Hydroxyapatite 3D-printed scaffolds with Gyroid-Triply periodic minimal surface porous structure:Fabrication and an in vivo pilot study in sheep

Bone repair is a major challenge in regenerative medicine, e.g. for large defects. There is a need for bioactive, highly percolating bone substitutes favoring bone ingrowth and tissue healing. Here, a modern 3D printing approach (VAT photopolymerization) was exploited to fabricate hydroxyapatite (HA) scaffolds with a Gyroid-“Triply periodic minimal surface” (TPMS) porous structure (65% porosity, 90.5% HA densification) inspired from trabecular bone. Percolation and absorption capacities were analyzed in gaseous and liquid conditions. Mechanical properties relevant to guided bone regeneration in non-load bearing sites, as for maxillofacial contour reconstruction, were evidenced from 3-point bending tests and macrospherical indentation. Scaffolds were implanted in a clinically-relevant large animal model (sheep femur), over 6 months, enabling thorough analyses at short (4 weeks) and long (26 weeks) time points. In vivo performances were systematically compared to the bovine bone-derived Bio-OssⓇ standard. The local tissue response was examined thoroughly by semi-quantitative histopathology. Results demonstrated the absence of toxicity. Bone healing was assessed by bone dynamics analysis through epifluorescence using various fluorochromes and quantitative histomorphometry. Performant bone regeneration was evidenced with similar overall performances to the control, although the Gyroid biomaterial slightly outperformed Bio-OssⓇ at early healing time in terms of osteointegration and appositional mineralization. This work is considered a pilot study on the in vivo evaluation of TPMS-based 3D porous scaffolds in a large animal model, for an extended period of time, and in comparison to a clinical standard. Our results confirm the relevance of such scaffolds for bone regeneration in view of clinical practice. Statement of significance: Bone repair, e.g. for large bone defects or patients with defective vascularization is still a major challenge. Highly percolating TPMS porous structures have recently emerged, but no in vivo data were reported on a large animal model of clinical relevance and comparing to an international standard. Here, we fabricated TPMS scaffolds of HA, determined their chemical, percolation and mechanical features, and ran an in-depth pilot study in the sheep with a systematic comparison to the Bio-OssⓇ reference. Our results clearly show the high bone-forming capability of such scaffolds, with outcomes even better than Bio-OssⓇ at short implantation time. This preclinical work provides quantitative data validating the relevance of such TMPS porous scaffolds for bone regeneration in view of clinical evaluation.</p

Identification de protéines interagissant avec les facteurs de transcription AP-2 et contribuant à la surexpression du gène ERBB2 dans le cancer du sein.

Le cancer du sein est le cancer le plus fréquent chez la femme (Boyle and Ferlay, 2005; Ferlay et al., 2007). Même si les traitements sont de plus en plus efficaces, il est responsable d’environ 130 000 décès en Europe. Environ 20-30% des cancers du sein surexprime le gène ERBB2. Cette surexpression confère à la cellule un profil tumoral très agressif, et résistant aux chimiothérapies conventionnelles. L’étude des facteurs responsable de la surexpression du gène ERBB2 dans les cancers du sein est le thème principal de recherche du laboratoire d’oncologie moléculaire. Notre laboratoire, a étudié les mécanismes moléculaires responsables de la surexpression du gène ERBB2 dans les cancers du sein. Nous avons montré que la surexpression est la conséquence d’une stimulation de la transcription et non de la stabilisation de l’ARN (Pasleau et al., 1993).Pour étudier la régulation de la transcription un fragment de 6 kb du promoteur du gène ERBB2 a été cloné et séquencé. Différentes régions régulatrices ont été identifiées (Grooteclaes et al., 1994). Plusieurs sites de liaison pour des facteurs AP-2 ont été identifiés (Delacroix et al., 2005; Grooteclaes et al., 1999; Vernimmen et al., 2003a). La fixation des facteurs au promoteur a été vérifiée par Chromatin Immuniprecipitation (ChIP assay) (Begon et al., 2005; Delacroix et al., 2005).Afin de mieux comprendre le fonctionnement des facteurs de transcription AP-2, nous avons cherché les protéines interagissant avec ce facteur et contribuant à la surexpression d’ERBB2 dans des lignées de cancer du sein. Précédemment, Dominique Begon a montré l’interaction de la protéine YY1 avec le facteur de transcription AP-2alpha et leur implication sur le promoteur du gène ERBB2 (Begon et al., 2005). La première partie de mon travail a été de mettre en corrélation par immunohistochimie l’expression des protéines YY1 et AP-2alpha avec la surexpression d’ERBB2 dans des tumeurs primaires. Nous avons également voulu étudier l’effet d’une diminution des protéines YY1 et AP-2 sur l’expression d’ERBB2, à l’aide de siRNAs (Voir article 1 en annexe, (Allouche A and Nolens G. et al., 2008). La deuxième partie de ce travail a porté sur l’identification d’autres protéines pouvant interagir avec les protéines AP-2. Après purification, les protéines Ku70 et Ku80 furent identifiées. Nous avons voulu étudier l’effet de cette interaction sur l’activité des protéines AP-2 (alpha et gamma;) et sur l’expression d’ERBB2 (voir article 2 en annexe)

