Comparisons of Emu Necrotic Femoral Head Micro Structure Repaired in Two Different Methods / 中国医学科学院学报

<p><b>OBJECTIVE</b>To compare emu necrotic femoral head micro structure repaired in two different methods.</p><p><b>METHODS</b>Fifteen adult emus were divided into 3 groups (all n=5), and the right femoral head was selected to research. The first group was the control group; in the second group, femoral head necrosis was made by cryogen with liquid nitrogen; and in the third group, femoral head necrosis was made by local pure ethanol injection. Right femurs were taken for micro CT examination,then femoral head micro structures were compared among these three groups.</p><p><b>RESULTS</b>No infection or unexpected death was found in all groups. Compared with normal group, necrotic femoral heads in cryogen group showed that bone mineral density significantly reduced after repaire (P=0.015), trabecular space significantly reduced (P=0.001), bone volume fraction significantly enlarged (P=0.036), bone surface/volume fraction (P=0.032) and trabecular numbers (P=0.002) significantly enlarged; trabecular thickness showed no significant difference (P=0.060). Compared with control group, necrotic femoral heads in ethanol group showed that bone mineral density significantly enlarged after repaire (P=0.001), trabecular thickness (P=0.003) and bone surface/volume fraction (P=0.022) significantly enlarged, trabecular space (P=0.001) and bone volume fraction (P=0.001) significantly reduced; the trabecular numbers showed no significant difference (P=0.143). Compared with ethanol group, necrotic femoral heads in cryogen group showed significant lower bone mineral density after repair (P=0.001), significantly lower bone volume fraction (P=0.001), significantly lower trabecular thickness (P=0.001), significantly higher bone surface/volume fraction (P=0.022) and higher trabecular numbers (P=0.003); the trabecular space showed no significant difference (P=0.398).</p><p><b>CONCLUSION</b>Different repair methods make reconstructed femoral head weight bearing area have different bone structure and bone mineral density, along with different bone trabecular quality.</p>

Study of animal model of osteonecrosis induced by local ethanol injection in emu / 中国医学科学院学报

<p><b>OBJECTIVE</b>To establish a new animal model of osteonecrosis of the femoral head by local ethanol injection in emu.</p><p><b>METHODS</b>Eight milliliter ethanol was injected slowly to the operated femoral head with customized probe in twenty adult male emus. Postoperatively, hip magnetic resonance imaging was performed at 1, 4, 8, 12 weeks. After emus were sacrificed, the femurs were collected for micro-computed tomography and histological analysis.</p><p><b>RESULTS</b>No emu demonstrated signs of infection or died unexpectedly. Magnetic resonance imaging examination showed broad edema at proximal femur at 1(th) week, and the edema decreased with time, till local edema at femoral head at the 12(th) week. Histological images showed human-like osteonecrotic changes with active bone repair. There were significant differences in trabecular structure and bone mineral density between the operated and intact femoral heads. No collapse was found 6 months after the operation.</p><p><b>CONCLUSIONS</b>This emu model of femoral head osteonecrosis by local ethanol injection can progress to early stage osteonecrosis. The different repair methods may have certain correlation with the results of osteonecrosis of the femoral heads.</p>

Combined effects of NEL-like type 1 gene and zoledronate in preventing collapse of the femoral head / 中国医学科学院学报

