John A. Hunt

The biocompatibility of novel starch-based polymers and composites: in vitro studies.

Studies with biodegradable starch-based polymers have recently demonstrated that these materials have a range of properties. which make them suitable for use in several biomedical applications, ranging from bone plates and screws to drug delivery carriers and tissue engineering scaffolds. The aim of this study was to screen the cytotoxicity and evaluate starch-based polymers and composites as potential biomaterials. The biocompatibility of two different blends of corn-starch, starch ethylene vinyl alcohol (SEVA-C) and starch cellulose acetate (SCA) and their respective composites with hydroxyapatite (HA) was assessed by cytotoxicity and cell adhesion tests. The MTT assay was performed with the extracts of the materials in order to evaluate the short-term effect of the degradation products. The cell morphology of L929 mouse fibroblast cell line was also analysed after direct contact with polymers and composites for different time periods and the number of cells adhered to the surface of the polymers was determined by quantification of the cytosolic lactate dehydrogenase (LDH) activity. Both types of starch-based polymers exhibit a cytocompatibility that might allow for their use as biomaterials. SEVA-C blends were found to be the less cytotoxic for the tested cell line, although cells adhere better to SCA surface. The cytotoxicity test also revealed that SCA and SEVA-C composites have a similar response to the one obtained for SCA polymer. Scanning electron microscopy (SEM) analysis showed that cells were much more spread on the SCA polymer and LDH measurements showed a higher number of cells on this surface.

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.

Variation of Postprandial Plasma Glucose, Palatability, and Symptoms Associated With a Standardized Mixed Test Meal Versus 75 g Oral Glucose

OBJECTIVE To compare within-subject variability of plasma glucose measured 2 h after a glucose tolerance test (GTT) with that of plasma glucose measured 2 h after administration of a standardized test meal (diabetes screening product [DSP], Ceapro, Edmonton, Alberta, Canada) and to determine the relationship between the two sets of plasma glucose measurements. RESEARCH DESIGN AND METHODS Plasma glucose and insulin responses of 36 overnight-fasted subjects (10 lean normal, 9 obese normal, 9 with impaired glucose tolerance [IGT], and 8 with mild diabetes) were studied on eight different mornings after they consumed 75 g oral glucose or 50 g carbohydrate from the DSP. Each test meal was repeated four times by each subject. Within-subject coefficients of variation (CVs) (CV = 100 × SD/mean) of plasma glucose concentrations 2 h after administration of the GTT and DSP were compared by repeated measures ANOVA and linear regression analysis. RESULTS Mean plasma glucose 2 h after administration of the DSP (D) was linearly related to that 2 h after the GTT (G): G = 1.5 × D − 1.6 (r = 0.97, P < 0.0001). The CV of 2-h plasma glucose was significantly lower after administration of the DSP, 10.5 ± 1.0%, than after the GTT, 12.7 ± 1.18% (P = 0.025). The effect of test meal on CV differed in different groups of subjects (P = 0.018), with the largest difference found in IGT subjects, in whom the CV after DSP administration was 47% < after the GTT (P = 0.0005). The DSP was significantly more palatable and produced fewer adverse symptoms than the GTT. CONCLUSIONS Plasma glucose concentrations measured 2 h after DSP administration are closely related to those measured 2 h after the GTT but are more consistent than the 2-h post-GTT concentrations within the critical IGT range. This finding suggests that measurement of plasma glucose 2 h after administration of the DSP may allow more precise discrimination among normal glucose levels, IGT, and diabetes than measurement of plasma glucose 2 h after the GTT.

Effects of plasma treated PET and PTFE on expression of adhesion molecules by human endothelial cells in vitro

