Biological response against foreign implants often leads to encapsulation, possibly resulting in malfunction of implants devices. The aim of this study was to reduce the foreign body reaction by surface modification of biomaterials through layer-by-layer deposition of type I collagen (COL)/hyaluronic acid (HA) multilayer films. Polydimethylsiloxane (PDMS) samples were coated with alternative COL and HA layers with different layers. We found that the
Biological response against artificial implants leads to encapsulation of implants, which often causes malfunction of implants or patients’ agonies due to capsular contracture [
Macrophages can be considered as a hallmark due to their critical roles in guiding through the entire wound healing process [
Natural polymers are usually thought to be biocompatible and suitable for biomedical applications. Among natural polymers, collagen (COL) and hyaluronic acid (HA), the main components in the extracellular matrix, are frequently used to prepare biomimicking coatings to improve the biocompatibility of biomedical devices [
The aim of the current study was to evaluate the modification of COL/HA on a polydimethylsiloxane (PDMS), a silicone that is widely used in breast implants, for reducing the foreign body reaction. PDMS substrates were modified with COL and HA by a simple layer-by-layer (LBL) deposition. This technique is based on alternate adsorption of positively and negatively charged macromolecules to build an ultrathin polyelectrolyte multilayer (PEM) film onto a substratum [
Reagents were received from Sigma-Aldrich (St. Louis, USA) unless specified otherwise. Ethidium homodimer-1 (EthD-1) was purchased from Fluka, USA. N-(1-Naphthyl)-ethylenediamine dihydrochloride (NED) was purchased from R.D.H. (Germany). Porcine collagen (3.0 mg/mL, pH ~ 2.3), obtained from SunMax biotechnology (Tainan, Taiwan), was purified from specific pathogen free porcine skin and contained 96–98% type I collagen with the remainder being type III collagen. Medical grade hyaluronic acid (Mw 1,500 kDa) was received from Maxigen Biotechnology Inc. (Taiwan). Polydimethylsiloxane (PDMS, Sylgard 184) was purchased from Dow Corning (USA).
Cell culture medium consisted of high glucose
Part A/Part B of Sylgard 184 was mixed at ratio of 10 : 1 to prepare PDMS elastomers according to the manufacturer’s instruction. To prepare samples for
PDMS substrates were treated with radio frequency glow discharge oxygen plasma (50 W, 70 mtorr, 2 min, 23.6 sccm) in order to enhance their hydrophilicity [
The deposition of COL/HA multilayer films was analyzed by quartz crystal microbalance (QCM). The QCM apparatus and quartz crystals with a resonance frequency of ca. 9 MHz were purchased from ANT Inc. (Taipei, Taiwan). The gold electrodes for the QCM head were first immersed in 10 mM 3-mercapto-1-propansulfonic acid solution in deionized water at room temperature for 1 day in order to create a negatively charged sulfonated substrate to facilitate the deposition of a layer of polyethylene imine (PEI, MW 750,000). Then, the functionalized substrates were alternately immersed in HA and COL solutions each for 10 min until the desired bilayer numbers were reached. After being proceeded with sterilization in 70% ethanol and air-dried, the QCM heads deposited with COL/HA multilayer films were inserted into the QCM apparatus for resonance frequency measurement. The resonance frequency was recorded after equilibrium at which the frequency shift rate was within 0.1 Hz/min. The adsorbed mass was calculated from frequency shift according to the Sauerbrey equation [
Raw 264.7 murine monocyte/macrophage cell line was obtained from Food Industry Research and Development Institute (Hsin Chu, Taiwan) and cultured under 37°C in 5% CO2 atmosphere by standard sterile cell culture procedures. The cell seeding densities were 6 × 104 cells/cm2 for cell adhesion/cytotoxicity analysis and 3 × 105 cells/cm2 for nitric oxide (NO) generation experiment.
Cell adhesion was determined by an LDH assay, modified from a previous procedure [
Cytotoxicity of COL/HA multilayer films was evaluated by staining of cell culture with calcein AM (for living cells) and EthD-1 (for dead cells). The cells were cultured on different substrates for 24 h, followed by incubation with the fluorescent dye for 30 min at 37°C. The reagent was then rinsed with PBS and cellular uptake of the fluorescent dyes was analyzed by fluorescent microscopy. The excitation and emission wavelengths for calcein AM were 488 nm and 505–530 nm, respectively, while those for EthD-1 were 543 nm and 560 nm, respectively.
The extent of activation in RAW 265.7 cells adhering on the substrates was evaluated by nitric oxide (NO) release. First, cells were seeded and incubated on the substrates for 12 h. Then the culture medium supplemented with lipopolysaccharide (LPS) replaced the culture medium for another 24 h of culture. Next, the cell medium was transferred into 96-wells plate and analyzed using Griess reagent (Promega, USA). In brief, equal volume of 1% sulfamide in 5% phosphoric acid in deionized water was added to the cell medium, followed by an addition of 0.1% NED solution for reaction. NO release was analyzed by the absorbance of 570 nm.
The animal procedure followed the ethical guidelines of Care and Use of Laboratory Animals (National Taiwan University, National Institutes of Health Publication number 85-23, revised 1985) and was approved by the Animal Center Committee of National Taiwan University. Prior to implantation, the samples were sterilized in 70% ethanol and then soaked in sterilized PBS. Male Wistar rats weighting ~500 g were first anesthetized by intravenous injection of ketamine. The bilateral sides of the backs of rats were shaved, depilated, and disinfected with 70% iodine. Next, the samples were then subcutaneously inserted into incisions made on the bilateral sides, as shown in Figure
(a) A schematic sketch of the implanted sites in rats. The white circles represent the implanted samples. (b) A schematic sketch of the tissues around an implant, where the epidermis side towards strata cornea is denoted as the epidermal layer and the dermal side towards hypodermis is denoted as the dermal layer.
