Nelly Muller

Ioannis Sarantitis

MD Marie Rouanet

MD Louis De Mestier

MD Christopher Halloran

PhD William Greenhalf

MD PhD Claude Férec

PhD Emmanuelle Masson

MD Philippe Ruszniewski

MD Philippe Lévy

MD PhD John Neoptolemos

MD Louis Buscail

MD PhD Vinciane Rebours
email: vinciane.rebours@aphp.fr

Vinciane Pr

Rebours

Natural history of SPINK1 germline mutation related-pancreatitis

Keywords: CFTR, cystic fibrosis transmembrane conductance regulator gene, CP, chronic pancreatitis, DM, diabetes mellitus, EPI, Exocrine Pancreatic Insufficiency, EUS, endoscopic ultrasound, IP, idiopathic pancreatitis, PDAC, pancreatic ductal adenocarcinoma, SPINK1, Serine Protease Inhibitor Kazal Type 1, SRP, SPINK1-related pancreatitis, CTRC, chymotrypsin C, PRSS1, serine protease SPINK1, genetic, pancreatitis, diabetes, pancreatic exocrine insufficiency, pancreatic cancer Research

Introduction

Advances in the field of clinical genetics and the availability of genetic testing at a larger scale in the general population have led to major progress in understanding the pancreatic inflammatory process and more particularly the causes of pancreatitis. The role of genetic variants is now better understood, providing an explanation of why some patients develop chronic pancreatitis early in life or why, on the other hand, only 5% of chronic alcohol drinkers will develop pancreatic disorders. Over the last two decades, several genes and multiple variants have been reported to have an effect on pancreatic homeostasis and act as potential risk factors for pancreatic inflammation. In everyday practice, however, only few genes, serine protease 1 (PRSS1), cystic fibrosis transmembrane conductance regulator gene (CFTR), serine protease inhibitor Kazal type 1 gene (SPINK1), chymotrypsin C gene (CTRC) and carboxypeptidase A1 (CPA1) are regularly analyzed in cases of idiopathic recurrent acute or chronic pancreatitis. Genetic testing for these cases should be performed within a context of a multidisciplinary approach, including genetic counselling when appropriate. PRSS1, encoding for cationic trypsinogen, is responsible for hereditary pancreatitis, an autosomal dominant disease with penetrance up to 93% [START_REF] Rebours | The natural history of hereditary pancreatitis: a national series[END_REF] . Its role in hereditary pancreatitis is wellestablished and is not a matter of debate. However, the role of heterozygous variants of CFTR, CTRC and SPINK1 genes in pancreatitis remains controversial. The prevalence of these variants in the general population is as high as 4% for CFTR, suggesting that there are other genetic and environmental modifying factors. SPINK1 (Serine Protease Inhibitor Kazal type 1), is a 4-exon gene located on chromosome 5, encoding for a 56 amino-acid protein. Its role is to maintain the integrity of exocrine pancreatic tissue by inhibiting prematurely activated intra-pancreatic trypsin, accounting for inhibition of 20% of trypsin activity [START_REF] Laskowski | Protein inhibitors of proteinases[END_REF] . Over 30 SPINK1 variants have been reported, the p.N34S mutation being the most frequently reported. An association between SPINK1 variants and pancreatitis was first reported by Witt et al. in 2000, who described the presence of the p.N34S variant in 18/85 (21%) of children with idiopathic pancreatitis (IP) [START_REF] Witt | Mutations in the gene encoding the serine protease inhibitor, Kazal type 1 are associated with chronic pancreatitis[END_REF] . Further studies reported SPINK1 mutations in 6.4% to 43% of patients with idiopathic pancreatitis [START_REF] Pfützer | SPINK1/PSTI polymorphisms act as disease modifiers in familial and idiopathic chronic pancreatitis[END_REF][START_REF] Threadgold | The p.N34S mutation of SPINK1 (PSTI) is associated with a familial pattern of idiopathic chronic pancreatitis but does not cause the disease[END_REF][START_REF] Truninger | Mutations of the serine protease inhibitor, Kazal type 1 gene, in patients with idiopathic chronic pancreatitis[END_REF][START_REF] Chen | Mutations of the pancreatic secretory trypsin inhibitor (PSTI) gene in idiopathic chronic pancreatitis[END_REF][START_REF] Midha | Idiopathic chronic pancreatitis in India: phenotypic characterisation and strong genetic susceptibility due to SPINK1 and CFTR gene mutations[END_REF] . However, the prevalence of heterozygous p.N34S mutation in the general population was estimated to be 1-2%, suggesting that the presence of SPINK1 p.N34S variant alone is not sufficient to explain the development of chronic pancreatitis (CP) [START_REF] Truninger | Mutations of the serine protease inhibitor, Kazal type 1 gene, in patients with idiopathic chronic pancreatitis[END_REF] . Therefore, the SPINK1 p.N34S variant can be considered as a disease modifier rather than a causative factor, leading to chronic pancreatitis, when additional risk factors for pancreatic inflammation such as environmental/lifestyle (alcohol or tobacco consumption) or genetic (known or yet unidentified) are present.

The natural history of SPINK1-related pancreatitis (SRP) is currently inadequately described and previous literature reports are still controversial. This is due to a variety of reasons including: limited number of small studies describing cohorts of 6 to 48 patients; genetic variants of unknown significance were included; evaluation for other risk factors is sometimes limited to tobacco consumption; finally, the frequent coexistence of other potential causes of pancreatitis [START_REF] Truninger | Mutations of the serine protease inhibitor, Kazal type 1 gene, in patients with idiopathic chronic pancreatitis[END_REF][START_REF] Midha | Idiopathic chronic pancreatitis in India: phenotypic characterisation and strong genetic susceptibility due to SPINK1 and CFTR gene mutations[END_REF][START_REF] Sandhu | Presence of SPINK-1 variant alters the course of chronic pancreatitis[END_REF] did not allow for clear conclusions to be drawn regarding the role of SPINK1 variants in pancreatitis. Furthermore the association between coexistent SPINK1 mutations and other germline mutations in genes involved in pancreatic diseases (trans-heterozygosity) needs to be further elucidated [START_REF] Sofia | Trans-heterozygosity for mutations enhances the risk of recurrent/chronic pancreatitis in patients with Cystic Fibrosis[END_REF] . The aim of this study was to describe epidemiological, genetic, clinical and pancreatic morphological features in a large, multicentre European cohort of patients with chronic pancreatitis who carry the minor allele of SPINK1 codon 34, and to compare these characteristics with a control population consisting of patients with idiopathic pancreatitis who carry wild-type SPINK1.

Patients and Methods

Patients

All consecutive SPINK1 mutation carriers with pancreatic symptoms from two French centers (Beaujon University Hospital, Clichy, and Rangueil University Hospital, Toulouse) and from the Royal Liverpool University Hospital, England, were identified from the local prospective cohorts dated from January 1 st 2000 to June 1 st 2018 and were included in this study.

SPINK1-related pancreatitis group

Inclusion criteria -Presence of at least one SPINK1 germline mutation -Pancreatic-related manifestations defined as acute or recurrent acute pancreatitis, chronic pancreatic pain, chronic pancreatitis or exocrine or endocrine pancreatic insufficiency with no other evident cause of pancreatitis.

-Extensive workup performed in order to rule out other causes of pancreatic disorders including clinical examination, biochemical analysis and imaging investigations including transabdominal ultrasound, CT scan, MRI (including MRCP, MR pancreas-specific protocol, and secretin-stimulated MRCP when indicated) or endoscopic ultrasound (EUS).

Indications for genetic testing

Genetic screening was performed when one or more of the following conditions were met: 1) Acute and recurrent acute pancreatitis with no identifiable cause following extensive investigations, 2) Chronic pancreatic pain, 3) Morphological abnormalities consistent with chronic pancreatitis and no identified cause 4) First attack of acute pancreatitis in young patients (< 35 years); or 5) In the case of a family history of pancreatitis.

Genetic testing included search for mutations in genes known to be associated with pancreatic inflammation: SPINK1 gene, cationic trypsinogen gene (PRSS1) and cystic fibrosis transmembrane regulator (CFTR). CTRC gene analysis was routinely performed only at the Beaujon and Toulouse centers.

Exclusion criteria

-All patients with potential causes of pancreatitis were excluded: chronic alcohol consumption (daily alcohol intake > 3 units (24 g of pure alcohol) per day in men, or > 2 units (16 g of pure alcohol) per day in women; hypercalcaemia >3 mmol/L; hypertriglyceridemia >10 mmol/L; ductal obstruction (ampullary adenoma, intraductal papillary mucinous neoplasm, cystic pancreatic neoplasm); posttraumatic duct stenosis or autoimmune pancreatitis.

-Presence of a PRSS1 mutation, because of the high penetrance, hence it would be adequate as a sole risk factor to explain the phenotype. Patients with CFTR or CTRC mutations or pancreas divisum were not excluded.

Control group, Idiopathic pancreatitis

The control group consisted of all patients with IP from Beaujon and Liverpool Hospitals.

Inclusion criteria were the same as for the SRP group but without any causes of pancreatitis after comprehensive workup including genetic testing for the PRSS1, SPINK1, CFTR and CTRC genes.

Data collection

The records of all included patients were retrospectively reviewed for data collection. The following data were collected: -General characteristics (age, gender, smoking status and number of pack-years, alcohol consumption as number of units of alcohol consumed per day, family history of PDAC, recurrent acute or chronic pancreatitis).

-Genetic characteristics (presence and type of SPINK1, PRSS1, CFTR or CTRC mutations).

-Clinical characteristics: date of first clinical manifestation (i.e. acute pancreatitis, pancreatic pain, exocrine or endocrine pancreatic insufficiency, cholestasis or PDAC); date of detection of imaging abnormalities including parenchymal or ductal calcifications, other ductal abnormalities, mass lesions, pseudocysts, porto-venous thrombosis; date of SRP diagnosis; date of diabetes mellitus (DM) diagnosis and type of treatment required; date of exocrine insufficiency diagnosis; date and cause of death.

-Endoscopic or surgical procedures performed for the management of acute and chronic pancreatitis-related complications.

