Ays ¸e

Gürel Bog

Bog Ays ¸e Gürel

Research on first language attrition of morphosyntax in adult bilinguals

Keywords: First language attrition, morphosyntactic attrition, vulnerable features, language interference, adult bilingualism

This article presents a selective review of previous research findings on first language (L1) attrition. The review is intentionally limited in scope as it only discusses studies on morphosyntactic attrition in the L1 grammar of adult bilinguals. To this end -and in order to present the most current line of research in this field -I first report on studies that appeared in three recent collections of papers on L1 change or attrition. I then discuss findings reported in these three volumes as well as those reported elsewhere, in order to identify morphosyntactic features that are more vulnerable to change due to L2 interference.

I Introduction

First language (L1) attrition is a multi-dimensional phenomenon. The body of attrition research includes studies in child L1 attrition (normally categorized as incomplete L1 acquisition) as well as non-pathological adult L1 attrition. L1 attrition research also includes sociolinguistic studies investigating the loss of ethnic minority languages, which in some language contact situations are replaced by a dominant language for social or political reasons. Most of these studies examine language attrition/death as an intergenerational process [START_REF] Dorian | Language loss and maintenance in language contact situations[END_REF].

However revealing child L1 attrition and intergenerational societal level-language change are, I deal with neither of these phenomena in this article. Rather, I define the term 'language attrition' as referring narrowly to second language (L2)-induced L1 change or restructuring in an individual speaker's grammar [START_REF] Pavlenko | L2 influence on L1 late bilingualism[END_REF]. The three collections under review include work that assumes various wider definitions of attrition; I leave open the question of the propriety of such definitions and focus on research contained in these three volumes that address morphosyntactic attrition in the L1 grammar of adult bilinguals.

One of the earliest examples of scientific interest in L2-interference dependent L1 attrition is Weinreich (1963: 1), who defines the interference phenomenon as 'deviation from the norms of either language which occurs in the speech of bilinguals as a result of their familiarity with more than one language, i.e. as a result of language contact.' The term interference here does not imply mere additions to an L1 grammatical inventory but rather the rearrangement or restructuring of patterns that result from the introduction of foreign elements (the phonemic system, morphology, syntax and vocabulary) into the L1 grammar. This review article aims to identify unstable areas of the L1 morphosyntax that are more likely to undergo attrition in language contact situations, and to discuss the impact of a dominant L2 as a possible cause of this grammatical change in the L1.

In the first book under review, the interference phenomena that appear in the form of L2 transfer into the L1 are discussed in the context of [START_REF] Cook | The poverty-of-the-stimulus argument and multi-competence[END_REF] multi-competence model, according to which L2 users' knowledge of either the L1 or the L2 is typically not identical to that of monolingual native speakers of L1 and L2 (Cook, 2002: 5-6).

It is commonly observed that most L2 users fail to attain native-like competence in L2 even after years of exposure, generally due to persistent L1 transfer (e.g. [START_REF] Bley-Vroman | The logical problem of foreign language learning[END_REF]. Therefore, the view that L2 users' knowledge of the L2 differs from that of native speakers is less controversial than the suggestion that L2 users' L1 knowledge diverges from that of monolingual L1 speakers in various respects. From the perspective of L1 attrition, this implies that native L1 speakers will develop different L1 representations as they become L2 users. Accordingly, L2-dependent change or loss in L1 grammatical features is expected to be a natural consequence of bilingualism. The extent of this change is, of course, subject to individual variations (Köpke and Schmid in Schmid et al., 2004;Köpke in Köpke et al., 2007).

The multi-competence model assumes that the L1 and L2 of the bilingual speaker form a language super-system at some level rather than exist as completely isolated systems (Cook, 2003: 2). Therefore, an intriguing task for researchers is to determine the linguistic content and organization of this super-system that includes a merged set of features from different languages. The two languages are schematized within an integration continuum that ranges from total separation to total integration (Cook, 2002: 11). The important point here is that the relationship between the L1 and L2 might change from one domain to another. For example, an L2 user might have interconnected vocabulary but entirely separate syntax (Cook, 2002: 12). It is also possible that, even within the same linguistic domain, the integration continuum might apply to linguistic features differentially. For example, some L1 and L2 syntactic features might be kept totally separate in the bilingual mind; other syntactic features might undergo complete integration (for similar findings, see [START_REF] Gürel | Linguistic characteristics of second language acquisition and first language attrition: Overt versus null pronouns[END_REF]. The question of why the bilingual mind cannot always keep linguistic information that comes from different language sources stable and separate deserves to be investigated as a distinct topic. What is of interest here is the pattern of integration of the two languages. How are L1 and L2 features rearranged when the two systems are integrated? It is crucial to identify possible linguistically or psycholinguistically determined arrangements of the two languages [START_REF] Köpke | Activation thresholds and non-pathological first language attrition[END_REF]Gürel, 2004;Gürel in Köpke et al., 2007;Köpke in Köpke et al., 2007; Paradis in Köpke et al., 2007;Sharwood Smith in Köpke et al., 2007). Nevertheless, it is not certain how far we can go in formulating 'hypotheses about the fate of certain linguistic features in sufficiently abstract terms as to be applicable to any language' (Andersen, 1982: 84).

II Review of the books

1 Cook, 2003 This book consists of 13 chapters. Each chapter examines L2 effects on L1 grammars in different areas such as collocations (Chapter 2 by Batia Laufer); narratives (Chapter 3 by Aneta Pavlenko); pragmatic formulations of requests (Chapter 4 by Jasone Cenoz); L1 morphology, lexicosemantics and idioms (Chapter 5 by Scott Jarvis); deviant L1 expressions and code-switching (Chapter 6 by Graeme Porte); the extent of L1 productivity and lexical diversity (Chapter 7 by Jean-Marc Dewaele and Aneta Pavlenko); L1 linguistic representations in bilingual children (Chapter 8 by Victoria A. Murphy and Karen J. Pine); L1 middle constructions (Chapter 9 by Patricia Balcom); L1 syntactic processing (Chapter 10 by Vivian Cook, Elisabet Iarossi, Nektarios Stellakis and Yuri Tokumaru); and L1 interpretation of pronominals (Chapter 11 by Teresa Satterfield). Chapter 12 by Ulrike Jessner introduces a 'dynamic model of multilingualism' to account for language acquisition and language loss. In Chapter 13, Istvan Kecskes and Tunde Papp discuss the effects of L2 in the form of foreign language classroom input on the L1 grammar. Given the focus of this review, a closer look at Chapters 3, 5, 6, 9, 10, 11 is necessary to examine in detail the morphosyntactic attrition data presented in these chapters.

In Chapter 3, Pavlenko discusses L2 English influence on L1 Russian, based on oral narratives collected from 30 late Russian-English bilinguals. Russian L2 users of English tend to use imperfective verbs and indeterminate verbs of motion in contexts that require perfective and determinate verbs. Some reflexive verbs that subcategorize for adjectives in the instrumental case are simplified in such a way that the reflexive particle of the verb is omitted and the adjective appears in the nominative case, as in English. Nouns and subject pronouns are case-marked incorrectly in a way that indicates that the sixcase system in Russian is reduced to the three-case system of English.

In Chapter 5, Jarvis discusses L2 English influence on L1 Finnish grammar of an adult immigrant in the USA. Jarvis identifies 15 L2-induced deviant patterns. Spontaneous speech data reveals that L2 English influences the participant's selection of incorrect prepositions in L1 Finnish. However, none of these deviant patterns appear in the film-retelling task. In the grammaticality judgement task, the participant accepts some deviant structures, including incorrect use of prepositions. The participant also tends to change various word order patterns in Finnish into the L2's canonical SVO, suggesting that the rigid word order of the L2 influences the L1 word order pattern.

In the subsequent chapter, Porte examines the L2 Spanish influence on L1 English in spoken data collected from three English teachers living in Spain. It is predicted that English language teachers might experience change in their L1 grammar by being exposed to defective English input from non-native students for so many years. Results reveal that instances of code-mixing are fewer than expected and mostly occur in highly-specific terms without true L1 equivalents. Thus, no attrition effects are found even after many years of residence in the L2 country. This finding is in line with Gürel (Köpke et al., 2007; see also below) that also finds no sign of L1 attrition in native English speakers.

Balcom's Chapter 9 discusses L2 English influence on L1 French grammar in the formation of middle-verb constructions (e.g. the bomb exploded), where English has more constraints than French. French middle constructions have a clitic pronoun (se), while English does not. French allows a variety of time, place and manner adverbials, while English middles can occur only with certain adverbials of manner. Unlike English, French allows impersonal subjects with middle constructions. The grammatical subject in French middles does not have to be affected by the action referred to by the verb. Results from a grammaticality judgement task reveal that compared to monolingual French speakers, 12 bilingual Francophone university students judge grammatical sentences to be ungrammatical significantly more frequently. This implies that the L1 grammar tends to become more conservative under the influence of L2. This contrasts with earlier suggestions that when the L2 offers a more restrictive grammar than the L1 (i.e. a subset of the L1), no attrition effects are expected [START_REF] Gürel | Linguistic characteristics of second language acquisition and first language attrition: Overt versus null pronouns[END_REF].

In Chapter 10, Cook, Iarossi, Stellakis and Tokumaru examine the L2 English effects on syntactic processing of Japanese, Spanish and Greek.

The prediction is that word order -the main cue in English -will override the weight of case, animacy and agreement cues in other languages. A total of 95 bilinguals and 67 monolinguals are tested. Results reveal that bilinguals in all three L1 groups behave differently from monolinguals. Even more interestingly, the Japanese bilingual group demonstrates more over-reliance on animacy and plurality cues compared with Japanese monolinguals. This result does not indicate L2 influence but suggests a changed L1 grammar in the L2 user, as predicted by the multi-competence model.

