Real Estate

Bisphosphonate derivatized polyurethanes resist calcification

Bisphosphonate derivatized polyurethanes resist calcification
of 11
All materials on our website are shared by users. If you have any questions about copyright issues, please report us to resolve them. We are always happy to assist you.
Related Documents
  Biomaterials 22 (2001) 2683–2693 Bisphosphonate derivatized polyurethanes resist calcification Ivan Alferiev a , Narendra Vyavahare a , Cunxian Song a , Jeanne Connolly a , John TravisHinson a , Zhibin Lu a , Sruthi Tallapragada a , Richard Bianco b , Robert Levy a, * a Di  v ision of Cardiolo g  y, The Children’s Hospital of Philadelphia, Abramson Research Bld  g ., 3516 Ci  v ic Center Bl  v d, Philadelphia, PA 19104, USA b The Department of Sur g ery, The Uni  v ersity of Minnesota, Minneapolis, MN 55455, USA Received 6 September 2000; received in revised form 2 January 2001; accepted 8 January 2001 Abstract Calcification of polyurethane cardiovascular implants is an important disease process that has the potential to compromise thelong-term function of devices such as polymer heart valves and ventricular assist systems. In this study we report the successfulformulation and characterization of bisphosphonate-derivatized polyurethanes, hypothesized to resist implant calcification based onthe pharmacologic activity of the immobilized bisphosphonate. Fully polymerized polyurethanes (a polyurea–polyurethane and apolycarbonate polyurethane) were modified (post-polymerization) with bromoalkylation of the hard segments followed byattachment of a bisphosphonate group at the bromine site. These bisphosphonate-polyurethanes resisted calcification in rat 60 daysubdermal implants compared to nonmodified control polyurethane implants, that calcify. Bisphosphonates-modified polyurethaneswere also studied in circulatory implants using a pulmonary valve cusp replacement model in sheep. Polyurethane cusps modifiedwith bisphosphonate did not calcify in 90 day implants, compared to control polyurethane cusps implants, that demonstratednodular surface oriented calcific deposits. It is concluded that bisphosphonate modified polyurethanes resist calcification both insubdermal implants and in the circulation. This novel biomaterial approach offers great promise for long-term blood streamimplantation with calcification resistance. # 2001 Elsevier Science Ltd. All rights reserved. Keywords:  Heart valves; Prostheses; Mineralization; Biomaterials 1. Introduction Polymeric heart valve prostheses have been used inboth experimental [1–6] and clinical studies [7–11].These constructs, which have been fabricated frompolyurethane and other elastomers, have all failed dueto either thrombosis or calcification or both [1–11].However, the attraction of the potential desirablefeatures of a polymer heart valve remains. Heart valvesfabricated from elastomers such as polyurethane, canhypothetically be designed with tri-leaflet functionality,and anatomic and fluid dynamic characteristics identicalto human heart valves. Furthermore, there are a largenumber of candidate polymers with appropriate me-chanical and durability characteristics. Despite this,there has been no clinical success with polymeric heartvalves.Bisphosphonates are well-established pharmaceuticalsthat have potent effects modulating mineralization [12– 20]. Bisphosphonates are now widely used for prevent-ing, and promoting the regression of osteoporosis[12,13]. These agents also have potent activity inpreventing pathologic calcification, with a significantrisk of associated osteomalacia due to unwanted bone-targeted side-effects [14]. Prior work by our group hasdemonstrated inhibition of glutaraldehyde fixed bio-prosthetic heart valve leaflet calcification with systemicbisphosphonate administration, but with significantbone toxicity [13]. However, local administration of bisphosphonates in this same model system usingpolymeric controlled release completely prevented calci-fication without adverse effects [15–17]. This concept of local efficacy led to our recent investigations concerningsynthetic