Incomplete conversion, an ongoing challenge facing photopolymerized methacrylate-based polymers, affects leachables as well as the resulting polymer network. As novel polymers and composites are developed, methods to efficiently screen cell response to these materials and their properties, including conversion, are needed. In this study, an in vitro screening methodology was developed to assess cells cultured directly on cross-linked polymer networks. A gradient in methacrylate double bond conversion was used to increase the experimental throughput. A substrate of 2,2-bis[4-(2-hydroxy-3-methacryloxypropoxy)phenyl] propane (BisGMA) and triethylene glycol dimethacrylate (TEGDMA) was prepared with a conversion ranging from 43.0% to 61.2%. Substrates aged for 7 days had no significant differences in surface roughness or hydrophilicity as a function of conversion. Leachables were detectable for at least 7 days using UV absorption, but their global cytotoxicity was insignificant after 5 days of aging. Thus, RAW 264.7 macrophage-like cells were cultured on aged substrates to evaluate the cell response to conversion, with possible contributions from the polymer network and local leachables. Conversions of 45% and 50% decreased viability (via calcein/ethidium staining) and increased apoptosis (via annexin-V staining). No significant changes (p>0.05) in tumor necrosis factor-α and interleukin-1β gene expression, as measured by quantitative, real-time reverse transcription-polymerase chain reaction, were seen as conversion increased. Thus, conversions greater than 50% are recommended for equimolar BisGMA/TEGDMA. The ability to distinguish cell response as a function of conversion is useful as an initial biological screening platform to optimize dental polymers.

Incomplete conversion, an ongoing challenge facing photopolymerized methacrylate-based polymers, affects leachables as well as the resulting polymer network. As novel polymers and composites are developed, methods to efficiently screen cell response to these materials and their properties, including conversion, are needed. In this study, an in vitro screening methodology was developed to assess cells cultured directly on cross-linked polymer networks. A gradient in methacrylate double bond conversion was used to increase the experimental throughput. A substrate of 2,2-bis[4-(2-hydroxy-3-methacryloxypropoxy)phenyl] propane (BisGMA) and triethylene glycol dimethacrylate (TEGDMA) was prepared with a conversion ranging from 43.0% to 61.2%. Substrates aged for 7 days had no significant differences in surface roughness or hydrophilicity as a function of conversion. Leachables were detectable for at least 7 days using UV absorption, but their global cytotoxicity was insignificant after 5 days of aging. Thus, RAW 264.7 macrophage-like cells were cultured on aged substrates to evaluate the cell response to conversion, with possible contributions from the polymer network and local leachables. Conversions of 45% and 50% decreased viability (via calcein/ethidium staining) and increased apoptosis (via annexin-V staining). No significant changes (p>0.05) in tumor necrosis factor-alpha and interleukin-1beta gene expression, as measured by quantitative, real-time reverse transcription-polymerase chain reaction, were seen as conversion increased. Thus, conversions greater than 50% are recommended for equimolar BisGMA/TEGDMA. The ability to distinguish cell response as a function of conversion is useful as an initial biological screening platform to optimize dental polymers.

We have developed a bioreactor-based millifluidic technique that allows for dynamic culture conditions and measurement of the fluid flow impinging upon a three-dimensional tissue engineering scaffold. Chondrocytes in scaffolds have been shown to require mechanical stimulation to produce an extracellular matrix that resembles native cartilage. This study investigates the effect of pulsatile flow on chondrocyte response in a model poly(ethylene glycol) dimethacrylate hydrogel. Bovine chondrocytes were encapsulated in the hydrogel and cultured for 7, 14 and 21 days at pulsatile flow frequencies of 0.5 Hz (15ml/min) and 1.5Hz (17ml/min). The scaffolds cultured under dynamic conditions were compared to those cultured under static (non-flow) conditions. Quantitative real-time reverse transcription polymerase chain reaction was used to quantify collagen type I, collagen type II and aggrecan gene copy numbers as markers for chondrocyte phenotypic expression. Histological sections stained with hematoxylin & eosin, and Alcian blue confirmed chondrocyte morphology and matrix formation. Interestingly, regulation of the collagen type II gene was particularly sensitive to the flow conditions. The understanding of the cell response to encapsulation and flow could be used to identify the appropriate culture conditions necessary to design and develop hydrogel carriers to promote the formation of extracellular matrix as well as to further our knowledge of chondrocyte mechanobiology.

