ORIGINAL PAPER
The effect of gold clusters on the autoxidation
of poly(3-hydroxy 10-undecenoate-co-3-hydroxy octanoate)
and tissue response evaluation
Derya Burcu Hazer&Baki Hazer
Received: 19 December 2009 / Accepted: 19 February 2010 / Published online: 19 March 2010 # Springer Science+Business Media B.V. 2010
Abstract We describe the synthesis and characterization of gold clusters embedded into poly (3-hydroxy octanoate-co-3-hydroxy-10-undecenoate) (PHOU), and evaluated the tissue response of the material upon implantation onto muscle tissue (rat). For this aim, PHOU was obtained by Pseudomonas oleovorans from octanoic acid (OA) and 10-undecenoic acid (UA) with a weight ratio of 50:50. The unsaturated co-polyester film in which HAuCl4 was
dispersed was exposed to air at room temperature to produce gold clusters embedded into cross-linked PHOU. The cross-linking kinetics of the gold catalyzed PHOU autoxidation was followed by sol-gel analysis. In vivo tissue reactions of the cross-linked PHOU embedded gold clusters were evaluated by subcutaneous implantation in rats. The rats appeared to be healthy throughout the implantation period. No symptom such as necrosis, abscess or tumor genesis was observed in the vicinity of the implants. Retrieved materials varied in their physical appearance after 6 weeks of implantation. AFM and SEM micrographs of the PHOU containing gold clusters were also taken.
Keywords Gold clusters . Poly (3-hydroxy octanoate-co-3-hydroxy-10-undecenoate) . Autoxidation .
In vivo biocompatibility
Introduction
Poly (3-hydroxy alkanoate)s, PHAs are a class of reserve polyesters produced by a large number of bacteria when subjected to metabolic stress. [1–6] Pseudomonas oleovorans is a very versatile bacterium for PHA production because it can produce medium-long chain length polyesters (m-lclPHA) [7, 8] from a wide variety of carbon substrates, including phenyl, bromine, arylalkyl, ester, branched alkyl, and vinyl groups [9–17]. Biosynthetic and chemical modification reactions of the PHAs have been performed to improve the mechanical and thermal properties for medical and industrial applications [18, 19]. Especially, the vinyl groups of the PHOU were most attractive for further chemical modifications in view of the epoxidation, hydroxylation, carboxylation and amine derivatization. [20–26] Cross linking reactions of the unsaturated side chains of these PHAs can make them non-sticky, easily handled material. [27–31]
Inorganic–organic hybrid PHAs were obtained by link-ing a polyhedral oligomeric silsesquioxane containlink-ing seven isobutyl groups and one mercapto propyl group (POSS-SH) via a free radical addition reaction to the side-chain double bonds of PHOU. [32] The covalent linking of POSS-SH to PHOU increased the heat stability and the glass transitions and melting points could be tailored between 48 and 120 °C. Encapsulated small metal particles, especially gold and silver, possesses a number of unique attributes, including size monodispersity, core-shell pro-cessability, air stability, and intriguing optical, electronic, magnetic, catalytic and chemical/biological properties. [33–39] Gold plays a special role in nanoscience and nanotechnology, due to the fact that gold is the most stable noble metal at the scale, so the designers of any nano-device requiring metallic components are likely to favor-ably consider gold. [40,41]
D. B. Hazer
Department of Neurosurgery, Çankırı Government Hospital, 18100 Çankırı, Turkey
e-email: burcuhazer2003@yahoo.com B. Hazer (*)
Department of Chemistry, Zonguldak Karaelmas University, 67100 Zonguldak, Turkey
e-mail: bkhazer@karaelmas.edu.tr e-mail: bhazer2@yahoo.com DOI 10.1007/s10965-010-9413-5
Biomaterials have an enormous impact on human health care. Applications include medical devices, diagnostics, drug delivery systems, and tissue engineering [42]. Bio-compatibility of biomaterials is usually tested in two ways: in vitro and in vivo. Following the implantation of any tested item or material into a living organism (in vivo), aseptic tissue inflammation develops at the site of implan-tation [43, 44]. Macrophages are important regulators in this aseptic inflammation process. They penetrate into the leukocyte barrier and phagocytize cell detritus and produce degradation products from the tissue and implanted material [45–47]. When a biodegradable polymer (including PHA) is implanted, macrophages and foreign body giant cells phagocytize and resorb the polymer and the biodegradation process is initiated [48–50]. Besides phagocytosis, macro-phages also initiate the formation of granulation tissue. The granulation tissue response is characterized by fibroblast infiltration and neoformation of blood capillaries [51].
