Photoactive Chitosan Switching on Bone-Like Apatite Deposition

Jan 19, 2010 - Molecular interactions, characterization and photoactivity of Chlorophyll a/Chitosan/2-HP-β-Cyclodextrin composite films as functional...
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Biomacromolecules 2010, 11, 309–315

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Photoactive Chitosan Switching on Bone-Like Apatite Deposition Valeria Chiono,† Piergiorgio Gentile,*,† Francesca Boccafoschi,†,‡ Irene Carmagnola,† Momchil Ninov,§ Ventsislava Georgieva,§ George Georgiev,§ and Gianluca Ciardelli† Department of Mechanics, Politecnico di Torino, Corso Duca degli Abruzzi 24, 10129 Torino, Italy, Department of Clinical and Experimental Medicine, University of Eastern Piedmont, Via Solaroli 17, 28100 Novara, Italy, and Laboratory of Water-Soluble Polymers, Polyelectrolytes and Biopolymers, Faculty of Chemistry, University of Sofia, James Bourchier Blvd 1, 1164 Sofia, Bulgaria Received October 13, 2009; Revised Manuscript Received November 25, 2009

The work was focused on the synthesis and characterization of the chitosan-g-fluorescein (CHFL) conjugate polymer as a biocompatible amphiphilic water-soluble photosensitizer, able to stimulate hydroxyapatite deposition upon visible light irradiation. Fluorescein (FL) grafting to chitosan (CH) chains was confirmed by UV-vis analysis of water solutions of FL and CHFL and by Fourier transform infrared spectroscopy (FTIR-ATR) analysis of CHFL and CH. Smooth CHFL cast films with 4 µm thickness were obtained by solvent casting. Continuous exposure to visible light for 7 days was found to activate the deposition of calcium phosphate crystals from a conventional simulated body fluid (SBF 1.0×) on the surface of CHFL cast films. EDX and FTIR-ATR analyses confirmed the apatite nature of the deposited calcium phosphate crystals. CHFL films preincubated in SBF (1.0×) solution under visible light irradiation and in the dark for 7 days were found to support the in vitro adhesion and proliferation of MG63 osteoblast-like cells (MTT viability test; 1-3 days culture time). On the other hand, the mineralization ability of MG63 osteoblast-like cells was significantly improved on CHFL films preincubated under visible light exposure (alkaline phosphatase activity (ALP) test for 1, 3, 7, and 14 days). The use of photoactive biocompatible conjugate polymer, such as CHFL, may lead to new therapeutic options in the field of bone/dental repair, exploiting the photoexcitation mechanism as a tool for biomineralization.

Introduction A bone regenerative composite material should mimic the nanostructure and chemical composition of the natural bone tissue, which is a complex assembly of parallel type I collagen nanofibrils (17-20 wt %) and HA crystals (69-80 wt %) precipitated on their surface.1 Bone substitutes should ideally bond to living bone and be mechanically compatible with the surrounding tissue. The essential requirement for a material to bond to living bone is the formation of an apatite layer on its surface upon contact with the in vivo environment.2 In vivo bioactivity can be predicted from in vitro tests using a simulated body fluid (SBF) with a similar ion concentration to that of human blood plasma. In our experiments, the original SBF solution was used having the same composition proposed in the work by Kokubo et al.3 This conventional SBF solution is highly supersaturated with respect to apatite and has been proposed to the Technical Committee ISO/TC150 of International Organization for Standardization as a solution for in vitro measurement of apatite-forming ability of implant materials. A material that rapidly forms apatite on its surface when immersed in SBF will bond to living bone in a short period after implantation. This method can thus be used for screening the bioactivity of materials before in vivo animal testing and is valid for materials that does not induce toxic or antibody reactions. Recently, biomimetic deposition of calcium phosphate from SBF has been shown to be an effective method for coating many * To whom correspondence should be addressed. Tel.: +39-011-5646969. Fax: +39-011-5646999. E-mail: [email protected]. † Politecnico di Torino. ‡ University of Eastern Piedmont Chemistry. § University of Sofia.