This study investigated four different connective tissue cell types to determine which cell type should be the source for seeding a tissue-engineered anterior cruciate ligament (ACL) replacement. Cells derived from the ACL, medial collateral ligament (MCL), achilles tendon (AT), and patellar tendon (PT) of New Zealand White rabbits were isolated and cultured. Each cell type was cultured in vitro after seeding on three-dimensional (3-D) braided polymer scaffolds and on tissue culture polystyrene that served as a control. Samples were evaluated and compared for their morphology, proliferation, and gene expression of fibronectin, type I and type III collagen. Scanning electron microscopy (SEM) photomicrographs verified cell attachment of all four types of connective tissue fibroblasts to the scaffolds. Preliminary results comparing proliferation indicate that cells obtained from the PT and AT have the fastest proliferation. Whereas gene expression of the phenotypic markers measured using real-time reverse transcription polymerase chain reaction (RT-PCR) indicates ACL cells have the highest gene expression for the matrix markers. This leads to the question of which cell type should be the cell source for tissue-engineering of ligament, the highly proliferating cells or the differentiated matrix producing cells. This study would suggest that ACL differentiated matrix producing cells are the most suitable cells for further study and development of a tissue-engineered ligament.

This study presented a well-characterized biocompatibility profile of dental bonding agents in a mouse macrophage in vitro model. The cellular response to four different formulations of dental bonding resins and the cell viability was determined. Materials were prepared by photopolymerization and the unreacted monomers were extracted in a buffered medium. Murine macrophages were incubated in the extract medium for 24 h. Cellular viability was assessed by fluorescence microscopy and Wst-1 assay, while flow cytometry was used to quantify the apoptotic response. As an indicator of inflammatory responses, real-time polymerase chain reaction was utilized to quantify elevated cytokine production. These responses were monitored by quantifying the levels of tumor necrosis factor alpha (TNFα) and interleukin-1 β (IL-1β) produced by the cells. Murine macrophage cells exposed to the unfilled resin systems containing glycidyl ether of bisphenol A (Bis-GMA) and 2-hydroxyethyl methacrylate (HEMA) had the most adverse response and cells exposed to the filled Bis-GMA and HEMA resin system were the most viable at photopolymerization times. Other resin systems displayed intermediate levels of viability when compared with the untreated control. The levels of IL-1β were significantly elevated in all samples.

The biocompatibility of metallic alloys is critical to the success of many orthopedic therapies. Corrosion resistance and the immune response of the body to wear debris products ultimately determine the performance of these devices. The establishment of quantitative tests of biocompatibility is an important issue for biomaterials development. We have developed an in vitro model to measure the pro-inflammatory cytokine production and in this study investigated the cellular responses induced by nitrogenated and 316L stainless steel alloys in both particulate and solid form. We utilized a murine macrophage cell line, RAW 264.7, to characterize and compare the mRNA profiles of TNF-α and IL-1β in these cells using real time-polymerase chain reaction (RT-PCR). Fluorescence microscopy and flow cytometry were used to probe the viability of the population and to examine the apoptotic pathway. The goals of this work were to develop improved measurement methods for the quantification of cellular inflammatory responses to biomaterials and to obtain data that leads to an enhanced understanding of the ways in which the body responds to biomaterials. Using these techniques, we observed evidence for an association between the upregulation of IL-1β and reversible apoptosis, and the upregulation of TNF-α and irreversible apoptosis.

Two-dimensional thin films consisting of homopolymer and discrete compositional blends of tyrosine-derived polycarbonates were prepared and characterized in an effort to elucidate the nature of different cell responses that were measured in vitro. The structurally similar blends were found to phase separate after annealing with domain sizes dependent on the overall composition. The thin polymer films were characterized with the use of atomic force microscopy (AFM), water contact angles, and time-of-flight secondary ion mass spectrometry (TOF-SIMS) and significant changes in roughness were measured following the annealing process. Genetic expression profiles of interleukin-1beta and fibronectin in MC3T3-E1 osteoblasts and RAW 264.7 murine macrophages were measured at several time points, demonstrating the time and composition-dependent nature of the cell responses. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) depicted upregulation of the fibronectin gene copy numbers in each of the blends relative to the homopolymers. Moreover, the interleukin-1beta expression profile was found to be compositionally dependent. The data suggest strongly that optimal composition and processing conditions can significantly affect the acute inflammatory and extracellular matrix production responses.

INTRODUCTION Tissue engineering applications involve developing materials to guide cellular response in the repair of missing or damaged tissue. We have been developing combinatorial methods for characterizing the functional dependence of cellular responses on material properties. Two strategies were pursued: gradient libraries, where samples are prepared with systematic variations in material properties, and combinatorial libraries, where concepts of fulland partial-factorial design are used to screen the interplay between biochemical, material, and cellular variables. A brief overview of our work in these areas will be presented. Cellular responses to gradient libraries of polymer crystallinity, blend composition, and surface energy have been investigated. We have characterized proliferation and matrix production of MC3T3-E1 osteoblasts as a function of material variables and are developing methods for measuring these responses using fluorescence microscopy. Design-of-experiment methods have been used to characterize the interactions of chondrocytes with peptide-functionalized alginates. Using these methods we have also characterized the influence of growth factors known to be important in cartilage development in vitro. The influences of insulin-like growth factor-I (IGFI), transforming growth factorβ (TGF-β), and basic fibroblast growth factor (bFGF) were investigated as a function of alginate functionalization and cell density.