Gold nanoparticles have drawn particular attention due to their biocompatibility, which makes them attractive for use in vivo as nanoscale biomaterials [52–54]. Gold nanoparticles are known to decrease neovascularization which is an important step in the inflammation process by inhibiting Vascular Endothelial Growth Factor (VEGF165) [55]. The gold nanoparticles can also catalyze nitric oxide (NO) production from endogenous S-nitrosothiols (RSNO)s in blood serum. The process is ascribed to the formation of the Au-thiolate on the surface of gold nanoparticles. Therefore, whenever the gold nanoparticles are used as a probe in the living body, a drug, or a component in drug excipient, it might trigger the oxidative stress in the microenvironment, accordingly. [56] Noble metal-based nanoparticles can kill cancer cells synergistically with conventionally used radiotherapy. [57] For selective cell apoptosis, the external photon energy with narrow-spectrum light, e.g., near-infrared light, can activate these gold nanoparticles, which resonate with the light, and then convert the light energy to heat, thereby rupturing the targeted cancer cell, but not the healthy tissues adjacent to the tumor. [58]
This work represents a novel gold-catalyzed oxidation of side-chain vinyl-terminated bacterial polyesters. The potential use of this polymer in a wide variety of biomedical applications was assessed through in vivo biocompatibility tests. Gold nanoparticles might aggravate foreign body reaction via increasing NO synthesis, but on the other hand, the inflammation subsided via inhibiting neovascularization. Therefore with this study we have tried to illuminate the
impact of gold catalysis of on the bacterial polyester on the foreign body reaction and tissue inflammation.
Experimental
Synthesis of microbial copolyesters
The unsaturated copolyester was prepared according to the procedure described in our recent papers. [26, 27] Briefly, Pseudomonas oleovorans were grown on octanoic acid and 10-undecenoic acid for the synthesis of PHOU, with the weight ratio of 50:50. The double bond content of the unsaturated co-polyester was found to be 11 mol% for PHOU. The molecular weights of the polyester was found to be Mn=64,000, Mw=311,000 for PHOU.
Preparation of PHA films containing AuNPs
All chemicals and solvents were supplied from Aldrich. THF was distilled before use.
A series of gold nanoparticles embedded into PHOU were prepared by modifying a previously reported proce-dure [59,60]. In a typical procedure, a PHA sample (0.2 g) was dissolved in 10 mL of THF. To this solution was added HAuCl4 (0.020 g) and vigorously stirred at room
temper-ature for 10 min. The solution was poured into a Petri dish (=4 cm) and the solvent was allowed to evaporate leaving a pink colored, thin polymer film. The solvent cast film was exposed to air for the autoxidation process. Sol-gel analysis of a piece of polymer film sample was carried out to determine the amount of cross-linked polymer formed at different time intervals. Cross-linked polymer was isolated by sol-gel analysis in chloroform. The gold content in the hybrid polymer was analyzed when cross-linked portion was more than 60 wt%.
Polymer characterization
1
H NMR spectra were recorded in CDCl3at 17 °C with a
tetramethylsilane internal standard using a 400 MHz NMR AC 400 L.1H NMR spectra of the swollen cross-linked co-polyesters in CDCl3 were also taken. 1H NMR technique
was also used to calculate double bond mol% of unsaturated PHA co-polyesters. The double bond content of the unsaturated polyester was calculated from the 1H NMR spectra using the equation given below:
The FT-IR spectra of the PHA films cast from CHCl3
solutions were recorded using a JASCO model 300E FT-IR spectrometer.
