New polymers for drug delivery systems in orthopaedics: in vivo biocompatibility evaluation

Biomedicine & Pharmacotherapy 58 (2004) 411–417 www.elsevier.com/locate/biopha

Original article

New polymers for drug delivery systems in orthopaedics: in vivo biocompatibility evaluation G. Giavaresi a,c,*, M. Tschon a, V. Borsari a, J.H. Daly b, J.J. Liggat b, M. Fini a,c, V. Bonazzi a,c, A. Nicolini d, A. Carpi d, M. Morra e, C. Cassinelli e, R. Giardino a,c a

Department of Experimental Surgery, Istituti Ortopedici Rizzoli, Via di Barbiano, 1/10, 40136 Bologna, Italy b Department of Pure and Applied Chemistry, University of Strathclyde, Glasgow, United Kingdom c Department of Scientific Research and Development, ANT, Italia ONLUS Foundation, Bologna, Italy d School of Internal Medicine, University of Pisa, Pisa, Italy e Nobil Bio Ricerche Srl, Villafranca d’Asti, Asti, Italy Received 21 July 2004 Available online 25 August 2004

Abstract The use of biodegradable polymers for drug delivery systems excluded the need for a second operation to remove the carrier. However, the development of an avascular fibrous capsule, reducing drug release, has raised concern about these polymers in terms of tissue-implant reaction. Five novel polymers were evaluated in vivo after implantation in the rat dorsal subcutis and compared to the reference polycaprolactone (PCL). Poly(cyclohexyl-sebacate) (PCS), poly(L-lactide-b-1,5-dioxepan-2-one-b-L-lactide) (PLLA-PDXO-PLLA), two 3-hydroxybutyrate-co-3-hydroxyvalerate copolymers (D400G and D600G), and a poly(organo)phosphazene (POS-PheOEt:Imidazole) specimens were histologically evaluated in terms of the inflammatory tissue thickness and vascular density at 4 and 12 weeks from surgery. The highest values of inflammatory tissue thickness were observed in D600G (P < 0.01), PCS (P < 0.001) and PLLA-PDXO-PLLA (P < 0.001) at 4 weeks, while POP—PheOEt:Imidazole showed the lowest value of inflammatory tissue thickness (P < 0.05) at 12 weeks. D400G, D600G, PLLA-PDXO-PPLA and POP—PheOEt:Imidazole showed higher (P < 0.001) values of vascular density near the implants in comparison to PCL at 4 weeks. Finally, D400G and D600G increased their vessel densities while POP—PheOEt:Imidazole and the synthetic polyester PLLA-PDXO-PLLA presented similar vessel density values during experimental times. These different behaviours to improve neoangiogenesis without severe inflammatory tissue-responses could be further investigated with drugs in order to obtain time-programmable drug delivery systems for musculoskeletal therapy. © 2004 Elsevier SAS. All rights reserved. Keywords: Drug delivery systems; Polymers; Polyesters; Polyphosphazenes; Animal model; Implants

1. Introduction During the last decade, the use of biodegradable polymers in the controlled-release field as carriers of drug molecules has increased considerably. A literature search of scientific publications (MEDLINE1) showed that polymers have been used to develop drug delivery systems for musculoskeletal pathologies to carry proteins and growth factors for bone * Corresponding author. E-mail address: [email protected] (G. Giavaresi). 1 MEDLINE Search Strategies: From 1972 to 2004 (“Bone and Bones” [MeSH] OR “Cartilage” [MeSH]) and (“Drug delivery system” [MeSH] and “Polymers” [MeSH])]. 0753-3322/$ - see front matter © 2004 Elsevier SAS. All rights reserved. doi:10.1016/j.biopha.2004.08.001

regeneration (54% of papers found through MEDLINE search), antibiotics to treat mainly osteomyelitis (22%), antiresorptive (5%) and antineoplastic (4%) drugs, and that also multifunctional devices, which combine mechanical functions and drug delivery, have been studied to improve bone tissue regeneration, fixation and augmentation (15%). The local delivery of drugs by biodegradable polymers has the advantages of avoiding systemic side effects, assuring high local levels of the drugs, avoiding a second operation to remove the carrier, and the need for recurrent injections or intravenous maintenance [15]. Although these carrier systems eliminate the significant toxicity associated with systemic drug administration, the application of synthetic polymeric materials in vivo has inevitably raised serious concern

