Development of a biomimetic bioreactor for regenerative endodontics research.

In the context of regenerative endodontics research with the development of biomaterials, this work aimed to develop and test a prototype biomimetic bioreactor of a human tooth. The bioreactor was designed to reproduce a shaped dental canal connected with a cavity reproducing the periapical region and irrigated through two fluidic channels intended to reproduce the apical residual vascular supply. A test biomaterial composed of polylactic acid/polycaprolactone-tannic acid (PLA/PCL-TA) was produced by electrospinning/electrospraying and calibrated to be inserted in a dental canal. This biomaterial was first used to evaluate its imbibition capacity and the oximetry inside the bioreactor. Then, Dental Pulp Stem Cells (DPSCs) were cultured on PLA/PCL-TA cones for 1-3 weeks in the bioreactor; afterward cell adhesion, proliferation, and migration were histologically assessed. Complete imbibition biomaterial was obtained in 10 min and oximetry was stable over time. In the bioreactor, DPSCs were able to adhere, proliferate and migrate onto the surface and inside the biomaterial. In conclusion, this bioreactor was used successfully to test a biomaterial intended to support pulp regeneration and constitutes a new in vitro experimental model closer to clinical reality.

Highly Structured 3D Electrospun Conical Scaffold: A Tool for Dental Pulp Regeneration.

New procedures envisioned for dental pulp regeneration after pulpectomy include cell homing strategy. It involves host endogenous stem cell recruitment and activation. To meet this cell-free approach, we need to design a relevant scaffold to support cell migration from tissues surrounding the dental root canal. A composite membrane made of electrospun poly(lactic acid) nanofibers and electrosprayed polycaprolactone with tannic acid (TA) microparticles which mimics the architecture of the extracellular matrix was first fabricated. After rolling the membrane in the form of a 3D conical scaffold and subsequently coating it with gelatin, it can be directly inserted into the root canal. The porous morphology of the construct was characterized by SEM at different length scales. It was shown that TA was released from the 3D conical scaffold after 2 days in PBS at 37 °C. Biocompatibility studies were first assessed by seeding human dental pulp stem cells (DPSCs) on planar membranes coated or not coated with gelatin to compare the surfaces. After 24 h, the results highlighted that the gelatin-coating increased the membrane biocompatibility and cell viability. Similar DPSC morphology and proliferation on both membrane surfaces were observed. The culture of DPSCs on conical scaffolds showed cell colonization in the whole cone volume, proving that the architecture of the conical scaffold was suitable for cell migration.

Maxillary Sinus Lift with Beta-Tricalcium Phosphate (ß-TCP) in Edentulous Patients: A Nanotomographic and Raman Study.

Sinus lift elevation restores bone mass at the maxilla in edentulate patients before the placement of dental implants. It consists of opening the lateral side of the sinus and grafting beta-tricalcium phosphate granules (ß-TCP) under the olfactory membrane. Bone biopsies were obtained in five patients after 60 weeks. They were embedded undecalcified in poly(methyl methacrylate) (pMMA); blocks were analyzed by nanocomputed tomography (nanoCT); specific areas were studied by Raman microspectroscopy. Remnants of ß-TCP were osseointegrated and covered with mineralized bone; osteoid tissue was also filling the inner porosity. Macrophages having engulfed numerous ß-TCP grains were observed in marrow spaces. ß-TCP was identified by nanoCT as osseointegrated particles and as granules in the cytoplasm of macrophages. Raman microspectroscopy permitted to compare the spectra of ß-TCP and bone in different areas. The ratio of the ~820 cm-1 band of pMMA (-CH2 groups) on the ν1 phosphate band at 960 cm-1 reflected tissue hydration because water was substituted by MMA during histological processing. In bone, the ratio of the ~960 cm-1 phosphate to the amide 1 band and the ratio ν2 phosphate band by the 1240-1250 amide III band reflect the mineralization degree. Specific bands of ß-TCP were found in osseointegrated ß-TCP granules and in the grains phagocytized by the macrophages. The hydration degree was maximal for ß-TCP phagocytized by macrophages. Raman microspectroscopy associated with nanoCT is a powerful tool in the analysis of the biomaterial degradation and osseointegration.

