Pedro Lopes Granja PhD Thesis Biomimetic Natural Materials for Bone Regeneration ‘Tese submetida & Faculdade de Engenharia da Universidade do Porto, para candidatura 4 obtengao do grau de Doutor em Ciéncias de Engenharia Faculdade de Engenharia Universidade do Porto Porto, Portugal July 2000This thesis was supervised by: In Portugal: Professor Mario A. Barbosa Faculdade de Engenharia, Universidade do Porto In France: Professor Charles Baquey Faculté de Medicine, Université Bordeaux 2 Professor Berard De Jéso Faculté des Sciences, Université Bordeaux 1 The host institutions of this thesis were: Laboratorio de Biomateriais, INES — Instituto de Engenharia Biomédica Universidade do Porto, Portugal INSERM U.443, Université Bordeaux 2, France Laboratoire de Chimie des Substances Vegetales (LCSV), Institut du Pin Université Bordeaux 2, France The research described in this thesis was financially supported by: Portuguese Foundation for Science and Technology (FCT), under the Programme PRAXIS XXI, scholarship ref. PRAXIS XXW/BD/3389/94 Ministry of Science, Portugal +P. rane, Porto 200 (Al ga rence No prt of is pubaton may be rprcicad ard 8 rer sytem o aramid in ny kam By ary meant: eocronc.sectosab, mzaete toe, mechan, photcoeyva, cording OF eters, without writen permssen Hm 19 cooranonnerThis thesis was based on the following publications: Book chapters or international refereed Journals: Granja PL, Barbosa MA, Pouységu L, De Jéso B, Baquey C. Cellulose phosphates as biomaterials. In: Otenbritte RM, editor. Frontiers in Biomedical Polymers 2. Lancaster, PA, USA: Technomic Press; 1999. p. 195-225. Granja PL, Pouységu L, Pétraud M, De Jéso B, Baquey C, Barbosa MA. Cellulose phosphates as biomaterials. Synthesis and characterization of highly phosphorylated cellulose gels. Submitted, 2000. Granja PL, Pouységu L, Deffieux D, Daudé G, De Jéso B, Labrugére C, Baquey C, Barbosa MA. Cellulose phosphates as biomaterials. Surface chemical modification of regenerated cellulose hydrogels. Submitted, 2000. Granja PL, Pouységu L, De Jéso B, Rouais F, Saquey C, Barbosa MA. Cellulose phosphates as biomaterials. Mineralization of chemically modified regenerated cellulose hydrogels. Submitted, 2000. Granja PL, Ribeiro C, De Jéso B, Baquey C, Barbosa MA. A simple method to induce mineralization on high swelling materials. Submitted, 2000. Granja PL, De Jéso 8, Bareille R, Rouais F, Baquey C, Barbosa MA. Cellulose phosphates as biomaterials. In vitro biocompatibility studies. Submitted, 2000. Granja PL, De Jéso B, Bareille R, Rouais F, Baquey C, Barbosa MA. Mineralization of regenerated cellulose hydrogels induced by Human osteoprogenitor cells. Submitted, 2000. Fricain JC, Granja PL, Barbosa MA, De Jéso B, Barthe N, Baquey C. Cellulose: phosphates as biomaterials. in vivo biocompatibility studies, Submitted, 2000.Communications in Conference Proceedings: Granja PL, Baquey C, Pouységu L, Bareille R, De Jéso 8, Barbosa MA, Cellulose-based biomaterials for bone regeneration: Hemi-synthesis and characterisation. Polymers in Medicine and Surgery (PIMS'96). Glasgow, UK: Institute of Materials; 1996. p. 73-80. Fricain JC, Granja PL, Barrias C, De Jéso 8, Barbosa MA, Baquey C. Etude de Tintegration osseuse dans le condyle femorale de lapins d'impiants de cellulose et de cellulose phosphatee. 2nd International Symposium on Advanced Biomaterials (ISAB) and 7th Intemational Academy of Shape Memory Material for Medical Use (!~ASMU), Montréal, Canada; June 2000. Granja PL, Ribeiro CC, Rouais F, Pouységu L, De Jéso 8, Baquey C, Barbosa MA, Influence of the pre-incubation in calcium on the mineralization of unmodified and phosphorylated cellulose. Biomineralization of Implant Materials. Lisbon, Portugal; June 2000, Granja PL, Ribeiro CC, Rouais F, Pouységu L, De Jéso 8, Baquey C, Barbosa MA. Influence of the phosphorylation degree on the mineralization of cellulose phosphate hydrogels. BioEng’00. Coimbra, Portugal; May 2000. Granja PL, Pouységu L, Bareille R, De Jéso 8, Saquey C, Barbosa MA. Bioactivty of cellulose phosphate biomaterials. Intemational Conference on Biopolymer Technology. Coimbra, Portugal; September 1999. Granja PL, Bareille R, Rouais F, De Jéso B, Barbosa MA, Baquey C. Influence of the phosphorylation degree on the attachment of human osteoprogenitor cells to cellulose phosphates. 15th European Conference on Biomaterials. Bordeaux, France; September 1999, Granja PL, Pouységu L, De Jéso 8, Baquey C, Barbosa MA. Cellulose phosphates as biomaterials. Mineralization studies. 13th European Conference on Biomaterials. Gothenburg, Sweden; September 1997. Granja PL, Pouységu L, De Jéso B, Baquey C, Barbosa MA. Synthesis and characterization of cellulose phosphates for biomedical applications. Gordon Research Conference on Biodegradable Polymers. Barga, Italy; May 1997. Granja PL, Pouységu L, Pétraud M, De Jéso 8, Baquey C, Barbosa MA. Cellulose phosphates as biomaterials. A nuclear magnetic resonance investigation, 2nd Symposium on Frontiers in Biomedical Polymers - Biomaterials and Drug Delivery ‘Systems. Eilat, Israel; April 1997. Granja PL, De Jéso B, Baquey C, Poustis J, Barbosa MA. Biomimetic natural materials for bone regeneration, Junior-Euromat 96. Lausanne, Switzerland; August 1998.. to my parents, Hugo and Manela. A thousand league joumey always starts with a single step. Chinese proverbPCRMOWI GOMES: Acknowledgements This is one of the most difficult items to be written in such a work. It seems to me rather unlikely to carry out a PhD without being proficiently supported by several people and institutions, as well as highly motivated. | had this privilege of working in three renowned institutions, with outstanding scientific supervisors and with remarkable colleagues. As much as | could write here would never be enough to show how grateful | ‘am to those people and institutions for their encouragement and support. The Biomaterials Laboratory of Instituto de Engenharia Biomédica (INEB, U. Porto, Portugal), the host institution, INSERM U.443 (U. Bordeaux 2), and Laboratoire de Chimie des Substances Végételes (LCSV, from INSTITUT DU PIN, U. Bordeaux 1), have always placed at my disposal every necessary mean, and have hosted me in a very friendly way, with high scientific level and good working environment. Professor Mério Barbosa encouraged me into this fascinating scientific field, and has inspired all my experimental work. | also owe him my scientific culture, which was built upon the involvement in scientifc events as well as in the laboratony’s daily life. And these are just some of the reasons for which | will always be grateful to him. | owe Professor Charles Baquey the warm welcome in his laboratory, his friendship, availabilty and sharing of a wide scientific knowledge. | owe Professor Berard De Jéso the affectionate reception | was provided with, his friendship and availability to teach me the necessary steps leading to the development of my work. Laurent Pouységu is an every time friend, who was patient enough to teach a metallurgist some organic chemistry, sometimes at the expense of his own work, and always with a smile. Laurent was an exceptional bench colleague, with whom | have leamt much. Although not directly involved in this work, Rui Reis is an ever since friend, to whom | also owe a part of my scientific culture, gained in many continuous and highly relevant discussions. Cristina Ribeiro was a kind of inspiration, since the beginning, due to her scientific competence as well as personal attitude, She has also significantly contributed to the enrichment of my experimental work. | owe Jean-Christophe Fricain the carying out of every animal experiments and his honest friendship. Reine Bareille allowed me,to develop biological experiments, with patience and scientific competence. Frangois Rouais and Michel Pétraud gave me their kind support in many experiments as well as their friendship. Denis Deffieux hosted me in his laboratory and was also a true friend during the most part of my staying in Bordeaux. | am thankful to Anne Hochedez for several characterization studies as well as for a pleasant daily empathy in the lab. ‘Some colleagues and friends from INEB, by their own works, have provided me with a considerable enriching knowledge of my own work. | would also like to thank Isabel Biomimetic Natural Materials for Bone Regeneration, Pedro L. Granjaite Acknowledgements Amaral, Cristina Bartias, Anténio Pedro Fonseca, José Paulo Pereira, Isabel Pamplona @ Ana Leite for their friendship, and recognize their influence on me. Although not influencing my experimental work so directly, other colleagues and friends from the three laboratories have also greatly contributed with their help and friendship, allowing me to overcome every possible difficulty. Besides Olga Paiva and ‘Ana Paula Filipe, whose friendship goes beyond laboratory relationship, | am deeply grateful to Carlos Fonseca, Cristina Martins, Denilson Perez, Fabio Palumbo, Felix Koehler, Gabriela Afonso, Gilles Sebe, Januério Lima, Jean Claude Lartigue, Jean-Yves Ellie, Jiang Ji, Joo Lopes, Manuela Brés, Marie Christine Durrieu-Porté, Huy-Loc Nguyen, Nicole Mendoza, Monique Rouais, Pedro Sé, Sophie Verrier, Susana Sousa and Virginia Fonseca, ‘Several other colleagues and friends were also very important during this work. | am very grateful to them all. At INEB: Ana Paula Pego, Ana Queiroz, Aurélio Campitho, Carmo Pereira, Conrado Aparicio, Daniel Rodriguez, Femando Jorge Monteiro, José Cavalheiro, José Domingos Santos, Judite Barbosa, Maria Ascensdo Lopes, Maria Pia Ferraz, Miriem Langhari, Roldéo Santos and Simone Morais; at INSERM: Alain Deschamps, Anthony Duncan, Berard Dupuy, Bruno Brisson, Frangoise Lefebvre, Frank Villars, Joelle Amédée, Josette Gautreau, Michel Rabaud, Nicole Barthe, Patrick Guiton e Sandrine Dutoya; and at INSTITUT DU PIN: Alain Castellan, Bemadette Guillabert, Bernard Delmond, Boris Monset, Christelle Faveau, Christian Baudry, Christian Servens, Eric Virol, Fabienne tbalot, Francoise Mercier, Frederique Ham- Pichavant, Gerard Daudé, James Dédier, Jean-Michel Lasnier, Marianne Vialemaringe, Marie Heléne Lescure, M. Ricarrere, Michélle Dupiré, Pascale Destribats, Patrick Pardon, Pierre Martineau, Vincent Darcos e Xavier Delrieu. From the Dept. Metallurgical Eng. (U. Porto): all my teachers and the technicians, that educated me and provided me with the necessary skills for the development of this work; Rui Correia from Dept. Eng. Cerémica @ do Vidro (U. Aveiro); Ricardo Livio and Carlos Sa from CEMUP (U. Porto); Jean Patrick Chenu from DETERCA (Département de Techniques et de Recherches Chirurgicalles Appliquées, U. Bordéus 2); N.B. Chanh from the Crystallography Laboratory (U. Bordéus 1); Christinne Labrugére from ICMCB (U. Bordéus 1); Ana Mota from Fac. Medicina Dentéria (U, Porto); Michel Harribey from HEXABIO (Bordeaux); Jean-Yves Elie from Association BIOMAT (Bordeaux); Rosério Soares and Artur Ferreira from Laboratério Central de Anélises (U. Aveiro): and Hannu Merikkalio, from Cellomeda (Finland). | would especialy ike to show my gratitude to Manela, Thanking may be indirectly proportional to the familiarity we have with the person we want to thank, maybe because itis so difficult to spell the right words. Thank you for everything, My fiends have also provided me with the sometimes necessary distraction, ‘Supported and encouraged my work, basically during the most dificult times. At last, but above all, my parents and brother, and my family in general, o whom | ‘owe everything | am. There are no words to express what | feel, except a profound pride in taking part in their ives. ‘Accounting for the fact that my work took a considerable time, | fear | might be forgetting someone. For the fact I sincerely apologize. ‘Biomimetic Natural Materials for Bone Regeneration, Pedro L. GranjaContents Acknowledgements #1 Contents, Abstract «v Résumé « vi Resumo «ix Foreword xi List of abbreviations + xiii 4, INTRODUCTION «1 1.1, Biologically functional biomaterials «1 1.2, Biomaterials for orthopedic applications «8 1.3. Cellulose in biomedical applications » 12 4.4, Cellulose phosphates: A review « 15 1.4.1. Preparation « 16 1.4.1.1. Reaction with inorganic phosphates « 16 1.4.1.2. Reaction with organic, phosphorus-containing reagents * 19 1.4.1.3, Reaction starting from cellulose derivatives « 19 1.4.1.4, Graft copolymers «21 1.42. Properties «21 1.4.2.1, Flame retardancy «22 1.4.2.2. lon exchange capacity «23 14.23, Viscosity «23 1.4.2.4, Water sorption «23 1.4.3. Applications «23 1.4.3.1, Flame retardants « 24 1.4.3.2. lon exchangers «24 1.4.3.3, Water sorbents and thickening agents « 30 1.4.3.4, Miscellaneous biomedical applications » 30 Biomimetic Natural Materials for Bone Regeneration, Pedro L. Granjave Contents 1.4.4. Perspectives of application as biomaterials «31 1.4.4.1. Biomaterials promoting mineralization, for orthopedic applications + 32 1.4.4.2. Support for the immobilization of bioactive species «34 1.4.43. Miscellaneous biomedical applic 1.5. Main objectives of the present research «34 2. SYNTHESIS AND PHYSICO-CHEMICAL CHARACTERIZATION « 57 2.1. Cellulose-based biomaterials for bone regeneration: Hemi-synthesis and characterization » 59 2.2. Cellulose phosphates as biomaterials «69 2.3. Cellulose phosphates as biomaterials. Synthesis and characterization of highly phosphorylated cellulose gels » 69 2.4, Cellulose phosphates as biomaterials. Surface chemical modification of regenerated cellulose hydrogels » 107 3. BIOLOGICAL BEHAVIOR EVALUATION +121 3.1. Cellulose phosphates as biomaterials. Mineralization of chemically modified regenerated cellulose hydrogels » 123 3.2, A simple method to induce mineralization on high swelling materials « 135 3.3. Cellulose phosphates as biomaterials. In vitro biocompatibility studies » 147 3.4, Mineralization of regenerated cellulose hydrogels induced by Human osteoprogenitor cells + 163 3.5. Cellulose phosphates as biomaterials. In vivo biocompatibility studies » 175 4. GENERAL DISCUSSION « 189 4.1. Synthesis of cellulose phosphates « 189 4.2. Characterization of phosphorylated cellulose « 190 4.3, Mineralization studies » 198 44, Invitro behavior in cultured bone cells « 199 4.5. In-vivo behavior « 202 5. CONCLUSIONS AND FUTURE WORK «213 5.1. Synthesis of cellulose phosphates «213 5.2. Function of cellulose phosphates » 213 53. Future work © 215 ‘Appendix - Definitions + 217 Biomimetic Natural Materials for Bone Regeneration, Pedro L. GranjaAbstract The present research is aimed at investigating the applicability of cellulose phosphate as a biomaterial for orthopedic applications. Cellulose phosphate (CP) has been used for decades in the treatment of Ca metabolismrelated diseases, such as renal stones, due to its high Ca binding capacity, associated with lack of toxicity and indigestibilty. It has also been widely used in affinity chromatography, ‘owing to its ability to specifically bind biologically active species, such as enzymes and peptides. Adequate biomaterials for orthopedic applications should consist in biomimetic structures capable of promoting the regeneration of bone tissue without inducing adverse reactions and assuring its physiological functions. Que to their capability of binding Ca and eventually growth factors, CP can be envisaged as a promising altemative biomaterial, capable of promoting an adequate healing response once implanted, In the present work, CP was synthesized according to an optimization of the HsPOUP20s/EtsPOuhexanol method. Reaction parameters investigated were the sweling pre-treatment, the sequence of addition of reagents, temperature and time of