Nick Rhodes

Intervertebral Disc Cell–Mediated Mesenchymal Stem Cell Differentiation

Low back pain is one of the largest health problems in the Western world today, and intervertebral disc degeneration has been identified as a main cause. Currently, treatments are symptomatic, but cell‐based tissue engineering methods are realistic alternatives for tissue regeneration. However, the major problem for these strategies is the generation of a suitable population of cells. Adult bone marrow–derived mesenchymal stem cells (MSCs) are undifferentiated, multipotent cells that have the ability to differentiate into a number of cell types, including the chondrocyte‐like cells found within the nucleus pulposus (NP) of the intervertebral disc; however, no method exists to differentiate these cells in an accessible monolayer environment. We have conducted coculture experiments to determine whether cells from the human NP can initiate the differentiation of human MSCs with or without cell–cell contact. Fluorescent labeling of the stem cell population and high‐speed cell sorting after coculture with cell–cell contact allowed examination of individual cell populations. Real‐time quantitative polymerase chain reaction showed significant increases in NP marker genes in stem cells when cells were cocultured with contact for 7 days, and this change was regulated by cell ratio. No significant change in NP marker gene expression in either NP cells or stem cells was observed when cells were cultured without contact, regardless of cell ratio. Thus, we have shown that human NP and MSC coculture with contact is a viable method for generating a large population of differentiated cells that could be used in cell‐based tissue engineering therapies for regeneration of the degenerate intervertebral disc.

Sodium channel protein expression enhances the invasiveness of rat and human prostate cancer cells

Expression of Na+ channel protein was analysed in established cell lines of rat and human prostatic carcinoma origin by flow cytometry using a fluorescein‐labelled polyclonal antibody. In many cell lines examined, the obtained frequency distribution profiles were bimodal and identified a subpopulation of cells which expressed high levels of Na+ channel protein. A significant positive correlation was demonstrated between the proportion of channel‐expressing cells and the functional ability of individual cell lines to invade a basement membrane matrix in vitro. In addition, two transfectant cell lines containing rat prostate cancer genomic DNA were found to express significantly elevated levels of Na+ channel protein when compared with the original benign recipient cell line. Enhanced Na+ channel expression by two metastatic derivatives of these transfectant cells directly correlated with increased invasiveness in vitro. These studies strongly support the hypothesis that expression of Na+ channel protein and the metastatic behaviour of prostatic carcinoma cells are functionally related, either by endowing the membranes of these cells with specialised electrophysiological properties (e.g. enhancing their motility and/or secretory activities) and/or by perturbing endogenous mechanisms regulating ionic homeostasis within the cells.

Autologous In Vivo Adipose Tissue Engineering in Hyaluronan-Based Gels—A Pilot Study

BACKGROUND There is a major clinical need for strategies for adequately reconstructing the soft tissue defects found after deep burns, tumor resection, or trauma. A promising solution is adipose tissue engineering with preadipocytes, stem-cell derived precursors of the adipose tissue, implanted within biomaterials. This pilot study evaluated hyaluronan gels mixed with autologous undifferentiated preadipocytes in a pig model for their potency to generate new fat. MATERIALS AND METHODS Preadipocytes were isolated from intra-abdominal pig fat by collagenase digestion, plated on fibronectin-coated culture dishes in Dulbeccos modified Eagle medium/Hams F12 (Biochrom, Berlin, Germany) combined with 10% pig serum, expanded, and mixed with hyaluronan gel. Two types of gels with varying degrees of amidation of the carboxyl groups were tested (HYADD3, HYADD4). Cell-loaded gels and unseeded controls were injected subcutaneously into the ears of three pigs, explanted at 6 wk, and analyzed histologically. RESULTS Both cell-loaded specimens were detected macroscopically. They demonstrated a slight volume effect with limited stability after 6 wk. Unloaded HYADD3 and HYADD4 controls could not be identified at the time of explantation. Histology of HYADD3 revealed islets of mature adipocytes and vessels embedded in fat tissue surrounded by gel. In contrast, no fat formation was found in HYADD4 gels when implanted in the ear. CONCLUSIONS Histological findings demonstrate that HYADD3 is a promising gel for generating adipose tissue. Even though HYADD3 might be a potential material for the reconstruction of small tissue defects, the question remains as to whether the adipose tissue within the gel is attributable to preadipocyte maturation or ingrowth from neighboring tissue.