Repair of calvarial bone defects in mice using electrospun polystyrene scaffolds combined with ß-TCP or gold nanoparticles.

Non-biodegradable porous polystyrene (PS) scaffolds, composed of microfibers, have been prepared by electrospinning for the reconstruction of large bone defects. PS microfibers were prepared by incorporating ß-TCP grains inside the polymer or grafting gold nanoparticles surface functionalized with mercaptosuccinic acid. Cytocompatibility of the three types of scaffolds (PS, ß-TCP-PS and Au-PS) was studied by seeding human mesenchymal stem cells. Biocompatibility was evaluated by implanting ß-TCP-PS and Au-PS scaffolds into a critical size (4mm) calvarial defect in mice. Calvaria were taken 6, 9, and 12 weeks after implantation; newly formed bone and cellular response was analyzed by microcomputed tomography (microCT) and histology. ß-TCP-PS scaffolds showed a significantly higher cell proliferation in vitro than on PS or Au-PS alone; clearly, the presence of ß-TCP grains improved cytocompatibility. Biocompatibility study in the mouse calvaria model showed that ß-TCP-PS scaffolds were significantly associated with more newly-formed bone than Au-PS. Bone developed by osteoconduction from the defect margins to the center. A dense fibrous connective tissue containing blood vessels was identified histologically in both types of scaffolds. There was no inflammatory foci nor giant cell in these areas. AuNPs aggregates were identified histologically in the fibrosis and also incorporated in the newly-formed bone matrix. Although the different types of PS microfibers appeared cytocompatible during the in vitro experiment, they appeared biotolerated in vivo since they induced a fibrotic reaction associated with newly formed bone.

Polystyrene scaffolds based on microfibers as a bone substitute; development and in vitro study.

We created non-resorbable porous scaffolds of polystyrene by electrospinning usable as a bone grafting material. Aligned and random fibers were prepared with a diameter ranging from 1 to 4.5µm. Influence of microfiber diameter and alignment were determined by culturing MC3T3 osteoblast-like cells and evaluation of adherence, proliferation and differentiation at day 14 and 28 on the scaffolds. Scanning electron microscopy (SEM), nanocomputed tomography (nanoCT) and confocal microscopy were used to observe microfibers and morphology of cells seeded on the scaffolds. Nile Red was used to label the fibers, DAPI for nuclear staining and calcein for the calcium/phosphate deposits. MC3T3 were more adherent on the randomly distributed fibers having the highest diameter. MC3T3 proliferated equally on scaffolds made with aligned fibers but cell density was lower on random fibers with the smaller diameter. Alkaline phosphatase activity (a marker of osteoblastic differentiation) was not influenced by the fibers apart from on random fibers with the smallest diameter. Calcospherites also developed at the surface of the fibers in long term culture. Cytometric determination of the nuclei shape factors evidenced that cells were elongated along the main direction of fibers only on the aligned fibers. This study shows that porous scaffolds based on microfibers allow adhesion, spreading, orientation and proliferation of cells. STATEMENT OF SIGNIFICANCE: We prepared polystyrene porous scaffolds composed of microfibers as a bone substitute by electrospinning. Polystyrene is a cytocompatible and non-resorbable polymer which can support osteoconduction. Scaffolds with different micro-diameters and orientation, (aligned and random) were seeded with osteoblast-like cells to evaluate cell adherence, proliferation and differentiation. Characterization of microfibers and cell morphology was done by scanning electron microscopy, nanocomputed tomography and confocal microscopy. We evidenced that initial adherence of cells was increased on randomly disposed fibers with a high diameter (3.5µm). Cell proliferation and differentiation seems not to be influenced by fiber diameter and orientation, apart from random fibers of 1µm diameter which had a lower cell attachment. Morphometric analysis of cell nuclei showed that cells were stretched along the aligned fibers.

