Expression of TRAIL and the death receptors DR4 and DR5 correlates with progression of degeneration in human intervertebral disks.

Intervertebral disks degenerate far earlier than other musculoskeletal tissues and apoptosis has been suggested to have a vital function in promoting the degeneration process that is strongly associated with back pain. However, the molecular mediators of apoptosis in the intervertebral disk are poorly understood. Fas/FasL, TRAIL/DR4, TRAIL/DR5 and TNF-alpha/TNFR1 are ligand/receptor pairs of the tumor necrosis factor/nerve growth factor family, which are able to induce apoptosis by trimerization of the receptor by its corresponding ligand. We investigated which of these molecules are expressed in intervertebral disks and whether their expression correlates to disk degeneration. Intervertebral disks from 28 donors (age 12-70 years) suffering from scoliosis, vertebrae fracture or disk degeneration were scored histologically for degeneration and analyzed for gene expression of FasL/Fas, TRAIL/DR4, TNF-alpha/TNFR1 and caspase 8. Protein expression of FasL and TRAIL was assessed by immunohistology and apoptotic cell death was quantified by poly(ADP-ribose) polymerase (PARP) p85 staining. Isolated disk cells were analyzed by flow cytometry for Fas, FasL, TRAIL, DR4 and DR5 expression. Gene expression of TRAIL (P=0.002) and caspase 8 (P=0.027) significantly correlated with degeneration. TRAIL expression further correlated with cellularity (P=0.04), muccoid matrix changes (P=0.009) and tears and cleft formation (P=0.019). FasL and TRAIL expression was confirmed by immunohistology and PARP cleavage was significantly associated with degeneration (P=0.027). Flow cytometry on isolated disk cells revealed correlations between DR4 and degeneration (P=0.014), DR4/DR5 double-positive cells and degeneration (P=0.019), as well as DR5 and changes in tissue granularity (P=0.03). This is the first study that shows that intervertebral disk cells express TRAIL, DR4 and DR5, which correlate to the degenerative state of the disk. Therefore, disk cells inherit the molecular machinery to induce and undergo cellular apoptosis, and the frequency of cytokine expression suggests that the TRAIL/DR4/DR5 axis is an important molecular mediator of apoptosis induction in disk tissue.

Vascular endothelial growth factor gene-activated matrix (VEGF165-GAM) enhances osteogenesis and angiogenesis in large segmental bone defects.

UNLABELLED: Healing of fractures is dependent on vascularization of bone, which is in turn promoted by VEGF. It was shown that 0.1 and 1 mg of pVEGF165-GAM led to a significant increase in vascularization and bone regeneration in defects that would otherwise have led to atrophic nonunions. INTRODUCTION: One reason for lack of bone healing in nonunions is the absence of vascularization. In skeletogenesis, which is tightly linked to angiogenesis, vascular endothelial growth factor (VEGF) promotes the vascularization of the growth plate and transformation of cartilage to bone. We postulate that a gene-activated matrix (GAM), created with a plasmid coding for human VEGF165, coated on a collagen sponge could efficiently accelerate bone healing in large segmental defects. MATERIALS AND METHODS: Sixty New Zealand white rabbits received a 15-mm critical size defect on one radius, which was filled with either 0.1 or 1 mg plasmid-DNA as GAM. Radiographs were obtained every 3 weeks. After 6 or 12 weeks, animals were killed. New bone was measured by microCT scans. Vascularity was measured using anti-CD31 staining of endothelial cells in 18 regions of interest per implant. RESULTS: Scaffold and control plasmid showed no defect healing, whereas most of the animals in the VEGF groups showed partial or total bone regeneration. Significantly more bone was found in the VEGF groups, with no significant differences between the 0.1- and 1-mg groups. Immunohistochemical staining of endothelial cells revealed that the VEGF groups showed two to three times the number of vessels and a significantly larger endothelial area after 6 weeks. Twelve weeks after surgery, the amount of vascularization decreased, whereas more new bone was detectable. CONCLUSIONS: The rabbit critical size defect was appropriate in size to produce atrophic nonunions. We showed that angiogenesis and osteogenesis can be promoted by a VEGF165-GAM that is an appropriate tool to induce bone healing in atrophic nonunions.

