MD Yonathan Freund

MsC Marine Cachanado

MD Quentin Delannoy

MD Youri Yordanov

MD Judith Gorlicki

MD Tahar Chouihed

MD Anne-Laure Féral-Pierssens

MD Jennifer Truchot

MD, PhD (10 Thibaut Desmettre

MD Celine Occelli

MD Xavier Bobbia

MD Mehdi Khellaf

MD Olivier Ganansia

MD Jérôme Bokobza

MD Frédéric Balen

MD Sebastien Beaune

MD Ben Bloom

MD Tabassome Simon

MD Alexandre Mebazaa

Effect of an emergency department care bundle on 30-day hospital discharge and survival among elderly patients with acute heart failure: the ELISABETH randomized trial

Keywords:

Question: Does an intervention aimed at improving guideline adherence for the treatment of acute heart failure, including intensive IV nitrate therapy and treatment of precipitating factors, improve hospital discharge and survival at 30 days? Findings: In this stepped-wedge cluster-randomized trial that included 503 patients aged 75 and older presenting to the emergency department with acute heart failure, implementation of an early and comprehensive care bundle compared with usual care improved guideline adherence, but had no significant effect on number of days alive and out of hospital at 30 days (median 19 in both groups).

Meanings: This emergency department care bundle did not improve 30-day outcomes among older patients with acute heart failure.

Introduction

Acute heart failure is one of the most common syndromes in older patients visiting the emergency department (ED). [START_REF] Ezekowitz | Acute heart failure in the emergency department: short and long-term outcomes of elderly patients with heart failure[END_REF] In an international observational study, which included patients aged 75 years and older from 2010 to 2013, acute heart failure was associated with a 10% mortality risk and up to 30% risk of hospital readmission at 30 days. [START_REF] Teixeira | Clinical presentation and outcome by age categories in acute heart failure: results from an international observational cohort[END_REF] International recommendations and clinical guidelines on acute heart failure include the use of moderate doses of diuretics, early initiation of nitrates, and non-invasive ventilation when indicated, along with the early detection and treatment of precipitating factors such as acute coronary syndrome, infection or atrial fibrillation. [START_REF] Mcmurray | ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure 2012: The Task Force for the Diagnosis and Treatment of Acute and Chronic Heart Failure 2012 of the European Society of Cardiology. Developed in collaboration with the Heart Failure Association (HFA) of the ESC[END_REF][START_REF] Mebazaa | Recommendations on pre-hospital & early hospital management of acute heart failure: a consensus paper from the Heart Failure Association of the European Society of Cardiology, the European Society of Emergency Medicine and the Society of Academic Emergency Medicine[END_REF][START_REF] Collins | Early management of patients with acute heart failure: state of the art and future directions--a consensus document from the SAEM/HFSA acute heart failure working group[END_REF] However, these recommendations are based on low levels of evidence. The cornerstone trials on small samples of patients with acute heart failure reported a clinical benefit with early high dose IV nitrates. [START_REF] Cotter | Randomised trial of high-dose isosorbide dinitrate plus low-dose furosemide versus high-dose furosemide plus low-dose isosorbide dinitrate in severe pulmonary oedema[END_REF][START_REF] Sharon | High-dose intravenous isosorbide-dinitrate is safer and better than Bi-PAP ventilation combined with conventional treatment for severe pulmonary edema[END_REF] Subsequent large scale trials testing novel agents with vasodilator properties failed to confirm improved outcomes, possibly because of effects besides vasodilation that might affect mortality risk. [START_REF] Kozhuharov | Effect of a Strategy of Comprehensive Vasodilation vs Usual Care on Mortality and Heart Failure Rehospitalization Among Patients With Acute Heart Failure: The GALACTIC Randomized Clinical Trial[END_REF][START_REF] Packer | Effect of Ularitide on Cardiovascular Mortality in Acute Heart Failure[END_REF] As a potential consequence, previous reports confirmed low adherence to these recommendations, leading to under-utilization of nitrates in the ED. [START_REF] Gorlicki | Low compliance to guidelines in the management of acute heart failure in emergency elderly patients: a multicenter pilot prospective study[END_REF][START_REF] Lemachatti | Temporal trends in nitrate utilization for acute heart failure in elderly emergency patients: A single-centre observational study[END_REF] The failure of recent acute heart failure studies to report clinical benefits with specific treatments may be attributable to both the delay from presentation to treatment because recent studies suggest that an early therapy is associated with better prognosis. and the lack of management of precipitating factors, because acute heart failure prognosis has been reported to be associated with the underlying precipitant of worsening heart failure. [START_REF] Arrigo | Effect of precipitating factors of acute heart failure on readmission and long-term mortality[END_REF][START_REF] Matsue | Time-to-Furosemide Treatment and Mortality in Patients Hospitalized With Acute Heart Failure[END_REF] Whether early ED management of congestion and precipitating factors improves outcomes is unknown.

The aim of this trial was to test the efficacy on clinical outcomes of an early, comprehensive and guideline-recommended care bundle on older patients with acute heart failure in the ED. [START_REF] Freund | Early and comprehensive care bundle in the elderly for acute heart failure in the emergency department: study protocol of the ELISABETH stepped-wedge cluster randomized trial[END_REF]

Methods

Study design

The protocol and statistical analysis plan of the ELISABETH trial is available in the Supplement 1, and has been previously published in detail. [START_REF] Freund | Early and comprehensive care bundle in the elderly for acute heart failure in the emergency department: study protocol of the ELISABETH stepped-wedge cluster randomized trial[END_REF] This was an unblinded superiority, stepped-wedge, cluster randomized clinical trial in France aimed at testing the effect of an early comprehensive care bundle for acute heart failure in older patients in the ED. Fifteen EDs in France participated in the study. The trial recruited from December 2018 to September 2019 and follow up ended in October 2019. The study was approved by an institutional review board (Comité de Protection des Personnes SOOM 2, Toulouse academic hospital, France). Written informed consent was sought for all patients before inclusion. If the patient lacked capacity to give consent, the emergency physician sought consent from a patient's relative. If no such relative was available, research staff were able to proceed to an emergency inclusion. For the latter, as soon as the patient's clinical condition allowed it, a clinical research technician or a physician informed him or her about the trial and sought written informed consent. The reporting of this trial followed the Consolidated Standards of Reporting Trials (CONSORT) statement extended to stepped-wedge cluster randomized trials. [START_REF] Hemming | Introducing the new CONSORT extension for stepped-wedge cluster randomised trials[END_REF][START_REF] Hemming | Reporting of stepped wedge cluster randomised trials: extension of the CONSORT 2010 statement with explanation and elaboration[END_REF]

Patients

Consecutive patients aged 75 and older with a clinical diagnosis of acute heart failure in the ED were eligible for inclusion in the trial. Acute heart failure diagnosis was made on the basis of at least one of acute or worsening dyspnea, or orthopnea and at least one of the following: bilateral pulmonary rales or peripheral edema, signs of pulmonary congestion on chest radiography or cardiac echography, or elevation in level of natriuretic peptides (BNP or NT pro BNP)

As a pragmatic study, whether the diagnosis of acute heart failure was confirmed was left to the discretion of the physician and echocardiography was not mandatory.

Patients were not included if they presented with another obvious cause of acute illness (e.g. ST elevation myocardial infarction or severe sepsis), a systolic blood pressure less than 100 mmHg, any contra-indication for nitrate therapy (severe mitral or aortic stenosis, or severe aortic regurgitation), or a known chronic kidney impairment that required dialysis. Since this trial focused on early treatment of acute heart failure, patients in whom time from ED presentation to inclusion was > 6h were also not included. Patients with no social security, who were incarcerated or under guardianship were also excluded.

