[Show abstract][Hide abstract] ABSTRACT: Hydrogels are a suitable scaffold material for a variety of tissue engineering applications. However, these materials have a weak structure and require reinforcement. Integrating electrospun fibers could strengthen material properties. This study created fibers and evaluated the influence of the presence of polar head groups within a polysaccharide backbone following functionalization: silated-hydroxypropyl methylcellulose (Si-HPMC). Electrospinning is a multi-parameter, step by step process that requires optimization of solution and process parameters to understand and control the process. Fibers were created for 2%–3% wt/v solutions in water and ethanol. Viscosities of solutions were correlated with spinnability. Variations on process parameters did not reveal major variation on fiber morphology. Once controlled, the process was used for HPMC/Si-HPMC mixture solutions. Solubilization and dilution of Si-HPMC were made with common solvents for electrospinning. Two forms of polymer conformation were electrospun: silanol ending and silanolate ending. Microstructures and resulting nanofibers were analyzed by scanning electron microscopy (SEM) and Energy Dispersive Analysis (EDX). The results show the feasibility of our strategy for creating nanofibers and the influence of polar head groups on electrospinnability.
[Show abstract][Hide abstract] ABSTRACT: The nutritional benefits of pomegranate have attracted great scientific interest. The pomegranate, including the pomegranate peel, has been used worldwide for many years as a fruit with medicinal activity, mostly antioxidant properties. Among chronic diseases, osteoporosis, which is associated with bone remodelling impairment leading to progressive bone loss, could eventually benefit from antioxidant compounds because of the involvement of oxidative stress in the pathogenesis of osteopenia. In this study, with in vivo and ex vivo experiments, we investigated whether the consumption of pomegranate peel extract (PGPE) could limit the process of osteopenia. We demonstrated that in ovariectomized (OVX) C57BL/6J mice, PGPE consumption was able to significantly prevent the decrease in bone mineral density (-31.9%; p < 0.001 vs. OVX mice) and bone microarchitecture impairment. Moreover, the exposure of RAW264.7 cells to serum harvested from mice that had been given a PGPE-enriched diet elicited reduced osteoclast differentiation and bone resorption, as shown by the inhibition of the major osteoclast markers. In addition, PGPE appeared to substantially stimulate osteoblastic MC3T3-E1 alkaline phosphatase (ALP) activity at day 7, mineralization at day 21 and the transcription level of osteogenic markers. PGPE may be effective in preventing the bone loss associated with ovariectomy in mice, and offers a promising alternative for the nutritional management of this disease.
[Show abstract][Hide abstract] ABSTRACT: Statement of significance:
A major challenge in the design of biomaterial-based injectable bone substitutes is the development of cohesive, macroporous and self-setting calcium phosphate cement (CPC) that enables rapid cell invasion with adequate initial mechanical properties without the use of complex processing and additives. Thus, we propose a simple and effective strategy to prepare injectable macroporous CPCs through syringe-foaming using a hydrophilic viscous polymeric solution (silanized-hydroxypropyl methylcellulose, Si-HPMC) as a foaming agent, that simultaneously meets all the aforementioned aims. Evidence from our in vivo studies shows the existence of newly formed bone within the implantation site, indicating the feasibility and effectiveness of this foaming strategy, which could be used in various CPC systems using other hydrophilic viscous polymeric solutions.
Full-text · Article · Nov 2015 · Acta biomaterialia
[Show abstract][Hide abstract] ABSTRACT: Objectives To develop an animal model of mandibular osteoradionecrosis (ORN) using a high-energy radiation source (as used in human therapeutics) and to assess the role of tooth extraction on ORN development. Materials and methods (study design) Ten animals were irradiated with a single 35- or 50-Gy dose. Three weeks later, the second left mandibular molar was extracted from three animals in each group. Nine weeks after irradiation, the animals were euthanized, with an injection of contrast agent in the bloodstream to highlight vascularization. Mandibles were harvested and studied using micro-CT, histology, tartrate-resistant acid phosphatase activity and scanning electron microscopy. Results This study demonstrates that a single 50-Gy dose associated with molar extraction is necessary for ORN development. In these conditions, absence of healing of the mucosa and bone, dental effects, fibrosis, an increase in osteoclast activity and a decrease in vascularization were observed. We also determined that molar extraction increases the impact of the cellular effects of radiation. Conclusion The mandibular ORN animal model was validated after 50-Gy irradiation and molar extraction. The results of this study therefore support an animal ORN model and tissue engineering strategies will now be developed to regenerate bone for patients with head and neck cancer.
