The application of comparative proteomic analysis to screen proteins associated with mechanical properties of engineered cartilage: a preliminary study / 中华整形外科杂志

<p><b>OBJECTIVE</b>To study proteins correlated with the mechanical properties of engineered cartilage by screening significantly changed proteins during cartilage formation by comparative proteomic analysis.</p><p><b>METHODS</b>Human chondrocyte, cultured and expanded, were seeded onto a polyglycolic acid/polylactic acid (PGA/PLA) scaffolds. After 4 weeks of culture in vitro, the constructs were divided into three groups. There were 6 specimens in each group. For the regular in vitro culture group (A), the constructs were kept in culture at the original condition for an additional 6 weeks. For in vivo groups, the constructs were implanted subcutaneously into nude mice for either 6 weeks (B) or 12 weeks (C). All specimens were harvested for gross observation, average wet weight and volume measurement, histology, immunohistochemistry and biomechanics to evaluate the results. Meanwhile, comparative proteomic analysis was performed for each group, and those proteins involved in extracellular matrix with at least 2 folds up-regulation were chosen for further exploration. The correlations between Young's modulus and the relative content of the selected proteins were analyzed by Pearson correlation coefficient.</p><p><b>RESULTS</b>All these samples in the three groups eventually formed hyaline-like cartilage structure. Specimens in C and B groups were similar with adult articular cartilage in appearance, and had multiple mature lacuna in histology. However, those specimens in A group had loose texture with irregular hypertrophy lacuna. Specimens implanted for 12 weeks in vivo had better wet weight (372.5 +/- 35.4) mg and Young's modulus (8.68 +/- 2.65) MPa than those cultured in vivo for 6 weeks (346 +/- 34.5) mg, (3.25 +/- 1.24) MPa (P < 0.01). In group A, they were (184.4 +/- 12.28) mg and (0.7 +/- 0.23) MPa. This study had detected 44 proteins in ECM by comparative proteomic analysis, then chosing the greatest ratio of 6 up-regulation proteins compared between C and A groups. The correlation results indicated the content of Decorin, Chondroadherin and Fibromodulin were linear correlation with the mechanical properties of engineered cartilage (P < 0.05).</p><p><b>CONCLUSIONS</b>Comparative proteomic analysis could provide large scale information of associated proteins, making it profit for advanced research on the relationship between extracellular matrix and mechanical properties of engineered cartilage by combination with tissue reconstruction techniques.</p>

Preliminary study of in vitro chondrogenesis by co-culture of chondrocytes and adipose-derived stromal cells / 中华整形外科杂志

<p><b>OBJECTIVE</b>To explore the feasibility of in vitro chondrogenesis by co-culture of chondrocytes and adipose-derived stromal cells (ADSCs) so as to confirm the hypothesis that chondrocytes can provide chondrogenic microenvironment to induce chondrogenic differentiation of ADSCs.</p><p><b>METHODS</b>Human ADSCs and porcine auricular chondrocytes were in vitro expanded respectively and then were mixed at the ratio of 7:3 (ADSCs: chondrocytes). 200 microl mixed cells (5.0 x 10(7)/ml) were seeded onto a polyglycolic acid/polylactic acid (PGA/PLA) scaffold, 8 mm in diameter and 2 mm in thickness, as co-culture group. Chondrocytes and ADSCs with the same cell number were seeded respectively onto the scaffold as positive control group and negative control group. 200 microl chondrocytes (1.5 x 10(7)/ml) were seeded as low concentration chondrocyte group. There were 6 specimens in each group. All specimens were harvested after in vitro culture for 8 weeks in DMEM plus 10% FBS. Gross observation, histology, immunohistochemistry, wet weight measurement and glycosaminoglycan (GAG) quantification were used to evaluate the results. Multiple-sample t-test statistics analysis was done to compare the difference of wet weight and glycosaminoglycan(GAG) content between the groups.</p><p><b>RESULTS</b>Cells in all groups had fine adhesion to the scaffold and could secrete extracellular matrix. In co-culture group and positive control group, cell-scaffold constructs could maintain the original size and shape during in vitro culture. At 8 weeks, cartilage-like tissue formed in gross appearance and histological features, and abundant type II collagen could be detected by immunohistochemistry. Wet weight and glycosaminoglycan(GAG) content of co-culture group were respectively (174 +/- 12) mg and (7.6 +/- 0.4) mg. There were respectively 75% (P < 0.01) and 79% (P<0.01) of those of positive control group. In negative control group, however, constructs shrunk gradually without mature cartilage lacuna in histology. In low concentration chondrocyte group, constructs also shrunk obviously with small amount of cartilage formation at the edge area of the construct, and wet weight was (85 +/- 5) mg, which was 37% (P<0.01) of that of positive control group.</p><p><b>CONCLUSIONS</b>Chondrocytes can provide chondrogenic microenvironment to induce chondrogenic differentiation of ADSCs and thus promote the in vitro chondrogenesis of ADSCs.</p>

