Increased cell senescence is associated with decreased cell proliferation in vivo in the degenerating human annulus

Background contextDuring disc degeneration, there is a well-recognized loss of cells. This puts the remaining cell population at high risk for any further decrease in cell function or cell numbers. Cell senescence has recently been shown to be present in the aging/degenerating human disc. Senescent cell are viable, metabolically active, persist, and accumulate over time, but cannot divide. Little is known about the relationship between renewal of the disc cell population via cell proliferation and disc cell senescence.PurposeTo determine the percentage of senescent cells and proliferating cells in the human annulus in vivo.Study design/settingHuman annulus specimens were obtained from surgical subjects and control donors in a study approved by the authors' Human Subjects Institutional Review Board.Patient sampleOne Thompson Grade I disc, 4 Grade II discs, 9 Grade III discs, and 12 Grade IV discs were studied.Outcome measuresThe percentages of senescent cells and the percentage of proliferating cells.MethodsImmunohistochemistry was used to detect senescent cells using an antisenescence-associated beta-galactosidase antibody, and an antiproliferation antibody (Ki67). An average of 410 cells/specimens was counted to determine the percent senescence, and an average of 229 cells was counted to determine the percent proliferation.ResultsCell proliferation was low in both surgical and control normal donor annulus tissue (4.09%+1.77 (26), mean+SD (n)). There was no significant difference in the percentage of proliferating cells for more degenerate discs versus healthier discs (4.7%+1.6 (21) for Grades III and IV vs. 5.3%+1.9 (5) for Grades I and II). More degenerated Grades III and IV discs contained significantly greater percentages of senescent annulus cells than did the healthier Grades I and II discs (44.4%+20.0 (21) vs. 18.8%+11.0 (5), respectively; p=.011). A significant negative correlation was present between the percentage of senescent cells versus the percentage of proliferating cells, r=?0.013, p=.013. No correlation was present between age and the percentage of senescent cells or age and the percentage of proliferating cells.ConclusionsBecause senescent cells cannot divide, senescence may reduce the disc's ability to generate new cells to replace cells lost to necrosis or apoptosis. Senescent cells also accumulate in the disc over time, such that their metabolic patterns may contribute to the pathologic changes seen in degenerating discs. Novel data presented here show a significant negative correlation between the percentage of senescent cells and the percentage of proliferating cells during disc degeneration. Molecular work is underway in our lab to help us determine whether senescent cells in the disc secrete factors that can result in decreased proliferation in neighboring cells.     

麝香调控退变大鼠颈椎间盘组织基因表达谱的研究

目的　分析麝香对退变椎间盘基因表达谱的调控作用。方法　建立颈椎间盘退变模型。从 9个月模型组、麝香治疗组大鼠颈椎间盘中抽提mRNA ,经反转录分别用Cy3、Cy5荧光标记 ,获得椎间盘cDNA的探针 ;cDNA探针与大鼠BiostarR - 4 0s基因表达谱芯片杂交 ,结果由激光扫描仪扫描并用软件进行图像分析、标准化处理、ratio值分析、聚类分析。结果　麝香组与模型组比较 ,11.1% (共 4 4 1条 )基因表达发生明显变化 ,其中 2 6 0条基因表达量上升(R >2 .0 ) ,182条基因表达量明显下降 (R <0 .5 )。经过信息学分析 ,在上述两张芯片上都有基因表达量明显变化的 90条 (R >2 .0或 <0 .5 ) ,其中基因表达量都明显上调的 2 5条 (R >2 .0 ) ,其中功能明确的基因有 3条 ;都明显下调的 2 0条 (R <0 .5 ) ,其中功能明确的基因有 5条 ;麝香发挥明显上调作用的共 13条 (由R <0 .5到R >2 .0 )。结论　麝香对退变椎间盘出现总体调控。筛选出大鼠退变椎间盘中相关基因 ,特别是有较多的细胞信号和传递蛋白类基因表达 ,说明细胞内外的信号传导介导了椎间盘退变过程。     