All patients were followed up at 30 days, either by hospital visit or by phone interview if already discharged. They were instructed to return to the same ED or hospital in the event of recurrent or worsening symptoms. A local clinical research assistant checked for return visits to the ED or admission to the hospital during the follow up period. The phone interview was performed using a structured questionnaire that recorded length of initial hospital stay, any return visit to the ED or admission to the hospital. When patients could not be contacted by phone, the patient's general practitioner was contacted. When it was not possible to contact the patient or their physician, death records were sought from the patient's birth town administrative record.

Randomization

After an initial control period (first step) of four weeks in all centers, one ED switched to the "intervention period" every two weeks. The order in which they switched was randomized. The first two weeks of the intervention period in each center was also a training period, in which a clinical research technician presented and detailed the care bundle and assisted emergency physicians in implementing it. After 32 weeks, all EDs were in the "intervention period" for the eight remaining weeks of inclusion (eFigure 1 in Supplement 2). Randomization of the order of the switch was performed by an independent statistician. At study commencement, EDs were classified on size based on their annual census. Randomization was stratified on cluster size (small, medium, large). Each set of 3 consecutive time periods would contain one small, one medium-sized, and one large site, in random order.

Intervention

This was an unblinded design, which was chosen because the intervention could not be performed in a blinded fashion for either physicians or patients.

In participating EDs and in most French EDs, patients are first seen by a triage nurse, then managed by a senior emergency physician, with or without the help of a trainee.

Only senior emergency physicians could include patients, and consequently the entirety of the ED medical management was completed by a senior emergency physician. In France, regular emergency medical services (EMS) cannot administer treatments, therefore no patients conveyed by regular EMS received any treatment before inclusion in the ED. However, some patients may have been transported by a physician-staffed EMS (Service mobile d'urgence-réanimation) where treatment could have been given. These patients are usually directly admitted to an intensive care unit. If that was not the case, patients in whom treatment was already started before inclusion were excluded.

The intervention of this trial consisted of a guideline-recommended care bundle for the early management of acute heart failure in the ED.

In the control period, patients' care was left to the discretion of the treating emergency physician (usual care). At the beginning of the trial, the recommendations for acute heart failure management were described to all emergency physicians, including moderate dose diuretics, high dose IV nitrates, search for and early treatment of most frequent precipitating factors, and non-invasive ventilation (NIV) when indicated.

In the intervention period, after a patient's inclusion, emergency physicians were instructed to follow the early care bundle for acute heart failure, with the help of a handover check-list. This care bundle mandates treatment initiation within one hour and completion within four hours of early treatment of pulmonary oedema with 40 mg (if naïve) or daily dose (if already on oral diuretics) of intravenous furosemide, IV nitrates in titration (given in 3 mg boluses every 5 minutes for one hour, titrated to a systolic blood pressure (BP) above 100 mmHg), detection and treatment of precipitating factors (specifically acute coronary syndrome, rapid atrial fibrillation and suspected infection). Suspicion of cardiac injury (based on troponin level measurement, echocardiography and electrocardiogram analysis) required introduction of antiplatelet therapy and referral to cardiologic intensive care unit for potential admission and coronary angiogram if indicated. Suspicion of respiratory tract infection (based on chest X-ray, C-reactive protein, procalcitonin and leucocytes level) required early introduction of antibiotics. Atrial fibrillation with a heart rate above 100 beats per minute required introduction of antiarythmic therapy (amiodarone, digoxin or beta-blockers as indicated). NIV was administered in the event of respiratory distress or hypercarbia with pH<7.35.

All treatments were to be initiated in the ED within the first hour of medical management, and for at least 4 hours. Their discontinuation was left to the discretion of the admitting physician.

Outcomes

The primary objective of the trial was to test the effect of an early comprehensive guideline-recommended care bundle on morbidity and mortality at 30 days in older patients. The primary endpoint was the number of days alive and out of hospital during the 30-day period after the ED visit. The choice of this endpoint allowed capturing the burden of acute heart failure in terms of mortality, hospital length of stay, and early readmission to the hospital, as recommended by the European Society of Cardiology consensus paper. [START_REF] Zannad | Clinical outcome endpoints in heart failure trials: a European Society of Cardiology Heart Failure Association consensus document[END_REF] Patients that died before day 30 were counted as having zero days alive and out of hospital. A return visit to the ED was considered as one day in the hospital. Details on the calculation of this endpoint were previously detailed. [START_REF] Freund | Early and comprehensive care bundle in the elderly for acute heart failure in the emergency department: study protocol of the ELISABETH stepped-wedge cluster randomized trial[END_REF] The secondary endpoints included 30-day all-cause mortality, 30-day cardio-vascular mortality, hospital readmission within 30 days, length of in-hospital stay truncated at 30 days, and changes of more than 2-fold in creatinine level from inclusion to day 30 or to discharge (whichever comes first). Creatinine was measured at day zero in the ED, and at discharge or day 30.

Power analysis

The full statistical plan and sample size calculation are available in Supplement 1.

Sample size was calculated under the superiority hypothesis. Based on previous cohort analysis, the mean number of days alive and out of hospital at 30 days was estimated at 14 (standard deviation [SD] 9). [START_REF] Gorlicki | Low compliance to guidelines in the management of acute heart failure in emergency elderly patients: a multicenter pilot prospective study[END_REF][START_REF] Arrigo | Effect of precipitating factors of acute heart failure on readmission and long-term mortality[END_REF][START_REF] Teixeira | Management of acute heart failure in elderly patients[END_REF] To be clinically relevant and consistent with previous literature, it was estimated that the intervention should improve this endpoint by 20% (i.e. three days at least). [START_REF] Kozhuharov | Effect of a Strategy of Comprehensive Vasodilation vs Usual Care on Mortality and Heart Failure Rehospitalization Among Patients With Acute Heart Failure: The GALACTIC Randomized Clinical Trial[END_REF][START_REF] Packer | Effect of Ularitide on Cardiovascular Mortality in Acute Heart Failure[END_REF] For a power of 80% and alpha at 5%, 283 patients needed to be included. Adding the cluster effect for this stepped-wedge design and assuming an intra-cluster correlation at 0.001, the required sample size was 454 patients. Taking into account a rate of lost to follow up of 10%, the necessary sample size was 500 patientstwo per cluster for each twoweek period (step). Due to acute heart failure seasonality and fewer cases in the summer period, we had to increase the length of the last inclusion step from four weeks to eight weeks in order to reach the target. [START_REF] Barnett | The seasonality in heart failure deaths and total cardiovascular deaths[END_REF][START_REF] Kaneko | Presentations and outcomes of patients with acute decompensated heart failure admitted in the winter season[END_REF]

Statistical analysis

The primary analysis included all patients who were randomized, with missing outcome data replaced using worst-case imputation (zero days alive and out of the hospital). Baseline characteristics were expressed as number (percentage) for categorical variables, and mean (standard deviation) or median (interquartile range,

[IQR]) for continuous variables depending on their distribution. Unadjusted differences and 95% CIs were calculated using using Wald method with continuity correction for binary variables and using Brookmeyer and Crowley method for continuous variables. Adjusted differences were calculated using a generalized linear regression mixed model with Bernoulli (binary) distribution and an identity link function, with intervention, time period and cluster size (categorical) as fixed effects and cluster as a random effect for binary variables and using quantile regression model for continuous variables. [START_REF] Machado | QREG2: Stata Module to Perform Quantile Regression with Robust and Clustered Standard Errors[END_REF] The 2-week training period for each site was analyzed as part of the intervention period. Primary and secondary outcomes were analyzed in all randomized patients with a primary outcome available. The number of days alive and out of hospital was analyzed using a generalized linear regression mixed model with negative binomial distribution with intervention, time period and cluster size as fixed effects and cluster as a random effect. [START_REF] Hussey | Design and analysis of stepped wedge cluster randomized trials[END_REF] A sensitivity analysis was performed on all patients that completed the trial. All-cause mortality at 30 days, cardiovascular mortality at 30 days and hospital readmission were compared between groups by using generalized linear regression mixed model with Poisson distribution. The logarithm of the number of patients was included as an offset term in the model. In surviving patients, the length of stay in hospital in days was compared between the two strategies by using a generalized linear regression mixed model with negative binomial distribution. For secondary outcome, fixed and random effects were defined as described previously. For non-hospitalized patients, length of hospital stay was counted as zero day. Percentage of patients with a change of more than 2-fold in creatinine between inclusion and 30 days were not analyzed because of the presence of a small number of cases and no or negligible differences between the two groups. Because of the potential for type 1 error due to multiple comparisons, the findings of analyses of secondary endpoints should be interpreted as exploratory.

