Hiroyoshi Yamasaki

Jérôme Louradour

Julie Hunter

Laurent Prévot

TRANSCRIBING AND ALIGNING CONVERSATIONAL SPEECH: A HYBRID PIPELINE APPLIED TO FRENCH CONVERSATIONS

Keywords: ASR, Transformer, French, Conversation

With the advent of transformer based models, the use of fully automated ASR-pipelines in a real-world context has come close to a reality. However, for faithful transcription of conversational speech, there remain challenges both in terms of the content predicted by these models (hallucinations, unintended normalizations of disfluencies and transcriptions of background noises) and in terms of alignment accuracy. In this paper we present a hybrid ASR-pipeline which augments transformer models with other algorithms in order to transcribe conversational data. Through experiments on two French datasets, we show that: 1) VAD preprocessing can significantly improve transcription quality as well as word level temporal alignment, 2) prompting can reduce unintended normalizations of disfluencies, 3) heuristic-based detection of untranscribed sounds can further improve alignment quality. We conclude that our hybrid pipeline is an efficient way to improve and augment existing ASR-models.

I. INTRODUCTION

While automatic speech recognition (ASR) has steadily improved over the past few decades for a variety of tasks and domains, conversational speech has remained a challenge. Until recently, word error rate (WER) had been so high that for tasks requiring accurate transcripts, manual transcription was often more efficient than correcting ASR output. The situation changed dramatically with the introduction of Whisper [START_REF] Radford | Robust speech recognition via large-scale weak supervision[END_REF], a transformer-based, end-to-end model trained on massive amounts of speech data. Other models followed, including models supporting additional languages [START_REF] Pratap | Scaling speech technology to 1,000+ languages[END_REF], [START_REF] Zhang | Google usm: Scaling automatic speech recognition beyond 100 languages[END_REF], and diverse extensions of the Whisper model [START_REF] Klein | Faster whisper transcription with ctranslate2[END_REF], [START_REF] Bain | Whisperx: Time-accurate speech transcription of long-form audio[END_REF]. This significant improvement in ASR models promises to greatly facilitate the production of conversational datasets, eliminating the central bottleneck for downstream linguistic study and modeling of conversational speech. Still, applying Whisper to conversational corpora presents certain challenges: Whisper (i) easily registers and transcribes background voices in cases where speakers have individual WE GRATEFULLY ACKNOWLEDGE SUPPORT FROM THE ANR-FUNDED PROJECT, SUMM-RE (ANR-20-CE23-0017) microphones but are in the same room (the standard scenario for conversational corpora such as ICSI [START_REF] Janin | The ICSI meeting corpus[END_REF], AMI [START_REF] Carletta | The AMI meeting corpus: A preannouncement[END_REF] and CID [START_REF] Blache | The corpus of interactional data: A large multimodal annotated resource[END_REF]), (ii) hallucinates content (inserts words not in the audio signal), (iii) frequently omits disfluencies and discourse markers, which are common in spontaneous speech, and (iv) either lacks word-level alignment (original model) or struggles with alignment when there are untranscribed disfluencies or other noises (more recent implementations).

In this paper, we present a pipeline designed to address the above challenges and facilitate transcription of conversational speech with a model like Whisper. We illustrate the impact of our pipeline on two datasets in French, a language which is well represented in the multilingual models but without large manually transcribed or corrected conversational corpora available for developing and training conversational models like AMI [START_REF] Carletta | The AMI meeting corpus: A preannouncement[END_REF] or Switchboard [START_REF] Godfrey | Switchboard: Telephone speech corpus for research and development[END_REF] for English. We hope this will open the door to the development of further conversational corpora and benchmarks in a wide variety of languages.

II. DATASET DESCRIPTION

We evaluate our pipeline on two French datasets: the Corpus of Interactional Data (CID) [START_REF] Blache | The corpus of interactional data: A large multimodal annotated resource[END_REF] and the newly created SUMM-RE corpus. CID features eight 1-hour long conversations between two friends talking about a given topic but without other constraints. SUMM-RE includes 300 roughly 20-minute conversations between 3-4 speakers, most of whom did not know each other before recording. 1 Participants in SUMM-RE were instructed to enter into a loosely guided role-play to simulate meeting-style interactions. Both corpora contain highly spontaneous interactional speech with a high rate of disfluencies [START_REF] Shriberg | Preliminaries to a theory of speech disfluencies[END_REF] and a complex mix of monologic (e.g., telling a story) and dialogic (e.g., negotiating the next topic to address) contributions.

CID was manually transcribed in its entirety and doublechecked. In addition, each transcript was manually segmented based on silences, yielding gold timestamps for the beginning and end of each speech segment. While most SUMM-RE transcripts have been produced automatically 1 While most recordings were made in a studio, Covid restrictions forced us to collect some data (<20%) via Zoom. Table I. Basic statistics for our two datasets using the pipeline described in this paper, a small subset of 10 meetings, SUMM-RE-sm, has been manually corrected at both the transcription and segment-timestamp level. Transcription and correction for both corpora were carried out with the Praat phonetic annotation tool [START_REF] Boersma | Speak and unspeak with praat[END_REF]. A summary of the data sets is provided in Table I. We will release SUMM-RE-sm with the paper and the rest of the SUMM-RE dataset at the end of the associated research project.

III. CHALLENGES

Background voices While participants in both CID and SUMM-RE wore individual headsets, their microphones often picked up the voices of other speakers when the participants were all in the same room and the ASR system thus transcribed the words of secondary speakers alongside those of the main speaker. The first challenge that we faced was to isolate the utterances of the main speaker in each individual speaker recording.

Hallucinations Like other transformer-based models, Whisper has a tendency to hallucinate. We found, for instance, numerous cases in which Whisper predicts "soustitres réalisés par [x].org" ("subtitles created by [x].org"). Other types of hallucinations involve getting stuck in loops of strings of words or just more standard cases of transcribing words that were not uttered in the conversation. Our second challenge was thus to find ways to keep Whisper from hallucinating.

Transcript "cleaning" Spontaneous conversation contains disfluencies and conversation-specific uses of discourse markers, as illustrated by (1-a):

(1) a. donc euh on a euh enfin j'ai contacté euh notre fournisseur so um we have um well I have contacted um our supplier b. j'ai contacté notre fournisseur I have contacted our supplier However, as shown in (1-b), Whisper has a tendency to delete discourse markers (in cyan) and disfluencies, including filled pauses (in blue) and fragments (in orange).

This not only has a negative impact on the evaluation of ASR output (by increasing deletions and complicating wordlevel alignment) but also removes information valuable for downstream linguistic tasks requiring richer representations of the dialogue dynamics. Our third challenge was therefore to find ways to help Whisper more faithfully transcribe conversational speech.

Signal alignment Precise word-level2 timestamps are useful for a variety of downstream tasks that require multimodal, text/audio representations, including discourse segmentation and dialogue act tagging. The original Whisper model, however, only provides timestamps for speech segments. And more recent implementations that add word-level timestamps struggle when there are untranscribed noises in the signal. Omitted disfluencies and discourse markers, for example, complicate the the idenification of timestamps for nearby words. Our fourth challenge, then, was to develop a means of accurately predicting word-level timestamps.

