Brent D. Brower-Toland

Mechanical disruption of individual nucleosomes reveals a reversible multistage release of DNA

The dynamic structure of individual nucleosomes was examined by stretching nucleosomal arrays with a feedback-enhanced optical trap. Forced disassembly of each nucleosome occurred in three stages. Analysis of the data using a simple worm-like chain model yields 76 bp of DNA released from the histone core at low stretching force. Subsequently, 80 bp are released at higher forces in two stages: full extension of DNA with histones bound, followed by detachment of histones. When arrays were relaxed before the dissociated state was reached, nucleosomes were able to reassemble and to repeat the disassembly process. The kinetic parameters for nucleosome disassembly also have been determined.

Chondrocytic differentiation of mesenchymal stem cells sequentially exposed to transforming growth factor‐β1 in monolayer and insulin‐like growth factor‐I in a three‐dimensional matrix

This study evaluated chondrogenesis of mesenchymal progenitor stem cells (MSCs) cultured initially under pre‐confluent monolayer conditions exposed to transforming growth factor‐β (TGF‐β), and subsequently in three‐dimensional cultures containing insulin‐like growth factor I (IGF‐I). Bone marrow aspirates and chondrocytes were obtained from horses and cultured in monolayer with 0 or 5 ng of TGF‐β1 per ml of medium for 6 days. TGF‐β1 treated and untreated cultures were distributed to three‐dimensional fibrin disks containing 0 or 100 ng of IGF‐I per ml of medium to establish four treatment groups. After 13 days, cultures were assessed by toluidine blue staining, collagen types I and II in situ hybridization and immunohistochemistry, proteoglycan production by [Chondrocytic differentiation of mesenchymal stem cells sequentially exposed to transforming growth factor‐β1 in monolayer and insulin‐like growth factor‐I in a three‐dimensional matrix35S]‐sulfate incorporation, and disk DNA content by fluorometry. Mesenchymal cells in monolayer cultures treated with TGF‐β1 actively proliferated for the first 4 days, developed cellular rounding, and formed cell clusters. Treated MSC cultures had a two‐fold increase in medium proteoglycan content. Pretreatment of MSCs with TGF‐β1 followed by exposure of cells to IGF‐I in three‐dimensional culture significantly increased the formation of markers of chondrocytic function including disk proteoglycan content and procollagen type II mRNA production. However, proteoglycan and procollagen type II production by MSCs remained lower than parallel chondrocyte cultures. MSC pretreatment with TGF‐β1 without sequential IGF‐I was less effective in initiating expression of markers of chondrogenesis. This study indicates that although MSC differentiation was less than complete when compared to mature chondrocytes, chondrogenesis was observed in IGF‐I supplemented cultures, particularly when used in concert with TGF‐β1 pretreatment.

Insulinlike growth factor-I gene therapy applications for cartilage repair

Cartilage function after resurfacing with cell-based transplantation procedures or during the early stages of arthritic disease may be bolstered by the addition of growth factor genes to the transplanted tissue. Insulinlike growth factor-I maintains chondrocyte metabolism in normal cartilage homeostasis and has been shown to improve cartilage healing in vivo. Given the relatively short half-life of insulinlike growth factor-I in biologic systems, however, maintenance of effective concentrations of this peptide has necessitated either very high initial doses or repeated treatment. Delivery of the insulinlike growth factor-I gene, using a deleted adenovirus vector, specifically targeting graftable articular chondrocytes, bone marrow-derived chondroprogenitor cells, or synovial lining cells, may provide more durable insulinlike growth factor-I fluxes to articular tissues. Cultured equine articular chondrocytes, mesenchymal stem cells, synovial explants, and synovial intimal cells were readily transfected with an E1-deleted adenoviral vector containing equine insulinlike growth factor-I coding sequence. Optimal viral concentrations for effective transduction were 100 multiplicities of infection in synoviocytes, 500 multiplicities of infection in chondrocytes, and 1000 multiplicities of infection in mesenchymal stem cells. Production of insulinlike growth factor-I ligand varied from 65 ng/mL to 246 ng/mL in medium from chondrocytes and synovial explants, respectively. For chondrocytes, these concentrations were sufficient to produce significant stimulation of cartilage matrix gene expression and subsequent proteoglycan production. Moreover, cells in infected cultures maintained a chondrocytic phenotype and continued to express elevated insulinlike growth factor-I levels during 28 days of monolayer culture. Minimal synthetic activity, other than insulinlike growth factor-I ligand synthesis, was evident in synovial cultures. These experiments suggest several avenues for insulinlike growth factor-I supplementation of articular cartilage, including preimplantation adenoviral-insulinlike growth factor gene transfer to chondrocytes or chondroprogenitor cells, and direct injection of adenoviral-insulinlike growth factor to transfect the synovial structures in situ.