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about these polymers in terms of tissue-implant interaction [13,23,28]. In particular, the development of an avascular fibrous capsule around implants, a chronic inflammatory response, leads to a reduction of drug release secondary to the increased distance between the system and circulation [29]. A variety of available degradable polymers is limited because of the stringent requirements of biocompatibility, and the necessity to be free from degradation related toxic products (e.g., monomers, stabilizers, polymerisation initiators, emulsifiers). Few polymers have been approved by the FDA, such as aliphatic polyesters including poly-lactic acid (PLA), poly-glycolic acid (PGA) and poly-dioxanone (PDS), which are already used routinely for producing drug delivery devices [7,17,22,26]. These polyesters, with poly(caprolactone) (PCL), polyanhydrides and polyphosphazenes (POPs), substituted with amino acid esters are the most extensively investigated polymers, essentially because of their good hydrolyzability and biocompatibility [1,2,13,23,28,32]. POPs constitute a family of greatly diverse performance materials with interesting technological properties (chemical, optical, electrical and mechanical). Their properties depend both on the characteristics of the inorganic altering P = N backbone and on the features of the substituent groups attached to P atoms. Being able to change the substituent groups easily without altering their chemical reactivity permits specific properties to be obtained, which are potentially transferable to the final composite materials. Recently, the natural polyhydroxyalkanoate polyester, 3-hydroxybutyrate-co-3-hydroxyvalerate (3HB-3HV) marketed under the Biopol™ trademark, thanks to its good biocompatibility [9,14], has been proposed for use in controlled drug delivery systems [3,6,10–12,16,27,30,35]. The 3HB3HV copolymers, produced from renewable resources by bacterial fermentation, have been used until now for packaging applications, hygienic, agricultural and biomedical products in the form of cast film, sheet, fibres and tubes. Degradation time of 3HB-3HV polymers ranges from weeks to over a year, but in vivo it is slower than that normally seen in microbiological environments and is thought to be mediated by non-specific esterases [25,36]. In order to develop polymers whose mechanical performance, biodegradation kinetics and chemical characteristics can be tailored to meet the demands of specific applications such as the capability to improve neoangiogenesis around an implanted drug delivery system, various copolymers were realised by adding polymers such as hydroxyethyl methacrylate, methacrylic acid, acrilamide or ethylene glycol [18,33]. Novel biodegradable polyesters based on (a) 1,4cyclohexanediol and a range of linear aliphatic dicarboxylic acids; and (b) the ring opening polymerisation of the cyclic monomer (1,5-dioxepan-2-one) (DXO) to PLLA (“Nanobiotechnology and Medicine” 5th Framework European Project—QLK3-CT-2000-01500) have been synthesised and characterized. The present study investigated and compared the in vivo behaviour of drug-unloaded synthetic polyesters, PCS and

PLLA-PDXO-PLLA, two natural 3HB-3HV polyesters, and a poly(organo)phosphazene with another already used polymer (polycaprolactone) by evaluating the local tissue response after subcutaneous implantation in rats in terms of inflammatory tissue presence and microvessel density. 2. Materials and methods 2.1. Polymers The different types of polymers tested were rectangular sheet specimens, 10 mm in length, 3 mm in width, and about 0.5 mm in thickness as follows. The specimens were sterilized by ethylene oxide (ethylene oxide: 400 mg/l; humidity rate: 50–60%; cycle of 12 h and a subsequent cycle of forced degasification of 24 h) according to UNI-EN 550 and ISO 11135 rules. 2.1.1. Poly(cyclohexyl-sebacate) (PCS) The synthetic polyester PCS was melt processed at 90 °C for 10 min under an inert nitrogen atmosphere using stoichiometric ratios of monomers obtained by Sigma-Aldrich Company Ltd. (United Kingdom) according to the procedure described by Braun and Hempler [4]. 2.1.2. Poly(L-lactide-b-1,5-dioxepan-2-one-b-L-lactide) (PLLA-PDXO-PLLA) PLLA-PDXO-PLLA is a triblock copolymer film, a middle block of 400 1,5-dioxepane-2-on (DXO) units and two side blocks of 100 lactide units each, synthesised as described by Ryner et al. [24], belonging to a class of physically cross-linked polymers, where the PLLA side blocks (hydrophobic) from several chains phase separate (demix) into crystalline microdomains cross-linking the amorphous central PDXO blocks (hydrophilic). PLLA-PDXO-PLLA was synthesised by ring opening polymerisation of the cyclic monomer DXO in order to obtain bioresorbable products with good elastic properties [21]. The polymerisation reaction was initiated by an aliphatic cyclic tin alkoxide, 2,2dibutyl-2-stanna-1,4-dioxacyclopentane, from which the polymerisation proceeds symmetrically in two directions and forms a new type of triblock copolymer. All reactions have to be carried out under strict oxygen and moisture-free conditions. 2.1.3. 3HB-3HV polymers (D400G and D600G) Two grades of Biopol™ biodegradable polyester containing 8% (D400G) and 12% (D600G) of hydroxyvalerate component were obtained from Monsanto plc (United Kingdom). Both these grades contain 1% boron nitride as a nucleating agent. Thin films of the polymers, 0.5 mm in thickness, were obtained by melt pressing at 175 °C for 15 min for D400G, and 170 °C for 10 min for the D600G system [5,8]. 2.1.4. Poly(organo)phosphazene (POP—PheOEt:Imidazole) Poly(organo)phosphazene substituted with phenylalanine ethylester (PheOEt) and imidazole (80/20 molar ratio) was

