Ex Vivo Toxicity of Commonly Used Topical Antiseptics and Antibiotics on Human Chondrocytes.

Topical povidone-iodine, chlorhexidine, bacitracin, and vancomycin are commonly used antiseptic and antimicrobial agents to reduce risk and treat surgical site infections in numerous orthopedic procedures. Chondrocytes potentially may be exposed to these agents during operative procedures. The impact of these topical agents on chondrocyte viability is unclear. The goal of this study is to determine human chondrocyte viability ex vivo after exposure to commonly used concentrations of these topical antiseptic and antimicrobial agents. Human osteochondral plugs were harvested from the knee joint of a human decedent within 36 hours of death. Individual human osteochondral plugs were exposed to normal saline as a control; a range of concentrations of povidone-iodine (0.25%, 0.5%, and 1%), chlorhexidine (0.01% and 0.5%), and bacitracin (10,000 units/L, 50,000 units/L, and 100,000 units/L) for 1-minute lavage; or a 48-hour soak in vancomycin (0.16 mg/mL, 0.4 mg/mL, and 1.0 mg/mL) with nutrient media. Chondrocyte viability was evaluated with a live/dead viability assay at 0, 2, 4, and 6 days after exposure to bacitracin at 0, 3, and 6 days). Control subjects showed greater than 70% viability at all time points. Povidone-iodine, 0.5% chlorhexidine, and vancomycin showed significant cytotoxicity, with viability dropping to less than 40% by day 6. Chondrocytes exposed to 0.01% chlorhexidine maintained viability. Chondrocytes exposed to bacitracin showed viability until day 3, when there was a large drop in viability. Commonly used topical concentrations of povidone-iodine, vancomycin, and bacitracin are toxic to human chondrocytes ex vivo. A low concentration of chlorhexidine appears safe. Caution should be used when articular cartilage may be exposed to these agents during surgery. [Orthopedics. 2022;45(5):e263-e268.].

How to select the appropriate method(s) of cytotoxicity analysis of mammalian cells at biointerfaces: A tutorial.

Many individuals perform cell viability assays as a measure of biocompatibility whether the focus of their research is on novel drug discovery, development of novel biomedical devices, or the study of biointerfacial phenomena. In this tutorial paper, the most commonly used methods available to users to perform biocompatibility testing are discussed. This includes a brief introduction into the benefits and drawbacks of the techniques, including which are best used as screening assays, which are better suited to experienced users, the relative cost of the assays per unit, and what detection techniques are most appropriate for use in conjunction with the assays. In addition to helping users ensure the rigor and reproducibility of their research design, this tutorial is meant to assist reviewers of interdisciplinary journals (such as Biointerphases itself), whose expertise is in other areas of this research but do not have the experience with cell-based assays themselves.

Exploring the anomalous cytotoxicity of commercially-available poly(N-isopropyl acrylamide) substrates.

Poly(N-isopropyl acrylamide) (pNIPAM) is a stimulus-responsive polymer that has been of great interest to the bioengineering community. When the temperature is lowered below its lower critical solution temperature (∼32 °C), pNIPAM rapidly hydrates, and adherent cells detach as intact cell sheets. This cell-releasing behavior in a physiologically relevant temperature range has led to NIPAM's use for engineered tissues and other devices. In a previous study, however, the authors found that although most techniques used to polymerize NIPAM yield biocompatible films, some formulations from commercially-available NIPAM (cpNIPAM) can be cytotoxic. In this work, the authors investigate the reasons underlying this anomaly. The authors evaluated the response of a variety of cell types (e.g., bovine aortic endothelial cells, BAECs; monkey kidney epithelial cells, Vero cells; and mouse embryonic fibroblasts, 3T3s) after culture on substrates spin-coated with sol-gel (spNIPAM) and commercially-prepared (cpNIPAM). The relative biocompatibility of each cell type was evaluated using observations of its cell morphology and function (e.g., XTT and Live/Dead assays) after 48 and 96 h in culture. In addition, the substrates themselves were analyzed using NMR, goniometry, and XPS. The authors find that all the cell types were compromised by 96 h in culture with cpNIPAM, although the manner in which the cells are compromised differs; in particular, while Vero and 3T3 cells appear to be undergoing cytotoxic death, BAECs undergo apoptic death. The authors believe that this result is due to a combination of factors, including the presence of short chain oligomers of NIPAM in the commercially-available preparation. This work will provide valuable insights into the cytotoxicity of commercially-prepared polymer substrates for this type of bioengineering work and therefore into the applicability of cells grown on such surfaces for human subjects.