different types of biomaterials because complex three-dimensional shapes can be evenly coated.4,5 In addition, the structure of biomimetic calcium phosphate may more closely match the structure of natural bone mineral and, therefore, enhance implant fixation and bone regeneration.6 The deposition of HA crystals upon immersion in SBF has been generally attributed to ionic interactions between the scaffold surface and the SBF medium;7 moreover, if the material of study is bioactive, HA deposition has been found to occur within 7-14 days of immersion in conventional SBF.8 In this work, an innovative method to promote the deposition of biomimetic calcium phosphate was proposed based on the use of a photoactive conjugate polymer for the photodynamic stimulation of hydroxyapatite deposition from SBF fluid (or blood plasma). Water-soluble copolymers containing chromophoric groups have recently attracted a considerable interest as potential photosensitizers in several fields, including biomedical (as drug carriers, sensitizers, sensors)9 and environmental applications.10-12 In previous papers, several water-soluble photosensitizers able to absorb light in the near-UV visible spectral region have been investigated, generally containing naphthalene, anthracene, phenanthrene, or carbazole pendant groups and able to act as photocatalysts.13 Few general studies have also been carried out on photosensitizers containing porphyrine14 or xanthene chromophores, such as Rose Bengal.15,16 Until now, polymer photosensitizers have never been applied for photodynamic induction of biomineralization. The photosensitizer synthesized in this work was based on a natural biocompatible polymer, chitosan (CH), and a xanthene chromophore, fluorescein (FL; Scheme 1). CH is a linear

10.1021/bm901169v  2010 American Chemical Society Published on Web 01/19/2010

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Scheme 1. Chemical Formula of CHFL Conjugate Polymer Obtained by the Grafting of FL Chromophore to CH Glucosamine Units via Amide Bondsa

a In the chemical formula, the acetylated original units of CH were omitted.

polysaccharide composed of randomly distributed β-(1-4)linked D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit). It is produced from N-deacetylation of chitin. It is a low cost, biodegradable, biocompatible material with antibacterial and antiviral properties and has been widely used for pharmaceutical purposes and, in other fields, as a clarification agent for wastewater, an ingredient for food products (wine, beer, cheese), and a wet strength additive in the paper industry.17 The use of CH for tissue engineering as a scaffolding material has also been reported.18 However, CH has been reported to lack bioactivity, which severely limits its biomedical applications.19,20 FL is a fluorophore commonly used in microscopy, in a type of dye laser as the gain medium,21 in forensics and serology to detect latent blood stains,22 and in dye tracing.23 FL has an absorption maximum at 494 nm, an emission maximum at 521 nm (in water), and an isosbestic point (equal absorption for all pH values) at 460 nm. FL derivatives have been frequently grafted to CH substrates for analysis purposes or to obtain probes for pH/temperature evaluation in biological systems. For instance, CH nanoparticles for ocular drug delivery have been functionalized with FL to monitor their in vivo behavior in the rabbit cornea and conjunctiva by confocal microscopy and spectofluorimetry.24 On the other hand, CH-FL conjugates have been found to provide temperature and pH sensitivities, similar to those of pure FL, with the advantages of long-term stability and a fast equilibrium response.25 The aim of the current work was the development of a highly biocompatible photosensitizer, which becomes bioactive (i.e., able to activate hydroxyapatite deposition from a conventional SBF solution on the surface of the conjugate polymer film) by irradiation with visible light. The switch-on mechanism of HA deposition on the photoactive film surface, which has never been documented before, will allow the design of new material formulations to be used in the orthopedic and dental/craniofacial implant fields.

Experimental Section Materials. Filtered CH FG 90 (CH, lot No. TM661) was supplied from Primex Ingredients ASA (Karmsund, Norway). The degree of deacetylation (DDA ) 89.5%) was determined by NMR spectrum. The number average molecular weight (210 ( 10 kDa) and polydispersity index (Mw/Mn ) 2.17) of the polymer were determined, based on the