modifications of polyurethane.Previous research from our laboratory has investi-gated bisphosphonate modified polyeurethanes, inwhich ethane hydroxy bisphosphonate was covalentlylinked to polyurethane through an epoxy derivatization[19]. Although these materials appeared promising in rat *Corresponding author. Tel.: +1-215-590-6119; fax: +1-215-590-5454. E-mail address: (R. Levy).0142-9612/01/$-see front matter # 2001 Elsevier Science Ltd. All rights reserved.PII: S 0142 -9612(01 )0 0010-2  subdermal studies, this formulation was not suitable forcirculatory implants.In the present series of experiments, we report theformulation, characterization, and successful in vivo useof a new family of polyurethanes, based on hardsegment activation with bromo-alkyl derivatization.These bromo-alkyl activated polyurethanes have beencovalently derivatized with bisphosphonates. The goalsof the present study were as follows.(1) To formulate bisphosphonate derivatized polyur-ethanes based on hard segment alkylation chemis-try.(2) To characterize these new polymers in terms of theirbisphosphonate content, stability of bisphospho-nate binding, and water absorption properties.(3) To evaluate their efficacy for calcification resistancewith in vivo studies using both rat subdermalimplants, and sheep circulatory implants. 2. Methods 2.1. Materials The following candidate polyurethanes were investi-gated for their calcification properties and bispho-sphonate derivatization. A polyurea polyurethane(Biospan, Polymer Technology Group Medical LLC,Berkeley, CA) and a polycarbonate polyurethane(Bionate 80A, from the same manufacturer) werestudied. Vinylidene-bisphosphonic acid (VBP) tetraso-dium salt was obtained from Albright & Wilson UKLtd. (England). The salt was transformed into the freeacid using cation exchange with Dowex-50. 2.2. Polyurethane modifications and characterization The derivatization of polymers with bisphosphonategroups was carried out in dimethylacetamide solutionsin two steps (Scheme 1) as previously published[20]. First, auxiliary 6-bromohexyl groups were attachedto the urethane nitrogens of polymers hard segmentsvia base-induced alkylation with 1,6-dibromohexaneat low temperature (  5 to 0 8 C) using the followingprocedure. The polyurethanes dissolved in  N  , N  -dimethylacetamide (DMA) were mixed with a largeexcess of 1,6-dibromobutane, cooled to  5 to  8 8 C, andthe polyanions (with the negative charges localized onthe urethane nitrogens) were generated by addition of lithium diisopropylamide solutions. After 2h at 0 8 C, thereactions were complete. The polyurethanes containingpendant 6-bromohexyl groups were precipitated withcold methanol, purified by washing, and dried. Thebromoalkylated polymers were then reacted with thiol-containing bisphosphonate in the form of a tetrabuty-lammonium salt (containing 1.5 Bu 4 N-cations for eachbisphosphonate group) to ensure the solubility in DMA.The reaction conditions were as follows. The bro-moalkylated polyurethanes were dissolved in DMA,mixed with DMA-solutions of thiol-bisphosphonate (ina 10-fold excess), and the thiol groups were activatedtowards the nucleophilic substitution of Br by additionof tetrabutylammonium tetraborate. After 1h at 0 8 C thereactions were complete. The thiol-bisphosphonate wasprepared by reacting VBP with a large excess of 1,3-propanedithiol at 120 8 C (Scheme 2) as describedpreviously [20]. The values of polyurethanes’ bispho-sphonate modifications were determined using phos-phorus analysis combined with  31 P NMR (BrukerAvance DMX 400, 101.97MHz), which indicated Scheme 1. Hard segment alkylation, with subsequent covalent binding of a thiol-bisphosphonate.Scheme 2. Thiol-bisphosphonate synthesis. I. Alferie v  et al. / Biomaterials 22 (2001) 2683–2693 2684  uniform phosphorus peaks at ca. 19 ppm characteristicof 2,2-diphosphonoethylthio groups [20].Water absorption by unmodified and bisphosphonatepolyurethanes was studied using the following metho-dology: Polymer films (0.15mm thick, weighing from 30to 100mg) were soaked in water for 1–2 days, wipedfree of droplets