Karen M. Haberstroh

Endothelial and vascular smooth muscle cell function on poly(lactic-co-glycolic acid) with nano-structured surface features.

Biomaterials that successfully integrate into surrounding tissue should match not only the tissues mechanical properties, but also its topography. The cellular response to a biomaterial may be enhanced in synthetic polymer formulations by mimicking the surface roughness created by the associated nano-structured extra-cellular matrix components of natural tissue. As a first step towards this endeavor, the goal of the present in vitro study was to use these design parameters to develop a synthetic, nano-structured, polymeric biomaterial that promotes cell adhesion and growth for vascular applications. In a novel manner, poly(lactic-co-glycolic acid) (PLGA) (50/50wt% mix) was synthesized to possess a range (from micron to nanometer) of surface features. Reduction of surface features was accomplished by treating conventional PLGA with various concentrations of NaOH for select periods of time. Results from cell experiments indicated that, compared to conventional PLGA, NaOH treated PLGA enhanced vascular smooth muscle cell adhesion and proliferation. However, PLGA prepared by soaking in NaOH decreased endothelial cell adhesion and proliferation compared to conventional PLGA. After further investigation, this finding was determined to be a result of chemical (and not topographical) changes during polymer synthesis. Surface chemistry effects were removed while retaining nano-structured topography by using polymer/elastomer casting methods. Results demonstrated that endothelial and smooth muscle cell densities increased on nano-structured cast PLGA. For these reasons, the present in vitro study provided the first evidence that nano-structured surface features can significantly improve vascular cell densities; such design criteria can be used in the synthesis of the next-generation of more successful tissue-engineered vascular grafts.

Selective bone cell adhesion on formulations containing carbon nanofibers.

Bone cell adhesion on novel carbon nanofibers and polycarbonate urethane/carbon nanofiber (PCU/CNF) composites is investigated in the present in vitro study. Carbon nanofibers have exceptional theoretical mechanical properties (such as high strength to weight ratios) that, along with possessing nanoscale fiber dimensions similar to crystalline hydroxyapatite found in physiological bone, suggest strong possibilities for use as an orthopedic/dental implant material. The effects of select properties of carbon fibers (specifically, dimension, surface energy, and chemistry) on osteoblast, fibroblast, chondrocyte, and smooth muscle cell adhesion were determined in the present in vitro study. Results provided evidence that smaller-scale (i.e., nanometer dimension) carbon fibers promoted osteoblast adhesion. Adhesion of other cells was not influenced by carbon fiber dimensions. Also, smooth muscle cell, fibroblast, and chondrocyte adhesion decreased with an increase in either carbon nanofiber surface energy or simultaneous change in carbon nanofiber chemistry. Moreover, greater weight percentages of high surface energy carbon nanofibers in the PCU/CNF composite increased osteoblast adhesion while at the same time decreased fibroblast adhesion.

Nanostructured Polymer/Nanophase Ceramic Composites Enhance Osteoblast and Chondrocyte Adhesion

Osteoblast (bone-forming cell) and chondrocyte (cartilage-synthesizing cell) adhesion on novel nanostructured polylactic/glycolic acid (PLGA) and titania composites were investigated in the present in vitro study. Nanostructured polymers were created by chemically treating micron-structured PLGA with select concentrations of NaOH for various periods of time. Dimensions of ceramics were controlled by utilizing either micron or nanometer grain size titania. Compared with surfaces with conventional or micron surface roughness dimensions, results provided the first evidence of increased osteoblast and chondrocyte adhesion on 100 wt% PLGA films with nanometer polymer surface roughness dimensions. Results also confirmed other literature reports of enhanced osteoblast adhesion on 100 wt% nanometer compared with conventional grain size titania compacts; however, the present study provided the first evidence that decreasing titania grain size into the nanometer range did not influence chondrocyte adhesion. Finally, osteoblast and chondrocyte adhesion increased on 70/30 wt% PLGA/titania composites formulated to possess nanosurface rather than conventional surface feature dimensions. The present study, thus, provided evidence that these nanostructured PLGA/titania composites may possess the ability to simulate surface and/or chemical properties of bone and cartilage, respectively, to allow for exciting alternatives in the design of prostheses with greater efficacy.