Definitions

The diagnosis of CP was based on the presence of, at least, one of the following: pancreatic calcifications diagnosed by CT scan or endoscopic ultrasonography (EUS), moderate to marked pancreatic ductal abnormalities on gadolinium-enhanced MRI with magnetic resonance cholangiopancreatography or EUS or presence of exocrine pancreatic insufficiency. Isolated mild CP stigmata on EUS (heterogeneity of the pancreatic parenchyma, hyperechoic foci without shadowing) were also considered as CP. Acute pancreatitis was defined by acute abdominal pain with increased serum pancreatic enzyme levels over three times the upper limit of normal values. Pancreatic pain was defined as typical pancreatic pain without elevation of serum pancreatic enzyme or typical pancreatitis lesion at imaging procedure. It was described either as continuous (with no pain-free intervals) or episodic. Exocrine pancreatic insufficiency (EPI) was diagnosed in the case of clinical steatorrhea, faecal elastase-1 concentration lower than 100 µg/g of stool or a need for long-term oral pancreatic enzyme supplements. Diabetes mellitus was diagnosed if two venous blood fasting glucose concentrations were recorded ≥ 126 mg/dL (6.99 mmol/l) or if one was recorded ≥ 11 mmol/l post prandially. Insulin requirement was defined by the inefficacy of adequate diet (diet without sugar) and oral drugs (biguanides, sulfonylurea, alpha-glucosidase inhibitors) in preventing hyperglycemia. Cholestasis was defined as increased alkaline phosphatase levels 1.5 times above the upper limit of normal values associated with a dilated common bile duct. Pseudocyst was defined according to the Atlanta classification, as a round and ovoid collection of pancreatic fluid enclosed by a wall of fibrous or granulation tissue, which arises as a result of acute or chronic pancreatitis, occurring at least 4 weeks after onset of symptom [START_REF] Banks | Classification of acute pancreatitis--2012: revision of the Atlanta classification and definitions by international consensus[END_REF] . Acute venous thrombosis was defined as the presence of a cruoric thrombus in the porto-mesenteric system or the splenic vein as evidenced by imaging procedures. PDAC were all histologically proven. The number of cigarettes smoked per day and duration of smoking were expressed as pack-years. The follow-up period was defined as the interim between the date of the first symptom related to pancreatic disease and the date of the last visit or death.

Genetic data

Mutation screening of the SPINK1 and PRSS1 gene was performed by pyrosequencing, denaturing High Performance Liquid Chromatography (DHPLC) as previously described [START_REF] Maréchal | Two novel severe mutations in the pancreatic secretory trypsin inhibitor gene (SPINK1) cause familial and/or hereditary pancreatitis[END_REF][START_REF] Maréchal | Identification of a novel pancreatitis-associated missense mutation, R116C, in the human cationic trypsinogen gene (PRSS1)[END_REF] , or by High Resolution Melting (HRM) analysis: four primer pairs (Table 1 supplementary data) were designed to amplify the four exons and their immediate flanking sequences. PCR was performed in a 10 μl reaction mixture containing 4 μl of LightScanner® Master Mix (Idaho Technology Inc., Salt Lake City, UT), 0.3 µM of each primer (Table 1 supplementary data) and 50 ng of DNA. Cycling conditions consisted of an initial denaturation step at 94°C for 2 minutes, followed by 40 cycles of 30 seconds at 94°C, 30 seconds at a specific hybridization temperature (Table 1 supplementary data), 30 seconds at 72°C, and a final cycle of 30 seconds at 94°C and 30 seconds at 25°C. After amplification, melting analysis was performed on LightScanner® System (Idaho Technology Inc.). Samples showing a positive profile were sequenced using the BigDye™ Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) to identify mutations [START_REF] Maréchal | Two novel severe mutations in the pancreatic secretory trypsin inhibitor gene (SPINK1) cause familial and/or hereditary pancreatitis[END_REF][START_REF] Maréchal | Identification of a novel pancreatitis-associated missense mutation, R116C, in the human cationic trypsinogen gene (PRSS1)[END_REF] .

Exons and exon/intron junctions of the CFTR gene were screened by DHPLC [START_REF] Marechal | Complete and rapid scanning of the cystic fibrosis transmembrane conductance regulator (CFTR) gene by denaturing high-performance liquid chromatography (D-HPLC): major implications for genetic counselling[END_REF] or high-resolution DNA melting analysis [START_REF] Audrezet | Validation of high-resolution DNA melting analysis for mutation scanning of the cystic fibrosis transmembrane conductance regulator (CFTR) gene[END_REF] as previously described. Samples showing abnormal profiles were sequenced as previously described [START_REF] Marechal | Complete and rapid scanning of the cystic fibrosis transmembrane conductance regulator (CFTR) gene by denaturing high-performance liquid chromatography (D-HPLC): major implications for genetic counselling[END_REF][START_REF] Audrezet | Validation of high-resolution DNA melting analysis for mutation scanning of the cystic fibrosis transmembrane conductance regulator (CFTR) gene[END_REF] .

All exons of the CTRC gene and their immediate flanking sequences were analyzed by direct sequencing as previously described 16 .

Statistical analysis

Continuous variables were expressed as median interquartile range [IQR]) and compared using the Mann-Whitney test. Qualitative variables were expressed as frequencies (percentages) and compared using Chi² test or Fisher's exact test, whichever most appropriate. The age of onset and the prevalence of pancreatitis-associated symptoms and complications were compared between the SRP and the control groups. The actuarial risks of occurrence of chronic pancreatitis, exocrine pancreatic insufficiency, diabetes mellitus and cancer were estimated using the Kaplan-Meier method, and were compared between the SRP and control groups using a log-rank test. Durations and survivals were expressed as their medians with 95% confidence intervals. Genotype-phenotype correlations were analyzed by comparing the risk of developing pancreatic symptoms or complications in heterozygous versus homozygous p.N34S patients and in heterozygous p.N34S patients with co-mutations (CFTR and CTRC) versus heterozygous p.N34S patients without comutations (CFTR and CTRC), using univariate Cox regression hazard models. All analyses were two-sided. Any p-value <0.05 was considered significant. All analyses were performed using Prism® (version 6, Graphpad™) and SPSS® (version 20, IBM™).

Results

Population

Overall, 209 patients (male, 51.2%) were included in the SRP group and 302 patients with idiopathic pancreatitis (male, 48.0%) in the control group (Table 1). The median follow-up was 8.3 years (3.7-17.4) in the SRP group vs 5.3 years (2.5-8.8) in the control group (p <0.001). The percentage of smokers and ex-smokers was higher in the control group than in the SRP group (46.3% vs. 33.8%, p=0.005), as well as the median number of pack-years smoked (15 vs. 8, p<0.0001). The median age at last contact or death was 34.5 years (24.4-48.3) in the SRP group versus 50.1 years (34.9-61.5) in controls.

Genetic characteristic

Within the SRP group, most patients had missense mutations (200/209, 95.7%) and 6 patients had deletions (2.9%). Finally, 3 patients (1.4%) had nonsense mutations due to frameshift, including 2 unrelated patients who had a previously undescribed exon 3 mutation (p.C61FfsX2). Of the 200 patients with specific missense mutations, exon 1 was involved in 4 patients (2%), exon 3 in 182 patients (91%), exon 4 in 5 patients (2.5%), intron 2 in 3 patients (1.5%), intron 3 in 10 patients (5 %). Among them, 5 had compound heterozygous mutations. The most frequent specific mutation was p.N34S (exon 3), which was heterozygous or homozygous in 159 patients and 22 patients, respectively (79.5% and 10.5%). One patient had a previously undescribed exon 1 mutation (p.S16R). Other mutations, less frequent, were found in 28 patients. All data are showed in the supplementary data section, table 2. Of the 209 SRP patients only 207 and 55 patients were tested for CFTR and CTRC mutations respectively. A CFTR or CTRC mutation was found in 44 (21.3%) and 5 (9.1%) patients respectively. Of note, 2 patients from a same family (mother and son) had a triple SPINK1-CFTR-CTRC mutation. Among patients with CFTR mutations, mutations were mild (n=17), severe (n=6), composite heterozygote severe /mild (n=3), composite heterozygote with 2 severe mutations (n=1), uncertain (n=2).

Onset of symptoms

The median age at onset of symptoms was 20.1 years (95% CI, 17.5-22.8) in the SRP group and 41.2 years (95% CI, 35.2-45.2) in the IP group (p< 0.001). In the SRP group, the proportion of symptomatic patients (i.e. pancreatic pain, steatorrhea etc) was 49.8% at 20 years, 71.1% at 30 years, 85.1% at 40 years and 92.0% at 50 years (Figure 1). The median delay between the onset of symptoms and the diagnosis of SPINK1 mutation was 3.7 years (1-11.3).

Among the SRP patients, the existence of a p.N34S homozygous mutation was associated with a statistically non-significant tendency for earlier onset of symptoms compared to p.N34S heterozygous mutation (HR=1.47, 95% CI [0.93-2.33], p=0.10). Among the group of p.N34S heterozygous patients, the existence of a co-mutation on the CFTR or CTRC genes tended to be associated with a younger age at onset of symptoms (HR=1.31, 95% IC [0.89-1.92], p=0.18).

Acute pancreatitis and pancreatic pain

Regarding the presentation of pancreatitis, the proportion of patients who had at least one episode of acute pancreatitis was similar in both groups, although the median number of acute pancreatitis episodes was higher in the SRP group, with no difference in terms of severity (Table 1). The proportion of patients with pancreatic pain was higher in the SRP group than in the IP group (79.8% vs. 62.6%, p<0.001), but there was no difference regarding the type of pain, i.e., episodic vs. continuous (Table 1).

Chronic pancreatitis

About 70% of SRP patients had signs of CP, versus 54% in the IP group (p<0.001). The SRP patients more frequently developed ductal abnormalities (72.5% vs. 48.6%, p<0.001) and pancreatic calcifications (74.3% vs 64.8 %) but this result didn't reach statistical significance (p=0.07). The median age of CP diagnosis was younger, of approximately 20 years in the SRP group [34.3 (95% CI, 30.8-37.8) years vs. 57.3 (95% CI, 53.7-60.9) years, p<0.001]. The cumulative incidence of CP signs in SRP patients was 16.1% at 20 years, 38.5% at 30 years, 56.7% at 40 years and 76.0% at 50 years (Figure 2a). The median delay between the first symptoms and the diagnosis of CP was similar in both groups [6.9 years in the SRP group (95% CI, 4.5-9.3) vs. 5.9 years (95% CI 4.2-7.6) years in the IP group]. Conversely to the IP group, tobacco consumption in SRP group was not associated with earlier CP diagnosis (Figure 2b). The proportion of patients with intra-abdominal spleno-portal venous thrombosis and/or pseudocysts was similar (Table 1).

The median age at which CP was diagnosed was 34.6 years (95% CI, 31.7-37.4) and 28.7 years (95% CI, 12.4-45.0) in heterozygous and homozygous p.N34S patients, respectively (HR=1.50, 95% CI [0.89-2.52], p=0.13). Among the p.N34S heterozygous patients, the presence of co-mutations did not influence the occurrence of CP (HR=1.25, 95% IC [0.78-2.0], p=0.36).

Exocrine pancreatic insufficiency

At the end point, the proportion of patients with steatorrhea was similar in both groups. The median delay between the first symptoms and the diagnosis of EPI was longer in the SRP group than in the control group [25. 3a, the proportion of SRP patients with EPI was 5.3% at 20 years, 14.7% at 30 years, 28.3% at 40 years and 52.4% at 50 years. Conversely to the control group, tobacco consumption in SRP group was not associated with earlier onset of EPI (Figure 3b). The median age at EPI onset was similar in heterozygous and homozygous p.N34S patients (HR=1.08, 95% CI [0.51-2.29], p=0.84). No difference was observed due to concomitant mutations in CFTR or CTRC genes.

