Weibiao Huang

Expression and localization of estrogen receptor-β in annulus cells of the human intervertebral disc and the mitogenic effect of 17-β-estradiol in vitro

BackgroundRecent evidence suggests that estrogens exert effects in different tissues throughout the body, and that the estrogen receptor β (ERβ) may be important for the action of estrogen (17-β-estradiol) on the skeleton. The cellular localization of ERβ in the human intervertebral disc, however, has not yet been explored.MethodsHuman disc tissue and cultured human disc cells were used for immunocytochemical localization of ERβ. mRNA was isolated from cultured human disc cells, and RT-PCR amplification of ERβ was employed to document molecular expression of this receptor. Cultured human disc cells were tested to determine if 17-β-estradiol stimulated cell proliferation.ResultsIn this report data are presented which provide evidence for ERβ gene expression in human intervertebral disc cells in vivo and in vitro. Culture of annulus cells in the presence of 10-7 M 17-β-estradiol significantly increased cell proliferation.ConclusionsThese data provide new insight into the biology of cells in the annulus of the intervertebral disc.

Optimizing Collagen Scaffolds for Bone Engineering: Effects of Cross-linking and Mineral Content on Structural Contraction and Osteogenesis.

Introduction: Osseous defects of the craniofacial skeleton occur frequently in congenital, posttraumatic, and postoncologic deformities. The field of scaffold-based bone engineering emerged to address the limitations of using autologous bone for reconstruction of such circumstances. In this work, the authors evaluate 2 modifications of three-dimensional collagen-glycosaminoglycan scaffolds in an effort to optimize structural integrity and osteogenic induction. Methods: Human mesenchymal stem cells (hMSCs) were cultured in osteogenic media on nonmineralized collagen-glycosaminoglycan (C-GAG) and nanoparticulate mineralized collagen-glycosaminoglycan (MC-GAG) type I scaffolds, in the absence and presence of cross-linking. At 1, 7, and 14 days, mRNA expression was analyzed using quantitative real-time -reverse-transcriptase polymerase chain reaction for osteocalcin (OCN) and bone sialoprotein (BSP). Structural contraction was measured by the ability of the scaffolds to maintain their original dimensions. Mineralization was detected by microcomputed tomographic (micro-CT) imaging at 8 weeks. Statistical analyses were performed with Student t-test. Results: Nanoparticulate mineralization of collagen-glycosaminoglycan scaffolds increased expression of both OCN and BSP. Cross-linking of both C-GAG and MC-GAG resulted in decreased osteogenic gene expression; however, structural contraction was significantly decreased after cross-linking. Human mesenchymal stem cells-directed mineralization, detected by micro-CT, was increased in nanoparticulate mineralized scaffolds, although the density of mineralization was decreased in the presence of cross-linking. Conclusions: Optimization of scaffold material is an essential component of moving toward clinically translatable engineered bone. Our current study demonstrates that the combination of nanoparticulate mineralization and chemical cross-linking of C-GAG scaffolds generates a highly osteogenic and structurally stable scaffold.

Genetic markers of osteogenesis and angiogenesis are altered in processed lipoaspirate cells when cultured on three-dimensional scaffolds.

Background: Liposuction-derived stem cells (processed lipoaspirate) have recently been shown to be capable of differentiating into bone. Most studies on osteoblastic growth and differentiation have been conducted in a conventional two-dimensional culture system; however, in native bone, osteoblasts are situated in a three-dimensional configuration. There have been limited studies of processed lipoaspirate behavior in three-dimensional systems. The authors studied the influence a three-dimensional scaffold has on the expression of genes related to osteogenesis and angiogenesis in processed lipoaspirate cells. Methods: One million processed lipoaspirate cells were seeded onto two-dimensional poly(l-lactide-co-glycolide) films or in three-dimensional poly(l-lactide-co-glycolide) scaffolds and incubated in osteogenic medium up to 21 days. RNA was extracted and analyzed with quantitative real-time polymerase chain reaction. Results: When an inert three-dimensional poly(l-lactide-co-glycolide) scaffold was introduced, the pattern and sequence of gene expression changed significantly. Processed lipoaspirate cells cultured onto three-dimensional scaffolds had increased expression of interleukin-8 and vascular endothelial growth factor compared with two-dimensional controls at early time points. Osteogenesis markers—alkaline phosphatase, collagen type I, osteocalcin, osteonectin, and osteopontin—were significantly up-regulated in three-dimensional cultures relative to two-dimensional controls after 24 hours and persisted throughout the 21 days. Conclusions: In human processed lipoaspirate cells, the introduction of a three-dimensional scaffold significantly enhances gene markers of angiogenesis and osteogenesis. On three-dimensional scaffolds, processed lipoaspirate cells first up-regulate genes involved with vascular ingrowth and then those involved in bone formation. We believe these differences will significantly impact the design of a bone graft substitute for clinical application.