Ku proteins interact with activator protein-2 transcription factors and contribute to ERBB2 overexpression in breast cancer cell lines

INTRODUCTION: Activator protein-2 (AP-2) alpha and AP-2 gamma transcription factors contribute to ERBB2 gene overexpression in breast cancer. In order to understand the mechanism by which the ERBB2 gene is overexpressed we searched for novel AP-2 interacting factors that contribute to its activity. METHODS: Ku proteins were identified as AP-2 alpha interacting proteins by glutathione serine transferase (GST)-pull down followed by mass spectrometry. Transfection of the cells with siRNA, expression vectors and reporter vectors as well as chromatin immunoprecipitation (ChIP) assay were used to ascertain the implication of Ku proteins on ERBB2 expression. RESULTS: Nuclear proteins from BT-474 cells overexpressing AP-2 alpha and AP-2 gamma were incubated with GST-AP2 or GST coated beads. Among the proteins retained specifically on GST-AP2 coated beads Ku70 and Ku80 proteins were identified by mass spectrometry. The contribution of Ku proteins to ERBB2 gene expression in BT-474 and SKBR3 cell lines was investigated by downregulating Ku proteins through the use of specific siRNAs. Depletion of Ku proteins led to downregulation of ERBB2 mRNA and protein levels. Furthermore, reduction of Ku80 in HCT116 cell line decreased the AP-2 alpha activity on a reporter vector containing an AP-2 binding site linked to the ERBB2 core promoter, and transfection of Ku80 increased the activity of AP-2 alpha on this promoter. Ku siRNAs also inhibited the activity of this reporter vector in BT-474 and SKBR3 cell lines and the activity of the ERBB2 promoter was further reduced by combining Ku siRNAs with AP-2 alpha and AP-2 gamma siRNAs. ChIP experiments with chromatin extracted from wild type or AP-2 alpha and AP-2 gamma or Ku70 siRNA transfected BT-474 cells demonstrated Ku70 recruitment to the ERBB2 proximal promoter in association with AP-2 alpha and AP-2 gamma. Moreover, Ku70 siRNA like AP-2 siRNAs, greatly reduced PolII recruitment to the ERBB2 proximal promoter. CONCLUSIONS: Ku proteins in interaction with AP-2 (alpha and gamma) contribute to increased ERBB2 mRNA and protein levels in breast cancer cells

Prospective cohort study comparing outcomes between vacuum extraction and second-stage cesarean delivery at a Ugandan tertiary referral hospital