Influence of in vivo or vitro microenvironment on the mechanical properties and histological structure of tissue engineered cartilage / 中华整形外科杂志

<p><b>OBJECTIVE</b>To investigate the influence of in vivo or vitro microenvironment on the mechanical properties and histological structure of tissue engineered cartilage, and to provide the appropriate parameters for cartilage construct in vitro.</p><p><b>METHODS</b>Human fetal articular chondrocytes were cultured and expanded in vitro, the passage 2 chondrocytes were seeded at the density of 6 x 10(7) cells/cm3 to cylindric dimensional scaffolds made by polyglycolic acid (PGA) and polylactic acid (PLA). These constructs were cultured in vitro for 4 weeks. After 4 weeks, part of samples were implanted subcutaneously into nude mice for 6 and 12 weeks, the others continued to be cultured in vitro. All specimens were harvested after 6 and 12 weeks, and evaluated by gross observation, histology, histochemistry, ultrastructure and mechanical test.</p><p><b>RESULTS</b>All specimens in vivo and vitro eventually formed good shape hyaline cartilage. The constructs in vivo group was white color with smooth surface, and had better mechanical properties than those in vitro, by TEM we can observe the thick and striated collagen fibers in regularly arranged collagen fibril which was similar to adult articular cartilage. The constructs in vitro group was yellow color with coarse surface, the appearance and ultrastructure was similar to fetal articular cartilage. Specimens implanted for 12 weeks in vivo had better compressive modulus(38.28 +/- 3.95) MPa and collagen diameter (41.58 +/- 2.78) nm than those cultured in vitro at the same time (4.12 +/- 0.63) MPa, (15.83 +/- 1.70) nm (P < 0. 01).</p><p><b>CONCLUSIONS</b>The structure and function of human tissue engineered cartilage became mature gradually from vitro to vivo, thick and striated collagen fibrils net similar to adult articular cartilage can be formed in constructs of vivo group,increased collagen cross-linking might be the reason that their mechanical properties been greatly improved.</p>

The study on the role of the soluble factors secreted by engineered cartilage in inducing bone marrow stromal cells chondrogenesis / 中华整形外科杂志

<p><b>OBJECTIVE</b>To study the role of the soluble factors secreted by tissue engineered cartilage in promoting bone marrow stromal cells (BMSCs) chondrogenesis as an important aspect.</p><p><b>METHODS</b>Porcine BMSCs, chondrocytes and dermal fibroblasts were respectively in vitro expanded and then seeded onto the polyglycolic acid/polylactic acid (PGA/PLA) scaffold. After 3 days, they were indirectly co-cultured by transwell. BMSCs-scaffold constructs were co-cultured with chondrocytes-scaffold constructs as experiment group (Exp), while co-cultured with fibroblasts-scaffold constructs as control group. BMSCs with the same cell number were seeded onto the scaffolds as another control group. There were 3 specimens in each group. All specimens were harvested after in vitro indirect co-culture for 8 weeks. Gross observation, histology, immunohistochemistry and RT-PCR were used to evaluate the results.</p><p><b>RESULTS</b>The BMSCs-scaffold constructs co-cultured with chondrocytes-scaffold shrunk gradually during in vitro culture, but formed the mature lacuna structures and metachromatic matrices, collagen II expression could be observed by immunohistochemistry and RT-PCR examination. In the control group, the constructs shrunk greatly during in vitro culture and showed mainly fibrous tissue.</p><p><b>CONCLUSIONS</b>The soluble factors secreted by chondrocytes can solely induce chondrogenic differentiation of BMSCs and thus promote the in vitro chondrogenesis of BMSCs.</p>