A Perkin Elmer Atomic Absorption Spectrometer (AAS)—Analyst 800 was used to determine gold wt % in the graft copolymer samples.
Scanning electron micrographs were taken on a JEOL JXA-6335 FS scanning electron microscope (SEM) with semi quantitative-Inca-energy dispersive X-ray spectrosco-py (EDS) for elemental analysis. The specimens were frozen under liquid nitrogen then fractured, mounted, and coated with palladium, gold, and carbon. The SEM was
operated at 15 kV, and the electron images were recorded directly from the cathode ray tube on a Polaroid film.
An AFM (Atomic Force Microscope) Q-Scope instrument with a Universal SPM (Scanning Probe Microscope) was used for taking surface images of the samples.
Swelling experiments of the pure cross linked PHA samples were carried out in chloroform. The polymer films were allowed to remain in solvent for 24 h at room temperature and the swollen films were weighed as soon as they were taken out from the solvent. The swelling degrees of the polymers at equilibrium were determined using the equation of swelling ratio, qv[61].
qv¼ Volume of swollen polymer Vswollen polymer
=Volume of dry polymer Vdry polymer
Vdry polymer ¼ mdry polymer=density of dry polymer
Vswollen polymer¼ mswollen polymer mdry polymer
=density of chloroform
þ Vdry polymer
Densities of the dry PHAs were measured in a 25 cm3 uncertified BLAUBRAND picnometer NS10/19 with ther-mometer at 29 °C using water as non-solvent to fill up the picnometer. Density of PHOU-5050 was 1.021 g/cm3.
In vivo implantation
Female albino Wistar rats with an average weight of 250 g were used in this study. They were anaesthetized with intraperitoneal injection of 0.1 mL/kg alphazyn and 0.3 mL/kg ketamin mixture. All procedures were carried out in compli-ance of Hacettepe University Ethical Commitee. The polymer films were prepared in dimensions 10×12×0.3 mm. Then, they were sterilized by ethylene oxide gas with an exposure time of 8 h. Under an operating microscope (Zeiss, 3,5*), a 5 cm midline incision was made on the back of the rat. The spinous processes of the vertebrae were the landmark to standardize the placement of different types of polymers in all rats. All the polymer blocks were placed in subcutaneous pockets created by blunt dissection and 7.0 nylon sutures (Ethilon, Ethicon) were used to anchor the grafts to the fascia in order to prevent the risk of displacement.
Graft harvesting
The rats were sacrificed on the 7th day and on the 45th day postoperatively. Then polymer samples attached to the
subcutaneous tissue and muscle fascia were harvested from each animal and each block was then analyzed for histological examination and SEM evaluation. The larger piece of samples was immediately fixed in a 10 wt% formalin solution for several days prior to processing. They were then embedded in paraffin wax, cut into 5 μm thick sections and stained with hematoxylin-eosin and Mason’s trichrome. Several days later, physical and histological microscopic findings were evaluated.
Histological observation
Polymer specimens were harvested with the gluteal muscle underneath on indicated days and were fixed and embedded in paraffin and sectioned at 5 µm in thickness. The sections were prepared to cut and stained with hematoxylin-eosin (HE) and Masson’s trichrome sepa-rately at 7th and 45th days of operation. The histolog-ical sections were observed by using an opthistolog-ical microscope with different magnifications. The type and the intensity of the inflammatory reaction were deter-mined and the extent of healing was analyzed in terms of collagen synthesis, coating presence and degree of tissue infiltration into the implant. Interpretation of inflammatory reaction in different polymer groups was performed by using a new scale which is a modified
form of Marious et al. [49]. In the scale, we have added giant cell count and newly formed blood vessels. For each polymer sample, the connective tissue capsule formation was analyzed and the thickness of the capsule surrounding the polymer sample was measured from four different standardized areas, one on each side of the implant. The final value was recorded as an average of these four different measurements. The fibroblast prolif-eration and inflammatory reactions were analyzed in these capsules and scored according to 4-point evaluation scales [62].