Communications Mark-Houwink-Sakurada equation, using the literature values of K ) 1.57 × 10 mL/g, R ) 0.79, and qMHS ) 0.95.26 FL was used as received from Alfa Aesar without further purification. The solvents (methanol, acetic acid, and acetone) were supplied from Sigma-Aldrich and were of analytical grade. Synthesis of Chitosan-g-Fluorescein (CHFL) Conjugate Polymer. CHFL was synthesized by a reported method.10 Briefly, CH amidation with FL was performed in 1 vol % acetic acid solution. CH (0.5 g) was dissolved in acetic acid solution (50 mL) at room temperature by continuous stirring for 1 day. After, a FL solution (50 mg FL in 5 mL methanol) was added dropwise over a period of 1 h. To complete the amidation, the mixture was intensively stirred using a magnetic stirring bar for 48 h. After, the mixture was kept 12 h under vacuum at 80 °C to dehydrate the CH salt formed and obtain amide linkages between CH and FL. The product was rinsed with methanol and acetone in sequence, and after its dissolution in water, it was exhaustively dialyzed (Sigma-Aldrich, cellulose tubing, cutoff 1200014000 g/mol) against water, basic (pH 8.2-8.5) and acid (pH 5.0-5.5), alternating (volume ratio: 1:10), for several days to remove unbound FL and CH. The dialysis was finished when no FL residues were detected by measurement of UV-vis spectrum of the dialysate (using UV-vis-NIR CARY 500 SCAN equipment.). The conjugate polymer was subsequently freeze-dried at -20 °C (SCANVAC Coolsafe). The content of FL in the CHFL conjugate polymer was determined by UV-vis spectroscopy to be 0.43 mol % (molar ratio with respect to the glucosamine unit of CH). Preparation and Characterization of Cast Films. CH cast films were produced by casting a few drops of a 1% (w/v) CH solution in 1% (v/v) acetic acid onto glass slides with 1.2 cm diameter. After drying in a vented oven (Micra9, Isca), film samples were washed until neutral pH of the washing solution. CHFL was dissolved in demineralised water at 37 °C, obtaining a solution with 0.75% (w/v) concentration. A few drops of the solution were cast on glass slides with, which were then left under a vented hood until complete evaporation of the solvent. Film samples were characterized for their morphology and composition by scanning electron microscopy (SEM, Philips 525 M, Acceleration Voltage 15 kV, Everhardt-Thornley detector) and energy dispersive X-ray (EDX) analyses, using the FEI QUANTA INSPECT 200 apparatus, equipped with EDAX Genesis Software, on specimens previously coated with Ag. Film samples were also analyzed for Fourier transform infrared-attenuated total reflectance spectroscopy (FTIR-ATR; diamond crystal with a penetration depth of 1.66 µm) in a PerkinElmer Spectrum One Spectrometer in the 4000-800 cm-1 wavenumber range. Bioactivity Tests in Simulated Body Fluid (SBF). SBF (1.0×) with a pH of 7.4 at 36.5 °C, buffered with 50 mM tris(hydroxymethyl)aminomethane and 45 mM hydrochloric acid, was prepared by the dissolution of reagent-grade chemicals in distilled water according to the method described by Kokubo et al.3 The inorganic ion concentrations (mM; Na+ 142.0, K+ 5.0, Mg2+ 1.5, Ca2+ 2.5, Cl- 148.0, HCO34.2, HPO42- 1.0, SO42- 0.5) were close to those found in human blood plasma.3 The SBF solution was metastable and calcium phosphate did not precipitate without external stimulation. In vitro bioactivity tests using SBF were performed on CH and CHFL cast films (0.5 × 0.5 cm2). Each sample was put into a transparent polystyrene vial containing 10 mL of SBF. Samples were incubated at 37 °C in a CH250 ACS Angelantoni apparatus. The SBF medium was refreshed at 2 day time intervals. In the case of the experiments carried out in the dark, vials were kept covered with Al during incubation, while for the experiments under irradiation with visible light, vials were incubated under the light of a lamp (W91575 lamp, 3.5 W, 220 V; emission maxima at 470 and 570 nm wavelength) belonging to the incubator equipment. Samples were withdrawn from SBF after 7 days incubation, then they were washed in deionized water to remove watersoluble salts, and, finally, dried for further analyses. Surface morphology and composition were evaluated by SEM and EDX analyses on

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Figure 1. FTIR-ATR spectra of the CH and CHFL films.