Effect of substrate storage conditions on the stability of "Smart" films used for mammalian cell applications.

When poly(N-isopropyl acrylamide) (pNIPAM) is tethered to a surface, it can induce the spontaneous release of a sheet of mammalian cells. The release of cells is a result of the reversible phase transition the polymer undergoes at its lower critical solution temperature (LCST). Many techniques are used for the deposition of pNIPAM onto cell culture substrates. Previously, we compared two methods of deposition (plasma polymerization, and co-deposition with a sol-gel). We proved that although both were technically appropriate for obtaining thermoresponsive pNIPAM films, the surfaces that were co-deposited with a sol-gel caused some disruption in cell activity. The variation of cell behavior could be due to the delamination of pNIPAM films leaching toxic chemicals into solution. In this work, we assessed the stability of these pNIPAM films by manipulating the storage conditions and analyzing the surface chemistry using X-ray photoelectron spectroscopy (XPS) and contact angle measurements over the amount of time required to obtain confluent cell sheets. From XPS, we demonstrated that ppNIPAM (plasma polymerized NIPAM) films remains stable across all storage conditions while sol-gel deposition show large deviations after 48 h of storage. Cell response of the deposited films was assessed by investigating the cytotoxicity and biocompatibility. The 37°C and high humidity storage affects sol-gel deposited films, inhibiting normal cell growth and proper thermoresponse of the film. Surface chemistry, thermoresponse and cell growth remained similar for all ppNIPAM surfaces, indicating these substrates are more appropriate for mammalian cell culture applications.

Optimization of electrospun poly(N-isopropyl acrylamide) mats for the rapid reversible adhesion of mammalian cells.

Poly(N-isopropyl acrylamide) (pNIPAM) is a "smart" polymer that responds to changes in altering temperature near physiologically relevant temperatures, changing its relative hydrophobicity. Mammalian cells attach to pNIPAM at 37 °C and detach spontaneously as a confluent sheet when the temperature is shifted below the lower critical solution temperature (∼32 °C). A variety of methods have been used to create pNIPAM films, including plasma polymerization, self-assembled monolayers, and electron beam ionization. However, detachment of confluent cell sheets from these pNIPAM films can take well over an hour to achieve potentially impacting cellular behavior. In this work, pNIPAM mats were prepared via electrospinning (i.e., espNIPAM) by a previously described technique that the authors optimized for cell attachment and rapid cell detachment. Several electrospinning parameters were varied (needle gauge, collection time, and molecular weight of the polymer) to determine the optimum parameters. The espNIPAM mats were then characterized using Fourier-transform infrared, x-ray photoelectron spectroscopy, and scanning electron microscopy. The espNIPAM mats showing the most promise were seeded with mammalian cells from standard cell lines (MC3T3-E1) as well as cancerous tumor (EMT6) cells. Once confluent, the temperature of the cells and mats was changed to ∼25 °C, resulting in the extremely rapid swelling of the mats. The authors find that espNIPAM mats fabricated using small, dense fibers made of high molecular weight pNIPAM are extremely well-suited as a rapid release method for cell sheet harvesting.

Skin irritation testing of antimicrobial conjugated electrolytes.

Each year, the United States spends about $20 billion to treat people who have been infected with antibiotic resistant bacteria. Even so, the development of new antibiotics has slowed considerably since the mid-20th century. As a result, researchers are looking into developing synthetic compounds and materials with antimicrobial activities such as those made by the Schanze and Whitten groups [ACS Appl. Mater. Interfaces 3, 2820 (2011)]. Previously, they have demonstrated that poly(phenylene ethynylene) (PPE) based electrolytes and oligomeric end-only phenylene ethynylene (EO-OPE) based electrolytes possess strong biocidal activity. However, before the PPE and OPE can be used with humans, skin irritation tests are required to ensure their safety. In this work, in vitro skin assays are used to predict in vivo irritation. Tissues were conditioned for 24 h, exposed to test substances for 1 h, and then tested for viability using colorimetric and cytokine assays. Concentrations up to 50 µg/ml were tested. Viability assays and cytokine (IL-1α) assays demonstrated that the two polymers, three symmetric oligomers, and three "end only" oligomers were nonirritants. In addition, electrospun mats consisting of several promising compounds, including poly(caprolactone), were evaluated. Therefore, all test substances are conservatively classified as nonirritants after a 1 h exposure time period.

Poly(N-isopropyl acrylamide)-coated surfaces: Investigation of the mechanism of cell detachment.