IV. PIPELINE

At a high level, the first part of our pipeline is designed to isolate the contributions of the main speaker in order to reduce the impact of both background voices and silences, which are correlated with hallucinations, on ASR quality. The next part focuses on ASR, exploiting three important additions to the original Whisper model: a prompt that encourages Whisper to transcribe disfluencies and discourse markers, word-level timestamps, and a method for mitigating the impact of untranscribed words and sounds on wordlevel alignment. Motivation for our pipeline choices comes from tests on CID and SUMM-RE-sm, but also on a small subset of SUMM-RE-sm consisting of roughly 60-90 second extracts of 10 of the files from SUMM-RE-sm. This smaller subset was used to reduce the search space for what we wanted to explore in more depth on the larger datasets.

IV-A. Isolating the main speaker

To isolate contributions from the main speaker, we tested three approaches: (i) inter-pausal-unit (IPU) detection using the SPPAS annotation toolkit [START_REF] Bigi | Sppas-multi-lingual approaches to the automatic annotation of speech[END_REF], (ii) repurposing the Pyannote speaker diarization model [START_REF] Bredin | pyannote.audio: neural building blocks for speaker diarization[END_REF], [START_REF] Bredin | End-to-end speaker segmentation for overlap-aware resegmentation[END_REF] for main speaker detection, and finally, (iii) combining SPPAS and Pyannote.

The SPPAS IPU-detection algorithm [START_REF] Bigi | Search for interpausal units: application to cheese! corpus[END_REF] allows for an optimized threshold value for each file. First, it calculates the root-mean-square (RMS) of the intensity inside a sliding time window of duration 20 ms and then calculates the threshold value Θ = min +µ -1.5σ where min is the minimum, µ is the mean, and σ is the standard deviation for RMS values. Once Θ is calculated, IPU intervals are determined to be those that exceed both Θ and a specified minimum duration (500ms in our case). Silences longer than a given length (100ms in our case) and IPUs that fall under the minimum are treated as silences.

The second approach uses Pyannote to predict speaker turns and labels. We identify the loudest speaker as the main speaker, except in two types of cases that complicate this assumption. First, background speakers sometimes make short but very loud utterances. We tried to eliminate these by applying a minimum duration of 0.3 seconds. Second, all speakers with a relative intensity above a given threshold (0.8 dBFS) were taken as candidates for main speaker, but sometimes, a single speaker could be assigned two different labels. To remove these cases, we added a secondary filter with intensity threshold of 0.5 dBFS and duration threshold of 0.2 seconds. For the current data set we additionally performed manual verification/correction of the main speaker assignment (2 files out of 39 modified).

Our third approach exploits the complementary benefits of SPPAS and Pyannote. For each case in which SPPAS predicts an interval that overlaps an interval predicted by Pyannote, we take the IPU to be the maximum interval on which the models agree. This approach shows significantly fewer false positives than either system alone, where errors in SPPAS are due to background voices with an intensity too close to that of the main speaker and false positives in Pyannote are linked to similarity in voice quality (e.g., similar age and gender, etc.). 3

IV-B. Passing segments to Whisper

As preliminary investigations showed that hallucinations are often triggered by silences or non-speech sounds, we decided to feed Whisper segments that had been separately identified as speech (either IPUs or segments selected by a VAD). While such segments can be processed independently by Whisper, we found that it was more efficient to first glue together the relevant segments of audio, separated by intervals of silence, and then pass the whole audio to Whisper (later recovering the original timestamps). We also applied post-processing to remove non-Latin characters, emojis, URLs and any remaining sentences about subtitles.

IV-C. Prompting Whisper

Like other transformer-based models, Whisper can condition its prediction on a given prompt. As it has the particularity of chunking audio files into 30-second segments for transcription, we developed a customized prompt and injected it for the first chunk and then continually reinjected it for the other chunks to maintain its effect throughout the audio. We tried dozens of different prompts that illustrated a variety of disfluencies in order to encourage Whisper to output the disfluencies that were present in the audio signal. The following prompt, involving discourse markers, repetitions, and filled pauses, was the most successful: Bon. Ben je crois euh je vois ce que euh tu veux dire. Hum tu tu tu euh ben tu me diras. 4Hum tu tu tu euh ben tu me diras. Um you you you uh well you'll tell me (literally: you me will tell)

IV-D. Signal alignment

Several strategies are possible to recover plausible wordlevel timestamps. We considered three:

1) Using the external Wav2Vec2 [START_REF] Baevski | wav2vec 2.0: A framework for self-supervised learning of speech representations[END_REF] model that predicts character probabilities for each speech frame, and performing an alignment based on Dynamic Time Warping (DTW) [START_REF] Giorgino | Computing and visualizing dynamic time warping alignments in R: The dtw package[END_REF] on these probabilities for the characters predicted by Whisper. This is the approach adopted by WhisperX [START_REF] Bain | Whisperx: Time-accurate speech transcription of long-form audio[END_REF]. 2) Performing DTW on combined cross-attention weights (of the decoder attending to the encoded speech signal) for each predicted token. This is the approach adopted by Whisper-Timestamped [START_REF] Louradour | whisper-timestamped[END_REF], Faster Whisper [START_REF] Klein | Faster whisper transcription with ctranslate2[END_REF] and OpenAI-Whisper [START_REF] Radford | Robust speech recognition via large-scale weak supervision[END_REF]. 3) Analyzing the probability distribution over timestamps at the end of each word, as in [START_REF] Jianfch | Stabilizing timestamps for whisper[END_REF], [START_REF] Gerganov | Whisper.cpp[END_REF]. Preliminary experiments showed that (3) produces very inaccurate timestamps. We therefore restricted our attention in subsequent experiments to the systems using (1) or [START_REF] Pratap | Scaling speech technology to 1,000+ languages[END_REF].

Even when using the prompt described above in IV-C, untranscribed disfluencies remain. These together with other non-transcribed acoustic noises degrade the prediction of the timestamps for nearby words that are successfully transcribed. Based on the observation that combined crossattention weights often form a concave curve around the prediction of each transcribed token, Whisper-timestamped [START_REF] Louradour | whisper-timestamped[END_REF] proposes an option to make timestamps more accurate around untranscribed disfluencies and other noises. The heuristics consist in applying a peak detection on the portion of cross-attention weights that correspond to the first token of each word. If several peaks are detected in the vicinity of a single token, the region corresponding to the first peaks is marked as silence, and the start of the last peak is chosen as the start of the word. We adopt this approach to further refine predictions of start timestamps.

V. EVALUATION METRICS

In our experiments, described in Section VI, we evaluated transcription accuracy in terms of Word Error Rate (WER), and its decomposition into Deletion (Del), Insertion (Ins) and Substitution (Sub) rates. We also used the two following metrics to take into account timestamp predictions.

V-A. F1-score

To measure both word errors and timestamp accuracy with a single metric, we use the harmonic mean of precision and recall metrics introduced in [START_REF] Bain | Whisperx: Time-accurate speech transcription of long-form audio[END_REF]. For each word in the ground truth transcript, we extend the interval determined by its start and end time by adding a collar of 20 ms 5 before and after it. If the model correctly predicts the word and predicts that the word overlaps this extended gold interval, then it is considered a true positive. If the word falls entirely out of the expanded window or if the model fails to predict the word, it is classified as a false negative. Any word predicted by the ASR and for which there is no corresponding word in the gold transcript is counted as a false positive. 6

V-B. Average timestamp difference T-δ

To evaluate the quality of word timestamps, we first identified the set of words that were correctly predicted. Using DTW [START_REF] Giorgino | Computing and visualizing dynamic time warping alignments in R: The dtw package[END_REF], we aligned the tokens based on the distance between timestamped words, which we calculated as the sum of the Levenshtein edit distance and the absolute difference between start/end timestamps (in seconds). We found that using DTW with such a hybrid distance calculation is more robust than a simple Levenshtein alignment, for instance in cases where some words are repeated but the repetition only appears in the ground truth or in the prediction.