In Chapter 11, Satterfield investigates the effects of non-pro-drop characteristics of L2 English on the pro-drop setting of L1 Spanish within the Minimalist framework [START_REF] Chomsky | The minimalist program[END_REF]. Satterfield discusses Lipski's data (1996) that reveal that transitional Spanish-English bilinguals tend to lose the contrastive function of overt pronouns in Spanish. In a sentence such as Ella i hablaba el ingles que *ella i / pro i sabía ('She spoke the English that she knew'), bilinguals permit the overt pronoun to appear in contexts that monolingual Spanish speakers typically reserve for pro, so that ella takes on the null subject's unmarked and non-contrastive interpretation. Satterfield argues that given the number of checking operations involved, while contrastive overt pronouns generate the highest cost, non-contrastive overt pronouns and null subjects generate lower cost. Thus bilinguals exhibit low incidence of referential pro and the more frequent use of overt subjects unspecified for focus. It is important to note that this analysis is based on Lipski's preliminary data that involved so-called transitional bilinguals: a group reported to have learned English before adolescence without schooling in Spanish. Therefore, it is highly questionable whether Satterfield's analysis can be applied to attrition in mature L1 Spanish grammar (see [START_REF] Gürel | Linguistic characteristics of second language acquisition and first language attrition: Overt versus null pronouns[END_REF] for the influence of a non-pro-drop L2 on a pro-drop L1, see [START_REF] Tsimpli | First language attrition and syntactic subjects: a study of Greek and Italian near-native speakers of English[END_REF].

Given the focus of this volume, all of the reviewed papers make valuable contributions in locating L2 effects on aspects of the L1 grammar. However, due to a lack of quantification of data, the extent of L2 effects cannot be clearly specified in most of the studies reported. Nevertheless, the overall picture suggests that as has been shown for other linguistic domains, L1 morphosyntax is vulnerable to L2 effects, and L2-dependent changes generally involve simplification or reduction of morphologically-marked distinctions or categories as well as elimination of costly syntactic operations in the L1 (compare Andersen, 1982).

2 Schmid, Köpke, Keijzer and Weilemar, 2004 This volume presents a collection of studies that deal with theoretical and methodological aspects of L1 attrition. In the introductory chapter, Barbara Köpke and Monika S. Schmid extensively review past and present attrition research while offering solutions to some of the main problems in this field.

Below is a general outline of the rest of the chapters in the book. This is followed by a selective discussion of some of the papers that address L1 morphosyntactic attrition.

Part I starts with Aneta Pavlenko's paper on L2-induced L1 attrition in adult bilinguals. In Chapter 2, Antonio F. Jiménez Jiménez examines language attrition from a socio-cultural perspective. In Chapter 3, Jean-Marc Dewaele analyses L2 users' perceived language attrition and its effects on self-rated proficiency in L1 production of emotions. Evelyn P. Altenberg and Robert M. Vago's Chapter 4 discusses the advantages and disadvantages of using grammaticality judgement tasks in acquisition and attrition research.

In the first chapter of Part II, Kutlay Yag ˘mur addresses L1 attrition of Turkish in Australia. In Chapter 2, Miriam Ben-Rafael discusses L1 attrition of French in Israel. In the third paper, Matthias Hutz examines L1 data from a German immigrant in the USA. In the last chapter, Valérie A.G. Ventureyra and Christopher Pallier present neurolinguistic data from young Korean adoptees who were immersed in L2 French.

The first three chapters of Part III include studies that are conducted within a generative grammar perspective. The first paper by Ays ¸e Gürel looks at L2-English-induced L1 attrition of Turkish in the context of pronominal binding. Bede McCormack's study also looks at attrition of binding properties, but examines attrition of L2 English reflexives in a L1 Japanese context. Silvina Montrul's chapter compares the characteristics of eroded L1 Spanish grammars of early Spanish-English bilinguals and the incomplete L2 grammars of L2 Spanish learners with respect to the semantic interpretations of the preterit/imperfect aspectual opposition. The last two chapters include studies carried out within Myers-Scotton and Jake's (2001) Abstract Level and 4-M models: Steven Gross examines L1 German attrition in late German-English bilinguals, and Elena Schmitt presents data from Russian-English bilingual children to argue that language loss is a process of gradual replacement of L1 structures with L2 structures (compare [START_REF] Andersen | Determining the linguistic attributes of language attrition[END_REF]Pavlenko in Schmid et al., 2004). In the concluding chapters, Schmid provides an annotated bibliography of language attrition research, and then presents suggestions and points to the future direction in this field.

Papers included in this volume all provide an in-depth analysis of some aspect of the language attrition phenomenon. In the rest of this section, I discuss in more detail those papers that offer direct implications for the attrition of L1 morphosyntax.

Pavlenko's paper provides a taxonomy of cross-linguistic influence, according to which L1-L2 contact might lead to processes such as:

• borrowing: addition of L2 elements to the L1;

• restructuring: deletion of certain L1 rules and/or incorporation of L2 elements into L1 resulting in substitutions or simplifications; • convergence: creation of a unitary system, distinct from both L1 and L2; • shift: a move from L1 structures to approximate L2 structures;

• attrition: loss of some L1 elements due to L2. Each process is observed in various linguistic domains, including morphosyntax. Pavlenko notes that statements of L1 attrition of morphosyntax require convincing evidence of attrition in L2 users' comprehension, production and metalinguistic judgements of L1 rules (for similar suggestions, see Altenberg and Vago in Schmid et al., 2004).

In another paper, Yag ˘mur reflects on his earlier research on L1 attrition of Turkish among first generation immigrants in Australia (Yag ˘mur, 1997). He reports that Turkish attriters demonstrate morphological errors such as double pluralization after a quantifier, as in *çok kitaplar ('many books'). Yagmur also tests relative clauses keeping with a regression assumption, according to which complex constructions such as relativization are acquired late, and, therefore, are lost early in language contact situations. Results reveal that when compared to the monolinguals in Turkey, the attriters (even those with low L2 proficiency) are found to be slower in producing these constructions, possibly due to L1 processing difficulties experienced after an extended stay in the L2 environment.

In the subsequent chapter, Ben-Rafael discusses the effects of L2 Hebrew on the L1 French grammar of adults living in Israel. An analysis of speech data reveals that due to the influence of Hebrew, the conditional form is expressed by the future tense, the distinction between passé composé and imparfait is not always maintained, the subjunctive mode is often disregarded, and indefinite articles are omitted. Ben-Rafael notes that grammatical changes observed in the data are often similar to certain registers of spoken French. Therefore, reduction and simplification processes may reflect tendencies inherent in the French language, triggered by contact with Hebrew. As for syntactic features that remain unchanged, French grammatical features without equivalents in Hebrew (e.g. word order, the number of negators) resist L2 influence.

Hutz discusses a case study based on a corpus consisting of letters written between 1939 and 1994 by a German immigrant who settled in the USA in adulthood with no prior L2 English knowledge. Hutz reports no strong morphological attrition and very few deviations in L1 word order. The morphosyntactic stability found 57 years after immigration is remarkable. Nevertheless, it is important to note that Hutz's study involves one participant, and that the written data on which the whole analysis is based might involve a high degree of monitoring and self-correction.

In search of syntactic attrition, Gürel discusses L1 data from Turkish speakers who were exposed to English in adulthood and who have been living in North America for a prolonged period of time. The study investigates possible attrition effects in binding properties of overt and null pronouns in pro-drop L1 Turkish under the influence of nonpro-drop L2 English. Attrition and monolingual groups are compared in three different tasks: written interpretation, truth-value judgement and picture identification. The attrition group allows more bound variable readings for the overt pronoun o than do monolinguals. This is a clear effect of L2 English because in constructions such as Murat i onun *i sinemaya gideceg ˘ini söyledi ('Murat i said that he i would go to the movies'), the Turkish overt pronoun o, unlike the English pronoun he, cannot be coreferential with the sentential subject. This suggests that the disjointness requirement of the overt pronoun o is not strictly followed by L1 attriters due to L2 English. However, the attrition group maintains the binding features of the other overt pronoun kendisi and the null pronoun, for which English has no corresponding forms.

Gross adopts the language production model of Myers-Scotton and Jake (2001) to analyse production data from six adult German immigrants who were exposed to English in the USA. Data support the model in the sense that content morphemes (e.g. nouns, verbs, idioms) are found to be more vulnerable to attrition than early system morphemes (e.g. determiners, plural -s, reflective markers). Yet, early system morphemes are more vulnerable than late system morphemes such as subject-verb agreement and case markers.

Overall, the morphosyntactic studies reviewed in this volume indicate considerable L2 influence on the mature L1 grammars of adult bilinguals. To summarize:

• Restructuring of the L1 grammar is possible due to extensive L2 exposure combined with less accessible L1 input. • When L1 and L2 have corresponding forms, the L2 form with all its morphosyntactic features can override the L1 form that is not used as frequently.

• When L1 and L2 have no corresponding forms, L2-interference dependent change is not relevant. • Complex L1 forms might be processed with difficulty as a result of long-term disuse.

3 Köpke, Schmid, Keijzer and Dostert, 2007 The most recent volume includes articles that provide theoretical foundations and perspectives for language attrition research. The 2004 volume came out of the First International Conference on First Language Attrition and focused on the methodological issues. In contrast, the 2007 volume reflects the theme of the Second International Conference on First Language Attrition and focuses on the role of theoretical models for the study of language attrition. This 13-chapter book starts with an introduction by Monika S. Schmid and Barbara Köpke, who emphasize the necessity of evaluating L1 attrition within a broader perspective of bilingualism. They suggest that, provided that attrition studies are conducted within a more theoretically-oriented frame (as in the case of L2 acquisition research), examination of L2-induced changes in the L1 grammar can provide valuable insights into linguistic research in general.

In the first chapter, Köpke examines biological, cognitive and external factors that might -individually or as a cluster -impact L1 attrition. In Chapter 2, Mike Sharwood Smith presents Modular Online Growth and Use of Language, a model that tries to integrate both linguistic and psycholinguistic accounts of language transition in the context of language acquisition and attrition. In Chapter 3, Kees de Bot suggests that some of the main characteristics of a dynamic systems model can explain language attrition.

Chapters by Carol Myers-Scotton (Chapter 4), Ianthi Maria Tsimpli (Chapter 5) and Ays ¸e Gürel (Chapter 6) comprise individual studies investigating morphosyntactic attrition within a certain linguistic framework. I discuss these studies in more detail after giving an overview of the rest of the book.

In Chapter 7, Michel Paradis discusses various constructs from a neurolinguistic theory of bilingualism and their implications for language attrition. In Chapter 8, on the basis of data from German-English and German-Dutch bilinguals, Schmid argues that not all types of L1 use help maintain the L1.