A method for classifying metabolites in topological pathway analyses based on minimization of pathway number.

Metabolic pathway analysis based on the concept of elementary flux mode is a valuable tool for reconstruction of bacterial metabolisms and in predicting optimal conversion yields in biotechnology. However, pathway analysis of large and highly entangled metabolic networks meets the problem of combinatorial explosion of possible routes across the networks. Here we propose a method for coping with this problem by suitably classifying metabolites as external or internal. External metabolites are considered to have buffered concentrations while internal metabolites have to fulfil a balance condition at steady state. For many substances such as nutrients and excreted products, there are biochemical reasons to classify them as external. In addition, other substances (especially at central branching points) can operationally be considered external in order to avoid combinatorial explosion. We suggest to find such a classification of metabolites that minimizes the number of elementary flux modes (pathways). This is motivated by the objectives of finding such a description of the system that reduces as much as possible the amount of necessary data and of removing the ambiguity and arbitrariness in the classification of metabolites in an automated, systematic way. For networks of moderate size, the solution to this combinatorial minimization problem can be found by exhaustive search. To tackle also larger systems, a stochastic optimization program based on the Metropolis algorithm was developed. Both methods are applied, for illustration, to several reaction schemes including a larger network representing glutathione metabolism.

Enhanced biochemical and biomechanical properties of scaffolds generated by flock technology for cartilage tissue engineering.

Natural cartilage shows column orientation of cells and anisotropic direction of collagen fibers. However, matrices presently used in matrix-assisted autologous chondrocyte implantation do not show any fiber orientation. Our aim was to develop anisotropic scaffolds with parallel fiber orientation that were capable to support a cellular cartilaginous phenotype in vitro. Scaffolds were created by flock technology and consisted of a membrane of mineralized collagen type I as substrate, gelatine as adhesive, and parallel-oriented polyamide flock fibers vertically to the substrate. Confocal laser scan microscopy demonstrated that mesenchymal stem cells (MSCs) adhered and proliferated well in the scaffolds and cell vitality remained high over time. Articular chondrocytes seeded in a collagen type I gel into flock scaffolds deposited increasing amounts of proteoglycans and collagen type II over time. MSC-seeded flock scaffold constructs under chondrogenic conditions deposited significantly more proteoglycans and collagen type II than MSC collagen type I gel constructs only. Biomechanical testing revealed higher initial hardness of flock scaffolds than that of a clinically applied collagen type I/III scaffold combined with superior relaxation and an increasing hardness in MSC-loaded flock biocomposites during chondrogenesis. In conclusion, flock technology allows fabrication of scaffolds with anisotropic fiber orientation that mediates superior biomechanical and biochemical composition of tissue engineering constructs for cartilage repair.

Matrix metalloprotease inhibitors suppress initiation and progression of chondrogenic differentiation of mesenchymal stromal cells in vitro.

Mesenchymal stromal cells (MSC) are an attractive source for cell therapy and tissue engineering of joint cartilage. Common chondrogenic in vitro protocols, however, induce hypertrophic markers like COL10A1, matrix metalloproteinase 13 (MMP13), and alkaline phosphatase (ALP) reminiscent of endochondral bone formation. To direct MSC toward articular chondrocytes more specifically, a better understanding of the regulatory steps is desirable. Proteases are important players in matrix remodeling, display inhibitory effects on growth plate development and MMP13 inhibition prevented hypertrophy of bovine chondrocytes. The aim of this study was to evaluate whether the activity of proteases and MMPs, especially MMP13, is crucial for the transition of MSC toward mature chondrocytes and could allow to selectively influence aspects of early and late chondrogenic differentiation. Protease inhibitors were added during MSC chondrogenesis and stage-specific markers were assessed by histology, qPCR, and ALP quantification. Chondrogenesis was little affected by leupeptin, pepstatin, or aprotinin. In contrast, broad spectrum pan-MMP inhibitors dose dependently suppressed proteoglycan deposition, collagen type II and type X staining, ALP activity, and reduced SOX9 and COL2A1 expression. A selective MMP13 inhibitor allowed chondrogenesis and showed only weak effects on ALP activity. In conclusion, transition of MSC toward mature chondrocytes in vitro depended on molecules suppressed by pan-MMP inhibitors identifying chondrogenic differentiation of MSC as a sophistically regulated process in which catabolic enzymes are capable to directly influence cellular fate. In future therapeutic applications of diseased joints, the tested MMP13-specific inhibitor promises suppression of collagen type II degradation without imposing a risk to impair MSC-driven regeneration processes.