No preview · Article · Nov 2015 · Journal of Cranio-Maxillofacial Surgery
[Show abstract][Hide abstract] ABSTRACT: Vascularization is essential for many tissues and is a main requisite for various tissue-engineering strategies. Different techniques are used for highlighting vasculature, in vivo and ex vivo, in 2-D or 3-D including histological staining, immunohistochemistry, radiography, angiography, microscopy, computed tomography (CT) or micro-CT, both stand-alone and synchrotron system. Vascularization can be studied with or without a contrast agent. This paper presents the results obtained with the latest Skyscan micro-CT (Skyscan 1272, Bruker, Belgium) following barium sulphate injection replacing the bloodstream in comparison with results obtained with a Skyscan In Vivo 1076. Different hard and soft tissues were perfused with contrast agent and were harvested. Samples were analysed using both forms of micro-CT, and improved results were shown using this new micro-CT. This study highlights the vasculature using micro-CT methods. The results obtained with the Skyscan 1272 are clearly defined compared to results obtained with Skyscan 1076. In particular, this instrument highlights the high number of small vessels, which were not seen before at lower resolution. This new micro-CT opens broader possibilities in detection and characterization of the 3-D vascular tree to assess vascular tissue engineering strategies.
[Show abstract][Hide abstract] ABSTRACT: Treatment of carcinomas of the upper aerodigestive tract often requires external radiation therapy. However, radiation affects all the components of bone, with different degrees of sensitivity, and may produce severe side effects such as mandibular osteoradionecrosis (ORN). Intraosseous vascularization is thought to be decreased after irradiation, but its impact on total bone volume is still controversial. The aim of this study was to compare intraosseous vascularization, cortical bone thickness, and total bone volume in a rat model of ORN versus nonirradiated rats, using a micro-computed tomography (micro-CT) analysis after intracardiac injection of a contrast agent. The study was performed on 8-week-old Lewis 1A rats (n = 14). Eleven rats underwent external irradiation on the hind limbs by a single 80-Gy dose. Three rats did not receive irradiation and served as controls for statistical analysis. Eight weeks after the external irradiation, all the animals received a barium sulfate intracardiac injection under general anesthesia. All samples were analyzed with the micro-computed tomography system at a resolution of 5.5 μm. The images were later processed to create 3D reconstructions and study vascularization, bone volume, and cortical thickness. Data from irradiated and nonirradiated rats were compared using the Kruskal-Wallis test. No animal died after irradiation. Nineteen irradiated tibias and six nonirradiated tibias were included for micro-CT analysis. The vessel percentage was significantly lower in irradiated bones (p = 0.0001). The distance between the vessels, a marker of vascular destruction, was higher after irradiation (p = 0.001). The vessels were also more altered distally after irradiation (p = 0.028). Cortical thickness was severely decreased after irradiation, sometimes even reduced to zero. Both trabecular and cortical structures were destroyed after irradiation, with wide bone gaps. Finally, both total bone volume (p = 0.0001) and cortical thickness (p = 0.0001) were significantly decreased in irradiated tibias compared to nonirradiated tibias. These results led to multiple spontaneous fractures in the irradiated group, and the destruction of intraosseous vessels observed macroscopically with the radiographic preview. This study revealed the impact of radiation on intraosseous vasculature and cortical bone with a micro-CT analysis in a rat ORN model. Hypovascularization and osteopenia are consistent with the literature, contributing a morphological scale with high resolution. Visualization of the vasculature by micro-CT is an innovative technique to see the changes after radiation, and should help adjust bone tissue engineering in irradiated bone.