The introduction of tissue engineering strategies for the repair and replacement of human body components extends the application and importance of biomaterials. Implanted biomaterials frequently evoke inflammatory responses that are complex and not well understood at present. The goals of this work were to develop improved measurement methods for the quantification of cellular inflammatory responses to biomaterials and obtain data that lead to an enhanced understanding of the ways in which the body responds to the introduction of biomaterials. To evaluate the biocompatibility of materials, we established a system that allows for the analysis and quantitation of cellular inflammatory responses in vitro. In this study, the inflammatory responses of murine macrophages (RAW 264.7) were analyzed. The cells were incubated with polymethylmethacrylate (PMMA) microspheres in the presence and absence of lipopolysaccharide (LPS) at 8 and 18 h. The analysis of the genetic material obtained from the cells was quantitated using real-time reverse transcription polymerase chain reaction (RT-PCR). The cell populations treated with LPS or PMMA microspheres singly resulted in an elevation of cytokine levels compared to the untreated control. LPS resulted in a 258-fold increase, while PMMA resulted in an 87.9-fold increase at 8 h. RAW 264.7 cells incubated with LPS and PMMA particles demonstrated a synergistic effect by producing a marked increase in the level of cytokine expression, 336-fold greater than that of the untreated control at 8 h. Fluorescence microscopy studies that assessed cellular viability were also performed and are consistent with the RT-PCR results.

The conjugation of the protein transduction domain (PTD) from the HIV-1 Tat protein to shell cross-linked (SCK) nanoparticles is a method to facilitate cell surface binding and transduction. In the previous report, the preparation, derivatization, and characterization of peptide-functionalized SCK nanoparticles were reported in detail. Following assembly, the constructs were evaluated in vitro and in vivo to obtain a preliminary biocompatibility assessment. The effects of SCK exposure on cell viability were evaluated using a metabolic 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and a fluorescent apoptosis assay. Furthermore, stages of apoptosis were quantified by flow cytometry. Although higher levels of peptide functionalization resulted in decreased metabolic function as measured by MTT assay, significant apoptosis was not observed below 500 mg/L for all the samples. To evaluate the potential immunogenic response of the peptide-derivatized constructs, a real-time polymerase chain reaction (RT-PCR) system that allows for the in vitro analysis and quantification of the cellular inflammatory responses tumor necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL1-beta) was utilized. The inflammatory response to the peptide-functionalized SCK nanoparticles as measured by RT-PCR show statistically significant increases in the levels of both TNF-alpha and IL1-beta relative to tissue culture polystyrene (TCPS). However, the measured cytokine levels did not preclude the further testing of SCKs in an in vivo mouse immunization protocol. In this limited assay, measured increases in immunoglobulin G (IgG) concentration in the sera were minimal with no specific interactions being isolated, and more importantly, none of the mice (>50) subjected to the three 100 microg immunization protocol have died. Additionally, no gross morphological changes were observed in postmortem organ histology examinations.

This study investigated four different connective tissue cell types to determine which cell type should be the source for seeding a tissue- engineered anterior cruciate ligament (ACL) replacement. Cells derived from the ACL, medial collateral ligament (MCL), achilles tendon (AT), and patellar tendon (PT) of New Zealand White rabbits were isolated and cultured. Each cell type was cultured in vitro after seeding on three-dimensional (3-D) braided polymer scaffolds and on tissue culture polystyrene that served as a control. Samples were evaluated and compared for their morphology, proliferation, and gene expression of fibronectin, type I and type III collagen. Scanning electron microscopy (SEM) photomicrographs verified cell attachment of all four types of connective tissue fibroblasts to the scaffolds. Preliminary results comparing proliferation indicate that cells obtained from the PT and AT have the fastest proliferation. Whereas gene expression of the phenotypic markers measured using real-time reverse transcription polymerase chain reaction (RT-PCR) indicates ACL cells have the highest gene expression for the matrix markers. This leads to the question of which cell type should be the cell source for tissue-engineering of ligament, the highly proliferating cells or the differentiated matrix producing cells. This study would suggest that ACL differentiated matrix producing cells are the most suitable cells for further study and development of a tissue-engineered ligament. Published by Elsevier Ltd.