Results and discussion
Autoxidation kinetics of the PHOU films with gold nanoparticles
In order to study the effect of the gold clusters on the cross-linking, the unsaturated polyester (PHOU) which contains double bonds at the end of some side chains was used (Scheme 1). PHOU copolyester was biosynthesized from the equal mixture of octanoic acid and 10-undecenoic acid using Pseudomonas oleovorans.
Scheme 1 Autoxidation of the PHOU in the presence of the gold NPs formed by the reduction of Au3+by the radicals occurred during the autoxidation
Run no. Film preparation AuNPs in autoxidized-PHA PHOU HAuCl4 Thickness b(wt%) Size (nm)
(g) (wt%) (mm) 22 1.6 22 1 12 800–1,000 23 1.3 12 1 10 800–1,000 40 1.2 – 1 – – 201 0.2 0.50 0.1 200 202 0.2 0.97 0.1 264 205 0.2 2.4 0.1 1.9 100–300 210 0.2 4.7 0.1 3.1 333 220 0.2 9.1 0.1 7.1 74–148 230 0.2 12.9 0.1 9.4 65–217 240 0.2 17.7 0.1 14 90–136 191a 2.0 20.0 1 7.7 800–1,000
Table 1 Autoxidation results of the PHOU films containing gold clusters
a
Reduced with NaBH4in MeOH. b
Inspiring by gold catalyzed oxidation reactions, we have used the gold clusters in the cross-linking reactions of the unsaturated microbial polyesters during the autoxidation process. Effect of the gold clusters on the autoxidation process of the PHOU has been discussed merely, in this work.
Generally, free radicals that are naturally generated in situ during the autoxidation process are used as reducing agents. Free-radical-induced metal NPs synthesis is well studied [63–66]. The presence of several in-situ-generated free radicals from the air oxygen such as ROO•, RO• and R• (R = PHA side chain) during autoxidation of drying oils
[67–69] could be useful for the reduction of metal salts to synthesize metal nanoparticles (MNPs) in situ. These radicals reduce the metal salt to metal (Scheme 1). This process does not require heating, and moreover the system is mild, renewable and non-toxic in nature.
Gold nanoparticles were formed into the PHOU films from auric acid reduced to metal by free radicals arising by the reaction with air oxygen. Formation of the cross-links was followed for several days by means of sol-gel analysis of a piece of PHA-film in chloroform. The amount of gold clusters was also analyzed by Atomic Absorbtion Spec-troscopy. The amount of gold clusters is in relation with the auric acid used in the feeding. Analysis results and experimental conditions of the PHOU films containing AuNPs have been listed in Table1.
As cross-linking improves, the gold nanoparticles are confined in the PHOU network and the gold nanoparticles are not released into the solution, but suspended in the
Fig. 1 Photographs of PHA-gold composite materials (#201 under yellow light, 201, 202, 205, 210, 220, 230 and 240 under white light) in CHCl3
Fig. 2 Autoxidation kinetics of the PHOU films with gold clusters. Effect of the gold nanoparticles formation: Reducing agent of the salt of gold is free radicals for #22 and # 23; sodium borohydride for #191. Original PHOU without gold clusters (#40)
Fig. 3 a. Variation of the cross-linked polymer formation against autoxidation time of the series of the PHOU films with gold clusters (#201–#240) and without gold clusters (#40). The effect of the gold content on the induction period of the autoxidation. b. Variation of the swelling degree of the cross-linked polymer against autoxidation time of the series of the PHOU films with gold clusters (#201–#240) and without gold clusters (#40)
swollen cross-linked polymer film. Figure 1 shows the photographs of the swollen polymer films containing AuNPs in chloroform. Yellow coloured PHOU gels with AuNPs can be seen.