Although there is a great deal of research focused on cell sheet engineering from polymers such as poly(N-isopropyl acrylamide) (pNIPAM), the biocompatibility of pNIPAM surfaces and the nature of cellular detachment from this polymer is still unclear. The most extensive study of the mechanism of detachment proposed a two-step process, with a first (passive) phase involving hydration of pNIPAM chains, and the second (active) phase involving cellular metabolism. However, a number of studies performed successful cell sheet detachment from pNIPAM-grafted surfaces at low temperatures which calls this hypothesis into question. Furthermore, although it has been demonstrated that low-temperature cell sheet detachment using pNIPAM-grafted surfaces is less destructive than other methods of detachment, it has not been investigated if cell sheet detachment removes a portion of pNIPAM from the surfaces as well. It is essential to know if any fragments of the polymer are removed along with the cells, as small polymer fragments could have cytotoxic effects on the cells. This is especially important if these cells are used for the generation of tissues used for transplantation. In this work, the mechanism of cell detachment from pNIPAM coated surfaces is investigated by testing how temperature and presence of an adenosine triphosephase inhibitor affect cellular detachment. Surface initiated atom transfer polymerization (ATRP) was utilized to synthesize thermoresponsive atrpNIPAM surfaces. pNIPAM surfaces were labeled to assess whether cell sheet detachment from pNIPAM is accompanied by the removal of pNIPAM from the substrate itself. Using a semipermeable superstrate, cell sheets were transferred to a secondary culture dish to assess whether cell detachment resulted in any pNIPAM removal. In addition, the function of the transplanted bovine aortic endothelial cells was assessed by determining whether they would proliferate and grow on a new secondary substrate.

Synthesis and optimization of fluorescent poly(N-isopropyl acrylamide)-coated surfaces by atom transfer radical polymerization for cell culture and detachment.

Although there are many stimulus-responsive polymers, poly(N-isopropyl acrylamide) (pNIPAM) is of special interest due to the phase change it undergoes in a physiologically relevant temperature range that leads to the release of cells and proteins. The nondestructive release of cells opens up a wide range of applications, including the use of pNIPAM for cell sheet and tissue engineering. In this work, pNIPAM surfaces were generated that can be distinguished from the extracellular matrix. A polymerization technique was adapted that was previously used by Mendez, and the existing protocol was optimized for the culture of mammalian cells. The resulting surfaces were characterized with X-ray photoelectron spectroscopy and goniometry. The developed pNIPAM surfaces were further adapted by incorporation of 5-acrylamidofluorescein to generate fluorescent pNIPAM-coated surfaces. Both types of surfaces (fluorescent and nonfluorescent) sustained cellular attachment and produced cellular detachment of ∼90%, and are therefore suitable for the generation of cell sheets for engineered tissues and other purposes. These surfaces will be useful tools for experiments investigating cellular detachment from pNIPAM and the pNIPAM/cell interface.

In vitro cytotoxicity of antimicrobial conjugated electrolytes: interactions with mammalian cells.

An estimated 19 000 deaths and $3-4 billion in health care costs per year in the United States are attributed to methicillin-resistant Staphlococcus aureus (MRSA) infections. Certain conjugated phenylene ethynylene (CPE)-based polymers (PPE) and oligomers (OPE) have been demonstrated to exhibit dark and light-activated antimicrobial activity. Until recently, the relative cytotoxicity of these PPEs and OPEs toward mammalian cells haas been unknown, limiting the applications for which they may be used (e.g., reducing and/or preventing the spread of untreatable bacterial strains). In this work, we examine the toxicity of CPEs to mammalian cells using cytotoxicity assays of cellular monolayers. Eight CPEs, two PPEs and six OPEs, were selected for these studies based on their biocidal activity, and diversity of repeat unit number and functional groups. Briefly, two cell types were exposed to CPEs at concentrations ranging from 1-100 ug/mL for 24 h. We find that concentration largely determines the resulting viability of cells, although at intermediate concentrations (5-10 ug/mL), the effect of light on light-activated compounds is very important. Furthermore, we find that the longer-chained compounds are cytotoxic at much higher concentrations, and therefore have the widest range of concentrations available for potential applications.

Assessment of cytotoxicity of (N-isopropyl acrylamide) and poly(N-isopropyl acrylamide)-coated surfaces.