A new porcine in vivo animal model of disc degeneration: response of anulus fibrosus cells, chondrocyte-like nucleus pulposus cells, and notochordal nucleus pulposus cells to partial nucleotomy.

STUDY DESIGN: In vivo animal study. OBJECTIVES: To describe a new porcine disc degeneration model, and to analyze disc remodeling and degeneration after nucleotomy with special view to the different nucleus pulposus (NP) cell types. SUMMARY OF BACKGROUND DATA: Thus far, predominantly smaller animals were used for disc degeneration models; however, such small discs were inappropriate to investigate cell implementation therapies. Though notochordal cells (NCs) are important for disc formation and maintenance, differences in the amount of NCs between human and animal discs have often been neglected. METHODS: Twenty-four Goettingen minipigs underwent partial nucleotomy with a 16G biopsy cannula, to remove approximately 10% of total NP volume. Animals were followed up for 3, or 24 weeks and analyzed by radiographs, MRIs, (immuno)histology, gene expression analysis, and biomechanical testing. RESULTS: Three weeks after nucleotomy disc height was reduced by 26%, and magnetic resonance imaging signal intensity by 40%. At 24 weeks disc height was decreased by 32%. Increased degenerative changes were found in a histodegeneration score 3 and 24 weeks after nucleotomy, as well as considerable NP scarification after 3 weeks. In controls, cytokeratin-8 immunohistochemistry identified NCs in proximity to chondrocyte-like NP cells at approximately equal ratio. After nucleotomy, NCs were considerably reduced to <10% of total NP cells. Matrix genes were upregulated, except for aggrecan that decreased to 35% of initial values 3 weeks after nucleotomy. Matrix degrading factors (matrix metalloproteinases 13 and 3) were continuously upregulated, whereas transcripts of their inhibitors (tissue inhibitors of matrix metalloproteinase 2 and 3) were downregulated. No significant changes in segmental spinal flexibility or bone density were found after nucleotomy. CONCLUSION: We introduced a new disc degeneration model with relatively large discs that could be used for cell therapeutic approaches. The study gives further information about disc remodeling after nucleotomy and indicates the relevance of an altered cellular composition for the development of disc degeneration.

Accelerated intervertebral disc degeneration in scoliosis versus physiological ageing develops against a background of enhanced anabolic gene expression.

Molecular consequences of long-term deformation and altered mechanical loading of intervertebral disc (IVD) tissue in scoliosis have yet to be elucidated. We hypothesized that histological disc degeneration is faster in scoliosis than in normal ageing and that this is reflected by an altered gene expression profile. A semiquantitative histodegeneration score (HDS) revealed significantly enhanced degeneration in scoliosis (HDS 5.3) versus age-matched control IVDs (HDS 2.25; p = 0.001). Gene expression analysis by cDNA array and RT-PCR demonstrated higher mRNA levels for extracellular-matrix molecules like aggrecan, biglycan, decorin, lumican, chondromodulin, and COL2A1 in scoliotic discs versus normal discs of identical degeneration score. No differences were evident for catabolic molecules like MMP3, MMP13, MMP17, and TIMP1. In sum, morphologic disc degeneration was accelerated by about 2 decades in scoliosis versus physiological ageing and developed against a background of stronger anabolic matrix metabolism at younger age or in response to the altered mechanical environment of the tissue.