Research into fetal development and medicin

Model-Based Design to Enhance Neotissue Formation in Additively Manufactured Calcium-Phosphate-Based Scaffolds

peer reviewedIn biomaterial-based bone tissue engineering, optimizing scaffold structure and composition remains an active field of research. Additive manufacturing has enabled the production of custom designs in a variety of materials. This study aims to improve the design of calcium-phosphatebased additively manufactured scaffolds, the material of choice in oral bone regeneration, by using a combination of in silico and in vitro tools. Computer models are increasingly used to assist in design optimization by providing a rational way of merging different requirements into a single design. The starting point for this study was an in-house developed in silico model describing the in vitro formation of neotissue, i.e., cells and the extracellular matrix they produced. The level set method was applied to simulate the interface between the neotissue and the void space inside the scaffold pores. In order to calibrate the model, a custom disk-shaped scaffold was produced with prismatic canals of different geometries (circle, hexagon, square, triangle) and inner diameters (0.5 mm, 0.7 mm, 1 mm, 2 mm). The disks were produced with three biomaterials (hydroxyapatite, tricalcium phosphate, and a blend of both). After seeding with skeletal progenitor cells and a cell culture for up to 21 days, the extent of neotissue growth in the disks’ canals was analyzed using fluorescence microscopy. The results clearly demonstrated that in the presence of calcium-phosphate-based materials, the curvature-based growth principle was maintained. Bayesian optimization was used to determine the model parameters for the different biomaterials used. Subsequently, the calibrated model was used to predict neotissue growth in a 3D gyroid structure. The predicted results were in line with the experimentally obtained ones, demonstrating the potential of the calibrated model to be used as a tool in the design and optimization of 3D-printed calcium-phosphate-based biomaterials for bone regeneration

Women’s, partners’ and healthcare providers’ views and experiences of assisted vaginal birth: a systematic mixed methods review

Background When certain complications arise during the second stage of labour, assisted vaginal delivery (AVD), a vaginal birth with forceps or vacuum extractor, can effectively improve outcomes by ending prolonged labour or by ensuring rapid birth in response to maternal or fetal compromise. In recent decades, the use of AVD has decreased in many settings in favour of caesarean section (CS). This review aimed to improve understanding of experiences, barriers and facilitators for AVD use. Methods Systematic searches of eight databases using predefined search terms to identify studies reporting views and experiences of maternity service users, their partners, health care providers, policymakers, and funders in relation to AVD. Relevant studies were assessed for methodological quality. Qualitative findings were synthesised using a meta-ethnographic approach. Confidence in review findings was assessed using GRADE CERQual. Findings from quantitative studies were synthesised narratively and assessed using an adaptation of CERQual. Qualitative and quantitative review findings were triangulated using a convergence coding matrix. Results Forty-two studies (published 1985–2019) were included: six qualitative, one mixed-method and 35 quantitative. Thirty-five were from high-income countries, and seven from LMIC settings. Confidence in the findings was moderate or low. Spontaneous vaginal birth was most likely to be associated with positive short and long-term outcomes, and emergency CS least likely. Views and experiences of AVD tended to fall somewhere between these two extremes. Where indicated, AVD can be an effective, acceptable alternative to caesarean section. There was agreement or partial agreement across qualitative studies and surveys that the experience of AVD is impacted by the unexpected nature of events and, particularly in high-income settings, unmet expectations. Positive relationships, good communication, involvement in decision-making, and (believing in) the reason for intervention were important mediators of birth experience. Professional attitudes and skills (development) were simultaneously barriers and facilitators of AVD in quantitative studies. Conclusions Information, positive interaction and communication with providers and respectful care are facilitators for acceptance of AVD. Barriers include lack of training and skills for decision-making and use of instruments

The combined immunodetection of AP-2α and YY1 transcription factors is associated with ERBB2 gene overexpression in primary breast tumors

