Formation of sparsely tethered bilayer lipid membrane on a biodegradable self-assembled monolayer of poly(lactic acid).

The utilization of biomimetic membranes supported by advanced self-assembled monolayers is gaining attraction as a promising sensing tool. Biomimetic membranes offer exceptional biocompatibility and adsorption capacity upon degradation, transcending their role as mere research instruments to open new avenues in biosensing. This study focused on anchoring a sparsely tethered bilayer lipid membrane onto a self-assembled monolayer composed of a biodegradable polymer, functionalized with poly(ethylene glycol)-cholesterol moieties, for lipid membrane integration. Real-time monitoring via quartz crystal microbalance, coupled with characterization using surface-enhanced infrared absorption spectroscopy and electrochemical impedance spectroscopy, provided comprehensive insights into each manufacturing phase. The resulting lipid layer, along with transmembrane pores formed by gramicidin A, exhibited robust stability. Electrochemical impedance spectroscopy analysis confirmed membrane integrity, successful pore formation, and consistent channel density. Notably, gramicidin A demonstrated sustained functionality as an ion channel upon reconstitution, with its functionality being effectively blocked and inhibited in the presence of calcium ions. These findings mark significant strides in developing intricate biodegradable nanomaterials with promising applications in biomedicine.

Advances in Functionalization of Bioresorbable Nanomembranes and Nanoparticles for Their Use in Biomedicine.

Bioresorbable nanomembranes (NMs) and nanoparticles (NPs) are powerful polymeric materials playing an important role in biomedicine, as they can effectively reduce infections and inflammatory clinical patient conditions due to their high biocompatibility, ability to physically interact with biomolecules, large surface area, and low toxicity. In this review, the most common bioabsorbable materials such as those belonging to natural polymers and proteins for the manufacture of NMs and NPs are reviewed. In addition to biocompatibility and bioresorption, current methodology on surface functionalization is also revisited and the most recent applications are highlighted. Considering the most recent use in the field of biosensors, tethered lipid bilayers, drug delivery, wound dressing, skin regeneration, targeted chemotherapy and imaging/diagnostics, functionalized NMs and NPs have become one of the main pillars of modern biomedical applications.

Digital Light Processing-3D Printing of Thermoset Materials with High Biodegradability from Amino Acid-Derived Acrylamide Monomers.

Six acrylamide resins, derived from l-phenylalanine and l-leucine, are designed for application in digital light processing (DLP) printers to obtain biodegradable thermoset polymers. The acrylamide copolymers are prepared under light irradiation at 405 nm and thermal post-curing processes. Low molecular weight poly(ethylene glycol)diacrylate (PEGDA) and N,N-dimethylacrylamide (DMAM), both liquid resins, are used as co-monomers and diluents for the amino acid-derived acrylamide solubilization. The presence of two phenylalanine units and two ester groups in the acrylamide monomer accuses a fast degradation rate in hydrolytic medium in 90 days. The residual products leached in the aqueous media prove to be non-cytotoxic, when 3D-printed samples are cultured with osteoblast cells (MG63), which represents an advantage for the safe disposal of printer waste materials. The scaled-up pieces derived from l-phenylalanine and diethylene glycol, as amino acid-derived acrylamide (named compound C), PEGDA and DMAM, present high dimensional stability after DLP printing of complex structures used as testing samples. Layers of 50 µm of thickness are well cohesive having isotropic behavior, as demonstrated with tensile-strain measurements performed in X-Y-Z (plane) directions. The compound C, which contains phenylalanine amino acid, reveals a promising potential to replace non-biodegradable acrylate polymers used in prototyping systems.

Mechanical Properties of Smart Polypropylene Meshes: Effects of Mesh Architecture, Plasma Treatment, Thermosensitive Coating, and Sterilization Process.