<p><b>OBJECTIVE</b>To determine if combined therapy consisting of NEL-like type 1 gene (NELL-1) and zoledronate can prevent the collapse of the femoral head and stimulate the new bone formation in an animal model of osteonecrosis.</p><p><b>METHODS</b>Ischemic osteonecrosis was surgically induced in 24 SD rats, whicih were equally randomly divided into three groups: combination group, treated with both NELL-1 and zoledronate; sham operation group; and placebo group, treated with normal saline solution. The animals were killed 5 weeks after surgery. Radiography, MicroCT, histology, and immunohistochemistry were performed to analyze the results.</p><p><b>RESULTS</b>Morphologically, the femoral head was at good shape in the combination group, while mildly flattened femoral head was seen in the placebo group. No heterotopic ossifications were observed in each group. MicroCT assessment showed significantly higher total and bone mineral volume in the combination group than in the placebo group (P<0.01), whereas no such significant difference was found when compared with the sham operation group(P>0.05). Histological assessment showed more active osteoblast activity and reduced osteoclast activity in the combination group compared with placebo group.</p><p><b>CONCLUSION</b>A combination of NELL-1 and zoledronate can decrease the femoral head deformity while stimulating bone formation in a traumatic rat osteonecrois model, showing a potential to reverse the osteonecrosis.</p>

In vitro cartilage production using an extracellular matrix-derived scaffold and bone marrow-derived mesenchymal stem cells / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Cartilage repair is a challenging research area because of the limited healing capacity of adult articular cartilage. We had previously developed a natural, human cartilage extracellular matrix (ECM)-derived scaffold for in vivo cartilage tissue engineering in nude mice. However, before these scaffolds can be used in clinical applications in vivo, the in vitro effects should be further explored.</p><p><b>METHODS</b>We produced cartilage in vitro using a natural cartilage ECM-derived scaffold. The scaffolds were fabricated by combining a decellularization procedure with a freeze-drying technique and were characterized by scanning electron microscopy (SEM), micro-computed tomography (micro-CT), histological staining, cytotoxicity assay, biochemical and biomechanical analysis. After being chondrogenically induced, the induction results of BMSCs were analyzed by histology and Immunohisto-chemistry. The attachment and viability assessment of the cells on scaffolds were analyzed using SEM and LIVE/DEAD staining. Cell-scaffold constructs cultured in vitro for 1 week and 3 weeks were analyzed using histological and immunohistochemical methods.</p><p><b>RESULTS</b>SEM and micro-CT revealed a 3-D interconnected porous structure. The majority of the cartilage ECM was found in the scaffold following the removal of cellular debris, and stained positive for safranin O and collagen II. Viability staining indicated no cytotoxic effects of the scaffold. Biochemical analysis showed that collagen content was (708.2-44.7) µg/mg, with GAG (254.7 ± 25.9) µg/mg. Mechanical testing showed the compression moduli (E) were (1.226 ± 0.288) and (0.052 ± 0.007) MPa in dry and wet conditions, respectively. Isolated canine bone marrow-derived stem cells (BMSCs) were induced down a chondrogenic pathway, labeled with PKH26, and seeded onto the scaffold. Immunofluorescent staining of the cell-scaffold constructs indicated that chondrocyte-like cells were derived from seeded BMSCs and excreted ECM. The cell-scaffold constructs contained pink, smooth and translucent cartilage-like tissue after 3 weeks of culture. We observed evenly distributed cartilage ECM proteoglycans and collagen type II around seeded BMSCs on the surface and inside the pores throughout the scaffold.</p><p><b>CONCLUSION</b>This study suggests that a cartilage ECM scaffold holds much promise for in vitro cartilage tissue engineering.</p>

Three-dimensional deformation field measurement system based on micro-CT images / 医用生物力学

Objective To develop a novel measurement system composed of micro-CT, mechanical loading device and digital volume correlation (DVC) technique, so as to measure the three-dimensional microstructural deformation field in bone tissue. Methods Uniaxial compression was applied on the specimen with the micromechanical loading device, and CT scans were also conducted while maintaining the same loads; then sequential CT images were matched and searched accordingly by DVC method to calculate the micro-displacement in the specimen along three directions before and after loading; repeated scanning of zero-displacement and rigid body translation were used to evaluate the accuracy and precision of the system. The three-dimensional distribution of displacement field in bovine cancellous bone was measured by the system. Results The result from repeated scanning of zero-displacement showed that the highest accuracy of measurement was performed in the loading direction and the precision was less than tenth of the CT resolution. The result of rigid body translation showed that the standard deviation was 0.001~0.002 μm. For cancellous bone specimen under the load of 600 N, the range of micro-displacement was 100.35~110.25 μm, with multilayer field distribution. Conclusions The accuracy and precision of this measurement system can meet the requirement of DVC method. It is proved that this system can be used for measuring the three-dimensional micro-deformation field in the cancellous bone and as a measurement platform for investigating the relationship between deformation distribution and structural response in bone tissue for the future research.