Surface modification of a segmented polyetherurethane using a low-powered gas plasma and its influence on the activation of the coagulation system

A medical grade segmented polyetherurethane (PEU) was treated with a low-powered gas plasma using O(2), Ar, N(2) and NH(3) as the treatment gases. Changes in the surface functional group chemistry were studied using X-ray photoelectron spectroscopy. The wettability of the surfaces was examined using dynamic contact angle measurements and the surface morphology was evaluated using atomic force microscopy. The influence of the surface modification to the polyurethane on the blood response to the polyetherurethane was investigated by measuring changes in the activation of the contact phase activation of the intrinsic coagulation cascade. The data demonstrate that the plasma treatment process caused surface modifications to the PEU that in all cases increased the polar nature of the surfaces. O(2) and Ar plasmas resulted in the incorporation of oxygen-containing groups that remained present following storage in an aqueous environment. N(2) and NH(3) plasmas resulted in the incorporation of nitrogen-containing groups but these were replaced with oxygen-containing groups following storage in the aqueous environment. In all plasma treatments there was a lowering of contact phase activation compared to the untreated surface, the N(2) and NH(3) treatments dramatically so.

Macrophage subpopulation differentiation by stimulation with biomaterials

Macrophages were elicited by the subcutaneous implantation of ultra high molecular weight polyethylene (UHMWPE) for periods of 2, 7, and 14 days in rats. Exudates of varying volumes were produced that was comprised of granulocytes, monocytes, immature and mature macrophages, and T-lymphocytes. No B-lymphocytes were observed at any time periods. Cell types were identified by their granularity and positivity to the following antibodies: leucocyte common antigen (LCA, pan leucocyte); CD11b/c (macrophage/monocyte); CD5 (T-lymphocyte); CD45RA (B-lymphocyte); HIS48 (granulocyte); ED2 (mature macrophage); and MCP-1 (monocyte chemoattractant protein 1). Monocytes isolated from control rat blood demonstrated a size slightly larger than that of granulocytes but with less granularity. Their size and granularity were followed over increasing time periods. The macrophages elicited by UHMWPE showed a similar pattern, with the exception of an apparently highly granular subpopulation with volumes similar to that of granulocytes but significantly more granular. The granular macrophage subset had a very high degree of ED2 and MCP-1 positivity, and their proportion, compared with other macrophages, was greatest at 2 days. The high MCP-1 expression was accounted for by MCP-1 molecules bound to the surface of a small proportion of macrophages that were activated. It is postulated that this subpopulation was responsible for the synthesis of the MCP-1 and could indicate a mechanism by which monocytes are attracted to the site of an implanted material.

Plasma recalcification as a measure of contact phase activation and heparinization efficacy after contact with biomaterials

The rate of plasma clotting was measured in order to investigate two different processes. In both cases normal, pooled platelet-poor plasma was used as a substrate for measurement of clotting. The intrinsic coagulation pathway was studied by bringing a variety of biomaterials into contact with a plasma aliquot and observing the rate of clotting diminish by virtue of factor XII activation. The efficacy of heparinization was investigated by measuring the increase in clotting time of a plasma aliquot during biomaterial contact. In both cases, clotting time was measured turbidometrically. Marked differences in intrinsic pathway activation were observed between a variety of materials. There were clear differences between the materials and the negative and positive controls. The assay showed that heparinized materials could be distinguished from non-heparinized materials and a non-activated plasma control.