reaction, and the relative quantities of reagents. The synthesis was optimized using microcrystalline cellulose. For the first time, cellulose triphosphate is reported, which opens the possibilty of using these materials more efficienty in many applications where the functionality is directly related to the phosphate content. It was demonstrated that the present technique promotes covalent binding of phosphate groups to the cellulose backbone, and only monoesters are obtained. The reaction seemed to allow regioselective substitution in the C-6 carbon for low phosphate contents. CP thus obtained was essentially amorphous, presented high water swelling, and seemed resistant to gamma sterilization. This synthesis route was effectively applied to the surface modification of regenerated cellulose hydrogels (RCH), resulting in highly phosphorylated products, with increased surface roughness and hydrophilicity. The assessment of the mineralization of CP in simulated physiological conditions, ie., the ability to induce the formation of an apatite layer, showed that only the Ca salt of CP promoted formation of apatite, and the interface between the polymer and the mineral layer was continuous. The Ca salt of surfaces phosphorylated for 8 hours promoted higher mineralization extents than the Ca salt of surfaces treated for shorter or longer periods. The pre-incubation of unmodified RCH in Ca also resulted in the homogeneous formation of an apatite layer, although in this case the interface between the polymer and the mineral layer was not continuous. in vitro biocompatibility studies in cultured bone cells showed that CP is Biomimetic Natural Materials for Bone Regeneration, Pedro L. Granja Abstract «Vvvie Abstract not cytotoxic, independently of the phosphate content. However, CP promoted poor rates of cell attachment, proliferation and diferentiation, which were attributed to the negative charge, associated with the high hydrophilicity of the cellulose derivative. On the contrary, unmodified RCH promoted high rates of cell attachment, proliferation and differentiation. in this case, an apatite layer was observed between the cellulose surface and the cell layer, which was attributed to the synthesis of extracelluiar matrix by the osteoblastic cells. Finally, animal implantation studies in rabbits revealed the biocompatibility of both unmodified and phosphorylated cellulose, as well as their osteoconductive properties. A full osseointegration could not be observed, although some remodeling activity of the bone tissue was observed when CP was used. ‘Biomimetic Natural Materials for Bone Regeneration, Pedro L. GranjaRésumé Ce travail de recherche est décié & étude du phosphate de cellulose en vue de son utilisation comme biomatériau pour des applications orthopédiques. Le phosphate de cellulose (PC) est utilisé depuis des maladies liées au métabolisme du Ca, tels que les calculs rénaux, en raison de sa grande capacité a fixer le Ca, de sa non-toxicité et de sa non-assimilation par Torganisme. Il a également été largement utilisé en chromatographie par affinté en raison de sa capacité & lier spécifiquement des espéces biologiquement actives, telles que des enzymes et des peptides. Les biomateriaux destines aux applications orthopédiques doivent présenter une structure biomimétique capable de promouvoir la régénération du tissu osseux sans provoquer de réactions défavorables et en assurant les fonctions physiologiques de los. Grace & sa capacité de fixation du Ca et par la suite de facteurs de croissance, le PC peut étre envisagé comme un biomatériau alternatif prometteur, susceptible, une fois implanté, de promouvoir une régénération adéquate des tissus. ‘Au cours de ce travail, le PC a été synthétisé selon une optimisation de la méthode HsPOuP20s/EtsPOuhexanol, Les paramétres de réaction étudiés ont été le pré-gonflement de la cellulose, la séquence d'addition des réactifs, la température et le temps de réaction, ainsi que les quantités relatives de réactifs. La synthase a été optimisée en utlisant de la cellulose microcristaline. Du triphosphate de cellulose est ici décrit pour la premiére fois, ce qui offre la possibiité dutiiser ces matériaux plus efficacement dans de nombreuses applications pour lesquelles la fonctionnalité est directement liée a la teneur en phosphate. Nous avons démontré que cette procédure permet la formation d'une liaison covalente entre les groupes phosphate et la chaine cellulosique et que seuls des monoesters sont obtenus. Cette méthode semble conduie @ une substitution régiosélective sur l'atome de carbone C-6 lorsque les teneurs en phosphate sont faibles. Le PC ainsi obtenu est essentiellement amorphe, gonfle considérablement dans eau et semble résister & la stériisation gamma. Cette synthase a été appliquée avec succés a la modification de surface d'hydrogels de cellulose régénérée (HCR), conduisant 8 des produits fortement phosphorylés, caractérisés par une augmentation de la rugosité et de "hydrophilie de surface. Liévaluation de ia minératisation des PC dans des conditions physiologiques simulées, clest 2 dire leur capacité & induire la formation d'une couche ¢'apatite, a montré que seuls les sels de Ca de PC induisent la formation d'apatite, interface observée entre le polymére et la couche minérale étant continue. L’étendue de snnies dans le traitement de minéralisation la plus importante est obtenue avec le sel de Ca de surfaces Biomimetic Natural Materials for Bone Regeneration, Pedro L. Granja Résumé viville Résumé phosphorylées pendant 8 heures, contrairement aux sels de Ca de surfaces soumises a des traitements plus courts ou plus longs. La pré-incubation dHCR non modifiés dans le Ca a également donné la formation homogéne dune couche apatite, bien que dans ce cas, linterface entre le polymere et la couche minérale ne soit pas continue, Des études in vitro de biocompatibilté dans des cultures de cellules osseuses ont montré que le PC n'est pas cytotoxique, indépendamment de sa teneur en phosphate. Cependant, le PC conduit @ de faibles pourcentages dattachement, de prolifération et de différentiation des cellules ; ce comportement a été attribué a la charge négative associée une forte hydrophilie du dérivé cellulosique. Par contre, les HCR non modifiés donnent des pourcentages élevés Gattachement, de prolifération et de différentiation des cellules. Une couche apatite entre la surface de la cellulose et la couche de cellules a été observée ; elle proviendrait de la synthése d'une matrice extracellulaire par les ostéoblastes. Enfin, des études i de la cellulose non modifiée et de la cellulose phosphoryiée, ainsi que leurs Propriétés dostéoconduction. Une ostéointégration compléte n'a pu étre observée, bien que lactivité de remodelage du tissu osseux ait été observé lors de Tutiisation de PC. plantation animale chez le lapin ont révélé la biocompatibilté ‘Biomimetic Natural Materials for Bone Regeneration, Pedro L. GranjaResumo presente trabalho visa investigar a aplicabilidade do fosfato de celulose como biomaterial para aplicagdes ortopédicas. O fosfato de celulose (FC) é usado ha décadas no tratamento de doengas relacionadas com o metabolismo do calcio, tais, ‘como pedras renais, devido @ sua elevada capacidade de ligagdo ao Ca, associada sua nao toxicidade e nao assimilagao pelo organismo. © FC também tem sido pacidade para largamente utiizado em cromatografia por afinidade, devido a sua ligar especificamente substancias biologicamente activas, tais como enzimas @ éptidos. Um biomaterial adequado para aplicacSes ortopédicas deveria consistir numa estrutura biomimética capaz de promover a regenerago do tecido ésseo sem induzir reacgdes adversas, assegurando ainda as suas fungées fisiolégicas. Devido 4 sua capacidade de fixagao do Ca e eventualmente factores de crescimento, 0 FC foi concebido como um biomaterial altemativo promissor, susceptivel, apés implantagdo, de promover uma regeneragtio adequada dos tecidos. No presente trabalho, 0 FC foi sintetizado de acordo com uma optimizagdo do método HsPOW/Pz0¥EtsPOdhexanol. Os parametros de reaceao investigados foram © tratamento de pré-inchamento, a sequéncia de adicao dos reagentes, a temperatura © 0 tempo de reaceéo, @ as quantidades relativas de reagentes. A sintese foi optimizada utiizando celulose microcrstalina. O trfosfato de celulose & aqui descrito pela primeira vez, 0 que cria a possibilidade de uma utlizagdo mais eficaz destes materiais em diversas aplicagSes nas quais a funcionalidade esta directamente relacionada com 0 teor em fosfato. Foi demonstrado que a técnica presente promove uma ligagdo covalente entre os grupos fosfato e a cadeia celulésica, @ que apenas monosteres so obtidos. Este método pareceu proporcionar uma substituicdo regioselectiva no carbono C-6, quando os teores em fosfato sdo reduzidos. O FC obtido por esta via é essencialmente amorfo, incha consideravelmente em agua e parece resistente a esteriizacdo gamma, Esta sintese foi aplicada, de uma forma eficaz, como modificagao superficial de hidrogeis de celulose regenerada (HCR), tendo resultado em produtos com elevados teores em fosfato, caracterizados por uma aumento da rugosidade e da hidroflicidade, © estudo da mineralizago do FC em condicdes fisiolégicas simuladas, i capacidade para induzir a formagao de uma camada de apatite, demonstrou que apenas 0 sal de Ca do FC promove a formagao de apatite, e que a interface entre 0 polimero © a camada mineral é continua. O sal de Ca de superficies fosfatadas durante 8 horas promoveu taxas de mineralizacdo mais elevadas em comparacdo com sais de Ca de superficies tratadas durante periodos mais curtos ou mais longos. A pré-incubagéo de HCR nao modificados em Ca resultou igualmente na Biomimetic Natural Materials for Bone Regeneration, Pedro L. Granja Resumo ixxe Resumo formago homogénea de uma camada de apatite, apesar de, neste caso, a interface entre 0 polimero e@ a camada mineral ndo ser co biocompatibilidade in vitro com células ésseas demonstraram que o FC ndo & citotéxico, independentemente do teor em fosfato. No entanto, o FC promoveu uma fraca adeséo, proliferagdo e diferenciagdo celulares, 0 que foi atribuido carga negativa, associada a elevada hidroflicidade do derivado celulésico. Pelo contrario, (0s HCR no modificados promoveram elevadas taxas de adesdo, proliferagao diferenciagao celulares. Foi observada uma camada de apatite entre a superficie da celulose e a camada celular, 0 que foi atribuido a sintese de matriz extracelular pelas células osteoblasticas. Por fim, estudos de implantagao animal em coelhos wa. Estudos de revelaram a biocompatibilidade tanto da celulose ndo modificada como também da fosfatada, bem como as suas propriedades osteocondutoras. Nao foi observada um ‘osseointegragao completa, apesar de terem sido observados indicios de actividade de remodelago, quando foi utilizada celulose fosfatada. Biomimetic Natural Materials for Bone Regeneration, Pedro L. GranjaForeword Cellulose is a natural polymer used for varied biomedical applications. In orthopedic surgery the use of implanted synthetic materials is now common practice as an answer to a great number of pathological situations. Cellulose has firstly been proposed as a biomaterial for orthopedic applications by the biomaterials group (INSERM U.443) from the University Bordeaux 2, in France. A new type of regenerated cellulose hydrogels was developed and patented. it was demonstrated that these materials are well tolerated in vivo but they lack full integration with the bone tissue. {n the present work, cellulose phosphates are proposed as new biomaterials for orthopedic applications. These materials are expected to be biocompatible and able to promote bone regeneration. Bone is a composite material, basically consisting in an organic phase of collagen and an inorganic phase of calcium phosphate mineral. Cellulose phosphates were then envisaged as materials capable of mimicking the natural matrix of bone, by providing phosphate functionalities in which calcium phosphate mineral can be formed. For this purpose, a collaboration was established between the INSERM U.443, specialized in the evaluation of the biological behavior of biomaterials, the Institut du Pin (University Bordeaux 1), specialized in cellulose chemistry, and the Biomaterials Laboratory of INEB (University of Porto), which is specialized in interfacial chemistry of biomaterials. The first chapter of the thesis consists in a brief overview on some recent concepts regarding biologically functional biomaterials, and especially biomaterials, for bone regeneration, as well as the biomedical applications of cellulose. Cellulose phosphates are reviewed in terms of synthesis routes, properties and applications. Prospects of application of cellulose phosphates as biomaterials are emphasized. ‘The second and third chapters describe the experimental part of the investigation, in the form of a compilation of works already published or submitted for publication in intemational refereed journals. In the second chapter, the chemical routes used to prepare and characterize cellulose phosphates are described. Finally, the biological behavior of cellulose phosphates, in vitro using cultured human osteoprogenitor cells, and in vivo through animal implantation, is described in tye third chapter. Atlast, the thesis concludes with a general discussion and future perspectives. Biomimetic Natural Materials for Bone Regeneration, Pedro L. Granja Foreword © xiAbbreviations. « xii List of Abbreviations AGP - Amorphous calcium phosphate ALP - Alkaline phosphatase AmCP - Cellulose phosphate NH salt ATP - Adenosine triphosphate ATR-FTIR - Attenuated Total Reflectance-FTIR BaCP - Cellulose phosphate Ba salt BeCP - Cellulose phosphate Be salt BMP - Bone morphogenetic protein CA - Cellulose acetate CAB - Cellulose acetate butyrate CaCP - Cellulose phosphate Ca salt Ca-P - Calcium phosphate CAP - Cellulose acetate propionate CAPh - Cellulose acetate phosphate CCP - Cellulose chloroethy! phosphate CDA - Cellulose diacetate COP - Cellulose diethyl phosphate CelL-P - Cellulose phosphate (better designated as cp) CMC - Carboxymethyicellulose CN - Cellulose nitrate COL |- Type I collagen CP - Cellulose phosphate CP/MAS - Cross Polarization/Magic Angle Spinning rl - Crystallinity Index CRV- Cellulose regenerated by the viscose process (see RCH) CRV-P - Phosphorylated CRV CRV-P-Ca - CRV-P calcium salt CS - Cellulose sulfate CTA.