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

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

Biocompatibility of stereolithography 3D-printed calcium-phosphate scaffolds for bone regeneration

Objectives: The aim of this study was to evaluate the in vitrobiocompatibility and in vivobehaviors of two CaP based scaffolds manufactured by stereolithography (SLA) 3D printing technology. Methods: Pellets and macro-porous scaffolds were manufactured respectively for the in vitroand the in vivoexperiments both in pure hydroxyapatite (HA) and in biphasic CaP (HA:60-TCP:40.) The in vitro cytocompatibility was assessed by cell culture using a human osteoblastic-like cell line (MG-63). MTS assays were performed in order to evaluate cell viability. Additionally, the cell morphology and growth were characterized using SEM as well as DAPI and Actin staining. In vivobiocompatibility, newly formed bone and biodegradability of the experimental scaffolds were evaluated 3 and 6 months after implantation in a subperiosteal cranial rat model. The sample were subjected to microtomography for quantitative analyses and then, to paraffin histology for a descriptive analysis. Results: In vitro, cell viability over 75% was found in both group and no statistical difference was found between the 2 biomaterials (p= 0,07). The cells displayed similar behavior, in terms of morphology and cell growth, independently of the scaffold type. The in vivo experiments revealed an excellent osteointegration of the scaffolds and a very limited biodegradability. After 3 months, the bone volume inside pores achieved 72,78% (± 17,87%) and 60,76% (± 23,78%) respectively for HA and HA-TCP. The histological analysis did not reveal signs of inflammation and highlighted close contacts between newly formed bone and the experimental biomaterials. Conclusions: This study emphasized that SLA-3D printing of CaP based biomaterials might be relevant for intra oral bone regeneration. The cytocompatibility and in vivobiological performances in terms of graft dimensional stability and osseointegration was demonstrated. However, further experiments in larger models should be conducted

Detection of activator protein 2 alpha (AP-2α), Yin Yang 1 (YY1), and ERBB2 by immunohistochemistry in breast tumors

Case with low immunoreactivity for AP-2α. Tumor sample expressing high level of AP-2α protein in more than 80% of the nuclei.Tumor with low immunoreactivity for YY1. Case expressing high level of YY1 protein in more than 80% of the nuclei. Case with no ERBB2 membrane staining, scored as IHC 0. Tumor with partial weak membrane staining, scored as IHC 1+. Case with ERBB2 score of 2+. Tumor with thick circumferential ERBB2 membrane staining, scored as IHC 3+.<p><b>Copyright information:</b></p><p>Taken from "The combined immunodetection of AP-2α and YY1 transcription factors is associated with gene overexpression in primary breast tumors"</p><p>http://breast-cancer-research.com/content/10/1/R9</p><p>Breast Cancer Research : BCR 2008;10(1):R9-R9.</p><p>Published online 24 Jan 2008</p><p>PMCID:PMC2374961.</p><p></p