The original analysis plan was intended to study different extensions of the models as sensitivity analyses, including both a time x cluster interaction as a fixed effect, and also as random effects. However, due to the structure of the data (low proportion of events by cluster by period), these sensitivity analyses could not be performed. All superiority tests were two-sided and P values <0.05 were considered significant. SAS V.9.4 software (SAS Institute Inc., Cary, NC) and Stata version 16 (Stata Corp), were used for statistical analyses.

Results

Study population

Fifteen EDs participated in the trial and 503 patients were recruited. Among them, one patient was under guardianship and was excluded, five patients withdrew their consent, three were lost to follow-up and the primary endpoint was missing for one patient. Therefore, 502 patients were included in the primary analysis: 303 in the control group and 199 in the intervention group. A total of 493 patients completed the trial and were included in the sensitivity analysis (Figure 1, eFigure 1 and eTable 1).

The mean number of patients recruited by center and by step was respectively 34 (12) and 31 (20). The median age was 87 (IQR 81 -91) years, 298 (59%) were women, and 269 (54%) had known chronic cardiac failure. Patients' baseline characteristics were similar between the two groups and are reported in table 1.

Pulmonary congestion was similar between the two groups: mean oxygen saturation was 90.6% (6.7) versus 91.1% (6.9), mean respiratory rate 26.2 (6.7) versus 25.9 (7.3) per min in intervention and control groups respectively, and mean systolic blood pressure was 155 (29) mmHg in the intervention group and 149 (28) mmHg in the control group.

Treatment in the ED

In the intervention group, patients were significantly more frequently treated by IV nitrates (96% vs 25%, adjusted difference 71% [95%CI 62% to 80%]), with higher median dose (27 mg vs 4 mg at four hours of initial management; adjusted difference 24mg [95%CI 13.5 to 34.1]) and more frequently treated by diuretics (98% vs 90%, adjusted difference 6.8% [95%CI 0.5 to 13.0]), (table 2). A precipitating factor was present in 45% of patients, and was significantly more frequently treated in the ED in the intervention group than in the control group (59% vs 32% overall, adjusted difference 31% [95%CI 14% -48%]), (eTable 2).

Study endpoints

Among the 502 patients included in the primary analysis, the median number of days alive and out of hospital at 30 days was 19 (IQR 0 -24) in the intervention group and 19 (IQR 0 -24) in the control group (adjusted difference -1.9 [95%CI -6.6 to 2.8]), adjusted ratio = 0.88 [95% CI 0.64 to 1.21], p=0.44) (table 3). The intra-class correlation coefficient was 0.057. A sensitivity analysis of patients that completed the trial exhibited similar results (eTable 3 and eTable 4). There was no statistically significant difference in all-cause mortality at day 30 (8.0% in the intervention vs 9.7% in the control, adjusted difference 4.1% [95%CI -17.2 ; 25.3], adjusted risk ratio aRR 1.17 [95% CI 0.53 to 2.57]; reversal in direction due to the adjustement for covariates) (Figure 2), median length of initial hospital stay (8 days [IQR 5 -21] vs 8 days [ IQR 5 -16] in the intervention and control groups respectively, adjusted difference 2.5 [95%CI -0.9 to 5.8] adjusted ratio=1.22 95%CI [0.94 to 1.59]), unscheduled readmission (14% vs 16% in the intervention and control groups respectively, adjusted difference -1.3% [95%CI -26.3% to 23.7%], adjusted risk ratio 0.96 [95%CI 0.48 to 1.95]) or acute kidney injury at 30 days (1.0% vs 1.4% in the intervention and control groups respectively) (table 3).

There was a similar rate of nitrate use in the control group (eFigure 2) and treatment of precipitating factors in the 5 centers randomized to later crossover than in the usual care period (17% vs 15% respectively).

Discussion

In this stepped-wedge, cluster randomized trial that included 502 older patients with acute heart failure, an early and comprehensive, guideline-recommended care bundle resulted in a significantly increased use of IV nitrates and more frequent treatment of precipitating factors but did not significantly improve the number of days alive and out of hospital at 30 days, nor any other early outcome.

For the past few decades, IV nitrates have been recommended for the early management of patients with acute heart failure. [START_REF] Cotter | Randomised trial of high-dose isosorbide dinitrate plus low-dose furosemide versus high-dose furosemide plus low-dose isosorbide dinitrate in severe pulmonary oedema[END_REF][START_REF] Sharon | High-dose intravenous isosorbide-dinitrate is safer and better than Bi-PAP ventilation combined with conventional treatment for severe pulmonary edema[END_REF] However, the failure of further trials to confirm any clinical benefit of IV nitrates in patients with acute heart failure resulted in subsequent moderate level of evidence for this recommendation, hence the reported low adherence in older patients. [START_REF] Lemachatti | Temporal trends in nitrate utilization for acute heart failure in elderly emergency patients: A single-centre observational study[END_REF] Several studies suggested that a more comprehensive and early management of patients with acute heart failure in the ED could improve prognosis, because prognosis could be dependent on underlying conditions and precipitating factors. [START_REF] Teixeira | Management of acute heart failure in elderly patients[END_REF][START_REF] Chioncel | Acute heart failure congestion and perfusion status -impact of the clinical classification on in-hospital and long-term outcomes; insights from the ESC-EORP-HFA Heart Failure Long-Term Registry[END_REF] The implementation of the tested care bundle resulted in a significantly higher compliance with guideline-recommended therapy both in the treatment of acute pulmonary congestion and of the precipitating factors.

The control group in this study received similar treatments to those reported in a recent multicenter observational study. [START_REF] Gorlicki | Low compliance to guidelines in the management of acute heart failure in emergency elderly patients: a multicenter pilot prospective study[END_REF] In that study, only 34% of patients with acute heart failure received IV nitrates, and very few were treated for precipitating factors in the ED. In France, IV nitrates are the only nitrates used for patients with acute heart failure, and no other nitrates were used in this study, or in France in general. [START_REF] Gorlicki | Patient care pathways in acute heart failure and their impact on in-hospital mortality, a French national prospective survey[END_REF] More patients in the intervention group had to be admitted to an intensive care department. Whether this was the result of adverse effect of the intervention or of a more proactive approach in this group is unknown.

In the present trial, it is possible there could have beena Hawthorne effect in that control patients would receive similar treatments to intervention patients, because physicians would be aware that the patients were in the trial, and aware their management decisions were being recorded. That this was not the case could be due to the stepped-wedge cluster-randomisation methodology. [START_REF] Gorlicki | Low compliance to guidelines in the management of acute heart failure in emergency elderly patients: a multicenter pilot prospective study[END_REF] There was also a low risk of contamination in sites randomized to later crossover, with a similar rate of nitrate use and treatment of precipitating factors. Furthermore, these results showed an intraclass correlation coefficient estimation of 0.05, higher than the one anticipated, which reduced statistical power. However, it seems unlikely that this would have change results on the primary outcome.