Repair of calvarial defects with human umbilical cord blood derived mesenchymal stem cells and demineralized bone matrix in athymic rats / 中华整形外科杂志

<p><b>OBJECTIVE</b>To investigate the feasibility of using human umbilical cord blood derived mesenchymal stem cells (UCB-MSCs) and demineralized bone matrix (DBM) scaffolds to repair critical-sized calvarial defects in athymic rats.</p><p><b>METHODS</b>Human UCB-MSCs were isolated, expanded and osteogenically induced in vitro. Osteogenic differentiation of UCB-MSCs was evaluated by Alizarin Red staining and measurement of calcium content respectively, and then the cells were seeded onto DBM scaffolds. Bilateral full-thickness defects (5 mm in diameter) of parietal bone were created in an athymic rat model. The defects were either repaired with UCB-MSC/DBM constructs (experimental group) or with DBM scaffolds alone (control group). Animals were harvested at 6 and 12 weeks post-implantation respectively, and defect repair was evaluated with gross observation, micro-CT measurement and histological analysis.</p><p><b>RESULTS</b>Micro-CT showed that new bone was formed in the experimental group at 6 weeks post-implantation, while no sign of new bone formation was observed in the control group. At 12 weeks post-transplantation, scaffolds had been degraded almost completely in both sides. It was shown that an average of (78.19 +/- 6.45)% of each defect volume had been repaired in experimental side; while in the control side, only limited bone formed at the periphery of the defect. Histological examination revealed that the defect was repaired by trabecular bone tissue in experimental side at 12 weeks, while only fibrous connection was observed in the control group.</p><p><b>CONCLUSIONS</b>Tissue-engineered bone composed of osteogenically-induced human UCB-MSCs on DBM scaffolds could successfully repair the critical-sized calvarial defects in athymic rat models.</p>

Residual undifferentiated embryonic stem cells in embryoid bodies / 上海第二医科大学学报

Objective To explore the residual undifferentiated mouse embryonic stem cells (ESCs) in embryoid bodies. Methods Mouse R1 and Oct-4-GFP transgenic ESCs were firstly cultured in suspension to form embryoid bodies (EBs). Twenty days later, EBs were digested into single cells and then re-plated in standard ESC culture condition. The morphology of residual undifferentiated cells in EBs was observed, and surface makers and in vitro redifferentiation potency of residual cells were examined by flow cytometry and immunofluoreseent staining. The residual cells were expanded and subcutaneously injected into nude mice, and the specimens were harvested from the injection site for histological analysis 6 weeks after injection. Results There were residual undifferentiated ESCs in EBs differentiated for 20 days, which displayed clonal morphology and expressed undifferentiated cell markers of ESCs, including SSEA1, CD31, CD9 and Oct-4. The cells could be differentiated to form EBs again, and could be re-expanded from secondary EBs. The residual cells were able to form teratoma at the injection site, and mature endoderm, mesoderm and ectoderm tissues could be found in teratoma tissues. Conclusion There are residual undifferentiated ESCs after differentiation of ESCs into EBs. The residual ESCs can differentiate again in vitro and in vivo, and can residue again in the in vitro differentiation.