Influence of the α1-adrenergic antagonist, doxazosin, on noradrenaline-induced modulation of cytoskeletal proteins in cultured hyperplastic prostatic stromal cells

Doxazosin, an α1‐adrenergic antagonist, inhibits sympathetic contraction of prostatic stromal smooth muscle cells and is used in the relief of obstructive benign prostatic hyperplasia (BPH). In vitro application of noradrenaline stimulates expression of cytoskeletal filaments, particularly actin and myosin, by prostatic stromal cells, thus enhancing their differentiation towards smooth muscle cells. This study examined the possible role of doxazosin in reversing this phenotypic modulation as well as in inhibiting smooth muscle cell contraction.

Influence of wall shear rate on parameters of blood compatibility of intravascular catheters.

Three polymeric materials (silicone, PVC and nylon) were compared in an in vitro perfusion model, whereby 5 ml whole blood were perfused along 1 m lengths of polymeric tubing of 1 mm internal diameter at wall shear rates of up to 1000 s-1. Perfusion took place at 37 degrees C for 30 min. The polymers were investigated for platelet activation, granulocyte secretion, complement activation and contact phase activation. These parameters were also analysed in static contact for comparison. All the parameters measured displayed a dependence on wall shear rate. In all the materials studied, platelet adhesion and platelet activation increased with increasing flow rate. Granulocyte elastase release increased slightly with increasing flow rate up to 300 s-1. Complement activation was greatest for PVC at 1000 s-1, greatest for nylon at 100 s-1, but there was no measurable difference at either rate for silicone. All samples caused an increase in clotting time with increasing wall shear rate. PVC was the most platelet compatible material, nylon the worst. Silicone caused least contact phase activation, PVC and nylon the most.

The use of flow perfusion culture and subcutaneous implantation with fibroblast-seeded PLLA-collagen 3D scaffolds for abdominal wall repair

Highly cellularised 3D-tissue constructs designed to repair large, complex abdominal wall defects were prepared using poly (lactic acid) (PLLA)-collagen scaffolds in vitro using a flow perfusion bioreactor. The PLLA-collagen scaffolds had a unique structure consisting of a collagen sponge formed within the pores of a mechanically stable knitted mesh of PLLA. The effect of the flow perfusion bioreactor culturing conditions was investigated in vitro for 0, 7, 14 and 28 days on scaffolds seeded with dermal fibroblasts. The cultured constructs were subsequently studied subcutaneously (SC) in an in vivo animal model. The results of in vitro studies demonstrated that the perfusion system facilitated increased cell proliferation and homogenous distribution in the PLLA-collagen scaffolds compared to static conditions. A highly cellularised 3D-tissue construct was formed by 7 days incubation under perfusion conditions, with increased cellularity by the 28 day time point. The in vivo model demonstrated that implanting constructs with high cellularity resulted in exceptional cell stabilisation, with the survival of implanted cells and expression of the phenotypically-relevant extracellular matrix proteins collagen types I and III, studied by fluorescence in situ hybridisation (FISH) and immunohistochemistry. The implantation of this porous PPLA-collagen scaffold seeded with dermal fibroblasts following in vitro maturation using a flow perfusion bioreactor system suggests a significant advance over current state-of-the-art procedures for the reconstruction of large, complex abdominal wall tissue defects.

Derivation and performance of an entirely autologous injectable hydrogel delivery system for cell-based therapies

A host-derived hydrogel has been designed and validated as an entirely autologous, injectable delivery system for cells with potential for cell-based therapies and tissue engineering applications. Each individual has components in their blood from which can be formed a mechanically stable hydrogel having the capacity to maintain cellular phenotype and support cellular proliferation of multiple cell types through several culture passages ex vivo. The hydrogel can be triggered to gel at the time of implantation into the patient through an injection system that facilitates a liquid injection of components of the donor plasma and cells into the site of interest. This results in stable ectopic tissue formation at the site of implantation. Our studies have demonstrated excellent integration of the neotissue with host tissues with maintenance of the phenotype of implanted cells whilst observing minimal host innate immune cell recruitment. These findings could provide the fundamental basis for new hydrogel-based biomaterial therapies, overcoming the histocompatibility factors associated with implantable biomaterials whilst providing a stable three dimensional medium for cellular growth both in vivo and ex vivo.