Regarding treatment of acute pulmonary congestion, these results are consistent with those from the GALACTIC trial in which early and sustained vasodilatation therapy was not significantly associated with improved outcomes. The present trial also tested a comprehensive approach, with earlier implementation of the intervention: patients were randomized within 6 hours of ED presentation and the care bundle was initiated during the first hour of medical management. With a significantly higher rate of treatment of precipitating factors, this trial is complementary to the GALACTIC trial and others that tested intervention during the first days of admission for acute heart failure. [START_REF] Kozhuharov | Effect of a Strategy of Comprehensive Vasodilation vs Usual Care on Mortality and Heart Failure Rehospitalization Among Patients With Acute Heart Failure: The GALACTIC Randomized Clinical Trial[END_REF] This trial focused on older patients, and it is likely that in these patients, who often present with comorbidities, the prognosis is not driven by pulmonary edema but rather by precipitating factors and underlying conditions [START_REF] Kozhuharov | Effect of a Strategy of Comprehensive Vasodilation vs Usual Care on Mortality and Heart Failure Rehospitalization Among Patients With Acute Heart Failure: The GALACTIC Randomized Clinical Trial[END_REF][START_REF] Packer | Effect of Ularitide on Cardiovascular Mortality in Acute Heart Failure[END_REF][START_REF] Arrigo | Effect of precipitating factors of acute heart failure on readmission and long-term mortality[END_REF][START_REF] Felker | Diuretic Strategies in Patients with Acute Decompensated Heart Failure[END_REF] .

Given the study findings, a more specific geriatric care pathway may need to be considered.

Limitations

This study has several limitations. First, only patients aged 75 and older were included, since this trial focused on a more homogeneous phenotype of patients. [START_REF] Senni | Congestive heart failure in the community: a study of all incident cases in Olmsted County, Minnesota[END_REF] However, this population may still include both patients with preserved and reduced systolic cardiac function. This parameter was not assessed in included patients, and it is possible that the intervention may cause different outcomes in different phenotypes of patients. The present trial included mostly wet and warm patients, the most common phenotype of acute heart failure. [START_REF] Chioncel | Acute heart failure congestion and perfusion status -impact of the clinical classification on in-hospital and long-term outcomes; insights from the ESC-EORP-HFA Heart Failure Long-Term Registry[END_REF] Second, a selection bias may also have occurred since some patients may have refused to be included in the study, and some eligible patients may have not been screened by the emergency physicians. Since this number was not recorded, the extent of this bias cannot be ascertained.

Third, the intervention included high-intensity IV nitrates, with a median of 27 mg of isosorbide dinitrate in the intervention group vs 4mg in the control group in the first four hours. The optimal dose may lie somewhere between these two numbers.

Fourth, the rate of hypotension that may have been caused by this treatment was not recorded, since the objective was to evaluate the overall effect of the bundle.

However, this risk is limited as the intervention included close monitoring of BP, and withholding nitrate therapy if the systolic BP dropped below 100 mmHg.

Fifth, the potential use in the ED of oral or topical nitrates, was not recorded in this study. However, in France, only IV nitrates are used for acute heart failure, and the proportion of patients previously treated with topical nitrates for chronic heart failure was similar in both groups (6%). Sixth, a short term prognosis endpoint (30 days) was chosen to capture the overall effect of the tested care bundle and this endpoint may have been influenced by post-ED therapy in the ward that is not standardized.

Seventh the rate of treatment of acute coronary syndrome was low in both groups, suggesting a suboptimal treatment of this precipitating factor, which in turn may have limited the benefit of the intervention.

Conclusion

Among older patients with acute heart failure, use of a guideline-based comprehensive care bundle in the emergency department compared with usual care did not result in a statistically significant difference in the number of days alive and out of the hospital at 30 days. Further research is needed to identify effective treatments for acute heart failure in older patient. a Differences were adjusted for intervention, time period and cluster size (categorical) as fixed effects and cluster as a random effect.

b Furosemide was the only iv diuretic used in participating centers.

c Cumulative dose of nitrates over the period of time that includes boluses (first hour) and infusion (between first hour and 4 th hour), among patients that were treated with nitrates.

d Given the small numbers, no analysis was performed for the dual antiplatelet agents variable.

e Including cardiac intensive care unit. 3: Study endpoints in the primary analysis. IQR: interquartile range. RR: risk ratio a Difference, ratios and risk ratios were adjusted for time period and cluster size (categorical) as fixed effects and cluster as a random effect. The difference is expressed as intervention minus control, and the ratio intervention/control. b Given the small numbers, no analysis was performed for the 2-fold rise in creatinine level variable.

Figure 1 :

 1 Figure 1: Flow of patients and clusters in the ELISABETH trial. The order in which

Figure 2 :

 2 Figure 2: Cumulative incidence of all-cause mortality. Shading for the 95%

Table 1 :

 1 Mortality data were missing for 4 patients who are not included in this analysis baseline characteristics. SD: standard deviation. ARB: angiotensin receptor blockade.

	Variable		Intervention (n=199)	Usual Care (n=303)	Normal values
			n	n
	Age, median (IQR), y		199 87.0 [81.0 ; 90.0]	303 87.0 [81.0 ; 91.0]
	Sex		199	303
	Women, n (%)		112 (56.3)	186 (61.4)
	Men, n (%)		87 (43.7)	117 (38.6)
	Comorbidities, n (%)	a	199	303
	Chronic pulmonary disease	39 (19.6)	46 (15.2)
	Chronic heart failure		111 (55.8)	302 158 (52.3)
	Chronic kidney disease		49 (24.6)	73 (24.1)
	Diabetes		54 (27.1)	92 (30.4)
	Myocardial infarction		80 (40.2)	91 (30.0)
	Vital signs at
	randomization, mean	199	303
	(SD)
	Heart rate, /min		86.3 (23.5)	86.6 (23.8)
	Oxygen saturation, (%)	185 90.6 (6.7)	286 91.1 (6.9)

b a Comorbidities were recorded by the emergency physician during emergency department visit. b Laboratory results were obtained as part of Emergency Department care.

Table 2 :

 2 Management in the Emergency Department. IQR, interquartile range;

Table

Acknowledgment:

The research was funded by a grant from Programme Hospitalier de Recherche Clinique -PHRC 2017 (Ministère de la Santé, Paris, France). The sponsor was Assistance Publique -Hôpitaux de Paris. The funders of the study had no role in design and conduct of the study; collection, management, analysis, and interpretation of the data; and preparation, review, or approval of the manuscript or the decision to submit for publication.

TS received fees for conferences or counselling and/or research grants from most companies marketing statins, from MSD, from AMGEN and from Sanofi. AM received speaker's honoraria from Orion, Otsuka, Philips, Roche and Servier. AM received fee as member of advisory board and/or Steering Committee and/or research grant from 4TEEN4, Adrenomed, Epygon, Neurotronik, Roche, Sanofi and Sphyngotec.

All other authors declare they have no conflict of interest with this study.

Data access

Yonathan Freund and Tabassome Simon had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. All data of the study can be provided upon request to the corresponding author. Data sharing statement: See online supplement 2.

Authors' contribution

YF conceived the study. YF, AM, MC and TS contributed to the study protocol. QD, SD, YY, JG, TC, ALF, JT, TD, CO, XB, MK, OG, JB, FB, SB included patients. MC provided statistical analysis. YF, AM and BB interpreted the results. YF drafted the manuscript. AM, BB, TS and MC provided substantial revisions. All authors agree to be accountable for all aspects of the work.