Tissue-engineering bone with ADSCs and coral scaffold for repairing of cranial bone defect in canine / 中华整形外科杂志

<p><b>OBJECTIVE</b>To investigate the application of tissue-engineering bone with ADSCs (adipose-derived stem cells) and coral scaffold for repairing of cranial bone defect in canine.</p><p><b>METHODS</b>Autologous ADSCs isolated from canine subcutaneous fat were expanded, osteogenically induced, and seeded on coral scaffolds. Bilateral full-thickness defects (20 mm x 20 mm) of parietal bone were created (n = 7). The defects were either repaired with ADSC-coral constructs (experimental group) or with coral alone (control group). Radiological, gross, biomechanical and histological observations were done to evaluate the bone regeneration.</p><p><b>RESULTS</b>Three-dimensional CT scan showed that new bones were formed in the experimental group at 12 weeks after implantation, while coral scaffolds were partially degraded in the control group. By radiographic analysis at 24 weeks post-transplantation, it showed that an average repair percentage of each defect was (84.19 +/- 6.45)% in experimental group, and (25.04 +/- 18.82)% in control group (P < 0.01). The maximum compression loading was (73.45 +/- 17.26) N in experimental group, and (104.27 +/- 22.71) N in control group (P <0.01). Histological examination revealed that the defect was repaired by typical bone tissue in experimental group, while only minimal bone formation with fibrous connection in the control group.</p><p><b>CONCLUSIONS</b>The tissue-engineering bone with autologous osteogenic ADSCs and scaffold could successfully repair the cranial defects in canine models.</p>

Preliminary study on tissue-engineered cartilage with human dermal fibroblasts co-cultured with porcine chondrocytes in vitro / 中华整形外科杂志

<p><b>OBJECTIVE</b>To explore the feasibility of constructing tissue-engineered cartilage with human dermal fibroblasts (HDFs) in vitro.</p><p><b>METHODS</b>Porcine articular chondrocytes and HDFs were isolated and in vitro expanded respectively. Then they were mixed at the ratio of 1:1 (chondrocytes: fibroblasts) . The mixed cells were seeded onto polyglycolic acid (PGA) scaffold at the ultimate concentration of 5.0 x 10(7)/ml as co-culture group. Chondrocytes and HDFs at the same ultimate concentration were seeded respectively onto the scaffold as chondrocyte group ( positive control group) and fibroblast group ( negative control group). The specimens were collected after in vitro culture for 8 weeks. Gross observation, histology and immunohistochemistry were used to evaluate the results.</p><p><b>RESULTS</b>In chondrocyte group, the cell-scaffold constructs could maintain the original size and shape during in vitro culture. The new formed cartilage-like tissue had typical histological structure and extracellular matrix staining similar to normal cartilage. In co-culture group the constructs shrunk slightly at 8 weeks, cartilage-like tissue formed and GAG could be detected for strong expression by Safranin O staining. Furthermore, using the specific identification, a few HDFs derived cells were found to form lacuna structure at the peripheral area of cartilage-like tissue. In fibroblast group, the constructs deformed and shrunk gradually without mature cartilage lacuna in histology.</p><p><b>CONCLUSION</b>The 3D-co-culture system can effectively induce the differentiation of HDFs to chondrocytes. The tissue-engineered cartilage can be constructed in vitro with the 3D-co-culture system.</p>

Constructing of smooth muscle layers of tissue engineered blood vessel in a bioreactor / 中华整形外科杂志

<p><b>OBJECTIVE</b>To investigate the feasibility of in vitro constructing tissue engineered blood vessels in bioreactor.</p><p><b>METHODS</b>Cell-PGA (polyglycolic acid) complex was constructed by seeding smooth muscle cells isolated from canine carotid artery on PGA unwoven fibers. The cell-PGA complex was cultured in a bioreactor (pulse rate = 75/min, radical distension < 5%). After three or six weeks in vitro culture, engineered tissues were harvested and tested.</p><p><b>RESULTS</b>Grossly,the experimental groups showed a tubular structure with a round lumen and good elasticity. Histological staining revealed smooth muscle fibers layers and dense elastic fibers presented in the engineered vessel wall. Bands of smooth muscle fibers and continuous endothelial cells layer were detected by the immuno-histological staining. In contrast, the control group took on poor elasticity, collapsed lumen and pale surface in the gross observation. In addition, its arrangement of smooth muscle fibers and elastic fibers was random and disorganized by histological observation, which was also confirmed by the immunohistological staining. The structure of 6 weeks TEBVs was more mature than that of 3 weeks and the biomechanical property of dynamic ones was as much as 60% of the normal one.</p><p><b>CONCLUSIONS</b>Blood vessel with good elasticity can be constructed in a bioreactor by tissue engineering approach.</p>