specimens previously coated with Ag. Moreover, FTIR-ATR analysis was carried out in the 4000-600 cm-1 wavenumber range. In Vitro Cell Tests. In vitro cell tests were performed on CHFL films after a preincubation of the samples in SBF for 7 days in the dark and under visible light irradiation, according to the previously reported procedure. Before cell seeding, material samples (10 × 10 mm size) were sterilized in a 70% ethyl alcohol solution (EtOH, Sigma) for 30 min, washed in PBS, and incubated with cell culture medium for 3 h. The medium was then discarded. MG-63 human osteoblast-like cells (ATCC, Rockville, MD) were grown in a controlled atmosphere (5% CO2; T ) 37 °C) in Iscove’s modified Dulbecco’s medium supplemented with 10% fetal calf serum (Sigma, Italy), 2 mM L-glutamine (Sigma, Italy), penicillin (100 U/mL), and streptomycin (100 µg/mL; Sigma, Italy) and 0.1 mM nonessential amino acids (NEAA, Lonza, Italy) for 3 weeks. Cells from up to three passages were used for all experiments. A total of 20000 cells/cm2 were seeded onto the film specimens and allowed to adhere and proliferate up to 14 days. MTT (3-Dimethylthiazol-2,5-diphenyltetrazolium Bromide) Colorimetric Assay. After culturing cells for 24 h and 3 days, the medium was removed; 10% MTT solution (5 mg · mL-1 in DMEM; Sigma, Italy) was added to the cell monolayers; the multiwell plates were incubated at 37 °C for an additional 4 h. After discarding the supernatants, the dark blue formazan crystals were dissolved by adding 100 µL of dimethylsulfoxide (DMSO, Sigma, Italy) and quantified spectrophotometrically (Secomam, Anthelie light, version 3.8, Contardi, Italy) at 570 nm. The results are reported as optical density units. Cells seeded on a cell culture plate, adequately treated for cell adhesion, were used as controls. The mean and the standard deviations were obtained from three different experiments. Samples were also analyzed under a fluorescence microscope (Leica DM2500). Mineralization Test. The alkaline phosphatase activity was measured using a colorimetric assay with p-nitrophenyl phosphate (Sigma, Italy). After 1, 3, 7, and 14 days, culture media from experimental groups were discarded and p-nitrophenyl phosphate substrate was added. The samples were incubated at 37 °C for 30 min in the dark. The

reaction was stopped with 1 N NaOH (Sigma, Italy), and the absorbance was read at 495 nm using a microplate reader. The mean and the standard deviation were obtained from three different experiments. Statistical Methods. All quantitative data were presented as mean ( standard deviation, unless otherwise noted. Statistical analysis was carried out using single-factor analysis of variance (ANOVA). A value of p < 0.05 was considered statistically significant.

Results and Discussion CHFL conjugate polymer was obtained as an orange powder. CHFL was soluble in water at a pH value lower than 7 up to a concentration of 1 g/L. FITR-ATR spectra of CHFL and CH were compared (Figure 1). In the CH FTIR-ATR spectrum, characteristic bands of CH were detected at 3400-3500 cm-1 wavelength range (N-H and O-H stretching), at 1644 cm-1 (amide I), and at 1586 cm-1 (amide II). Bands at 1150 and 1062 cm-1 (C-O-C asymmetric and symmetric stretching, respectively) and at 1025 cm-1 (C-O stretching vibrations of alcohol moieties) are typical of the CH saccharide structure. The amide I and amide II peaks of CHFL FTIR-ATR spectrum were shifted to lower frequencies (respectively, from 1644 to 1636 cm-1 and from 1586 to 1570 cm-1), which suggests a decrease in association via hydrogen bonds.27 On the other hand, the band at 826 cm-1 wavelength, attributed to the out-of-plane bending vibrations of C-H bonds belonging to aromatic rings of FL molecules, was visible in the FTIRATR spectrum of CHFL. The absorption band associated with the amide bonds between CH chains and FL (amide I band) partially overlapped with amide I and amide II bands of CH component. However, the shifting of amide I and amide II absorption to lower wavelengths for CHFL conjugate polymer as compared to pure CH, suggesting a decrease in association

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Figure 2. SEM micrographs and EDX spectra of films: CH upper surface (a) and fractured section (c); CHFL upper surface (b) and fractured section (d); bar ) 10 µm.