Poly(N-isopropyl acrylamide) (pNIPAM) is one of the most popular stimulus-responsive polymers for research. It is especially of great interest in the field of tissue engineering. While it is known that the NIPAM monomer is toxic, there is little conclusive research on the cytotoxicity of the polymer. In this work, the relative biocompatibility of the NIPAM monomer, pNIPAM, and pNIPAM-coated substrates prepared using different polymerization (free radical and plasma polymerization) and deposition (spin coating and plasma polymerization) techniques was evaluated using appropriate cytotoxicity tests (MTS, Live/Dead, plating efficiency). Four different mammalian cell types (endothelial, epithelial, smooth muscle, and fibroblasts) were used for the cytotoxicity testing. The pNIPAM-coated surfaces were evaluated for their thermoresponse and surface chemistry using X-ray photoelectron spectroscopy and goniometry. We found that while cell viability on pNIPAM surfaces decreases when compared to controls, the viability also seems to be deposition type dependent, with sol-gel based pNIPAM surfaces being the least biocompatible. Long term experiments proved that all pNIPAM-coated surfaces were not cytotoxic to the four cell types evaluated in a direct contact test. Plating efficiency experiments did not show cytotoxicity. Cellular sensitivity to pNIPAM and to the NIPAM monomer varied depending on cell type. Endothelial cells consistently showed decreased viability after 48 hours of exposure to pNIPAM extracts and were more sensitive than the other cell lines to impurities in the polymer.

ARGET-ATRP synthesis and characterization of PNIPAAm brushes for quantitative cell detachment studies.

Stimuli responsive (or "smart") polymer brushes represent a non-toxic approach for achieving release of biofouling layers. Thermo-responsive poly(N-isopropylacrylamide) (PNIPAAm) polymer brushes have been shown to modulate bacterial adhesion and release through transition between temperatures above and below the lower critical solution temperature (LCST ~32 °C) of PNIPAAm in water. In this article, we describe a convenient method to synthesize grafted PNIPAAm brushes over large areas for biological studies using a relatively simple and rapid method which allows atom transfer radical polymerization (ATRP) in presence of air using the activator regenerated electron transfer (ARGET) mechanism. PNIPAAm brushes were characterized using X-ray photoelectron spectroscopy, time-of-flight secondary ion mass spectroscopy, Fourier transform infrared spectroscopy, ellipsometry, and contact angle measurements. Our studies demonstrate that uniform, high purity PNIPAAm brushes with controlled and high molecular weight can be easily produced over large areas using ARGET-ATRP. We also report the use of a spinning disk apparatus to systematically and quantitatively study the detachment profiles of bacteria from PNIPAAm surfaces under a range (0-400 dyne/cm(2)) of shear stresses.

Biological cell detachment from poly(N-isopropyl acrylamide) and its applications.

Over the past two decades, poly(N-isopropyl acrylamide) (pNIPAM) has become widely used for bioengineering applications. In particular, pNIPAM substrates have been used for the nondestructive release of biological cells and proteins. In this feature article, we review the applications for which pNIPAM substrates have been used to release biological cells, including for the study of the extracellular matrix (ECM), for cell sheet engineering and tissue transplantation, the formation of tumorlike spheroids, the study of bioadhesion and bioadsorption, and the manipulation or deformation of individual cells. The articles reviewed include submissions from our own group as well as from those performing research in the field worldwide.

A low-cost, rapid deposition method for "smart" films: applications in mammalian cell release.

The "smart" polymer poly (N-isopropyl acrylamide), or pNIPAM, has been studied for bioengineering applications. The polymer's abrupt change in hydrophobicity near physiologic temperatures makes it ideal for use as a substrate in many applications, including protein separation and prevention of biofouling. To tether pNIPAM, many techniques such as plasma deposition, have been utilized, but most are expensive and require long equipment calibration or fabrication periods. Recently, a novel method for codepositing this smart polymer with a sol-gel, tetraethyl orthosilicate (TEOS), was developed. In this work, we adapt this technique for applications in mammalian cell attachment/detachment. In addition, we compare the effects of the pNIPAM/TEOS ratio to functionality using surface analysis techniques (XPS and contact angles). We found the optimal ratio to be 0.35 wt % pNIPAM/TEOS. Cell detachment from these substrates indicate that they would be ideal for applications that do not require intact cell sheets, such as biofouling prevention and protein separation, as this technique is a simple and affordable technique for pNIPAM deposition.

Fabrication and Characterization of Thermoresponsive Films Deposited by an RF Plasma Reactor.