Diabetes mellitus (DM)

At the end of follow-up, there was a similar proportion of patients with DM in the SRP (17.3%) and in the IP (23%) groups (Table 1). The severity of DM was similar, with a comparable proportion of patients requiring insulin. DM occurred after a median delay of 37.7 (95% IC, 33.3-42.1) years following the onset of symptoms in the SRP group vs. 30 4a). Conversely to the control group, tobacco consumption in the SRP group was not associated with earlier DM onset (Figure 4b). Homozygous carriers for p.N34S mutation did not appear to be at increased risk of diabetes, in comparison with heterozygous carriers for p.N34S mutation (HR=0.27, 95% CI [0.04-2.02], p=0.20). Among heterozygous p.N34S patients, the presence of concomitant mutations in CFTR or CTRC genes did not influence the occurrence of diabetes either (HR=1.56, 95% IC [0.60-4.04], p=0.36).

Pancreatic ductal adenocarcinoma

Seven patients (3.3%) and 3 patients (0.99%) developed PDAC in the SRP and IP groups respectively (p=0.1), at a median age of 60 vs 66 years. In both groups, all patients with PDAC had morphological signs of chronic pancreatitis (missing data for one patient in each group). All but one patient had calcifications (one missing data) in the SRP group, none in the control group. All patients in the control group were smokers (3/3) versus 5/7 in SRP group. In the SRP group, the median tobacco consumption was higher in patients with PDAC (15 pack-years) than in the whole group (8 pack years). The actuarial risk of developing PDAC was 0.8% before 50 years, 11.9% at 60 years, 27.7% at 70 years and 51.8% at 80 years in the SRP group and was significantly higher than in the control group (p<0.001) (Figure 5). Patients in the SRP group had a risk of developing PDAC 12 times higher than the patients in the control group (Cox HR 12.0 (3.0-47.8), p<0.001). Among the SRP with PDAC, 5 had p.N34S heterozygous mutation (including one with CTRC and CFTR co-mutations) and 2 had a SPINK1 deletion. The presence of co-mutations did not influence the occurrence of PDAC (HR=1.56, 95% IC [0.60-4.04], p=0.36).

Death

Seven patients died in SRP group (4 of PDAC, one of cardiac amyloidosis, 2 of unknown cause) and 20 in the control group. There was no increased risk of death from any cause among the SRP patients (HR=0.48, 95% CI [0.19-1.18], p=0.11).

Discussion

We described herein one of the largest cohorts of patients with SRP. By the age of 50 years old, 76% of patients with SRP had morphological signs of CP, 52.4% had EPI and 26.3% had DM. In comparison with patients with idiopathic pancreatitis, patients in the SRP group presented with more episodes of acute pancreatitis, more pancreatic pain, had more frequent morphologic signs of CP. In addition, they had a significantly higher actuarial risk of developing chronic pancreatitis (Figure 2a), EPI (Figure 3a), DM (Figure 4a) and PDAC (Figure 5). These results might have been influenced by the longer follow-up in the SRP group and by the mismatch of disease proportions between the two groups. The presence of more patients with chronic pancreatitis in the SPINK1 related-pancreatitis group should have lead to an increased incidence of pancreatic insufficiencies and cancer. However, these clinical complications, except from PDAC, systematically occurred from 15 to 20 years earlier in the SRP group than in the control group. However, some authors [START_REF] Truninger | Mutations of the serine protease inhibitor, Kazal type 1 gene, in patients with idiopathic chronic pancreatitis[END_REF][START_REF] Midha | Idiopathic chronic pancreatitis in India: phenotypic characterisation and strong genetic susceptibility due to SPINK1 and CFTR gene mutations[END_REF] previously reported no clinical or morphological difference between patients with SRP and patients with ICP. Sandhu et al. [START_REF] Sandhu | Presence of SPINK-1 variant alters the course of chronic pancreatitis[END_REF] reported an earlier onset and more frequent acute bouts of pancreatitis in 13 patients with SRP compared to 35 patients with ICP. However, these studies included limited populations and SRP patients frequently had other causes of CP.

Recently, a large Chinese series aimed to assess the risk of chronic pancreatitis and complications in case of rare pathogenic variants in the CFTR, SPINK1, PRSS1 and CTRC genes. Authors included 1061 Chinese CP patients divided into three subgroups (idiopathic CP, alcoholic CP and tobacco related CP) and 1196 controls. Mutation-positive patients had earlier median ages at disease onset and at diagnosis of diabetes mellitus and steatorrhea than mutation-negative IP patients. Pathogenic variants were present in 57.1, 39.8, and 32.1% of the following subgroups: idiopathic, alcohol and tobacco. Even if this cohort is large and well explored by genetic testing, some issues have to be addressed. The definitions of the subgroups can be debated. A CP was considered as tobacco related if the intake was up to 2 pack-years; tobacco intake was not excluded in the alcohol group and patients with idiopathic pancreatitis could have alcohol consumption under 80g/day. No specific analysis was performed according to the genetic type and for patients with SPINK1 variants especially. In our study, we had very selective criteria to obtain the more characterized populations with no potential cofounded factors as alcohol intake for example. [START_REF] Zou | SPINK1, PRSS1, CTRC, and CFTR Genotypes Influence Disease Onset and Clinical Outcomes in Chronic Pancreatitis[END_REF] We found limited genotype-phenotype correlations. There was no influence of the genetic alterations (homozygous vs heterozygous p.N34S mutations, presence of co-mutations) on the age of onset of pancreatic complications although there was statistical tendency for earlier onset of symptoms in patients with homozygous p.N34S mutation or co-mutations (CFTR and CTRC in addition to SPINK1). This result is consistent with those reported by Drenth et al [START_REF] Drenth | Mutations in serine protease inhibitor Kazal type 1 are strongly associated with chronic pancreatitis[END_REF] describing a population of 14 SPINK1 mutation carriers including two patients with homozygous p.N34S mutation. The patients included in this SRP cohort had no other known causes of pancreatic disease (especially we excluded patients with a consumption of alcohol greater than 3 units per day), which strongly suggests that their pancreatic disease were related to the SPINK1 mutations. This is consistent with a recent Italian meta-analysis which reported that the presence of p.N34S mutation increases nine times the overall CP risk in a European population (OR 9.7, 95% CI [7.9-11.9]) [START_REF] Leo | Meta-analysis of the impact of SPINK1 p.p.N34S gene variation in Caucasic patients with chronic pancreatitis. An update[END_REF] .

However, SPINK1 mutations are frequent in the general population (1-2%) but not constantly associated with pancreatitis [START_REF] Pfützer | SPINK1/PSTI polymorphisms act as disease modifiers in familial and idiopathic chronic pancreatitis[END_REF] . The pattern of inheritance and the reasons explaining the development of SRP in only a small proportion of SPINK1 mutation carriers is unclear. This is also underlined by the fact that the p.N34S mutation may not have functional consequences in vitro [START_REF] Kereszturi | Minigene analysis of intronic variants in common SPINK1 haplotypes associated with chronic pancreatitis[END_REF] . Hence, yet unidentified environmental or genetic factors are likely to be associated with SPINK1 mutations for the development of pancreatic symptoms. Among the patients in the SRP group, 21.3% and 9.1% had a co-mutation involving CFTR or CTRC, which are well recognized causative or facilitating mutations for the development of CP [START_REF] Castellani | Consensus on the use and interpretation of cystic fibrosis mutation analysis in clinical practice[END_REF][START_REF] Szmola | Chymotrypsin C (caldecrin) promotes degradation of human cationic trypsin: Identity with Rinderknecht's enzyme Y[END_REF] . These rates are much higher than in the general population (4% for CFTR). This advocates that mutations in the SPINK1 gene might facilitate -rather than causepancreatic disease, when associated with other cofactors, some of them being still unidentified. Similarly, Sofia et al. [START_REF] Sofia | Trans-heterozygosity for mutations enhances the risk of recurrent/chronic pancreatitis in patients with Cystic Fibrosis[END_REF] recently reported compound heterozygosity, involving various genes including SPINK1, enhanced the risk for CP in patients with cystic fibrosis. Surprisingly, tobacco consumption was not associated with an earlier onset of CP, EPI or DM in the SRP group in comparison with the control group. One explanation could be that there was quantitatively and qualitatively significantly less smokers in the SRP group than in the control group. Moreover, the influence of tobacco exposure on the onset of the first symptoms could not be evaluated. Indeed, one could imagine that following SRP diagnosis, patients with medical follow-up were most likely strongly advised to quit smoking which finally ends up in a better natural history of the disease. Tobacco then falsely appears as a protective factor.

Conversely to PRSS1 mutations, for which the risk of PDAC is now clearly identified [START_REF] Rebours | The natural history of hereditary pancreatitis: a national series[END_REF][START_REF] Howes | Clinical and genetic characteristics of hereditary pancreatitis in Europe[END_REF] , the association between the presence of a SPINK1 mutation and the development of PDAC has been controversial. Several studies reported that SPINK 1 did not significantly increase the risk of PDAC 24- 26 . Nevertheless, those studies did not have the sufficient statistical power nor follow-up to estimate life-long actuarial risk. In the present study, the risk of PDAC was 12 times higher in the SRP patients than in the IP patients and the actuarial risk of PDAC was also higher in the SRP patients than in the IP patients (p<0.001, figure 5b). Although this estimation relied on a low number of PDAC, it may well be that p.N34S is associated with cancer.

This study has several limitations, such as its retrospective design. Moreover as the role of SPINK1 mutations in pancreatitis is controversial (facilitating gene more than causative) it would have been interesting to test other susceptibility genes (ie: CEL-HYB, CTRB [START_REF] Johansson | The role of the carboxyl ester lipase (CEL) gene in pancreatic disease[END_REF][START_REF] Rosendahl | Genome-wide association study identifies inversion in the CTRB1-CTRB2 locus to modify risk for alcoholic and non-alcoholic chronic pancreatitis[END_REF] ) which was impossible in this retrospective study. We were unable to explain why the number of deaths was higher in the control group than in the SRP group because causes of deaths in the first group were not known. SPINK1 mutations are considered as strong candidates for contributing to the pathogenesis of tropical calcific pancreatitis [START_REF] Paliwal | Genetic and phenotypic heterogeneity in tropical calcific pancreatitis[END_REF] , an entity for which the risk of PDAC is considered elevated [START_REF] Chari | Risk of pancreatic carcinoma in tropical calcifying pancreatitis: an epidemiologic study[END_REF][START_REF] Ramesh | Surgery in tropical pancreatitis: analysis of risk factors[END_REF] . In our study the identification of risk factors for the development of PDAC was not possible because of a limited number of patients with PDAC. However, we observed that all the patients who developed PDAC had morphological signs of CP and a heavy smoking consumption (median 15 pack-years), which are two major risk factors of PDAC. Finally, the only patient who developed PDAC before 50 years had a first-degree familial history of PDAC. Large prospective cohorts would be necessary to be precise, in the population of SPINK1 mutation carriers, the risk factors for PDAC in order to identify a subgroup of patients who could benefit from PDAC screening. It is highly possible that the development of PDAC is a consequence of pancreatic inflammation (partly assessed by the presence of calcifications) rather than the SPINK1 mutation itself. Given the results of this study we would propose a close surveillance of patients over 50 year old, smokers, with morphological signs of severe CP, or in any patients with a familial history of PDAC, as for other germline mutations predisposing to PDAC. The modalities of such a screening is yet to be determined but should be performed accordingly to the International Cancer of the Pancreas Screening (CAPS) guidelines [START_REF] Canto | International Cancer of the Pancreas Screening (CAPS) Consortium summit on the management of patients with increased risk for familial pancreatic cancer[END_REF] .