No preview · Article · May 2015 · Calcified Tissue International
[Show abstract][Hide abstract] ABSTRACT: Bone repair is an important concept in tissue engineering, and the ability to repair bone in hypotrophic conditions such as that of irradiated bone, represents a challenge for this field. Previous studies have shown that a combination of bone marrow and (BCP) was effective to repair irradiated bone. However, the origin and role played by each cell type in bone healing still remains unclear. In order to track the grafted cells, the development of an animal model that is immunotolerant to an allograft of bone marrow would be useful. Furthermore, because the immune system interacts with bone turnover, it is of critical importance to demonstrate that immunosuppressive drugs do not interfere with bone repair. After a preliminary study of immunotolerance, cyclosporin-A was chosen to be used in immunosuppressive therapy. Ten rats were included to observe qualitative and quantitative bone repair 8 days and 6 weeks after the creation of bone defects. The defects were filled with an allograft of bone marrow alone or in association with BCP under immunosuppressive treatment (cyclosporin-A). The results showed that there was no significant interaction of cyclosporin-A with osseous regeneration. The use of this new immunotolerant rat model of bone marrow allograft in future studies will provide insight on how the cells within the bone marrow graft contribute to bone healing, especially in irradiated conditions.
No preview · Article · Feb 2015 · Calcified Tissue International
[Show abstract][Hide abstract] ABSTRACT: Trabecular bone and its micro-architecture are of prime importance for health. Changes of bone micro-architecture are linked to different pathological situations like osteoporosis and begin now to be understood. In a previous paper , we started to investigate the relationships between bone and vessels and proposed some indices of characterization for the vessels issued from those used for the bone. Our main objective in this paper is to qualify the classical values used for bone as well as those we proposed for vessels according to different acquisition parameters and for several thresholding methods used to separate bone vessels and background. This study is also based on vessels perfusion by a contrast agent (barium sulfate mixed with gelatin) before euthanasia on rats. Femurs and tibias as well as mandibles were removed after rat's death and were imaged by microCT (Skyscan 1272, Bruker, Belgium) with a resolution ranging from 18 to 3μm. The so obtained images were analyzed with various softwares (NRecon Reconstruction, CtAn, and CtVox from Bruker) in order to calculate bone and vessels micro-architecture parameters (density of bone/blood within the volume), and to know if the results both for bone and vascular micro-architecture are constant along the chosen pixel resolution. The result is clearly negative. We found a very different characterization both for bone and vessels with the 3μm acquisition. Tibia and mandibles bones were also used to show results that can be visually assessed. The largest portions of the vascular tree are orthogonal to the obtained slices of the bone. Therefore, the contrast agent appears as cylinders of various sizes.
[Show abstract][Hide abstract] ABSTRACT: Self-injection of high-dose buprenorphine is responsible for well-described complications. In 2011, we have been alerted by unusual but serious cutaneous complication among injection buprenorphine users. A prospective data collection identified 30 cases of necrotic cutaneous lesions after injection of filtered buprenorphine solution, among which 25 cases occurred following injection of buprenorphine generics. The main goal of our study was to put forward particularities that could explain the cutaneous complications, by qualitatively and quantitatively confronting particles present in Subutex and generics solutions. We used the same protocol that injected-buprenorphine users: generic or subutex tablets were crushed in sterile water and filtered through 2 filters commonly used (cotton-pad and sterifilt). Solutions were analyzed by laser granulometry, flow cytometry and scanning electron microscopy. We have highlighted the wide variation of the quantity and the size of the particles present in solution between the two drugs after cotton-pad filtration. The proportion of particles <10 µm is systematically higher in the generic solutions than with Subutex. All of the insoluble particles found in generic solutions contain silica, whereas non- organic element was to be identified in the insoluble particles of Subutex. One skin biopsy obtained from one patient who developed a necrotic lesion after intravenous injection of filtrated solution of buprenorphine generic, shows non-organic elements. Identification of particles in situ enables us to confirm the presence of silica in the biopsy. Actually the monitoring of patient receiving generic of buprenorphine must be strengthened.