Effect and mechanism of zoledronate on prevention of collapse in osteonecrosis of the femoral head / 中国医学科学院学报

<p><b>OBJECTIVE</b>To observe the effect and mechanism of zoledronate on prevention of collapse in an animal model of osteonecrosis.</p><p><b>METHODS</b>Ischemic osteonecrosis was surgically induced in 16 SD rats (which were further divided into zoledronate group and placebo group); another 8 rats were used as sham surgery group (n=8). The animals were killed 5 weeks after surgery. Radiographic, Micro-CT, histological, and immunohistochemical assessments were performed.</p><p><b>RESULTS</b>Radiographic assessment showed better preservation of the femoral head shape in the zoledronate group than in the placebo group but not significantly different from the sham surgery group. Micro-CT assessment showed higher total volume, bone volume, and total mineralized content in the zoledronate group(all P0.05). Compared with the placebo group, the zoledronate group had reduced osteoclast and osteoblast activity, as confirmed by histological examinations.</p><p><b>CONCLUSION</b>Zoledronate can decrease the femoral head deformity by reducing the osteoclast activity while suppressing new bone and vessels formation in a rat model of traumatic osteonecrosis, and therefore may delay the collapse of femoral head.</p>

Research advances in experimental animal models of osteonecrosis / 中国医学科学院学报

Osteonecrosis is a common disease, mainly affecting femoral head. Good animal models are helpful in research on the pathologic mechanism of osteonecrosis and the exploration of effective treatment. Although it is relatively easy to establish animal models of early osteonecrosis of femoral head using various approaches, it is difficult to develop an animal model that mimics the full range of osteonecrosis of femoral head. In this paper, we reviewed the current researches on experimental animal models of osteonecrosis, with an attempt to provide evidences for choosing the appropriate animal models and find the way of future development.

Evaluation of an extracellular matrix-derived acellular biphasic scaffold/cell construct in the repair of a large articular high-load-bearing osteochondral defect in a canine model / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Osteochondral lesion repair is a challenging area of orthopedic surgery. Here we aimed to develop an extracellular matrix-derived, integrated, biphasic scaffold and to investigate the regeneration potential of the scaffold loaded with chondrogenically-induced bone marrow-derived mesenchymal stem cells (BMSCs) in the repair of a large, high-load-bearing, osteochondral defect in a canine model.</p><p><b>METHODS</b>The biphasic scaffolds were fabricated by combining a decellularization procedure with a freeze-drying technique and characterized by scanning electron microscopy (SEM) and micro-computed tomography (micro-CT). Osteochondral constructs were fabricated in vitro using chondrogenically-induced BMSCs and a biphasic scaffold, then assessed by SEM for cell attachment. Osteochondral defects (4.2 mm (diameter) × 6 mm (depth)) were created in canine femoral condyles and treated with a construct of the biphasic scaffold/chondrogenically-induced BMSCs or with a cell-free scaffold (control group). The repaired defects were evaluated for gross morphology and by histological, biochemical, biomechanical and micro-CT analyses at 3 and 6 months post-implantation.</p><p><b>RESULTS</b>The osteochondral defects of the experimental group showed better repair than those of the control group. Statistical analysis demonstrated that the macroscopic and histologic grading scores of the experimental group were always higher than those of the control group, and that the scores for the experimental group at 6 months were significantly higher than those at 3 months. The cartilage stiffness in the experimental group (6 months) was (6.95 ± 0.79) N/mm, 70.77% of normal cartilage; osteochondral bone stiffness in the experimental group was (158.16± 24.30) N/mm, 74.95% of normal tissue; glycosaminoglycan content of tissue-engineered neocartilage was (218 ± 21.6) µg/mg (dry weight), 84.82% of native cartilage. Micro-CT analysis of the subchondral bone showed mature trabecular bone regularly formed at 3 and 6 months, with no significant difference between the experimental and control groups.</p><p><b>CONCLUSION</b>The extracellular matrix-derived, integrated, biphasic scaffold shows potential for the repair of large, high-load-bearing osteochondral defects.</p>