Smart polypropylene (PP) hernia meshes were proposed to detect surgical infections and to regulate cell attachment-modulated properties. For this purpose, lightweight and midweight meshes were modified by applying a plasma treatment for subsequent grafting of a thermosensitive hydrogel, poly(N-isopropylacrylamide) (PNIPAAm). However, both the physical treatment with plasma and the chemical processes required for the covalent incorporation of PNIPAAm can modify the mechanical properties of the mesh and thus have an influence in hernia repair procedures. In this work, the mechanical performance of plasma-treated and hydrogel-grafted meshes preheated at 37 °C has been compared with standard meshes using bursting and the suture pull out tests. Furthermore, the influence of the mesh architecture, the amount of grafted hydrogel, and the sterilization process on such properties have been examined. Results reveal that although the plasma treatment reduces the bursting and suture pull out forces, the thermosensitive hydrogel improves the mechanical resistance of the meshes. Moreover, the mechanical performance of the meshes coated with the PNIPAAm hydrogel is not influenced by ethylene oxide gas sterilization. Micrographs of the broken meshes evidence the role of the hydrogel as reinforcing coating for the PP filaments. Overall, results confirm that the modification of PP medical textiles with a biocompatible thermosensitive hydrogel do not affect, and even improve, the mechanical requirements necessary for the implantation of these prostheses in vivo.

Multimodal Biomedical Implant with Plasmonic and Simulated Body Temperature Responses.

This work presents a novel nanoparticle-based thermosensor implant able to reveal the precise temperature variations along the polymer filaments, as it contracts and expands due to changes in the macroscale local temperature. The multimodal device is able to trace the position and the temperature of a polypropylene mesh, employed in abdominal hernia repair, by combining plasmon resonance and Raman spectroscopy with hydrogel responsive system. The novelty relies on the attachment of the biocompatible nanoparticles, based on gold stabilized by a chitosan-shell, already charged with the Raman reporter (RaR) molecules, to the robust prosthesis, without the need of chemical linkers. The SERS enhanced effect observed is potentiated by the presence of a quite thick layer of the copolymer (poly(N-isopropylacrylamide)-co-poly(acrylamide)) hydrogel. At temperatures above the LCST of PNIPAAm-co-PAAm, the water molecules are expulsed and the hydrogel layer contracts, leaving the RaR molecules more accessible to the Raman source. In vitro studies with fibroblast cells reveal that the functionalized surgical mesh is biocompatible and no toxic substances are leached in the medium. The mesh sensor opens new frontiers to semi-invasive diagnosis and infection prevention in hernia repair by using SERS spectroscopy. It also offers new possibilities to the functionalization of other healthcare products.

Smart Design of Sensor-Coated Surgical Sutures for Bacterial Infection Monitoring.

Virtually, all implantable medical devices are susceptible to infection. As the main healthcare issue concerning implantable devices is the elevated risk of infection, different strategies based on the coating or functionalization of biomedical devices with antiseptic agents or antibiotics are proposed. In this work, an alternative approach is presented, which consists of the functionalization of implantable medical devices with sensors capable of detecting infection at very early stages through continuous monitoring of the bacteria metabolism. This approach, which is implemented in surgical sutures as a representative case of implantable devices susceptible to bacteria colonization, is expected to minimize the risk of worsening the patient's clinical condition. More specifically, non-absorbable polypropylene/polyethylene (PP/PE) surgical sutures are functionalized with conducting polymers using a combination of low-pressure oxygen plasma, chemical oxidative polymerization, and anodic polymerization, to detect metabolites coming from bacteria respiration. Functionalized suture yarns are used for real-time monitoring of bacteria growth, demonstrating the potential of this strategy to fight against infections.

Preparation and Characterization of Functionalized Surgical Meshes for Early Detection of Bacterial Infections.

Isotactic polypropylene (i-PP) nonabsorbable surgical meshes are modified by incorporating a conducting polymer (CP) layer to detect the adhesion and growth of bacteria by sensing the oxidation of nicotinamide adenine dinucleotide (NADH), a metabolite produced by the respiration reactions of such microorganisms, to NAD+. A three-step process is used for such incorporation: (1) treat pristine meshes with low-pressure O2 plasma; (2) functionalize the surface with CP nanoparticles; and (3) coat with a homogeneous layer of electropolymerized CP using the nanoparticles introduced in (2) as polymerization nuclei. The modified meshes are stable and easy to handle and also show good electrochemical response. The detection by cyclic voltammetry of NADH within the interval of concentrations reported for bacterial cultures is demonstrated for the two modified meshes. Furthermore, Staphylococcus aureus and both biofilm-positive (B+) and biofilm-negative (B-) Escherichia coli cultures are used to prove real-time monitoring of NADH coming from aerobic respiration reactions. The proposed strategy, which offers a simple and innovative process for incorporating a sensor for the electrochemical detection of bacteria metabolism to currently existing surgical meshes, holds considerable promise for the future development of a new generation of smart biomedical devices to fight against post-operative bacterial infections.