- Cellulose triacetate CuGP - Cellulose phosphate Cu salt DEAE - Diethylaminoethyl cellulose DMA - N-dimethyl acetamide DMF - .N-dimethylformar DMSO - Dimethyisulfoxide DP - Degree of Polymerization DS - Degree of Substitution EC - Ethylcellulose ECM - Extracellular matrix ECTEOLA - cellulose derivative prepared with epichlorohydrin and triethanolamine EDS - Energy Dispersive Spectroscopy ELISA - Enzyme-linked immunosorbent assay EVAL - Ethylene Vinyl Alcohol FCS - Fetal Calf Serum FTIR - Fourier Transform infrared GH - Growth hormones GLA - Gamma-carboxyglutamic acid GRAS - Generally Recognized as Safe HAp - Hydroxyapatite HEC - Hydroxyethylcellulose HMC - Hydroxymethylcellulose HOP - Human osteoprogenitor HPC - Hydroxypropylcellulose HPLC - High performance liquid chromatography HPMC - Hydroxypropyimethylceliulose IGF - Insulin-like growth factor IMOM - Iscove modified Dubelcco's medium MAS - Magic angle spinning MC - Methyiceliulose MCC - Microcrystaline cellulose MCC-P - Phosphorylated MCC MgCP - Cellulose phosphate Mg salt MPC - 2-methyacryloyloxyethyl phosphorylcholine MTT - Mitochondrial tetrazolium test NaCMC - Sodium carboxymethyicellulose NaCP - Cellulose phosphate Na salt NaCS - Sodium cellulose sulfate NCP - Non-collagenous proteins NICP - Cellulose phosphate Ni salt NMMNO - A-methylmorpholine N-oxide NMR - Nuclear Magnetic Resonance OC - Osteocalcin ‘Biomimetic Natural Materials for Bone Regeneration, Pedro L. GranjaAbbreviations « xiv OCP - Octacalcium phosphate PA - Polyamide (nyton) PAN - Polyacrylonitrile PbCP - Cellulose phosphate Pb salt PBS - Phosphate-buffered saline PDMS - Poly(dimethylsiloxane) (silicone) PE - Polyethylene PEEK - polyetheretherketone PEOIPBT - poly(ethylene oxide)/poly(butylene terephtalate) PET - Poly(ethylene terephtalate) (Dacron®) PGA - Polyglycolic acid PHEMA - Poly(2-hydroxyethyl methacrylate) PLA- Polylactic acid PLGA - copolymer of PLA and PGA PMMA - Poly(methyl metacrylate) PP - Polypropilene PS - Polysuifone PTFE - Polytetrafluoroethylene (Teflon®) PTH - Parathyroid hormone PU - Polyurethane PVC - Poly(vinyl chloride) RCH - Regenerated cellulose hydrogels (see CRV) SBF - Simulated body fluid ‘SDS-PAGE - Sodium dodecy! sulfate- polyacrylamide gel electrophoresis ‘SEM- Scanning electron microscopy TCPS - Tissue culture polystyrene TEAE - triethylaminoethyl cellulose TGF- - Transforming growth factor-B ‘TMS - Trimethytsily! UHMWPE - Ultra-high molecular weight PE UI- Unique identitier number, from Mediine XPS - X-ray Photoelectron Spectroscopy XRD - X-ray diffraction Biomimetic Natural Materials for Bone Regeneration, Pedro L. Granja“4 Chapter 1 Introduction 4.4, BIOLOGICALLY FUNCTIONAL BIOMATERIALS Biomaterials science contributes considerably to improve the quality of lfe of an increasing amount of people. New fields of application are constantly being found due to technical as well as conceptual achievements. Many diseases can now be treated due to implanted biomaterials, although a great number of problems associated to their in situ behavior have always accompanied every successful approach. A biomaterial, or biomedical material, is defined as a material intended to interface with biological systems to evaluate, treat, augment or replace any tissue, organ or function of the body." ‘The historical pathway of biomaterials science has known profound changes in its relatively short time of existence. The most important changes were due to new philosophical concepts and not to technical improvements. The latter were many and varied but were always a consequence of new ways of reasoning biomaterials. Originally, the first biomaterials were not designed as such, but they were found useful due to their bioinertness (e.g. stainless steel)? Their immediate clinical perform- ance was generally satisfactory, and their development as biomaterials was based on a trial- ‘and-error optimization approach rather than being engineered to produce the desired interfacial reaction* Later on, new reasoning imposed the development of bioactive materials, je, materials exhibiting a particular biological activity rather than being bioinert.’**"" Besides the appearance of new biomaterials, research on the field started to focus on the development of specially conceived biomaterials, designed for a particular application. At present, materials in clinical utilization can be considered in an intermediate state between the previous concept of bicinertness and of bioactive materials. Table 1.1 lists selected applications of biomaterials as well as the main types of materials currently used. The most well known example of a bioactive material that is in clinical use is probably hydroxyapatite, This so-called bioactive ceramic is a synthetic material with the same composition of the main mineral constituent of bone tissue and, as such, has been reported as capable of inducing bone regeneration" The wide range of biomaterials presently available for clinicians vary from the so-called traditional materials, like metals, ceramics, glasses and glass-ceramics, polymers ‘and their composites, to the less traditional ones, such as hydrogels, biodegradable materials and natural materials. Despite the usefuiness of all these materials for many clinical applications, it can be easily seen in Table 1.1 that the same material is used for several distinct applications, thus indicating lack of specific behavior. In fact, research on the field is far ahead and new directions point out to the use of much more intricate structures, providing biological reactions, biotechnology, molecular biology and materials sci- ence, in an effort of synthesizing biologically functional biomaterials.*°"° specific by combining Biomimetic Natural Materials for Bone Regeneration, Pedro L. Granja2e Chapter 1. introduction Table 1.1. Selected applications of biomaterials and examples of main types of materials curently used in their manufacture #4243824? ‘SysteriApplication Examples Types of materials Skeletal Joint replacements (hip, knee)* Devices for fracture fixation: plates, wires, pins, screws and nails Bone cement Bony defect repair ‘Astifcial tendon and ligament Dental implant for tooth fixation Maxilofacial reconstruction Breast augmentation or reconstruction Cosmetic replacements ‘Avtifcial skin, burn dressings Blood vessel Heart valve Reproductive Integumentary Circulatory Catheter Tota artificial heart", Cardiac pacemaker Hydrocephalus shunt Middle ear ossicte Intraocular lenses Contact lenses Coatings for tablets or capsules, Nervous Vision Controlled drug dotivery transdermal systems, microcapsules, implants General surgery Sutures Adhesives Hemostatic agents (gauzes) Extracorporeal Dialysis membrane Ti Talloy, stainless steel, UHMWPE, PMMA, ‘alumina, zirconia, carbon, PA, PP Stainless stee!, Co-Cr alloy, Ti alloy, PEEK, carbon, PA, PLA, PGA PMMA HAp, collagen, bioactive glasses and glass-ceramics PTFE, PET, PE, carbon Noble metal aloys, porcelain, Ti, alumina, HAp, PMMA, UHMWPE, epoxy resin HAp, PU, PVC, PMIMA, collagen, PTFE, PET, PE, PDMS, POMS, silicone gel, PU PMMA, POMS Reprocessed collagen, slicone-collagen, PU, alginate PET, PTFE, collagen, PU, PVC Reprocessed collagen, stainless steel, Co-Cr alloy, carbon, PET, polyacetal PDMS, PTFE, PU PU, PVC, PE, Tialloy Poms HAp, carbon PHEMA, PMMA, POMS ‘CAB, POMS-acrylate, PHEMA, PMMA-PDMS Alginates, cellulose derivatives, PLA, PGA, chitosan, collagen PA, PET, PTFE, PU, PLA, PGA, chitin, chitosan Fibrn, collagen Collagen, gelatin, chitosan, oxidized cellulose Cellulose derivatives, PAN, PS, EVAL Devi consisting of distinct pats that are made From diferent materia; CA - Celloee acetal; GAB - Celulose acetato buat: EVAL Etylene vinyl alcohol: Hap - Hysroxyapatte; PA -Polyaige (oon); PAN - Polyenontie; POMS - Poh(simetiylsilxane) (sllcone) PE - Polyetyfene; PEEK - Polyethetetherkelone; PET - Polyethylene terepiaat) (Dacron®), PGA - Plygycalic acid (biodegradable); PHEMA - Poly2shydronethy methacyate) (hyrogel); PLA -Poliactic aid (biodegradable): PUMA -Paty(metin metacryate); PP -Polproplns; PS - Pelysuifone; PTFE - Poltevafuoroethyene (Teton; PU - Polyurethane: PVC - Ptjvnl chloride); UHMVVPE - Ulvahigh molecular weght ovetylene, Many of the biomaterials listed in Table 1.1 constitute the best, or the only, currently available option for a given clinical situation, thus providing an effective treatment that was not previously available, ‘or a considerable improvement in patients’ quality of life. For instance, wound or burn dressings are used to promote regeneration of skin, facilitate healing and help minimizing adverse long-term cosmetic effects in patients that have suffered from severe ‘bum injuries. Artifcial tendons and ligaments are used to replace the anterior cruciate ligament (a ligament connecting the femur to the tibia through the knee joint), which is usually ruptured during sports activities. Total knee or hip replacements are ‘Biomimetic Natural Materials for Bone Regeneration, Pedro L. Granjanecessary to substitute the damaged joints of patients suffering from osteoarthritis and rheumatoid arthritis. Contact lenses and comeal implants are extensively used in ophthalmology. Vascular grafts are used to replace, for example, the aorta in patients with pathological conditions. Several types of dental implants are used to substitute damaged teeth. Breast and facial implants are used for cosmetic and reconstructive surgery. And many lite of an more examples exist related to the increasing amount of people throughout the world. The growing life span in modem western countries is a consequence of the recent achievements in the treatment of disease which, in part, were due to the developments in biomaterials science.” On the other hand, the enhanced life span associated with the decrease in birth rate, has raised several additional problems, namely the increase in the proportion of elder people in the growing population, whose organism naturally does not function as well as before and shows consequent increased needs for health care, and mainly tissue and organ replacement.”"? The total of organ transplants has increased considerably over the last number decade, as well as the number of patients on a waiting list, the time they spend waiting, and the number of deaths while waiting.'® For instance, it was estimated that, only in the USA, 7.6 mi patients per year (1998) rely on nonbiological implant materials to replace the structure and function of damaged tissues or organs." ‘At present, pathologies like tissue and organ deformities, or diseases caused by trauma, are treated by transfer of grafts from healthy donor tissue to the damaged by organ transplantation, or recurring to medical devices. All these alternatives have advantages and jisadvantages.*"**“7 Autograft_is the solution preferred by clinicians. since as it comes from a different location of the same individual, it is immunologically safe, thus limiting rejection concems. However, their unavailability, morbidly of the donor site, which can cause various subsequent A tissue, Biomimetic Natural Materials for Bone Ret Chapter 1. introduction « 3 painful problems, and fimitaions conceming the size and shape constitute. disadvantages.*"*“6"7 The problems of unavailability and shape and size can be partly overcome by using allografts since they come from major deceased donors, after processing for removing bioactive molecules. The immune response and the possibilty of transmission of viral constitute problems related = to.—this alternative.*"****" Several drawbacks also exist Telated to the use of biomaterials (whether alone or device), namely their dissimilarity to natural tissues or organs, incapabilty to adapt to the changing demands of human activity, namely growing and ageing, poor biocompatibility, infection susceptibility, immune responses, thrombus formation, elicitation of cellular degradation, and their limited service expectancy due to friction, wear and degradation in the warm, humid and corrosive environment of the human body.“ Since the fundamental objective of medicine is to understand and correct the disease process, it is lear that the use of artificial materials, as they are used today, does not accomplish that objective. However, as previously pointed out examples, the organ approach is considerably more effective than drug therapy or corrective medicine in the treatment of conditions like joint or dental fallure, cardiac valve disease, arterial obstruction and cataract, among many others, although no fully effective clinical solutions are yet available for restoring structure and function of damaged tissues and organs. The awareness of all these problems has driven biomaterials scientists diseases constituting a medical in some artificial to conceive new strategies, often totally antagonist to previous concepts, i.e., instead of trying to see if existing materials work in the body, the specific needs for a particular application are being fully investigated to develop biologically functional biomaterials, which would provide longer- ical benefit and fewer complications. in order term neration, Pedro L. Granja4.6 Chapter 1. Introduction Table 1. their applications. Biologically active molecule forimmobiization Examples of biologically active species that may be immobilized on or within polymeric biomaterials, as well as, Application —_—_— Proteinpeptide Enzymes, antibodies, antigens, cell achesion molecules Saccharides ‘Sugars, oligosaccharides, polysaccharides Lipids Fatty acids, phopholipis, glycolipids Drugs ‘Antithrombogenic agents, anticancer agents, antibiotics, contraceptives, drug antagonists, peptide/protein drugs Ligands Hormone receptors, cell surface receptors Nucleic acids, nucteotides ‘Single or double-stranded DNA, RNA Cells [As a result of this quest for the holy healing biomaterial, several new scientific concepts have emerged that strongly correlate with biomaterials, science, but where the input from the health sciences has the paramount role. For instance, a term now widely used is tissue engineering, which is defined as the persuasion of the body to heal itself, through the delivery to the appropriate sites of molecular signals, cells and supporting structures.’ It constitutes a good example of the enthusiasm originated by recent developments: in the past, a biomaterial conceived to interact properly with a living cell is now designated as a material for tissue engineering. This enthusiasm may lead to the feeling that it concems the same old concepts with a different name, but in fact differences are considerable and encouraging. Present biomaterials are intended to biologically function properly, and hence the required degree of specificity at a molecular level involves the incorporation of biologically active species, in such a way that the boundary between modem biomaterials science and biology is hardly distinguishable. Pioneer studies on the interaction of biologically active species with materials lead to the presently Bioreactors, boseparations, biosensors, diagnostic assays, biocompatible surfaces, affinty separations, targeted drug delivery, cell culture Antibacterial surfaces, thrombo-resistant surfaces ‘Thrombo-resistant surfaces ‘Thrombo-resistant surfaces, drug delivery systems Call culture, bicartfcial organsitissue engineering DNA probes, gene therapy Bioreactors, bioartificial organstissue engineering biosensors available technologies, allowing the preparation of structures consisting in material, cells and proteins, thus providing a cocktail ready to promote a specific 512405259 biological response after implantation. The ultimate goal would be to reconstitute living tissues and organs from specialized cells and intercellular matrix molecules to form higher ordered structures that closely resemble their natural counterparts. Examples of biologically active molecules that may be immobilized on or within polymeric biomaterials, as well as their applications, are listed in Table 1.2. Of course they are widely Used in conjugation or mixtures with each other. The interaction of those species with biomaterials surfaces is usually responsible for the biological behavior of implanted materials. For instance, the control of the protein monolayer adsorbed at the moment of implantation is of Paramount importance. When a biomaterial is implanted, the conformation of this protein layer is governed by non-specific interactions with the surface, thus resulting in a broad range of possible interactive sites, which in tum result in the simultaneous activation of a number of receptors on the cell membrane.” This seems to be closely Biomimetic Natural Materials for Bone Regeneration, Pedro L. Granjarelated to the fact that biomaterials presently used induce the same host response, usually ending-up with fibrous capsule formation involving the implant On the contrary, in biological systems, cone ligand specifically triggers a particular process. Hence, the successful development of biologically functional biomaterials seems to be closely related to their capability of inhibiting non-specific reactions, by switching on precise healing pathways via recognition events, thus activating signaling pathways. Biomaterials possessing specially designed receptor sites are encouraging. Molecular self-assembly is an example of a new approache that allows the development of surfaces with high degrees of specificity + on gucste omer nanan + colpoaets Ap renin Oy ersten @ croc Figura 1.1. Schemate iustaton of the concept of immunesolaien nvehed in the wansplataton of senogenictesue encapsulated in semipermeable polymer membranes.