Methionine balanced diet improves performances and biosynthetic traffic in mammary epithelial cell

Eric Chanat, Sophie Lemosquet, A Leduc, L Bahloul, Marion Boutinaud

 (INRA, 2007). Goats (n=23) were slaughtered and mammary tissue was processed for Western blotting using secretory compartment specific markers. In goats fed Met balanced diets, milk protein yield and casein content significantly increased (P = 0.01), as was previously observed in cow. The amount of the endoplasmic reticulum (ER) markers Calnexin and ERLIN2 decreased (20%, P ≤ 0.05) in goats fed the LE diet. Met balanced diets had the opposite effect on both markers (20%, P ≤ 0.05) and on protein disulfide isomerase (45%, P ≤ 0.05). These observations are in agreement with a positive effect of Met on the activity of the ER, the site where milk specific proteins and lipids are synthesized. On the other hand, a specific marker of the exit site of the Golgi apparatus and secretory vesicles formation (AP1) decreased with the LE diet (25%, P ≤ 0.05) and its highest level was found in goats fed Met balanced diet at AE supply. Concerning ßCOP, a marker of intra Golgi transport, its variation clearly evoked a decrease in membrane transport at LE diets. These data show that energy level has a direct impact on membrane traffic in the secretory pathway of mammary epithelial cell while suggesting that Met improves ER activity and has the tendency to further promote intracellular transport of milk components and, ultimately, their secretion.

  E. Chanat*, S. Lemosquet*, A. Leduc*, L. Bahloul ‡, M. Boutinaud* *INRAE, Agrocampus Ouest, PEGASE. 16, Le clos, 35590, Saint-Gilles, France ‡CERN, Adisseo France S.A.S. 6, route noire, 03600 Commentry, France Methionine balanced diet improves performances and biosynthetic traffic in mammary epithelial cell Methionine (Met) or energy supplementation increase milk protein and fat yields in cow. Here we investigated whether this could be explained by increasing flow of milk components in the secretory pathways of mammary epithelial cells. Multiparous Alpine goats (mid lactation, n=47), grouped by levels of expression of CSN1S1, were assigned to 4 treatments in a randomized complete block design. Treatments were: LE (Low Energy), LEMet (LE, balanced Met), AE (Adequate Energy) and AEMet (AE, balanced Met) for 5 weeks. Diets consisted in a fixed amount of hay per group with individual amount of energy concentrates (LE vs. AE: 1.47 vs. 1.54 Mcal/kg DM). Isopropyl ester of 2hydroxy-4-methylthio butanoic acid (HMBi 0.24% concentrate DM) was incorporated in the LEMet and AEMet concentrates based on 100% cow Met requirement


Davar Ali
email: daverali@karabuk.edu.tr

Mehmet Ozalp
email: mozalp@karabuk.edu.tr

Sebastien B G Blanquer
email: sebastien.blanquer@umontpellier.fr

Selis Onel

A CFD analysis

Keywords: Bone scaffolds, Permeability, Wall shear stress, Minimal surface architectures, Lattice-based architectures

come

Permeability and fluid flow-induced wall shear stress in bone scaffolds with TPMS and lattice architectures:

Introduction

Culturing cells from the patient in scaffolds before their actual implantation into tissues is a promising approach that can accelerate the treatment process [START_REF] Chen | Bioreactors for tissue engineering[END_REF]. Stem cells have been used extensively in such processes for differentiation to other phenotypes [START_REF] Cheng | Promoting osteogenic differentiation in pre-osteoblasts and reducing tibial fracture healing time using functional nanofibers[END_REF][START_REF] Sierra-Sanchez | Epithelial In vitro Differentiation of Mesenchymal Stem Cells[END_REF] due to their ability to respond to both biochemical and biophysical cues around them. The phenotype of stem

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Journal Pre-proof cells can be regulated depending on the signals they receive [START_REF] Miyashita | Mechanical forces induce odontoblastic differentiation of mesenchymal stem cells on three-dimensional biomimetic scaffolds[END_REF][START_REF] Yang | Combinatorial Extracellular Matrices for Human Embryonic Stem Cell Differentiation in 3D[END_REF]. Biophysical signals involve those that arise from the changes in the mechanical properties of the matrix and substrate, such as strain and stiffness, substrate topography, hydrostatic pressure, fluid flowinduced shear stress, and electromagnetic field [START_REF] Prendergast | Biophysical stimuli on cells during tissue differentiation at implant interfaces[END_REF][START_REF] Maul | Mechanical stimuli differentially control stem cell behavior: morphology, proliferation, and differentiation[END_REF][START_REF] Govey | Biophysical Regulation of Stem Cell Differentiation[END_REF].

Wall shear stress (WSS) plays a significant role in the differentiation and proliferation of cells in perfusion bioreactors that include relatively more dynamic cell culture systems [START_REF] David | A Perfusion Bioreactor for Engineering Bone Constructs: An In Vitro and In Vivo Study[END_REF].

Differences in shear stress create a ubiquitous environmental signal that can be sensed by stem cells during differentiation or circulation in a perfusion bioreactor [START_REF] Toh | Fluid shear stress primes mouse embryonic stem cells for differentiation in a self-renewing environment via heparan sulfate proteoglycans transduction[END_REF]. The contribution of shear stress to the differentiation of pluripotent stem cells toward endothelial [11], cardiac [START_REF] Sargent | Hydrodynamic modulation of embryonic stem cell differentiation by rotary orbital suspension culture[END_REF], hematopoietic [START_REF] Adamo | Biomechanical forces promote embryonic haematopoiesis[END_REF], and osteoblast [START_REF] Reilly | Mesenchymal stem cell responses to mechanical stimuli[END_REF] phenotypes has been widely reported. A study on the role of WSS during the initial deposition of cells on the scaffolds [START_REF] Melchels | The influence of the scaffold design on the distribution of adhering cells after perfusion cell seeding[END_REF] has shown that local WSS is the main factor controlling the extent of cell deposition, which is strongly influenced by the architecture of the pore network of the scaffold. Evaluation of the degradable behaviors of scaffolds, specifically of biodegradable polymers employed as implants in the body, is another important issue for clinical applications. Studies on the degradation effects of flow behavior of fluids in polymeric scaffolds of varying porosity and permeability have shown that lower values of porosity and permeability caused higher rates of degradation with shorter scaffold life due to higher initial stiffness [START_REF] Li | The effect of mechanical loads on the degradation of aliphatic biodegradable polyesters[END_REF]. Therefore, determining and prediction of fluid flow-induced shear stress on the walls of a scaffold can be an effective step in controlling the perfusion in cell culture processes.

WSS is defined as the force per unit area applied by the wall surface on the fluid in a direction on the local tangent plane [START_REF] Katritsis | Wall Shear Stress: Theoretical Considerations and Methods of Measurement[END_REF]. Its measurement is not practical due to the need for embedding very sensitive sensors on the walls [START_REF] Amili | Evaluation of a film-based wall shear stress measurement technique in a turbulent channel flow[END_REF][START_REF] Liu | An improved wall shear stress measurement technique using sandwiched hot-film sensors[END_REF]. This difficulty becomes more critical in the case of tissue scaffolds that possess a porous structure in the micro scale [START_REF] Ali | Permeability and fluid flow-induced wall shear stress of bone tissue scaffolds: Computational fluid dynamic analysis using Newtonian and non-J o u r n a l P r e -p r o o f Journal Pre-proof Newtonian blood flow models[END_REF][START_REF] Ali | Finite element analysis of mechanical behavior, permeability and fluid induced wall shear stress of high porosity scaffolds with gyroid and latticebased architectures[END_REF].