Chapters 9 and 10 investigate L1 attrition in early bilinguals. Christophe Pallier presents data from Korean adoptees in France to argue that a critical period in language acquisition is a result of the stabilization of neural connections through the language learning experience itself, not the result of a loss of neural plasticity. In Chapter 10, Rosalie Footnick presents a case study of a bilingual to argue that an apparently forgotten language can be recovered through hypnosis.

The last three chapters examine the role of identity, ideology, emotions and attitudes in L1 attrition. In Chapter 11, Petra Presher explores adult immigrants' own perception of their bilingual and bicultural identity in relation to L1 attrition. In Chapter 12, Miriam Ben-Rafael and Monika S. Schmid compare the changes in the L1s of French and Russian immigrants in Israel. In Chapter 13, Antonio F. Jiménez Jiménez discusses the use of the stimulated recall protocol in the study of L1 attrition.

In the rest of this section, I return to the three studies on L1 attrition of morphosyntax. Carol Myers-Scotton's Chapter 4 reports on a study of Xhosa-English bilinguals in South Africa to investigate the linguistic steps that lead to language shift. Speech samples analysed within the 4-M model did not reveal any considerable signs of L1 attrition of grammatical morphemes. Code-switching found in the speech samples may be a step on the way to language shift, but Myers-Scotton's data does not conclusively show that shift is inevitable.

In Chapter 5, Tsimpli presents a Minimalist approach to selectivity in morphosyntactic attrition. She argues that the attrition process can affect interface features but not syntax proper. First, the author discusses the results of the production and interpretation tasks reported in [START_REF] Tsimpli | First language attrition and syntactic subjects: a study of Greek and Italian near-native speakers of English[END_REF] that looks at L2 English-induced changes in L1 Greek grammars of late L2 learners living in Britain for a minimum of six years. The Greek data involving an interface phenomenon -namely (in)definite preverbal and postverbal Determiner Phrase (DP) subjectsis analysed. Results of the production task reveal that the attrition group, under the influence of English, produces more preverbal subjects in contexts where the non-attrited group strongly prefers postverbal subjects. However, the same effect is not clearly observed in the interpretation task. Second, Tsimpli discusses results of an online grammaticality judgement task reported in [START_REF] Kaltsa | First language attrition in Greek[END_REF] pilot study that investigates L1 case-marking on definite and indefinite DPs of Greek-Swedish and Greek-German early bilinguals. Attrition effects found in these early bilinguals are possibly due to incomplete acquisition of Greek.

Gürel's chapter compares the results of a L2 Turkish-induced L1 English attrition study with the results of a L2 English-induced L1 Turkish attrition study reported in [START_REF] Gürel | Linguistic characteristics of second language acquisition and first language attrition: Overt versus null pronouns[END_REF], which accounts for selective L1 attrition through the subset-superset relations of the L1 and L2. In the present study, the attrition group consists of 15 English native speakers who have learned Turkish in adulthood and whose length of stay in Turkey ranged from 10 to 35 years. Attrition effects in binding interpretations of pronouns and reflexives are tested through two tasks: written interpretation and truth-value judgement. Results reveal no evidence for the L2 Turkish influence in the L1 English grammar, disconfirming predictions drawn from the Subset Model of language attrition. Although [START_REF] Gürel | Linguistic characteristics of second language acquisition and first language attrition: Overt versus null pronouns[END_REF] study demonstrates L2 English effects in the L1 grammar of Turkish native speakers living in North America, the same L2 effects are not found in the L1 grammar of native English speakers living in Turkey. This difference is attributed to the frequency of L1 use: an important psycholinguistic factor in language attrition (compare Schmid in Köpke et al., 2007).

III General discussion

1 The integration of two languages in the bilingual mind: Is the outcome predictable? Data from both L2 acquisition and L1 attrition seem to suggest that language representation of bilinguals is subject to change. Various changes occur in the content of both grammars over time due to changing input conditions and due to continuous interaction of the two linguistic systems [START_REF] Sharwood Smith | First language attrition and the parameter setting model[END_REF]Paradis in Köpke et al., 2007). This observation had led many researchers to propose a multicompetence view of bilingualism that considers the bilingual mind as an entity in its own right, not as a composite of two monolingual minds [START_REF] Cook | The poverty-of-the-stimulus argument and multi-competence[END_REF]. Accordingly, integration of two languages is assumed to be an inevitable consequence of bilingualism, creating a single conjoined system different from monolingual versions of either language (Cook, 2003: 8).

The application of these relatively new concepts into the domain of L1 attrition brings more questions than answers: What determines the extent of integration or the linguistic content of the merged system? Does this integration lead to the creation of a unitary system, distinct from both L1 and L2 (Cook, 2003) or to the incorporation of L2 rules into the L1 [START_REF] Pavlenko | L2 influence on L1 late bilingualism[END_REF]Pavlenko in Schmid et al., 2004)? Which L1 features are more affected by this integration [START_REF] Gürel | Linguistic characteristics of second language acquisition and first language attrition: Overt versus null pronouns[END_REF][START_REF] Tsimpli | First language attrition and syntactic subjects: a study of Greek and Italian near-native speakers of English[END_REF]Gürel in Köpke et al., 2007;Tsimpli in Köpke et al., 2007)? Are changes in L1 features internally-induced (i.e. L2-independent)? In other words, is attrition of some L1 features the result of inherent unsteadiness (weakness) that makes them more susceptible to erosion [START_REF] Seliger | Deterioration and creativity in childhood bilingualism[END_REF]1996;Ben-Rafael in Schmid et al., 2004;[START_REF] Sorace | Selective optionality in language development[END_REF]? Alternatively, can we consider attrition the consequence of a particular manner in which two languages interact? In other words, is L1 attrition an L2-induced phenomenon that emerges when the L2 grammar becomes the source of indirect positive evidence due to the lack of L1 input (Seliger, 1991;[START_REF] Sharwood Smith | First language attrition and the parameter setting model[END_REF][START_REF] Gürel | Linguistic characteristics of second language acquisition and first language attrition: Overt versus null pronouns[END_REF][START_REF] Schmid | First language attrition, use, and maintenance: the case of German Jews in Anglophone countries[END_REF]? We can consider a third possibility in which both internally-and externally-induced changes occur at the same time. In other words, certain L2 features trigger attrition only in inherently unstable L1 features.

Indeed, in externally-induced attrition (i.e. L2-induced attrition) there is some selectivity. Attrition does not apply across the board. Only certain L2 forms can trigger change in some L1 properties. For example, only relatively less complex L2 forms encroach on the L1 and accelerate change or attrition in the corresponding L1 properties [START_REF] Andersen | Determining the linguistic attributes of language attrition[END_REF][START_REF] Seliger | Deterioration and creativity in childhood bilingualism[END_REF][START_REF] Schmid | First language attrition, use, and maintenance: the case of German Jews in Anglophone countries[END_REF]. The belief that less complex L2 features replace more complex L1 features is generally linked to a reduction or simplification rule in attrition [START_REF] Andersen | Determining the linguistic attributes of language attrition[END_REF][START_REF] Seliger | Deterioration and creativity in childhood bilingualism[END_REF]1996). For example, in [START_REF] Seliger | Deterioration and creativity in childhood bilingualism[END_REF]1996) 'redundancy reduction principle', reduction is associated with some form of 'markedness' in the sense that less marked L2 forms are more likely to replace more marked forms in the L1, whereas less marked L1 forms appear more resistant to attrition. In this account, grammatical forms that are more complex and have a narrow linguistic distribution are considered marked. This follows that integration of two languages brings about a competition between L1 and L2 rules that are linguistically comparable. Thus, L2-induced change or attrition is only relevant when the L1 and L2 have equivalent forms that are in competition [START_REF] Köpke | Activation thresholds and non-pathological first language attrition[END_REF]Ben-Rafael in Schmid et al., 2004;Gürel, 2004;Gürel in Köpke et al., 2007;Köpke in Köpke et al., 2007;Paradis in Köpke et al., 2007; see also [START_REF] Andersen | Determining the linguistic attributes of language attrition[END_REF]. This is in line with Altenberg's (1991: 204) suggestion that L1 and L2 similarity is a necessary condition for L2-induced L1 attrition.

The idea that some linguistic features are inherently more problematic (i.e. hard to acquire and easy to lose) is also proposed in recent Minimalist approaches to attrition. For example, [START_REF] Tsimpli | First language attrition and syntactic subjects: a study of Greek and Italian near-native speakers of English[END_REF], [START_REF] Sorace | Selective optionality in language development[END_REF] and Tsimpli (in Köpke et al., 2007) suggest that extensive L2 use accompanied by infrequent L1 contact will only influence 'soft' interface constraints, which are associated with the mapping of syntax, lexical semantics, pragmatics, and information structure. In contrast, 'hard' constraints that are purely syntactic in nature will be retained in L1 attrition (Sorace, 2005: 55). Accordingly, null subjects -which result from the specification of [Ϫinterpretable] features (e.g. case and agreement on verbs) -will not be affected by attrition, whereas the distribution of null and overt subjects, which is determined by [ϩinterpretable] (or syntactic/pragmatic) features (e.g. [ϩTopic Shift]), will be subject to attrition. It is true that purely syntactic features are more resistant to attrition. However, this does not mean that syntactic attrition is never possible. As documented in all three volumes reviewed here and in previous studies, attrition is not limited to the interface between syntactic and discourse/pragmatic knowledge. Restructuring is possible in various domains of morphosyntax such as word order [START_REF] Schaufeli | Word order patterns in contact: Turkish in the Netherlands[END_REF]Jarvis in Cook, 2003), relative clause formation [START_REF] Seliger | Deterioration and creativity in childhood bilingualism[END_REF]; Yag ˘mur in Schmid et al., 2004), the formation of middle constructions (Balcom in Cook, 2003), the aspectual system [START_REF] Polinsky | American Russian: language loss meets language acquisition[END_REF]Pavlenko in Cook, 2003), the binding domain [START_REF] Gürel | Linguistic characteristics of second language acquisition and first language attrition: Overt versus null pronouns[END_REF], the pronominal system [START_REF] Gürel | Linguistic characteristics of second language acquisition and first language attrition: Overt versus null pronouns[END_REF][START_REF] Tsimpli | First language attrition and syntactic subjects: a study of Greek and Italian near-native speakers of English[END_REF]Tsimpli in Köpke et al., 2007), case and plural morphology [START_REF] Larmouth | Differential interference in American Finnish cases[END_REF][START_REF] Polinsky | American Russian: language loss meets language acquisition[END_REF]Pavlenko in Cook, 2003;Yag ˘mur in Schmid et al., 2004), indefinite articles (Ben-Rafael in Schmid et al., 2004) and verbal agreement (Ben-Rafael in Schmid et al., 2004).