Poly(N-isopropyl acrylamide) (pNIPAM) undergoes a sharp property change in response to a moderate thermal stimulus at physiological temperatures. In this work, we constructed a radio frequency (RF) plasma reactor for the plasma polymerization of pNIPAM. RF deposition is a method that coats surfaces of any geometry producing surfaces that are sterile and uniform, making this technique useful for forming biocompatible films. The films generated are characterized using X-ray photoelectron spectroscopy (XPS), contact angles, cell culture, and interferometry. We find that a plasma with a decreasing series of power settings (i.e., from 100W to 1W) at a pressure of 140 millitorr yields the most favorable results.

Altered calcium dynamics in cardiac cells grown on silane-modified surfaces.

Chemically defined surfaces were created using self-assembled monolayers (SAMs) of hydrophobic and hydrophilic silanes as models for implant coatings, and the morphology and physiology of cardiac myocytes plated on these surfaces were studied in vitro. We focused on changes in intracellular Ca(2+) because of its essential role in regulating heart cell function. The SAM-modified coverslips were analyzed using X-ray Photoelectron Spectroscopy to verify composition. The morphology and physiology of the cardiac cells were examined using fluorescence microscopy and intracellular Ca(2+) imaging. The imaging experiments used the fluorescent ratiometric dye fura-2, AM to establish both the resting Ca(2+) concentration and the dynamic responses to electrical stimulation. A significant difference in excitation-induced Ca(2+) changes on the different silanated surfaces was observed. However, no significant change was noted based on the morphological analysis. This result implies a difference in internal Ca(2+) dynamics, and thus cardiac function, occurs when the composition of the surface is different, and this effect is independent of cellular morphology. This finding has implications for histological examination of tissues surrounding implants, the choice of materials that could be beneficial as implant coatings and understanding of cell-surface interactions in cardiac systems.

Understanding the force-vs-distance profiles of terminally attached poly(N-isopropyl acrylamide) thin films.

In this work, we examine the interaction between thin films composed of terminally anchored poly(N-isopropyl acrylamide) (PNIPAAm) immersed in water and test surfaces. Understanding this force of interaction can be important when using PNIPAAm surfaces in biotechnological applications such as biological cell cultures. The two novel contributions that are presented here are (1) the use of a recently developed self-consistent field (SCF) theory to predict the force-vs-distance profiles, and (2) the use of a modified polymer scaling theory to estimate the wet film thickness from experimental force-vs-distance profiles. SCF theory was employed to model the equilibrium structure of the uncompressed PNIPAAm chains, and the force between a compressed polymer film and a test surface as a function of wall separation distance. The parameters that were varied include temperature, polymer molecular weight, and surface coverage. The force-vs-distance profiles obtained at low and high temperatures with the SCF theory were in qualitative agreement with the experimentally measured profiles reported in the literature. We also compared the results of our SCF theory to the Alexander de Gennes scaling theory and found agreement at large separation distance. We also propose a method to estimate the wet polymer film thickness from a force-vs-distance profile obtained from an atomic force microscope measurement. The main novelties of this approach are that we employed a density functional theory corrected version of scaling theory proposed by McCoy et al. [McCoy, J. D.; Curro, J. G. J. Chem. Phys. 2005, 122, 164905], and we provide equations to account for various geometries of AFM tips.

Comparison of native extracellular matrix with adsorbed protein films using secondary ion mass spectrometry.

In the past decade, the temperature-responsive behavior of poly(N-isopropyl acrylamide) (pNIPAM) has come to be recognized as a convenient method for the nondestructive harvest of confluent cell layers. Recently, we have utilized this nondestructive cell harvest method as a means to ascertain the nature of the extracellular matrix (ECM) secreted from cells. In this work, we compare the ECM obtained after cell liftoff to individual ECM proteins adsorbed directly onto RF-plasma-deposited pNIPAM (ppNIPAM). Using X-ray photoelectron spectroscopy, we find that the composition of ppNIPAM post-cell liftoff surfaces is consistent with those of the ppNIPAM post-protein adsorption surface, both of which differ from control surfaces. Using principal component analysis of positive-ion time-of-flight secondary ion mass spectrometry (ToF-SIMS) data, we show that the major ECM proteins examined can effectively be identified from their amino acid compositions. By comparing the positive-ion ToF-SIMS data from each of the ppNIPAM post-protein adsorption surfaces to that of ppNIPAM post-cell liftoff, we find that ppNIPAM post-cell liftoff surfaces are distinctly separate from fibronectin (FN). This result is consistent with our previous observation using immunoassay that FN is clearly associated with the cell sheet after low-temperature liftoff from ppNIPAM.