Direct adenovirus-mediated insulin-like growth factor I gene transfer enhances transplant chondrocyte function

Cell-based cartilage-resurfacing procedures may be enhanced by the addition of insulin-like growth factor I (IGF-I) to the transplant biomatrix. Given the relatively short half-life of IGF-I in biological systems, however, maintenance of effective concentrations of this peptide necessitates either high initial doses, or repeated treatment. This study investigated IGF-I delivery via adenoviral gene therapy, targeting graftable articular chondrocytes. Cultured articular chondrocytes were infected with an E1-deleted adenoviral vector containing IGF-I-coding sequence under CMV promoter control. Increased adenovirus-IGF-I concentrations resulted in coordinate increase in IGF-I mRNA and ligand expression; however, chondrocyte matrix synthesis was maximized by the lower adenovirus-IGF-I concentration (100 MOI) without additional increase at 200 or 500 MOI. Using 100 MOI, infected monolayers produced medium IGF-I content of at least 10 ng/ml in each 48-hr period for 28 days, reaching a day 4 peak concentration of 66 +/- 4.0 ng/ml. These concentrations were sufficient to produce significant stimulation of normal cartilage matrix gene expression. The concentration of secreted matrix products in medium from infected monolayers was increased up to 8-fold over uninfected control cultures. Moreover, compared with uninfected cultures, cells in infected cultures were more resistant to de-differentiation over time under serum-starved conditions, maintaining a normal chondrocyte molecular phenotype for at least 28 days. These data indicate that cultured chondrocytes are readily transduced by recombinant adenoviral vectors. The adenoviral-IGF transgene is abundantly expressed and its product secreted at therapeutic concentrations for at least 28 days, resulting in increased matrix biosynthesis and maintenance of the chondrocytic phenotype. Combined, this information suggests that there may be significant value in preimplantation adenoviral-IGF gene therapy for chondrocytes destined for cartilage resurfacing.

Exogenous insulin-like growth factor-I stimulates an autoinductive IGF-I autocrine/paracrine response in chondrocytes.

The modulation of insulin‐like growth factor‐I (IGF‐I) gene expression by chondrocytes following exogenous IGF‐I supplementation of culture was assessed to examine the hypothesis that constitutive IGF‐I mRNA activity is suppressed by exogenous administration of IGF‐I to cartilage in situ. Chondrocytes in monolayer culture were treated with 0, 10 or 100 ng/ml IGF‐I for 48 h and resultant IGF‐I and matrix gene expression patterns were assessed by quantitative polymerase chain reaction (qPCR) and northern blotting, respectively. Effective translation of proteoglycan (PG) as a response to IGF‐I was determined by dimethylmethylene blue (DMMB) dye‐binding assay. To determine the temporal nature of the IGF‐I autocrine/paracrine response to exogenous IGF‐I, chondrocyte cultures were treated with 100 ng/ml IGF‐I and the IGF‐I mRNA response was assessed at 0, 4, 12, 24, 48 and 72 h. Significant increases in chondrocyte density and in PG synthesis occurred during treatment of chondrocyte cultures with 10 and 100 ng/ml IGF‐I. Persistent exposure of chondrocytes to 100 ng/ml IGF‐I resulted in maximal IGF‐I mRNA response at 24 h, with declining message accumulation at 48 and 72 h. These data suggest that IGF‐I induces an autoinductive IGF‐I autocrine/paracrine transcriptional response. The clinical ramifications of these findings include support for the use of exogenous IGF‐I for cartilage repair where it could conceivably amplify and extend the effect of exogenous IGF‐I beyond the transitory persistence of supplemental IGF‐I ligand in repair constructs.

Parathyroid hormone‐related peptide and Indian hedgehog expression patterns in naturally acquired equine osteochondrosis

Early changes in parathyroid hormone‐related peptide (PTH‐rP) and Indian hedgehog (Ihh) expression were examined in equine articular osteochondrosis (OC) as a model of a naturally acquired dyschondroplasia. Cartilage was harvested from OC‐affected femoropatellar or scapulohumeral joints from immature horses and normal control horses of similar age. PTH‐rP expression levels were assessed by semi‐quantitative PCR, in situ hybridization, and immunohistochemistry. Ihh protein expression levels were assessed by immunohistochemistry. Elevated PTH‐rP protein and mRNA expression were identified in the deeper layers of affected articular cartilage and the fibrous tissue of interposing clefts. These changes were confined to the chondrocytes in the OC‐affected cartilage, which had significantly increased PTH‐rP protein and mRNA expression when compared to control cartilages. Ihh protein expression showed similar distribution as PTH‐rP in the deeper layers of articular cartilage; however, only a trend for increased Ihh immunostaining was evident in the OC cartilage when compared to the normal cartilage. Increased PTH‐rP expression in pre‐hypertrophic chondrocytes of diseased OC cartilage suggests a possible link between this peptide and the delayed ossification, which is a consistent histologic alteration in OC. More evidence is necessary to determine the role of Ihh in articular cartilage and if a similar feedback cycle exists as previously described for the growth plate.

Gene mediated insulin-like growth factor-I delivery to the synovium.