In another approach to attrition, it has been suggested that the outcome of the linguistic integration of two languages can be predicted in a principled way by taking account of L1/L2 subset relationships [START_REF] Gürel | Linguistic characteristics of second language acquisition and first language attrition: Overt versus null pronouns[END_REF]Gürel in Köpke et al., 2007). In cases where the L2 has broader grammatical options with respect to a particular linguistic property (i.e. L2 forms a superset of L1), the options of the L1 are broadened on the model of L2 as restrictions tend to neutralize in language attrition. In a sense, in language contact situations, the language with fewer restrictions will replace the language with more restrictions. This prediction has been confirmed by studies in which restrictions on binding interpretations of pronouns are found to be loosened [START_REF] Gürel | Linguistic characteristics of second language acquisition and first language attrition: Overt versus null pronouns[END_REF]. Nevertheless, Gürel (in Köpke et al., 2007) reports that so long as psycholinguistic conditions for language attrition are not present, patterns of syntactic attrition predicted by this subset-superset model are not observed.

Characteristics of morphosyntactic attrition

Within the current state of L1 attrition research it is not yet possible to specify the exact linguistic nature of morphosyntactic attrition, mostly due to the incomparability of cross-linguistic data (see below). Nevertheless, attrition studies reviewed in the three volumes as well as those published elsewhere seem to suggest that morphosyntactic attrition in mature L1 grammars of bilinguals follows a pattern. The pattern reflects both internally-and externally-induced linguistic change that is triggered and/or accelerated by the interaction of two languages [START_REF] Schmid | First language attrition, use, and maintenance: the case of German Jews in Anglophone countries[END_REF]. In both types of change, the predicted tendency is towards reduction of morphosyntactic complexity as well as loosening of morphosyntactic restrictions, which will eventually give the speaker a smaller number of linguistic constructions and devices [START_REF] Andersen | Determining the linguistic attributes of language attrition[END_REF]Satterfield in Cook, 2003). Among the common examples of morphosyntactic reduction or simplification are loss of case morphology, loss of gender-marking and adjective/noun convergence, omission of indefinite articles, elimination of relative pronouns, reduction of allomorphic variation, simplification of verbal agreement, use of lexemes instead of bound morphemes to encode grammatical relations, simplification of word order, reduction of restrictions in the binding domain, and elimination of the perfective/imperfective aspectual distinction [START_REF] Schmid | First language attrition, use, and maintenance: the case of German Jews in Anglophone countries[END_REF].

Methodological issues

It is important to note that although there is considerable evidence of morphosyntactic change, some research findings do not reveal significant L1 attrition in adult grammars (Porte in Cook, 2003;Hutz in Schmid et al., 2004;Gürel in Köpke et al., 2007;Myers-Scotton in Köpke et al., 2007;see also Balcom in Cook, 2003;Pavlenko in Cook, 2003). It is commonly acknowledged that attrition is dependent on a variety of internal and external factors. Therefore, it is natural to find a high degree of variability in the extent of language attrition that occurs in mature grammars [START_REF] Schmid | First language attrition, use, and maintenance: the case of German Jews in Anglophone countries[END_REF]Köpke and Schmid in Schmid et al., 2004;Köpke in Köpke et al., 2007). Nevertheless, contradictory results can be attributed to methodological problems [START_REF] Schmid | First language attrition, use, and maintenance: the case of German Jews in Anglophone countries[END_REF]Köpke and Schmid in Schmid et al., 2004;Schmid and Köpke in Köpke et al., 2007). As Schmid (2002: 29) notes, there are three major methodological considerations in attrition research: linguistic features to be examined; data collection procedure; and determination of what counts as evidence for attrition.

Frequently, contradictory results are due to researchers' different conceptualizations of these three issues. Furthermore, as Schmid and Köpke (Köpke et al., 2007: 4) note, 'the field of attrition is still far less extensive, less theoretically sophisticated' and most attrition research is of a rather descriptive nature. Small data size and lack of proper quantification are the major problems of attrition studies. This makes it difficult (if not impossible) to compare the findings of existing research studies. In the absence of such comparability, major questions in attrition research will remain unanswered.

IV Conclusions

In this article, I reviewed three volumes on L1 change/attrition and discussed recent studies with a focus on morphosyntactic attrition in order to identify L1 features that are more vulnerable to attrition. Research findings suggest that however slow and limited, L1 attrition of morphosyntax is possible. Nevertheless, it is highly selective and can be both internally and externally induced. Therefore, predicting the most vulnerable features is not always easy. This requires a full understanding of the pattern of interaction between bilinguals' two languages. To identify the integration of the two languages, a holistic view of bilingualism has to be adopted. It is not possible to characterize L2-induced L1 attrition in isolation; we also need to identify the course of L2 acquisition of the bilingual (Schmid and Köpke in Köpke et al., 2007). What is lost and what is maintained in the L1 might be directly related to what is acquired and what is not acquired in the in L2 [START_REF] Gürel | Linguistic characteristics of second language acquisition and first language attrition: Overt versus null pronouns[END_REF][START_REF] Sorace | Selective optionality in language development[END_REF]. As suggested by dynamic systems theory, we may discover individual factors in language attrition, but the course of change cannot be predicted unless we measure the exact impact of each individual variable upon the complete interconnectedness of systems (de Bot in Köpke et al., 2007).

Acknowledgements

I am grateful to Margaret Thomas for her helpful suggestions and patience throughout the process. I would also like to thank an anonymous Second Language Research reviewer for comments. All errors are mine.


Ankur Raval

CHHAYA ENGINEER AND Animesh Choubey

Devesh Kothwala

Surface Conditioning of 316LVM Slotted Tube Cardiovascular Stents

Keywords: coronary stents, electropolishing, passivation, balloon expandability

The surface quality of coronary stents has a significant influence on its biocompatibility. Therefore, surface polishing is of paramount importance in the production and application of stents. In the present study, electropolishing is performed on 316LVM steel slotted tube coronary stents. Additionally, acid pickling, as a pretreatment of electropolishing, is also conducted. Gravimetric analysis of the stents (weight loss and strut width change) in the process of acid pickling and electropolishing are done. Qualitative roughness measurements are made to evaluate the stent surface. Electropolished stents are passivated causing chromium enrichment on the surface of the material, thereby enhancing its corrosion resistance. Passivated and electropolished samples are examined using energy dispersive spectrometry. Balloon expanded and crimped profiles of the passivated stents are qualitatively analyzed.

INTRODUCTION

S tents are tubular intravascular devices, which are placed within the blood vessels to structurally hold open the vessel. The device can be used to maintain the patency of a blood vessel, immediately after intravascular treatments, and to reduce the likelihood of the development of restenosis [START_REF] Serruys | A Comparison of Balloon Expandable Stent Implantation with Balloon Angioplasty in Patients with Coronary Artery Disease[END_REF][START_REF] Puel | Early and Late Assessment of Stenosis Geometry after Coronary Arterial Stenting[END_REF]. Figure 1 shows a typical coronary stent profile featuring the main strut, the subsidiary strut, and the link. It is highly desirable for the surface of the stent to be extremely smooth so that it can be inserted easily and experience low-friction travel through the tortuous vessel pathway prior to implantation. A roughened outer surface may result in increased frictional obstruction during insertion and excess drag during travel to the stenotic site as well as damaging the endothelium lining of the vessel wall [START_REF] Williams | Surface Interactions[END_REF]. A rough surface may cause frictional resistance to such an extent as to prevent travel to the desired distal locations. It may also cause damage to the underlying inflation balloon [START_REF] Williams | Surface Interactions[END_REF]. Surface roughness also influences the amount of protein adherence as it determines the contact area [START_REF] Andrade | Plasma Protein Adsorbtion: The Big Twelve[END_REF]. Immediately following and in concert with the adsorbed proteins after stent implantation, the stent surface properties will determine platelet and leucocyte adherence. Fibrogen, immunoglobulins, complement factors, and coagulation factors, all influence platelet activation and aggregation, as well as neutrophil and macrophage adhesion and activation [START_REF] Spector | The Local Tissue Response to Biomaterials[END_REF]. As such, the surface properties determine thrombogenicity, inflammation, and vascular wound-healing.

As the surface characteristics influence the biocompatibility of the stents directly, a smooth surface obtained after acid pickling and electropolishing can minimize thrombosis effectively, and also has a potential beneficial effect on neointimal hyperplasia [START_REF] De Scheerder | Metallic Surface Treatment Using Electrochemical Polishing Decreases Thrombogenicity and Neointimal Hyperplasia of Coronary Stents[END_REF]. Electropolishing is a process in which a metallic surface is smoothened by polarizing it anodically in an adequate electrolyte. It is classified into two processes: anodic leveling and anodic brightening [START_REF] Maganio | An Impedance Study of Stainless Steel Electropolishing[END_REF]. Anodic leveling results from the difference in the dissolution rate between the peaks and valleys on a rough metal surface depending on the current distribution or masstransport conditions. On the other hand, anodic brightening is associated with the suppression of the influence of the metal microstructure on the dissolution rate. A smooth electropolished surface, which appears bright to the naked eye, results from a combination of these two factors [START_REF] Maganio | An Impedance Study of Stainless Steel Electropolishing[END_REF]. The aim of the current research work is to investigate and determine the optimal conditions for acid pickling and electropolishing of 316LVM coronary stents. Also, an attempt has been made to characterize stents by weight and strut width measurements. Passivation of electropolished stents is done to increase its corrosion resistance in severe blood-contacting environments. Stents must have a high expandability. This can also be a high ratio between crimped and expanded diameters to allow for the smallest diameter of the system. Hence, crimped and expanded balloon profiles of the passivated stents are also observed.