°* Several promising approaches are now being investigated to develop biologically functional biomaterials, for varied applications. A concept that is being largely investigated for a wide range of applications is the use of composites of synthetic materials with living cells (also called organoids’), to accelerate implant integration within the body. For instance, in the case of blood-contacting materials, thrombosis usually ‘encountered. The incorporation of heparin, agents is among problems still Chapter 1. Introduction « 5 like phospholipids, adsomtion of passivating proteins like albumin, or seeding with endothelial cells on material surface have proven to be effective in reducing thrombosis and enhancing biocompatibility."*7"** _Self-assembled lipid microstructures have been proposed for the encapsulation and delivery of hemoglobin, avoiding many of the inherent toxicities associated with the delivery of free hemoglobin.” The treatment of diabetes, a major disease affecting many people, is another example. Its treatment requires insulin and pancreas transplants have shown to be unsuccessful. As an altemative, the administration, transplantation of pancreatic islets, for continuing secretion of insulin, is under investigation.‘70294° Immunosupression cell therapy, of immuncisolation, uses microcapsules or hollow fibers to entrap cells or other biofunctional agents before implantation in the host organism, in order to limit communication between the cell in the device and the host organism, thus providing selective exchange of substances with the surrounding milieu and ing undesirable immune responses (like inward transport of leukocytes) (Fig. 4.4),422227340208" 1n the case of diabetes, secretion of insulin to the organism is provided by pancreatic islets, entrapped adequate immunosuppressive device (e.g. calcium alginate ‘surrounded with poly-L-¥ysine).70% ‘Another promising approach that may lead to the treatment of previously untreatable diseases is nerve regeneration. Nerve cells, unlike other cells, do not have the capability to proliferate." However, recent studies have demonstrated thelr capability to reestablishing continuity across defect sites 5° Some biomaterials have been investigated ‘as matrices for nerve regeneration, by facilitating the elongation of nerve calls, although an adequate solution has not been reported yet.**? Schwan cells seeded in biodegradable nerve guidance channels containing growth factors, neural support cells and genetically engineered cells seems to be a promising approach aimed at regeneration or nerves (Fig. 1.2). a in an promoting ‘Biomimetic Natural Materials for Bone Regeneration, Pedro L. Granja6 © Chapter 1. Introduction Filing of tube by blood derved fd and protein (ay 1) Invasion of cable by Schwann cels, foroblasts and endothe cals (days 7-14) ‘Another example is found in gene therapy, which provides the transfer of genetic material to the cells of a recipient to induce the release of a specific bioactive substance, or the transplantation of genetically modified cells to achieve a therapeutic benefit (Fig. 1.3)" This strategy has been proposed to the treatment of genetic diseases, acquired infections and gene augmentation in cancer, using viral transfer methods, physical transfer methods with liposomes, and genetically engineered cells transfer methods." The sequencing of the human genome, which can lead to a direct understanding of the Biomimetic Natural Materials for Bone eo ‘Sroonor Paso eles Oiuecle youth roughwal ‘aceon Figure 1.2. a) Nerve regeneration through a ‘uidance channel” b) Schematic ilustration of a nerve guidance channel and some of the possible strategies for influencing nerve regeneration.* provide the ultimate too! to healing biomaterials in many of the existing human pathologies. In order to be prepared for the yet unclear possiilies ctiginated by this process, materials possessing adequate structural and chemical functionalities for providing an appropriate housing for the delivery of genes are necessary.“ Many more interesting and encouraging approaches @ combination of biomaterials and biologically active species, and can be found in the literature.**"* 108253555954 The case of orthopedic applications will bbe analyzed in detail in the next section, Regeneration, Pedro L. GranjaGena caver Ineo etree vweutesinietran Ine recombinant ‘ruses or iposones othe ing tense recombinant ret congas Gone gn cars gee canted migo-pojeios Chapter 1. Introduction «7 “Transplant san gata win goetcaly| ‘rode krteocyae se gory fear trciats note hepaiccyes serataty meted planted encapeites serncaly mote rect geetensy mee’ centres Transplant senate ay na wt geetcaly Figure 1.3. The two principal strategies for gene delivery." In the past decade, we have witnessed a real revolution not only in the minds of biomaterials scientists but also conceming the general public interest in this field. The threat of having living cells implanted in their body, or re-implanted, whether or not genetically modified, has risen unmatched public attention, along with its consequent legal and ethical issues. Recent mediatic legal issues related, for example, to the eventual toxicity of silicone in breast implants*"*"5*" and PVC in blood bags,"**? have frightened companies at such an extent that the former is no longer commercialized in some countries and the latter is to be prohibited in many thers, even though, both examples represent huge markets. Fear from the general public, health authorities and practitioners to new, not fully tested solutions raises ethical issues that immensely prolong the period between successful clinical trials and approval by health authorities. Hence, it seems wise to develop future strategies to enhance public knowledge of the new developments and their real consequences, or otherwise the application of scientific progresses and many people's lives may be at risk. Despite these encouraging examples, because of them, many complex biomaterial issues or ‘Biomimetic Natural Materials for Bo are stil to be addressed for the forthcoming decades. The achievement of biologically functional biomaterials seems to be closely related to our ability to address. the -—following jesues:tS*2172422380041 + Adequate biodegradable and bioresorbable for and organization of various cell types, and other bioactive substances (e.g. growth controlling factors). Their degradation should match the rate of tissue regeneration, in order to build physiologically functional body parts that can be implanted and that stimulate the natural healing process; ‘+ Nanostructures and macromolecular assemblies that mimic natural structures or create new matrices in vitro colonization ones: + Environmentally materials; (or stimuli) responsive + Permselective membranes for transplantation of allogenic or xenogenic tissue protected against immune rejection by preventing immune recognition of the implant; Regeneration, Pedro L. Granja86 Chapter 1. introduction + Surfaces promoting selective cell achesion and protein adsorption, as well as specific cellular behaviors and phenotypic responses; + Matrices for targeted controlled release of bioactive substances, including matrices for gene therapy in which the gene expression is enhanced; + Manipulation of the humoral and cellular immune responses to transplants in order to improve allograft or xenograft tolerance: + Minimally invasive, dynamically responsive, and adaptable to growth and ageing techniques; + Better correlations between in vitro tests and in Vivo responses, as well as minimization of human and animal experimentation, through improvements in modeling and simulation; + Understanding of the mechanisms by which extracellular matrix components induce cell signaling and subsequent cellular responses; + Translate from lab to clinic and enhance general public awareness of new developments. 1.2, BIOMATERIALS FOR ORTHOPEDIC APPLICATIONS, In a materials science perspective, bone can be Understood as a composite material constituted by cells, the organic and inorganic matrices, and the extracellular matrix (ECM),"**457°7° Bone cells can be further subdivided in osteoblasts (the bone forming cells), osteocytes (the main cells of fully formed bone), and osteoclasts (the multinucleated giant cells responsible for bone resorption). “2457 ® The organic matrix (approximately 35% of the dry weight of bone) is mainly constituted by type | collagen (approximately 90%)."***7°% Collagen is, organized in fibers in which deposited. The inorganic matrix (approximately 60- 70% of the dry weight of bone) provides minerals for homeostasis, such as Ca, P, Na and Mg, and it consists of hydroxyapatite (HAp), with the structural formula of Cayo(PO,)e(OH)2.02 111642487020 The extracellular matrix (ECM) collagenous proteins, which are responsible for Providing the linking structure and function of cells, alized tissue is composed by non- ‘Biomimetic Natural Materials for Bone with soluble signaling factors, thus affecting cell anchorage, proliferation and phenotype expression. '824870% The mineralization of hard tissue, ie., the formation of the mineral phase into the organic matrix, is a matter of great relevance concerning materials for orthopedic applications and itis further discussed in chapter 2.2. Bone tissue is assuring functional properties, such as mechanical support and protection for the organs, and a site of attachment for muscles used in locomotion, as well responsible for as physiological activities, such as responsiveness the ability to regenerate in response to injury and is continuously to metabolic needs. Bone has being remodeled, ie., chan 19 the equilibrium between osteogenesis (formation of new bone) and resorption, *842457020 Bone diseases are many and varied. As previously mentioned, current available options for treatment of orthopedic diseases include grafts and synthetic materials, with th and drawbacks. In the treatment of some bone diseases, biomaterials play an important role and constitute an outstanding improvement in the quality of life of a large amount of patients, who would otherwise be confined to their most basic activities, and limited to a painful fe. Such diseases include skeletal loss due to trauma or removal of tumors, fractures and joint malfunctioning. To date, mainly metals, ceramics and polymers have been used in orthopedic applications,*#**" ‘S7RIBE59® as indicated in Table 1.1. Bioglasses*”” "245 and polymericeramic composites, such as UHMWPEVHAp”*5"" and PLAY or PLGA/HAp" composites, are also in clinical utilization, although in a minor scale since they constitute relatively respective advantages recent developments. ‘Skeletal loss can be treated using bone fillers. Calcium phosphate powders (mainly hydroxyapatite or Brtricalcium phosphate), reprocessed bovine collagen (xenograft or injectable materials made of calcium phosphates in a viscous polymeric vehicle are used for this purpose. *“"9872.76.08.29 Easy-to-treat fractures can be treated by non- surgical procedures, like immobilization with plaster. In the case of difficult-to-treat fractures, biomaterials Regeneration, Pedro L. Granjain the form of external or intemal fixation devices are used, Failure of fixation devices can be due to reasons so varied as overload, fatigue, corrosion and — loosening.°*“967275555 Biodegradable polymers with adequate mechanical properties constitute promising altematives since their use does not imply implant retrieval, avoiding a second surgical intervention, besides eliminating the problem of the presence of a foreign body in the organism. Bene: Capsular oament ricuar cartilage Bone: Figure 1.4, Schematic ilustration of a joint” Joint replacements present much more of a challenge to biomaterials scientists than fracture fixation, since joints (e.g. hip. knee, ankle, shoulder, elbow, finger, and intervertebral discs) consist in highly complex structures involving contacting parts made of distinct tissues (Fig. 1.4), and are load- bearing applications.*4“9*57272%59999 — Uniike fracture fixation devices, joint replacements are permanent implants, which makes their implantation an imeversible process. Examples of clinical situations that may lead to the need for joint replacement are osteoarthritis and ostoporosis. In both diseases, joint replacement, through the currently available techniques, only serves to alleviate the problem and is very far from representing a solution. In osteoarthritis, joint degeneration is caused by destruction of the articular cartilage (due to ageing or acute injury), resulting in severe pain and loss of motion, among effects,'5424570 7808009182 other deleterious Chapter 1, Introduction «9 Cartilage, contrarly to bone tissue, is a non- vascularized tissue with very limited capacity for repair, and therefore the damage is permanent and often progressive. Available treatments are based in antiinflammatory medication, arthrodesis (fusion of the joint), and joint replacement."*<2ss727888099182 Osteoporosis causes generalized bone mass loss. Available treatments are scarce, ineffective and strong efforts are put on prevention. Medication is preferred to joint replacement since, in many cases, bone is too fragile to withstand the surgical procedure,"*2457975825 Unfortunately, in the case of a fracture, namely in the femur, bone has lost the capacity to regenerate and a prosthesis. ‘must be implanted. Long-term implanted joint replacements is strongly dependent of load transfer characteristics at the interface between bone and implant, and this fact depends on implant design, #245, 707686 survival of ‘material properties and fixation metho ‘Overload or shielding from load-transfer may result in bone resorption and subsequent loosening of the implant'$2457975852° additionally, implant removal can be anticipated, which represents a problem since it should not require destruction of a large ‘amount of surrounding tissues. Bone implants for joint replacements may be fixed by diferent methods: mechanical interlocking, fixation, direct chemical bonding, or combinations between them,.*4"-04s7278ene20080 Mechanical interlocking is achieved by press-fiting the implant and using a bone cement - usually poly(methyl methacrylate) ~ for immediate fixation. This solution has proved to cause several problems like necrosis due to heating in the polymerization of the resin, toxicity of wear debris and methacrylate ‘monomers, and loosening at both new interfaces - bonefimplant and resin/implant.“**° Biological fixation consists in using textured or porous surfaces, allowing bone to grow in the interstices. Pores 100 to 350 um sized can accommodate bone ingrowth.’** Direct chemical bonding occurs when there is chemical similarity between bone and the implant surface, as for instance in metalic implants coated with hydroxyapatite.