Catalyst effect of the gold NPs [70,71] are well known. Gold is effective for oxidation reactions. In particular it catalyses alkene epoxidation [72]. To see the catalyst effect of the gold clusters and to prepare implant material, a series Fig. 4 a SEM micrographs and
semi-quantitative EDS analysis of the gold nanoparticles embedded into PHOU samples (201, 205, 210, 220, 230 and 240). XRD spectra obtained by the EDS analysis for the hybrid polymer films contain charac-teristic gold signals at 1.7 and 2.2 keV. b AFM micrographs of PHA sample #23. Size of gold micro spheres is 800–1,000 nm
of the PHOU cast films containing HAuCl4varied from 12
to 22 wt% (#22, #23 and #191 in Table1) were exposed to air at room temperature for the autoxidation process leading to formation of gold nanoparticles (NPs) and a network structure. The autoxidation time versus cross-linked poly-mer (wt%) and swelling degree were plotted. Typical plots of the autoxidation time versus cross linked polymer and swelling ratio were shown in Fig. 2. In this Figure, the initial cross-linking has started on the 70th day of the autoxidation for the pure PHOU (Induction period is the time when cross linking starts.) (induction period is 70 days) while the induction period of the PHOU samples with AuNPs was 2–3 days. Typical catalyst effect of the gold NPs on the oxidation process was observed in this way. We have also calculated autoxidation rate constants by using the plots in Fig.2. Taking the first swelling degree as qvo and that of the following as qv, from the plot of the
Lnqvo/qv versus autoxidation time, autoxidation rate
con-stants were calculated by using the beginning slope of the linearity as 0.042 and 0.046 day−1 for pure PHOU and PHOU having gold clusters: # 22, respectively. The AuNPs in the PHOU samples shortened the induction period to 2– 3 days from 70 days while they did not influence the cross-linking reaction rate.
Is the preparation method of the gold nanoparticles into the PHOU films affected on the autoxidation process? To answer to this question, in the case of sample #191, gold nanoparticles were in the beginning formed by the addition of an external reducing agent, NaBH4, to the polymer
solution containing auric acid [63]. Then the solvent casted film containing AuNPs was exposed to air for the autoxidation process. When we compare the autoxidation kinetics of the sample #191 with #22 (#22 is one of the samples with AuNPs formed by free radicals arose during Fig. 4 (continued)
Fig. 5 In vivo visual appearence of the PHAs on the 0 (a), 7th (b) and 45th (c) days implantation: original-PHOU (#40), PHOU with gold nps (#22), PHOU with gold (#23). There is a fine capsule formation around PHOU with gold nps on 7th and 45th days of implantation. The edges of the polymer samples (#23 and #22) get rounded on day 45th of implantation, which is a sign of biodegradation. b: bubbles, C: capsule
autoxidation), we have noticed that the gold catalyzed autoxidation process was independent of the method of the gold cluster preparation.
The effect of film thickness on the gold catalyzed oxidation was also evaluated from the data in Table 1. When we compare the autoxidation kinetics of the samples #22 with the film thickness 1 mm and #240 with the film thickness 0.1 mm, we see that they have the same induction period.
In another scenario, we designed a set of experiments to determine the the effect of the amount of the Au nano-particles on the induction period. We prepared a series of PHOU films containing HAuCl4 varying from 0.5 to
17.7 wt% (#201, #202, #205, #210, #220, #230, #240 in Table 1). Figure 3 shows the autoxidation kinetics with various degrees of cross-linked polymer (Fig. 3a) and swelling degree (Fig. 3b) against the amount of gold clusters (wt%). PHOU films containing 2.4 and more wt% of gold nanoparticles were cross-linked immediate after 3 days of autoxidation (induction period is 3 days), while PHOU films containing 0.5 and 1.0 wt% of gold nano-particles were cross-linked after 15 days of autoxidation (induction period is 15 days). Higher gold content causes shorter induction period. As the gold content decreases, the induction period lasts longer. As we mentioned before, in the case of original PHOU without gold clusters, an
induction period of 70 days was observed. We have also evaluated swelling and cross linking values of the PHOU samples at the end of the 120 days of autoxidation time. In view of the lower swelling and the higher crosslinking for the control PHOU sample without gold, PHOU samples containing AuNPs indicated less dense crosslinking structure because their network cells are full of gold clusters.