Computational simulations of biomaterials have recently gained significant attention with the

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Journal Pre-proof capacity to provide immediate results as an alternative and economical approach compared to experimental analyses [START_REF] Ali | Finite element analysis of boron nitride nanotubes' shielding effect on the stress intensity factor of semielliptical surface crack in a wide range of matrixes using RVE model[END_REF][START_REF] Gómez | Design and properties of 3D scaffolds for bone tissue engineering[END_REF]. Numerous computational fluid dynamics (CFD) analyses have been conducted to evaluate the permeability and WSS of various types of scaffolds [START_REF] Melchels | The influence of the scaffold design on the distribution of adhering cells after perfusion cell seeding[END_REF][START_REF] Marin | The inter-sample structural variability of regular tissueengineered scaffolds significantly affects the micromechanical local cell environment[END_REF][START_REF] Thondapu | Endothelial shear stress 5 years after implantation of a coronary bioresorbable scaffold[END_REF][START_REF] Truscello | Prediction of permeability of regular scaffolds for skeletal tissue engineering: A combined computational and experimental study[END_REF]. Lesman and et al. employed CFD simulations to show that cellular growth within 3D scaffolds caused the WSS to increase due to contraction in the channels of the scaffolds during perfusion cell culturing [START_REF] Lesman | Modeling of Flow-Induced Shear Stress Applied on 3D Cellular Scaffolds: Implications for Vascular Tissue Engineering[END_REF]. Zhao et al. studied the parametric impacts of the geometric features of scaffolds, such as architecture, pore size, and porosity on the WSS in the pores employing a fluid-structure interaction model for various cases of loading during fluid perfusion, mechanical compression, as well as a combination of the two [START_REF] Zhao | Quantification of fluid shear stress in bone tissue engineering scaffolds with spherical and cubical pore architectures[END_REF]. Their results showed that specifically pore size substantially influenced the mechanical stimulation within the scaffold and combined application of fluid perfusion with mechanical load amplified the WSS significantly. Marin and Lacroix examined the effects of inter-sample structural variability of regular scaffolds on the magnitude and distribution of WSS using CFD calculations [START_REF] Marin | The inter-sample structural variability of regular tissueengineered scaffolds significantly affects the micromechanical local cell environment[END_REF]. Their results showed that geometric inconsistencies instigated high variability in velocity and WSS among the samples.

Another parameter that plays an important role in the success of scaffolds is their intrinsic permeability [START_REF] Gómez | Design and properties of 3D scaffolds for bone tissue engineering[END_REF]. A permeable scaffold should allow for unhindered flow and diffusion of cell culture media through its pores in order to supply oxygen and nutrition to the cells [29].

Prediction of the permeability in scaffolds using computational simulations is a common practice in tissue engineering [START_REF] Ali | Permeability and fluid flow-induced wall shear stress of bone tissue scaffolds: Computational fluid dynamic analysis using Newtonian and non-J o u r n a l P r e -p r o o f Journal Pre-proof Newtonian blood flow models[END_REF][START_REF] Truscello | Prediction of permeability of regular scaffolds for skeletal tissue engineering: A combined computational and experimental study[END_REF]. In a previous study, we conducted a finite element analysis to identify and compare the effects of two different structures, namely gyroid and rectangular lattice structures, on the permeability, fluid-induced WSS, elastic modulus, and compressive strength in highly porous scaffolds. Our results signified the potent effects of porosity on the permeability and the WSS, where higher porosity caused the permeability to increase while WSS, elastic modulus, and compressive strength values decreased [START_REF] Ali | Finite element analysis of mechanical behavior, permeability and fluid induced wall shear stress of high porosity scaffolds with gyroid and latticebased architectures[END_REF].

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In the design of bone scaffolds, the common goal in general is to mimic the structure of the trabecular (cancellous or spongy) bone [START_REF] Torres-Sanchez | Porosity and pore size effect on the properties of sintered Ti35Nb4Sn alloy scaffolds and their suitability for tissue engineering applications[END_REF] found in the human body. The trabecular bone has a greatly porous structure, accordingly, the design of high porosity scaffolds is the main criterion in bone tissue engineering. In response to this necessity of bone scaffolds, the triply periodic minimal surface (TPMS) and the lattice-based architectures are the two groups of scaffolds that are used widely [START_REF] Ali | Finite element analysis of mechanical behavior, permeability and fluid induced wall shear stress of high porosity scaffolds with gyroid and latticebased architectures[END_REF][START_REF] Al-Ketan | Topology-mechanical property relationship of 3D printed strut, skeletal, and sheet based periodic metallic cellular materials[END_REF]. However, the effects of geometrical parameters on the permeability and WSS for different scaffold architectures are critical and still remain less understood due to the diversity of scaffold structures. Therefore, analyzing and comparing the permeability and WSS of these two groups of scaffolds can be a very effective step in selecting and designing bone scaffolds.

In this study, we built eight different scaffolds computationally with a constant porosity of 80%. We used the TPMS and lattice-based structures with each based on four different geometries. These scaffolds were then exploited for quantification of their permeability and WSS using CFD analysis.

Materials and Methods

Scaffold models

Total of eight scaffolds were designed using the SolidWorks 2017 software. Four of the scaffolds were built in the lattice-based architecture using one of the double-diamond, truncated-octahedron [START_REF] Rodríguez-Montaño | Comparison of the mechanobiological performance of bone tissue scaffolds based on different unit cell geometries[END_REF], octet [START_REF] Egan | Computationally designed lattices with tuned properties for tissue engineering using 3D printing[END_REF] and one optimized-lattice [START_REF] Sercombe | Failure modes in high strength and stiffness to weight scaffolds produced by Selective Laser Melting[END_REF] structures. A strut size of 200 µm was selected for the scaffolds [START_REF] Van Bael | The effect of pore geometry on the in vitro biological behavior of human periosteum-derived cells seeded on selective laser-melted Ti6Al4V bone scaffolds[END_REF]. The other four scaffolds were designed using the TPMS architecture in one of the double-diamond [START_REF] Rittman | Direct visualisation of lipid bilayer cubic phases using Atomic Force Microscopy[END_REF], gyroid [START_REF] Blanquer | Surface curvature in triply-periodic minimal surface architectures as a distinct design parameter in preparing advanced tissue engineering scaffolds[END_REF], schwarz-primitive [START_REF] Lee | Regularized Dirac delta functions for phase field models[END_REF] and F-RD [39] type geometries. The primary surfaces in TPMS scaffolds were created using the k3dsurf (k3dsurf.sourceforge.net) software application, which has the capability of visualizing parametric equations in 3-D and more. The outputs of this software, in the form of mesh files with .obj extension, were imported to the SolidWorks software and were thickened

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Journal Pre-proof geometrically to obtain the desired porosity. A wall thickness of 100 µm was selected for TPMS scaffolds [START_REF] Ali | Finite element analysis of mechanical behavior, permeability and fluid induced wall shear stress of high porosity scaffolds with gyroid and latticebased architectures[END_REF]. Trigonometric functions of x, y and z that were used in building the TPMS scaffold structures are shown in table 1.

TABLE 1 Parametric equations for TPMS structures

Governing equations in CFD analysis

Navier-Stokes relation for fully developed flow of an incompressible fluid with constant density and viscosity was used in the CFD analysis [START_REF] Vossenberg | Darcian permeability constant as indicator for shear stresses in regular scaffold systems for tissue engineering[END_REF]: WSS, represented by 𝜏 𝜔 , is the tangential drag force exerted by the fluid flowing across the surface of the scaffolds [START_REF] Egger | Development and Characterization of a Parallelizable Perfusion Bioreactor for 3D Cell Culture[END_REF] and is given by:

𝜏 𝜔 = µ 𝜕𝑢 𝜕ℎ ( 2
)
where u denotes the flow velocity and h represents the x-, y-, and z-directions [START_REF] Gooch | Encyclopedic Dictionary of Polymers[END_REF].

Permeability, k, of the scaffolds was calculated using Darcy's law [START_REF] Gómez | Design and properties of 3D scaffolds for bone tissue engineering[END_REF]:

k = QμL A∆P (3)
where Q, , µ, A, and represent the inlet fluid flow rate (m 3 /s), dynamic fluid viscosity (kg/m.s), model length (m), inlet cross section area (m 2 ), and pressure drop (Pa), respectively.