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prepared as described by Veronese et al. [32]. Briefly, polydichlorophosphazene was dissolved in toluene and a dry tetrahydrofuran solution of phenylalanine ethylester hydrochloride, imidazole and triethylamine was added drop-wise under stirring. The mixture was filtered and the polymer was precipitated from organic solution by the addition of n-hexane, dissolved in toluene and precipitated again in n-heptane. 2.1.5. Polycaprolactone (PCL) TONE™ P787 PCL (Union Carbide Corp., USA) was used for preparing flat samples by injection moulding. PCL was used as reference material. 2.2. Study design2 Sixteen Sprague–Dawley female rats (Charles River SpA, Calco, Italy), 3-month-old and 200 ± 50 g b.w., were housed under controlled conditions and offered a standard pellet diet and water ad libitum. After performing a dorsal skin incision, six subcutaneous pockets spaced 10 mm were obtained by blunt dissection, 10 mm from the line of incision, three on the right side and three on the left of the spine, under general anaesthesia (intramuscular injection of 87 mg/kg ketamine and 13 mg/kg xilazine). The specimens were placed one in each pocket for each polymer, and non-resorbable stitches were used to suture skin. No abnormal findings, including local, systemic and behavioural abnormalities, were observed in the postoperative period. Four and 12 weeks from surgery, animals were pharmacologically euthanised with an intracardial injection of 1 ml Tanax (Hoechst AG, Frankfurt-am-Mein, Germany), under general anaesthesia. Dorsal skin containing all implants (eight specimen for each tested material at each experimental time) was placed fur side down and a 15-mm diameter trephine was used to excise each 10-mm diameter disc, complete with some surrounding tissue. Biopsies were fixed in 4% paraformaldehyde in phosphate buffered saline (pH 7.4) and processed through to wax and sections cut at 6 µm thickness. Then, sections were stained with Mallory’s trichrome. 2.3. Histology and histomorphometric analyses Up to six images (2714 × 2012 pixels) were grabbed on the long flat edges from each side of the implant at 10× and 50× magnification by using a transmission light Olympus BX41 microscope. Image analysis was performed by means of the QWIN image analysis software (Leica Imaging Systems Ltd, Cambridge, England). 2 The experiment was performed in compliance with European and Italian Laws on animal experimentation, ISO 10993-2, the principles stated in the “Guide for the Care and Use of Laboratory Animals” and the Animal Welfare Assurance No. #A5424-01 by the National Institute of Health (NIH-Rockville Maryland USA).

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The biological response parameters assessed included: (a) the thickness of inflammatory tissue and fibrous capsule, measured 12 times for each side at random locations along the length of the implant; and (b) the vascular density determined by counting the number of microvessel profiles (10 µm diameter or smaller) present within a distance of 100 µm from the implant surface (number of vessels/mm2). Vessels were defined as having an identifiable endothelial layer in cross, longitudinal, and oblique section, or by the presence of intraluminal red blood cells. 2.4. Statistical analysis Statistical evaluation of data was performed using the software package SPSS/PC+ Statistics™ 12.1 (SPSS Inc., Chicago, IL USA). Data are reported as mean ± standard deviations (SD) at a significance level of P < 0.05. After having verified normal distribution and homogeneity of variance, the non-parametric Kruskall–Wallis test, followed by the Monte Carlo methods to compute probability, was used to highlight any significant difference between tested polymers and reference material.