Effect of donor characteristics, technique of harvesting and in vitro processing on culturing of human marrow stroma cells for tissue engineered growth of bone.

The aim of this study was to assess the effect of donor characteristics and the technique of harvesting and in vitro processing on the efficacy of culturing of human mesenchymal stem cells (hMSCs) for tissue engineered growth of bone. Cultures of hMSCs were derived from iliac crest bone marrow aspirates (21 donors, age 11-76) and from cancellous bone grafting material (32 donors, age 13-84). Age had no significant effect on the ability to isolate and culture hMSCs, although the failure rate was 55.6% in donors beyond the age of 60, while it varied between 14.3% and 22.2% in donors under 60 years of age. Male and female donors had comparable failure rates (27.3% and 28.6%, respectively). Culturing of hMSCs was successful in 90.4% of marrow aspirates from 21 donors and in 62.5% of cancellous bone specimens from 35 donors. This difference was statistically significant (P=0.023). Regression analysis confirmed that at simultaneous testing of the three variables, only the source of cells significantly affected the result (P=0.043). Morphological evaluation of the unfractionated primary population showed a change in cell shape of the adherent cells from a triangular into thin spindle-shaped elongated form, which remains until confluence. When the cultures were exposed to osteoinductive medium, various morphotypes expressing different levels of alkaline phosphatase and secreting different amounts of mineral were evident. Morphology of marrow stroma cells (MSCs) from marrow aspirates was not different from MSCs derived from cancellous bone specimens. Expression of osteogenic markers in MSCs as shown by PCR as well, did not differ between the two sources. It is concluded that marrow aspirates and cancellous bone specimens produce comparable populations of MSCs. However, bone marrow aspirates from donors under the age of 60 years rather than cancellous bone chips are favourable for isolation and expansion of hMSCs for tissue engineered growth of bone.

Matrix-assisted cell transfer for intervertebral disc cell therapy.

Cell therapy seems to be a promising way to reconstitute degenerated discs. We elucidate the basic aspects of intervertebral disc (IVD) cell therapy to estimate its potential in disc regeneration. Cell transfer efficiency and survival was quantified by luciferase expression after injection of recombinant cells into healthy, nucleotomized or mechanically degenerated rabbit IVDs in vitro, in situ or in vivo. A two-component fibrin matrix was adapted to allow injection of a fluid cell suspension that quickly polymerizes in IVDs. Thirty-five to fifty percent of matrix injected cells remained in the nucleus and transition zone in contrast to a rapid loss of medium-injected cells. Nucleotomy, which reduces intradiscal pressure, was crucial to the survival of the transferred cells over 3 days and nutritional enrichment of the fibrin matrix with potent biomolecules from serum significantly enhanced cell viability. In conclusion, advanced matrix substitutes are needed for efficient transfer and improved cell survival in the low-nutrient intradiscal environment to further improve disc cell therapy.

Induction of intervertebral disc-like cells from adult mesenchymal stem cells.

The potential of adult mesenchymal stem cells (MSCs) to differentiate towards cartilage, bone, adipose tissue, or muscle is well established. However, the capacity of MSCs to differentiate towards intervertebral disc (IVD)-like cells is unknown. The aim of this study was to compare the molecular phenotype of human IVD cells and articular chondrocytes and to analyze whether mesenchymal stem cells can differentiate towards both cell types after transforming growth factor beta (TGF beta)-mediated induction in vitro. Bone marrow-derived MSCs were differentiated in spheroid culture towards the chondrogenic lineage in the presence of TGF beta(3) dexamethasone, and ascorbate. A customized cDNA-array comprising 45 cartilage-, bone-, and stem cell-relevant genes was used to quantify gene expression profiles. After TGF beta-mediated differentiation, MSC spheroids turned positive for collagen type II protein and expressed a large panel of genes characteristic for chondrocytes, including aggrecan, decorin, fibromodulin, and cartilage oligomeric matrix protein, although at levels closer to IVD tissue than to hyaline articular cartilage. Like IVD tissue, the spheroids were strongly positive for collagen type I and osteopontin. MSC spheroids expressed more differentiation markers at higher levels than culture-expanded IVD cells and chondrocytes, which both dedifferentiated in monolayer culture. In conclusion, mesenchymal stem cells adopted a gene expression profile that resembled native IVD tissue more closely than native joint cartilage. Thus, these cells may represent an attractive source from which to obtain IVD-like cells, whereas modification of culture conditions is required to approach the molecular phenotype of chondrocytes in hyaline cartilage.