“"424#72788800:099 biota, Biomimetic Natural Materials for Bone Regeneration, Pedro L. Granja10» Chapter 1. Introduction Bone properties as a material have been extensively investigated and, although depending fon many factors ike moisture, age, site, and orientation of sample (due to its anisotropy), itis evident that its mechanical properties are quite different from the ones of materials currently used as orthopedic implants.**72***% For instance, the widely used metals are significantly stifer than bone, causing mismatching and thus being Chondrocytes Polymer Scatfold ~—-# In vitro tissue culture Pei Spier task Microgravity an poreeaar = +e 2 ey responsible for stress shielding and subsequent painful experiences to patients. On the contrary, ceramics are not adequate due to lack of flexibility Polymers, on the other hand, are flexible but usually not stiff to withstand mechanical requirements. Thus, the association of polymers enough and ceramics as composites appears as a alleviate this, promising alternative to problem, 72878857 b Jee Seti) Figure 1.5. a) Model system for tissue engineering. Chondrocytes isolated {rom a harvested cartilage sample are seeded on a synthetic, biodegradable Polymer scaffold, and cultured in vitro in peti dishes, spinner flasks, or microgravity bioreactors prior to in vivo implantation, @.9., to form Subcutaneous neocartlage or to repair damaged articular cartilage." b) IMustration depicting a tissue engineered construct. A biodegradable polymer Provides @ scaffold for parenchymal cells and support colls. As cells proliferate, they generate their own extracellular matrix However, bone is much more complex than a simple material and bone substitution cannot be restricted to a purely mechanical problem. As described previously, besides the organic and mineral phases, bone also contains cells, proteins and blood vessels. Thus, it seems evident that bone substitution will never be solved by a purely ‘materials science driven approach. In fact, a major health problem persists, since the existing devices are not effective and, in the best case, patients will need one or more additional surgeries to replace malfunctioning implants. Hence, better devices providing better solutions are necessary. Despite the past and present great controversy about the ideal properties of bone replacement materials, it seems that the concept of tissue engineered constructs, made of porous biodegradable polymers pre-colonized with bone cells before implantation, gathers most of favorable opinions (Fig. 1.5). However, important key issues Biomimetic Natural Materials for Bone Regeneration, Pedro L. Granjaremain to be clarified, like the one concerning what is more important to promote bone apposition, or osseointegration.'*°*"*"' On the one hand, many research efforts have been directed through biomaterials promoting growth and phenotype expression, which seems to be an opinion mainly diffused among biology-driven efforts,"*18177277-0% "8 On the other hand, many other works have the development of osteoblasts adhesion, focused on the development of biomaterials capable of inducing the formation of an apatitic layer, ie, that mineralize, which seems to be a popular concept mainly among materials science-oriented studies.*"°°""? it was already shown that surfaces promoting anchorage and differentiation, provide the necessary conditions for apatitic mineral to be formed.”™""""* In addition, many studies have shown that osteoblasts adhere and differentiate at a good extent on calcium phosphates,""*""® And to increment the controversy, some studies have shown that calcium phosphates, and namely HAp, inhibit osteoblast adhesion and osteoblasts proliferation and are digested by macrophages. "21 This is a good example of the great complexity of this particular scientific field. Seemingly, the two concepts are correct and should be unified, which means that the successful development of new biomaterials is unlikely without a close cooperation between biology and materials science. Thus, the future of orthopedic biomaterials, in concept, appears to be no different from the examples of biologically functional materials described in section 1. Several attempts are ongoing to prepare tissue engineered constructs for orthopedic applications (Fig. 1.5). Requirements for those structures include the ability to promote the proliferation and growth of viable bone cells expressing their phenotype, thus allowing the formation.of new bone in a 3D porous s2t945202009.74,7277.9999.322-125 structure, ‘An encouraging example can be easily selected from present literature, Promising substrates for tissue engineered bone tissue appear to be porous biodegradable scaffolds made from polymers belonging to the family of the poly(a-hydroxy) acids, namely poli(tactide), poli(glycolide), and their Chapter 1. Introduction «14 copolymers, mainly due to their good biocompatibility, good interaction with bone cells, adjustable biodegradation in the human body into metabolic residues, commercial availabilty, among other adequate properties."2 "52072229 However, ‘some complications were reported in vivo, namely inflammatory reactions at the site of implantation, or even tissue necrosis, which were attributed to the release of lactic acid and subsequent local tissue acidification. "21820'28124:27'29% Other polymers, although not so widely investigated, also seem promising for this namely, polyphosphazenes, poly (propylene fumarate), and poly(ethylene oxide)/poly (butylene terephtalate) (PEOIPBT), Polyphosphazenes have a phosphorus- nitrogen backbone, degrade in metabolic products and promote osteoblast-like cells growth."* in vitro, cell growth and degradation rates can be modulated by varying the nature of the hydrolytically unstable side chain.'" Composites poly (propylene fumarate)(tricalcium phosphate are also biodegradable and possess adequate mechanical properties, although preliminary animal studies showed a mild intial inflammatory response, ‘$""2*% PEOIPBT copolymers also promote bone cells growth and promote bone ingrowth in vivo, although degradation seems to be slower than desirable." 9 Some non biodegradable alternatives have also shown to be useful in applications where the tissue has a low capacity to regenerate." For cartilage repair, instead of osteoblasts, chondrocytes have been used with PLA, PGA and their combinations, and collagen, in the form of membranes, also showed promising resuts."* Polymerizable calcium alginate and poly(ethylene oxide) gels, seeded with cells, were used as injectable products, which constitute an interesting approach."® Protein-based materials are being investigated for many varied biomedical applications." Many other key issues still remain to be fully elucidated.”7-%"*" it has been shown that osteoblasts can be obtained mesenchymal stem cells isolated from bone marrow, However, it seems unlikely that cells alone will be effective, unless extracellular matrix is present. The in vitro maintenance of the purpose, in vitro from ‘Biomimetic Natural Materials for Bone Regeneration, Pedro L. Granja412 © Chapter 1. Introduction osteoblastic phenotype, practical clinical applications, also needs to be improved. Bone morphogenetic proteins have proven to be essential so that bone cells organize into tissues and organs, and therefore should be integrated in future implant materials,*"* although no adequate delivery systems seem to function property for that purpose, as a whole integrated system Furthermore, mechanical stability during bone regeneration, which is a matter of paramount importance, must be carefully addressed in clinical studies, required for 1.3, CELLULOSE IN BIOMEDICAL APPLICATIONS Cellulose is a natural occurring polymer, or biopolymer, since it is produced by plants, as well as by microorganisms. The use of natural polymers as structural materials is not new. Nature itself has always used, for instance, cellulose to provide the structure of higher plants, chitin as the exoskeleton of several mollusks, keratin for thermoinsulation in hair, collagen for mechanical support in connective tissues, and sik in spiderwebs. ‘The socio-economic situation of the modern world has raised the interest in natural polymers. Oil embargos and high prices, associated with the concerns of oil long-term availability, are favorable to the replacement of oilderived polymers by Polymers derived from Environmental concems are also playing an increasingly important role, contributing to the growing interest in natural polymers due to their biodegradability, low toxicity and low disposal costs. The usually low manufacture costs of biopolymers, felated to their large agricultural availability and renewability, are also-an advantage. Furthermore, their versatility of chemical structures and their well- known chemistry allow the development of advanced functionalized materials that can match several varied requirements. In addition, the rapid renewable resources. advancement in understanding of fundamental biosynthetic through genetic manipulations will provide tailoring of biopolymer pathways structure and function, thus opportunities for these polymers.“*" In the field, the degradation of natural polymers into physiological metabolites makes them, excellent candidates for every kind of application where they match required properties, such as drug delivery. Cellulose is the world’s most abundant natural, renewable and biodegradable polymer. It enhances. the comfort and quality of our life so profoundly that, our world is hardly imaginable without it. For Man- kind, cellulose has always been a paramount ‘source of cotton for textiles, and of wood for shelter creating new biomedical and paper. The early bes itself walks along with the history of studies on cellulose structure.""” In 1838, Anselme Payen isolated a substance which was afterwards named cellulose. The discoveries of — gun-cotton (oitrocellulose), in 1846 by Christan F. Schénbein, and of celluloid® (the first useful plastic), in 1869 by John W. Hyatt, were two major contributions, which changed the modern world considerably. “° ing of polymer science H20H cH,0) on HO 3 H CH,0H Figure 1.8. The cellulose molecule - (CeHieOs)s- in its chair configuration. Itis out of the scope of the present text o review the chemistry of cellulose. However, it is. worth mentioning several excellent books that have consecutively reviewed this useful and stl intriguing material, and those are part of the present references." The complexicity, associated with the many useful products resulting from cellulose, has encouraged a probably Unmatched amount of research work, which is even considered as the starting point of polymer science Cellulose is the B(1-+4) polymer of anhydro- glucose (Fig. 1.6). Polysaccharides, cellulose, have some excellent properties which structural including Biomimetic Natural Materials for Bone Regeneration, Pedro L. Granjamake them the polymer group with the longest and widest medical applications experience:"**16+'# nontoxicity (monomer residues are not hazardous to health), water solubility or high swelling ability by simple chemical modification, stability to pH variations, and a broad variety of chemical structures. This versatility makes these materials able to overcome some disadvantages like low me- chanical, temperature and chemical stability, and proneness to microbial and enzymatic degradation, which, in some cases, can be used as an advantage. In the biomedical field, CaCP > AmCP > CP; the amount of residue followed in the same order; the flame retardancy presented the inverse order; and the efficiency in flame retardancy decreased in the order AMCP > CP > NaCP = Cacp.*" Biomimetic Natural Materials for Bone Regeneration, Pedro L. Granja1.4.2.2. lon-exchange capacity The ion-exchange capacity of CP. i.e. cation- exchange ability, hasbeen extensively investigated 2"223827 2542022075207 0302206 Most of the applications proposed for these materials are due to this property, and are described in section 1.4.3.2. CP has a high capacity {or binding metal ions as well as biomolecules. The specificity for binding some metals preferentially to others, when they are in competition, has been shown in many studies. For example, Rocha ef al have shown in aquatic humic substances, that metal ions were incorporated in CP in the following order Cu > Pb > Mn > Ni> Cd. In a detailed study, Lawton and Phillips showed that CP binds K preferably to Na, and demonstrated that ions are bound in the hydrated form to CP.* inoue et al. reported that binding of metals ions to CP is pH- dependent: Mg and Hg were not adsorbed in acidic conditions but adsorption was very effective in alkaline media.*° They also showed that CP could be further reused since ions adsorbed could be easily removed by washing with HC1.™® 1.42.3. Viscosity CP monoesteres are anionic derivatives that yield higher viscosity, clearer, and more stable jersions than unmodified celluloses. Increasing substitution results in higher cold water swelling. Addition of salts causes thinning of the viscous pastes. As with most polyelectrolytes, the viscosity is influenced by pH. The solubility of CP in water or aqueous alkali is rather more the exception than the rule, due to the previously mentioned crosslinking reaction, and requires special procedures for the reaction itself and for the subsequent product isolation and Purification,?"” Whistler and Towle prepared CP monoesters with high P contents, yielding viscous solutions that were described as thixotropic. Viscosity of the solutions was temperature-dependent to some extent but this effect was lowered by addition of salts?" Chapter 1, Introduction» 23 1.4.2.4. Water sorption The water sorption of CP was investigated in several studies revealing that these materials can be used as efficient sorbents ™%2%8251a0745 Lassen et al. described highly absorbent fibers with fast wicking rates. Filatova and Novichkova proposed the purification of aqueous acid solutions with CP due to its high water sorbency.™? Saito et al, prepared superabsorbent wood derivatives through phosphorylation after oxidation, yielding transparent hydrogels with water sorption of 115 H20/g sampie.°#* Leshchenko ef al. investigated the interaction between CP and water." Hydrophilicity studies showed that the heat of wetting decreased markedly with increased humidity (equally for NaCP, CacP and FeCP)™**" The considerable swelling observed can be enhanced in the salt forms, being directly related with the hydration capacity of the cation.» The state of water absorbed on CP and its Na and Ca salts, was studied by NMR.“ Water sorption of CP and its salts was higher than unmodified cellulose, as would be expected. It was suggested that adsorption of the first water molecules occurs by a similar mechanism to the ‘one observed on cellulose: hydrogen bonds are formed with -OH groups; swelling follows increasing amounts of water sorption, leading to weakening of hydrogen bonds due to the increasing distance between the -OH groups of neighboring chains. “* In the Na and Ca salt forms, lower water sorption was observed, as translated by a decrease in line width as a function of the water content. 