3D-Printed Polymer-Infiltrated Ceramic Network with Antibacterial Biobased Silver Nanoparticles.

This work aimed at the antimicrobial functionalization of 3D-printed polymer-infiltrated biomimetic ceramic networks (PICN). The antimicrobial properties of the polymer-ceramic composites were achieved by coating them with human- and environmentally safe silver nanoparticles trapped in a phenolated lignin matrix (Ag@PL NPs). Lignin was enzymatically phenolated and used as a biobased reducing agent to obtain stable Ag@PL NPs, which were then formulated in a silane (γ-MPS) solution and deposited to the PICN surface. The presence of the NPs and their proper attachment to the surface were analyzed with spectroscopic methods (FTIR and Raman) and X-ray photoelectron spectroscopy (XPS). Homogeneous distribution of 13.4 ± 3.2 nm NPs was observed in the transmission electron microscopy (TEM) images. The functionalized samples were tested against Gram-positive (Staphylococcus aureus) and Gram-negative (Pseudomonas aeruginosa) bacteria, validating their antimicrobial efficiency in 24 h. The bacterial reduction of S. aureus was 90% in comparison with the pristine surface of PICN. To confirm that the Ag-functionalized PICN scaffold is a safe material to be used in the biomedical field, its biocompatibility was demonstrated with human fibroblast (BJ-5ta) and keratinocyte (HaCaT) cells, which was higher than 80% in both cell lines.

Dual-Responsive Polypropylene Meshes Actuating as Thermal and SERS Sensors.

Polypropylene (PP) surgical meshes, with different knitted architectures, were chemically functionalized with gold nanoparticles (AuNPs) and 4-mercaptothiazole (4-MB) to transform their fibers into a surface enhanced Raman scattering (SERS) detectable plastic material. The application of a thin layer of poly[N-isopropylacrylamide-co-N,N'-methylene bis(acrylamide)] (PNIPAAm-co-MBA) graft copolymer, covalently polymerized to the mesh-gold substrate, caused the conversion of the inert plastic into a thermoresponsive material, resulting in the first PP implantable mesh with both SERS and temperature stimulus responses. AuNPs were homogeneously distributed over the PP yarns, offering a clear SERS recognition together with higher PNIPAAm lower critical solution temperature (LCST ∼ 37 °C) than without the metallic particles (LCST ∼ 32 °C). An infrared thermographic camera was used to observe the polymer-hydrogel folding-unfolding process and to identify the new value of the LCST, connected with the heat generation by plasmonic-resonance gold NPs. The development of SERS PP prosthesis will be relevant for the bioimaging and biomarker detection of the implant by using the plasmonic effect and Raman vibrational spectroscopy for minimally invasive interventions (such as laparoscopy), to prevent patient inflammatory processes. Furthermore, Raman sources have been proved to not damage the cells, like happens with near-infrared irradiation, representing another advantage of moving to SERS approaches. The findings reported here offer unprecedented application possibilities in the biomedical field by extrapolating the material functionalization to other nonabsorbable polymer made devices (e.g., surgical sutures, grapes, wound dressings, among others).

Novel Biobased Epoxy Thermosets and Coatings from Poly(limonene carbonate) Oxide and Synthetic Hardeners.

In the area of coating development, it is extremely difficult to find a substitute for bisphenol A diglycidyl ether (DGEBA), the classical petroleum-based raw material used for the formulation of epoxy thermosets. This epoxy resin offers fast curing reaction with several hardeners and the best thermal and chemical resistance properties for applications in coatings and adhesive technologies. In this work, a new biobased epoxy, derived from poly(limonene carbonate) oxide (PLCO), was combined with polyetheramine and polyamineamide curing agents, offering a spectrum of thermal and mechanical properties, superior to DGEBA-based thermosets. The best formulation was found to be a combination of PLCO and a commercial curing agent (Jeffamine) in a stoichiometric 1:1 ratio. Although PLCO is a solid due to its high molecular weight, it was possible to create a two-component partially biobased epoxy paint without the need of volatile organic compounds (i.e., solvent-free formulation), intended for use in coating technology to partially replace DGEBA-based thermosets.