Experimental study of electroporation-mediated plasmid gene expression in skin and incisional wound / 中华整形外科杂志

<p><b>OBJECTIVE</b>To explore the feasibility of electroporation mediated gene transfer in rat incisional wound.</p><p><b>METHODS</b>12 Sprague-Dawley rat's dorsal skins were electroporated (800 voltages in amplitude with 6 square wave pulses, each lasting 20 milliseconds with 200 millisecond interval) after injection of plasmid DNA (1 microg/microl, in 100 microl PBS) containing enhanced green fluorescence protein (EGFP) gene. Electroporated skins were incisionally wounded 24 hours after electroporation. Specimens were harvested at day 2, 4, 6, 14, then EGFP expression in dermis was observed and quantitatively analyzed with integrated optical density (IOD) followed by H&E staining.</p><p><b>RESULTS</b>Electroporation can mediate EGFP expression in epidermis, dermis and panniculus muscle. The expression level in dermis was the highest at day 2 (IOD = 3.50 +/- 1.45) and disappeared at day 14. EGFP expression was not found in dermis if no electroporation applied after plasmid injection (IOD = 0).</p><p><b>CONCLUSION</b>Electroporation can mediate plasmid gene expression in incisional wound efficiently and widely.</p>

Application of adipose-derived cells in reconstruction of tissue engineered cartilage in vitro / 中华整形外科杂志

<p><b>OBJECTIVE</b>To explore the feasibility of application of adipose-derived cells (ADCs) in reconstruction of tissue engineered cartilage in vitro.</p><p><b>METHODS</b>Adipose tissue were obtained from human liposuction aspirate (19 cases, 31.5 +/- 5.8 years old). ADCs were isolated by collagenase digestion from liposuction aspirates. 3rd passage cells were seeded into PLGA scaffolds. The copolymer constructs were cultured in conditioned or non-conditioned medium in vitro for 4 weeks. The constructs were evaluated though gross morphology, histology, and immunohistochemistry.</p><p><b>RESULTS</b>The cell-polymer constructs kept its original shape in the induced group, but lost its original shape in the non-induced group. The scaffold group were collapsed. Histologically, the induced groups showed dense cellularity and lacunae-containing cells embedded in a basophilic matrix, while non-induced groups showed connective tissue-like morphology. Collagen and proteoglycan deposition was revealed by Massons's trichome and Safranin' O staining, and minor collagen II expression in the matrix was detected by immunohistochemistry staining in the induced group. They were all negative in the non-induced groups.</p><p><b>CONCLUSIONS</b>Although ADCs included many kinds of cells, it is feasible to use ADCs as seeds cells for reconstruction of tissue engineered cartilage.</p>

The experimental study of creating a new rat scarring model by inserting absorbable gelatin sponge into rats' excisional wounds / 中华整形外科杂志

<p><b>OBJECTIVE</b>To explore the possibility of creating a rat new scar model by inserting gelatin sponge into rat excisional wounds.</p><p><b>METHODS</b>Two full-thickness wounds were created in each of total 49 SD rats. In the Experimental group (n = 19), a regular incisional wound (1 cm) was created on the left side, and an excisional wound of 1.0 cm x 0.2 cm was created on the right side with a gelatin sponge inserted. In control 1 group (n = 15), an excisional wound with sponge insertion was created on both sides of rats. In control 2 group (n = 15), two excisional wounds were created on both sides, and only one side wounds were inserted with a sponge. Animals were sacrificed at various time points for different examinations.</p><p><b>RESULTS</b>The wound/scar width increased 4 - 11 times in inserted wounds than in regular incisional wounds (P < 0.01), with an obvious delay of epithelialization. No difference in wound/scar width was found in both sides of wounds of control 1 group at various locations. In contrast to the linear scar of sponge-inserted wounds, contracted and irregular scar was found in non-inserted wounds of control 2 group.</p><p><b>CONCLUSIONS</b>Gelatin sponge insertion can create a thick linear scar in rat wounds, and thus provides a new model for scar research.</p>