1.4.3. Applications As previously described, CP has proved to be a material with excellent water sorbency, retardancy and ion-exchange properties. Several applications have been envisaged taking advantage of this latter capacity, namely related to biological sciences: separation and purification of complex biochemical compounds, extraction of metals from aqueous solutions, stabilization of donated blood, and prevention of calcific diseases (e.g. renal flame Biomimetic Natural Materials for Bone Regeneration, Pedro L. Granja24 « Chapter 1. Introduction stones), due to its ability to anchor calcium ions. More recently, some works gave some promising insights regarding the possibility of using phosphorylated cellulosics for inducing the formation of calcium phosphates. NaCP is manufactured and commercialized by several companies, namely Merck, Fluka, Whatman (P11 powder; filter paper P 81 with ion-exchange capacity of 18.0 uEgicm’), BioRad (Cellex powder), Sigma, Serva (P-Cellulose), and James River Company (Berlin, NH, USA) Some applications proposed for CP do not ‘match the following categorized sections but are worth mentioning: filer for enhancing crosslinking of an epoxy resin (20% CP), fibers for removal of NHs from air,” filler for improving electrorheological activity in oil dispersions," lubricant for metal plastic working,™* and additive to promote resistance to diffusion of motor fuel vapors or the like. 1.4.3.1. Flame retardants ‘The use of phosphorylated cellulosics as flame retardants was specially investigated for textiles 29924248285 25825027 283.205 610,32122324308.60- at 215282267280 although the application on paper wood™##7288288209°5°55 haye also been widely reported. The Ban-Flame process, one of the first commercially feasible flameproofing procedures for cotton fabric, was based on the phosphorylation of cellulosic sheets, fibers, roving or yam by the urealHsPO, method.”"** As previously described, phosphorylation of cellulosic textiles provides flame retardancy but some degradation also occurs. Vigo et al, have phosphorylated textile products, with up to 4.73% P (containing also residual Cl and N), which promoted flame retardancy, as well as moderate increase in the wet and dry wrinkle recovery, although decreases in tensile strength and resistance to laundering were also observed * #7 lon-exchange with Ca during laundering reduced flame retardancy.**7" Shimizu showed that after- treatment with stannic chloride prevented undesirable adsorption of heavy metal ions, keeping flame retardancy.™° Yeadon et al. reported that Biomimetic Natural Materials for Bone Rey flame weathering for 12 months.” Bandyopadhyay et al, reported that flame retardancy was maintained even after washing in hard water, and was improved by aftertreatment with Zr, Ti and Sb.*** Blanchard ct al obtained flame retardant textiles with improved resiliency through phosphorylation, and with addition of polyethylene and elastomeric polymer emulsions.** Textiles obtained were durable- press and had smooth drying properties, Bajal obtained similar results.*? Gallagher reported high retention of tearing and breaking strengths, and retardancy was maintained even after good nonsoiling properties” Katsura ef al investigated the thermal degradation and the flame retardancy properties of phosphorylated cotton, and they showed that the addition of organics solvents, such as HCONMe2, was successful in promoting a smaller decrease in tensile strength.¥°2#91832" Clermont proposed flame-resistant CP as fillers in plastics, paper, paints and coatings, as packing or gasket material, and as a textile fiber component or coating. Touey and Kingsport suggested the use of these materials as sizing, finishing or dye- dispersing agents as well as detergent additives." The high solvent resistance was highlighted as Useful in many of these applications. Tanno et al investigated the use of CP for providing flame retardancy to electrolyte solutions for electrolytic capacitors." Takayasu reported the preparation of fire-resistant phosphorylated wood boards using guanidine phosphate and other reagents (see chapter 1.4.1.1) 1.4.3.2. lon-exchangers lonexchange celluloses are prepared by grafting of substituent groups with basic or acidic Properties. Several ion-exchange celluloses are available commercially. They are particularly useful inthe chromatographic separation of. polyelectrolytes, especially for biochemical research, due to their several advantages over conventional ion-exchange resins, namely a finer structure, thus possessing a larger surface, and a porous structure, which allows the rapid entrance land attachment of large molecules." For this application, low DS values are required since it is neration, Pedro L. GranjaTable 4.5. Extraction of metals from aqueous solutions using cellulose phosphate. Metal Ref. —_—_—_—_—_—_—— Ag 246, 286, 368 AL 286, 369 Ba 246,370 Be 370 Bi 286, ca od 216, 246, 254, 269, 298, 949, 968, 371-975 286, 269, 338, 268, 376 Ce 286, 330, 935, Co 246, 275, 286, 330, 97 cr 320, 368, 376, 37 cs 217,275 Cu 271, 286, 298, 330, 336, 368, 376-379, 380-382 Eu 275 Fe 286, 380, 335, 343, 368, 372, 375, 380 Ga 369 HE 383, Hg 246 Ho 330 In 369 K 298, 973,374 La 286, 330 Ng 248, 368, 970, 373 Ma 275, 298, 996, 274, 381 Mo 269,275 Na 215,246, 298, 343 Ni 269, 286, 336, 268, 370, 981, 382 Pb 269, 271, 286, 336, 370, 371, 37, 381 Sb 286, 356, 359 Sn 286 Sr 217,275 Th 395, 383-385 Ti 335,359, 360 nT 369 U 269, 335, 383 vo 278,377 W 269, 275,386 Y 275,330 Zn 286, 330, 368, 37 Zr 335,388, 359, 383 desirable that products do not dissolve or swell excessively. CP is a well-known cation-exchanger due to its anionic character. Other cation-exchange include suifoethyl, _sulfomethyl, carboxymethyl phosphonomethyl and oxidized celluloses (Chapter 1. Introduction « 25 Anion-exchange celluloses include aminoethyl, __iethylaminoethy!_ (DEAE), ‘viethylaminoethyl (TEAE), and ECTEOLA (prepared epichlorohyarin triethanolamine) celluloses. Cellulose ion-exchangers should never be died before use since desweliing causes undesirable close approach of the cellulose chains. CP are available commercially as cation- as previously pointed out, Their excellent properties for ion-exchange were widely investigated for varied applications, such as the cones described below. celluloses, with and ‘exchangers, Peper Pechkovskaya and Zosim prepared phosphorylated cellulose paper with ion-exchange properties." Anderson and Hearon showed that electrostatic printing paper providing good line resolution and a minimum background could be prepared from phosphorylated bleached sulfite pulp (17.49% P).” Bemardin showed that phosphorylated paper was adequate for catamenial its good water absorbency associated with ion-exchange properties, thus providing reduction of the alkaline pH during menstruation.** Goldman et al. also reported the usefulness of CP as additives for producing diapers ‘and sanitary napkins, owing to their high water sorbency and cation-exchange capacity."” tampons due to Extraction of metals from solutions One of the most extensively investigated applications of CP is the separation of metal ions from solutions. This field provides a large number of economically relevant applications like purification of solutions from radioisotopes, separation of metal ions in competition from solutions, purification of wastewaters, recovery of metals from dissolved minerals, spot tests for evaluation of purity in solutions, among others. Table 1.5 lists metal ion binding on CP, as reported in the literature. Klemm et al. reported the separation of radioisotopes such as Sr and "Cs." from wastewaters used in electrodeposition was Extraction of heavy metals Biomimetic Natural Materials for Bone Regeneration, Pedro L. Granja26 © Chapter 1. Introduction reported by Meisch and Gaver, as well as in several other works using sclutions.°*'**”*" inoue et al, reported exceptional adsorption of Fe”, Fe”, ‘Ag’ and Bi", despite the use of very low P contents. Binding of metals ions to CP was pH dependent: Mg and Hg were not adsorbed in acidic conditions but adsorption was very effective in alkaline media. The materials could be further reused many times since metal ions adsorbed could be easily removed by washing with HCI Bilba and Fisel reported the sorption and separation of Al, Tl, Ga, Id from HCkwater-methanol or ethanol solutions. Preferential thorium recovery from monazite (dissolved mineral) was reported by Head et al°* and Audsley et al. Head et al, “° described the effective separation of the pairs of ions Bi-Pd, Cu-Cd, Cu-Co, Cu-Ni Luneva et al described CP as effective sorbents (99.99% sorption) for purification of technogene waters Containing radionucleotides and active components of sorption membranes for the separation of alkaline metals.” Kember developed a high sensitivity spot test for evaluation of high purty water, using a Phosphorylated cellulose paper forthe determination of Cu and Fe.” Due to its high cation-exchange capacity, Etmolenko ef al. have investigated the use of CP for stabilization of donated blood by preventing its coagulation in storage.*"*" Blood collected from blood banks can be stabilized by passage through a column of CP to remove Ca, K, and Mg ions. No Contamination of the blood by CP was observed and its mon toxicity was confirmed in vivo on mice?" Antonova et al. used NaCP for the Preparation of decalcinated gelatinol for blood preservation. Buglov ef al. prepared citrate-free blood used for transfusions, by passing blood through a CP ion-exchange resin.°”? Hydrodynamic sorbent resistance was negligible. The product was described as highly thermostable, and hence easily autoclavable. Blood was partly deprived from Ca, Mg, K, thrombocytes (5-15%), leukocytes (0-10%) and an increase in inorganic P of ca. 1.9 mg % was observed. Sano and Shimomura prepared membranes of CA (98.7% water) and CP (1.80% P), with selective permeability, ion-exchange capacity of 1.00 mequivig, 91% impermeabilty for NaCl and 0.490 Lidm* day permeability for water. Membranes containing CP were also prepared by Hata and Yokota." The purification of wine, by removal of undesirable Ca, Mg, Fe and K ions, without altering its flavor, has been investigated. Ogorodnik et al. studied the removal of Fe and Ca, and concluded that wine products obtained presented good stability against formation of Fe-containing precipitates and had improved organoleptic —properties.°7375°"2 Furthermore, mousy flavor could be eliminated, and wine was protected from cloudiness, without affecting bouquet or taste, 72975 Owing to its high calcium-binding capacity, associated with its good lack of toxicity and indigestibibilty in the human body, CP has been widely studied to treat and prevent diseases related to calcium metabolism, such as renal stones. Hypercatciuria, hypercalcemia (abnormal concentration of Ca ions in blood), idiopathic hypercalcemia of infants (associated with renal dysfunction), hyperparathyroidism (associated to an increase of parathyroid secretion, promoting an increase of serum Ca, decrease of serum P, and increase of Ca and P excretion) calcinosis cutis, (calcification in the skin and subcutaneous tissue), and nephrolthiasis (renal stones) are among the treated with cellulose phosphate therapy in its sodium form (NaCP). Urine of patients forming calcium stones is supersaturated with calcium oxalate and phosphate. NaCP binds calcium in the intestinal tract, decreasing the amount in the urine without altering calcium balance. Several clinical studies have confirmed of urinary calcium levels and decrease of renal stone formation upon treatment with Na CP? pak et al? have reported successful treatment of patients with hypercalcemia with NaCP, and Burke et al the treatment of idiopathic infantile ephrocalcinosis using chlorothiazide"™ or prednisolone“ with CP. Marks showed that CP was effective in treating calcinosis cutis, especialy in diseases that can be normalization hypercalcemia with combination with a low calcium diet." In many studies, it was shown that, during treatment, indicators like serum Ca, Na, K, Mg, Cu, Zn and Fe, ‘Biomimetic Natural Materials for Bone Regeneration, Pedro L. Granjaparathyroid hormone secretion, as well as serum ALP and bone density, did not change significantly remained — within the range, BASS7AR44749 However, studies have also shown that with CP therapy, if no magnesium supplements or restriction of dietary oxalate are performed, this medicine is nearly ineffective, since hyperoxaluria as well hypomagnesuria may develop, which predispose to stone formation.“ pak ef al, have reported similar observations in the treatment of patients with hypercaicemia.‘” Dent ef al. highlighted the need for Mg supplementation therapy.“"° On the other hand, Cook stated that Mg supplementation could, in part, be responsible for the decrease in stone formation, since Mg is recognized as a potent inhibitor of crystal formation in urine.“ Berstad et al, reported an additional increase of urinary phosphorus excretion, which was attributed to partial hydrolysis of CP. ‘A commercial product, Caleitind? (NaCP), is available to prevent the formation of Ca-containing kidney stones where other medical and surgical procedures are inappropriate. it is used in patients whose bodies absorb too much Ca from their food. NaCP works by combining with the Ca and other minerals in food, in the gastrointestinal tract, thus preventing Ca from reaching the kidney, where the stones are formed. It is worthy ‘mentioning the indications and contraindications of this medicine, It should be taken strictly as directed {not more, not more often, and not for a longer time). It should be taken with meals mixed in a full glass of water, soft drink or fruit juice. Additional uid doses (without the medicine) should be taken every hour while awake. Use is not recommended for children. Some additional care must be taken when using this medicine: pregnant women usually need more calcium in their diet and hence should ‘ot take it; it should not be taken in conjunction with Ca or Mg supplements, or Ca- or Mg-containing antacids or laxatives, since excess levels of Ca in the body may prevent it from working property; in patients with bone diseases, the administration of CP may make it even worse, since it holds Ca and ther minerals; it may cause the body to hold water or normal several as Chapter 1. Introduction « 27 vitamin C, spinach (or other dark green leafy vegetables), chocolate, brewed tea, thubarb, milk, or milk products (cheese, ice cream, yogurt) should not be consumed while taking NaCP since, due to their high oxalate content, they may increase the chance of calcium oxalate kidney stones; eating salty foods might increase the chance of unwanted effects. Side effects reported include: more commonly, abdominal or stomach discomfort, loose bowel movements of diarthea and, less commonly, convulsions, drowsiness, loss fof appetite, mood or mental changes, muscle spasms or twitching, nausea or vomiting, trembling, unusual tiredness or weakness.“ Immobilization technology The use of cellulose as chromatography sorbent has proved to be effective for binding cations as well as biologically active substances (afinity chromatography). 