The aim of this study was to evaluate the expression of adhesion molecules on the surface of human endothelial cells in response to the systematic variation in materials properties by the ammonia plasma modification of polyethylene terephthalate (PET) and polytetrafluorethylene (PTFE). These adhesion molecules act as mediators of cell adhesion, play a role in the modulation of cell adhesion on biomaterials and therefore condition the response of tissues to implants. First and second passage human umbilical vein endothelial cells (HUVECs) were cultured on plasma treated and untreated PET and PTFE. HUVECs grown on polystyrene tissue culture coverslips and HUVECs stimulated with tumour necrosis factor (TNF-alpha) were used as controls. After 1 day and 7 days, the expression of adhesion molecules platelet endothelial cell adhesion molecule-1 (PECAM-1), intercellular adhesion molecule-1 (ICAM-1), Integrin alphavbeta3, vascular cell adhesion molecule-1 (VCAM-1), E-selectin, P-selectin and L-selectin were evaluated using flow cytometry and immunohistochemistry. There was a slight increase in positive cell numbers expressing the adhesion molecules ICAM-1 and VCAM-1 on plasma treated PET and PTFE. A significant increase in E-selectin positive cells on untreated PTFE was demonstrated after 7 days. Stimulation with TNF-alpha demonstrated a significant increase in the proportion of ICAM-1. VCAM-1 and E-selectin positive cells. Almost all cells expressed PECAM-1 and integrin alphavbeta3, on both materials and controls but did not express P- and L-selectin on any surface. When second passage cells were used, the expression of the adhesion molecules ICAM-1 and VCAM-1 was markedly increased on all surfaces but not with TNF-alpha. These significant differences were not observed in other adhesion molecules. These results were supported by immunohistochemical studies. The effects of plasma treated PET and PTFE on cell adhesion and proliferation was also studied. There was a 1.3-fold increase in cell numbers adhered on ammonia plasma treated PET compared to untreated PET and a 5.5-fold increase in cell numbers on treated PTFE compared to untreated PTFE after 1 day. This is significantly different when analysed statistically. After 7 days, cell number increased significantly on all surfaces compared to 1 day, except for untreated PTFE which conversely reduced by 41%. Cell number on the surface of untreated PET was no different to treated PET on days 1 and 7 when second passage cells were used. The study has shown that the plasma treatment of PET and PTFE with ammonia improves the adhesion and growth of endothelial cells and slightly upregulates the expression of adhesion molecules. This surface modification should promote colonisation of an artificial vascular prosthesis by endothelial cells and make it less vulnerable to immune system cells of the recipient. In addition, it should be considered which passage of cells is used due to the different adhesion features of different passages of HUVECs on untreated PET.

Quantitative assessment of the response of primary derived human osteoblasts and macrophages to a range of nanotopography surfaces in a single culture model in vitro

The effect of nanotopography on a range of Ti oxide surfaces was determined. Flat Ti, 3%, 19%, 30% and 43% topography densities of 110 nm high hemispherical protrusions were cultured in contact with primary derived human macrophages and osteoblasts in single culture models. Prior to introduction of the test substrate the phenotype and optimum conditions for in vitro cell culture were established. The cellular response was investigated and quantified by assessments of cytoskeletal development and orientation, viable cell adhesion, cytokine production and release and RT-PCR analysis of osteogenic markers. The tested nanotopographies did not have a statistically significant effect on viable cell adhesion and subsequent cytoskeletal formation. Surface chemistry was the dominant factor as established via incorporation of a tissue culture polystyrene, TCPS, control. The topography surfaces induced a release of chemotactic macrophage activation agents at 1 day in conjunction with stress fibre formation and a subsequent fibronectin network formation. Osteoblasts migrated away from the topography surfaces to the exposed TCPS within the wells during the 7-day period.

Hydrogels for tissue engineering and regenerative medicine

Injectable hydrogels have become an incredibly prolific area of research in the field of tissue engineering and regenerative medicine, because of their high water content, mechanical similarity to natural tissues, and ease of surgical implantation, hydrogels are at the forefront of biomedical scaffold and drug carrier design. The aim of this review is to concisely summarise current state-of-the-art in natural and synthetic hydrogels with respect to their synthesis and fabrication, comparing and contrasting the many chemistries available for biomedical hydrogel generation using both biologic and synthetic base materials. We then discuss these hydrogels in the specific instance of several pertinent areas of TERM which have been specifically selected to demonstrate how this versatile class of materials can be modified to augment damage and disease of a seemingly limitless array of adult tissues.

Effect of biomaterial surface charge on the inflammatory response: evaluation of cellular infiltration and TNF alpha production.