Table 1: Population characteristics and pancreatic complications in both groups

Figure 1 :Figure 2 :Figure 3 :Figure 4 :Figure 5 :

 12345 Figure 1: Cumulative incidence of symptoms depending on age

  0 (95% IC, 20.2-29.8) years vs. 15.0 (95% IC, 10.3-19.7) years, p=0.007] However, the age of onset of EPI in the SRP group was about 15 years younger than in the control group [median age, 49.5 (95% IC, 44.5-54.6) years vs. 65.2 (95% IC, 62.1-68.3) years, p<0.001]. As shown in Figure

  .6 (95% IC, 17.3-43.8) years in the IP group (p=0.002). The age of DM onset was approximately 15 years younger in the SRP group than in the control group [68.5 (95% CI, 56.2-80.7) vs. 82.3 (95% IC, 68.4-96.2), p=0.018]. The proportion of SRP patients with DM was 7.8% at 30 years, 13.4% at 40 years, 26.3% at 50 years and 43.4% at 60 years (Figure

Table 1 : Population characteristics and pancreatic complications in both groups

 1

	*Expressed as median and range
		SPINK1-related	Idiopathic pancreatitis	p-value
		pancreatitis group		group
		Missing values	N=209	Missing values	N=302
	Male gender, n (%)		107 (51.2)		145 (48)	0.48
	Follow-up from the date of first symptoms*	8	8.3 (3.7-17.4)	1	5.3 (2.5-8.8)	<0.001
	Associated mutations, n (%)
	CFTR	2	44 (21.3)
	CTRC	154	5 (9.1)
	Alcohol consumption, n (%)	3	33 (16)	2	158 (52.3)	<0.001
	Tobacco consumption, n (%)	8	68 (33.8)	2	139 (46.3)	0.005
	Number of pack-year*	19	8 (0.5-80)	25	15 (0.5-140)	<0.0001
	Acute pancreatitis, n (%)	4	167 (81.5)		230 (76.2)	0.15
	Acute pancreatitis with organ failure, n (%)	9	10 (5)	20	24 (8.5)	0.14
	Number of acute pancreatitis*	19	8 (5-15)	25	15 (8-30)	<0.001
	Pancreatic pain, n (%)	1	166 (79.8)		189 (62.6)	<0.001
	Type of pancreatic pain, n (%)
	Episodic		131 (78.9)		134 (70.9)	0.08
	Continuous		35 (21.1)		55 (29.1)
	Chronic pancreatitis, n (%)	3	144 (69.9)	2	162 (54)	<0.001
	Ductal abnormalities, n (%)	5	103 (72.5)	55	53 (48.6)	<0.001
	Calcifications, n (%)	3	107 (74.3)		105 (64.8)	0.07
	Pseudocyst(s), n (%)	4	34 (16.6)	53	55 (22.1)	0.14
	Thrombosis, n (%)	2	19 (9.2)	2	34 (11.3)	0.44
	Cholestasis, n (%)	2	14 (6.8)	101	30 (15.9)	0.008
	Steatorrhea, n (%)	1	77 (37)	2	110 (36.7)	0.97
	Diabetes mellitus, (%)	1	36 (17.3)	2	69 (23.0)	0.12
	Diabetes mellitus with insulin requirement, n (%)		25 (69.4)	21	29 (60.4)	0.39
	Endoscopic treatment, n (%)	1	32 (15.4)	0	42 (13.9)	0.64
	Surgical treatment, n (%)	1	35 (16.8)	0	53 (17.5)	0.83
	Pancreatic ductal adenocarcinoma, n (%)	0	7 (3.3)	0	3 (0.99)	0.10
	Pancreatic cancer and tobacco consumption		5/7		3/3	<0.0001

Grant support: none

Declaration of interest: No conflicts of interest exist

Authors' contribution

Nelly Muller and Vinciane Rebours: Study design and concept, data acquisition, data analysis, statistical analysis, and manuscript drafting. Louis de Mestier, Ioannis Sarantitis Christopher Halloran and William Greenhalf: data analysis, statistical analysis, and manuscript drafting. Marie Rouanet, Claude Férec, Emmanulle Masson, Philippe Ruszniewski, Philippe Lévy, John Neoptolemos and Louis Buscail: data acquisition and Critical revision.


R Agniel

E Pauthe

J Massera

Michel Boissière
email: michel.boissiere@u-cergy.fr

A Houaoui

I Lyyra

Dissolution, bioactivity and osteogenic properties of composites based on polymer and silicate or borosilicate bioactive glass

Keywords: Bioactive Glass, Composite material, Osteogenic differentiation

Introduction

Bioresorbable polymers have been used widely in the past decades as pins, plates and screws in orthopaedic, cranial and oral surgery [START_REF] Schwach | In vitro and in vivo degradation of lactic acid-based interference screws used in cruciate ligament reconstruction[END_REF][START_REF] Claes | New bioresorbable pin for the reduction of small Gony fkagm.ents: design[END_REF][START_REF] Suuronen | Comparison of absorbable self-reinforced poly-L-lactide screws and metallic screws in the fixation of mandibular condyle osteotomies: An experimental study in sheep[END_REF][START_REF] Santos | Bioresorbable Polymers for Tissue Engineering[END_REF]. The bioresorption of the implants enables leaving the fixation in place until it degrades in the body, releasing non-toxic dissolution byproducts which are then metabolised [START_REF] Pietrzak | Bioresorbable implants -practical considerations[END_REF]. However, the bioresorbable polymers developed thus far clinically, are found to degrade at slow rate and lack osteoconductive properties [START_REF] Schwach | In vitro and in vivo degradation of lactic acid-based interference screws used in cruciate ligament reconstruction[END_REF][START_REF] Walton | Long-term in vivo Degradation of Poly-L -lactide (PLLA) in Bone[END_REF].

Bioceramics are a class of materials grouping all traditional nearly inert ceramics such as, Al2O3 and ZrO2, and include calcium phosphate ceramics and silicate bioactive glass (BAG). These demonstrate properties extending from bioresorbable to bioactive class A. Clinically, β-TCP (bioresorbable) and synthetic hydroxyapatite (HA) (bioactive) are the more widely used traditionally [START_REF] Schwach | In vitro and in vivo degradation of lactic acid-based interference screws used in cruciate ligament reconstruction[END_REF][START_REF] Shimazaki | Comparative study of porous hydroxyapatite and tricalcium phosphate as bone substitute[END_REF][START_REF] Ohura | Resorption of, and bone formation from, new ?-tricalcium phosphate-monocalcium phosphate cements: Anin vivo study[END_REF], but their slow dissolution rate is a limiting factor [START_REF] Hasegawa | A 5-7 year in vivo study of high-strength hydroxyapatite/poly(l-lactide) composite rods for the internal fixation of bone fractures[END_REF][START_REF] Verheyen | Hydroxylapatite/poly(L-lactide) composites: An animal study on push-out strengths and interface histology[END_REF]. Indeed, concerns have been addressed pertaining to the limited resorption of those ceramics, in-vivo, when used in cements [START_REF] Ishikawa | Calcium Phosphate Cement[END_REF]. BAG is a sub-category of ceramics showing not only osteoconduction, as in synthetic HA, but also osteoinduction [START_REF] Hench | The story of Bioglass®[END_REF]. These glasses are commercialized mainly for hard tissue reconstruction, but they also show significant ability to bond to soft tissue [START_REF] Day | Assessment of polyglycolic acid mesh and bioactive glass for soft-tissue engineering scaffolds[END_REF]. However, shaping the glass into its final shape is, as for all ceramics, challenging.

The quest for bioresorbable implants which are osteoconductive for use in the treatment of traumas in the skeletal system is still ongoing and presents significant challenges still. To overcome some of the drawbacks of single materials, composites have been developed. Here, the focus will be on polymer/BAG composites, as previous studies have demonstrated that fast release of ions from BAG compensated for the decrease in pH due to the rapid degradation of Poly (Lactic acid-co Glycolic Acid) (PLGA) [START_REF] Li | pH-compensation effect of bioactive inorganic fillers on the degradation of PLGA[END_REF]. By adding BAG to (Poly (D,L-Lactic Acid) (P(D,L)LA) or Poly (Lactide-co-Caprolactone) (PLCL)) the mechanical properties increases as well as precipitation of an HA layer at the composite scaffold surface [START_REF] Tainio | In Vitro Degradation of Borosilicate Bioactive Glass and Poly(l-lactide-co-ε-caprolactone) Composite Scaffolds[END_REF][START_REF] Maquet | Preparation, characterization, and in vitro degradation of bioresorbable and bioactive composites based on Bioglass®-filled polylactide foams: Porous Polylactide/Bioactive Glass Composites[END_REF]. These studies used the solvent-casting method. Vergnol et al. developed composites based on P(L,DL)A/45S5 BAG. In this study, glass particles (3.5µm average diameter) were mixed with polymer dissolved in acetone. The pellets obtained were then injected into molds at 145°C and 150 bars of pressure. The presence of the BAG not only increased the rate of degradation of the polymer, but also significantly promoted new bone formation in this animal study [START_REF] Vergnol | In vitro and in vivo evaluation of a polylactic acid-bioactive glass composite for bone fixation devices: POLYLACTIC ACID-BIOACTIVE GLASS COMPOSITE FOR BONE FIXATION DEVICES[END_REF]. However, there was a large loss in polymer molecular weight reported over the course of the sample processing.