MATERIALS AND METHODS

Materials

The original material used in this study was 316LVM (low carbon vacuum remelted steel) slotted tube coronary stents produced from a stainless steel tube by laser precision cutting (Sahajanand Laser Technologies Pvt. Ltd., India). The stents were approximately 16 mm long. Their outer diameter was about 1.72 mm. The wall thickness of the stent was 110 mm. The as-received samples were initially cleaned using distilled water in an ultrasonic agitation bath for 15 min and dried in air. Percutaneous Transluminal Coronary Angioplasty (PTCA) balloon catheter used during the study was procured from Arthesis, France (Figure 2). It was made of Nylon-12 material with PTFE-coated stainless steel hypotube shaft. Two radiopaque markers were located underneath the balloon, which fluoroscopically marked its working length. The dimensions of the balloons used were 3.0 Â 17 mm and 3.5 Â 17 mm. Of these dimensions, 3.0 and 3.5 designates the balloon diameter (in mm), when expanded at recommended expansion pressure (6 atm) provided by the manufacturer and 17 mm indicates the length of the balloon.

Acid Pickling

Laser processing is done at high temperature and this often leaves debris and slag material attached to the stent. Acid pickling is an effective method for the chemical removal of surface oxides and other contaminants from metallic materials by immersion in an aqueous acid solution [START_REF] Hudson | Pickling of iron and Steel[END_REF]. Alkaline soap wash by a spray of soap water (approximately 2 mL of concentrated alkaline liquid cleaner mixed in 200 mL of deionized water) was given to the laser-cut stents to remove dust particles, oil, grease, and slag. The stents were then cleaned with a spray of deionized water to remove soap. To remove any residual grease or oil from the stent surface, it was dipped in 25 mL of trichloroethylene (AR grade) in a culture tube for 11-12 h. After degreasing, the stents were washed with deionized water using an ultrasonic cleaner for 10 min followed by drying in an oven at 90-95 C. For acid pickling, two different bath compositions were explored (Table 1a). The samples were pickled for a different amount of time for both the processes and by the observation of the change of the surface state of these specimens, a time was chosen which caused the complete removal of oxide scales from the inner and outer surfaces and the cutting zone. In acid pickling with HNO 3 bath, a glass beaker containing the stents in acid solution was placed on a thermostat, maintaining the temperature at 80-90 C. In step one, the stents were pickled for a varying amount of time 1, 3, and 5 h and washed with a spray of deionized water. In step two, for the further removal of loosened oxide scales, electrolysis (see Table 1b) was carried out at room temperature for 20-45 s.

In the second process (Table 1a), the pickling of stents was performed by immersing the stents in an acid solution containing HF, HNO 3 , and H 2 O. The acid beaker was kept in an ultrasonic cleaner for 10 min to allow degassing. The temperature of the water bath in the ultrasonic cleaner was maintained at 35-40 C throughout the process. The glass beaker containing the stents dipped in acid pickling solution was subjected to ultrasonic agitation. After the specified time (5, 10, and 15 min) of cleaning, the heat controller, timer and ultrasonic cleaner were switched off. Pickled stents were given a deionized water wash and dried by means of a hot air drier (Philips Flex-1000, Japan).

Electropolishing

The electropolishing device was self-designed, as illustrated in Figure 3. A 1000 mL glass beaker was used as a cell. A DC rectifier (Testronix 92D) was used for power supply (30 V and 10 A maximum). Electropolishing is a process by which metal is removed from a workpiece by the passage of electric current when the workpiece is immersed in a liquid medium, i.e. electrolyte. The workpiece (316LVM stents) were connected to the anodic terminal and 304 steel ring was used as the cathode. Both anodic and cathodic terminals were submerged in the solution, forming a complete electrical circuit. The current applied is a direct current. The composition of the electrolyte is summarized in Table 2. The optimized parameters for electropolishing are listed in Table 3. The temperature was maintained between 70 and 80 C and the bath was stirred for about 40 min with a magnetic stirrer (Remi 1MLH) during the preparation of the solution for the complete dissolution of the chemicals. Voltage and current are co-related by the Ohm's law. When a current passes through an electrolyte, a liquid layer of anodic dissolution products is formed on the surface of the anode. This layer has a higher viscosity and greater electrical resistivity than the bulk of the electrolyte. The thickness of the liquid layer on a rough surface differs from one area to the next. The current density is nonuniform as a result of such a nonuniform liquid layer; i.e. it is higher on peaks than crevices. Thus, peaks dissolve more rapidly than crevices, this, therefore produces a surface leveling effect on the stents [START_REF] Hudson | Pickling of iron and Steel[END_REF]. The stents during electropolishing were handled carefully using latex gloves (sterilized with ethylene oxide) and were stored in an airtight container.

Gravimetric Analysis of Stents

Weights of the as-cut, acid pickled, and electropolished stents were measured with an electronic analytical balance (Citizen CX-265). Measurement of widths of the stent strut was carried out before and after electrochemical polishing using a light optical microscope (SZX-12, Olympus-Japan). Six measuring sites of the stent struts were arbitrarily chosen to conduct the width measurement, and the mean value was calculated. Percentage weight loss and width reduction of the stents were also estimated.

Passivation 316 L stainless steel has an intrinsic corrosion resistance because of its ability to form a protective oxide or passive layer on its surface by alloying >12% chromium with iron, nickel, and molybdenum [START_REF] Barbosa | The Pitting Resistance of AISI 316 Stainless Steel Passivated in Diluted Nitric Acid[END_REF]. This protective layer is thought to consist of a heterogeneous chromium oxide film (Cr 2 O 3 ) along with elemental iron, nickel, and their respective oxides. In general, this passive layer protects the steel from oxidation. However, when subjected to extremely corrosive environments, the heterogeneous film is attacked and chemical reduction occurs. When the oxide film layer is destroyed beyond regeneration, localized corrosion takes place in the bulk alloy and reaction by-products are generated [START_REF] Barbosa | The Pitting Resistance of AISI 316 Stainless Steel Passivated in Diluted Nitric Acid[END_REF][START_REF] Walls | In Situ Observation of the Oxidation and Reduction Processes on Fe-Cr Alloys[END_REF]. The corrosion resistance at the steel surface can be enhanced by increasing the amount of chromium that can form Cr 2 O 3 in the presence of an oxidizing environment.

In the present study, passivation solution was made by adding 35 mL of nitric acid (AR grade) to 70 mL of deionized water in a glass beaker. The beaker containing the solution was heated to 75 C (AE3 C) using a thermostat. Stents were dipped in the solution after the desired temperature was achieved and kept there for 30-35 min. A carbonate wash was provided to (30 g of sodium carbonate added to 200 mL of deionized water) neutralize the nitric acid followed by the cleaning of stents with deionized water to remove any traces of carbonate. The stents were kept in isopropyl alcohol solution to avoid any microbial contamination on the surface and dried with a hot air drier in the filter area. Care was taken to keep the drier in such a position that it draws air from the laminar airflow supply. Passivated surfaces were examined by SEM-EDAX (Philips XL30, Japan).

Balloon Expandability of Stents

To ensure uniform expandability and mechanical strength of the stents after surface treatment, they were subjected to balloon expandability test. Passivated stents were crimped on the balloon by means of automatic crimping machine (Machine Solutions, MSI-500, USA). The crimped stents were expanded by expansion device (Medtronic, Skimed-Sedat, USA) used by interventional cardiologists during angioplasty. The pressure gauge was attached with the expansion device to measure the pressure applied to the balloon. Sterile fluid was pumped in to assist balloon expansion. Balloons of diameters 3 and 3.5 mm were used. Stents crimped on these balloons were expanded at a rated pressure of 6 atm. The balloon was kept dilated on manufacturers' recommended pressure for approximately 45-60 s and then deflated. The procedure was repeated twice with a 1-2 min rest period between each dilation. The crimped and dilated stent diameters were measured with a micrometer and their profile was inspected by stereooptical microscope (SZX-12, Olympus-Japan) and scanning electron microscopy.

RESULTS AND DISCUSSION

As-received Stents

Figure 4 shows the morphology of the laser-cut zone of as-cut stent 'as received'. The slag is clearly visible in the scanning electron micrograph. Considerable roughness was observed in the laser-cut zone.

Pickling of Stents

Figure 5 shows the scanning electron micrographs of stents pickled by HNO 3 þ electrolysis (Figure 5(a)) and HF þ HNO 3 process (Figure 5(b)). The slag in the laser-cut zone was not removed totally in the case of pickling with HNO 3 þ electrolysis process; also the oxides produced by the laser cutting process still adhered to the surface of the stents. Immersion time (5 h) could not be exceeded with this solution to avoid excessive stock removal. Whereas, after pickling for a short period of 15 min with HF þ HNO 3, the slag could be effectively removed from the laser-cut zone. Therefore, it can be considered that pickling with HF þ HNO 3 is more suitable for 316LVM slotted tube coronary stents. Acid pickling with both the processes could remove the products covering the outer surface of the stent formed while producing the tubing, thus resulting in a rough outer surface. It has been reported that nitric-hydrofluoric acid solution is used for removing both metallic contamination, welding, and heat-treating scales [START_REF]ASTM Standard: A 380-02[END_REF]. The mixture of HF þ HNO 3 directly penetrates through the crevices of the scale layer and starts dissolving it. By combining the effect of ultrasonic waves, the pickling efficiency increases and complete removal of the scale can be observed in a short span of time. Ultrasonic waves loosen and remove contaminants from deep recesses and other inaccessible areas [START_REF] Hudson | Pickling and Descaling[END_REF]. Cavitation in the pickling solution produced by the high-frequency sound waves causes micro agitation of the cleaning media in even tiny recesses of the stent, making the scales to remove completely from the entire surface area.

Electropolishing

Figure 6 shows scanning electron micrographs of electropolished stents after acid pickling in HF þ HNO 3 bath. Evidently, after electropolishing a considerably smooth surface was revealed (Figure 6(a)). The surface quality was largely improved compared to the pickled stent. The roughness on the laser-cut zone has been entirely removed (Figure 6(b)). Therefore, from a comparison of the polished and as-received stents, it can be considered that this selected condition of electropolishing is suitable for the improvement of the surface roughness of the stents.

Removal of Stent Material

Table 4 presents the average weight and percentage weight loss of the as-received, acid pickled, and electropolished stents. Table 5 illustrates the width and percentage width reduction of stent struts. The as-received stent has a weight of 27.8 mg and a strut width of about 150 AE 5 mm. After pickling in the selected acid solution (HF þ HNO 3 ) at 45 C for 15 min, the weight loss was 7.91%. The strut width decreased to 145 AE 5 mm. The width reduction was thus 3.33%. After polishing using an electropolishing bath (Table 2) at the condition presented in Table 3, the weight of the stent became 20.7 mg and the strut width was reduced to 110 AE 5 mm. Figure 7 gives a plot between the weight of stent and the number of stents. It shows a variation of weight after acid picking, electropolishing, and weight loss in electropolishing, for 24 stents examined.