A new animal model of osteonecrosis induced by focal alternative cooling and heating insults / 中国医学科学院学报

<p><b>OBJECTIVE</b>To establish a new animal model of osteonecrosis of the femoral head(ONFH) with improved consistency and incidence of femoral head collapse for studies on the mechanism of osteonecrosis. and on the assessment of treatment effectiveness.</p><p><b>METHODS</b>Twenty adult male emus were used. Guide instrumentation was constructed to position the customized probe just articularly and at the proximal part of the femoral head. An alternating focal liquid nitrogen freezing and radiofrequency heating was applied. At 2, 4, 8, 12 and 16 weeks after surgery, hip magnetic resonance imaging (MRI) was performed. Before the emus were sacrificed, barium sulfate was infused to lower extremities for microangiography. The femoral samples were scanned by micro-computed tomography (Micro-CT) and evaluated histologically.</p><p><b>RESULTS</b>No bird demonstrated signs of infection or died unexpectedly. Hip MRI showed changes massive edema at the 4th week, increasingly localized abnormal signals at the 8th'" week, and femoral head collapse at the 12'h week. Micro-CT scans and histological images at the 16th week showed human-like osteonecrotic changes with impaired local blood supply. Bone mineral density of the collapsed head was (380. 31 + 28. 12) mg/cm3 and trabecular spaces were (0. 86 ±0.32) mm; both were significantly lower than those in the control side, which were (415.75 41.28) mg/cm3 and (1. 17 ± 0. 17) mm, respectively (P < 0. 05). Bone volume fraction of the collapsed head was(47.28 ± 17. 14)% and trabecular thickness was (506. 17 ± 220. 58) p.m; both were significantly higher than those at control side, which were (30. 92 ± 4. 01)% and (325. 50 ±44. 53) pm, respectively (P <0. 05). The microangiography at the 16th week showed that vessel volume fraction was (0. 315 ± 0. 055)% , which was significantly higher than the collapsed side [ (0. 142 ± 0. 059)% ] (P <0. 05).</p><p><b>CONCLUSIONS</b>The emu model of fem-oral head osteonecrosis was successfully established using focal alternating cooling and heating insults. The models, with improved consistency and incidence of femoral head collapse, can be used in studies on the mechanism of osteonecrosis and on the assessment of treatment effectiveness.</p>