The decrease in molecular weight could not only be assigned to the processing temperature but also to the presence of the BAG particles [START_REF] Kometani | Quantitative Analysis for Polymer Degradation in the Extrusion Process[END_REF]. The mechanical properties of the scaffold were also found to decrease drastically over the short immersion time in vitro. The loss in mechanical properties seemed to be correlated with the loss in the mineral phase, which is assumed to be fast given the rapid dissolution rate of the small BAG particles. Another study by Niemelä et al. presents self-reinforced composites based on P(L,DL)A/13-93 BAG, made by twin-screw extrusion at temperatures varying between 190 and 195°C. In this study, the particle size was between 50 and 125µm. It is noteworthy, that not only 13-93 is slower dissolving than 45S5 (at similar particle size), but also the larger particle size will further slowdown the dissolution rate of the inorganic phase [START_REF] Niemelä | Self-reinforced composites of bioabsorbable polymer and bioactive glass with different bioactive glass contents. Part II: In vitro degradation[END_REF][START_REF] Brink | The influence of alkali and alkaline earths on the working range for bioactive glasses[END_REF]. Degradation of the self-reinforced composites was evaluated in PBS and the results supported the effect of glass dissolution on the polymer degradation rate [START_REF] Niemelä | Self-reinforced composites of bioabsorbable polymer and bioactive glass with different bioactive glass contents. Part II: In vitro degradation[END_REF]. The 13-93 particles contained in the self-reinforced composite create a porosity which induces a degradation of the polymer due to acidic dissolution products coming from the environment. The use of 13-93 in the composites appeared to retain higher polymer molecular weights than when using faster dissolving glasses such as 45S5 or S53P4. To overcome the slow dissolution rate of 13-93, boron can replace the silica in the structure, yielding a borosilicate glass. The resulting borosilicate and borate glasses, based on the 13-93 composition, show faster in-vitro dissolution and also faster conversion into HA [START_REF] Brown | Effect of borate glass composition on its conversion to hydroxyapatite and on the proliferation of MC3T3-E1 cells[END_REF][START_REF] Ojansivu | The effect of S53P4-based borosilicate glasses and glass dissolution products on the osteogenic commitment of human adipose stem cells[END_REF]. While high boron content was associated with a decrease in the cell proliferation rate, it was also found to stimulate osteogenic commitment and upregulate endothelial markers [START_REF] Ojansivu | The effect of S53P4-based borosilicate glasses and glass dissolution products on the osteogenic commitment of human adipose stem cells[END_REF]. In-vivo and in-vitro studies have shown the promising nature of borosilicate glasses [START_REF] Niemelä | Self-reinforced composites of bioabsorbable polymer and bioactive glass with different bioactive glass contents. Part II: In vitro degradation[END_REF][START_REF] Fu | Silicate, borosilicate, and borate bioactive glass scaffolds with controllable degradation rate for bone tissue engineering applications. II. In vitro and in vivo biological evaluation[END_REF]. The use of borate and borosilicate glasses as a secondary phase in a polymeric matrix has not yet been widely studied. Taino et al. produced PLCL (Poly(L-lactide-co-ε-caprolactone)/borosilicate glass composites, with varying content of 125-250 µm glass particles using the solvent-casting method. These scaffolds were then foamed by supercritical CO2. Degradation of the polymer was linked to the dissolution of the glass [START_REF] Tainio | In Vitro Degradation of Borosilicate Bioactive Glass and Poly(l-lactide-co-ε-caprolactone) Composite Scaffolds[END_REF].

Marquardt et al. studied the processing of fibrin / borate glass composites obtained by mixing fibrin with glass microfibers 0.5 -10 µm diameter, or with rods of 50 -200 µm diameter which were placed on a fibrin scaffold prior to polymerization. The materials obtained were able to support directed axon growth [START_REF] Marquardt | Effects of borate-based bioactive glass on neuron viability and neurite extension: Borate-based Bioactive Glass[END_REF].

However, the effect of boron substitution for silica, in the glass composition, on the composite physico-chemical properties and cell / material interaction has not yet been studied.

Added to this, based on previously reported results, the use of glass 13-93 and its boroncontaining counterpart might be suitable in maintaining polymer integrity and composite mechanical properties in-vitro while supporting osteogenesis. Therefore, we have developed PLA/BAG composites using 13-93 as control and 13-93B20 with 20% SiO2 replaced by B2O3.

To investigate the in vitro dissolution behavior of our composites, they were immersed in TRIS buffer solution. Ion release from the glass and change in the polymer molecular weight were quantified. The mechanical properties of the composites were studied during the immersion.

The bioactivity, assumed to be related to the precipitation of a HA layer at the surface of the material when immersed in aqueous solution, was assessed in Simulated Body Fluid (SBF), the procedure usually used for testing BAG [START_REF] Maçon | A unified in vitro evaluation for apatite-forming ability of bioactive glasses and their variants[END_REF]. Preliminary cell experiments were done to assess cell activity and ability of these two BAG to promote osteogenesis by culturing C2C12 myoblastic cells at the surface of composite discs. Cell proliferation and morphology were studied as well as presence of myosin and/or osteopontin which were tracked by immunostaining. C2C12 cells capacity to synthesize their mineral matrix was analyzed with Alizarin Red S staining. The aim of this study was to assess if these cells were able to commit to an osteoblastic lineage in presence of the BAG. A 4 mm nozzle was used and the rods were pulled using a caterpillar. The speed of the caterpillar was adjusted to obtain 3 mm diameter rods.

The rods of each composition (PLA as a control; PLA/13-93; PLA/13-93 B20) were analyzed by Thermogravimetric Analysis (NETZSCH, Leading Thermal Analysis, STA449F1) to measure their glass content. All tests were performed in an Alumina (Al2O3) crucible and in a N2 atmosphere. 10 mg of sample were heated from 25 °C to 1100 °C at a rate of 10 °C/min. At set times after immersion, the concentration of elements released from the composites was studied by diluting 5mL of the immersion solution in 45mL of ultra-pure water for ion analysis. ICP-OES (Agilent technologies 5110) was employed to quantify P (λ = 253.561 nm), Ca (λ = 422.673 nm), Mg (λ = 279.553 nm), Si (λ = 250.690 nm), B (λ = 249.678nm), K (λ = 766.491 nm) and Na (λ = 589.592 nm) concentrations in the solution after sample immersion. Measurements were made on four separate samples at each set time for each composite and the results presented as mean ± standard deviation (SD). The rods were then rinsed with acetone and dried.

Rod cross section analysis

After immersion in TRIS, the samples were embedded in resin and then polished to observe their cross section using Scanning Electron Microscopy (SEM -GEMINISEM 300 from Zeiss).

Molecular weight of the polymer

Molecular weights of PLA after the samples processing and at various immersion times were determined by Gel Permeation Chromatography (GPC) (Merck Hitachi Lachrom 7000 series) consisting of a pump, a refractive index detector and two Waters Styragel columns (HR5E and HR1). Tetrahydrofuran (THF) was used as eluent at a flow rate of 1 mL/min at 35°C. For each material, an amount of ≃7,5mg of polymer (cross-section of the rod) was weighed and immersed in 5mL of THF until complete dissolution. The solution was then filtered and analyzed. Molecular weights were calibrated using polystyrene standards. The measurements were conducted in four separate samples at each time points for each composition and the results are presented as mean ± SD.

Mechanical properties of the composites

The mechanical properties of the composite rods post-processing and after immersion (wet) were tested on the Instron 4411 (Instron Ltd., High Wycombe, UK) using a 3-point bending and shear test at room temperature. At least four parallel samples of each composite type were tested. For the bending test, the properties were studied with a crosshead speed of 5 mm.min -1 and a bending span of 42 mm. For the shear test [START_REF] Niemela | Self-reinforced composites of bioabsorbable polymer and bioactive glass with different bioactive glass contents. Part I: Initial mechanical properties and bioactivity[END_REF], the crosshead speed was 3 mm.min -1 . The measurements were conducted in the four samples at each set point for each composite and the results are presented as mean ± SD.

Composites bioactivity

Immersion in Simulated Body Fluid (SBF)

SBF was prepared following the methodology [START_REF] Kokubo | Solutions able to reproducein vivo surface-structure changes in bioactive glass-ceramic A-W3[END_REF] from the standard ISO/FDIS 23317 as described by Kokubo et al. The samples were cut to obtain rods of 7cm (≃650 mg) which were immersed in 12mL SBF solution for up to 2 weeks at 37°C in a shaking incubator (HT Infors

Multitron) with at a speed of 100rpm. During the experiment, the solution was not refreshed so that calcium phosphate could be measured. The ion concentration in the solution according to immersion time was measured as previously described. Measurements were made on four separate samples at each set time for each composite and the results presented as mean ± SD.

Rod surface analysis

The reactive layer at the rod surface after immersion in SBF was observed by SEM (GEMINISEM 300 from Zeiss) and its composition was analyzed by Energy-Dispersive X-ray spectroscopy (EDX Quantax from BRUKER). The Infrared (IR) absorption spectra of the composites immersed in SBF were also recorded using a Bruker Alpha FTIR in Attenuated Total Reflectance (ATR) mode to see the effect of the dissolution on their structural properties.

The measurements were performed on dry samples. All IR spectra were recorded within the range 399-4000 cm -1 with a resolution of 2 cm -1 and 32 accumulation scans.

Cell analysis

Disk preparation

PLA, PLA/13-93 and PLA/13-93B20 disks were obtained by compression molding (Nike Hydraulics ZB110, Eskilstuna, Sweden) of a piece of the rods for the cellular tests. The rods were compressed under 10-20 MPa pressure at 140 °C for 1 min, then the mold was cooled down with compressed air and 14mm disks were cut from the plates obtained. These disks were then sterilized by gamma irradiation (25 kGy) at BBF sterilisations service GmbH (Germany).

All experiments were performed in 24-well plates and the disks were washed with PBS prior to use.

Cell culture

Myoblastic C2C12 cells were cultured in DMEM Glutamax supplemented with 10 % Fetal Bovine Serum (FBS) and 1 % penicillin/streptomycin, in an humidified atmosphere of 5 % CO2 at 37 °C.

Cell proliferation and morphology

To compare the behavior of C2C12 cells on the different samples, cell proliferation was studied using CyQUANT Cell Proliferation Assay kit (Invitrogen, Life Technologies). Around 7700 cells/disk were seeded on the 14mm disks in 24-well plate and the medium was changed every 2 days. Cleaned and sterilized microscope glass slides were used as controls. After 1, 2, 4 and 7 days of culture, the cells were lysed with 400 µL 0.1 % Triton-X100 (Sigma-Aldrich)

buffer and conserved at -80 °C. After one freeze-thaw-cycle, three 20 μl aliquots of each lysate were pipetted on to a black 96-well plate (Corning) and mixed with 180 μl working solution (CyQUANT GR dye and cell lysis buffer). The fluorescence was then measured at 520nm with a Spectrofluorimeter Xenius XM (SAFAS).

The morphology of the cells on the different samples was observed after 48 h of culture.

The same number of cells was seeded on the disks and after 48 h, the cells were fixed with 3 % (w/v) para-formaldehyde solution dissolved in PBS (Sigma Aldrich) for 15 min, then permeabilized with 0.1 % (v/v) Triton X-100 (Sigma Aldrich) for 10 min. Non-specific binding sites were blocked by incubating the disks in PBS containing 1 % Bovine Serum Albumin (BSA) for 1 h. The cytoskeleton and nuclei of the cells were stained respectively with 1:500 FITC-labelled phalloidin (Sigma Aldrich P5282) and 1:1000 4',6-Diamidino-2-phenylindole dihydrochloride (DAPI, Sigma Aldrich D9542) in PBS-BSA 0.5 % for 1 h. Each incubation with antibodies was performed in the dark in a humid atmosphere. Samples were then washed in PBS-BSA 0.5 %, mounted in Prolongold (Invitrogen), and observed under a LSM710 confocal microscope (Carl Zeiss).

Cell differentiation

The expression of a late myoblastic marker (myosin heavy chain) and an early osteoblastic marker (osteopontin) were studied using specific antibodies. Around 600 C2C12 cells were seeded on the disks and cultured for 14 days. Cleaned and sterilized microscope glass slides were used as controls. Cells were then labelled with mouse anti-myosin heavy chain (MHC, 1:1000, Millipore 05716), and rabbit anti-osteopontin (1:500, Millipore AB10910) diluted in PBS containing 0.5 % BSA. Primary antibodies are revealed using Alexa Fluor 488 or Alexa Fluor 568-conjugated goat anti-mouse or anti-rabbit antibodies both 1:400 in PBS-BSA 0.5 % (Invitrogen) as secondary antibodies. Samples were observed using a LSM710 confocal microscope (Carl Zeiss).