of water, weighed and dried in vacuo.Water absorption was calculated from the differencebetween the wet and dry weights. The stability of covalently bound bisphosphonate was assessed byincubating films of bisphosphonate-modified polyur-ethanes at pH 7.4 (0.05m HEPES) and 37 8 C for 9months, followed by decomposition with boiling H 2 SO 4 containing small amounts of HNO 3  and analyses forphosphorus according to established procedures [21].Infrared spectra (Fourier transform, FTIR) of unim-planted polyurethane films, and explants, both ratsubdermal and sheep circulatory, were obtainedusing a prot ! eeg ! ee 460 ESP FT-IR spectrometer withInspect IR-FT-IR micro sampling and imaging acces-sory supported by OMNIC software for spectralanalysis including subtraction and deconvolution (Nico-let, Madison, WI). 2.3. Animal implant studies Rat subdermal implants were carried out using ourestablished calciphylaxis model [19]. In brief, dihydro-tachysterol (DHT, 1mg in 1ml of corn oil) wasadministered with an oral-gastric tube to Sprague– Dawley rats (males, 150g in weight) 24h prior to sub-dermal implant surgery. Polyurethane samples (100 m min thickness, and 1  1cm) were implanted in subdermalpouches over the ventral abdominal wall. Animals were Fig. 1.  31 P NMR of bisphosphonate-modified polyurethanes (101.97MHz) (A) Biospan (0.091mmol/g of BP-groups); (B) Bionate (0.112mmol/g of BP-groups). Solvent: a mixture of DMA and DMSO-d 6 , 3:1 by volume. The signals of bisphosphonate groups are broadened due to the influence of polymer surrounding. Only one characteristic  31 P signal (at  d ¼ 19 : 0 2 19 : 5ppm) is observed in the spectra, with the chemical shift corresponding toSCH 2 CH(PO 3 H 2 ) groups.Table 1Polyurethane characterizationPolymer type Productname a MW (Da) Waterabsorption (%)Bisphosphonatecontent (initial)(mmol/g)Bisphosphonatecontent (final,9 months) (mmol/g)Polyureapolyurethane Biospan 214 000 1.5  } } Polyureapolyurethane } bisphosphonate modifiedBiospan 214 000 22.6 0.091 0.092Polycarbonate polyurethane Bionate 242 000 0.8  } } Polycarbonate polyurethanebisphosphonate modified } ABionate 267 000 7.2 0.087 Not determinedPolycarbonate polyurethanebisphosphonate modified } BBionate Not determined 15.0 0.112 0.115 a All unmodified polymers were manufactured by the Polymer Technology Group, Medical LLC, Berkeley, CA I. Alferie v  et al. / Biomaterials 22 (2001) 2683–2693  2685  under general anesthesia using ketamine and xylazine aspreviously published [18,19]. The implant duration was60 days, and implants were retrieved following eutha-nasia using a carbon dioxide overdose.A sheep model of polyurethane-pulmonary cuspreplacement to study blood material interactions wasused, in which under general anesthesia, and cardiopul-monary bypass, the right anterior pulmonary leaflet of  Fig. 2. Infrared surface scans of polyurethane films demonstrating Fourier transform infrared spectra (FTIR) spectra before and after derivatizationand rat subdermal implantation for polyurea–polyureathane (Biospan) and polycarbonate–polyurethane (Bionate). Results demonstrating uniquespectra for the parent polyurethanes without bulk changes following either derivatization or implantation. (A) Biospan spectra: (a) bisphosphonatemodified and unimplanted, (b) bisphosphonate modified, rat subdermal explant, (c) non-modified, unimplanted, (d) non-modified, rat subdermalexplant (B) Bionate spectra: (a) bisphosphonate modified and unimplanted, (b) bisphosphonate modified, rat subdermal explant, (c) non-modified,unimplanted, (d) non-modified, rat subdermal explant. I. Alferie v  et al. / Biomaterials 22 (2001) 2683–2693 2686  the sheep pulmonary valve apparatus was removed, andreplaced with a 1.5  1.5cm polyurethane flap sewn in,as a replacement valve cusp. Animals were recovered,and after 90 days were subjected to euthanasia with anoverdose of barbiturates, followed by retrieval of thepulmonary outflow track, for explant analyses. 