Fluid properties and boundary conditions in CFD

A 1000 kg/m 3 density and a 0.0037 Pa.s dynamic viscosity (for cell culture media with 5% wt/wt dextran) were assigned to the fluid properties [START_REF] Sinha | Endothelial cell alignment as a result of anisotropic strain and flow induced shear stress combinations[END_REF]. We assumed an entirely liquid cell culture media with no solid particles for ease of calculations. The inlet velocity was selected as 0.1 mm s ⁄ suitable for flow in bone scaffolds. The walls of the scaffolds were assumed to be hydrophilic and the no-slip condition was applied [START_REF] Truscello | Prediction of permeability of regular scaffolds for skeletal tissue engineering: A combined computational and experimental study[END_REF][START_REF] Voronov | Computational modeling of flow-induced shear stresses within 3D salt-leached porous scaffolds imaged via micro-CT[END_REF]. The outlet gauge pressure was adopted as zero [START_REF] Marin | The inter-sample structural variability of regular tissueengineered scaffolds significantly affects the micromechanical local cell environment[END_REF][START_REF] Truscello | Prediction of permeability of regular scaffolds for skeletal tissue engineering: A combined computational and experimental study[END_REF][START_REF] Lesman | Modeling of Flow-Induced Shear Stress Applied on 3D Cellular Scaffolds: Implications for Vascular Tissue Engineering[END_REF] (Figure 2). The pressure drop used in equation (3) to calculate the permeability and WSS of the scaffolds was computed using the Ansys Fluent software.

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Journal Pre-proof CFD analysis was performed assuming steady-state fluid flow in 3-D scaffold geometries that were meshed using tetrahedral elements [START_REF] Gómez | Design and properties of 3D scaffolds for bone tissue engineering[END_REF]. In order to ensure the independence of the results to mesh size, the sensitivity of the pressure drop in the inlet to the number of elements for each model was analysed. Table 3 represents the number of elements in each scaffold.

The residual sensitivity criterion for convergence was set as 1e-5.

Results

Fluid flow dynamics within scaffolds

Simulation results for pressure profile in all scaffold geometries have shown that the highest pressure occurred in the inlet and gradually tended to zero in the outlet region confirming the boundary conditions used in the models. Figure 3 illustrates the pressure profile within the 3-D geometries in the counter format.

Generally, the exerted pressure in lattice-based scaffolds is lower than that observed in TPMS models. An intra-group comparison in lattice-based scaffolds showed that the highest pressure was observed in the octet model and the lowest was observed in the optimum-lattice model. Amongst the geometries used in the TPMS scaffolds group, the highest pressure was observed in the double-diamond structure while the lowest was marked in the gyroid model.

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To determine the reliability and precision of the CFD models, their pressure drop, ∆P, responses to the changes in inlet velocity were investigated for four different inlet velocities, namely 0.1, 0.25, 0.5, and 1 mm/s [START_REF] Truscello | Prediction of permeability of regular scaffolds for skeletal tissue engineering: A combined computational and experimental study[END_REF]. Figure 4 illustrates the logarithmic curve-fitting plots of the assigned inlet velocity and the ∆P obtained for each of the models. In all scaffolds, the pressure drop correlated extremely well with the inlet velocity with R 2 > 0.999.

FIGURE 4

Correlation between assigned inlet velocity and calculated ∆P in the scaffolds.

Permeability

The permeability of each scaffold was calculated with reference to the average ∆P at the inlet derived from the CFD models and Darcy's law using equation (3). Figure 5 shows the calculated permeability values for each scaffold.

J o u r n a l P r e -p r o o f For the case of similar porosity, lattice-based scaffolds showed substantially higher permeability than did the TPMS models except for the gyroid architecture. To validate the CFD results, the permeability value obtained for the gyroid model in this study as 5.36*10 -9 m 2 was compared to that (6.7*10 -9 m 2 ) derived in our previous work [START_REF] Ali | Finite element analysis of mechanical behavior, permeability and fluid induced wall shear stress of high porosity scaffolds with gyroid and latticebased architectures[END_REF] using only two unitcells with the periodic boundary condition. The difference found was about 25%, which was caused by an underestimation of the magnitude of the pressure drop in the previous model by using limited number of unit-cells.

WSS

The WSS profile contours on the walls within all scaffolds are shown in Figure 6. Given that the WSS changes linearly with the velocity gradient (equation 2), the maximum WSS was found to occur along the narrower regions of the channels in the scaffold causing higher fluid flow rates. For all models, the calculated maximum WSS varied between 41.5 and 81.4 mPa.

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The range of average WSS values that would initiate osteogenic differentiation of mesenchymal stem cells (MSC) used in the literature [START_REF] Zhao | Quantification of fluid shear stress in bone tissue engineering scaffolds with spherical and cubical pore architectures[END_REF] is between 0.1-10 mPa and an average WSS smaller than 15 mPa is often recommended for differentiation of MSCs to osteoblasts and osteocytes in perfusion bioreactor culture systems [START_REF] Li | Effects of Flow Shear Stress and Mass Transport on the Construction of a Large-Scale Tissue-Engineered Bone in a Perfusion Bioreactor[END_REF][START_REF] Olivares | Finite element study of scaffold architecture design and culture conditions for tissue engineering[END_REF][START_REF] Zhao | Multiscale fluid-structure interaction modelling to determine the mechanical stimulation of bone cells in a tissue engineered scaffold[END_REF].

Discussion

In this work, we conducted a computational study to understand the effects of scaffold architecture on the permeability and WSS using eight different models with the same porosity (80%). The calculated values of the permeability were in the range of 1.85 -5.62 × 10 -9 𝑚 2 .

These results are in agreement with the value, 5.13 × 10 -9 𝑚 2 , that has been found for human trabecular bone permeability [49]. Although porosity is usually described as the main factor affecting the permeability of scaffolds [START_REF] Gómez | Design and properties of 3D scaffolds for bone tissue engineering[END_REF]29,[START_REF] Rahbari | Predicting permeability of regular tissue engineering scaffolds: scaling analysis of pore architecture, scaffold length, and fluid flow rate effects[END_REF], the results of this study suggest that under the condition of similar porosity, scaffold geometry is another important factor.

The simulation results shown in Figure 5 indicate that the permeability of the lattice-diamond model is three times as large as that of the double-diamond scaffold calculated at the same porosity. This concludes that the permeability of scaffolds is highly dependent on their architecture as well as on their porosity. Table 4 provides the differences in the geometric parameters in dimensionless form used for each of the eight scaffolds in this study, which can be used to understand the substantial discrepancy in the calculated permeability of scaffolds with different architectures. D/d represents the ratio of the widest pore size to the narrowest and A/A0 represents the ratio of the area with no obstacles to the total inlet area. The area with no obstacles within the scaffolds can be described as the cross-sectional area, through which the fluid flows from inlet to outlet without encountering any walls.

J o u r n a l P r e -p r o o f As it can be seen in Table 4, the highest permeability value was found for the lattice-diamond structure while the lowest value was obtained for the double-diamond model, which possess the lowest and the maximum D/d values, respectively. Therefore, it can be concluded that increasing the extent of variations in the channel size, which implies an increase in tortuosity, in the TPMS scaffolds, has caused their permeability to decrease. Accordingly, it is logical to expect that the octet model will be the second most permeable scaffold amongst the latticebased group of scaffolds, but on the contrary it is found to possess the lowest permeability amongst them. This is where the ratio given by 𝐴/𝐴 0 needs to be considered as the second factor affecting the permeability of the scaffolds. The octet model, due to its very low value of 𝐴/𝐴 0 , presented the minimum permeability amongst the lattice-based scaffolds. With regards to both groups of scaffolds, the results imply that the scaffold permeability was primarily affected by any variations in the channel size. The existence and/or absence of straight-gaps along the flow streamline between the inlet and outlet locations was the second factor affecting the scaffold permeability.