Biocompatibility evaluation of electrospun aligned poly (propylene carbonate) nanofibrous scaffolds with peripheral nerve tissues and cells in vitro / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Peripheral nerve regeneration across large gaps is clinically challenging. Scaffold design plays a pivotal role in nerve tissue engineering. Recently, nanofibrous scaffolds have proven a suitable environment for cell attachment and proliferation due to similarities of their physical properties to natural extracellular matrix. Poly(propylene carbonate) (PPC) nanofibrous scaffolds have been investigated for vascular tissue engineering. However, no reports exist of PPC nanofibrous scaffolds for nerve tissue engineering. This study aimed to evaluate the potential role of aligned and random PPC nanofibrous scaffolds as substrates for peripheral nerve tissue and cells in nerve tissue engineering.</p><p><b>METHODS</b>Aligned and random PPC nanofibrous scaffolds were fabricated by electrospinning and their chemical characterization were carried out using scanning electron microscopy (SEM). Dorsal root ganglia (DRG) from Sprague-Dawley rats were cultured on the nanofibrous substrates for 7 days. Neurite outgrowth and Schwann-cell migration from DRG were observed and quantified using immunocytochemistry and SEM. Schwann cells derived from rat sciatic nerves were cultured in electrospun PPC scaffold-extract fluid for 24, 48, 72 hours and 7 days. The viability of Schwann cells was evaluated by 3-[4,5-dimethyl(thiazol-2-yl)-2,5-diphenyl] tetrazolium bromide (MTT) assay.</p><p><b>RESULTS</b>The diameter of aligned and random fibers ranged between 800 nm and 1200 nm, and the thickness of the films was approximately 10 - 20 µm. Quantification of aligned fiber films revealed approximately 90% alignment of all fibers along the longitudinal axis. However, with random fiber films, the alignment of fibers was random through all angle bins. Rat DRG explants were grown on PPC nanofiber films for up to 1 week. On the aligned fiber films, the majority of neurite outgrowth and Schwann cell migration from the DRG extended unidirectionally, parallel to the aligned fibers. However, on the random fiber films, neurite outgrowth and Schwann cell migration were randomly distributed. A comparison of cumulative neurite lengths from cultured DRGs indicated that neurites grew faster on aligned PPC films ((2537.6 ± 987.3) µm) than randomly-distributed fibers ((493.5 ± 50.6) µm). The average distance of Schwann cell migration on aligned PPC nanofibrous films ((2803.5 ± 943.6) µm) were significantly greater than those on random fibers ((625.3 ± 47.8) µm). The viability of Schwann cells cultured in aligned PPC scaffold extract fluid was not significantly different from that in the plain DMEM/F12 medium at all time points after seeding.</p><p><b>CONCLUSIONS</b>The aligned PPC nanofibrous film, but not the randomly-oriented fibers, significantly enhanced peripheral nerve regeneration in vitro, indicating the substantial role of topographical cues in stimulating endogenous nerve repair mechanisms. Aligned PPC nanofibrous scaffolds may be a promising biomaterial for nerve regeneration.</p>

Study on differentiation of rat adipose tissue-derived stromal cells into Schwann-like cells / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To investigate the phenotypic, molecular and biological characteristics of adipose tissue-derived stromal cells (ADSCs) differentiated alonely a Schwann cells (SCs) lineage and to provide a new cells' seed source for nerve tissue engineering or cell therapy.</p><p><b>METHODS</b>Cultured ADSCs were isolated from SD rats and the undifferentiated ADSCs were confirmed by detection of MSC-specific cell-surface markers. The ADSCs were differentiated along a glial cell lineage using an established cocktail of growth factors. Following differention, we used immunofluorescene staining and RT-PCR to evaluate the characteristics of differentiated WJMSCs.</p><p><b>RESULTS</b>ADSCs were successfully isolated from the rats' fat tissue. The isolated ADSCs expressed CD29, CD90 but not CD34, CD44 nor CD45. Osteogenic differentiation was detected by Alizarin red staining and adipogenic differentiation was comfirmed by Oil-red O staining. ADSCs treated with a mixture of glial growth factors adopted a spindle-like morphology similar to Schwann cells. Immunocytochemical staining and RT-PCR analysis revealed that the treated cells expressed the glial markers S100, P75 and glial fibrillary acidic protein indicative of differentiation.</p><p><b>CONCLUSION</b>ADSCs can be differentiated into cells that are Schwann-like in terms of morphologic features and phenotype and could be suitable Schwann-cell substitutes for nerve repair in clinical applications.</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>

Fabrication and characteristics of oriental poly (lactic-co-glycolic acid) scaffold: a preliminary study / 中华外科杂志