Passivation

The SEM-EDAX analysis provided data on the elemental and oxide surface chemistries of the treated samples. Table 6 compares the chromium to iron (Cr/Fe) ratios for surfaces modified by electropolishing and passivation for five samples. The ratios for the passivated samples were higher than the values obtained for the electropolished samples. Specifically, the average Cr/Fe ratio for the passivated samples was 0.41 versus 0.29 for the electropolished samples. The higher surface chromium on the passivated samples might contribute to a higher surface corrosion resistance than that of the electropolished samples because the removal of surface iron and metallic contaminants reduces the potential for reactive-site corrosion.

Balloon Expandability of Stents

Figure 8 shows the stent crimped on a balloon. The scanning electron micrograph shows a perfect crimping profile. Overlapping of any of the struts was not observed. Links and subsidiary struts are found to be absolutely straight. For the stent mounted on 3.0 and 3.5-mm diameter balloon, the crimped diameter of the stent was 1.016 and 1.041 mm, respectively. Figure 9(a) and (b) illustrate the expansion profile of the stents. The figures reveal that, at 6 atm of applied pressure the stents were fully to a diameter of approximately 3.0 mm (using the balloon of 3 mm diameter) and 3.5 mm (using the balloon of 3.5 mm diameter). Appropriate straightening of the alternate link between two subsequent cells could be observed. An expanded profile also revealed that none of the struts were disproportionately dilated. Proper opening of the main and subsidiary struts at an expansion pressure of 6 atm could also be observed from the scanning electron micrograph. Figure 10 shows the optical micrographs of as-received stent and dilated stents of 3 and 3.5 mm diameter. No foreshortening (reduction in length upon expansion) was observed after balloon expansion to these diameters. Foreshortening was measured by comparing the length of the dilated stents to that of the undilated ones. No distinguishable reduction in length of the expanded stents was observed. This can be attributed to low stress concentration in the longitudinal direction and uniform stress distribution, owing to smooth stent surface topography obtained after electropolishing.

CONCLUSIONS

Scanning electron micrographs revealed that acid pickling with HF þ HNO 3 caused a considerable decrease in roughness of the cutting zone. The slag was effectively removed and a clean stent surface was obtained for electropolishing. Surface quality of 316LVM slotted tube coronary stent is largely improved by means of electropolishing performed with optimized parameters. Compositional analysis of electropolished and passivated stent surfaces showed a higher Cr/Fe ratio for passivated stents, indicating enhanced corrosion resistance of these stents in severe blood contacting human body environments. To ensure the mechanical strength of the stent after material removal during surface treatment, balloon expanded and crimped profiles of the stents were observed. The original laser-cut stents were found to be too rigid to be balloon expandable, whereas, passivated coronary stent could very easily be crimped on the balloon and subsequently dilated. Uniform expansion profile was observed in scanning electron micrographs. Optical images depicted almost zero foreshortening of the dilated stents.

Figure 1 .

 1 Figure 1. Expanded stent profile showing: (a) main strut; (b) subsidiary strut; and (c) link.

Figure 2 .

 2 Figure 2. Balloon used for expanding stents in the arteries (Arthesis, France).

Figure 3 .

 3 Figure 3. Self-designed electrochemical polishing device.

Figure 4 .

 4 Figure 4. Surface topography of laser-cut zone and outer surface.

Figure 5 .

 5 Figure 5. Surface topography of stents pickled by: (a) HNO 3 þ electrolysis; and (b) HF þ HNO 3 process.

Figure 6 .

 6 Figure 6. Surface topography of stents electropolished after pickling in HF þ HNO 3 bath.

Figure 7 .

 7 Figure 7. Plot showing variation of stent weight.

Figure 8 .

 8 Figure 8. Scanning electron micrograph showing a stent crimped on a

Figure 9 .

 9 Figure 9. Scanning electron micrographs showing the profile of a stent dilated to (a) 3 mm and (b) 3.5 mm.

Figure 10 .

 10 Figure 10. Optical micrographs showing (a) as-received stent; (b) 3 mm dilated and (c) 3.5 mm dilated stent geometry.

Table 1a .

 1a Composition of pickling solutions.

		Component	Amount (mL)
	1	HNO 3	150
	2	HF (48%)	1
		HNO 3 (70%)	9
		Deionized water	90

Table 1b .

 1b Composition of electrolysis solution.

	Component	Amount
	H 2 SO 4	20 mL
	Deionized water	180 mL
	NH 2 CS.NH 2 (thiourea)	0.5 g

Table 2 .

 2 Composition of electrolyte for electropolishing.

	Component	Amount
	H 3 PO 4 (ortho-phosphoric acid)	325 mL
	H 2 SO 4	125 mL
	Deionized water	50 mL
	Na 5 O 10 P 3 (sodium tripolyphosphate)	1.0 g

Table 3 .

 3 Parameters for electropolishing.

	Parameter	Value
	Temperature range	70-80 C
	Voltage	8.0 V
	Current	0.4 A
	Time	80 s

Table 5 .

 5 Width and %width reduction of stent strut before and after electropolishing.

			Width
	Sample	Width (mm)	reduction (%)
	As-received	150 AE 5	-
	Acid pickled	145 AE 5	3.33
	Electropolished	110 AE 5	24.14

Table 4 .

 4 Weight and % weight loss of stents before and after electropolishing.

		Average	Weight
	Sample	weight (mg)	loss (%)
	As-received	27.8	-
	Acid pickled (HF þ HNO 3 )	25.6	7.91
	Electropolished	20.7	19.18

Table 6 .

 6 Cr/Fe ratio for electropolished and passivated stent samples.

	Sample	1	2	3	4	5
	Electropolished (Cr/Fe ratio)	0.28	0.30	0.34	0.29	0.27
	Passivated (Cr/Fe ratio)	0.42	0.40	0.41	0.40	0.43

ACKNOWLEDGMENT

The authors wish to thank Mr. Rajesh Vaishnav, Technical Director, Sahajanand Medical Technologies Pvt. Ltd. for providing the technical support. They also express their sincere gratitude to Mr. Dhirajlal Kotadia, Chairman, Sahajanand Group of Companies, for providing financial assistance to carry out the research work.


C Giordano
email: carmen.giordano@polimi.it

V Sanginario

L Ambrosio

L Di

M Santin

Chemical-Physical Characterization and in vitro Preliminary Biological Assessment of Hyaluronic Acid Benzyl Ester-Hydroxyapatite Composite

ABSTRACT: HYAFF Õ 11 is a biocompatible, biodegradable benzyl ester of hyaluronic acid. However, in order to use it for orthopedic application, its mechanical performance needs to be improved. In this study, a novel composite based on HYAFF Õ 11 polymer matrix reinforced with hydroxylapatite (HA) has been developed. Its advantage is having a similar component of the mineral phase of bone resulting in favorable osteoconductive properties. The present study has examined the compressive mechanical and surface chemical-physical properties of the novel HYAFF Õ 11-HA composite. Preliminary biological investigations, including pH and cytotoxicity studies of the material extracts, have also been performed using an in vitro primary human osteoblast-like cell model. Moreover, protein, especially fibronectin adsorption has been investigated following incubation in culture medium and human plasma. The results show a grainy surface topography composed mainly of C, P, and Ca, with a Ca/P atomic ratio indicating HA on the composite surface. Mechanical analysis shows an improvement of the compressive properties of HYAFF11 matrix, both in the dry and swollen states, with values in the range of that of spongy bone. No cytotoxic effects and no inhibition of cell proliferation have been observed in the presence of the material extracts with pH values within acceptable ranges for cell vitality. Protein studies reveal a similar pattern, but a higher amount of fibronectin following incubation in human plasma when compared with culture medium. The results show that the novel HYAFF Õ 11-HA composite shows a great potential for application in orthopedic fields, especially as vertebral trabecular bone substitute.

KEY WORDS: hydrogel, composite, surface analysis, stress analysis, cell response, protein adsorption, biocompatibility, osteoblast, in vitro tests, cell culture.

INTRODUCTION

M usculoskeletal conditions are becoming an increasing health concern in aging population. Common treatments in fracture repair and total joint replacement still use traditional methods, such as autograft and allograft for bone repair, both of which have limitations.

The most commonly used biomaterials for orthopedic applications are natural and modified natural materials, as well as metals, ceramics, and synthetic polymers. In order to achieve specific properties not attainable by single-phase materials, it is now possible to design composites combining polymers, ceramics, and metals in order to obtain tailor-made devices for applications in various biomedical fields. Synthetic polymeric composites are very attractive biomaterials because of their similarities with most of the structural living tissue, composed of macromolecular composites.

Owing to their biodegradability, high biocompatibility, and nontoxicity, many natural polysaccharides have more recently been used for biomedical applications. Particular attention has been given to the largest glycosaminoglycans (GAG), hyaluronic acid or hyaluronan, a linear anionic polysaccharide composed of repeating disaccharide units of glucuronic acid and N-acetyl-glucosamine with the following molecular structure: -[D-glucuronic acid (1--3) N-acetyl-D-glucosamine (1--4)] n - [START_REF] Laurent | Hyaluronan[END_REF]. Hyaluronic acid is heterogeneously distributed in various tissues, where it plays diverse roles with regard to structural, rheological, physiological, and biological functions in the body. Physical and biological characteristics of hyaluronan in its purified form, such as water solubility, rapid resorption rate, and short half-life in the tissues, limit its possible biomedical applications. As a result, several attempts have been made to modify the molecular structure of high molecular weight purified hyaluronan in order to obtain a more stable solid material, preserving its biocompatibility and noninflammatory properties [START_REF] Campoccia | Semisynthetic Resorbable Materials Form Hyaluronan Esterification[END_REF].