24" Guthrie some applications of CP for ion-exchange and, besides of the applications, the chromatographic purification of polysaccharide hydrolases from fungi (due to its fesistance to hydrolysis by cellulases) was mentioned. Kennedy and Cabral reviewed substrates for the immobilization of enzymes and described CP as useful for the immobilization of aminoacyl-tRNA synthetases (by ionic binding)" Several other enzymes were immobilized on CP. Bioactive substances, other than enzymes, were on CP, tke pararosaniline for the development of an optical formaldehyde sensor.” Lipase was tied to be immobilized on cationic-exchange CP resins but the fixation yield was relatively low, as well as the residual activity of the enzyme. In this type of investigation, the enzymes bound to CP invariably kept their specific activity, although variable recoveries have been reported. CP chromatography was successfully used as one of the steps in the purification of phosphatidylinositol kinase," and aldose“? The specific binding between CP and aldoase was emphasized, since binding occured despite the fact that this enzyme is known to bind a number of phosphate-containing reviewed some previously mentioned also successfully immobilized Biomimetic Natural Materials for Bone Regeneration, Pedro L. Granja28 » Chapter 1. Introduction ligands, which were also in the medium, in competition with the substrate." CP was used to purify AMP deaminase from the gill of the mullet Chelon labrous R.,°* from dogfish erythrocytes, ** from pig kidney, and from porcine heart and skeletal muscle.” AMP phosphodiesterase was purified from rat liver by chromatography on CP, ECTEOLA-cellulose, HAp, a theophylline affinity matrix and HPLC on OEAE-substituted column. isolated by CP Phospholipase C was chromatography from porcine lymphocytes, from rat brain," and from from human platelets." In the first case, further purification was necessary by phenyl Sepharose. = and._—HAp chromatographies,“ while in the second, Aff-gel was used with CP, and followed by HPLC steps.“ In the latter case, purification to homogeneity was achieved on Fast Q-Sepharose, CP, heparin- agarose, phenyl Sepharose, Superose 12, OEAE- SPW and Hap.“ Duck heart muscle myocardial adenylate purified by CP chromatography, fractionation by sulfate, chromatography on Sephadex G-25 and ion-exchange chromatography on DEAE- cellulose.“ Lactoferrin from human whey was successfully isolated (96% purity) by a process consisting in direct adsorption on CP by batch extraction and elution by a stepped salt and pH gradient.“ Mine et al, have developed a liquid chromatographic method using electrochemical detection for the deaminase was ‘ammonium simultaneous quantification of histamine and N tau- methylhistamine in rat brain, in which CP fibrous cation-exchangers served to the purification step.“* ‘The purification of creatine kinase from bovine heart mitochondria was cartied out by concentration on CP gel and gel fitration on Sephacryl $-300.* Pyrimidine nucleoside monophosphate kinase was purified (ca. 90% purification) by affinity chromatography on Biue Sepharose and CP.” A procedure for the purifcation of enolpyruvylshikimate S-phosphate synthase from Escherichia coli was described, by substrate elution of the enzyme from a CP column. In order to investigate the immunoreactivity of the two alloenzymes of glucosephosphate isomerase, they were purified from skeletal muscle murine of several mouse strains by substrate elution on cP.“? Phosphorylase kinase was subjected to limited proteolysis by subiiisin and the hydrolysate was separated by CP chromatography.‘ Alpha-D- ‘mannose:beta-1,2,N-acetylglucosaminyltransferase was isolated ftom swine trachea mucosa, by chromatography on DEAE-cellulose, CP and Sephacryl $300 columns and by affinity chromatography on a Sepharose 48 column containing covalently bound —_ovomucoid.“** Tropomyosin kinase from chicken embryos was isolated using DEAE-cellulose, CP and gel fitration chromatography." Calmodulin (sine 115) Nemethyltransferase from Paramecium tetraurelia was purified using dialysis, CP chromatography, Reactive Red 120 agarose chromatography and calmodulin-Sepharose affinity chromatography.“ Chromatography on DEAE- followed by FPLC on Ni-lminodiacetate-agarose, or reverse-phase HPLC on a C-4 column was used to isolate pNixc, a Ni? binding protein present in Xenopus laevis cocytes and embryos.“ CP chromatography was used to separate human lung mast cell tryptase isoforms, “** ‘and two isoenzymes of choline acetyltransferase from squid head ganglia." Bovine milk acid phosphatase was purified by ion-exchange chromatography on Ambertite CG-50 resin, followed by Sephadex C-50, CP and affinity chromatography ‘on alphat-casein-Sepharose.“” Rabbit muscle Glucosephosphate isomerase was isolated using cp.“* The activities of nuclear protein kinases during the cell cycle of Hela $3 cells were investigated by separating them in fractions through CP chromatography.“ Alpha-L-iduronidase from pig liver was purified by chromatography on CP, concavalin A-Sepharose 48, heparin-Sepharose 48, Toyopearl HW-55, Sephadex G-100 and chelating Sepharose 68 charged with cupric ions.“ Human skin tryptase was purified using octyl ‘Sepharose CL-48.— hydrophobic affinity chromatography, and Sephacryl S-200 gel ftration, followed by ociylSepharose CL-4B or CP ion- exchange chromatography." Ribonuclease from sequential cellulose and CP, human liver was obtained using a CP column.“ Human urine ribonuclease 1 was purified by column chromatographies on DEAE-Sepharose CL-6B, CP ‘Biomimetic Natural Materials for Bone Regeneration, Pedro L. Grarja| ‘and CMC, followed by gel fitrations on Bio-Gel P- 400 and Sephadex G-75 and finally to a homogeneous state by SDS-PAGE.“° Glycogen synthase kinase-3 was purified from bovine caudal epididymal sperm extracts by chromatography with DEAE-cellulose, AffGel blue and CP, allowing its identification.“ After extraction from tumip roots, peroxidase isoenzymes were isolated using CP chromatography.“ Kotake et al. used a phosphorylated gauze to immobilize (make insoluble) the antibiotic substance chlortetracycline.“* Granerus et al. used a CP column for the final separation step, prior to HPLC, to isolate telemethylimidazoleacetic acid in urine.**” West and Strohfus reported the immobilization of fungall cells on CP for pullulan production, which is useful as food additive, flocculant, blood plasma substitute, among others.“ Shih and Martin reported the chemical linkage of nucleic acids and efficient isolation (immobilization) of specific sequences using CP.“ Single-stranded RNA was coupled to CP by the carconyidiimidazole method, in order to isolate complementary DNA.“ Peterson and Sober described the use of CP chromatography for the separation of proteins, namely horse carbon monoxide hemoglobin, which was adsorbed in considerable amounts, at pH 7.02" Using a combination of CP, DNA-cellulose and DNA affinity chromatographies, Du et al. purified the 19-base pair transcriptional regulatory element from the promoter region of the rat betaé subunit gene.*” Wendler and Grossbach described a blotting procedure specific protein-DNA interactions using a non-selective electrophoretic sequence-specific preserving transfer of proteins from acetic acid-urea gels onto nitrocellulose containing CP ester, thus preserving their ability to interact specifically with DNA and avoiding exposure to dodecyl sulfate ions normally used.“ Holmquist and Schroeder reported the separation of peptides from human hemoglobin on CP, and highlighted the lack of contamination of the eluent by CP as an advantage over other available methods.“ Peptides obtained were highly pure but recovery rates obtained were better suited for acidic (Chapter 1, Introduction «29 than for basic peptides.“ They also reviewed other well succeeded applications of CP for affinity chromatography, such as the isolation of carbamyl phosphate synthetase from frog livers, and peptides from the peptic and chymotryptic hydrolysates of ‘egg and lysozyme.‘ Sugihara et al. also proposed the separation of peptides by CP chromatography, Using as example the application to the analysis of a human hemoglobin variant”? Resuits indicated substantial efficiency and reproducibility as well as @ high loading capacity, and they concluded that CP coupled with molecular-sieve chromatography on microcrystalline polyacryiamide gels, was a versatile technique for the effective separation of peptides, since using CP alone several peptides eluted together. This high recoveries and ease of sample collection and preparation, and was found to be specially suited for handing a quantity of peptides for determination of the amino acid sequence.” Thomaidis of al. used CP equilibrated with acetate buffer at pH 5.0 for the extraction of free nucleotides from plant tissue, including UDP- glucose, resulting in good recoveries.” CP chromatography was also used for the fractionation of human serum y-globulins, in a reproducible and simple way, although further fractionation was necessary to obtain pure fractions.‘"* Nevertheless, fractions obtained were physiologically active, as shown, for example, by the ability to stimulate phagocytosis of staphylococci by autologous polymorphonuclear teucocytes."® Ashkar ef al showed a specific phosphoryl binding site in swine kidney phosphofructokinase, by investigating its binding ability to phosphoryl groups in CP columns, among other phosphorylated species."”* The purification of S-carboxymetylated high-sulphur proteins from mouse hair alpha-keratins was reported using ion-exchange chromatography on cP at p26.” Hydroxamic acids produced by yeast cells of Histoplasma capsulatum were isolated by extraction and cation-exchange chromatography with CP.""® Kim et al, have studied albumin adsorption on phosphorylated cellulose surfaces and found increased adsorption compared procedure provided to unmodified Biomimetic Natural Materials for Bone Regeneration, Pedro L. Granja30 © Chapter 1. Introduction cellulose.” On the contrary, Habbaba et al reported that phosphorylated cellulose does not adsorb albumin," which seems to be more in agreement with the fact that usually highly hydrophilic surfaces are not very attractive to protein adsorption.“** 1.4.3.3. Water sorbents and thickening agents ‘The high water sorbency of CP can be used for several applications. Lassen ef al. proposed their use for highly absorbent fibers with fast wicking rates, for disposable diapers, sanitary products, wipers and the like. Filatova and Novichkova found them useful for the purification of aqueous containing 20-40% to Bernardin, phosphoric acid solutions, H:PO.™° According ccatamenial tampons can be prepared from CP, due to their good absorbency associated with ion- exchange properties, reduction of the alkaline pH during menstruation. "* Goldman et al, also reported the usefulness of CP as additives for producing diapers and sanitary napkins, owing to their high water absorption and cation-exchange capacity.” Shamoo showed that adequate which are suitable for CP could be used as additive for improving counting efficiency in liquid scintilators, by absorbing and/or emulsifying water that is present (when the isotope to be detected is an aqueous solution). Touey and Kingsport suggested the use of CP as thickening agents to stabilize emulsions or to suspend materials, as additives to form capsules or as binding agents in making tablets and the like, for the food, cosmetic and pharmaceutical industries.”"* 1.4.3.4, Miscellaneous biomedical applications Besides applications such as immobilization of bioactive substances and treatment of calcium metabolism-related diseases (described in section 1.4.3.2), some other biomedical applications of CP were investigated, due to its intrinsic properties. Regioselectively substituted (in the C-2,3 position) CP were recently observed to inhibit the activation of detrimental proteins after hemodialysis membranes.""” incorporation in Soluble cellulose acetate phosphates were incorporated into a cellulose hemodialysis membrane and subsequently deacetylated. Low anticloting activity was observed, but the detrimental activity of blood proteins (C3A activity) was also. efficiently suppressed.”* Hayashi and Matsukawa reported the use of CP as adequate additives for encapsulation in the preparation of walls of capsules by complex coacervation, using gelatin, CMC and CP.“ Ermolenko ef al reported the use of CP for the preparation of a hemostatic agent which promotes acceleration of wound healing.“* COrlianskaia et al. also investigated the use of CaCP ‘as hemostatic agent after resection of the spleen ‘and showed its biocompatibility as well as good hemostatic and plastic properties for use in the plasty of veins. * With the aim at investigating the deposition of ‘bone mineral by synthetic implant materials, Mucalo ct al, have studied the ability of cellulose (cotton) phosphorylated by the urea/HPO, method to induce the formation of apatite, in simulated physiological conditions.“*” It was hypothesized that such materials could allow the design of coated materials that more closely resemble the function of the tissue they are replacing. They showed that only the Ca form of GP (CaGP) is capable of inducing the formation of carbonated calcium deficient apatite, but not materials only phosphorylated.“***” They also used cellulose phosphorylated by the Urea/phosphorous acid method, and have obtained comparable results.** Li et al. have reported similar, findings using bamboo (composed mainly by cellulose) phosphorylated by the urealHsPO. and DMF-HsPO, methods.“ Conceming the latter method, bamboo was previously treated with NaOH. This material was selected due to its mechanical and macrostructural similarity with bone tissue. The pre-treatment with calcium to form CaCP was desciibed as inevitable for the formation of apatite to occur.” The DMF/HsPOx method provided a material presenting a better interaction with the apatite layer formed, in terms of rate of formation and adhesion. In both studies, from Mucalo et al” and Li et al,“ it was shown that unmodified ‘Biomimetic Natural Materials for Bone Regeneration, Pedro L. Granjacellulose was not effective in inducing the formation of apatite Shimabayashi et al. have investigated the formation of hydroxyapatite (HAp) in the presence of insoluble phosphorylated cellulose, in aqueous solutions.“ It has been demonstrated that insoluble CP fibers promoted the formation of HAP, presumably by providing the heterogeneous nucleation sites for the crystal growth through binding of Ca ions.“® High amounts of CP inhibited the formation of HAp, seemingly through excessive consumption Ca ions. Water-soluble phosphorylated polymers ‘monomers (phosphorylated PVA and serine, respectively), used for comparison, inhibited the formation of HAP through binding to the initial crystals and inhibition of their growth, due to electrostatic repulsion to inorganic phosphate ions, which are necessary for mineral propagation. they studied the influence of bovine serum albumin (BSA) on the formation of HAp from solutions, in the presence of CP fibers. it was shown that albumin inhibited the formation HAp by adsorbing to the aystal nucleus and thus retarding inorganic phosphate uptake by electrostatic and steric repulsion. The presence of CP enhanced the inhibitory effect of BSA and this fact was attributed to the adhesion of amorphous calcium phosphate (a precursor phase of HAp) precipitate particles on CP fibers. Okada et al, prepared CP, mineralized it with HAp as described in the previous paragraphs, and promoted the incorporation of Ag on these materials in order to study their antibacterial properties using Bacillus subtilis. It was demonstrated that the adhesion of the bacteria was inversely proportional to the amount of Ag incorporated and hence these materials were proposed as virus and bacteria fiters.* Other studies have attributed an antibacterial effect to CP alone by demonstrating their ability to inhibit bacterial growth. 