The series of the PHOU samples with confined gold clusters in the network structure were also analyzed by taking SEM micrographs. Figure 4a shows SEM micro-graphs and semi-quantitative EDS analysis of the gold nanoparticles embedded into PHOU samples (#201, 205, 210, 220, 230 and 240). XRD spectra obtained by the EDS analysis for the hybrid polymer films contain characteristic gold signals at 1.7 and 2.2 keV. AFM micrographs of the samples #23 are shown in Fig.4b. As the materials undergo phase transitions, they often have changes in their surface structure that can be readily imaged with an AFM.
As we mentioned before, film thickness is not influenced on the gold catalyzed oxidation kinetics. However, film thickness influences the size of gold cluster. Table 1 also shows the size of gold cluster in the PHOU composites. Spherical gold clusters with 0.8–1.0 μm were observed in the PHOU films of 1 mm thickness (#22 and #23, the SEM pictures are not shown), while 64–333 nm gold nano-Table 2 Histochemical finding of biocompatibility of different polymer types
Sample # Implant time(day) Collagen Inflammatory cells Giant cell Neovasculerization Capsule thickness (mm) Property
22 7. + 96 0 + 0.110 Loose 45. +++ 58 1 ++ 0.240 Moderate 23 7. + 132 7 ++ 0.160 Loose 45. + 62 1 ++ 0.080 Loose 40 7 ++ 86 0 +++ 0.280 Dense 45 ++ 98 1 +++ 0.420 Dense
Fig. 6 Sample #23 (PHOU-Au-gel-23), a: very thin capsule and no prominent cellular infiltra-tion, notice the degradation of the whole polymer block. Trichrome staining, 10× magni-fication. b: Same sample on 45th day; the capsule get thicker but inflammatory cell infiltration is still less, notice the degraded polymer in pieces and giant cell neighboring the polymer pieces, Trichrome staining, 40× magnification. P: polymer, C: capsule
particles were observed into the PHOU films of 0.1 mm thickness (#201, 202, 205, 210, 220, 230, 240).
In vivo implantation studies Physical appearance observation
Poly-3-hydroxy alkanoates have been widely used as biomaterials [73]. In this study we have compared the biocompatibility and biodegradation properties of the polymer samples #40 (PHOU-gel), #22 and #23 through aspects of histological sectioning. Film samples #23 and #22 (PHOU with gold nps) and pure PHOU-gel sample (#40 as control in Table1) were placed on the back of the rat in similar fashion with our previous study [74]. Throughout the implantation period antibiotics were not used. The rats were healthy, and no symptoms such as necrosis, abscess or tumor genesis was observed in the vicinity of the implants. These films were harvested on the 7th and 45th days of implantation. Figure5shows in vivo visual appearance of the PHAs on the 7th (a) and 45th (b) days implantation: PHOU-gel (#40), PHOU dispersed gold NPs (#22), PHOU dispersed gold NPs (#23). On day 7th, there is a fine thin capsule formation surrounding the polymers #23 and #22 which may indicate that there is
fairly mild foreign body reaction. However the capsule covering sample 40,the original PHOU sample, is thicker than the gold catalyzed polymer sample and this might show that foreign body reaction for sample #40 is more pronounced than the gold catalyzed samples. On the 45th day of implantation there was a slight muscle tissue overriding the edges of the polymers #23 and #22 which seemed as if the film was buried deep in the muscle tissue and fused with the muscle tissue itself. This behavior might also be a strong indicator of high level of biocompatibility. There was also bubbling on the surface of the samples #23 and #22 which can be a sign of the beginning of biodegradation. But the surface of sample #40 was smooth and a thick capsule was covering the entire sample without any sign of degradation. However, as we will discuss later, in SEM analysis of the PHOU samples with and without gold clusters after implantation, there was slight cracks which may be attributed to biodegradation.