due to hydrogen bonding, was an indirect confirmation of the grafting. Moreover, FTIR-ATR spectrum of CHFL did not show characteristic bands of ester groups (ca. 1730 cm-1), confirming that FL units were grafted via amide bonds. However, the reaction between the hydroxyl groups of CH and the carboxylic groups of FL by dehydration may occur at some extent as previously documented.28 UV-vis absorption spectrum of an aqueous solution of CHFL (0.004% (w/v); data not shown) displayed the characteristic bands of FL chromophore. However, the absorption bands of CHFL water solutions displayed some broadening and a redshifting (absorption maximum at 481 nm wavelength) as compared to the ones of FL water solutions (absorption maximum at 475 nm wavelength). This feature confirmed that FL chromophores were covalently attached to the CH chains.17 For initial studies on CHFL conjugate polymer, cast films were produced on glass slides from solutions of CH and CHFL. Smooth CH and CHFL cast films with 4 µm thickness were obtained (Figure 2). EDX analysis was performed to confirm the chemical nature of the fractured sections of CH and CHFL films and to distinguish them from the glass support. EDS spectra showed the characteristic elements of CH and CHFL: carbon (C), nitrogen (N), and oxygen (O). CH and CHFL cast films were incubated in SBF medium at 37 °C for 7 days in the dark and under visible light exposure to study their bioactivity. CH film samples were found not to be bioactive after 7 days incubation in conventional SBF (Figure 3a,c). On the other hand, CHFL was not bioactive after incubation in SBF medium in the dark (Figure 3b), whereas the surface of CHFL film covered with calcium phosphate crystals with a “cauliflower-like” morphology after 7 days exposure to light in SBF medium (Figure 3d). EDX analysis carried out on the surface of CHFL films immersed in SBF for 7 days under continuous irradiation showed

that the Ca/P ratio of the deposited globular crystals was 1.61, which is very close to the value found for stoichiometric hydroxyapatite (1.67). Hence, EDX analysis confirmed the bioactivity of the CHFL photosensitizer under visible light irradiation. Finally, FTIR-ATR carried out on the surface of CHFL film after SBF treatment for 7 days under irradiation (Figure 4) confirmed the chemical nature of the deposited crystals. FTIRATR analysis showed weak bands associated with CHFL conjugate polymer at 1647, 1569, and 1460-1420 cm-1 wavenumbers and a very intense absorption peak centered at 1022 cm-1, which is typically associated with P-O stretching vibrations of HA crystals.29,30 The deposition of hydroxyapatite crystals on CHFL surfaces was demonstrated to occur after 7 days soaking in conventional SBF and switched on by visible light irradiation. The mechanism explaining the CHFL photobioactivity is currently under investigation. Two hypotheses for the deposition mechanism of HA, reported in the literature, could be applied also to explain the bioactivity of CHFL surfaces. The first theory considers the electrostatic attraction between oppositely charged ions (calcium and phosphate) from the SBF medium and a charged surfaceas the first event leading to the nucleation of HA-like crystallites.31 The second one assumes first the formation of negatively charged homogeneous nuclei of HA in the SBF solution that are then attracted by a positively charged film surface32 The latter phenomenon is defined as electrostatic adhesion and growth of HA particles (EAG process). The two types of mechanisms are based on electrostatic interactions between solution ions or crystallization nuclei and the material charged surface groups. The surface of CHFL films in contact with SBF medium is expected to have a positive charge due to the presence of the protonated amino groups of CH glucosamine units. However, electrostatic interactions

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Figure 3. SEM micrographs and EDX spectra of the surface of analyzed films after 7 days immersion in SBF (1.0×): (a) CH and (b) CHFL incubated in the dark; (c) CH and (d) CHFL incubated under visible light irradiation; bar ) 10 µm.

Figure 4. FTIR-ATR spectra of the surface of CHFL film after SBF treatment for 7 days under visible light irradiation.

between the positively charged groups of CHFL and solution ions or crystallization nuclei did not cause apatite deposition, as evidenced by the results of SBF test carried out for 7 days in the dark. The reason for this behavior could be the low protonation degree of CH at pH 7.4. Upon irradiation, electron and energy transfer mechanisms from the photosensitizer to nearby molecules (oxygen and water) occurred, leading to the formation of singlet oxygen, radical species, and superoxide radical anions.33 Electron transfer is also known to cause the formation of an instantaneous positive charge on the photosensitizer.33 The instantaneous formation of additional local positive charges on the substrate (on the chromophoric groups) via photoexcitation could be the reason for the enhanced attraction of negative ions or crystallization nuclei from SBF solution to the film surface. Viability of MG63-osteoblast-like cells preincubated in SBF for 7 days under visible light irradiation or in the dark was similar to the TCPS control after 24 h culture time, as shown

in Figure 5. After 3 days, viability of cells cultured on TCPS (p < 0.05) was significantly higher than for cells cultured on CHFL. No significant difference was detected between cell viability on CHFL films preincubated in SBF in the dark and under irradiation after 3 days culture time. The difference between the behavior of samples and the control could be associated with the higher proliferation rate of cells on TCPS. This hypothesis is confirmed by the analysis of cell morphology after actin-labeling with a fluorescent dye, showing well-adhered and spread cells on each CHFL substrate. No morphological differences were detected between cells adhered on CHFL substrates and TCPS (Figure 6). At each culture time, alkaline phosphatase (ALP) activity was enhanced with statistical relevance in the case of MG63osteoblast-like cells cultured on CHFL films preincubated in SBF for 7 days under continuous exposure to visible light as compared, evidencing that mineralization was stimulated by the preformed apatite coating of CHFL. However, after 14 days,