Human and mouse osteoprogenitor cells exhibit distinct patterns of osteogenesis in three-dimensional tissue engineering scaffolds.

Background: Understanding interspecies variation between animal models and humans is essential to develop tissue-engineered bone. The authors studied osteogenic and angiogenic marker expression in human and murine osteoblasts and mesenchymal stem cells. Methods: Three human cells (human mesenchymal stem cells, multilineage progenitor cells, and normal human osteoblasts) and three murine cells (MC3T3-E1, C3H10T1/2, and M2-10B4) were used. Cells were seeded onto poly-lactide-glycolic acid–coated tissue culture plates or three-dimensional poly-lactide-glycolic acid scaffolds, incubated in osteogenic medium, and harvested at 1, 4, and 7 days. mRNA expression was analyzed using quantitative real-time reverse-transcriptase polymerase chain reaction for osteogenic markers, including alkaline phosphatase, osteocalcin, bone sialoprotein, and core-binding factor alpha-1, and angiogenic markers, including vascular endothelial growth factor and interleukin-8. Data were analyzed using analysis of variance. Results: All human cells had significantly increased expression of osteogenic markers in three dimensions compared with two dimensions (alkaline phosphatase by 220 percent, osteocalcin by 323 percent, bone sialoprotein by 534 percent, and core-binding factor alpha-1 by 357 percent). However, all murine cells exhibited significant decreases in the expression of osteogenic markers in three-dimensional compared with two-dimensional cultures (alkaline phosphatase by 89 percent, osteocalcin by 64 percent, bone sialoprotein by 76 percent, and core-binding factor alpha-1 by 73 percent). In contrast, all human and murine cells showed markedly elevated expression of angiogenic factors interleukin-8 and vascular endothelial growth factor in three-dimensional compared with two-dimensional cultures. Measurement of alkaline phosphatase activity confirmed this pattern of osteogenic differentiation. Conclusions: In three-dimensional versus two-dimensional cultures, osteogenesis increased significantly in human cells but decreased in murine cells; angiogenesis increased regardless of species. Since three-dimensional cultures represent in vivo conditions more closely, this species variation has important translational implications to tissue-engineered bone research.

Pretreatment of poly(l-lactide-co-glycolide) scaffolds with sodium hydroxide enhances osteoblastic differentiation and slows proliferation of mouse preosteoblast cells.

Background: Surface topography is important in the creation of a scaffold for tissue engineering. Chemical etching of poly(l-lactide-co-glycolide) with sodium hydroxide has been shown to enhance adhesion and function of numerous cell types. The authors investigated the effects of sodium hydroxide pretreatment of three-dimensional poly(l-lactide-co-glycolide) scaffolds on the adhesion, differentiation, and proliferation of MC3T3-E1 murine preosteoblasts. Methods: MC3T3-E1 cells were seeded onto three-dimensional poly(l-lactide-co-glycolide) scaffolds with and without 1 M sodium hydroxide pretreatment. Cells were then cultured in osteogenic medium and harvested at varying time points for RNA extraction. Quantitative real-time reverse-transcriptase polymerase chain reaction was performed to measure mRNA expression of several osteogenic marker genes. In addition, cell numbers were determined at varying time points during the culture period. All experiments were performed in triplicate. Results: Pretreatment of three-dimensional poly(l-lactide-co-glycolide) scaffolds with sodium hydroxide resulted in statistically significant up-regulation of mRNA expression of alkaline phosphatase, bone sialoprotein, osteocalcin, and vascular endothelial growth factor during the first 10 days of culture. Histologic analysis demonstrated a striking increase in mineralized cell matrix deposition in the sodium hydroxide–treated group. Cell number was statistically higher in the sodium hydroxide–treated group immediately after cell seeding, suggesting improved adhesion. During the first 24 hours of culture, cells grew faster in the control group than in the sodium hydroxide–treated group. Conclusions: Chemical etching of poly(l-lactide-co-glycolide) scaffolds with sodium hydroxide strongly influences the behavior of MC3T3-E1 preosteoblasts in vitro by enhancing adhesion and differentiation and slowing proliferation. Sodium hydroxide treatment may represent a simple and inexpensive way of improving scaffolds for use in bone tissue engineering.