We then calculated the absolute difference between the predicted start/end time and the real start/end time for each of these correctly predicted words. The average timestamp difference, T-δ, is simply the average of these values.

VI. RESULTS

Table II shows, in the form of an ablation study, the results obtained from testing our pipeline on CID and SUMM-RE-sm. The systems we compare all use Whisper large-v2 as the core model. Our reference system uses IPUs obtained by combining SPPAS and Pyannote predictions (cf. Section IV-A) and then glued together as described in IV-B. It also uses the custom prompt introduced in IV-C, disfluency detection heuristics (cf. IV-D) and then Whisper-Timestamped as the ASR implementation.

Results of Table II:A show a clear reduction in insertions when Whisper receives information on IPUs (Reference) relative to the case where Whisper receives no information on speech segments (No VAD). We also see a notable improvement with the state of the art VAD Silero [START_REF] Silero | Silero vad: pre-trained enterprise-grade voice activity detector[END_REF], further underscoring the value of speech segments.

Insertions can be due to a) transcription of background speaker voices or b) hallucinations from Whisper. To distinguish these possibilities, we used gold IPUs to identify intervals of background speech and intervals of silences, as the latter are associated with hallucinations. We then used these intervals to filter (i.e., remove words from) transcripts that had been produced in three different settings: (1) without any VAD, (2) with the VAD Silero, and (3) with our pipeline. In particular, we compare results when (i) background voices are filtered out but silences remain, and (ii) both background voices and silences are filtered, leaving only the main speaker contributions. Figure 1 shows that (i) leads to a significant decrease in insertions when no particular attempt is made to isolate main speaker contributions (settings ( 1) and ( 2)), suggesting that most insertions can be traced to background speakers. Still, the reduction of insertions in these settings when we additionally filter silences suggests that a portion of the errors come from hallucinations.

Table II:B shows that using our custom prompt (cf. IV-C) significantly reduces the number of deletions, as more disfluencies and discourse markers are transcribed. It also slightly increases the insertion rate, but Figure 1 (right column) shows that this is not due to an increase in hallucinations. Whisper also offers the possibility of conditioning its prediction for a given chunk on that of the previous chunk. Table II:B shows that considering the previous chunk (previous) slightly lowers performance on all metrics except insertions, where we see only a subtle improvement.

Heuristics on cross-attention weights to refine the start times of words uttered after untranscribed sounds greatly improve the accuracy of T-δ, while they have no influence on other metrics. Using no prompt and no conditioning on the previous chunk slightly improves T-δ. This is because words recovered by use of our custom prompt are harder to align, which calls into question the reliability of T-δ when comparing models with different WER.

In terms of ASR implementation (Table II:C), WhisperX outperforms others in WER while Whisper-Timestamped has the best T-δ and overall F1-score. Faster Whisper has better T-δ than WhisperX but a poor WER. Differences in WER are most likely due to details relating to decoding heuristics.

We also tested the impact of applying IPUs as a postprocessing step to filter out words from ASR transcripts, instead of providing them as input to Whisper. Taking Silero (represented with (✻) in Table II:D) as the only VAD during pre-processing, we tested IPUs predicted by (i) SPPAS, (ii) Pyannote and (iii) our combined approach. We also checked (iv) filtering with combined IPUs after preprocessing with combined IPUs. To see if we could further improve alignment, we evaluated the impact of (v): supplementing case (iv) with a dedicated alignment tool, Julius [START_REF] Lee | Julius-an open source real-time large vocabulary recognition engine[END_REF]. We chose the SPPAS [START_REF] Bigi | Sppas-multi-lingual approaches to the automatic annotation of speech[END_REF] wrapper for Julius and an alignment model that had been trained on conversational French. Table II:D shows that using combined IPU after Silero yields a lower WER than using SPPAS or Pyannote alone. Using combined IPU post-processing does not lead to significant differences, suggesting that IPUs can be applied before or after the ASR model. Forced alignment with Julius also failed to improve performance.

VII. DISCUSSION

In our experiments, accurately detecting the main speaker had the greatest effect on both transcription accuracy and word-level alignment for all models. This was particularly evident on SUMM-RE, most likely because background speakers were more active than in dyadic CID. Postprocessing experiments suggest that this improvement holds regardless of whether IPUs are given as input to the ASR system or applied as filtering after the fact. The use of a custom prompt significantly reduces deletions, and heuristics for dealing with untranscribed noises clearly improve alignment. Noteworthy is the very high WER for out-of-the-box models suggesting that mainstream systems are not suitable for meetings and other natural, conversational data without modification. Our results show that various strategies and heuristics can be applied to enhance state of the art models.

VIII. CONCLUSION AND FUTURE WORK

The pipeline described in this paper is designed to address certain challenges that arise when applying high-performance, transformer-based ASR models to conversational speech. Our pipeline first exploits a combination of an intensity-based VAD and a slightly modified speaker diarization model in order to isolate the conversational contributions of the main speaker in a situation in which speakers have individual microphones. We show that this step allows for a significant reduction in insertions, which can be traced to background voices and hallucinations, and overall improvement in WER, F1-scores and word alignment. Our pipeline then employs a prompt that leads to improvements on ASR metrics by helping the model to predict disfluencies and discourse markers in the audio signal. Finally, we apply additional heuristics to reduce the impact of untranscribed words and other noises on word-level alignment.

Our approach is fairly generic and can be applied to any language. Moreover, the modular nature of our pipeline allows for a flexible combination of various components (ASR, IPU-detection, alignment) as new and improved models become available. Thus, the pipeline can be constantly improved even as the individual models become obsolete.

Future work could consider laughs, coughs and other paralinguistic sounds that are not currently addressed by our pipeline. These signals are not only of interest to researchers of human communication but may also confound ASR models and lead to erroneous transcriptions. Addressing this issue could thus further improve the performance.

Fig. 1 .

 1 Fig. 1. WER details for different pipeline settings and with different types of filtering for the predicted words.

Table II .

 II Evaluation of different ASR pipelines with word-level timestamps for SUMM-RE-sm and CID.