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Figure 5. (a) MG63-osteoblast-like cell viability measured by MTT tests on CHFL cast films preincubated in SBF (1.0×) for 7 days under visible light irradiation (“light”) and in the dark (“dark”) as a function of culture time (1-3 d). (b) ALP activity of MG63-osteoblast-like cells on CHFL cast films preincubated in SBF (1.0×) for 7 days under visible light irradiation (“light”) and in the dark (“dark”) as a function of time (1-14 d). In both cases, columns are the average values, whereas the bars represent standard deviations.

the ALP activity decreased for cells cultured on both CHFL samples to allow the HA mineral deposition by the enzymatic activity, as observable by microscopy analysis of the samples.

Conclusions A novel photoactive, biocompatible, water-soluble conjugate polymer was synthesized by grafting FL chromophore (0.43 mol %) to glucosamine units of CH via amide bonds. Cast film samples of CHFL conjugate polymer (and CH polymer) were not bioactive after 7 days immersion in conventional SBF in the dark. On the other hand, the continuous exposure to visible light of CHFL promoted the deposition of an apatite-like layer on the film surface after 7 days immersion in SBF. CHFL films coated with an apatite layer (obtained by photoactivation for 7 days in SBF) were found to significantly enhance the ALP activity of MG63-osteoblast like cells, showing that apatitecoated CHFL is suitable for bone tissue regeneration. Photoexcitation of the chromophore groups induces the formation of singlet oxygen, radical species, and superoxide anions from oxygen and water molecules of the nearby SBF medium. The photoexcitation mechanism is also known to induce the formation of local positive charges on the substrate surface due to electron transfer from the chromophoric groups to the nearby reactive species (oxygen and water). Photoexcitation could activate two different mechanisms of HA deposition: (i) the homogeneously nucleated negatively charged calcium phosphate crystals formed in SBF solution or (ii) the negative ions of SBF medium (mainly phosphate ions) could be electrostatically attracted by the positive local charges of the film surface. In accordance with this hypothesis, photoinduced bioactivity of CHFL polymers is expected to increase

Figure 6. Fluorescence microscopy images of MG63-osteoblast-like cell cultured for 3 days on (a) tissue culture plastic plate (TCPS); (b) CHFL film preincubated in SBF in the dark; and (c) CHFL film preincubated in SBF under visible light irradiation. Magnification is 20×.

(and the time for apatite deposition to decrease) with increasing the amount of grafted FL. Additional studies will be performed in the future to validate the proposed mechanism for HA photoinduced deposition. An innovative therapeutic approach could derive from the use of biocompatible photoactive conjugate polymers, such as CHFL, for bone/dental applications. As the bone-bonding ability of an implant is mediated by the formation of an apatite layer on the surface of biomaterials, photoactive conjugate polymers could be coated on originally nonbioactive implants to promote rapid in vitro apatite deposition by photoexcitation. Moreover, in vivo applications of the proposed photosensitizer could be possible by adopting the minimally invasive surgical techniques based on the use of illuminating optical fibers, which are nowadays available for bone cancer photodynamic therapy (PDT). The composition of CHFL could also be adjusted to reduce the necessary exposure time to visible light irradiation. However, in vivo applications of photoactive polymers for regenerative purposes are limited by concerns about the possible

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cytotoxicity of generated singlet oxygen and radical species toward living cells and tissues. Before any in vivo applications, a careful analysis of risk/benefits ratio arising from the use of photoactive polymers is necessary. On the other hand, improved formulations of the proposed photoactive conjugate polymer could be applied in dental medicine for the repair of teeth defects by local photostimulated deposition of biomimetic apatite. Acknowledgment. The work was funded by PhotoNanoTech project: Photozyme Nanoparticle Applications for Water Purification, Textile Finishing, Photodynamic Biomineralization and Biomaterials Coating, Contract Number 033168. The authors are grateful to Mauro Raimondi (Dept. of Chemical EngineeringPolitecnico di Torino) for his technical support during SEM analyses.

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