[Show abstract][Hide abstract] ABSTRACT: In the context of bone regeneration in an osteoporotic environment, the present study described the development of an approach based on the use of calcium phosphate (CaP) bone substitutes that can promote new bone formation and locally deliver in situ bisphosphonate (BP), directly at the implantation site. The formulation of a CaP material has been optimized by designing an injectable apatitic cement that (i) hardens in situ despite the presence of BP and (ii) provides immediate mechanical properties adapted to clinical applications in an osteoporotic environment. We developed a large animal model for simulating lumbar vertebroplasty through a two-level lateral corpectomy on L3 and L4 vertebrae presenting a standardized osteopenic bone defect that was filled with cements. Both 2D and 3D analysis of micro-architectural parameters demonstrated that implantation of BP loaded cement in such vertebral defects positively influenced microarchitecture of the adjacent trabecular bone. This biological effect was distance-dependent from the implant, emphasizing the in situ effect of the BP and its release from the cement. As a drug device combination, this BP-containing apatitic cement shows good promise local approach for the prevention of osteoporotic vertebral fractures through percutaneous vertebroplasty procedures.
No preview · Article · Nov 2014 · Acta Biomaterialia
[Show abstract][Hide abstract] ABSTRACT: The consequences of the treatment of the squamous cell carcinomas of the upper aerodigestive tract (bone removal and external radiation therapy) are constant. Tissue engineering using biphasic calcium phosphate (BCP) and mesenchymal stem cells (MSC) is considered as a promising alternative. We previously demonstrated the efficacy of BCP and total fresh bone marrow (TBM) in regenerating irradiated bone defect. The aim of this study was to know if adding MSC to BCP + TBM mixture could improve the bone formation in irradiated bone defects. Twenty-four Lewis 1A rats received a single dose of 20 Gy to the hind limbs. MSC were sampled from non-irradiated donors and amplified in proliferative, and a part in osteogenic, medium. 3 weeks after, defects were created on femurs and tibias, which were filled with BCP alone, BCP + TBM, BCP + TBM + uncommitted MSC, or BCP + TBM + committed MSC. 3 weeks after, samples were removed and prepared for qualitative and quantitative analysis. The rate of bone ingrowth was significantly higher after implantation of BCP + TBM mixture. The adding of a high concentration of MSC, committed or not, didn't improve the bone regeneration. The association BCP + TBM remains the most efficient material for bone substitution in irradiated areas.
No preview · Article · Aug 2014 · Journal of Materials Science Materials in Medicine
[Show abstract][Hide abstract] ABSTRACT: Maintenance of cell survival is essential for proper embryonic development. In the mouse, Notchless homolog 1 (Drosophila) (Nle1) is instrumental for survival of cells of the inner cell mass upon implantation. Here, we analyze the function of Nle1 after implantation using the Meox2tm1(cre)Sor mouse that expresses the Cre recombinase specifically in the epiblast at E5.5. First, we find that NLE1 function is required in epiblast cells, as Nle1-deficient cells are rapidly eliminated. In this report, we also show that the Meox2Cre transgene is active in specific tissues during organogenesis. In particular, we detect high Cre expression in the vertebral column, ribs, limbs and tailbud. We took advantage of this dynamic expression profile to analyze the effects of inducing mosaic deletion of Nle1 in the embryo. We show that Nle1 deletion in this context, results in severe developmental anomalies leading to lethality at birth. Mutant embryos display multiple developmental defects in particular during axial skeletal formation. We also provide evidence that axial defects are due to an increase in apoptotic cell death in the somite at E9.5. These data demonstrate an essential role for Nle1 during organogenesis and in particular during axial development.