171A: THREE DIMENSIONAL POLY-LACTIDE-GLYCOLIC ACID (PLGA) AND TYPE I COLLAGEN SCAFFOLDS EXERT AN OPPOSITE EFFECT ON OSTEOGENISIS IN HUMAN AND MOUSE OSTEOBLASTS

Introduction: Joint surfaces injuries arise from repetitive or traumatic high impact loading and are a common and potentially debilitating problem. Repair strategies focus on the creation of scar tissue, which is biomechanically inferior to articular cartilage. Previous studies have demonstrated the ability of tissue-engineered cartilage to bond to articular cartilage; however the ability to bond to subchondral bone is uncharacterized. The goal of this study is to characterize the bond formed between native cartilage, engineered cartilage and subchondral bone using histology and biomechanical testing.

168 DIFFERENTIATION OF MC3T3-E1 PREOSTEOBLASTIC CELLS ON TWO-DIMENSIONAL POLY(LACTIDE-CO-GLYCOLIDE) (PLGA) FILMS AND THREE-DIMENSIONAL PLGA SCAFFOLDS: A REAL-TIME REVERSE TRANSCRIPTASE-POLYMERASE CHAIN REACTION STUDY.

Purpose This study aimed to investigate the expression of a number of osteogenic and angiogenic markers in MC3T3-E1 cells cultured on two-dimensional (2-D) (PLGA films and three-dimensional (3-D) PLGA scaffolds. Methods MC3T3-E1 cells were incubated in mineralized medium in the absence or presence of 100 ng/mL recombinant human BMP-2 for 2 weeks. Cells were cultured either on 2-D PLGA films or in 3-D PLGA scaffolds and harvested on days 0, 1, 2, 3, 5, 7, 10, and 14. Total RNA was extracted and subjected to quantitative real-time reverse transcriptase-polymerase chain reaction analysis for expression of genes involved in osteogenesis and angiogenesis. Results Expression of BSP and OCN increased when cells cultured on 2-D PLGA films were subjected to differentiation medium containing ascorbic acid and beta-glycerol phosphate for a period of 2 weeks. Treatment with recombinant human BMP-2 resulted in further increases in expression of both genes after day 5. Cells cultured on 3-D PLGA scaffolds showed significantly slowed differentiation as measured by expression of the above two genes. Both genes remained highly responsive to BMP-2 treatment after day 5. OPN expression was elevated when cells were transplanted into 3-D scaffolds. Its expression in cells cultured in 3-D scaffolds was less responsive to BMP-2 treatment than on 2-D films. Expression of VEGF was increased as differentiation proceeded on 2-D films and was enhanced with BMP-2 treatment. Its expression was significantly induced when cells were cultured in 3-D scaffolds. A 2-week culture of cells in 3-D scaffolds resulted in decreased VEGF expression. BMP-2 inhibited VEGF expression in 3-D scaffolds at days 10 and 14. Conclusions The results indicate that MC3T3-E1 cells commit to osteogenic differentiation at a slower rate in 3-D scaffolds than in 2-D scaffolds.

THE EFFECT OF NAOH ON PROLIFERATION AND DIFFERENTIATION OF MC3T3-E1 PREOSTEOBLASTIC CELLS GROWN ON THREE-DIMENSIONAL POLY(L-LACTIDE-CO-GLYCOLIDE) SCAFFOLDS.: 475

Background In creating a biosynthetic scaffold that promotes osteogenesis surface topography is an important variable. It has been demonstrated that chemical etching of poly(l-lactide-co-glycolide) (PLGA) with NaOH enhances adhesion and function of numerous cells types in two-dimensional systems (hepatocytes, endothelial cells, vascular and bladder smooth muscle cells) and three-dimensional (3-D) systems (chondrocytes). This study compares the biological behavior of osteoblasts grown on NaOH-treated PLGA scaffolds and conventional PLGA scaffolds by measuring osteoblast adhesion, differentiation, and proliferation of MC3T3-E1 cells cultured on NaOH-treated 3D PLGA scaffolds and untreated scaffolds. Methods MC3T3-E1 mouse preosteoblast cells were seeded onto 3-D PLGA scaffolds ± 1 M NaOH for 10 minutes. Quantitative real-time RT PCR was performed to measure mRNA expression of osteogenic marker genes. In addition, cell numbers were determined at varying time points. Results Pretreatment of 3D PLGA scaffolds with NaOH resulted in statistically significant up-regulation of mRNA expression of alkaline phosphatase (ALP), bone sialoprotein (BSP), osteocalcin (OCN), and vascular endothelial growth factor (VEGF). A striking increase in mineralized cell matrix deposition in the NaOH group was also observed. Cell number was statistically higher in the NaOH group immediately after cell seeding, suggesting improved adhesion. During the first 24 hours of culture, cell proliferative rates were higher in the control group than the NaOH group. Conclusion Chemical etching of PLGA scaffolds with NaOH enhances adhesion and differentiation and slows proliferation of preosteoblast cells. NaOH treatment may represent an inexpensive way to improve scaffolds for use in bone tissue engineering.