Gene expression patterns in bone following mechanical loading

The advent of high‐throughput measurements of gene expression and bioinformatics analysis methods offers new ways to study gene expression patterns. The primary goal of this study was to determine the time sequence for gene expression in a bone subjected to mechanical loading during key periods of the bone‐formation process, including expression of matrix‐related genes, the appearance of active osteoblasts, and bone desensitization. A standard model for bone loading was employed in which the right forelimb was loaded axially for 3 minutes per day, whereas the left forearm served as a nonloaded contralateral control. We evaluated loading‐induced gene expression over a time course of 4 hours to 32 days after the first loading session. Six distinct time‐dependent patterns of gene expression were identified over the time course and were categorized into three primary clusters: genes upregulated early in the time course, genes upregulated during matrix formation, and genes downregulated during matrix formation. Genes then were grouped based on function and/or signaling pathways. Many gene groups known to be important in loading‐induced bone formation were identified within the clusters, including AP‐1‐related genes in the early‐response cluster, matrix‐related genes in the upregulated gene clusters, and Wnt/β‐catenin signaling pathway inhibitors in the downregulated gene clusters. Several novel gene groups were identified as well, including chemokine‐related genes, which were upregulated early but downregulated later in the time course; solute carrier genes, which were both upregulated and downregulated; and muscle‐related genes, which were primarily downregulated. © 2011 American Society for Bone and Mineral Research.     

AAV2-hVEGF165与AAV2-hTGFβ1对退变兔椎间盘纤维环细胞的生物学效应

目的采用基因治疗方法，把AAV2-hVEGF165和AAV2-hTGFβ1共转染兔纤维环细胞，观察其生物学活性，并进一步评价人血管内皮生长因子165（hVEGF165）和人转化生长因子β1（hTGFβ1）逆转椎间盘退变的可行性。方法分离并培养兔椎间盘纤维环细胞，以腺相关病毒（AAV2）为载体，分别携带hVEGF165和hTGFβ1eDNA，转染兔纤维环细胞。用Westernblotting检测hVEGF165和hTGFβ1的表达，并用同样的方法检测纤维环细胞中Ⅰ型胶原的表达。结果AAV2能够感染兔椎间盘细胞。用Westernblotting可以检测到AAV2-hVEGF165和AAV2-hTGFβ1在退变椎间盘细胞中的表达，并且，两种因子联合转染纤维环细胞时Ⅰ型胶原的表达要比分别转染是显著增高。结论hVEGF165和hTGFβ1共同转染培养的兔退变纤维环细胞，Ⅰ型胶原的表达显著增高。     

Expression and regulation of metalloproteinases and their inhibitors in intervertebral disc aging and degeneration

Background contextDestruction of extracellular matrix (ECM) leads to intervertebral disc degeneration (IDD), which underlies many spine-related disorders. Matrix metalloproteinases (MMPs), and disintegrins and metalloproteinases with thrombospondin motifs (ADAMTSs) are believed to be the major proteolytic enzymes responsible for ECM degradation in the intervertebral disc (IVD).PurposeTo summarize the current literature on gene expression and regulation of MMPs, ADAMTSs, and tissue inhibitors of metalloproteinases (TIMPs) in IVD aging and IDD.MethodsA comprehensive literature review of gene expression of MMP, ADAMTS, and TIMP in human IDD and reported studies on regulatory factors controlling their expressions and activities in both human and animal model systems.ResultsUpregulation of specific MMPs (MMP-1, -2, -3, -7, -8, -10, and -13) and ADAMTS (ADAMTS-1, -4, and -15) were reported in human degenerated IVDs. However, it is still unclear from conflicting published studies whether the expression of ADAMTS-5, the predominant aggrecanase, is increased with IDD. Tissue inhibitors of metalloproteinase-3 is downregulated, whereas TIMP-1 is upregulated in human degenerated IVDs relative to nondegenerated IVDs. Numerous studies indicate that the expression levels of MMP and ADAMTS are modulated by a combination of many factors, including mechanical, inflammatory, and oxidative stress, some of which are mediated in part through the p38 mitogen-activated protein kinase pathway. Genetic predisposition also plays an important role in determining gene expression of MMP-1, -2, -3, and -9.ConclusionsUpregulation of MMP and ADAMTS expression and enzymatic activity is implicated in disc ECM destruction, leading to the development of IDD. Future IDD therapeutics depends on identifying specific MMPs and ADAMTSs whose dysregulation result in pathological proteolysis of disc ECM.     