3. Results Neither degeneration nor necrosis was observed macroscopically around the implants. An inflammatory fibrous tissue around all tested polymers was observed at both experimental times, which was organized in a fibrillar extracellular matrix with fibroblasts, myofibroblasts and inflammatory cells, with more dense collagen fibrils oriented parallel to the implant surface. No ingrowth of connective tissue into the polymer film was observed except for POP—PheOEt:Imidazole whose film lost its shape and only small fragments with signs of resorption were recognizable, surrounded by a dense inflammatory sheath at both experimental times. Histomorphometric analysis of the thickness of the inflammatory tissue showed significant differences among groups at 4 (P < 0.005) and 12 weeks (P < 0.01) (Fig. 1). D400G and POP—PheOEt:Imidazole presented a similar tissue reaction to reference polymer at 4 weeks, while the highest significant values of inflammatory tissue thickness were observed in D600G (P < 0.01), PCS (P < 0.001) and PLLA-PDXO-PLLA (P < 0.001) at 4 weeks. The extent of inflammatory tissue around implants increased from 4 to 12 weeks for all tested polymers except for PCS and PLLAPDXO-PLLA (Figs. 1,2). At 12 weeks, PLLA-PDXO-PLLA showed the lowest significant value of inflammatory tissue thickness (P < 0.05). Regarding neoangiogenesis at four weeks (Fig. 3), D400G, D600G, PLLA-PDXO-PPLA and POP—PheOEt: Imidazole showed higher significant (P < 0.001) values of vessel density near the implants in comparison to PCL; the lowest significant result was obtained with PCS implants

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Fig. 1. Results of the inflammatory tissue thickness of all tested polymers implanted in the subcutaneous tissue of rats at 4 and 12 weeks (Mean ± SD; n = 8). Kruskal–Wallis multiple comparison tests to PCL at 4 weeks: ***, P < 0.001; **, P < 0.01; and at 12 weeks: °, P < 0.05.

(P < 0.01). A vast network of newly formed capillaries placed in the connective matrix near the fibrous tissue was well documented around the D400G, D600G, PLLA-PDXOPLLA and POP—PheOEt:Imidazole implants at 12 weeks (Fig. 2). The vessel density increased in D400G and D600G, while it remained stable in PLLA-PDXO-PLLA and POP—PheOEt: Imidazole between 4 and 12 weeks (Fig. 2). PCS showed the lowest significant vascular densities at both experimental times (P < 0.01).

4. Discussion The difficulties in treating pharmacologically musculoskeletal pathologies such as infections (osteomyelitis), metabolic bone diseases (osteoporosis) and tumors, and also because of the impaired vascularity commonly present in these situations, prompted researcher to find other therapeutic solutions, such as local drug delivery. For many years, the innovative treatment of osteomyelitis has been local antibiotic therapy by implantation of antibiotic-loaded polymethylmethacrylate beads. However the use of this nonbiodegradable polymer does have its limitations, above all the necessity to perform a second surgical procedure for its removal [18]. Alternatives to polymethylmethacrylate were found in biodegradable materials, such as poly (D,Llactide)cylinders, polyglycolide beads, polylactide-coglycolide beads, polyanhydrides sebacic acid and fatty dimmer beads, plaster of Paris, calcium hydroxyapatite beads, fibrin sealant, and bovine collagen type I [26]. Various biodegradable polymers have been characterized and experimentally tested for drug delivery systems [1–3,6,10–13,16,17,22,23,26–28,30,32,35], but one of the most important complications of currently used polymers is the occurring reaction to rapidly degrading materials. The current authors decided to use the PCL as reference material because (a) its degradation time is considerable longer than

that of other polymers, such as polylactic acid, remaining active as long as a year for drug delivery; and (b) PCL is degraded by acid hydrolysis and free radicals to the natural by-products CO2 and H2O. In addition, PCL has been successfully used in the release of anti-inflammatory agents and protein moieties in in vitro and in vivo experimental studies [12,21]. Many factors are claimed to determine the rate of degradation, including molecular weight, crystallinity, sample form, porosity and the site of implantation; most of them seem to influence also the degree and morphology of the induced fibrous tissue around implants. All tested polymers had the characteristic of controlling their backbone architectures by incorporating other molecules with the aim of tailoring their mechanical properties and biodegradation lifetime to meet the demands of specific applications [8,20,21,24]: • 1,4 cyclohexanediol acid provides a means of incorporating some rigidity in PCS chain while the linear sebacic diacids give some flexibility [8]; • PDXO increases PLLA wettability (the balance of hydrophilicity and hydrophobicity) with improved mechanical elasticity and cell adhesion [20,21,24]; • the increasing hydroxyvalerate copolymer content improves flexibility and ductility in D400G and D600G [5,31]; • the amino acid esters, phenylalanine ethylester, and sensitive side group, imidazole, modulate, respectively, the rate of the polymeric degradation and biocompatibility of POP—PheOEt:Imidazole [1,32]. Regarding the site of implantation, the current authors decided to perform implants in subcutaneous tissue because (a) the extent and duration of the inflammatory response evoked by subcutaneous implants anticipate the response to them in other specialized sites and (b) when a drug delivery system is used for musculoskeletal pathologies, it is exposed to bone and also various soft tissues and muscles, which generally show more severe responses than hard tissue [34].The present findings substantiate the view that these polymers are biocompatible enough to be used for biomedical applica-