	(A) Speech detection
	Silero (✻)	0.63	117	78.4	5.1	67.1	6.3	0.74	82	40.8	9.7	22.7	8.4
	No VAD	0.49	196	133.0	4.3	119.1	9.6	0.68	101	52.6	9.3	34.3	9.1
	(B) ASR details
	No prompt & no previous	0.80	94	23.6	14.1	4.7	4.8	0.77	71	29.1	20.9	2.2	5.9
	No prompt + previous	0.77	114	24.2	15.0	4.0	5.2	0.70	114	32.3	24.3	2.1	5.9
	No heuristics	0.82	204	18.8	8.1	5.8	4.8	0.81	105	22.9	13.5	3.0	6.4
	(C) ASR implementation
	WhisperX	0.80	157	18.3	7.9	6.1	4.3	0.80	90	21.4	13.3	3.5	5.7
	Faster Whisper	0.80	120	24.0	15.1	4.3	4.7	0.77	82	30.0	22.2	1.7	6.1
	(D) Post-processing
	Filter: (✻) SPPAS IPU	0.72	109	44.8	6.9	31.8	6.0	0.79	76	27.6	11.4	8.1	8.1
	Filter: (✻) Pyannote IPU	0.79	108	26.4	8.2	12.6	5.6	0.80	78	24.8	13.5	4.2	7.0
	Filter: (✻) combined IPU	0.81	105	21.5	8.8	7.3	5.3	0.80	76	23.8	14.0	3.0	6.8
	Filter: combined IPU	0.81	107	19.2	9.0	5.4	4.9	0.81	77	23.5	14.5	2.8	6.2
	Combined IPU + Julius	0.72	113	18.8	8.3	5.7	4.8	0.72	82	22.8	13.9	2.6	6.4

For "word"-level timestamps, we ignore here the difference between words and contractions of words such as "j'ai" (I've).

Note that as Pyannote detects mainly speech, non-speech sounds such as laughs, coughs etc. are not recognized by this approach, although they are generally included in conversation/dialogue-processing pipelines.

The prompt can be translated as follows: Bon. Ben je crois euh je vois ce que euh tu veux dire. Well. Well I think um I see what um you mean. (literally: what you want to say)

The original paper used 200ms, but a higher degree of precision is needed for downstream applications exploiting both signal and transcripts.

This metric does not measure a model's capacity for recognizing zones of silence, as there is no penalty for predicting that a word not only overlaps the desired (gold) interval but also covers the silences around that word.

IX. ACKNOWLEDGEMENTS

We would like to thank Roxane Bertrand for manually correcting transcripts and Océane Granier for correcting transcripts and collecting the original data.


MD Francesco Innocente

PhD Delia Mandracchia

MD Erman Pektok

PhD; Benjamin Nottelet

MD Jean-Christophe Tille

Sarra De Valence

MSc Giuseppe Faggian

MD Alessandro Mazzucco

MD Afksendiyos Kalangos

FECTS Robert Gurny

PhD ; Sarra De Valence

PhD Michael Moeller

MD, FAHA, Director, Beat H Walpoth
email: beat.walpoth@hcuge.ch

Paclitaxel-eluting biodegradable synthetic vascular prostheses: a step towards reduction of neointima formation?

Keywords:

procedures are required, partly because of aging of the population. [START_REF] Ounpuu | The impending global epidemic of cardiovascular diseases[END_REF] Up to now, blood vessels are replaced either by autologous veins or arteries or by biostable synthetic vascular grafts such as expanded polytetrafluoroethylene (e-PTFE) and polyethylene terephthalate (Dacron). These synthetic grafts show very good properties to replace large diameter vessels, but they lead to specific problems for small diameters (Ͻ6 mm) such as coronary artery bypass grafts. [START_REF] Teebken | Tissue engineering of small diameter vascular grafts[END_REF] In particular, they are prone to acute thrombosis when associated with reduced flow, which can lead to graft occlusion with fatal clinical outcome. Furthermore, their inadequate mechanical properties (compliance mismatch) and lack of biocompatibility (no endothelium) are responsible for the appearance of intimal hyperplasia, leading to a thickening of the arterial wall, mainly at the anastomoses, and a narrowing of the lumen. [START_REF] Pawlowski | Endothelial cell seeding of polymeric vascular grafts[END_REF] It is therefore clear that novel small-diameter vascular grafts with an acceptable clinical outcome are needed.

We recently proposed an optimized small-diameter degradable vascular graft based on poly(-caprolactone) (PCL) nano-and microfibres suitable for in vivo vascular regeneration. [START_REF] Nottelet | Factorial design optimization and in vivo feasibility of poly(caprolactone)-micro-and nanofiber-based small diameter vascular grafts[END_REF][START_REF] Pektok | Degradation and healing characteristics of small-diameter poly(-caprolactone) vascular grafts in the rat systemic arterial circulation[END_REF] Using the advantages of the electrospinning technique, which allows the preparation of extracellular matrixlike scaffolds, the slow degradation rate of PCL, and its better mechanical properties compared to other described synthetic or natural polymers, [START_REF] Nair | Biodegradable polymers as biomaterials[END_REF] we showed that the PCL grafts were successful as abdominal aorta replacements in rats up to 6 months. [START_REF] Pektok | Degradation and healing characteristics of small-diameter poly(-caprolactone) vascular grafts in the rat systemic arterial circulation[END_REF] They were fully patent at 24 weeks with no thrombosis, stenosis, or aneurysmal dilatation, and histological analyses revealed a homogeneous cellular infiltration associated with polymer degradation and extracellular matrix formation, as well as a rapid and complete endothelialization, though some neointima formation was observed. Nevertheless, this was significantly less pronounced than for ePTFE grafts. Yet it would be interesting to minimize neointima formation applying similar drug-eluting PCL grafts, which deliver an antiproliferative drug in situ.

Recently, stents coated with slow-releasing paclitaxel (PTX) have been used successfully in the experimental model and in patients to reduce in-stent restenosis by inhibiting the cellular proliferation attributable to high local drug concentration with minimal systemic side effects. [START_REF] Hong | Paclitaxelcoated Gianturco-Roubin (R) IIAl (GR (R) II) stents reduce neointimal hyperplasia in a porcine coronary in-stent restenosis model[END_REF][START_REF] Scho ¨mig | A meta-analysis of 16 randomized trials of sirolimus-eluting stents versus paclitaxel-eluting stents in patients with coronary artery disease[END_REF] Drug incorporation in fibers using standard or coaxial electrospinning of drug-loaded solutions have been described for various compounds and is promising for controlled drug delivery applications related to tissue engineering. Some groups prepared PTX-eluting fibers for biomedical applications based on PLA [START_REF] Zeng | Biodegradable electrospun fibers for drug delivery[END_REF] or PLGA. [START_REF] Xie | Electrospun micro-and nanofibers for sustained delivery of paclitaxel to treat C6 glioma in vitro[END_REF] However, recent reports have shown that these polymers do not possess the adequate mechanical properties to be used as vascular grafts in the systemic circulation. [START_REF] Walpoth | Biodegradable small calibre polydioxanone-based vascular prostheses: Potential as coronary bypass grafts[END_REF] Hence, our aim was to prepare and assess in vitro and in vivo, a PTX-eluting biodegradable vascular graft, which would combine the positive findings of our previously described PCL grafts [START_REF] Pektok | Degradation and healing characteristics of small-diameter poly(-caprolactone) vascular grafts in the rat systemic arterial circulation[END_REF] with the ability of the drug to prevent intimal hyperplasia formation.