Histology
As we have mentioned in our previous study [74], inflammatory cells such as polymorph nuclear cells and macrophages play an important role in the degradation and biocompatibility of different types of polymers on different Table 3 Swelling degrees (in CHCl3) of the PHA gels before and after implantation
Entry# Autoxdtn time, day Implant time (day) Crude polym. (g)
Crosslinked polymer (g) Swollen polymer, g Swelling degree, qv
23 225 – 0.048 0.040 0.400 7.1 7 0.037 0.022 0.525 7.3 45 0.036 0.025 0.334 9.3 270 – 0.022 0.016 0.193 8.4 40 235 – 0.050 0.048 0.422 6.2 7 0.020 0.019 0.160 6.0 45 0.057 0.055 0.405 5.3 330 – 0.068 0.066 0.152 1.9
Fig. 7 Sample #23, on day 7th of implantation. a Closer view of Fig.5a, the vessels under-neath the capsule close to the polymer block. Trichrome staining, 40× magnification. b: The giant cell formation neigh-bouring the vessels, sample #23 Hemotoxylen-Eosin staining, 40× magnification. P: polymer, e: endothelia, G: giant cell
stages of the inflammatory reaction. In this work, we have introduced a new modified form of the histological grading scale including neovascularization (Table2) [74].
All these cells are carried to the implantation site by new vessels; therefore neovascularization is an important com-ponent of the inflammation process [75]. When evaluating the capsule thickness, the sample #23 (PHOU-Au-gel) had the thinnest among all the polymer samples (0.080 mm, Table 2). Inflammatory cell reaction in each of the Au particle-containing polymer samples was fairly mild com-pared to the Au particle-free polymer sample. On the 7th day of implantation, a very thin capsule surrounded the polymer film with no prominent cellular infiltration (Fig. 6a). On the 45th day this capsule thickened but the cellular infiltration was still less (Fig.6b). These findings are strong indicators of the biocompatibility. When evalu-ating the host foreign-body cell reactions to composite materials, the specimens with gold particles appeared to have more foreign-body giant cells compared to the pure PHOU copolymer specimens. These multinucleated giant cells with recently formed capillaries were seen in close proximity to the surrounding fibrous tissue capsule in the implant-tissue border (Fig.6a and b).
These inflammatory cells are assumed to be involved in the degradation of the implanted polymers by releasing biologically active species such as free radicals into the area of implant. [76] Therefore these cellular activation in the implant-tissue border may be the main cause of the in vivo polymer degradation [77].
There is not much difference in collagen deposition in between Au particle-containing polymers and pure PHOU
samples. When the time dependent changes of capsule integrity is concerned, sample #40 had dense capsule at both 7th and 45th days, sample #22 had loose capsule at 7th days and moderate at 45th days and sample #23 had loose capsule integrity at both 7th and 45th days. Neovasculari-zation is vital in many of the pathological conditions such as wound healing and chronic inflammation via transport of the inflammatory cells to the region. [75–77] Bhattacharya et al. [55] had found out that gold nanoparticles inhibit proliferation of Human Umbilical Vein Endothelial Cells (HUVEC) via binding to Heparin-binding VEGF 165 and therefore inhibit neovascularization. In our study, new vessel formation is also found to be low in PHOU-Au samples when compared to the pure PHOU-gel sample #40 (Table 3). In sample #23 these vessels are seen in close proximity to the implant-tissue border (Fig. 7a), which is the initial site of foreign body reaction. Since major inflammatory cells are carried to the inflammation site by newly formed vessels, it is logical to find low cellular count accompanied by few blood vessel formations in implant tissue border of highly biocompatible material. [49, 62] Therefore, gathering the data we have collected in our study one can conclude that when gold particles are integrated into a polymer, the biocompatibility is increased. As we have mentioned in our previous study [74], inflammatory cells such as polymorph nuclear cells and macrophages play an important role in the biocompatibility of different types of polymers on different stages of inflammatory reaction. If a foreign body is placed inside of a living tissue, different population of these cells migrate to this area and try to restrict and discard this foreign body out of the living Fig. 8 SEM micrographs of the