The specimens were processed by standard histological techniques. Briefly, the PDMS samples along the surrounding tissues were first dehydrated by soaking in a series of ethanol solutions (from 0% to 95%; 30 min each) and 100% ethanol (overnight). The samples were then soaked in xylene before placing them in 60°C paraffin wax for 2 h, followed by cooling in an ice bath. The samples were then sliced in half in order to remove the PDMS samples from the tissues and then embedded in paraffin wax again. Tissue slides were prepared with microtome incision of embedded tissues into 6
The slides of the surrounding tissue on two sides of each sample, the epidermal layer towards skin and the dermal layer towards hypodermis (Figure
All statistical data were performed with the GraphPad InStat software (Instat 3.0, GraphPad Software, USA) by Student-Newman-Keuls multiple comparisons test.
QCM measurement was applied to investigate the development of COL/HA multilayered films in this study. However, the base substrate of QCM chips is gold, which is quite different from the PDMS substrate. Unfortunately, it is very difficult to duplicate the PDMS surface on the gold substrate. Therefore, the results from the QCM measurement are not necessary to represent the actual deposition of COL/HA multilayer films on the PDMS samples. Nevertheless, we think that the results could provide a clue for the growth of COL/HA multilayer films.
The development of COL/HA multilayer films showed an approximate linear growth on the adsorbed mass in respect of bilayer numbers between 45, 86, and 200
Mass accumulation of COL/HA multilayer films determined by QCM. The adsorbed mass was calculated from frequency shift according to the Sauerbrey equation.
Compared to the PDMS control, COL/HA multilayered films decreased the adhesion of RAW265.7 cells (Figure
The adhesion of RAW 264.7 cells on the untreated and COL/HA coated PDMS after 1 or 2 days of culture. Multilayer films of COL/HA are represented by [COL/HA]
Since the cease in cell proliferation on [COL/HA]20 may be due to potential cytotoxicity of the films, the live and dead staining was used to verify the liability of the cells on [COL/HA]20. We hardly found any dead cell on all COL/HA modified substrates, while more than 98% of the cells were found alive on the PDMS control. The result indicates that the decrease in cell numbers on the COL/HA deposited substrates is not due to cytotoxicity of the COL/HA multilayer coating.
The morphology of RAW265.7 cells on different substrates was observed using phase contrast microscopy (Figure
Microscopic images showed the morphology of RAW 264.7 cells on different substrates after 1 day of culture (200x for the large images and 400x for the inserted images). Arrows indicate the areas in the inserts. Multilayer films of COL/HA is represented by [COL/HA]
The decrease in cell affinity of COL/HA multilayered films with increasing bilayer numbers is consistent with the results on other polyelectrolyte multilayer (PEM) systems [
As shown previously, RAW265.7 cells adhering to the untreated PDMS showed a sign of activation with spreading and dendritic morphology, while the cells culturing on COL/HA multilayer films did not seem to be activated in terms of morphology. The production of NO by RAW265.7 cells was further evaluated on [COL/HA]20. LPS was added to stimulate macrophage activation, otherwise the NO production was too low to differentiate the effect of [COL/HA]20. The addition of LPS decreased cell numbers after 24 h of culture (Figure
RAW 264.7 cells were seeded on PDMS or 20 bilayer-COL/HA films ([COL/HA]20) for 12 h, followed by 24 h incubation with and without LPS induction (50 or 500 ng/mL). (a) The densities of RAW 264.7 cells; (b) microscopic images from the cells incubated without LPS or with 500 ng/mL LPS; (c) nitric oxide (NO) productions of RAW 264.7 cells.
The anti-inflammatory effect might come from the physical and chemical properties of COL/HA multilayered films. The reduction in cell attachment to COL/HA multilayered films may affect the subsequent cellular events such as proliferation and differentiation [
The untreated PDMS and [COL/HA]20-coated PDMS were implanted under bilateral sides of the backs of rats and explanted after 3 weeks of implantation. In the excised subcutaneous tissue surrounding implanted samples, it was observed that all samples were surrounded by fibrous capsules. The capsules identified as dense connective tissues that consist of fibrocytes, collagen, and blood vessels with some common inflammatory cells [
(a) Hematoxylin and Eosin staining of tissue sections around the PDMS and [COL/HA]20-coated implants at the epidermal layer towards stratum cornea and the dermal layer towards hypodermis after 3 weeks of implantation. Arrows indicated FBGCs. (b) Statistical analysis of the capsular thickness around implants.
Further analysis of capsular thickness indicated the formation of thinner capsules surrounding the [COL/HA]20 sample when comparing with the unmodified PDMS. The thickness of capsules around the epidermal and dermal sides of the [COL/HA]20 samples was decreased by 29.14% and 56.61%, respectively, compared to the same side of PDMS (Figure
This study demonstrates the potential of layer-by-layer COL/HA multilayer films to reduce the foreign body reaction against implants. Both COL and HA are biodegradable and will be resorbed quickly after implantation. In this study, we only evaluated the outcome after 3-week implantation, and thus long-term efficacy remains under investigation. Nevertheless, the properties of PEM films can be further tuned by adjusting deposition parameters such as the types of polyelectrolytes, pH, and ionic strength of polyelectrolyte solutions for finding better coatings. Furthermore, biomolecules such as anti-inflammatory drugs can be incorporated into PEM films to prevent capsule formation [
In this study, we demonstrated that COL/HA multilayer films reduced the adhesion, proliferation, and activation of macrophages
The authors declare that there is no conflict of interests regarding the publication of this paper.
The authors gratefully acknowledge financial support from National Science Council, Taiwan (Grant nos. 96-2218-E-002-009 and 97-3114-E-002-001).