A rat model was used to investigate the effect of net surface charge on polymer biocompatibility and its potential to modify and stimulate the inflammatory response. Poly(ether)urethane was taken as the base material and the net charge altered by introducing sulphonate ionic groups to the polymer backbone. Three differently charged poly(ether)urethanes were made with 10, 20, and 30% sulphonate substitution, giving a range of negative charge, with unmodified poly(ether)urethane used as a control. The polymers were implanted intramuscularly into rats for 2 days, and for 1, 2, and 12 weeks. After explantation, the cellular infiltration in the tissue surrounding the implants was evaluated using immunohistochemistry to stain for specific cell types: macrophages, neutrophils, lymphocytes, and the cytokine TNF alpha. In situ hybridization was used to detect expression of mRNA encoding TNF alpha. Stained sections were analyzed and the cellular response quantified using image analysis. Initially macrophages and neutrophils were observed around all the materials, but neutrophils were absent in all samples at 12 weeks. The 2-day time point had significantly more macrophages than the later time points. By 2 weeks the 20%-charged polymer elicited significantly less neutrophil infiltration than the other three polymers. In all samples where macrophages were observed, cells staining positive fore TNF alpha protein and message also were observed. No T or B lymphocytes were observed in the infiltrates around the materials at any time point. The results indicate that surface charge can influence the early phase acute inflammatory response to an implanted material.

Introducing dip pen nanolithography as a tool for controlling stem cell behaviour: unlocking the potential of the next generation of smart materials in regenerative medicine.

Reproducible control of stem cell populations, regardless of their original source, is required for the true potential of these cells to be realised as medical therapies, cell biology research tools and in vitro assays. To date there is a lack of consistency in successful output when these cells are used in clinical trials and even simple in vitro experiments, due to cell and material variability. The successful combination of single chemistries in nanoarray format to control stem cell, or any cellular behaviour has not been previously reported. Here we report how homogenously nanopatterned chemically modified surfaces can be used to initiate a directed cellular response, particularly mesenchymal stem cell (MSC) differentiation, in a highly reproducible manner without the need for exogenous biological factors and heavily supplemented cell media. Successful acquisition of these data should lead to the optimisation of cell selective properties of materials, further enhancing the role of nanopatterned substrates in cell biology and regenerative medicine. The successful design and comparison of homogenously molecularly nanopatterned surfaces and their direct effect on human MSC adhesion and differentiation are reported in this paper. Planar gold surfaces were patterned by dip pen nanolithography (DPN) to produce arrays of nanodots with optimised fixed diameter of 70 nanometres separated by defined spacings, ranging from 140 to 1000 nm with terminal functionalities of simple chemistries including carboxyl, amino, methyl and hydroxyl. These nanopatterned surfaces exhibited unprecedented control of initial cell interactions and subsequent control of cell phenotype and offer significant potential for the future.

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.

Biological responses to hydroxyapatite surfaces deposited via a co-incident microblasting technique

Hydroxyapatite (HA) is routinely used as a coating on a range of press-fit (cementless) orthopaedic implants to enhance their osseointegration. The standard plasma spraying method used to deposit a HA surface layer on such implants often contains unwanted crystal phases that can lead to coating delamination in vivo. Consequently, there has been a continuous drive to develop alternate surface modification technologies that can eliminate the problems caused by a non-optimal coating process. In this study two methods for creating a HA layer on metal alloys that employ micro-blasting have been evaluated to determine if the inclusion of an abrasive agent can enhance the in vitro and in vivo performance of the modified surface. The first method employs direct micro-blasting using HA as the abrasive media, while the second employs a simultaneous blasting with an alumina abrasive and coincident blasting with HA as a dopant. Whereas, both methods were found to produce a surface which was enriched with HA, the respective microstructures created were significantly different. Detailed surface characterisation revealed that the use of the abrasive produced disruption of the metal surface without producing detectable incorporation of alumina particles. Roughening of the metal surface in this way breached the passivating oxide layer and created sites which subsequently provided for impregnation, mechanical interlocking and chemical bonding of HA. The co-incident use of an alumina abrasive and a HA dopant resulted in a stable surface that demonstrated enhanced in vitro osteoblast attachment and viability as compared to the response to the surface produced using HA alone or the metal substrate control. Implantation of the surface produced by co-incident blasting with alumina and HA in a rabbit model confirmed that this surface promoted the in vivo formation of early stage lamellar bone growth.