<p><b>OBJECTIVE</b>To explore the method of fabricating oriental scaffolds and investigate the biocompatibility of the scaffolds as well as cells distribution within the scaffolds in vitro.</p><p><b>METHODS</b>The oriental poly (lactic-co-glycolic acid) (PLGA) scaffolds were fabricated with modified emulsion-phase separation method. The scaffolds were treated with plasma and then anchored with collagen I. Articular chondrocytes were loaded into the scaffolds. The growth status and distributing characteristic of the cells were investigated by environmental scanning electron microscope.</p><p><b>RESULTS</b>The scaffold was well compatible with the articular chondrocytes. The cells could reach to 2.5 mm depth with unilateral loading. The cells distributed evenly in the scaffold and lined along the inner pipes.</p><p><b>CONCLUSIONS</b>The oriental scaffold fabricated could significantly promote the distributing characteristics of the chondrocytes. The vertical alignment of the chondrocytes within the scaffold is closely similar to that of articular cartilage.</p>

Osmic acid staining of myelin sheath in normal and regenerated peripheral nerves / 中华创伤杂志(英文版)

<p><b>OBJECTIVE</b>To introduce a practical, economical, and time-saving method to stain (with osmic acid) the myelin sheath in normal and regenerated peripheral nerves.</p><p><b>METHODS</b>A total of 12 Sprague Dawley rats, weighing 250-320 g (mean equal to 276 g+/-38 g), were divided into two groups: a normal nerve group (n equal to 6) and a regenerated nerve group (n equal to 6). In the normal nerve group, the ventral and dorsal roots of L(4) to L(6) and their sciatic nerves were harvested for histological analysis. While in the regenerated nerve group, the right sciatic nerves were severed and then repaired with an epineurial microsuture method. The repaired nerves were harvested 12 weeks postoperatively. All the specimens were fixed in 4% paraformaldehyde and transferred to 2% osmic acid for 3-5 days. Then the specimens were kept in 75% alcohol before being embedded in paraffin. The tissues were cut into sections of 3 micromolar in thickness with a conventional microtome.</p><p><b>RESULTS</b>Under a light microscope, myelin sheaths were clearly visible at all magnifications in both groups. They were stained in clear dark colour with a light yellow or colorless background, which provided high contrast images to allow reliable morphometric measurements. Morphological assessment was made in both normal and regenerated sciatic nerves. The ratios of the myelin area to the fibre area were 60.28%+/-7.66% in the normal nerve group and 51.67%+/-6.85% in the regenerated nerve group, respectively (P less than 0.01).</p><p><b>CONCLUSIONS</b>Osmic acid staining is easy to perform and a very clear image for morphometrical assessment is easy to obtain. Therefore, it is a reliable technique for quantitative evaluation of nerve morphology.</p>

Influences of decellularization processes on immunogenicity of chemically acellular nerve allografts / 中华外科杂志

<p><b>OBJECTIVE</b>To investigate the relationship between immunogenicity and decellularization processes of chemically acellular nerve allografts.</p><p><b>METHODS</b>Adult Sprague Dawley rats were used as nerve donors and adult male Wistar rats used as nerve recipient hosts. 25 mm nerve segments were excised from SD rats' sciatic nerves. The nerve segments were decellularized via an improved chemical decelluarization treatment as follows: (1) nerve segments were rinsed with cold sterile Ringer's solution; (2) stabilized by pinning the ends to a thin plastic support, and submerged in 4% Triton-100 solution 12 h; (3) soaked into 3% sodium deoxycholate for 12 h; (4) washed in distilled water for 6 h. The procedures were repeated once again. The acellular nerve allografts from SD rats were sterilized by gamma irradiation and implanted into Wistar rats subcutanously. The control group was implantation of fresh nerve allografts from SD rats. The immunogenicity of acellular nerve allograft was tested by immunohistochemical examination of the intensity of CD3(+), CD4(+) and CD8(+) cells that infiltrated the allografts. Ulnar nerve segments were obtained from forearms of dogs and decellularized according to above procedures. According as the decellularization times, The ulnar nerve segments were divided into three subgroups: in group I, group II and group III, the nerve segments were decellularized repeatedly two, three and four cycles respectively. Each ulnar nerve segment was subdivided into five portions from proximal to distal end. The degrees of decellularization, demyelination and basal lamina integrity of extracellular matrix scaffold were observed with microscope and assessed by a score system. The immunohistochemical staining of GAG was observed.</p><p><b>RESULTS</b>The intensity of CD3(+), CD4(+) and CD8(+) T cells that infiltrated the allografts was greatly lower in acellular nerves than in fresh nerves. The mild cell-mediated host-graft immunorejection in acellular nerves was observed. On the decellularization procedures, the cells were completely extracted from nerves in all groups, but the myelin sheath were partially existed, and the GAG was present in the basal membrane of myelin sheath. In the score of demyelination, there were no statistical differences between groups (P > 0.05). The statistical difference of basal lamina integrity scores between group I and group II, group I and group III were significant (P < 0.05). As increasing the times of process, the degrees of disintegrity of basal lamina was significantly enhanced.</p><p><b>CONCLUSIONS</b>Although decellularization processes significantly reduce the cell-mediated immunorejection of acellular nerve allografts, it can induce mild immunoreaction all the same, the antigen that responsible for immunogenicity may be the residual component of GAG in myelin sheath.</p>