脂肪间充质干细胞向髓核样细胞诱导分化的初步研究

[目的] 定向诱导大鼠脂肪间充质干细胞分化为髓核样细胞,为椎间盘退变性疾病的细胞移植治疗奠定生物学基础.[方法] (1)200 g SD雄性大鼠断颈处死后,取腹股沟脂肪垫处脂肪,0.1%胶原酶消化,体外接种培养,同时采用极限稀释法纯化细胞;(2)利用流式细胞技术对细胞表面标记进行鉴定;(3)第3代细胞消化计数后,用1.2%海藻酸钠溶液悬浮,调整细胞密度至1×10~6/ml,滴入3.5%CaCl_2溶液形成微球,加入含TGF-beta1的诱导培养基,低氧条件下进行微球联合培养;(4)7 d后,取部分微球进行Alcian Blue染色并收集微球内细胞提取RNA,进行RT-PCR检测.[结果] (1)体外培养的脂肪间充质干细胞主要呈梭形和多角形,核大、核仁明显,平行排列生长或呈漩涡状生长;(2)流式细胞仪检测显示Sca-1、CD44的阳性率分别为95.2%、99.7%,CD45、CD11b的阳性率分别为0.4%、1.5%;(3)微球联合培养7 d后,Alcian Blue染色表明,实验组细胞外基质的生成明显多于对照组;(4)RT-PCR结果证实,髓核细胞标志基因和软骨细胞标志基因的表达,实验组明显高于对照组.[结论] (1)本实验方法可以获得大量均一的脂肪间充质干细胞;(2)脂肪间充质干细胞可以被定向诱导分化为髓核样细胞;(3)首次探讨了将脂肪间充质干细胞体外诱导后移植治疗椎间盘退变性疾病,为椎间盘退变性疾病的干细胞移植治疗提供了新的思路.     

Regional variations in certain cellular characteristics in human lumbar intervertebral discs, including the presence of alpha-smooth muscle actin.

An evaluation of the regional variation of certain cellular features in the human intervertebral disc (IVD) could lead to a better understanding of site-specific properties relative to degradation, response to injury, and healing processes. The objective of this study was to determine how cell density, cell morphology, cell grouping, and expression of a specific actin isoform varied with location and degeneration in the human disc. A total of 41 human L4-L5 and L5-S1 discs removed postmortem from 21 individuals were analyzed. The discs were graded for degeneration based on the Thompson scale and processed for evaluation. Microtomed sections from paraffin-embedded specimens were stained with hematoxylin and eosin or a monoclonal antibody to alpha-smooth muscle actin (alpha-SMA), an actin isoform often associated with contraction. A significant regional dependence was found for most of the measured parameters. A fourfold increase in cell density was found in proceeding from the nucleus pulposus (NP) to the outer annulus (OA) of the IVD. Approximately 30% of the cells in the NP were present in groups. Virtually all of the cells in the NP and 40% of those in the OA were round. Moreover, notable percentages (12-15%) of the cells in the NP and inner annulus (IA) contained alpha-SMA. Only pair density was found to be correlated with Thompson grade, with more degenerated specimens having higher values. A greater effect was also observed on the percentage of cells in groups. These findings provide the basis for future work to investigate the importance of cells in groups, the role of alpha-SMA in the disc, and the changes in these cellular characteristics in pathological disc conditions.     

动物模型椎间盘退变全程基因变化的对比

目的：新西兰大白兔的纤维环损伤制作腰椎间盘退变模型，用以证实和比较在人椎间盘退变中明显变化的基因变化情况。方法：2．0只新西兰大白兔L4，5及L5,6纤维环损伤作为实验组，4只作对照组未行损伤。2、4、8、40周分别行核磁共振及计算机扫描摄影拍片证实椎间盘退变情况同时取椎间盘行精确定量逆转录聚合酶链式反应检测金属蛋白酶(matrix metalloproteinase)MMP-1、MMP-2、MMP-3及它们的特意性抑制剂(tissue inhibitors of metalloproteinases)Timp-1、Timp-2的变化。结果：核磁共振及计算机扫描摄影证实了腰椎纤维环损伤后腰椎逐渐退变并与人类退变结果相似，基因表达情况MMP-1、MMP-2、MMP-3、Timp-1、Timp-2早期均上调，MMP-3、Timp-1在退变的晚期出现下调。结论：纤维环损伤可以成功制作出椎间盘退变的模型。在人类腰椎间盘退变中明显上调的基因在此退变模型椎间盘中被发现同样上调，从而在分子水平证实了动物退变模型与人类的相似性。     

Percutaneous plasma decompression alters cytokine expression in injured porcine intervertebral discs.