A Biosourced Epoxy Resin for Adhesive Thermoset Applications.

Biobased epoxy-derived raw materials will be essential for future coating and adhesive designs in industry. Here, a facile approach is reported towards the incorporation of limonene into an epoxy-functionalized polycarbonate and its crosslinking with a polyamine curing agent to obtain a thermoset material. For the first time, a solvent-borne adhesive with excellent film-forming, mechanical and adhesion strength properties is described.

3D-Printed Polymer-Infiltrated Ceramic Network with Biocompatible Adhesive to Potentiate Dental Implant Applications.

The aim of this work was to prepare and characterize polymer-ceramic composite material for dental applications, which must resist fracture and wear under extreme forces. It must also be compatible with the hostile environment of the oral cavity. The most common restorative and biocompatible copolymer, 2,2-bis(p-(2'-2-hydroxy-3'-methacryloxypropoxy)phenyl)propane and triethyleneglycol dimethacrylate, was combined with 3D-printed yttria-stabilized tetragonal zirconia scaffolds with a 50% infill. The proper scaffold deposition and morphology of samples with 50% zirconia infill were studied by means of X-ray computed microtomography and scanning electron microscopy. Samples that were infiltrated with copolymer were observed under compression stress, and the structure's failure was recorded using an Infrared Vic 2DTM camera, in comparison with empty scaffolds. The biocompatibility of the composite material was ascertained with an MG-63 cell viability assay. The microtomography proves the homogeneous distribution of pores throughout the whole sample, whereas the presence of the biocompatible copolymer among the ceramic filaments, referred to as a polymer-infiltrated ceramic network (PICN), results in a safety "damper", preventing crack propagation and securing the desired material flexibility, as observed by an infrared camera in real time. The study represents a challenge for future dental implant applications, demonstrating that it is possible to combine the fast robocasting of ceramic paste and covalent bonding of polymer adhesive for hybrid material stabilization.

Plasma-Functionalized Isotactic Polypropylene Assembled with Conducting Polymers for Bacterial Quantification by NADH Sensing.

Rapid detection of bacterial presence on implantable medical devices is essential to prevent biofilm formation, which consists of densely packed bacteria colonies able to withstand antibiotic-mediated killing. In this work, a smart approach is presented to integrate electrochemical sensors for detecting bacterial infections in biomedical implants made of isotactic polypropylene (i-PP) using chemical assembly. The electrochemical detection is based on the capacity of conducting polymers (CPs) to detect extracellular nicotinamide adenine dinucleotide (NADH) released from cellular respiration of bacteria, which allows distinguishing prokaryotic from eukaryotic cells. Oxygen plasma-functionalized free-standing i-PP, coated with a layer (≈1.1 µm in thickness) of CP nanoparticles obtained by oxidative polymerization, is used as working electrode for the anodic polymerization of a second CP layer (≈8.2 µm in thickness), which provides very high electrochemical activity and stability. The resulting layered material, i-PPf /CP2 , detects the electro-oxidation of NADH in physiological media with a sensitivity 417 µA cm-2 and a detection limit up to 0.14 × 10-3 m, which is below the concentration of extracellular NADH found for bacterial cultures of biofilm-positive and biofilm-negative strains.

Green Nanocoatings Based on the Deposition of Zirconium Oxide: The Role of the Substrate.

Herein, the influence of the substrate in the formation of zirconium oxide monolayer, from an aqueous hexafluorozirconic acid solution, by chemical conversion and by electro-assisted deposition, has been approached. The nanoscale dimensions of the ZrO2 film is affected by the substrate nature and roughness. This study evidenced that the mechanism of Zr-EAD is dependent on the potential applied and on the substrate composition, whereas conversion coating is uniquely dependent on the adsorption reaction time. The zirconium oxide based nanofilms were more homogenous in AA2024 substrates if compared to pure Al grade (AA1100). It was justified by the high content of Cu alloying element present in the grain boundaries of the latter. Such intermetallic active sites favor the obtaining of ZrO2 films, as demonstrated by XPS and AFM results. From a mechanistic point of view, the electrochemical reactions take place simultaneously with the conventional chemical conversion process driven by ions diffusion. Such findings will bring new perspectives for the generation of controlled oxide coatings in modified electrodes used, as for example, in the construction of battery cells; in automotive and in aerospace industries, to replace micrometric layers of zinc phosphate by light-weight zirconium oxide nanometric ones. This study is particularly addressed for the reduction of industrial waste by applying green bath solutions without the need of auxiliary compounds and using lightweight ceramic materials.