Mineralization was also assessed at 10 days and 14 days using Alizarin Red S stain (the calcium minerals stain red). The staining from a previously described protocol was adjusted [START_REF] Tirkkonen | The effects of vibration loading on adipose stem cell number, viability and differentiation towards bone-forming cells[END_REF] in that cells were fixed with paraformaldehyde for 15min at room temperature and stained with 2 % Alizarin red S (pH 4.1-4.3; Sigma-Aldrich) for 20 min at room temperature. The excess color was washed away with three consecutive water washes after which the samples were observed under an optical microscope.

Statistical analysis

Data were analyzed using GraphPad Prism Software. Statistical significance between groups is assessed by one-way analysis of variance (ANOVA). Experimental results are expressed as means ± standard deviation. Statistical significance is taken for values of p < 0.05.

Results and discussion

The aim of this study was to develop a polymer-BAG composite able to release ions beneficial for bone regeneration while maintaining, post-processing, the mechanical properties and the molecular weight of the polymer. As it has been reported that it is difficult to produce a polymer-bioglass ® (i.e. 45S5) composite, we decided to incorporate BAG 13-93 into a PLA matrix. As reported by Brink et al. the dissolution rate of the glass 13-93 is much slower than the typical BAG used clinically (i.e. 45S5 (Bioglass®) and S53P4 (BonAlive®) [START_REF] Brink | The influence of alkali and alkaline earths on the working range for bioactive glasses[END_REF]). To control glass dissolution, a second glass composite was tested [START_REF] Fu | Silicate, borosilicate, and borate bioactive glass scaffolds with controllable degradation rate for bone tissue engineering applications. II. In vitro and in vivo biological evaluation[END_REF], whereby 20% of the SiO2 in the 13-93 composition was replaced with B2O3. The targeted glass loading was 30 wt%. Thermogravimetric analysis was conducted (Figure S1) to assess the true glass loading in the polymer matrix post-processing as well as the homogeneity of the processed rods. Glass loading was 38±2 wt% and 35±4 wt% for the PLA/13-93 and PLA/13-93B20, respectively, demonstrating good control over the process used to produce the composites.

Characterization of the composites after processing

Before extrusion, the molecular weight of the PLA granules was measured to be ~530 kDa. As expected, after extrusion, the molecular weight of the pure PLA decreased about 40 %, while the PLA loaded with BAG decreased by ~50% regardless of the glass composition.

The decrease of the molecular weight recorded is not as high as that reported in the literature.

For example, Vergnol et al. show a decrease in molecular weight of ~90% when producing PLA-BAG composite processed by injection molding [START_REF] Vergnol | In vitro and in vivo evaluation of a polylactic acid-bioactive glass composite for bone fixation devices: POLYLACTIC ACID-BIOACTIVE GLASS COMPOSITE FOR BONE FIXATION DEVICES[END_REF]. Moreover, the PLA/S53P4 The mechanical properties of the PLA and its composites were measured in 3-point bending and shear. Both composites have an almost stable flexural modulus in wet and dry conditions but show a decrease in shear stress in both conditions, when compared to the polymer alone. The changes in the mechanical properties are only a function of the glass loading but not due to the nature of the glass. The decrease in the shear strength was not unexpected, since polymer glass composites are known to become weaker, and tend to become more brittle, as can be seen by an increase in their elastic modulus. Here, the decrease in the flexural modulus indicates an increase in the ductility. Such behavior, was already reported in self-reinforced polymer/BAG filaments [START_REF] Niemela | Self-reinforced composites of bioabsorbable polymer and bioactive glass with different bioactive glass contents. Part I: Initial mechanical properties and bioactivity[END_REF]. The loss in mechanical properties is probably due to the absence of chemical bonds between the glass and the polymer, leading to a loss of cohesivity.

Behavior of the composites in solution 2.1 Dissolution in TRIS

The co-degradation in TRIS of the polymer and glass was assessed by GPC and ICP-OES measurements and compared to that of the dissolution of the pure PLA rods. Figure 2 presents the concentration in Si A), Na B), K C), B D), Mg E), Ca F) and P G) post immersion in TRIS for various times. As the immersion solution was refreshed every week the results are presented cumulatively and normalized to the sample mass. As expected, the PLA alone does not exhibit any change in ion concentration. Upon immersion of the composites, the concentration in solution of the different elements increases, indicating that the glass particles are being hydrated and dissolve through the polymer. As expected, the addition of boron to the glass structures leads to faster initial ion release (up to ~10 days). However, at longer immersion times all curves seem to plateau for solutions containing the PLA/13-93B20 composites whereas the dissolution of the glass in the PLA/13-93 composite remains almost linear. It should be noted that; especially at longer immersion times, the standard deviation was higher in the case of the polymer containing the glass 13-93B20 than 13-93.

It is a known fact that upon immersion of a silicate glass in aqueous solution, initially ion exchange occurs followed by condensation and re-polymerization of a silica-rich layer [START_REF] Hench | The story of Bioglass®[END_REF].

If this layer is not uniformly formed at the surface of all glass particles, variations in ion release can be expected. All ion concentrations were normalized to the element contained in the starting rod to show clearly the extent of ion release.

G

Figure 3A) presents the percentage of ion released from glass 13-93 in TRIS solution. It is clear that glass dissolution is limited, whereby at 70 days less than 5% of each element is released into the solution. While the Si and P release seems to level-off at immersion times >20 days, all other constituents, i.e. Na, Ca, Mg and K seemed to be leached-out in an almost linear manner up to 70 days. Figure 3B) presents the release of ion from the glass 13-93B20, in % of initial mass of the elements. As seen in the case of the composites containing the glass 13-93, all ions are found to leach out into the solution. However, the difference in the dissolution profile of ions from the composites containing the glass 13-93B20 compared to ion release from composites containing the glass 13-93, is of interest.

1. While in Figure 3A) the Ca, K, Mg and Na dissolve, from the glass 13-93, at similar rate, a slower release of Ca was measured during dissolution of composites containing the glass 13-93B20 (Figure 3B). Furthermore, Ca release follows a similar dissolution profile to B as shown in Figure 3B).

2. The initial release rate for all ions is faster for composites containing the glass 13-93B20.

However, at extended immersion time (>15 days) a decrease in the release rate of all ions can be seen; whereas when immersing composites containing the glass 13-93 (Figure 3A)

only Si and P release rate slows down at extended immersion time, while all other ions show a linear release.

3. The final ion release content in the solution (at 70 days) is slightly lower in the case of the composite containing the glass 13-93B20 than in that containing the glass 13-93.

From the literature many interactions between the B2O3 and SiO2 in the glass were reported to happen. Some are reported below [START_REF] Wu | Sodium tracer diffusion and 11B NMR study of glasses of the type (Na2O)0.17(B2O3)x(SiO2)0.83-x[END_REF][START_REF] Du | Nature of Silicon-Boron Mixing in Sodium Borosilicate Glasses: A High-Resolution 11 B and 17 O NMR Study[END_REF][START_REF] Du | Solid-state NMR study of metastable immiscibility in alkali borosilicate glasses[END_REF][START_REF] Sun | Structural Roles of Boron and Silicon in the CaO-SiO2-B2O3 Glasses Using FTIR, Raman, and NMR Spectroscopy[END_REF]:

2Si-O -+B-O-B→Si-O-Si+2B-O - (1) Si-O -+B-O-B→Si-O-B+B-O - (2)

B

However, those reactions were not sufficient to explain the physical, thermal and structural changes associated with the substitution of the SiO2 with B2O3. In depth structural analysis showed preferential interaction between CaO and B2O3 as follow [START_REF] Lai | Structural investigation of calcium borosilicate glasses with varying Si/Ca ratios by infrared and Raman spectroscopy[END_REF]:

BO3 + CaO ↔ BO4 (3)
More recently, Yu et al. reported the medium-range structural organization of phosphorus bearing borosilicate glasses and the consequence of B/Si substitution [START_REF] Yu | Medium-Range Structural Organization of Phosphorus-Bearing Borosilicate Glasses Revealed by Advanced Solid-State NMR Experiments and MD Simulations: Consequences of B/Si Substitutions[END_REF]. Their findings support the increase in the polymerization of the silica network with increasing substitution ratio. Furthermore, they demonstrated a higher affinity of the phosphorus to bond with B rather than with Si. Therefore, while in typical silicate glass the majority of the phosphorus is present as Q 0 (zero bridging oxygen atom per PO4 unit), an increase in Q 1 units, bridging one oxygen atom per PO4 unit, was evidenced. Overall, the structural modification occurring in the glass network when substituting Si for B is in agreement with the dissolution behavior observed in Figure 3A) and B), i.e. boron and calcium are released at the same rate (Figure 3B) as typically seen in a congruent dissolution. Thus, it appears that the calcium preferentially interacts with the boron sub-network (congruent dissolution) rather than with the silicate sub-network (non-congruent dissolution). The lower, overall release of the ions (at long immersion time) is most likely related to the increase in the degree of polymerization of the silica network as shown in equations 1 and 2, where the number of non-bridging oxygen is expected to decrease with increasing the boron content. However, such structural analysis does not fit the dissolution behavior reported in the literature, which tend to demonstrate that the addition of B2O3 at the expense of SiO2 should increase the glass dissolution rate [START_REF] Huang | Kinetics and mechanisms of the conversion of silicate (45S5), borate, and borosilicate glasses to hydroxyapatite in dilute phosphate solutions[END_REF][START_REF] Yao | In Vitro Bioactive Characteristics of Borate-Based Glasses with Controllable Degradation Behavior[END_REF][START_REF] Liu | Bioactive borosilicate glass scaffolds: in vitro degradation and bioactivity behaviors[END_REF][START_REF] Lepry | Highly Bioactive Sol-Gel-Derived Borate Glasses[END_REF]. It is therefore possible to assume that either the increase in Si-O-Si bridges is associated with a disproportionation of the silicate structure, whereby, 2Q 2 ↔ Q 1 + Q 3 , and/or Si-O-B bridges form between the borate and silicate units. Both assumptions would be consistent with the increase in the initial dissolution rate of the glass in the composite containing the glass 13-93B20 and a progressive decrease in the ion release rate due to remnants of a more stable silicate. As seen in Figure 3A) and B), this leads to a higher ion concentration in the solution containing the borosilicate glass at a short immersion time, and saturation occurring at an earlier time than in the case of traditional silicate BAG such as 13-93. Finally, the increased linkages between the phosphorus structural unit and the more stable BO4 units proposed by Yu et al. [START_REF] Yu | Medium-Range Structural Organization of Phosphorus-Bearing Borosilicate Glasses Revealed by Advanced Solid-State NMR Experiments and MD Simulations: Consequences of B/Si Substitutions[END_REF] is confirmed by the lower phosphorus release profile in the case of the glass 13-93B20 than in 13-93.