Materials and Methods

In Vitro Studies

Preparation of Unloaded and PTX-Loaded Vascular Grafts by Electrospinning

Preparation of 2-mm inner diameter PCL grafts after an optimization process by a factorial design approach and the in vitro and in vivo assessments have been reported in detail elsewhere. [START_REF] Nottelet | Factorial design optimization and in vivo feasibility of poly(caprolactone)-micro-and nanofiber-based small diameter vascular grafts[END_REF][START_REF] Pektok | Degradation and healing characteristics of small-diameter poly(-caprolactone) vascular grafts in the rat systemic arterial circulation[END_REF] In a similar factorial design approach, various optimized PTX-loaded vascular grafts (PCL-PTX) were prepared, and the effect of different PTX concentrations on fiber morphology, graft tensile stress, and strain behaviors were investigated. The PTX-loaded vascular grafts were prepared like the nonloaded controls by electrospinning of PCL solutions (7.5, 9 and 12% ͓wt/vol͔) containing different amounts of PTX (0.25, 0.5, 0.75 and 1% ͓wt/wt͔). The generated fibers were collected on a rotating (4500 rpm) and translating (200 rpm, 4-cm amplitude) mandrel to form a nonwoven tubular PCL graft. The grafts were dried at 37°C under vacuum overnight. Residual solvents were analyzed with a Headspace Sampler (HP7694, Hewlett-Packard) coupled to a gas-chromatography system (GC System, 6850 Series, Agilent Technologies) equipped with an HP-1 capillary column (Agilent Technologies). All vascular grafts were ␥-sterilized at 25kGy before characterization and in vivo use.

Assessment of PTX Loading and Distribution in the Vascular Graft

The extent of drug loading and the incorporation efficiency were assessed by high performance liquid chromatography (HPLC) analysis on 2.5-cm long PTX-loaded vascular grafts with theoretical loadings of 0.25%, 0.5%, 0.75%, and 1% (wt/wt). Additionally, the homogeneity of PTX distribution along the vascular grafts (nϭ3) was verified by cutting 4 1-cm-long pieces from a 4-cm PTX-loaded graft, theoretically loaded with 0.5% and 0.75% (wt/wt).

PTX Release Studies In Vitro

Vascular grafts theoretically loaded with 0.5%, 0.75%, and 1% of PTX (nϭ3 for each graft type) were placed in vials with a measured amount of release medium of phosphate buffered saline (pHϭ7.4) containing 0.05% (vol/vol) Tween 20. The vials were kept at 37°C with rotational mixing at 100 rpm for 1, 5, and 16 hours and 1, 6, 8, 12, 14, 16, 20, and 27 days. At each time point, 1-mL aliquots of the release medium were taken for PTX quantification. The remaining release medium was aspirated and fresh release medium was added between each time point to maintain sink conditions.

Mechanical and Morphological Properties Evaluations

Fiber diameter and morphology of the grafts were investigated using a scanning-electron microscope (SEM; JEOL JSM-6400, Jeol Ltd). Tensile stress and strain were measured on complete vascular grafts using a mechanical testing bench from Schenck AG (Na ¨nikon, Switzerland) with a cross-head speed of 10 mm/min at room temperature.

In Vivo Studies

The PCL-PTX graft chosen for in vivo evaluation was made from a 12% w:v PCL solution with PTX drug-loading of 0.75% because it showed similar fiber morphology and mechanical properties to plain PCL grafts used previously. [START_REF] Nottelet | Factorial design optimization and in vivo feasibility of poly(caprolactone)-micro-and nanofiber-based small diameter vascular grafts[END_REF][START_REF] Pektok | Degradation and healing characteristics of small-diameter poly(-caprolactone) vascular grafts in the rat systemic arterial circulation[END_REF]

Experimental Rat Model

Our experimental rat model, including anesthesia, surgery, and the follow-up protocols for digital substraction angiography (DSA) imaging and sampling of the vascular graft, was described in details previously. [START_REF] Pektok | Degradation and healing characteristics of small-diameter poly(-caprolactone) vascular grafts in the rat systemic arterial circulation[END_REF] Briefly, 18 male Sprague-Dawley rats (375 g) were anesthetized and operated according to this protocol (Janvier S.A.S., Le Genest-St-Isle, France). PCL (nϭ9) and PCL-PTX (nϭ9) grafts were implanted randomly into the abdominal aorta of the rats and follow-up times were 3, 12, and 24 weeks (nϭ3 for each time point in each group). No anticoagulation or antiplatelet drugs were given. At the conclusion of the study period, DSA images were obtained after blood sampling for PTX blood levels, and rats were euthanized after explantation of the vascular graft with the adjacent abdominal aorta segments.

Histological Assessment and Morphometric Analysis

For histological investigations, explanted grafts with both anastomoses were fixed in 4% formaldehyde for 24 hours, cut into 2 longitudinal halves, and then embedded into paraffin. Histological sections of 4 m were stained with Hematoxylin-Eosin (H&E) for cellular ingrowth, neointima formation, and tissue reaction, Miller and Masson for elastin fibers and collagen deposition, and immunohistochemistry with an anti-CD31 antibody (Santa Cruz Biotechnology, Inc, PECAM-1 ͓M-20͔) for endothelial cells and neoangiogenesis.

Slides were also analyzed quantitatively by computed histomorphometry using a Leitz Medilx (Leica) motorized microscope, a Sony 3CCD color-video camera, and the software Leica Q-win standard version Y2.3 for image analysis. "Endothelial coverage" was defined as the length of endothelial cell layer on the luminal surface and expressed as the percentage of total graft length. "Neointima formation" was defined as the area between the endothelial layer and luminal surface of the prosthesis and calculated per unit length of graft (mm 2 /mm) and per unit length of neointima (mm 2 /mm) reflecting the mean thickness of neointima. "Transmural cellular ingrowth" was defined as the percentage of graft area which was penetrated densely by host cells from the adventitial tissue toward luminal surface. Additionally, SEM pictures were taken at 5 different locations with 3 different magnifications to assess the luminal surface and endothelial coverage.

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Circulation September 15, 2009

PTX Blood Levels

At the conclusion of the study, a blood sample was taken from each rat to assess the systemic PTX concentration. After sample treatment of the serum by an online column switching technique, PTX was quantified by HPLC with UV detection at 205 nm with a lowest detection limit of 500 ng/mL.

Statistical Analysis

Statistical tests were performed using SPSS (version 16.0). Results for continuous variables are expressed either as meanϮSD for in vitro results (Table andFigures 1 and2) or, as meanϮSEM for in vivo results (all other figures). Mann-Whitney U test was used to compare nonparametric values at each time point, as well as comparison between time points in the same group. At each time point and in each group, the sample size is 3 for the in vivo part. The level of significance was set at PϽ0.05.

Statement of Responsibility

The authors had full access to the data and take responsibility for its integrity. All authors have read and agree to the manuscript as written.

Results

In Vitro Studies

Mechanical Properties and Fiber Morphology

A complete mechanical and morphological study of the PCL-PTX compared to the nonloaded grafts was carried out. These results will not be presented in detail because they are beyond the scope of this article. Briefly, for the same PCL concentration, PTX-loaded grafts have higher maximal stress and strain values than nonloaded grafts, and PTX-loading had the same effect as a PCL concentration increase. In terms of morphology, fewer beads and larger fibers were found at higher PCL concentrations. Fiber diameters were 500Ϯ290 nm, 640Ϯ540 nm, and 1640Ϯ1000 nm for nonloaded grafts obtained with PCL concentrations of 7.5, 9, and 12% (wt/vol), respectively. When PTX was added into the PCL solutions, the beads disappeared and the fibers became 80% larger at PCL concentrations of 7.5 and 9%, whereas no significant differences in beads and/or fibers were observed at 12%.