Atmospheric pressure plasma liquid assisted deposition of polydopamine/acrylate copolymer on zirconia (Y-TZP) ceramics: a biocompatible and adherent nanofilm.

Polydopamine-ethylene glycol dimethacrylate copolymer is a biocompatible coating with cell adhesion promotion and antibiofilm properties. This copolymer has been successfully applied on metallic implants, such as stainless steel and titanium implants, using several deposition techniques (e.g. layer-by-layer, silane activation, chemical vapor deposition, or liquid-assisted plasma polymerization). However, its application in zirconia ceramic materials, which are widely used in dentistry and medicine, has never been described. In this work, polydopamine-ethylene glycol dimethacrylate copolymer has been deposited on ultra-smooth surfaces of yttria-stabilized zirconia discs (average roughness = 2.08 ± 0.08 nm) by using liquid-assisted atmospheric-pressure plasma-induced polymerization (LA-APPiP). After the polymerization, the nanometric coating (250 nm, measured by ellipsometry) had an average roughness of 79.85 ± 13.71 nm and water contact angle of 57.8 ± 2.2 degrees, consistent with the highly hydrophilic nature of the biocompatible copolymer, if compared to the pristine zirconia (72.7 ± 2.0 degrees). The successful covalent bonding of the copolymer with the zirconia surface, thanks to the previous activation of the substrate with oxygen plasma, was proved by X-ray photoelectron spectroscopy (XPS). The polymer composition has been investigated by XPS and Raman spectroscopies. The LA-APPiP technique has been proved to be an excellent method to produce homogenous films without the need to employ solvents and further purification steps. The new copolymer film allows the uniform growth of human osteoblast-like MG-63 cells, after 7 days of cell culture, as observed by fluorescence microscopy.

Smart design for a flexible, functionalized and electroresponsive hybrid platform based on poly(3,4-ethylenedioxythiophene) derivatives to improve cell viability.

Development of smart functionalized materials for tissue engineering has attracted significant attention in recent years. In this work we have functionalized a free-standing film of isotactic polypropylene (i-PP), a synthetic polymer that is typically used for biomedical applications (e.g. fabrication of implants), for engineering a 3D all-polymer flexible interface that enhances cell proliferation by a factor of ca. three. A hierarchical construction process consisting of three steps was engineered as follows: (1) functionalization of i-PP by applying a plasma treatment, resulting in i-PPf; (2) i-PPf surface coating with a layer of polyhydroxymethy-3,4-ethylenedioxythiophene nanoparticles (PHMeEDOT NPs) by in situ chemical oxidative polymerization of HMeEDOT; and (3) deposition on the previously activated and PHMeEDOT NPs coated i-PP film (i-PPf/NP) of a graft conjugated copolymer, having a poly(3,4-ethylenedioxythiophene) (PEDOT) backbone, and randomly distributed short poly(ε-caprolactone) (PCL) side chains (PEDOT-g-PCL), as a coating layer of ∼9 µm in thickness. The properties of the resulting bioplatform, which can be defined as a robust macroscopic composite coated with a "molecular composite", were investigated in detail, and both adhesion and proliferation of two human cell lines have been evaluated, as well. The results demonstrate that the incorporation of the PEDOT-g-PCL layer significantly improves cell attachment and cell growth not only when compared to i-PP but also with respect to the same platform coated with only PEDOT, constructed in a similar manner, as a control.

Polypropylene mesh for hernia repair with controllable cell adhesion/de-adhesion properties.