INTRODUCTION: Overexpression of the ERBB2 oncogene is observed in about 20% of human breast tumors and is the consequence of increased transcription rates frequently associated with gene amplification. Several studies have shown a link between activator protein 2 (AP-2) transcription factors and ERBB2 gene expression in breast cancer cell lines. Moreover, the Yin Yang 1 (YY1) transcription factor has been shown to stimulate AP-2 transcriptional activity on the ERBB2 promoter in vitro. In this report, we examined the relationships between ERBB2, AP-2alpha, and YY1 both in breast cancer tissue specimens and in a mammary cancer cell line. METHODS: ERBB2, AP-2alpha, and YY1 protein levels were analyzed by immunohistochemistry in a panel of 55 primary breast tumors. ERBB2 gene amplification status was determined by fluorescent in situ hybridization. Correlations were evaluated by a chi2 test at a p value of less than 0.05. The functional role of AP-2alpha and YY1 on ERBB2 gene expression was analyzed by small interfering RNA (siRNA) transfection in the BT-474 mammary cancer cell line followed by real-time reverse transcription-polymerase chain reaction and Western blotting. RESULTS: We observed a statistically significant correlation between ERBB2 and AP-2alpha levels in the tumors (p < 0.01). Moreover, associations were found between ERBB2 protein level and the combined high expression of AP-2alpha and YY1 (p < 0.02) as well as between the expression of AP-2alpha and YY1 (p < 0.001). Furthermore, the levels of both AP-2alpha and YY1 proteins were inversely correlated to ERBB2 gene amplification status in the tumors (p < 0.01). Transfection of siRNAs targeting AP-2alpha and AP-2gamma mRNAs in the BT-474 breast cancer cell line repressed the expression of the endogenous ERBB2 gene at both the mRNA and protein levels. Moreover, the additional transfection of an siRNA directed against the YY1 transcript further reduced the ERBB2 protein level, suggesting that AP-2 and YY1 transcription factors cooperate to stimulate the transcription of the ERBB2 gene. CONCLUSION: This study highlights the role of both AP-2alpha and YY1 transcription factors in ERBB2 oncogene overexpression in breast tumors. Our results also suggest that high ERBB2 expression may result either from gene amplification or from increased transcription factor levels

Mismatch-repair protein MSH6 is associated with Ku70 and regulates DNA double-strand break repair

MSH6, a key component of the MSH2–MSH6 complex, plays a fundamental role in the repair of mismatched DNA bases. Herein, we report that MSH6 is a novel Ku70-interacting protein identified by yeast two-hybrid screening. Ku70 and Ku86 are two key regulatory subunits of the DNA-dependent protein kinase, which plays an essential role in repair of DNA double-strand breaks (DSBs) through the non-homologous end-joining (NEHJ) pathway. We found that association of Ku70 with MSH6 is enhanced in response to treatment with the radiomimetic drug neocarzinostatin (NCS) or ionizing radiation (IR), a potent inducer of DSBs. Furthermore, MSH6 exhibited diffuse nuclear staining in the majority of untreated cells and forms discrete nuclear foci after NCS or IR treatment. MSH6 colocalizes with γ-H2AX at sites of DNA damage after NCS or IR treatment. Cells depleted of MSH6 accumulate high levels of persistent DSBs, as detected by formation of γ-H2AX foci and by the comet assay. Moreover, MSH6-deficient cells were also shown to exhibit impaired NHEJ, which could be rescued by MSH6 overexpression. MSH6-deficient cells were hypersensitive to NCS- or IR-induced cell death, as revealed by a clonogenic cell-survival assay. These results suggest a potential role for MSH6 in DSB repair through upregulation of NHEJ by association with Ku70

A research agenda to improve incidence and outcomes of assisted vaginal birth.

Access to emergency obstetric care, including assisted vaginal birth and caesarean birth, is crucial for improving maternal and childbirth outcomes. However, although the proportion of births by caesarean section has increased during the last few decades, the use of assisted vaginal birth has declined. This is particularly the case in low- and middle-income countries, despite an assisted vaginal birth often being less risky than caesarean birth. We therefore conducted a three-step process to identify a research agenda necessary to increase the use of, or reintroduce, assisted vaginal birth: after conducting an evidence synthesis, which informed a consultation with technical experts who proposed an initial research agenda, we sought and incorporated the views of women's representatives of this agenda. This process has allowed us to identify a comprehensive research agenda, with topics categorized as: (i) the need to understand women's perceptions of assisted vaginal birth, and provide appropriate and reliable information; (ii) the importance of training health-care providers in clinical skills but also in respectful care, effective communication, shared decision-making and informed consent; and (iii) the barriers to and facilitators of implementation and sustainability. From women's feedback, we learned of the urgent need to recognize labour, childbirth and postpartum experiences as inherently physiological and dignified human processes, in which interventions should only be implemented if necessary. The promotion and/or reintroduction of assisted vaginal birth in low-resource settings requires governments, policy-makers and hospital administrators to support skilled health-care providers who can, in turn, respectfully support women in labour and childbirth