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Fig. 2. Light microscopy of longitudinal sections (Mallory’s trichrome, original magnification 50×) of subcutaneously implanted polymers at 12 weeks: (a) PCS; (b) PLLA-PDXO-PLLA; (c) D400G; (d) D600G; (e) POP-PheOEt:Imidazole (f) PCL. The bottom of each image corresponds to the inflammatory tissue around implants: (a), (b) and (c) show an immature fibrous, with fibroblasts and little collagen; (d) and (f) present a granulous reactive tissue, containing both fibroblasts and inflammatory cells; (e) shows a dense reactive tissue with inflammatory cells and modest sign of connective tissue organization. Vast networks of newly formed capillaries (*) in the connective matrix are visible near implants. The majority of microvessels (→) had diameters of 10 µm or less and were predominantly capillaries; no large diameter vessels were included in the statistical analysis.

tions, but their capability to stimulate neoangiogenesis over time differed significantly. PCS determined the same tissue reaction at both experimental times in terms of inflammatory tissue thickness, significantly higher than PCL at 4 weeks, while a worsening in terms of vascular density was found from 4 to 12 weeks and in comparison to PCL. These results, besides the continuous release of chemical products, could be related also to a quicker degradation by surface erosion, as observed by Wang et al. in an in vivo study on poly(glycerolsebacate), and therefore PCS does not seem particularly suitable as a carrier for drug delivery systems. PLLA-PDXOPLLA behaved similarly to PCS for inflammatory tissue thickness, but its capability to stimulate neoangiogenesis remained stable during experimental times, even though it was lower than that of PCL at 12 weeks. In our opinion, it should be investigated if its neoangiogenetic capability could

remain stable longer than 12 weeks because it would make PLLA-PDXO-PLLA one of the most suitable biodegradable drug carriers.On the contrary, D400G and D600G increased in parallel both inflammatory tissue thickness and vascular density, presenting higher values than PCL. According to Gogolewski et al., these phenomena might be due to the continuous release of chemical products that are directly related to the increasing content of the valerate unit in the polymer chain [9]. The positive results in terms of vascular density could be in favour of a good functionality of a drug delivery system produced with D400G or D600G faced with the increasing development of a fibrous capsule, as well as lead to quick drug release.Finally, POP—PhEOt:Imidazole showed a mild tissue reaction quite similar to PCL with a little increase in vascular density at 4 weeks in comparison to PCL. These results, together with the low risk of immunoge-

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Fig. 3. Results of the vessel density of all tested polymers implanted in the subcutaneous tissue of rats at 4 and 12 weeks (mean ± S.D.; n = 8). Kruskal–Wallis multiple comparison tests to PCL at 4 weeks: ***, P < 0.001; **, P < 0.01; and at 12 weeks: °°°, P < 0.001; °°, P < 0.01.

nicity, indicate that POP-PhEOt:Imidazole could be another suitable biodegradable carrier, allowing the diffusion of the released drug [32]. In conclusion, the natural polyesters D400G and D600G increased their vessel densities while POP—PheOEt: Imidazole and the synthetic polyester PLLA-PDXO-PLLA presented similar vessel density values during experimental times. These different behaviours associated with the different inflammatory tissue-responses could be further investigated with antibiotics and anti-inflammatory drugs in order to obtain time-programmable drug delivery systems for musculoskeletal therapy.

[5]

[6] [7]

[8]

[9]

Acknowledgements This work has been partially supported by the EC under Contract QLK3-CT2000-01500 (NANOMED) and sponsored by EPSRC grant number GR/R51193/01. The authors thank Prof. Adam Curtis (Centre for Cell Engineering, University of Glasgow, Glasgow, United Kingdom), Dr Anders Wirsén (Department of Polymer Technology, Royal Institute for Technology, Stockholm, Sweden) and Prof. Francesco Maria Veronese (Department of Pharmaceutical Sciences, University of Padova, Padova, Italy) for supplying the PCL, PLLA-PDXO-PLLA and POP—PheOEt:Imidazole samples, respectively.

[10]

[11]

[12]

[13]

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