implanted fractural surfaces: a PHA sample #23 before im-plantation (bar shows 10μm), b: same sample after 45th days of implantation, cracking of the polymer seen on the upper left corner (bar shows 10μm). c: PHA sample #40 before im-plantation (bar shows 100μm), d: PHOU sample #40 after 45th days of implantation, cracking of the polymer is seen (bar shows 10μm)
organism. This restriction is done by the formation of capsule around the foreign body. Additionally these cells are gathered to the implantation area with newly formed vessels. These data reflects the degree of the biocompati-bility of this foreign structure. Therefore, if this foreign body is harmful or non compatible to the living organism, a more intense foreign body reaction is seen with an increase cell population and newly formed vessel count in the capsule. In our study, we have detected less cellular infiltration and lower blood vessel formation in the capsule of gold clustered PHO films compared to the pure ones. This means a better biocompatibility compared to unpro-cessed PHO films. Our data is also similar with other studies in literature. They have demonstrated the biocom-patible properties of gold nanoparticles such as non toxicity, non immunogenic and high tissue permeability without hampering cell functionality [78]. Consequently, in vivo results suggest improved biocompatibility upon inclusion of Au nanoparticles into the PHOU film which may be due to the specific biological response caused by the Au atoms gradually released from the degraded PHOU with Au NPs film after implantation. Further studies are naturally required to better understand.
Biodegradation
It is known that biodegradable polyesters are degraded through hydrolysis. [62] When a polymer film is implanted into the tissue, the first reaction in polymer structure is the water absorption of the polymer. [77] So swelling is the initial finding of biodegradation of a polymer. Table3lists the swelling degrees of the gels before and after implant. As observed from results shown in Figs.5,6,7and8, there are clear evidences of biodegradation of the PHOU film with Au clusters (#23). In consistency with this result, there was a considerable difference in between the swelling degrees of the polymer films before and after implantation for the sample #23. However the released Au atoms/ions never-theless remain undetermined. Several researches are stil being investigated.
Shelf life of the film samples with or without gold NPs is similar in both exposing to the air and in the implant position.
The SEM micrographs of PHOU with gold clusters were also taken after the 45th day of implantation. Figure8 shows SEM micrographs of the polymer films before and after implantation. It is considerable that on the 45th day of implantation slight cracking has started on the upper left corner of sample #23. Surprisingly, although there wasn’t any remarkable change in the gross appearance of sample #40 on 45th day (Fig.5c), SEM analysis revealed slight cracking of the polymer film on 45th day of implantation.
Conclusion
This work presents a new way to fabricate implantable structures, made of bacterial polyesters. We describe the gold-catalyzed oxidation of side-chain vinyl-terminated bacterial polyesters, and evaluated the tissue response of the material upon subcutaneous implantation into the rat. Gold nanoparticles in concentration higher than 2 wt% of initial HAuCl4 confined into the PHOU accelerated the
cross linking of the side chain double bonds during autoxidation. In vivo implantation results of the PHOU with gold clusters indicated that the biocompatibility was increased by the gold cluster inclusion of the PHOU sample. This novel implantable polyester containing gold NPs can be promising material for medical applications (ca. noble metal-based nanoparticles can kill cancer cells synergistically with conventionally used radiotherapy [57]).
Acknowledgement This work financially supported by grants from Zonguldak Karaelmas University Scientific Research Projects Commission grant# 2008-70-01-01 and TÜBİTAK grant# 108T423. The Authors wish to thank Kevin Cavicchi, Taner Erdoğan and Amitav Sanyal for their valuable discussions.
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