The feasibility of articular gene therapy using insulin‐like growth factor‐I transgene expression in synovial tissues was assessed in vitro by transfection of synovial explant and monolayer cultures. Synovial membrane was harvested from horses and distributed for explant culture in multiwell plates or digested for monolayer culture in multiwell plates and chamber slides. Synovial monolayers were cultured for 48 h after infection with 0, 100, 200, or 500 moi adenovirus‐IGF‐I (AdeIGF‐I) to establish an optimum dose. Explants were then either infected with AdeIGF‐I or adenoviral LacZ and cultured for 8 days, treated with 100 ng/ml recombinant IGF‐I as a positive control, or remained as uninfected untreated culture controls. Expression of IGF‐I in explants and monolayers was assessed by in situ hybridization and quantitative polymerase chain reaction (PCR), and translation confirmed by IGF‐I radioimmunoassay (RIA) and tissue immunoreaction. Effects of IGF‐I on synovial function was assessed by proteoglycan and hyaluronan assay, and northern blot assessment of decorin and collagen type I expression. Significant transgene expression in synovial cells was present for all AdeIGF‐I concentrations. Similarly, medium IGF‐I concentrations were significantly elevated in AdeIGF‐I infected synovial monolayer and explant cultures at all time points. Peak IGF‐I concentration of 246 ± 43 ng/ml developed in explant cultures on day 4; IGF‐I levels in control explant groups were unchanged over baseline values. In situ hybridization and immunolocalization for IGF‐I indicated focal IGF‐I expression in intimal and subintimal layers of infected explants, with diffuse immunoreaction throughout infected subintimal and fibrous layers. For monolayer cultures, intracellular immunoreaction to IGF‐I was markedly higher in infected cells, and was most prominent at 100 moi. Effects of IGF‐I on synoviocyte cultures were evident on northern blots, which showed decreased decorin expression and elevated type I collagen production in AdeIGF‐I infected monolayers. Proteoglycan concentration in the medium from explant cultures rose over the initial 4 days but was similar between treatment groups. The concentration of hyaluronan in medium from explant cultures did not differ significantly within or between treated and control groups during the 8‐day study period. These data indicate that IGF‐I can be successfully introduced to synovial structures by adenoviral vectors and results in effective IGF‐I ligand synthesis without untoward synovial morphologic effects.

Direct adenovirus-mediated IGF-I gene transduction of synovium induces persisting synovial fluid IGF-I ligand elevations.

Insulin-like growth factor-I (IGF-I) is one of the most influential growth factors in cartilage repair. Maintenance of adequate IGF-I levels after articular repair procedures is complicated by the short biological half-life of IGF-I in vivo. This study investigated the potential for more prolonged IGF-I delivery through direct adenoviral mediated transduction of synovial tissues in the metacarpophalangeal (MCP) joints of horses. The use of a large animal model provided a structurally similar and metabolically relevant corollary to the human knee. The complete IGF-I coding sequence was packaged into an E1–E3 deleted adenovirus-5 vector under cytomegalovirus promoter control (AdIGF-I), and injected at varying total joint doses to the MCP joints of 14 horses. Direct injection of 20 and 50 × 1010 AdIGF-I resulted in significant elevations of IGF-I in synovial fluid for approximately 21 days. Synovial tissue taken from injected joints at day 35 following injection and compared to tissue taken preinjection from the same joints revealed elevated synoviocyte IGF-I mRNA levels for the highest viral dose by in situ hybridization and real-time PCR techniques. AdIGF-I injections did not result in significant lameness, joint effusion or elevated total protein concentrations in the synovial fluid. Mild mononuclear infiltration of white blood cells was evident in histologic sections of the synovium in the second highest adenoviral IGF-I dose of 20 × 1010 particles. Cartilage biopsies taken from all injected joints did not reveal any significant changes in proteoglycan levels nor in histological morphology, which included chondrocyte cloning, architecture, cell type or toluidine blue staining, when compared to control joints. Based on these findings, gene transfer of IGF-I to the synovium of joints can result in significant and persistent elevations of IGF-I ligand in synovial fluid with minimal detrimental effects. Direct IGF-I gene therapy may offer a simple approach in treating patients with acute cartilage injury.

Use of optical trapping techniques to study single-nucleosome dynamics.

Publisher Summary This chapter describes the optical trapping system and experimental sample preparation techniques necessary to carry out dynamic structural analysis of individual nucleosomes in nucleosomal arrays. Optical trapping techniques are single-molecule techniques that allow mechanical manipulation of a nucleosomal DNA molecule under physiological solution conditions. Because the sample is immobilized, solution conditions and sample components can actually be varied during the course of experimentation. The experiments can be nondestructive, permitting repeated sampling of the same nucleosomal arrays. An added advantage of the use of an optical trap is the freedom to consider individual nucleosomal structure in the context of a nucleosomal array, rather than on isolated mononucleosomes. Optical trapping technology provides a useful addition to this repertoire of techniques. The chapter anticipates that single-molecule optical trapping experiments on chromatin structure can complement more traditional technologies, and aid in the elucidation of the structural role in chromatin of histone and nonhistone proteins and their post-translational modifications. Likewise, optical trapping methods can be adaptable to the study of enzymatic activities such as RNA polymerases and ATP-dependent chromatin remodelers operating on chromatin structure.