BACKGROUND CONTEXT: Discectomy is a surgical technique commonly used to treat bulging or herniated discs causing nerve root compression. Clinical data suggest discectomy may also help patients with contained discs and no clear neural compromise. However, the mechanisms of clinical efficacy are uncertain, and consequently bases for treatment optimization are limited. PURPOSE: To determine the effect of percutaneous plasma decompression on the histologic, morphologic, biochemical and biomechanical features of degenerating intervertebral discs. STUDY DESIGN: An adult porcine model of disc degeneration was used to establish a degenerative baseline against which to evaluate discectomy efficacy. OUTCOME MEASURES: Cytokines interleukin (IL)-1, IL-6, IL-8, and tumor necrosis factor (TNF)-alpha were measured from tissue samples using enzyme-linked immunosorbent assay. Histology and morphology images were rated for degenerative findings (of cells and matrix) in both the nucleus and annulus. Proteoglycan content was determined, and intact specimen stiffness and flexibility were measured biomechanically. Magnetic resonance images were collected for biomechanical specimens. METHODS: Using a retroperitoneal surgical approach, stab incisions were made in four or five lumbar discs per spine in 12 minipigs. Animals were allocated into one of three groups: 6-week recovery, 12-week recovery and percutaneous plasma decompression using an electrosurgical device at 6 weeks with recovery for 6 additional weeks. Four additional animals served as controls. RESULTS: Discs treated with discectomy had a significant increase in IL-8 and a decrease in IL-1 as compared with the 12-week, nontreated discs. There were no significant differences in morphologic and biomechanical parameters or proteoglycan content between treated discs and time-matched, nontreated discs. CONCLUSIONS: Our results demonstrate that percutaneous plasma discectomy alters the expression of inflammatory cytokines in degenerated discs, leading to a decrease in IL-1 and an increase in IL-8. Whereas both IL-1 and IL-8 have hyperalgesic properties, IL-1 is likely to be a more important pathophysiologic factor in painful disc disorders than IL-8. Therefore, the alteration in cytokine expression that we observed is consistent with this effect as a mechanism of pain relief after discectomy. In addition, given that IL-1 is catabolic in injured tissue and IL-8 is anabolic, our results suggest that a percutaneous plasma discectomy may be capable of initiating a repair response in the disc.     

Gene expression comparison between primary estrogen receptor-positive and triple-negative breast cancer with paired axillary lymph node metastasis

The aim of this study is to characterize and compare changes in gene expression patterns of paired axillary lymph node (ALN) metastases from estrogen receptor (ER)-positive and triple-negative (TNBC) primary breast cancer (PBC). Patients with stage 2-3 PBC with macrometastasis to an ALN were selected. Gene expression of 2567 cancer-associated genes was analyzed with the HTG EdgeSeq system coupled with the Illumina Next Generation Sequencing (NGS) platform. Changes in gene expression between ER/PR-positive, HER2-negative PBC, and their paired ALN metastases were compared with TNBC and their paired ALN metastases. Fourteen pairs of ER-positive and paired ALN metastasis were analyzed. Compared with the PBC, ALN metastasis had 673 significant differentially expressed genes, including 348 upregulated genes and 325 downregulated genes. Seventeen pairs of TNBC and paired ALN metastasis were analyzed. ALN metastasis had 257 significant differentially expressed genes, including 123 upregulated genes and 134 downregulated genes. When gene expression of the ALN for ER-positive PBC was compared to that of TNBC, 97 genes were upregulated in both, and 115 genes were similarly downregulated. Common upregulated genes were associated with cell death, necrosis, and homeostasis. Common downregulated genes were those of migration, degradation of extracellular matrix, and invasion. Although ER-positive PBC and TNBC have a distinct gene expression profiles and distinct changes from PBC to ALN metastases, a significant number of genes are similarly up- or downregulated. Understanding the role of these common genomic changes may provide clues to understanding the metastatic process itself.     