Nano-structured polymers enhance bladder smooth muscle cell function

It is the hypothesis of the present study that a biocompatible material which mimics the nanometer topography of native bladder tissue will enhance cellular responses and lead to better tissue integration in vivo. Previous in vitro studies have verified the ability to successfully reduce the surface feature dimensions of poly(lactic-co-glycolic acid) (PLGA) and poly(ether urethane) (PU) films into the nanometer regime via chemical etching procedures. Results from these studies also provided the first evidence that bladder smooth muscle cell adhesion was enhanced on chemically treated nano-structured polymeric surfaces compared to their conventional counterparts. Although cell adhesion is necessary for a biomaterials success, subsequent cell functions (such as long-term cell growth and proliferation) are also critical for tissue ingrowth and long-term implant survival. The present in vitro study, therefore, investigated the function of bladder smooth muscle cells on these novel, nano-structured polymers over the expanded periods of 1, 3 and 5 days. Results indicated that cell number was influenced by both surface roughness and surface chemistry changes; the important contributor, however, was increased nanometer surface roughness. This claim is supported by the fact that cell number was enhanced on nano-structured compared to conventional PLGA and PU once chemistry changes were eliminated using casting techniques.

Comparison of the boundary‐lubricating ability of bovine synovial fluid, lubricin, and Healon

Purified human umbilical hyaluronate and a commercial preparation of rooster comb hyaluronate (Healon) intended for intra-articular viscosupplementation did not demonstrate the same degree of boundary-lubricating ability as bovine synovial fluid or its purified lubricating mucin, lubricin (p < 0.01). Boundary lubrication was measured in vitro in an arthrotripsometer oscillating natural latex against polished glass under a load of 0.35 MPa with an entraining velocity of 0.37 mm/s. The two hyaluronate solutions possessed the same hyaluronate concentration as synovial fluid, but Healon was 4.5 times more viscous. Present practice of viscosupplementation therapy for degenerative joint disease is limited and fails to implicate the important role of synovial mucin. Boundary lubrication provided by synovial mucin, independent of its viscosity, is not replicated by hyaluronate hydrogels.

Enhanced functions of osteoblasts on nanostructured surfaces of carbon and alumina

It is of the utmost importance to increase the activity of bone cells on the surface of materials used in the design of orthopaedic implants. Increased activity of such cells can promote either integration of these materials into surrounding bone or complete replacement with naturally produced bone if biodegradable materials are used. Osteoblasts are bone-producing cells and, for that reason, are the cells of interest in initial studies of new orthopaedic implants. If these cells are functioning normally, they lay down bone matrix onto both existing bone and prosthetic materials implanted into the body. It is generally accepted that a successful material should enhance osteoblast function, leading to more bone deposition and, consequently, increased strength of the interface between the material and juxtaposed bone. The present study provided the first evidence of greater osteoblast function on carbon and alumina formulations that mimic the nano-dimensional crystal geometry of hydroxyapatite found in bone.

The effects of sustained hydrostatic pressure on select bladder smooth muscle cell functions

PURPOSE Normal bladder development is believed to depend on the active work of the bladder for storing and expelling urine. When high urinary diversion is performed in infants and the bladder no longer undergoes normal filling, bladder development may be altered, ultimately resulting in bladder dysfunction. To help better understand this relationship of bladder function with growth at the cellular level we developed a novel laboratory method for applying hydrostatic pressure to cell cultures, and we characterized the response of neonatal bladder smooth muscle cells to physiological levels of sustained hydrostatic pressure. MATERIALS AND METHODS Neonatal ovine smooth muscle cells staining positive for desmin and alpha-smooth muscle actin were exposed to pressures of 0.3 (controls), 2, 4, 6 and 8.5 cm. water for 1, 3, 5 and 7 days. At the end of the experiments the cells were fixed, stained and counted. Mitogenic activity of the supernatant media from bladder smooth muscle cells exposed to 8.5 cm. water for 5 days (conditioned media) was tested before and after treatments of heating, freezing, passing through a heparin-sepharose affinity chromatography column or after the addition of suramin, a nonspecific growth factor inhibitor. Statistical analysis was performed using Students t test with p <0.05 considered statistically significant. RESULTS Exposure of bladder smooth muscle cells to sustained hydrostatic pressures of 4, 6 and 8.5 cm. water resulted in increased cell proliferation. Differences became statistically significant (p <0.05) by day 5. Also, conditioned media contained mitogenic activity that was ablated by heating, freezing, passage through a heparin-sepharose affinity chromatography column or with the addition of suramin. CONCLUSIONS We have demonstrated a proliferative response of neonatal bladder smooth muscle after exposure to physiological levels of sustained hydrostatic pressure. This response is partially due to 1 or more transferable mitogenic factors. These data support the hypothesis that pressure associated with bladder filling is an important stimulus for detrusor development.