Temperature dependent activity and structure of adsorbed proteins on plasma polymerized N-isopropyl acrylamide.

Thorough studies of protein interactions with stimulus responsive polymers are necessary to provide a better understanding of their applications in biosensors and biomaterials. In this study, protein behavior on a thermoresponsive polymer surface, plasma polymerized N-isopropyl acrylamide (ppNIPAM), is investigated using multiple characterization techniques above and below its lower critical solution temperature (LCST). Protein adsorption and binding affinity are probed using radiolabeled proteins. Protein activity is estimated by measuring the immunological activity of an antibody adsorbed onto ppNIPAM using surface plasmon resonance. Conformation/orientation of the proteins is probed by time-of-flight secondary ion mass spectrometry (TOF-SIMS) and principal component analysis (PCA) of the TOF-SIMS data. In this work, we find that at low protein solution concentrations, ppNIPAM-treated surfaces are low fouling below the LCST, but protein retentive above it. The protein adsorption isotherms demonstrate that apparent affinity between soluble protein molecules and the ppNIPAM surface are an order of magnitude lower at room temperature than at 37 degrees C. Although direct protein desorption is not observed in our study when the surface temperature drops below the LCST, the binding affinity of surface adsorbed protein with ppNIPAM is reduced, as judged by a detergent elution test. Furthermore, we demonstrated that proteins adsorbed onto ppNIPAM are functionally active, but the activity is better preserved at room temperature than 37 degrees C. The temperature dependent difference in protein activity as well as TOF-SIMS and PCA study suggest that proteins take different conformations/orientations after adsorption on ppNIPAM above and below the LCST.

Cell sheet detachment affects the extracellular matrix: a surface science study comparing thermal liftoff, enzymatic, and mechanical methods.

This work compares the removal of bovine aortic endothelial cell (BAEC) monolayers via 1) low-temperature liftoff from a "smart polymer," plasma polymerized poly(N-isopropyl acrylamide) (ppNIPAM), 2) enzymatic digestion, and 3) mechanical dissociation from ppNIPAM surfaces. We examine the surfaces after cell removal by using X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectrometry (ToF-SIMS), immunostaining, and cell adhesion assay. Immunoassay results indicate that low-temperature liftoff nondestructively harvests the cell sheet and most of the underlying extracellular matrix (ECM), whereas enzymatic digestion and mechanical dissociation are damaging to both the cells and ECM. XPS results indicate that amide and alcohol groups attributed to proteins in the ECM are present on postliftoff surfaces. Principal component analysis (PCA) of ToF-SIMS data indicates that molecular ion fragments of amino acids are present on postliftoff surfaces. Finally, a cell adhesion assay seeding new cells on surfaces from which an initial layer of cells was removed via each of the three methods indicates that liftoff and mechanical dissociation leave behind surfaces that better promote cell adhesion. We conclude that the removal of BAEC cells via low-temperature liftoff from ppNIPAM-treated surfaces is less damaging to the ECM proteins remaining at the surface than the other methods.

Surface chemical and mechanical properties of plasma-polymerized N-isopropylacrylamide.

Surface-immobilized poly(N-isopropyl acrylamide) (pNIPAM) is currently used for a wide variety of biosensor and biomaterial applications. A thorough characterization of the surface properties of pNIPAM thin films will benefit those applications. In this work, we present analysis of a plasma-polymerized NIPAM (ppNIPAM) coating by multiple surface analytical techniques, including time-of-flight secondary-ion mass spectrometry (ToF-SIMS), contact angle measurement, atomic force microscopy (AFM), and sum frequency generation (SFG) vibrational spectroscopy. ToF-SIMS data show that the plasma-deposited NIPAM polymer on the substrate is cross-linked with a good retention of the monomer integrity. Contact angle results confirm the thermoresponsive nature of the film as observed by a change of surface wettability as a function of temperature. Topographic and force-distance curve measurements by AFM further demonstrate that the grafted film shrinks or swells depending on the temperature of the aqueous environment. A clear transition of the elastic modulus is observed at 31-32 degrees C. The change of the surface wettability and mechanical properties vs temperature are attributed to different conformations taken by the polymer, which is reflected on the outmost surface as distinct side chain groups orienting outward at different temperatures as measured by SFG. The results suggest that a ppNIPAM thin film on a substrate experiences similar mechanical and chemical changes to pNIPAM bulk polymers in solution. The SFG result provides evidence supporting the current theory of the lower critical solution temperature (LCST) behavior of pNIPAM.