Differential expression of matrix metalloproteinase 3 (MMP3) in preadipocytes/stromal vascular cells from nonobese nondiabetic versus obese nondiabetic Pima Indians

Prior microarray studies comparing global gene expression patterns in preadipocytes/stromal vascular cells isolated from nonobese nondiabetic versus obese nondiabetic Pima Indians showed that matrix metalloproteinase 9 (MMP9) is upregulated in obese subjects. The current study targeted analysis of nine additional MMP genes that cluster to a region on chromosome 11q22 that is linked to BMI and percent body fat. Differential-display PCR showed that MMP3 is downregulated in preadipocytes/stromal vascular cells from obese subjects, and real-time PCR showed that MMP3 expression levels are negatively correlated with percent body fat. To determine whether variants within MMP3 are responsible for its altered expression, MMP3 was sequenced, and seven representative variants were genotyped in 1,037 Pima subjects for association analyses. Two variants were associated with both BMI and type 2 diabetes, and two additional variants were associated with type 2 diabetes alone; however, none of these variants were associated with MMP3 expression levels. We propose that the MMP3 pathway is altered in human obesity, but this alteration may be the result of a combination of genetic variation within the MMP3 locus itself, as well as variation in additional factors, either primary or secondary to obesity, that regulate expression of the MMP3 gene.     

Comprehensive analysis of gene expression in testes producing haploid germ cells using DNA microarray analysis

The comprehensive changes in testicular gene expression before and after haploid germ cell differentiation were examined using microarray analysis. Approximately 14,000 expressed sequence tag (EST) clones of Mouse FANTOM Array ver.1 were hybridized with probes generated from mRNA of adult and juvenile (17 days postpartum) testes before the onset of spermiogenesis. Of 1315 genes that exhibited reproducible changes in expression (p < 0.05), 46% exhibited an increase of twofold or more in adults compared to juveniles, and 22% a decrease of twofold or more. The analysis not only confirmed the reported haploid-specific expression of several known genes, but also provided new information on the differential expression of various other genes, including upregulated genes such as Allc and Skd3 and downregulated genes such as hbb b1, before or after the onset of spermiogenesis. Based on the fundamental difference in expression profiles, and molecular functions of the encoded products, the genes were classified into several groups: postmeiotically upregulated genes encoding various enzymes, structural and regulatory proteins, and chaperones, and downregulated genes encoding haemoglobins and oxidation/reduction-related proteins or the machinery associated with protein synthesis, such as ribosomal proteins.     

Lovastatin prevents discography-associated degeneration and maintains the functional morphology of intervertebral discs

Background context

Discography is an important diagnostic approach to identify the painful discs. However, the benefit of discography, a procedure involving needle puncture and injection of the diagnostic agent into the intervertebral disc, is controversial and has been reported to be associated with accelerated degeneration.

Purpose

To investigate the effect of lovastatin on the prevention of degeneration caused by a discography simulation procedure in rat caudal discs.

Study design

In vivo study using rat caudal discs.

Methods

A single flexible 27-gauge needle puncture into rat caudal discs was performed under fluoroscopic monitoring. Different concentrations (0.1, 1, 5, and 10 μM) of lovastatin were prepared and injected into randomly chosen caudal discs. RNA expression of selected genes, histologic, and immunohistochemical staining were performed to evaluate the phenotypic effects of lovastatin on rat caudal discs.

Results

Simulation of the discography procedure by puncturing the rat caudal discs with a 27-gauge needle and injection of saline solution induced degenerative changes in the nucleus pulposus with minimal damage to the annulus fibrosus. Aggrecan, Type II collagen, and SOX9 expressions were upregulated, whereas Type I collagen expression was significantly suppressed in discs treated with 5 and 10 μM lovastatin. Discs treated with 5 and 10 μM lovastatin were subjected to alcian blue staining and immunohistochemistry that revealed higher levels of glycosaminoglycans and an increase in the number of cells producing S-100 proteins, Type II collagen, and bone morphogenetic protein-2 (BMP-2), respectively. The most effective phenotypic repair was observed in discs treated with 10 μM lovastatin.

Conclusions

Intradiscal administration of lovastatin solution upregulated the expressions of BMP-2 and SOX9 and promoted chondrogenesis of rat caudal discs after needle puncture and substance injection. Therefore, we suggest that lovastatin promotes disc repair and can be used as a potential therapeutic agent for biological repair of disc degeneration after the diagnostic discography procedure.     