Vascular endothelial growth factor (VEGF-A) expression in human mesenchymal stem cells: autocrine and paracrine role on osteoblastic and endothelial differentiation.

Angiogenesis is essential in bone fracture healing for restoring blood flow to the fracture site. Vascular endothelial growth factor (VEGF) and its receptor have been implicated in this process. Despite the importance of angiogenesis for the healing processes of damaged bones, the role of VEGF signaling in modulation of osteogenic differentiation in human mesenchymal stem cells has not been investigated in great detail. We examined the expression of VEGF-A and VEGFR-1 in human adult mesenchymal stem cells derived from trabecular bone (hTBCs). VEGF-A was found to be secreted in a differentiation dependent manner during osteogenesis. Transcripts for VEGF-A were also seen to be elevated during osteogenesis. In addition, transcripts for VEGF-A and the corresponding receptor VEGFR-1 were upregulated under hypoxic conditions in undifferentiated hTBCs. To investigate the signaling of VEGF-A on osteogenesis recombinant hTBCs were generated. High expression of VEGF-A stimulated mineralization, whereas high expression of sFLT-1, an antagonist to VEGF-A, reduced mineralization suggesting that VEGF-A acts as autocrine factor for osteoblast differentiation. In addition, VEGF-A secreted by hTBCs promotes sprouting of endothelial cells (HUVE) demonstrating a paracrine role in blood vessel formation. In summary, an in vitro analysis of transgene effects on cellular behavior can be used to predict an effective ex vivo gene therapy.

Stimulation of gene expression and loss of anular architecture caused by experimental disc degeneration--an in vivo animal study.

STUDY DESIGN: An external compression model was used to evaluate gene and protein expression in intervertebral discs with moderate disc degeneration. OBJECTIVE: To determine messenger ribonucleic acid and protein expression levels of relevant disc components. SUMMARY OF BACKGROUND DATA: An animal model of mechanically induced disc degeneration was developed and characterized histologically. However, little is known at the molecular level in moderate disc degeneration. METHODS: There were 8 New Zealand white rabbits subjected to monosegmental posterior compression to induce moderate disc degeneration. Twelve animals served as controls or sham controls. Discs were analyzed using immunohistochemistry for collagen type 1 (COL1), COL2, aggrecan, and bone morphogenetic protein-2/4 (BMP-2/4). For gene analysis, conventional and quantitative polymerase chain reactions were used for COL1A2, COL2A1, aggrecan, BMP-2, biglycan, decorin, osteonectin, fibromodulin, fibronectin, matrix metalloproteinase-13 (MMP-13), and tissue inhibitor of MMP-1. Gene expression for nontreated, sham-treated, and compressed discs was quantified in relation to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase. RESULTS: Immunohistochemistry of compressed discs showed a loss of anular architecture, and a significant reduction of BMP-2/4 and COL2 positive cells. Gene expression analysis showed a significant up-regulation of COL1A2, osteonectin, decorin, fibronectin, tissue inhibitor of MMP-1, BMP-2, and MMP-13 in compressed discs. CONCLUSIONS: Experimental moderate disc degeneration is characterized by a loss of BMP-2/4 and COL2 positive cells, although gene expression of disc constituents, catabolic enzymes, and growth factors is stimulated to reestablish disc integrity.