Figure 4 shows the cross sections of the composites before and after 10 weeks of immersion in TRIS. Before immersion, the BAG granules are fixed and appear to be maintained by the PLA matrix. After 10 weeks in TRIS, they are seen to be detached from the polymer, regardless of the glass composition, as expected post surface dissolution of the glass particles.

At the same time, the change in the polymer molecular weight was assessed according to time and is presented in Figure 5. Although the extrusion process affected the PLA molecular weight (Mw), it does not seem to be impacted by the interaction with the medium after immersion for up to 10 weeks. However, slow but linear molecular weight decrease is apparent during immersion of the composite. The composite containing the BAG 13-93, showed the fastest PLA degradation with a loss of ~45% of its molecular weight post-extrusion at 10 weeks at a rate of 1710 ± 96 Da / day (R 2 =0.99). The composite containing the BAG 13-93B20 showed a slower decrease, of ~29 % of its original molecular weight after 10 weeks of immersion at a rate of 1133 ± 82 Da / day (R 2 =0.98). In both cases, the degradation of the polymer is due to the interaction between glass dissolution by-products and PLA, most likely by alkaline hydrolysis of ester bonds at the glass/PLA interface [START_REF] Jong | New insights into the hydrolytic degradation of poly(lactic acid): participation of the alcohol terminus[END_REF]. Such behavior was, however, not reported by Maquet et al. with the dissolution of bioglass®-filled polylactic foams but occurred during dissolution of PLA/BAG composite in vivo [START_REF] Maquet | Preparation, characterization, and in vitro degradation of bioresorbable and bioactive composites based on Bioglass®-filled polylactide foams: Porous Polylactide/Bioactive Glass Composites[END_REF][START_REF] Vergnol | In vitro and in vivo evaluation of a polylactic acid-bioactive glass composite for bone fixation devices: POLYLACTIC ACID-BIOACTIVE GLASS COMPOSITE FOR BONE FIXATION DEVICES[END_REF]. Added to this, the difference in PLA degradation can be related to the more sustained ion release in the case of the composite containing the glass 13-93 as seen in Figure 3.

B

The mechanical properties (Young modulus and Shear stress) were measured on wet samples as a function of immersion time (Figure 6). While no significant changes in shear stress, within the accuracy of the measurement, were recorded, a net fall in the Young modulus can be seen after immersion for up to three days. As all the measurements were corrected to the swelling of the sample, such a drop can be attributed to the diffusion of water within the composite structure, most likely due to pores being formed during the processing. After the initial drop, the Young modulus does not seem to be, drastically, impacted by either the glass or the polymer dissolution/degradation.

Upon immersion of composites in TRIS buffer solution, a decrease in the average molecular weight was measured. However, in this study and in agreement with data reported by Vergnol et al, the decrease of the PLA molecular weight alone could not be correlated to changes in the mechanical properties [START_REF] Vergnol | In vitro and in vivo evaluation of a polylactic acid-bioactive glass composite for bone fixation devices: POLYLACTIC ACID-BIOACTIVE GLASS COMPOSITE FOR BONE FIXATION DEVICES[END_REF]. Yet, in Vergnol et al., a decrease in mechanical properties was recorded upon immersion of the BAG/PLA composite. The loss in mechanical strength is directly related to the composite mass loss with regards to the immersion time.

Figure 6: A) Young modulus reported from the bending test and B) Shear stress of wet PLA (∎), PLA/13-93 (•) and PLA/13-93B20 (▲) rods according to immersion time in TRIS.

Therefore, the dissolution of mineral phase dictated the change in mechanical properties. In our study, the larger particle size of BAG with slower dissolution rate than in Vergnol et al's work

were used [START_REF] Vergnol | In vitro and in vivo evaluation of a polylactic acid-bioactive glass composite for bone fixation devices: POLYLACTIC ACID-BIOACTIVE GLASS COMPOSITE FOR BONE FIXATION DEVICES[END_REF]. The low dissolution rate of the BAG used in this study implies that the composite keeps its integrity for at least 10 weeks.

The immersion of the composites in TRIS helped to understand the dissolution of both composites in solution. The 13-93B20 dissolves faster initially than the 13-93 and saturates more quickly to the same level as the 13-93 in the end. This dissolution leads to the decrease of the polymer molecular weight, but the mechanical properties stay almost stable.

Dissolution in SBF

Samples were also immersed in Simulated Body Fluid (SBF). As postulated by L.L.

Hench, the ability of a material to induce the precipitation of an hydroxyapatite layer at its surface is considered to be a sign of bioactivity [START_REF] Hench | The story of Bioglass®[END_REF].

Immersion in SBF was conducted over a two weeks period and the solution was not refreshed.

ICP-OES was used to quantify the ion concentration in the solution. The difference between the ion concentration in SBF and ion concentration post composite immersion was calculated (Figure 7).

The elements Si, K, Mg and B (for the PLA/13-93B20) show similar trends when immersed in TRIS where the 13-93B20 glass leaches out its ions at a faster rate initially and then stabilizes.

However, it is important to point out that i) the dissolution rate starts to slow down at an earlier immersion time in SBF than in TRIS and ii) while limited saturation was noticed, in the case of immersion of the PLA/13-93 in TRIS buffer solution, in SBF, saturation can also be seen for this glass. The Ca concentration seems to increase initially and then decreases with increasing immersion time, whereas the P concentration decreases constantly over dissolution time.

Generally, the decrease in Ca and P in SBF is associated with the precipitation of a calcium-phosphate reactive layer. The decrease in Ca and P is faster and starts at earlier time when the composites containing BAG 13-93B20 are immersed, indicating a faster and more rapid precipitation of the reactive layer in the case of this material. This is most likely due to the faster initial dissolution. While no significant change in the surface topography can be seen after immersion of the PLA in SBF, some spheres could be seen at the surface of the composites after 14 days. The spheres were small and sparsely dispersed at the surface of the composites containing the glass 13-93 (Figure 8D), while higher density of larger spheres (with a wide size distribution) were covering the composites containing the glass 13-93B20 (Figure 8F). This is in agreement with the faster reactive layer formation hypothesized from the ICP analysis for the boron containing composites.

EDX was performed on the spheres showed on Figure 8G) and an EDX spectra is presented in Figure 9A). The composition of the sphere is mainly Ca and P with a Ca/P ratio of ~1.6. This is a good indication that the calcium phosphate layer precipitating is hydroxyapatite. This was further confirmed by FTIR spectroscopy shown in Figure 9B). While only peaks related to the PLA structure where seen in the pure PLA and PLA/13-93 composite, prior and after immersion in SBF, whereas two peaks in the 400-600 cm -1 appeared after PLA/13-93B20 immersion in SBF. These peaks are characteristic of the 4 (P -O bending) and 2 (O -P -O bending) PO4 3- vibration in apatite structure [START_REF] Stanislavov | Structural features of hydroxyapatite and carbonated apatite formed under the influence of ultrasound and microwave radiation and their effect on the bioactivity of the nanomaterials[END_REF][START_REF] Baddiel | Spectra structure correlations in hydroxy and fluorapatite[END_REF]. This effect is partially overcome when working with 13-93B20. The particles are mainly embedded in PLA which slows the release rate due to the PLA diffusion barrier. However, one should keep in mind that despite 13-93B20 promoting more effective HA precipitation than 13-93, a similar reactivity to 45S5 or S53P4 has not yet been reached.

We developed PLA/BAG composites using a process that enables limiting the polymer degradation while maintaining mechanical properties which are of interest in bone tissue engineering. Furthermore, the inorganic filler dissolved when immersed in aqueous solution with kinetics function of the glass composition. The PLA/13-93B20 composite was also found to precipitate a hydroxyapatite layer upon immersion in SBF. Therefore, we decided to do preliminary cell experiments to assess if these composites have potentially osteogenic properties which are of utmost important in bone reconstruction.

C2C12 proliferation and morphology

The C2C12 cell system was chosen owing to its known dependence by adhesion to the substrate rigidity, and to the experimental ease in measuring the cellular response to the Bone Morphogenetic Protein 2 (BMP-2). In addition, this cell line is important and relevant in the progenitor cell system for bone tissue engineering [START_REF] Gand | Nanotemplated polyelectrolyte films as porous biomolecular delivery systems: Application to the growth factor BMP-2[END_REF].

Firstly, the proliferation of C2C12 cells on PLA and both composites was studied for up to 7 days (Figure 10).

Glass slides were used as a control. The cells proliferated with the characteristic profile of this cell phenotype on all substrates. At 4 days, the proliferation of C2C12 cells on the composites is significantly higher than on the PLA alone. Fu et al. and Eqtesadi et al. [START_REF] Fu | Silicate, borosilicate, and borate bioactive glass scaffolds with controllable degradation rate for bone tissue engineering applications. II. In vitro and in vivo biological evaluation[END_REF][START_REF] Eqtesadi | Effect of milling media on processing and performance of 13-93 bioactive glass scaffolds fabricated by robocasting[END_REF] have already demonstrated that 13-93 glass alone promotes cell adhesion and proliferation. It is interesting to point out, that at day 4, the cell count is statistically higher at the surface of the PLA/13-93 than on the PLA/13-93B20. This is certainly due to the release of boron from the borosilicate glass, which is known to decrease cell proliferation while promoting osteogenesis [START_REF] Ojansivu | The effect of S53P4-based borosilicate glasses and glass dissolution products on the osteogenic commitment of human adipose stem cells[END_REF]. It can also be seen that the morphology of murine C2C12 myoblasts after 48h of incubation (Figure 11), seem to spread more within the cytoskeleton of the cells with PLA/13-93 and PLA/13-93B20, than with PLA alone. This is confirmed by the measurements of the spreading surface of the cells on each material (Figure 11). The lower ability of the cells to spread on PLA can be correlated to slightly slower proliferation of cells at day 4 (Figure 10), when compared to proliferation on composites.

Thereby, both types of BAG do not present cytotoxic effects and when included in the PLA, they promote proliferation and adhesion of C2C12 cells compared to the PLA alone.

C2C12 differentiation

In order to study the capacity of C2C12 cells to commit to an osteoblastic lineage in presence of BAG, myosin heavy chain (late marker of myogenic differentiation) and osteopontin (early marker of osteoblastic differentiation) were stained after 14 days of incubation.

In Figure 12, it can be seen that the glass control (Figure 12B andC) and pure PLA (Figure 12F and G) have a high myosin expression but no osteopontin expression. The differentiation of C2C12 myoblasts into myotubes can be observed. With a BAG load into the PLA, myosin expression is decreased and osteopontin expression becomes visible (Figure 12J, K, N, O). This expression seems higher for the cells seeded on the composite containing 13-93B20 and myosin expression seems completely suppressed. Clearly, the osteopontin, marker of osteoblastic differentiation, is significantly expressed in cells cultured on the composites and this expression seems higher when using the borosilicate glass in PLA. However, the marker needed quantifying to confirm the effect of the boron included in the glass formulation compared to the silicate glass. C2C12 cells are often used to study osteodifferentiation in presence of BMPs [START_REF] Gand | Nanotemplated polyelectrolyte films as porous biomolecular delivery systems: Application to the growth factor BMP-2[END_REF][START_REF] Saito | Differentiation potential of osteoblast from cultured C2C12 cells on zirconia disk[END_REF]. Here, their capacity to differentiate into osteoblastic cells is exploited in the presence of BAG showing promising results for osteodifferentiation.

