METHODS</b>Series two-dimensional images of femoral head's specimen of dogs were obtained by micro-computed tomography (Micro-CT). According to these images, porous biomimetic biphasic calcium phosphate (BCP) ceramic scaffolds with oriented trabecular structure were fabricated by three-dimensional (3D) gel-lamination technology. And then, the three-dimensional structure of the scaffolds were reconstructed by computer according to Micro-CT images of these scaffolds and evaluated by three-dimensional parameters. These parameters included bone volume fraction (BVF, BV/TV), bone surface/bone volume (BS/BV) ratio, trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular spacing (Tb.Sp) and structure model index (SMI). The biomechanical properties and biocompatibility of these scaffolds were also evaluated in the study. Six scaffolds, which were combined with BMCs (bone mesenchymal cells, BMCs), were planted into the bone defect of six dogs' femoral head respectively.</p><p><b>RESULTS</b>There was no significant difference between trabecular samples and BCP scaffolds in BV/TV, Tb.Th, Tb.N, and Tb.Pf (P > 0.05). The trabecular system of the scaffold, which had some orientation, represented plate-like model. With a micro-porous porosity of 62%, the average compressive modulus and ultimate strength along the axis of the scaffolds reached (464.0 +/- 36.0) MPa and (5.6 +/- 0.8) MPa respectively. The results of animal test indicated that the trabeculae of these scaffolds were covered by a layer of new bone after 10 weeks of operation.</p><p><b>CONCLUSION</b>Porous BCP scaffolds have been produced with oriented microarchitectural features designed to facilitate vascular invasion and cellular attachment and with initial mechanical properties comparable to those of trabecular bone.</p>

An experimental study of demineralized bone matrix to repair bone defects as a scaffold of tissue engineering / 中国医学科学院学报

<p><b>OBJECTIVE</b>To evaluate application of the sponge of demineralized bone matrix (SDBM) in tissue engineering of bone.</p><p><b>METHODS</b>SDBM was prepared from long bone of rabbits. Bone marrow cells were flushed from the bone shaft of femurs of a two-month-old New Zealand white rabbit. After the cells were cultured for 9 days, the flasks were added into dexamethasone (10(-8) mol/L), beta-glycerophosphate sodium (10 mmol/L) and L-ascorbic acid (50 micrograms/ml). After 5 weeks, the cultured cells were collected and marked by 5-Bromo-2'-dexyouridine (BrdU). The grand sum of cells seeded on a piece of SDBM was about (4-6) x 10(6). The composites of cells and SDBM (tissue engineered chip, TEC) were implanted into muscles and bone defects of radius in rabbits. A standard procedure was applied to make a 10 mm long defect bilaterally in the radius of nine skeletally mature male New Zealand white rabbits. All of the 18 defects were randomly divided into three groups: group I, six defects were grafted by TEC; group II, six defects were grafted with SDBM alone; group III, six defects were empty.</p><p><b>RESULTS</b>The results of radiographic and histological evaluation showed that all of the defects were repaired in group I and group II at 6 weeks, none of the defects was repaired in group III. The results of BrdU staining showed that the staining was positive in group I, but negative in group II. Biomechanical test showed that the compressive ultimate strength (CUS) of new bone in TEC implanted group was comparable with normal radius (P = 0.623) and in SDBM implanted group was significant lower than normal radius (P = 0.038).</p><p><b>CONCLUSIONS</b>The TEC can form cartilage and bone tissue in muscles and repair segmental bone defects. SDBM is a kind of effective natural scaffold in tissue engineering of bone.</p>