Evaluation of PTX-Loading and Incorporation Efficiency

Studies have been carried out to analyze the extent of the PTX loading into the vascular grafts. PCL-PTX graft loading in the range from 0.2% to 1% (wt/wt) have been prepared successfully, with a high incorporation efficiency of 80% to 100% representing 50 to 200 g PTX respectively for 2.5-cm long grafts (Figure 1). PTX distribution was homogeneous along the grafts, with no significant difference in the amount of drugs recovered from different parts of the grafts (Table ). Therefore, the final product chosen for rat implantation had comparable PTX amount as reported for the Taxus and Express stents, which contain 151 g of PTX per 2.4 cm stent length (http://www.fda.gov).

In Vitro PTX Release Profile

The results of PTX release from the electrospun vascular grafts in vitro are summarized in Figure 2. All PCL-PTX vascular grafts with 3 different PTX concentrations demonstrated triphasic release profiles which is typical for a degradable polymer. The 3 phases correspond to (1) an "initial slight burst release" of around 20% of the incorporated PTX within hours, (2) followed by a "slower, sustained release" by a drug diffusion controlled process over the following 22 days, and which led to the cumulative release of around 20% to 30% after 6 days, and between 45% to 60% after 22 days, and (3) a "final plateau phase" with a much slower release, which may be governed by the release of the remaining PTX from slow degrading of PCL fibers or by a high interaction between drug and polymer.

In Vivo Studies

Experimental Rat Model PCL and PCL-PTX grafts were easily implanted into the rats using standard microsurgical techniques. No significant bleeding was observed in either group. All animals survived until the end of follow-up, and all grafts were patent in both groups. DSA revealed no significant stenosis, nor aneurysm formation in either group.

Histological Evaluation of PCL and PCL-PTX Grafts

PCL and PCL-PTX grafts were analyzed at all time points. Major differences were found between these 2 groups at 3 weeks. Firstly, PCL grafts were infiltrated from the adventitial side by fibroblasts, macrophages, and giant cells (Figure 3A) and showed an endothelialization with CD31-positive endothelial cells (data not shown) starting from both anastomoses (Figure 3B). On the contrary, PCL-PTX grafts had no cellular infiltration (Figure 3C) and no endothelial coverage on the luminal side (Figure 3D).

At 12 weeks, PCL-PTX grafts showed cellular infiltration and CD31-positive endothelial coverage (Figure 4D and4E; see inset) similar to PCL grafts (Figure 4A and4B). Neointima formation was minimal including 2 or 3 spindle-shaped cell layers in the PCL-PTX grafts without any elastin fibers (Figure 4F). Neointima was composed of 6 to 10 spindleshaped cell layers with homogenous elastin fiber deposition (Figure 4C) in the PCL group.

At 24 weeks, confluent endothelial coverage was found in both groups (by morphometry and SEM). No differences in cellular infiltration were observed in either group (Figure 5A and 5B). Neoangiogenesis was present in both groups (Figure 5C and5F). Neointima formation was different, with more cell layers and some elastin fibers in the PCL group compared to the PCL-PTX group. We observed comparable amount of collagen deposition within the graft in both groups, as demonstrated by Miller and Masson staining (Figure 5B and5E). As previously reported for PCL vascular grafts, [START_REF] Pektok | Degradation and healing characteristics of small-diameter poly(-caprolactone) vascular grafts in the rat systemic arterial circulation[END_REF] we observed chondroid metaplasia in both groups at 12 weeks within the neointima, but this was less important in the PCL-PTX group than the PCL group. In both groups, this disappeared almost completely at 24 weeks, leaving some neointima calcification (data not shown).

Morphometric Analysis

Quantitative morphometric comparison between the PCL and the PCL-PTX groups over time (3, 12, and 24 weeks) demonstrated a significantly delayed endothelialization in PCL-PTX grafts compared to the PCL grafts. However, at 24 weeks, 99% of the PCL-PTX grafts were covered by the endothelium (Figure 6A). Cellular infiltration was also reduced significantly at 3 weeks in the PCL-PTX grafts; additionally 37% and 35% of these grafts were infiltrated at 12 and 24 weeks, respectively. Despite the lack of statistical significance, the cellular infiltration of PCL-PTX grafts was less than that of PCL grafts at all time points (Figure 6B). Neointima formation was negligible in both groups at 3 weeks and showed a significant increase at 12 and 24 weeks in the PCL group, but remaining stable and significantly lower in the PCL-PTX group. Additionally, the area and mean thickness of neointima formation was reduced by more than half in the PCL-PTX grafts at 12 and 24 weeks when compared to PCL group (Figure 7A and7B).

Paclitaxel Blood Levels

None of the samples collected at the conclusion of the study showed a detectable PTX concentration, indicating a subclinical level of PTX in the systemic circulation (Ͻ500 ng/mL).

Discussion

Although intensive research has been initiated in recent years toward the preparation of small-diameter vascular grafts suitable for clinical use, none of this has led to a functional candidate yet. Among the various causes responsible for failure, thrombosis, aneurysms, and intimal hyperplasia are leading. [START_REF] Teebken | Tissue engineering of small diameter vascular grafts[END_REF][START_REF] Pawlowski | Endothelial cell seeding of polymeric vascular grafts[END_REF] As a consequence, it would be of great interest to dispose of novel vascular grafts having both very good mechanical properties [START_REF] Lee | Development of a composite vascular scaffolding system that withstands physiological vascular conditions[END_REF] and a nonproliferative therapeutic effect. Our approach aimed at preparing biodegradable PTXeluting small-diameter vascular grafts made of PCL for clinical applications. In fact, PTX is a well-known mitotic inhibitor used in cancer chemotherapy, but it is also used in some cardiovascular applications, like coronary stents, to prevent restenosis by limiting the growth of neointima. [START_REF] Drachman | Neointimal thickening after stent delivery of paclitaxel: Change in composition and arrest of growth over six months[END_REF] Its mechanism of action is related to the hyper-stabilization of microtubules through its binding with the ␤-subunit of tubulin, which results in a blocked mitotic cycle at the G2/M stage. [START_REF] Liu | A novel translymphatic drug delivery system: Implantable gelatin sponge impregnated with PLGA-paclitaxel microspheres[END_REF] Beside this antiproliferative effect, the stability of PTX during the electrospinning process, its solubility in the solvents used with PCL and its resistance to ␥-sterilization render it a valuable candidate for the formulation of novel vascular grafts.

However, one has to keep in mind that inclusion of drugs into nano-or microfibre polymers does influence their basic properties, both mechanically and morphologically. [START_REF] Zeng | Biodegradable electrospun fibers for drug delivery[END_REF] In addition, it has been demonstrated that the drug release from such fibers is strongly dependent on the nature of the incorporated drug. [START_REF] Zeng | Influence of the drug compatibility with polymer solution on the release kinetics of electrospun fiber formulation[END_REF] Because intimal hyperplasia tends to appear within the first months after implantation, it is of importance to have a slow and controlled release of PTX over this period of time, to ensure an adequate antiproliferative effect and to avoid a burst release of massive doses of PTX, which could result in an acute cytotoxicity for the surrounding tissues. Therefore, the influence of various PTX loadings into PCL vascular grafts was investigated in the present study, first in vitro for fiber morphology, mechanical properties, drug distribution, and drug release profiles; second, for in vivo effects of time-released PTX from the grafts implanted in the rat abdominal aorta.