Improved osteoblast viability in the presence of smaller nanometre dimensioned carbon fibres

Carbon nanofibres have been proposed as a possible new orthopaedic/dental implant material due to their unique mechanical, electrical, and cytocompatibility properties. Specifically, these fibres have dimensions (diameters ranging between 60 and 200?nm and aspect ratios of about 500) similar to hydroxyapatite crystals and collagen fibres found in bone. More importantly, previous in vitro studies have provided evidence that nanophase (?nm diameter) carbon fibres enhance osteoblast (the bone-producing cell) function over conventional (>100?nm diameter) carbon fibres and current orthopaedic implant materials such as titanium, Ti6Al4V, and CoCrMo. However, articulating components of orthopaedic implant materials may generate harmful wear debris. To determine, for the first time, the influence of carbon nanofibre wear debris on osteoblast viability, direct contact toxicity studies were performed in the present in vitro study. Not surprisingly, the results from direct-contact toxicity studies over a 24?h time period provided evidence of time-?and concentration-dependent cell viability decreases when exposed to carbon nanofibres. Most importantly, the results from this study provided the first evidence that nanophase carbon fibres were less detrimental to osteoblast viability compared to larger diameter conventional carbon fibres. For this reason, this in vitro study provided continuing evidence of the promise of nanophase materials (particularly, carbon nanofibres) in improving orthopaedic implant efficiency.

The role of polymer nanosurface roughness and submicron pores in improving bladder urothelial cell density and inhibiting calcium oxalate stone formation

Synthetic polymers have been proposed for replacing resected cancerous bladder tissue. However, conventional (or nanosmooth) polymers used in such applications (such as poly(ether) urethane (PU) and poly-lactic-co-glycolic acid (PLGA)) often fail clinically due to poor bladder tissue regeneration, low cytocompatibility properties, and excessive calcium stone formation. For the successful reconstruction of bladder tissue, polymer surfaces should be modified to combat these common problems. Along these lines, implementing nanoscale surface features that mimic the natural roughness of bladder tissue on polymer surfaces can promote appropriate cell growth, accelerate bladder tissue regeneration and inhibit bladder calcium stone formation. To test this hypothesis, in this study, the cytocompatibility properties of both a non-biodegradable polymer (PU) and a biodegradable polymer (PLGA) were investigated after etching in chemicals (HNO(3) and NaOH, respectively) to create nanoscale surface features. After chemical etching, PU possessed submicron sized pores and numerous nanometer surface features while PLGA possessed few pores and large amounts of nanometer surface roughness. Results from this study strongly supported the assertion that nanometer scale surface roughness produced on PU and PLGA promoted the density of urothelial cells (cells that line the interior of the bladder), with the greatest urothelial cell densities observed on nanorough PLGA. In addition, compared to respective conventional polymers, the results provided evidence that nanorough PU and PLGA inhibited calcium oxalate stone formation; submicron pored nanorough PU inhibited calcium oxalate stone formation the most. Thus, results from the present study suggest the importance of nanometer topographical cues for designing better materials for bladder tissue engineering applications.

Non-Uniform Flow Behavior in a Parallel Plate Flow Chamber Alters Endothelial Cell Responses

Arterial flow characteristics determine vessel health by modulating vascular endothelial cells. One system used to study these interactions is the parallel plate flow chamber. The present in vitro study quantified the uniformity of fluid flow across a parallel plate flow chamber and characterized plate-location dependent endothelial cell gene expression. More specifically, shear stress varied by as much as 11% across the chamber area, which caused non-uniform ecNOS (p < 0.05) and COX-2 (p < 0.05) mRNA expression across the plate area. Results herein suggest that chamber variations may result during construction or assembly, which ultimately affect flow-sensitive cell responses (including mRNA expression). Therefore, these limitations should be considered when reporting endothelial cell responses to fluid flow using parallel plate flow chambers.