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>

Comment on some spinal surgery / 中国医学科学院学报

Bone tumor metastases is relatively common. Approximately 5%-20% of spinal metastatic tumors will finally invade the spinal cord and exacerbate symptoms. The adoption of combined approach in recent years has significantly increased the survival rate. After excision of the tumors, internal fixation instrumentations are needed to stabilize the vertebrae. These procedures must be performed under the condition that the patients can tolerate the operation. Fused vertebra with instrumentation may cause degenerative diseases at the adjacent segments, which has been a problem of concern recently. Results of biomechanical tests indicated that these degenerative changes are related to the increased motion range of the neighboring segments. An old view is "the more rigid the instrumentation is, the better the results are", which has been disproved by clinical evidences. Improper use of internal spine fixation instrument should be avoided. Artificial intervertebral disc replacement can produce favorable short-time effects, however, its long-term effects and complications still requires further observations.

Acellular nerve allograft by chemical extraction in humans / 中华外科杂志

<p><b>OBJECTIVE</b>To develop a procedure by which Schwann cells and myelin in the peripheral nerve could be removed while the basal lamina tubes remained intact, and to obtain a thick and long acellular nerve allograft in humans.</p><p><b>METHODS</b>Four ulnar nerves 10.0 cm long and 4.0 - 5.0 mm in diameter were excised from a donated male body and cleaned from external debris. The nerves were treated with a solution of Triton X-100 and a solution of sodium deoxycholate at room temperature. After a final wash in water, the nerves were stored in phosphate-buffered saline (PBS, pH 7.2) at 4 degrees C. HE, luxol fast blue and fibrin staining were performed to visualize cells, myelin and basal membranes respectively and immunohistochemical staining was performed to visualize the presence of laminin, a Schwann cell lamina component, both in fresh and acellular nerve segments. To reveal overall structure better, methylene blue-fuchsin staining was performed in semithin section. The ultrastructure of acellular and fresh nerves were observed and photographed in a transmission electron microscope.</p><p><b>RESULTS</b>The acellular human ulnar nerve was white long cylinder with well elasticity and ductility. HE, myelin and fibrin staining revealed that cells, axons and myelin sheath were removed and basal membrane was preserved after extraction procedure. Staining for the presence of laminin showed that the Schwann cell basal lamina component were present in the nerves after chemical treatment. Methylene blue-fuchsin staining and transmission electron microscopy showed that the myelin sheaths were absent in the extracted nerve segments and empty basal lamina tubes remained in the endoneurium.</p><p><b>CONCLUSIONS</b>We developed an extracted procedure with the detergents of Triton X-100 and deoxycholate, by which cells, axons and myelin sheaths could be removed from a human ulnar nerve while the basal lamina tubes remain intact and a thick long acellular nerve allograft is obtained. The laminin, a Schwann cell basal lamina component, can be preserved in the acellular nerve.</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>