Construction of urethra mucosa structure in vitro by tissue engineering / 中华男科学杂志

<p><b>OBJECTIVE</b>To construct a structure of urethra mucosa in vitro by tissue engineering.</p><p><b>METHODS</b>Primary porcine urothelial cells (UC) were obtained from the porcine bladder by enzymatic digestion and detected by immunofluorescence and RT-PCR. Bladder acellular matrix grafts (BAMG) were prepared, used as the scaffold and then evaluated by HE staining, Masson's trichrome staining, immunohistology and scanning electron microscopy. After in vitro culture and amplification, the UCs were seeded on the luminal surface the BAMGs.</p><p><b>RESULTS</b>After 1 week of in vitro culture, the UCs formed a multilayer structure on the luminal surface of the BAMGs along the basement membrane. The tissue-engineered urothelium and BAMG complex was well formed and pan cytokeratins were positively expressed in the UCs on the scaffold.</p><p><b>CONCLUSION</b>By tissue engineering, the urethra mucosa structure can be rapidly constructed in vitro, which can be applied to the repair of such urethral defect as hypospadias and urethral stricture.</p>

Pilot study of using autologous bone marrow stromal cells and coral to repair canine segmental mandibular defects / 中华整形外科杂志

<p><b>OBJECTIVE</b>To repair segmental mandibular defects with autologous bone marrow stromal cells (BMSCs) engineered bone.</p><p><b>METHODS</b>Isolated BMSCs were expanded in vitro and osteogenic induced. In 12 canines, a 3 cm segmental mandibular defect at right mandible was created. 6 canine's defects were repaired with cell-scaffold constructs made from induced BMSCs and coral; others were repaired with coral as control. The engineered bone was evaluated by X-ray, CT, Dual Energy X-ray Absorptiometry (DXA), gross and histological examination, and biomechanical test post-operatively.</p><p><b>RESULTS</b>Induced BMSCs grew well on coral scaffold. At 12 weeks, X-ray showed more callus formed in experimental group, while evident scaffold duration in control group. At 32 weeks, gross observation, X-ray and CT demonstrated well bony-union in experimental group, while bony-nonunion in control group. Also DXA revealed significantly higher bone mineral density of experimental group than control group. Histologically, mature bone were commonly observed and there were bony healing in experimental group, while fibrous healing occurred in control group. Biomechanical test revealed no significant difference between experimental group and normal group.</p><p><b>CONCLUSIONS</b>Canine segmental mandibular defects can be repaired with the tissue-engineered bone generated by coral scaffold with autologous osteogenic BMSCs.</p>

The isolation, subculture and identification of human adipose derived endothelial progenitor cells / 中华整形外科杂志

<p><b>OBJECTIVE</b>To investigate the methods of isolating and identifying human adipose derived EPCs.</p><p><b>METHODS</b>The cells obtained from human lipoaspirates were plated on culture dishes coated with human fibronectin and were cultured in DMEM containing 2% FBS. Cells of passage 2 cultured in EGM-2 (2% FBS) served as the induced cells (experimental group), with cells cultured in DMEM (2% FBS) as the non-induced cells (control group) . Immunofluorescence was used to detect the expression of cell markers, including CD34, vWF and PECAM-1. FACS (fluorescence activated cell sorter) was used to quantitatively analyze the expression rate of cell markers (CD34, CD45, CD133 and PECAM-1). Fluorescence microscope was used to observe the function of taking up DiI-ac-LDL by the induced cells. To determine the ability of forming capillary-like structure in three-dimensional matrices, the induced cells were also cultured in methylcellulose.</p><p><b>RESULTS</b>The induced cells of passage 2 exhibited cobblestone morphology, similar to that of the endothelial cells. In contrast, these morphological changes were not observed in non-induced cells. Immunofluorescence detected expression of vWF, PECAM-1 in induced cells and CD34 in non-induced cells. FACS analysis showed (67.41 +/- 13.35)% of the induced cells expressed PECAM-1 and (6.73 +/- 2.21)% of the non-induced cells expressed PECAM-1 (P < 0.01), while (72.39 +/- 13.45)% of the non-induced cells expressed CD34 and (16.06 +/- 3.86)% of the induced cells expressed CD34 (P < 0.01). Fluorescence microscopy observed the induced cells took up low-density lipoprotein (LDL). The formation of "branch-like" structure confirmed their functional activity.</p><p><b>CONCLUSION</b>EPCs derived from human adipose may serve as another source of seeding cells for vascular tissue engineering.</p>