It is previously shown that a heterogeneous WSS distribution causes non-uniform cell differentiation within the scaffolds [START_REF] Marin | The inter-sample structural variability of regular tissueengineered scaffolds significantly affects the micromechanical local cell environment[END_REF][START_REF] Lesman | Modeling of Flow-Induced Shear Stress Applied on 3D Cellular Scaffolds: Implications for Vascular Tissue Engineering[END_REF][START_REF] Egger | Development and Characterization of a Parallelizable Perfusion Bioreactor for 3D Cell Culture[END_REF]. Therefore, understanding the distribution statistics of WSS on the scaffolds is crucial and necessary. The distribution curves and modality of WSS with respect to the percent area of the walls in the eight scaffolds with different architectures are shown in Figure 8. A coefficient of variance larger than zero in all models showed that the WSS was not distributed equally in any of the models (Figure 8). Amongst the models in both the TPMS and lattice-based architecture groups, the lattice-diamond model with the lowest coefficient of variance (0.42) showed a relatively more equal distribution of the WSS on the walls of the scaffold and the Schwartz-primitive model with the highest coefficient of variance (1.39)

showed a relatively less uniform distribution of WSS.

A skewness value that is higher than zero indicates that WSS values greater or smaller than the mean WSS were not distributed symmetrically and that the relative sizes of the two tails of the distribution curve are not similar. The gyroid model, which showed very small skewness (-0.01), presented a relatively more symmetrical WSS distribution than did the other scaffolds.

Kurtosis measures the tail-heaviness of a distribution and the calculated kurtosis values in In an overview of the permeability results, as well as the WSS distribution statistics (modality, standard deviation, coefficient of variance, skewness, and kurtosis), the latticediamond model presented an outstanding performance amongst the eight models. In addition, the lattice-diamond model, due to its architecture, only possesses convex surfaces, which may contribute to osteogenic differentiation enhancement of human MSCs [START_REF] Werner | Surface Curvature Differentially Regulates Stem Cell Migration and Differentiation via Altered Attachment Morphology and Nuclear Deformation[END_REF].

It is worth noting that in this simulation study, we considered the cell culture medium as a Newtonian fluid, i.e. constant viscosity liquid, with no solid particles for simplification of the system of governing equations. Cell culture media involving serum may behave as a non-J o u r n a l P r e -p r o o f

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Newtonian fluid and such shear rate dependent viscosity can cause changes in WSS distribution modality in the scaffolds [START_REF] Wyma | Non-Newtonian rheology in suspension cell cultures significantly impacts bioreactor shear stress quantification[END_REF]. It may be necessary to employ WSS quantification methods based on variable viscosity for studies involving non-Newtonian media.

Conclusion

We used CFD analysis to compare the permeability and the distribution statistics of fluidinduced WSS in high-porosity scaffolds, which are widely used in bone tissue engineering.

Results of the study can be summarized as follows:

-The permeability of scaffolds is governed by their architecture and any changes in the size of channels (narrowing) reduce permeability. Therefore, scaffolds with a less variable pore size are more permeable.

-The ratio of the area with no obstacles to the total inlet area, 𝐴/𝐴 0 , plays a significant role in determining the permeability of the scaffolds in addition to D/d, which represents the ratio of the widest pore size to the narrowest.

-WSS and its distribution in the scaffold are influenced by the architecture of the scaffold.

-It is not possible to find a correlation between the architecture of the scaffolds and their WSS distribution statistics, which suggests that the design of scaffolds with the desired WSS distribution is not a straightforward task in tissue engineering.

-The results of this comparative study shed more light on the effective selection of architecture in the design of highly porous scaffolds.

  TPMS architecture Equations f (x,y,z)=0 Gyroid cos(x)sin(y)+cos(y)sin(z)+cos(z)sin(x) Schwarz primitive cos(x)+cos(y)+cos(z) Double diamond cos(x)cos(y)cos(z)-sin(x)sin(y)sin(z) F-RD 3[cos(x)+cos(y)+cos(z)]+8[cos(x)cos(y)cos(z)] The model unit cells were repeated in the direction of the three axes (x, y, and z) four times to obtain a cubic model made-up of 64 unit cells of any of the architectures listed. Fluid domains in the 3-D structures were obtained by subtracting the geometrical parts from an enclosing box. All eight unit cells employed are isometric and have symmetric architecture in the x, y, and z axes. The geometrical parameters of the unit cells are presented in table 2.

Figure 1

 1 Figure1shows a representative picture of each scaffold created in this study including the

FIGURE 1

 1 FIGURE 1 The unit cells and the corresponding 3-D scaffolds with the solid (gray) and fluid

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  n a l P r e -p r o o f Journal Pre-proof ρ ∂u ∂t -μ∇ 2 u + ρ(u. ∇)u + ∇p = F , ∇. u = 0 (1) where , u, and µ represent the density (kg/m 3 ), velocity (m/s), and the dynamic viscosity of fluid (kg/m.s). ∇ denotes the del operator and p denotes the pressure (Pa). F represents the forces, such as gravity and centrifugal force, where F = 0 in the specific problem studied here [40, 41].

FIGURE 2 A

 2 FIGURE 2 A velocity of 0.1 mm s ⁄ and no-slip boundary condition were assigned at the

FIGURE 3 3 -

 3 FIGURE 3 3-D simulation results showing pressure profiles in counter form for a) lattice-

FIGURE 5

 5 FIGURE 5 Calculated permeability of each scaffold using Darcy's law.

FIGURE 6

 6 FIGURE 6 WSS contours for the a) lattice-diamond, b) octet, c) truncated-octahedron, d)

FIGURE 7

 7 FIGURE 7 Calculated values of average WSS in scaffolds with eight different structures

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  n a l P r e -p r o o f

FIGURE 8

 8 FIGURE 8 Histograms for WSS distribution in scaffolds with eight different architectures,

Figure 8

 8 Figure 8 shows the range of variation in WSS values in the scaffolds. The optimum-lattice

TABLE 2

 2 Geometrical parameters for the lattice-based and TMPS scaffold models. L, s, D, and d represent the model size, unit cell length, pore size in the widest area and pore size in the narrowest area, respectively.

	Model parameters (mm) J o u r n a l P r e -p r o o f Lattice-based scaffolds TPMS scaffolds Length in 𝑥, 𝑦, 𝑧 calculated in [-𝜋, 𝜋] boundary Lattice-diamond Truncated-octahedron Octet Optimized-lattice Double-diamond Gyroid Schwarz primitive F-RD
	L	3.80	4.64	5.60	4.00	10.16	6.24	4.00	7.36
	S	0.95	1.16	1.40	1.00	2.54	1.56	1.00	1.84
	D	0.49	0.80	0.79	0.51	1.17	0.68	0.90	0.48
	D	0.49	0.15	0.33	0.20	0.20	0.68	0.40	0.39

TABLE 3

 3 Number of elements in each scaffold in the CFD models

	Lattice-	Truncated-	Octet	Optimized-	Double-	Gyroid	Schwarz-	F-RD
	diamond	octahedron		lattice	diamond		primitive
	5645734	9107611	17549250	8341502	1441009	14658043 5062830	10989006

TABLE 4

 4 Architectural parameters in dimensionless form and the respective calculated permeability for each scaffold-geometry

	Model	Lattice-diamond	Octet	Optimize d-lattice	Truncated-octahedron	Double-diamond	F-RD	Schwarz-primitive	Gyroid
	𝐷/𝑑	1.00	2.38	2.56	5.26	5.88	3	2.27	1
	𝐴/𝐴 0 (%)	15.68	9.10	30.60	35.00	0.00	4.80	21.50	10.63
	Permeability (10 -9 𝑚 2 )	5.62	4.81	5.48	5.06	1.85	3.46	2.48	5.36

Highlights:

-Minimal surface and lattice-based architectures exhibit different flow behavior -Scaffold permeability can vary up to three times depending on the architecture -Scaffolds with minimal variation in channel size exhibit the highest permeability -Wall shear stress distribution does not correlate with scaffold architecture -Scaffold design with desired wall shear stress distribution is problematical -Architecture selection in designing highly porous scaffolds will affect bioactivity J o u r n a l P r e -p r o o f