The results obtained from PCL-PTX grafts are consistent with our previous study on nonloaded PCL grafts. Tensile stress values of PCL grafts were increasing with PCL concentration up to 12% (wt/vol), which corresponded to a maximum of 5 MPa, and decreased with further PCL concentration increase. [START_REF] Nottelet | Factorial design optimization and in vivo feasibility of poly(caprolactone)-micro-and nanofiber-based small diameter vascular grafts[END_REF] In this study, drug loading might have the same effect as an increase of the PCL concentration leading to better mechanical properties observed for electrospun grafts from low PCL concentrations. The factorial design process led to the preparation of an optimized vascular graft spun from a 9% PCL solution containing 0.5% of PTX. It should be noted that the tensile stress values of the drug-loaded grafts after ␥-sterilization were always above 2 MPa, which is the maximum stress value for the natural vessels of rats and pigs [START_REF] Clerin | Tissue engineering of arteries by directed remodeling of intact arterial segments[END_REF][START_REF] Cerqueira | Morphological and biomechanical study of abdominal aorta of rats submitted to experimental chronic alcoholism[END_REF] and is considered as a lower limit value for implanted synthetic grafts into the systemic circulation.

Along with the incorporation of PTX, an increase of the fiber diameters of 80% was observed, which led to higher tensile stress values of drug-loaded vascular grafts in comparison to the nonloaded ones.

PTX incorporation efficiency into the nanofibres was high (80% to 100%) and homogeneous along the graft. This is very important for a controlled drug release. As outlined above, optimized grafts with a PTX concentration as described for Taxus and Express stents were prepared and could be implanted successfully. PTX in vitro release was controlled over a time period of 30 days with only a little burst at the initial phase, and 60% of PTX was released over 22 days. It is assumed that the remaining PTX would be released in small concentrations along with time-dependent degradation of the PCL fibers. Previous human studies involving Taxus and Express stents demonstrated that low-dose PTX release over several days was efficacious, and completion of release was expected at 6 months. [START_REF] Grube | Six-and twelve-month results from a randomized, double-blind trial on a slow-release paclitaxel-eluting stent for de novo coronary lesions[END_REF] The results from our PTX-eluting PCL vascular grafts are promising, and may provide a step toward reduction of intimal hyperplasia in small-diameter grafts.

We preferred to use PTX as a suitable drug, for its known potent antiproliferative effect on the neointima formation, and its continuous local release from the graft. The analysis of blood samples confirmed that concentration of the antimitotic drug was below the detection limit in the systemic circulation, which may not be sufficient to create toxic side effects. Note the absence of endothelial cells in PCL-PTX grafts at 3 weeks. Pϭ0.046 at 3 weeks, Pϭ0.126 at 12 weeks, and Pϭ0.310 at 24 weeks. Note the significant increase in endothelialization for both the PCL and PCL-PTX grafts between 3, 12, and 24 weeks, respectively. B, Percentage of transmural cellular ingrowth at 3, 12, and 24 weeks. Note that the ingrowth is delayed in the PCL-PTX grafts. Pϭ0.046 at 3 weeks, Pϭ0.126 at 12 weeks, and Pϭ0.317 at 24 weeks. There is a significant increase in cell invasion of the PCL-PTX grafts between 3, 12, and 24 weeks, respectively (mean valuesϮSEM, nϭ3).

Nevertheless, the biological effect of PTX is well demonstrated histologically and leads to significant alterations mainly at the 3-and 12-week follow-up. The delayed endothelial coverage may represent a problem for early thrombogenicity. Virmani's group has shown similar results in drugeluting stents implanted in a rabbit model. [START_REF] Joner | Endothelial cell recovery between comparator polymer-based drugeluting stents[END_REF] Additionally, several well-documented clinical reports attribute the occurrence of coronary thrombosis after drug-eluting stent placement to the lack or incomplete endothelial coverage. [START_REF] Stone | Safety and efficacy of sirolimus-and paclitaxel-eluting coronary stents[END_REF] Finally, using a similar model in the rat, we have previously observed enhanced thrombus formation in synthetic vascular grafts treated by systemic or local administration of rapamycin. [START_REF] Walpoth | Prevention of neointimal proliferation by immunosuppression in synthetic vascular grafts[END_REF] However, in the present study, no occlusions were found, probably because of the high flow in the rat abdominal aorta. At 24 weeks, the endothelialization was nearly complete, indicative of a delay only in the PCL-PTX grafts. Simultaneously, the cellular ingrowth of the biodegradable prosthesis was also delayed, which may eventually delay the regeneration of an autologous arterial wall. This delay should not create any problems for the biomechanical properties of the graft during the vascular remodeling because PCL has a relatively slow degradation rate. Up to 6 months after implantation, no aneurysm formation was observed in our series. However, this hypothesis should be confirmed by other in vivo studies with longer follow-up.

Neointima formation, which normally progresses to intimal hyperplasia, and eventually stenosis and occlusion, is a major problem in small-diameter vascular grafts. Many attempts by several authors have been made to reduce intimal hyperplasia, such as the topical use of drug-releasing gel, including antimitotic drugs around the anastomosis, showing a reduction of intimal hyperplasia at the anastomosis with this method; however the method was relatively cumbersome, and drug release was not well controlled. [START_REF] Schachner | Local application of rapamycin inhibits neointimal hyperplasia in experimental vein grafts[END_REF] In our PCL-PTX grafts, a linear PTX release in vitro was observed over the first month. This in vitro drug release matches well the histological and morphological findings of neointima formation in vivo, which was significantly reduced after 12 weeks, compared to the control PCL grafts. This trend in histology and morphology extends to 24 weeks. Comparing the results of the present study with our previously published study in Circulation, which assessed PCL versus the classical ePTFE grafts, PCL-PTX grafts showed a delayed endothelialization similar to ePTFE grafts, but a significantly reduced neointima formation within the first 24 weeks when compared to ePTFE grafts. [START_REF] Pektok | Degradation and healing characteristics of small-diameter poly(-caprolactone) vascular grafts in the rat systemic arterial circulation[END_REF] For the above described positive effect of the PTX-loaded grafts, further studies with longer assessment periods are needed because we have to follow the biological effect of the drug release and its consequences up to 1 or more years to exclude a "late catch-up phenomenon" as described for drug-eluting stents. [START_REF] Park | Two-year follow-up of the quantitative angiographic and volumetric intravascular ultrasound analysis after nonpolymeric paclitaxel-eluting stent implantation: late "catch-up" phenomenon from ASPECT study[END_REF][START_REF] Daemen | Three-year clinical follow-up of the unrestricted use of sirolumus-eluting stents as part of the Rapamycin-Eluting Stent evaluated at Rotterdam Cardiology Hospital (RESEARCH) registry[END_REF] Furthermore, the delayed endothelialization of the small-diameter drug-eluting PCL grafts may cause thrombosis and occlusions in low-flow conditions.

Currently, a direct application of these results is not yet possible for coronary bypass grafting in humans. First, longer follow-up studies have to evaluate complete degradation of the polymer scaffold, and second, a more relevant small calibre graft model has to be assessed in a larger animal.

Conclusions

Excessive growth of neointima in vascular grafts leads to graft failure in the long-term. This is even more pronounced in small-diameter grafts, such as coronary bypass grafts. Local delivery of PTX in the coronary arteries by drugeluting stents has been shown to limit the growth of neointima, providing improved patency rates in the short-term. However, a "late catch-up phenomenon" has been described for such stents. PTX-loaded biodegradable vascular grafts may delay the excessive neointima formation in the grafts, whereas the scaffold degradation and an arterial regeneration process take place. Although topical application of antiproliferative drugs around vascular anastomoses have been reported with promising in vivo results, the use of a drug-eluting vascular graft has not yet been reported.