HYAFF Õ is a class of hyaluronan derivative polymer obtained by esterifying the free carboxyl group of glucuronic acid, with different types of alcohol (aliphatic, aryliphatic, cycloaliphatic, and others) in order to reduce the hydrophilic, negatively charged, carboxyl groups of the polyanionic polysaccharide, thereby increasing its hydrophobic components. This results in a decrease in solubility and a slight increase in structural rigidity of the macromolecule, caused by the interaction of hydrophobic group organized in hydrophobic patches. The benzyl ester of hyaluronan, termed HYAFF Õ 11, is a well-characterized HYAFF Õ polymer from both the chemical-physical and biological point of view [START_REF] Iannace | Thermomechanical Properties of Hyaluronic Acid Derived Products[END_REF]. The structure of HYAFF Õ 11 is characterized by the presence of numerous hydrophobic benzyl groups along the polymeric chain, probably organized in hydrophobic patches. This gives the molecule a more rigid and less mobile conformation, reducing its ability to interact freely with water.

In order to improve the mechanical performance of HYAFF Õ 11, a novel composite adopting calcium hydroxyapatite, Ca 10 (PO 4 ) 6 (OH) 2 , (HA) as reinforcement was designed. It is highly biocompatible and shows good bioaffinity by stimulating osteoconduction along its surface and being slowly replaced by host bone post implantation [START_REF] Legeros | Dense Hydroxyapatite[END_REF][START_REF] Ozawa | Evaluation of Implant Materials (Hydroxyapatite, Glass Ceramics, Titanium) in Rat Bone Marrow Stromal Cell Culture[END_REF][START_REF] De Groot | Calcium Phosphate Coatings for Medical Implants[END_REF].

The aim of this study is to develop and characterize this novel composite based on HYAFF Õ 11-HA 14/86 (w/w). Mechanical performance of the material with compressive tests in dry and wet conditions was evaluated. Surface chemical-physical properties, especially topography and stoichiometry composition, were also analyzed.

A preliminary biological examination was also performed to analyze the pH values and the cytotoxicity of the composite extracts after long-term incubation in culture medium. In addition, protein, especially fibronectin adsorption were investigated following incubations in culture medium containing fetal calf serum (FCS) and in human plasma, in order to simulate an in vivo environment.

MATERIALS AND METHODS

Materials

The composites used in this study were obtained using the fully benzylated ester of hyaluronic acid (HYAFF Õ 11, HATBA, batch 0.100.00, average Mw: 200,000 Da; Fidia Advanced Biopolymers (FAB), Albano Terme, Italy) reinforced with 86% (w/w) ratio of HA (batch N1904, particle diameter: 2-10 mm; P205 grade from IRC in Biomedical Material-QMW, London, UK).

A solution of 8% (w/w) HYAFF Õ 11 in dimethylsulfoxide (DMSO, Sigma-Aldrich) was prepared under a slow magnetic stirrer at room temperature (25 AE 2 C) in a glass container. The homogeneous mixture was mixed at room temperature with HA in order to obtain the final composition of HYAFF Õ 11-HA 14/86 (w/w) composite. The composite paste was placed in the selected shapes (discs, 14 mm diameter and 3 mm high, except for mechanical tests) and then washed with ethyl alcohol (95%, Ashland Chemical) for 7 days, three changes a day. The samples were washed with bidistilled H 2 O (Carlo Erba) for 7 days, three changes a day, and then dried under air for 3 days.

HYAFF Õ 11 alone was used as a control for compressive properties and protein analysis, and was prepared as described here.

Mechanical Characterization

All the tests were performed at least in triplicate.

Compressive Properties

Cylindrical specimens (6 mm diameter and 12 mm length) of HYAFF Õ 11 and HYAFF Õ 11-HA composite were tested (INSTRON testing machine model 4204; crosshead speed of 1 mm/min) according to ASTM D 695 in dry and swollen conditions.

Surface Chemical-Physical Characterization

Surface Topography

The samples were sputter coated (Emitech 500, 4 min at 20 mA, 10 À1 mBar) before examination under a Jeol JSM 5500 LV at an accelerating voltage of 15 keV.

Surface Chemistry

The samples were sputter coated (Emitech K550) and then analyzed by energy dispersive atomic X-ray (Philips XL 30; LaB 6 filament; EDAX CDU leap detector) for elemental analysis.

In vitro Biological Assays

Thermanox (TMX) discs (Life Technology) as negative, nontoxic control and polyvinylchloride (PVC) discs (Life Technology) as positive, toxic control were the controls used.

All the tests were performed at least in triplicate.

Elution pH Measures

The samples were placed into a 24-well tissue culture plate and incubated in 1 mL of culture medium at 37 C on a plate shaker. At the selected time points (1, 3, 7, 14, 21, 28, 35, and 42 days) the aqueous extracts were collected and replaced with 1 mL of fresh medium and the plate placed back at 37 C. The pH of the eluted medium was measured using a pH meter (Corning 240) in triplicates.

Elution Cytotoxicity Assays

Primary human osteoblast-like cells (HOBs) were isolated from trabecular bone using a previously described method [START_REF] Di Silvio | Osteoblasts[END_REF]. Briefly, the bone chips were cultured under sterile conditions until 'osteoid seams' were seen, then digested using collagenase (100 U/mL) (Sigma) and trypsin (300 U/mL) (Sigma) solution. Following isolation, expansion, and characterization, the obtained cells were used for the cytotoxicity tests.

The sterile samples were placed into a 24-well tissue culture plate incubated in 1 mL of culture medium at 37 C with 5% CO 2 in a humidified atmosphere. At the selected time points (1, 3, 7, 14, 21, 28 days) the aqueous extracts were collected in sterile condition, replaced with 1 mL of fresh medium and the plate was placed back in the incubator. One hundred microliters of 1 Â 10 5 cell/mL cell suspension were seeded into a sterile 96-well culture plate (Beckton Dickinson) and incubated until almost confluent, usually 24 h. The culture medium was then replaced with aqueous extract (100 mL/well) and the plates were further incubated at 37 C with 5% CO 2 in a humidified atmosphere for 72 and 48 h. Ten microliters of MTT (Sigma) solution (5 mg/mL in phenol-and ascorbic acid-free culture medium containing FCS) was added to each well, the plates were incubated for a further 4 h and then the medium was removed by inversion. One hundred microliters of DMSO (Merck) was added to each well and mixed for 20 min until the crystals were completely dissolved. Absorbance was measured by a Dynatech MR 700 plate reader, using a test wavelength of 570 nm and a reference wavelength of 630 nm.

Protein Adsorption Studies

The sterile samples were placed in a 24-well plate, washed in PBS (Oxoid Ltd) and then equilibrated with 1 mL of FCS-free culture medium for 30 min. The samples were then incubated either in a culture medium containing 10% FCS or in human plasma obtained from healthy human donor blood in static conditions at 37 C for 1 h. The specimens were removed, washed carefully in 1 mL of PBS for 5 min at room temperature, placed in a new 24-well plate and proteins were eluted for 1 h at 37 C by 200 mL of SDS-PAGE sample buffer: (2% (w/v) SDS, 5% (v/v) -mercaptoethanol, 10% (w/v) glycerol, 0.02% (w/v) bromophenol blue in 62.5 mM tris-HCl, pH 6.8). Supernatants were withdrawn and boiled for 3 min. After cooling, 20 mL of each elutions were analyzed by a 10% SDS-PAGE electrophoresis using a Bio-Rad Miniprotean II Module. Electrophoresis was performed at 100 V, 46 mA for 90 min according to Laemmli's method [START_REF] Laemmli | Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4[END_REF]. As controls, 1 : 10 diluted complete DMEM and 1 : 10 diluted human plasma were also used. Protein molecular weight was evaluated by loading SDS-PAGE standard (Bio-Rad, Mw range 205,000-7600 Da: cat. 161-0318). Polyacrylamide gels were stained for 1 h using a Silver Stain Plus Kit (Bio-Rad). Following electrophoresis, proteins were blotted onto nitrocellulose Hybond membranes (Amersham) using a Mini Trans Blot Module (Bio-Rad). Membranes were blocked with 1% (w/v) gelatin in PBS and incubated with 50 mg/mL primary antibody solution containing rabbit antibodies (Dako) against either human or bovine fibronectin (Fbn) overnight at room temperature. Goat anti-rabbit immunoglobulins conjugated with horseradish peroxidase (Dako), diluted 1 : 2000 in the same buffer, were used as secondary antibody. Bands were detected using 0.03% (w/v) 3-amino-9-ethylcarbazole and 0.03% (w/v) H 2 O 2 (Sigma-Aldrich) in 0.1 M sodium acetate (Sigma-Aldrich), pH 5.2, as substrates.

RESULTS

Mechanical Characterization

Compressive Tests

The compressive elastic modulus (E), the maximum compressive strength ( max ) and the maximum deformation (" max ) were evaluated for HYAFF Õ 11-HA composite and HYAFF Õ 11 samples in dry and swollen states (Table 1, Figure 1(a) and (b)).

The results showed an increase in the mechanical properties for the composites due to the reinforcing HA phase. For the HYAFF Õ 11, the fracture was not detectable for the plastic behavior of the materials. The same behaviors were obtained in the swollen state. In the latter case, a decrease in the compressive properties of both HYAFF Õ 11-HA and HYAFF11 HYAFF Õ 11 samples were observed, mainly due to the plasticizing effect of the absorbed water. The water content at the equilibrium state of HYAFF Õ 11 has been reported as 32 AE 2% [START_REF] Netti | Thermodynamics of Water Sorption in Hyaluronic Acid and its Derivative[END_REF] while for the HYAFF Õ 11-HA composite it was, 5 AE 1% due to the presence of HA.

Surface Topography

The samples showed a rough surface consisting of spherulites <10 mm in diameter (Figure 2).

Surface Chemistry

The EDAX analysis of the sample surfaces showed the following elements -Ca, P, C, Na, and Si. The Ca/P atomic ratio value of 1.75, obtained for the composite, was close to both the typical value of stoichiometric HA and the value obtained for the HA used for the test composite preparation. No traces of S were detected (Figure 3).

pH Determination of the Eluted Media

The results obtained showed no significant differences in the pH values of the elution collected, remaining approximately in the range of 7-8 from day 1 to day 42 of sample incubation in the culture medium.

Elution Cytotoxicity Assay

No evidence of cytotoxicity was observed after 48 h (Figure 4(a)) and 72 h (Figure 4

(b)) of incubation with HOB cells in the presence of

Protein Adhesion

A similar protein adsorption profile was observed following incubation of the materials in culture medium containing FCS (Figure 5 205 kDa (arrow 1) as well as protein adsorption in the range of 119-30 kDa (indicated range) and around 7.6 kDa (arrow 2). With the exception of the 205 kDa band, protein adsorption was visible in the above-mentioned ranges also for materials incubated in culture medium containing FCS.