Mineralization was investigated using Alizarin Red Staining after 10 and 14 days of culture. This product stains the mineral calcium. As 10 days appeared to be too short a time to distinguish the mineralization, only images after 14 days of incubation are presented in Figure 13. Figure 13 shows the glass, the PLA, and both composites post staining with Alizarin Red.

On the glass control and the PLA without cells (Figure 13A and B), no red staining could be observed with the cells (Figure 13E and F), the staining seen is attributed to the high cell density, and therefore gives an indication of background noise. On both composites without cells, a slight red coloration is observed (Figure 13C andD). As shown in Figure 7, the dissolution of the composites leads to a small amount of HA precipitation. This may well occur within the culture medium and, therefore, the slight red coloration may be due to either, Alizarin being trapped at the glass/PLA interface of stained HA mineral. When the PLA/13-93 and the PLA/13-93B20 are seeded with the cells, strong red staining is evident, showing mineral formation (Figure 13G andH). These results, when compared to the results obtained without cells, suggest that the mineral stained by the Alizarin is not due to the precipitation of HA but

Conclusion

Composites made of PLA and bioactive glasses (silicate 13-93 and borosilicate 13-93B20) were processed by twin-screw extrusion. This process led to composites with content in the organic phase consistent across the length of the filaments and in agreement with the expected loading. The choice of the glass composition, particle size, glass loading, extrusion temperature as well as pressure for extrusion notably reduced the thermal degradation of the PLA, when compared to previous studies performed with similar or other processing techniques. The presence of BAG particles within the PLA matrix leads to a more ductile, but more fragile material as seen in the case of self-reinforced polymer/BAG composites.

The dissolution of the glass is not impaired by the polymeric matrix. As hypothesized, the substitution of part of the SiO2 in the 13-93 glass composition with B2O3 leads to an increase in the initial dissolution rate. In turns, this leads to a higher level of hydroxyapatite precipitation. However, the dissolution of the BAG leads to an increased degradation rate of

Experimental 1 . Material preparation and characterization 1 . 1 .

 111 Bioactive glass (BAG) preparationBAG 13-93 and 13-93B20 were prepared from analytical grade K2CO3 (Alfa Aesar, Haverhill, USA), (Na2CO3, NH4H2PO4, (CaHPO4)(2(H2O)), CaCO3, MgO, H3BO3 (Sigma Aldrich, Saint-Louis, MS, USA) and Belgian quartz sand. The 100-gram batches of 13-93 and 13-93B20 were melted for 3 hours in a platinum crucible at 1425°C and 1275°C, respectively.The molten glasses were cast, annealed, crushed and finally sieved into 125-250µm particles.The glasses were dried at 125°C for 2 hours before use. The nominal oxide compositions of the glasses are given in

Figure 2 :

 2 Figure 2: Element concentrations of A) Si, B) Na, C) K, D) B, E) Mg, F) Ca and G) P in the dissolution products of PLA (∎), PLA/13-93 (•) and PLA/13-93B20 (▲) immersed in TRIS according to time.

Figure 3 :

 3 Figure 3: Release of ions from the A) PLA/13-93 and B) the PLA/13-93B20, immersed in TRIS according to time.

Figure 4 :Figure 5 :

 45 Figure 4: Observations of the cross section of the composites by SEM. A) and B) show the PLA/13-93 before immersion, C) and D) show the PLA/13-93 after 10 weeks of immersion in TRIS. E) and F) present the PLA/13-93B20 before immersion and G) and H) show the PLA/13-93B20 after 10 weeks of immersion in TRIS. Scale bar 200µm

FFigure 7 :Figure 8 :

 78 Figure 7: A) Si, B) K, C) B, D) Mg, E) Ca and F) P element concentrations released in SBF from the PLA (∎), PLA/13-93 (•) and PLA/13-93B20 (▲) according to immersion time. ΔElement = [Element] in SBF in presence of the sample -[Element] in SBF initial solution.

Figure 9 :

 9 Figure 9: A) EDX analysis of the nodules found on PLA/13-93B20 surface and B) FTIR analysis of the sample surfaces before and after 2 weeks of immersion.

Figure 10 :

 10 Figure 10: Proliferation of C2C12 cells cultured in DMEM complete medium on glass, PLA, PLA/13-93 and PLA/13-93B20 for 7 days, analyzed with a CyQUANT Cell Proliferation Assay kit, ***p<0.001, *p<0.05.

  772 ± 212 m 2 439 ± 77 m 2 1097 ± 358 m 2 892 ± 269 m 2

Figure 11 :

 11 Figure 11: Morphology observations of C2C12 cells in DMEM complete medium analyzed by immunostaining, Nuclei (DAPI -blue) and Actin (Phalloidin -green), after 48h of incubation on glass, PLA, PLA/13-93, PLA/13-93B20 (Scale bar 20µm). Under each image the spreading area of the cells is annotated on each sample after 48h of incubation.

Figure 13 :

 13 Figure 13: Mineralization of C2C12 cells in DMEM was studied with Alizarin red S staining after 14 days of incubation. The first line shows A) the glass, B) the PLA, C) the PLA/13-93 and D) the PLA/13-93B20 without any cells seeded. The second line represents E) the glass, F) the PLA, G) the PLA/13-93 and H) the PLA/13-93B20 with C2C12 cells seeded for 14 days. Scale bar 200µm

Table

  .

	atmosphere (Figure 1). The feed rates for the PLA and the BAG were fixed to 140g.h -1 and
	60g.h -1 respectively to obtain approximately 70wt. % of polymer and 30wt. % of glass in the
	composite. The processing temperatures and pressures are presented in Table 2. The polymer
	took about 5 minutes to produce in the extruder including melting and producing the final
	product. The production time is less when the extruder is filled in advance.
				Temperature (°C)
								Pressure (psi)
			Cylinder	Adapter	Die
	PLA rods		200	230		230		200
	PLA/13-93		190	200		220		150
	PLA/13-93B20		185	220		215		200
	Table 1: Nominal glass composition (mol%)
					mol%
	Glass
	Na2O	K2O	MgO	CaO	P2O5	SiO2	B2O3
	13-93	6.0	7.9	7.7	22.1	1.7	54.6	--
	13-93B20	6.0	7.9	7.7	22.1	1.7	43.7	10.9
	1.2. Sample Fabrication
	Medical grade Poly(Lactic Acid) (PLA) with a (L/DL) ratio of 70/30, with an inherent
	viscosity of 4.0dl/g was obtained from Evonik Nutrition & Care GmbH (Essen, Germany).
	PLA, PLA/13-93 and PLA/13-93B20 rods were produced by melt-extrusion using a co-rotating
	twin-screw extruder (Mini ZE 20*11.5 D, Neste Oy, Porvoo, Finland) under nitrogen
								6

Table 2 :

 2 Materials processing parameters

	Figure 1: PLA rod (up) and composite rod (down) obtained after extrusion

. Behavior of the PLA/BAG composites 2.1. Physico-chemical properties of the composites 2.1.1 Immersion in TRIS

	Tris(hydroxymethyl)aminomethane (TRIS) solution (50mM) was prepared by mixing
	ultra-pure TRIS (MP Biomedicals) and TRIS-HCl (Sigma Aldrich) in ultra-pure water and the
	pH was adjusted to 7.4 at 37°C. The rods were cut into lengths of 7cm (≃650 mg) which were
	immersed in 12mL of TRIS solution for up to 10 weeks at 37°C in a shaking incubator (HT
	Infors Multitron) at an orbital speed of 100rpm. To avoid saturation of the solution with the
	ions released from the composite, the TRIS buffer was refreshed each week.

Table 3

 3 presents the glass loading in each composite, with the mechanical properties of the composites and the molecular weight of the polymer included in each composite.

	Materials	Composites glass loading	Young modulus (GPa) Dry Wet	Shear stress (MPa) Dry Wet	PLA Mw before extrusion	PLA Mw after extrusion
		(wt%)	samples	samples	samples	samples	(kDa)	(kDa)
	Bulk PLA	--	--	--	--	--	526 ± 7	--
	PLA rods	--	3.5 ± 0.1	3.6 ±0.2	46.6 ± 0.8 50.1 ± 1.4	--	305 ±14
	PLA/13-93 rods	38 ± 2	3.6 ± 0.3	3.3 ± 0.3 34.0 ± 2.0 32.3 ± 1.4	--	248 ± 5
	PLA/13-
	93B20	35 ± 4	3.6 ± 0.7	3.1 ± 0.4 32.1 ± 1.5 32.1 ± 1.1	--	251 ± 15
	rods

Table 3 :

 3 Measured glass loading, mechanical properties and average molecular weight of the processed PLA and PLA/BAG composites (for wet samples, the mechanicals properties were measured after 10min of immersion in TRIS). composites were tested, but, during the processing the viscosity of the polymer drastically and rapidly decreased and the materials obtained were amber-like as reported by Vergnol et al. for the PLA/45S5. The reason for this rapid change in viscosity and subsequent thermal degradation of the PLA is not yet well understood. However, this phenomenon appears when processing PLA with fast reacting BAG and does not occur that readily with more stable glasses. Therefore, a hypothesis for the thermal degradation of PLA upon extrusion of composites using 45S5 and/or S53P4 may be due to the high intrinsic water content in the glass structure or the glass degradation/dissolution when in the polymer melts. The combination of PLA and the proposed BAG 13-93 processed by twin-screw reduces loss of molecular weight during melt processing.

Acknowledgement

The authors would like to acknowledge the Academy of Finland (Academy Research Fellow grant to JM), TTY-säätiö for IL financial support, the Institute for Advanced Studies (IAE) for enabling researcher mobility, Delphine Logeart-Avramouglou (Laboratory of Bioengineering and Biomechanics for Bone and Articulations, UMR 7052, CNRS, University Paris Diderot) for the kind gift of C2C12 cells and Annette Lane for proofreading this manuscript.

is produced by the cells through their metabolism in presence of the 13-93 or 13-93B20.

Therefore, the cells are conditioned by the composites to synthesize their mineral matter. the PLA, not affecting the mechanical properties which remained stable for at least 10 weeks.

Finally, the dissolution of the composites in SBF support the hypothesis that the developed composites are bioactive, especially in the case of the composite containing the glass 13-93B20.

The osteogenic response of the C2C12 myoblastic cells to both composites -PLA/13-93 and PLA/13-93B20 -was studied. Cells were grown and spread on the composites and the expression of the myosin and the osteopontin measured after 14 days. When cells were cultured on pure PLA, myosin expression was clearly observed while on composites, only osteopontin was expressed. The mineralization experiment showed that the cells in presence of the 13-93 and the 13-93B20 were able to synthesize their mineral matrix.

These composites are promising for bone application. Nevertheless, we feel that more studies are needed to quantify and confirm the effect of the borosilicate at the cellular level.

Conflict of interest

There are no conflicts to declare.

Supplementary data