Gene expression comparison between primary triple‐negative breast cancer and paired axillary and sentinel lymph node metastasis

Few studies examine the genomics of axillary lymph node (ALN) metastasis in triple‐negative breast cancer (TNBC). The aim was to characterize and compare gene expression patterns of primary breast cancers and paired ALN metastases. Patients with stage 2‐3 ER/PR negative, HER2 negative TNBC with ALN macrometastasis without neo‐adjuvant therapy were selected. Tumor‐specific area was isolated from breast and ALN tissue sections. Gene expression of 2567 cancer‐associated genes was analyzed with the HTG EdgeSeq system coupled with Illumina next‐generation sequencing (NGS). Seventeen pairs of TNBC and autologous ALN metastasis were analyzed. Compared with the primary, ALN metastasis had 257 statistically significant differentially expressed genes, including 123 upregulated genes and 134 downregulated genes. Notably, there was an upregulation of anti‐apoptosis and survival signaling genes (BIRC3, TCL1A, FLT3, and VCAM1) in the ALN metastasis. There was also an upregulation of chemotaxis genes (CCL19, CCL21, CXCL13, and TNFSF11). The most striking feature is the downregulation of genes known to regulate cell microenvironment interaction (MMP2, MMP 3, MMP 7, MMP 11, MMP14, COL1A1, COL1A2, COL3A1, COL5A1, COL5A2, COL6A6, COL11A1, and COL17A1). In TNBC, ALN metastases have a distinct gene expression profile. Genes associated with anti‐apoptosis, survival responses, and chemotaxis are upregulated, and genes associated with regulation of extracellular matrix are downregulated when compared to autologous primary cancer. TNBC cells metastatic to lymph nodes undergo a change in order to metastasize and survive in the new microenvironment, which may lead to insights into the metastatic process.     

Effects of diazoxide on gene expression in rat pancreatic islets are largely linked to elevated glucose and potentially serve to enhance beta-cell sensitivity

Diazoxide enhances glucose-induced insulin secretion from beta-cells through mechanisms that are not fully elucidated. Here, we used microarray analysis (Affymetrix) to investigate effects of diazoxide. Pancreatic islets were cultured overnight at 27, 11, or 5.5 mmol/l glucose with or without diazoxide. Inclusion of diazoxide upregulated altogether 211 genes (signal log(2) ratio > or =0.5) and downregulated 200 genes (signal log(2) ratio -0.5 or lower), and 92% of diazoxide's effects (up- and downregulation) were observed only after coculture with 11 or 27 mmol/l glucose. We found that 11 mmol/l diazoxide upregulated 97 genes and downregulated 21 genes. Increasing the glucose concentration to 27 mmol/l markedly shifted these proportions toward downregulation (101 genes upregulated and 160 genes downregulated). At 27 mmol/l glucose, most genes downregulated by diazoxide were oppositely affected by glucose (80%). Diazoxide influenced expression of several genes central to beta-cell metabolism. Diazoxide downregulated genes of fatty acid oxidation, upregulated genes of fatty acid synthesis, and downregulated uncoupling protein 2 and lactic acid dehydrogenase. Diazoxide upregulated certain genes known to support beta-cell functionality, such as NKX6.1 and PDX1. Long-term elevated glucose is permissive for most of diazoxide's effects on gene expression, the proportion of effects shifting to downregulation with increasing glucose concentration. Effects of diazoxide on gene expression could serve to enhance beta-cell functionality during continuous hyperglycemia.     

Cell-based tissue engineering for the intervertebral disc: in vitro studies of human disc cell gene expression and matrix production within selected cell carriers.

BACKGROUND CONTEXT: Little is known about how disc cells attach, proliferate and form extracellular matrix (ECM) within carrier materials. Such information is needed to help formulate criteria for successful cell-carrier interactions in tissue engineering. PURPOSE: To compare proliferation, ECM production and gene expression in annulus cells cultured in a variety of cell carrier materials with potential application in tissue engineering of the disc. STUDY DESIGN: Human intervertebral disc cells from the annulus were used in a prospective study of proliferation, ECM production and gene expression within selected cell carriers. METHODS: Annulus cells from discs of 29 individuals were tested in collagen sponge, collagen gel, agarose, alginate or fibrin gel formulations. In situ hybridization assessed ECM gene expression of Types I and II collagen, aggrecan and chondroitin-6 sulfotransferase. Cell proliferation, cell shape, attachment and ECM production were evaluated. RESULTS: Collagen sponges provided the best microenvironment for disc cell ECM production and gene expression. Although collagen gels often could support good cell growth, such constructs did not result in either abundant ECM production or ECM gene expression, as shown by in situ hybridization. Growth and ECM production and gene expression in alginate, agarose and fibrin microenvironments were inferior. CONCLUSIONS: Tissue engineering techniques open new therapeutic possibilities for use of autologous disc cells, but fundamental questions on how these cells interact with cell carriers are unexplored. Results provide novel data on disc cell gene expression within diverse microenvironments. The collagen sponge proved to be a superior microenvironment.