7° Cellulose membranes have been widely studied as substrates for bioreactors. However, they have been reported as poor substrates for culturing kidney ces.” Kim et af. have studied the effect of phosphate grafted cellulose membranes on the of and In another investigation, Chapter 1. Introduction © 34 adhesion of Chinese hamster ovary cells, in order to the use of CP membranes as improved cytocompatibitity.7? Using low phosphate contents, they reported a slight enhancement of cell adhesion with increasing phosphate content, as well as low aggregation of cells”? The influence of hydrophilicity was also assessed, showing no significant differences upon phosphorylation, but only an apolar liquid was used.” Cell adhesion in phosphorylated cellulose has not been comprehensively studied yet, although some studies exist using phosphorylated surfaces other than cellulose. Gunasekaram ef al. have investigated the behavior of Schwann cells on phosphorylated collagen and an enhancement of cell proliferatoion."™ However, phosphorylation of peptides is usually restricted to some of the aminoacids, leaving the remaining investigate bioreactors with reported functionalities of the protein available to behave differently. Phosphorylated proteins are known to play a key role in cell metabolism as they serve as ligands for kinases, and promote influx of Ca”, for example.“ Katayama at al. reported that phosphorylation of osteopontin enhanced adhesion of osteoclasts but not osteoblasts. * Yamamoto et al, have surface phosphorylated poly(ethylene terephtalate) polymers and found an enhancement of osteoblasts adhesion by the formation of a calcium phosphate mineral.” On the contrary, some studies support that calcium phosphate particles inhibit osteoblasts proliferation and this fact seems to be related to the release of excess inorganic phosphate and calcium ions to the cuture medium.""*"* it seems though that cell adhesion to phosphorylated surfaces needs to be further clarified. 1.4.4. Perspectives of application as biomaterials CP can be adequate biomaterials mainly taking advantage of their ion-exchange capacity, as described in section 1.4.3. In orthopedics, the ability of materials for apatite layer (mineralization) seems to be an advantage, and the calcium salt of CP (CaCP) seems to be adequate to inducing an Materials for Bone Regeneration, Pedro L. Granja32 © Chapter 1. Introduction accomplish this aim. CP has also shown to be a good substrate for the immobilization of bioactive substances, and this can be used for many different purposes in the biomaterials field. 1.4.4.1, Biomaterials promoting miner for orthopedic applications The ability of the calcium salt of CP to induce the formation of apatite has been demonstrated in some works, as described in chapter 1.4.3.4, Other works using other phosphorylated polymers have also shown similar results. Yokogawa et al, have used phosphorylated chitin and chitosan‘ and showed their ability to induce the formation of pre-incubation in Ca(OH) et al. grafted a phosphorylated monomer (methacryloxyethy| phosphate) on a polyethylene (PE) substrate, and compared its mineralization with another _anionie-grafted functionality (carboxylic group). They concluded that both surfaces induced mineralization, and the phosphorylated one promoted a lower extent of apatite after a Tretinnikov apatite formation, although bonding between the surface and the apatite formed was significantly higher in the case of the phosphorylated surface, indicating chemical bonding.*® If compared to the ‘works previously mentioned, where a pre-incubation in a calcium-containing solution was performed, mineralization observed in the present case was Poor (as seen in SEM micrographs), probably due to lack of nucle formed by interaction of calcium ions and phosphate grafted groups. In a subsequent work from the same research group, Kato et al, have used the same phosphorylated monomer grafted on PE but performed the pre-incubatio CaChz before mineralization assays." They showed a significantly different behavior, with the apattic layer formed covering the whole surface of materials." Dalas etal. investigated the deposition of apatite on polystyrene divinyl benzene copolymer and polystyrene alone, with grafied phosphate groups, and obtained increasing mineralization rates with increasing phosphate contents on the surface.“* In another investigation they used structurally distinct phosphorylated several polymers and showed that open copolymers yielded high rates of mineralization, observed using copolymers in which phosphate groups were not structure while mineralization was not accessible to the Ca ions in the solution.** In both studies Dalas ef al assays using the constant composition method, while in the works described before, materials were immersed in simulated plasma solutions, which ried out mineralization were renewed. This fact may be relevant for the ‘obtention of mineralization by Dalas et al,, in samples phosphorylated but not previously incubated in calcium. The constant composition method provides instant adjustment of the medium ionic composition, namely calcium and phosphate, and this may be responsible for a more specific interaction between calcium ions and phosphate groups of the polymeric surfaces, thus promoting the inital apatite nuclei formation. Tanahashi and Matsuda have studied apatite deposition on self- assembled monolayers of alkenethiols grafted with several distinct chemical functionalities, including phosphate groups.%™ They found that phosphate groups were the most effective and attributed mineralization to the interaction of phosphate functionalities with calcium ions. Bonel ef al. have prepared a material by co-precipitation of an organic phosphate (amino-2-ethyiphosphate) and an inorganic phosphate, obtaining a composite material of phosphates. Contrarly to the previously described phenomena, when phosphorylated species are in solution and do not constitute a solid support, they seem to inhibit the formation of apatite. Linde® and ‘Anderson and Mortis reviewed biomineralization ‘and the role of phosphoproteins (Fig. 1.8), which are also phosphate-containing organic species, was described according to this-__behavi Phosphoproteins (also referred as phosphoryns) exist in mineralized tissues as part of the non- collagenous proteins, with different degrees of phosphorylation, and possess high calcium-binding properties." The amino acid sequences of these and were successful in calcium r. proteins are mostly constituted by serine, including phosphoserine, and aspartic acid, thus being highly Biomimetic Natural Materials for Bone Regeneration, Pedro L. Granjanegatively charged by phosphate or carboxy! groups. Linde ef al. studied the mineralization of phosphoproteins in solution and grafted to agarose beads. They showed that, in solution, they inhibit mineralization, but promote it when immobiized on a surface, and suggested that phosphoproteins may be involved in the regulation of mineralization in vivo through this Several other independent studies have reported similar findings using, for instance, decalcified collagen complexed with phosphoproteins.“**"* In a similar way, membrane phospholipids, phosphated species, seem to play an active role in inducing mineralization through their calcium-binding abilty.°°*” Contrary to the previously described results obtained with insoluble phosphorylated chitin, Andrew etal, have also reported inhibition of apatite formation by phosphorylated water soluble chitin "* wich is in agreement with the resuits from Shimabayashi et al. (section 1.4.3.4), using water- soluble phosphorylated polymers and monomers." Wikiel et af. have used the constant composition ‘method to investigate the formation of apatite in the presence of synthetic phosphorylated pentapeptides mechanism.5 ‘another and also found a marked inhibition on mineralization." Nucleation of HAP i ° 1 ca o-P- 2s i ro og (On P-O io 1 \ 6 CO O-P-0 0-P-0 1 ca a Lo 8 \ ca ca” 4oN AON ° ° 0 ° O=P-0 O-P=-0 O=P=0 0-P-0 phosphoprotein substrata Figure 1.8. Schematic ilustration of the mechanism of formation of hydroxyapatite from a physiological solution (Containing calcium and phosphate ions) on ‘hosphoprotein substrate.” Chapter 1. Introduction » 33 The process of mineralization and the evaluation ofits influencing factors is a matter of great interest, particularly with a view to understanding mine- ralization-related diseases (reviewed in chapter 2.2). The natural choice of a bone replacing material should be collagen, but several problems make its wide use difficul, namely inticate biochemistry, leading to a prohibitive cost, and unsatisfactory mechanical properties.““**"" The possibility of contamination by pathogens must also be carefully Hence, must be investigated not only as implantable biomaterials addressed. new materials but also as substrates to investigate the mineraliza- phenomena. Biological molecules possessing phosphate functional groups, like phosphoproteins, play a paramount role in the fegulation of mineralization. Nevertheless, the phosphorylation process and its relationship with mineralization have not been clarified yet. Phosphate groups alone seem also to influence this process considerably. *"** In order to study the properties of these phosphorylated species in vitro, it has been necessary to develop efficient methods of synthesis, riot only of the naturally occurring molecules, but model tion-related physical and chemical also of analogues to be used as compounds. Cellulose is a candidate polymer for biomedical applications and phosphorylation can be regarded as the means to render it bioactive. Furthermore, although native cellulose is not digestible in living tissues, it can be made biodegradable through modifications that disturb its higher ordered structure, rendering the glycosidic susceptible to hydrolysis or attack by mammalian enzymes."*""” Phosphorylation can be a means to achieve that disturbance. The combination of biore- sorbability with mineralization induction ability, which opens the possiblity of having a material which, once implanted in hard tissues, could promote the formation of calcium phosphates and then be eliminated, possibly within a controllable kinetics, matching that of bone regeneration. linkage Biomimetic Natural Materials for Bone Regeneration, Pedro L. Granja34.6 Chapter 1. Introduction 1.4.4.2. Support for the immobilization of bioactive species ‘Among natural polymers that can be used as macromolecular supports, polysaccharides present some significant characteristics: they react with forming stable ly inert and ‘When active species molecules do not biologically active substances, compounds, and are pharmacologi nontoxic." contain functions complementary to those of the polymer, the latter can be functionalized, thus creating binding sites for biologically active species." Cellulose was one of the first materials to be used as a matrix for the covalent binding of bioactive species, as described in section 1.3. However, for many applications, its hydroxy! groups may not be reactive enough to form covalent bonds, between the bioactive substance and the support, A wide range of chemically modified cellulosics can be prepared, changing its properties significantly, allowing an equally wide range of covalent binding methods, in without previous activation. Which the enzyme usually binds mainly through ‘amino groups."** Many enzymes have already been combined, physically or chemically, with cellulose derivatives to yield biological functional systems in therapeutic, diagnostic and bioprocess applications. Phosphorylated cellulose is negatively charged, as ‘opposed to the positively charged ar Thus, it seems to be a suitable substrate for binding enzymes, proteins and other biologically active studies 10 groups. species, as demonstrated in several described in section 1.4.3.2. Cellulose phosphate, since it has highly reactive sites, can also be regarded as a possible substrate for the study of the mineralization phenomena, through the adsorption of biologically active species, like proteins, functional groups or metallic ions, onto its surface. Hence, the influence of several factors in mineralization can be studied selectively, or even in a combined form. The clinical advantages of this, the injectable form, as a membrane, as a dense or powdered kind of material can be numbertess, material, either as coating material or even for bone replacing or fling. Furthermore, cellulose phosphates are highly hydrophilic, which constitutes a popular and widely used strategy for sustained-elease drug delivery, with polysaccharides and their derivatives as the polymers of choice as the rate-controling carriers for these systems." The advantages of hydrogels for this systems include adequate controlled-release characteristics, due to adjustable water sorption and sorption rate, and good biocompatibility with biological tissues and fluids.**"”**5 Conventionally, hydrogels are suitable materials for administration of biologically active species from the standpoint of biocompatibility, because they generally produce ow stimulation of the tissue,'”® 1.4.4.2. Miscellaneous biomedical applications AAs previously described in sections 1.4.3.2 and 1.4.3.3, cellulose phosphate has several interesting properties for many biomedical Although NaCP is used for the treatment of calcium metabolism-related diseases, the other applications proposed were kept as laboratory curiosities. The anticlotting activity associated with other relevant properties for those applications conceming blood contact should be further investigated, since CP provement in highly demsinding fields, such as: transfusion medicine, as membranes with permeability for stabilization of donor blood, through removal of Ca, , Mg, leukocytes and thrombocytes; hemodialysis, through Inhibition of the activation of detrimental proteins; wound healing, as hemostatic agent; and as antibacterial agents or surfaces. applications. could constitute an selective 1.5, MAIN OBJECTIVES OF THE PRESENT RESEARCH ‘The present work consists basically in an attempt to synthesize cellulose phosphates and study their functionality as biomaterials for bone regeneration. ‘The objectives of the experimental pathways to synthesize and characterize cellulose phosphates consist inthe following: ‘Biomimetic Natural Materials for Bane Regeneration, Pedro L. Granja‘Synthesis of cellulose phosphates by the HsPOWP:0EtPOuhexanol method, through bulk modification of microcrystalline cellulose. The following reaction parameters should be optimized, in order to obtain high degrees of substitution: + Swelling pre-treatment, sequence of addition ‘of reagents, temperature and time of reaction, and relative quantities of reagents; Surface chemical modification of regenerated cellulose hydrogels, via phosphorylation; Physico-chemical characterization of cellulose phosphates by following properties: + Phosphate content, nature of grafting of phosphate groups, regioselectiviy of reaction, structure, morphology, crystallinity, calcium-binding capacity, resistance to water surface assessing the sterilization, sorption, roughness, and hydrophilicity; In order to investigate the functionality of cellulose phosphates as biomaterials for bone regeneration, the following studies are envisaged: + Mineralization conditions, i.e., the ability to induce the formation of an apatite layer, and characterization of the mineral formed as function of the phosphate content; + In vitro biocompatibility studies in cultured bone cells, as a function of the phosphate content, through evaluation of cytotoxicity, and cell attachment, proliferation and diferentiation; + In vivo biocompatibility studies, through animal implantation. in simulated physiological References 1 Wiliams OF, The Willams Dictionary of Biomaterials, Liverpool, UK: Liverpool University Press, 1999, Ratner BD. 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