Microarray analysis of dedifferentiation related gene expression of human chondrocytes cultured in vitro / 中华整形外科杂志

<p><b>OBJECTIVE</b>Dedifferentiation of chondrocytes during in vitro expansion is the major cause that limits the potential of chondrocytes for cartilage engineering. This study dissected dedifferentiation mechanism of in vitro cultured human chondrocytes by microarray analysis of gene expression changes.</p><p><b>METHODS</b>Spare human costal cartilage from ear reconstruction patients (n=3, aged 10-20) were digested with collagenase II to isolate human chondrocytes(HCC) and were expanded from P1 to P4. For microarray, RNA was isolated from the P1 and P4 cells and labeled with cy3 and cy5 fluorescence respectively as probes to hybridize a cDNA microarray( about 8300 genes). Those genes with 2-fold difference of expression were selected.</p><p><b>RESULTS</b>Microarray results showed that 56 genes were up-regulated at P4, including proteases, inflammatory factors, growth suppressor and apoptosis genes. Down-regulated 84 genes at P4 include extracellular matrices, protein anabolism, growth factors, cell cycle and cytoskeleton related genes.</p><p><b>CONCLUSIONS</b>These results provide insight into the mechanism of chondrocytes dedifferentiation. The characteristic appearance of dedifferentiation related genes expression includes increased level of stress response and inflammation. The down-regulation of anabolic metabolism related genes and up-regulation of proteases genes leaded to extra cellular matrix decrease. Decreased production of growth factors and increased apoptosis cause decreased cell proliferation of dedifferentiation chondrocytes.</p>

PDGF-BB initiates vascular smooth muscle-like phenotype differentiation of human bone marrow mesenchymal stem cells in vitro / 中华整形外科杂志

<p><b>OBJECTIVE</b>To investigate the feasibility of human bone marrow mesenchymal stem cells (hBMSCs) in vitro differentiation into vascular smooth muscle cells with induction of platelet-derived growth Factor BB (PDGF-BB).</p><p><b>METHODS</b>Bone marrow mesenchymal stem cells of adult healthy donors were separated from iliac crest aspiration and expanded in DMEM-LG medium. Cells at passage 1 were transferred to EGM-2 medium containing PDGF-BB (20 ng/ml) and cultured for 14 days. The expression of SM alpha-actin, SM calponin, SMMHC and SM 22alpha were detected by immunofluorescence and observed with fluorescence microscope. mRNA expression of SMalpha-actin, SM calponin, SMMHC as well as SM 22alpha was analyzed by RT-PCR. The method of Western-Blot was applied to determine protein expression of SM 22alpha. Cells with induction were observed for the expression of SM alpha-actin,SM calponin,SMMHC by FACs analysis.</p><p><b>RESULTS</b>With the induction of PDGF-BB, the morphology of cells changed to a spindle fibroblastic appearance. By fluorescence microscope observation, expression of SM alpha-actin, SM calponin and SMMHC was found intracellularly in PDGF-BB treated hBMSCs at 14 days. Western-Blot detection confirmed SM 22alpha expression by 14 days induction. RT-PCR of characteristic vascular smooth muscle cells related genes, such as SM alpha-actin, SM calponin, SMMHC and SM 22alpha revealed differentiation of vascular smooth muscle cells phenotype in monolayer culture upon stimulation with PDGF-BB for 14 days. The positive expression of SM alpha-actin, SM calponin and SMMHC in induced cells was significantly higher than that in non-induced cells (P < 0.05, n=3).</p><p><b>CONCLUSION</b>These results suggested hBMSCs could be differentiated into vascular smooth muscle cell phenotype with PDGF-BB induction in vitro.</p>