From the immunoblot analysis, fibronectin was undetectable for all the materials incubated in the culture medium containing FCS, but was clearly visible for samples incubated in human plasma (Figure 6).

DISCUSSION

In the present study, mechanical and surface chemical-physical characterization as well as a preliminary biological assessment were performed on a novel HA-reinforced hydrophilic polymer, based on HYAFF Õ 11 polymer matrix, HYAFF Õ 11-HA 14/86 (w/w). Appropriate mechanical properties are crucial requirements in the design of bone-substitute materials. Both HYAFF Õ 11 and HA are known to be bioactive, but when used alone their mechanical properties are not sufficient and hence, limit their use. Improvements in composite technology have resulted in the design of substitute materials with enhanced properties. The compressive tests have shown that it is possible to have a composite structure with compressive properties in the range of that of spongy bone. As presented in Table 1, the composite shows compressive properties in the range of vertebral trabecular bone [START_REF] Morgan | Dependence of Yield Strain of Human Trabecular Bone on Anatomic Site[END_REF]. The higher reduction of the HYAFF Õ 11-HA compressive properties in the swollen state is due to the poor adhesion between HA and HYAFF Õ 11, which is enhanced by the presence of water, and the presence of voids (Figure 2) generated during the preparation.

Surface topography affects important parameters involved in the cell-material interaction. Topography influences cell anchorage, orientation, alignment, and migration by recognizing surface features and reacting to them [START_REF] Curtis | New Depths in Cell Behaviour: Reactions of Cells to Nanotopography[END_REF][START_REF] Curtis | Reaction of Cells to Topography[END_REF]. Rugophilia has also been described to play a role, whereby a roughened surface increases cell, especially, osteoblast response to materials [START_REF] Harris | Development and Characterisation of a Conditionally Immortalised Human Fetal Osteoblastic Cell Line[END_REF][START_REF] Larsson | Bone Response to Surface Modified Titanium Implants with Different Oxide Thickness and Morphology[END_REF].

The HYAFF Õ 11-HA composite structure showed a grainy surface with exposed HA particles, thus indicating a potentially favorable surface topography for cellular adhesion and proliferation.

In addition to topography, surface stoichiometry is another important requirement for biomaterials to promote cellular adhesion. It has been reported that exposing HA increases the osteoconductive properties with material surfaces directly in contact with cells, creating a favorable surface for enhancing osteoblast adhesion [START_REF] Kasemo | Material-Tissue Interfaces: the Role of Surface Properties and Processes[END_REF][START_REF] Kasemo | Surface Science Aspects of Inorganic Biomaterials[END_REF][START_REF] Stephenson | The Effect of Hydroxyapatite Coating on Ingrowth of Bone into Cavities of an Implant[END_REF][START_REF] Ohgushi | Osteogenic Differentiation of Cultured Marrow Stromal Stem Cells on the Surface of Bioactive Glass Ceramics[END_REF][START_REF] Ohgushi | In vitro Bone Formation by Rat Marrow Cell Culture[END_REF][START_REF] Posner | Synthetic Amorphous Calcium Phosphate and its Relation to Bone Mineral Structure[END_REF][START_REF] Dalby | Initial Attachment of Osteoblasts to an Optimised HAPEX Topography[END_REF].

Surface stoichiometry analysis on the test samples showed the presence of the elements C, P, and Ca, the expected elements in the material composition. The detected traces of Na and Si might be due to impurities related to solvents/disposables used during the specimen preparation process or to the sample holders used before/during the EDAX analysis. The absence of S in HYAFF Õ 11-HA composite samples indicated that they were effectively purified from the toxic DMSO used during the composite sample preparation.

The results relating to the Ca/P atomic ratio of the composite sample surface indicated the presence of HA on the surface, suggesting a composite surface microenvironment suitable for HOB cell attachment. EDAX allows the analysis of a sample surface layer that can have a maximum thickness of a few nanometers, so the described results show that the preparation method adopted, phase inversion and casting, and also the high concentration of HA used allow HA particles to be present not only in the bulk but also on the composite surface. It should be noted, however, that the values obtained from the EDAX analysis were semiquantitative. This was a result of the analysis being performed directly on the 'as-cast' sample surface, which was irregular and during the analysis, each point was not equidistant from the EDAX microscope probe. The sample surfaces were not previously treated for smoothing, thus presenting a natural surface similar to that exposed to cells once implanted in vivo.

Cellular response is known to be affected by the release of leached products as a result of the degradation process stimulated upon incubation in an aggressive environment, such as that in the human body. It has been documented in the literature that pH changes of the surrounding environment affect cellular activity [START_REF] Davis | High Performance Biomaterials[END_REF][START_REF] El-Ghannam | A New Bioactive Glass Template for the Synthesis Bone-like Tissue in vitro[END_REF].

The pH measurements of the supernatant collected for the composite for all the selected time points showed pH values ranging from 7 to 8. It has been reported in literature that alkaline environment with pH values above 9 can affect cell activity [START_REF] El-Ghannam | A New Bioactive Glass Template for the Synthesis Bone-like Tissue in vitro[END_REF]. It can be concluded that the reactivity of the material with the surrounding fluids was able to create a favorable microenvironment for cell viability.

Furthermore, from the cytotoxicity tests performed, there was no indication of any considerable deterioration in cell viability as assessed with the long-term eluted extracts. The results showed that there was no release of toxic leachables from the composite eluted over the selected period of time.

In order to analyze the protein pattern on the sample surface more accurately, SDS-PAGE was performed on specimens incubated both in culture medium containing FCS and, to better simulate an in vivo environment, in human plasma. The presence of fibronectin, adhesion protein signalling for HOB cells, was analyzed. Fibronectin, once adsorbed onto a material surface, has been shown to mediate and increase cell adhesion and spreading [START_REF] Curtis | Reaction of Cells to Topography[END_REF].

The SDS-PAGE results showed a similar pattern of protein adsorption for the test HYAFF Õ 11-HA composite and the HYAFF Õ 11 when incubated in culture medium. These observations imply that the cellular response of the composite should be similar to that of the HYAFF Õ 11 control. Although the adsorption of proteins from medium supplemented with FCS was, as expected, lower than the clinically reflective incubation in plasma, the pattern of adsorption was not significantly different, suggesting a similar behavior of the composite when placed in an in vivo environment. This hypothesis is correlated by the immunoblotting of fibronectin. Fibronectin was not detected for the samples incubated in the culture medium containing FCS and for the culture medium itself, probably as a result of the dilution of FCS concentration in the culture medium to a level undetectable for Western Blot. It was however, clearly visible in those specimens incubated in human plasma.

CONCLUSIONS

Mechanical analysis showed that the presence of the HA improved the compressive properties of the HYAFF Õ 11 matrix both in the dry and the wet states, with values in the range of that of spongy bone.

HYAFF Õ 11-HA 14/86 (w/w) composite sample surface analysis showed a grainy topography, the presence of C, P, and Ca with small traces of Na and Si impurities, and a Ca/P atomic ratio close to the typical value of stoichiometric HA and to the value obtained for the HA used in sample preparation.

The in vitro cytotoxicity tests showed no evidence of toxic leachables from the composites and acceptable pH values for cell survival and proliferation.

Protein adhesion analysis showed a similar protein pattern in culture medium with FCS and human plasma; with a higher amount of fibronectin detected in the latter.

In conclusion, these results indicate that the novel HYAFF Õ 11-HA composite has potentials for a wider range of application in the field of orthopedics, especially as vertebral trabecular bone substitute.

  Characterization and Assessment of HYAFF Õ 11-HA Composite sample extracts at the selected time points, as shown by the comparison with the TMX and PVC control extracts.

  (a)) and human plasma (Figure 5(b)), respectively. The SDS-PAGE for the HYAFF Õ 11-HA specimen and the HYAFF Õ 11 samples, incubated in human plasma showed a band with molecular weight around

Figure 1 .

 1 Figure 1. Compressive stress-strain curve of HYAFF Õ 11-HA and HYAFF Õ 11, in (a) dry and (b) swollen states.

Figure 2 .

 2 Figure 2. Scanning electron micrograph of HYAFF Õ 11-HA composite sample surface.

Figure 3 .

 3 Figure 3. EDAX analysis of HYAFF Õ 11-HA composite sample surface.

Figure 4 .

 4 Figure 4. MTT test on HOB cells cultured (a) for 48 and (b) 72 h in culture medium previously incubated for 1, 3, 7, 14, 21, and 28 days with HYAFF Õ 11-HA composite samples (C).

Figure 5 .

 5 Figure 5. SDS-PAGE of samples incubated (a) in culture medium and (b) human plasma: lane 1: molecular weight standard; lane 2: HYAFF Õ 11-HA composite; lane 3: HYAFF Õ 11; lane 4: relative incubation medium. Arrows and arrow range indicate protein with a different adsorption profile on the tested surfaces (for details see text).

Figure 6 .

 6 Figure 6. Immunoblot of samples incubated in culture medium (lane 1: HYAFF Õ 11-HA composite; lane 3: HYAFF Õ 11; lane 5: culture medium) and human plasma (lane 2: HYAFF Õ 11-HA composite; lane 4: HYAFF Õ 11; line 6: human plasma).

Table 1 .

 1 Compressive properties of HYAFF Õ 11-HA and HYAFF Õ 11 in dry and swollen states.

		E (MPa)	c (MPa)	" (mm/mm)
	Material (Dry)
	HYAFF Õ 11	27 AE 5	-	-
	HYAFF Õ 11-HA (14/86)	340 AE 40	12 AE 1	0.24 AE 0.01
	Material (Swollen)
	HYAFF Õ 11	10 AE 2	-	-
	HYAFF Õ 11-HA (14/86)	70 AE 4	4 AE 1	0.31 AE 0.01

Characterization and Assessment of HYAFF Õ 11-HA Composite

ACKNOWLEDGMENTS

The authors wish to thank Neelam Gurav, Caroline Clifford, and Michael Kayser from Institute of Orthopaedics, UCL, London, UK and Matthew Dalby, University of Glasgow, UK, for their expertise and contribution to the present work, and the E.C. 5th FW Project: Novel Intervertebral Disc Prostheses, Contract No. G5RD-CT-2000-00267 for financial support.