2.4. Explant e v aluation Polyurethane samples were subjected to calciumanalyses using atomic absorption spectroscopy toevaluate calcium levels in acid hydrolysates [18,19].Phosphorous content was determined using molybdatecomplexation spectroscopy on samples of the same acidhydrolysates [18,19]. Scanning electron microscopy[18,19] was performed on mounted, carbon-sputteredexplants using a JEOL model 330 scanning electronmicroscope (JEOL, Tokyo, Japan). Elemental analysescarried out with the same instrument using backscatterX-ray methodology (EDAX) with X-ray microanalysissoftware (Quantex V, Smyrna, GA). Microscopicstudies were carried out on neutral buffered formalinfixed specimens of the explanted polyurethane materials.Hematoxylin and eosin stains were used to determinemorphology characteristics, and calcification-specificstaining used the von Kossa method. 2.5. Statistical methods Data were calculated as means  standard error (SE).The significance of differences was examined withStudent’s  t -test. Statistical significance was noted with  p 4 0 : 05. 3. Results 3.1. Polyurethane pre-implant characteristics Bisphosphonate derivatized polyurethanes in generalappeared physically comparable to non-derivatizedpolyurethanes. Water absorption capacity markedlyincreased with bisphosphonate derivatization, comparedto non-derivatized controls (Table 1). Bisphosphonatederivatized Biospan demonstrated the greatest waterabsorption capacity, increasing more than 15-fold frombaseline levels. Similarly, two examples of derivatizedBionate had roughly the same magnitude of changefrom control conditions. A stability study of the bindingof the bisphosphonates derivatized to each type of polyurethane to be used in implant studies was carriedout for a period of 9 months at 37 8 C at pH 7.4 in 0.05 m HEPES buffer (Table 1). No detectable change of phosphorus content in either type of polyurethane wasnoted after this period. Thus, covalently linked bispho-sphonate within either type of polyurethane studied, wasstable over time with virtually no dissociation. FTIRscans of unmodified and modified polyurethanes re-vealed no spectra differences due to the covalentattachment of bisphosphonate (Figs. 1 and 2). Thus,both types of polyurethane, polyurea and polycarbo-nate, were successfully derivatized with bisphosphonate,in a stable covalent attachment that did not result in anysignificant changes in chemical bonding characteristicsas noted by FTIR. 3.2. Rat explant results Implants of non-modified polyurea polyurethane, butnot polycarbonate polyurethane, calcified under condi-tions of DHT induced calciphylaxis. The polyureapolyurethane calcification was documented both withbulk analyses (Fig. 3), and scanning electron microscopystudies (Fig. 4). In general, the calcification was nodularin nature and completely surface-oriented. Bisphospho-nate modified polyurea polyurethane (Biospan) did notcalcify significantly, as documented both by chemicalanalyses and by scanning electron microscopy (Figs. 3and 4). Similarly, bisphosphonate derivatized polycar-bonate polyurethane (Bionate) also did not calcify in thesubdermal studies, as was true for the non-modifiedmaterial. FTIR spectra of non-calcified regions of polyurethane samples revealed no differences betweenmodified and non-modified, and in fact these spectra didnot differ from unimplanted samples (Fig. 2). However,calcific nodules could be scanned on the surface of thenonmodified Biospan explants, and these mineraldeposits have spectral characteristics in the IR rangeconsistent with the presence of hydroxyapatite and otherunidentified calcium phosphate mineral phases as well Fig. 3. Calcification of polyurethanes implanted subdermally in ratsinhibited by bisphosphonate modification (Biospan Mod. & Bionate-Mod). Results of 60 day explants.  p ¼ 0 : 05 Biospan vs. Biospanbisphosphonate modified. I. Alferie v  et al. / Biomaterials 22 (2001) 2683–2693  2687
Related Search
We Need Your Support
Thank you for visiting our website and your interest in our free products and services. We are nonprofit website to share and download documents. To the running of this website, we need your help to support us.

Thanks to everyone for your continued support.

No, Thanks