An experimental study on influence of concentration of fetal bovine serum on chondrogenesis of BMSCs / 中华整形外科杂志

<p><b>OBJECTIVE</b>To explore the chondrogenetic effect of induce media containing different concentrations of fetal bovine serum (FBS) on BMSCs differentiation in vitro and provide technical parameters for cartilage engineering in vitro.</p><p><b>METHODS</b>Passage 2 BMSCs of swine were seeded at the density of 5 x 10(7) cells/cm3 to disc-shaped PGA scaffolds with a diameter of 5mm and a thickness of 2mm. After 7days, the scaffolds were induced in media with TGF-beta1, IGF-I, dexamethasone, and different concentrations of FBS: 0% in A group, 5% in B group, and 10% in C group. Specimens were collected after 8 weeks for gross observation, size evaluation, wet weight, glycosaminoglycan (GAG) content, histology assessment, and immunohistology of type II collagen.</p><p><b>RESULTS</b>The compound of C group showed china-white color, hard and fine texture, no obvious change in size and shape, typical lacuna structures, cartilage specific ECM, and significantly higher wet weight and GAG content. The compound of B group showed reduced size, fewer lacuna structures and some cartilage specific ECM. And the compound of A group showed greatly reduced size, soft and loose texture, and no typical lacuna structure or cartilage specific ECM.</p><p><b>CONCLUSIONS</b>FBS was indispensable to chondrogenetic media for in-vitro tissue engineering of cartilage with BMSCs.</p>

Experimental study of construction of tissue engineered bone ectopically by human bone marrow mesenchymal stem cells / 中华整形外科杂志

<p><b>OBJECTIVE</b>To study the possibility and mechanism of construction of tissue engineered bone with human bone marrow mesenchymal stem cells (hBMSCs) as seeding cells and partially demineralized bone matrix (pDBM) as scaffold.</p><p><b>METHODS</b>hBMSCs are cultured and mutiplified. The 4th grade hBMSCs are seeded on the pDBM, the growth and adhesion of hBMSCs on pDBM are observed under scanning electro microscope. The adhesion efficiency is assessed. The complexes are implanted in the nude mice subcutaneously, the pDBM without cells as control. The grafts are taken out on the 8th and 12th week.</p><p><b>RESULTS</b>There is new bone formation on the 8th and 12th week in complex group. There is a layer of osteoblast like cells adhered on the surface of most of the new bone, which suggest the possibility of intramembranous ossification. There is no bone formation in control group.</p><p><b>CONCLUSIONS</b>Tissue engineered bone can be constructed with hBMScs and pDBM in vivo, and the mechanism of which could be intramembranous ossification.</p>

Repair alveolar cleft bone defects with bone marrow stromal cells / 中华整形外科杂志

<p><b>OBJECTIVE</b>To explore the feasibility of repairing alveolar cleft bone defects with bone marrow stromal cells.</p><p><b>METHODS</b>Total 7 patients of alveolar cleft were included in this study. The hBMSCs were isolated by percoll gradient centrifugation from patient's bone marrow aspirated from iliac crest. The hBMSCs were cultured in vitro and induced to become osteogenic cells in the DMEM medium containing 10% self-serum, beta-glycerophosphate (10 nmol/L) dexamethasone (10(-8) mol/L) , L-2-ascorbic acid(50 micromol/L), and 1, 25 (OH)2 VD3 (10 nmol/L). Induced hBMSCs of passage 3 were harvested and seeded onto partly demineralized allogenic bone matrix (pDBM) to form a cell-scaffold construct and in vitro co-culture for 1 week. The defects were repaired with the cell-scaffold construct. All cases were followed up for 3, 6 months post-operation as short-term evaluation and 1 to 3 years post-operation as long-term evaluation by three-dimensional computerized tomography (3D-CT) and clinical examination.</p><p><b>RESULTS</b>3D-CT demonstrated that engineered bone was formed in 3 months post-operation. Additionally, formed bone maintained stable up to 1 - 3 years without absorption.</p><p><b>CONCLUSIONS</b>Engineered bone can be used to repair clinical alveolar cleft bone defects.</p>