Herein, a versatile bilayer system, composed by a polypropylene (PP) mesh and a covalently bonded poly(N-isopropylacrylamide) (PNIPAAm) hydrogel, is reported. The cell adhesion mechanism was successfully modulated by controlling the architecture of the hydrogel in terms of duration of PNIPAAm grafting time, crosslinker content, and temperature of material exposure in PBS solutions (below and above the LCST of PNIPAAm). The best in vitro results with fibroblast (COS-1) and epithelial (MCF-7) cells was obtained with a mesh modified with a porous iPP-g-PNIPAAm bilayer system, prepared via PNIPAAm grafting for 2 h at the lowest N,N'-methylene bis(acrylamide) (MBA) concentration (1 mM). Under these conditions, the detachment of the fibroblast-like cells was 50% lower than that of the control, after 7 days of cell incubation, which represents a high de-adhesion of cells in a short period. Moreover, the whole system showed excellent stability in dry or wet media, proving that the thermosensitive hydrogel was well adhered to the polymer surface, after PP fibre activation by cold plasma. This study provides new insights on the development of anti-adherent meshes for abdominal hernia repair.

Electrospun Conducting and Biocompatible Uniaxial and Core-Shell Fibers Having Poly(lactic acid), Poly(ethylene glycol), and Polyaniline for Cardiac Tissue Engineering.

Electroactive and biocompatible fibrous scaffolds have been prepared and characterized using polyaniline (PAni) doped with dodecylbenzenesulfonic acid (DBSA) combined with poly(lactic acid) (PLA) and PLA/poly(ethylene glycol) (PEG) mixtures. The composition of simple and core-shell fibers, which have been obtained by both uniaxial and coaxial electrospinning, respectively, has been corroborated by Fourier-transform infrared and micro-Raman spectroscopies. Morphological studies suggest that the incorporation of PEG enhances the packing of PLA and PAni chains, allowing the regulation of the thickness of the fibers. PAni and PEG affect the thermal and electrical properties of the fibers, both decreasing the glass transition temperature and increasing the electrical conductivity. Interestingly, the incorporation of PEG improves the PAni-containing paths associated with the conduction properties. Although dose response curves evidence the high cytotoxicity of PAni/DBSA, cell adhesion and cell proliferation studies on PLA/PAni fibers show a reduction of such harmful effects as the conducting polymer is mainly retained inside the fibers through favorable PAni···PLA interactions. The incorporation of PEG into uniaxial fibers resulted in an increment of the cell mortality, which has been attributed to its rapid dissolution into the culture medium and the consequent enhancement of PAni release. In opposition, the delivery of PAni decreases and, therefore, the biocompatibility of the fibers increases when a shell coating the PAni-containing system is incorporated through coaxial electrospinning. Finally, morphological and functional studies using cardiac cells indicated that these fibrous scaffolds are suitable for cardiac tissue engineering applications.

Free-Standing Faradaic Motors Based on Biocompatible Nanoperforated Poly(lactic Acid) Layers and Electropolymerized Poly(3,4-ethylenedioxythiophene).

The electro-chemo-mechanical response of robust and flexible free-standing films made of three nanoperforated poly(lactic acid) (pPLA) layers separated by two anodically polymerized poly(3,4-ethylenedioxythiophene) (PEDOT) layers has been demonstrated. The mechanical and electrochemical properties of these films, which are provided by pPLA and PEDOT, respectively, have been studied by nanoindentation, cyclic voltammetry, and galvanostatic charge-discharge assays. The unprecedented combination of properties obtained for this system is appropriated for its utilization as a Faradaic motor, also named artificial muscle. Application of square potential waves has shown important bending movements in the films, which can be repeated for more than 500 cycles without damaging its mechanical integrity. Furthermore, the actuator is able to push a huge amount of mass, as it has been proved by increasing the mass of the passive pPLA up to 328% while keeping the mass of electroactive PEDOT unaltered.

The mechanism of adhesion and graft polymerization of a PNIPAAm thermoresponsive hydrogel to polypropylene meshes.

In this study, a commercial and fully flexible monofilament mesh has been used for the deposition of a thermosensitive hydrogel, generated by graft copolymerization of N-isopropylacrylamide (NIPAAm) and N,N'-methylene bis(acrylamide) (MBA) monomers. The mechanism of adhesion and graft copolymerization have been elucidated combining micro- and standard spectroscopy techniques such as Raman spectroscopy, FTIR spectroscopy and XPS, before and after the activation of the polypropylene (PP) fibre surface by using oxygen-plasma. The good covalent interactions among NIPAAm monomers and PP fibres, and the hydrogel chain growth in such flexible bidimensional structures, were demonstrated. Additionally, the thermoresponsive properties of PNIPAAm were obtained (VPTT behaviour). The bilayer system is stable below and above a low critical solution temperature (LCST) of 33.2 °C.