After our first report on biodegradable vascular prostheses in the arterial circulation, our present study is another step forward in this field. We first produced a PTX-loaded PCL vascular graft after a factorial design optimization process and performed in vitro tests to assess the mechanical and morphological properties of the graft after drug loading, the drug distribution in the graft body, and the early release from the graft. Our in vitro studies revealed that PTX loading influenced the mechanical and morphological properties of the PCL grafts, however this did not jeopardize their suitability for implantation. Then, the PCL-PTX grafts which showed the most relevant characteristics for in vivo application were chosen for implantation in the rat aorta. After 6 months follow-up, we observed the biological effects of local PTX release such as delayed endothelialization, limited neointima growth, and limited cellular infiltration. Despite these findings, the healing process of these biodegradable grafts was substantial, and the limitation of neointima forma-tion was prominent. Based on our initial findings, we speculate that PTX-eluting PCL vascular grafts may potentially lead to better clinically applicable grafts in the future.

Figure 3 .

 3 Figure 3. Histological analysis of PCL (A and B) and PCL-PTX (C and D) grafts at 3 weeks. A, Full thickness of PCL graft showing cellular infiltration of the external part (H&E, ϫ100 magnification). B, Endothelialization on the luminal side of a PCL graft (H&E, ϫ400 magnification). C, Full thickness of a PCL-PTX graft with no cellular ingrowth (H&E, ϫ100 magnification). D, No endothelial cell coverage on the luminal side of PCL-PTX graft (H&E, ϫ400 magnification).

Figure 2 .

 2 Figure 2. In vitro drug release profiles of PTX from PCL vascular grafts containing 0.5, 0.75, and 1% (wt/wt) PTX over 27 days in PBS pH 7.4-Tween 20 at 37°C. Data are expressed as an average of results obtained from 3 grafts at each time point (mean valuesϮSD, nϭ3).

Figure 4 .

 4 Figure 4. Histological analysis of PCL (A through C) and PCL-PTX (D through F) grafts at 12 weeks. A, Full thickness of PCL graft showing cellular infiltration toward the middle part (H&E, ϫ100 magnification). B, Neointima formation on the luminal surface of a PCL graft, indicated by a black arrow (H&E, ϫ200 magnification). C, Collagen deposition in the graft body (green) and elastin fibers in the neointima of a PCL graft (Miller and Masson staining, ϫ200 magnification). D, Full thickness of a PCL-PTX graft showing cellular infiltration until the middle part (H&E, ϫ100 magnification). E, Endothelialization (black arrow) of PCL-PTX graft (H&E, ϫ400 magnification). Inset shows a CD31 immunostaining with positive endothelial cells. F, PCL-PTX graft with collagen deposition within the graft and no neointima formation (Miller and Masson staining, ϫ400 magnification).

Figure 5 .

 5 Figure 5. Histological analysis of PCL (A through C) and PCL-PTX (D through F) grafts at 24 weeks. A, Luminal part of PCL graft with neointima and cellular infiltration (H&E, ϫ200 magnification). B, Elastin fibers in the neointima of PCL graft with collagen deposition in green (Miller and Masson staining, ϫ200 magnification). C, Macrophage and giant cell reaction with neoangiogenensis in PCL graft (H&E, ϫ400 magnification). D, Luminal part of PCL-PTX graft with little neointima and cellular infiltration (H&E, ϫ200 magnification). E, No elastin fibers in the neointima of PCL-PTX graft and with collagen deposition in green (Miller and Masson staining, ϫ200 magnification). F, Macrophage and giant cell reaction with neoangiogenesis in PCL-PTX graft (H&E, ϫ400 magnification).

Figure 6 .

 6 Figure 6. Morphometric cellular repopulation of PCL and PCL-PTX grafts. A, Percentage of endothelial coverage at 3, 12, and 24 weeks.Note the absence of endothelial cells in PCL-PTX grafts at 3 weeks. Pϭ0.046 at 3 weeks, Pϭ0.126 at 12 weeks, and Pϭ0.310 at 24 weeks. Note the significant increase in endothelialization for both the PCL and PCL-PTX grafts between 3, 12, and 24 weeks, respectively. B, Percentage of transmural cellular ingrowth at 3, 12, and 24 weeks. Note that the ingrowth is delayed in the PCL-PTX grafts. Pϭ0.046 at 3 weeks, Pϭ0.126 at 12 weeks, and Pϭ0.317 at 24 weeks. There is a significant increase in cell invasion of the PCL-PTX grafts between 3, 12, and 24 weeks, respectively (mean valuesϮSEM, nϭ3).

and carried out in conformity with the Guide for Care and Use of the Laboratory Animals (National Research Council, Washington, DC: National Academy Press; 1996).

	The experimental protocol has been approved by the
	Animal Experiments Ethical Committee of the University of Geneva
	(Protocol No. 06/52) and the Veterinary Office of State of Geneva,
	Switzerland (No. 1081/3232/11),

Table . Percentage of PTX Amount Found in Each of the 4 Cut Graft Pieces

 .

		0.5% (w:w)	0.75% (w:w)
	Graft Part	PTX-Loaded Grafts	PTX-Loaded Grafts
	1	25.95Ϯ1.0	27.05Ϯ1.6
	2	28.5Ϯ1.0	18.75Ϯ6.6
	3	20.95Ϯ2.1	28.15Ϯ1.2
	4	24.6Ϯ0.4	26.05Ϯ1.7

Mean Percentage of PTX Found in Each

Piece/Total PTX Amount in Full Grafts Figure

1

. Experimental PTX-loading for small diameter grafts prepared to contain 0.25%, 0.5%, 0.75%, and 1% (w/w) of the drug. Each curve corresponds to a defined PCL concentration of 7.5%, 9%, and 12% (mean valuesϮSD, nϭ3).

Figure 7 .

 7 Neointima formation of PCL and PCL-PTX grafts. A, Area of neointima formation per total graft length (mm 2 /mm). Neointima formation is negligible at 3 weeks and increases significantly at 12 and 24 weeks in the PCL grafts. In contrast, PCL-PTX grafts show a significantly lower neointima formation than PCL grafts. Pϭ0.317 at 3 weeks, Pϭ0.049 at 12 weeks, and Pϭ0.275 at 24 weeks. B, Area of neointima formation per neointima length (mm 2 / mm). Please note that PCL grafts show a significant neointima increase between 3, 12, and 24 weeks, respectively and remain higher than in PCL-PTX grafts. Pϭ0.317 at 3 weeks, Pϭ0.275 at 12 weeks, and Pϭ0.275 at 24 weeks (mean valuesϮSEM, nϭ3).

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Acknowledgments

The authors thank the following collaborators for their appreciated help and involvement in this study: Dr Wojciech Mrowczynski for his help with the statistical analyses; Dr Damiano Mugnai for performing some of the angiograms; Dr Marc Fathi for the analyses of the paclitaxel blood levels; Unn Lutzen for histological preparations. Additionally, we are grateful to Prof Thomas Perneger from the Centre de Recherche Clinique of the University Hospital of Geneva for the statistical advice he provided.

Sources of Funding

This study was partly supported by a grant from the Swiss National Science Foundation (320000-119822-1).

Disclosures

None.