3D Porous Architecture of Stacks of ß-TCP Granules Compared with That of Trabecular Bone: A microCT, Vector Analysis, and Compression Study.

The 3D arrangement of porous granular biomaterials usable to fill bone defects has received little study. Granular biomaterials occupy 3D space when packed together in a manner that creates a porosity suitable for the invasion of vascular and bone cells. Granules of beta-tricalcium phosphate (ß-TCP) were prepared with either 12.5 or 25 g of ß-TCP powder in the same volume of slurry. When the granules were placed in a test tube, this produced 3D stacks with a high (HP) or low porosity (LP), respectively. Stacks of granules mimic the filling of a bone defect by a surgeon. The aim of this study was to compare the porosity of stacks of ß-TCP granules with that of cores of trabecular bone. Biomechanical compression tests were done on the granules stacks. Bone cylinders were prepared from calf tibia plateau, constituted high-density (HD) blocks. Low-density (LD) blocks were harvested from aged cadaver tibias. Microcomputed tomography was used on the ß-TCP granule stacks and the trabecular bone cores to determine porosity and specific surface. A vector-projection algorithm was used to image porosity employing a frontal plane image, which was constructed line by line from all images of a microCT stack. Stacks of HP granules had porosity (75.3 ± 0.4%) and fractal lacunarity (0.043 ± 0.007) intermediate between that of HD (respectively 69.1 ± 6.4%, p < 0.05 and 0.087 ± 0.045, p < 0.05) and LD bones (respectively 88.8 ± 1.57% and 0.037 ± 0.014), but exhibited a higher surface density (5.56 ± 0.11 mm(2)/mm(3) vs. 2.06 ± 0.26 for LD, p < 0.05). LP granular arrangements created large pores coexisting with dense areas of material. Frontal plane analysis evidenced a more regular arrangement of ß-TCP granules than bone trabecule. Stacks of HP granules represent a scaffold that resembles trabecular bone in its porous microarchitecture.

Analysis of ß-tricalcium phosphate granules prepared with different formulations by nano-computed tomography and scanning electron microscopy.

Among biomaterials used for filling bone defects, beta-tricalcium phosphate (ß-TCP) is suitable in non-bearing bones, particularly in dental implantology, oral and maxillofacial surgery. When ß-TCP granules are placed in a bone defect, they occupy the void 3D volume. Little is known about the 3D arrangement of the granules, which depends on the nature and size of the granules. The aim of this study was to examine the 3D architecture of porous ß-TCP granules. Granules were prepared with different concentrations of ß-TCP powder in slurry (10, 11, 15, 18, 21, and 25 g of ß-TCP powder in distilled water). Granules were prepared by the polyurethane foam method. They were analyzed by nano-computed tomography (nanoCT) and compared with scanning electron microscopy (SEM). Commercial granules of hydroxyapatite-ß-TCP prepared by the same methodology were also used. The outer and inner architectures of the granules were shown by nanoCT which evidenced macroporosity, internal porosity and microporosity between the sintered grains. Macroporosity was reduced at high concentration and conversely, numerous concave surfaces were observed. Internal porosity, related to the sublimation of the polyurethane foam, was present in all the granules. Microporosity at the grain joints was evidenced by SEM and on 2D nanoCT sections. Granules presented a heterogeneous aspect due to the different mineralization degree of the sintered powder grains in the ß-TCP granules; the difference between hydroxyapatite and ß-TCP was also evidenced. NanoCT is an interesting method to analyze the fine morphology of biomaterials with a resolution close to synchrotron and better than microcomputed tomography.

Three-dimensional arrangement of ß-tricalcium phosphate granules evaluated by microcomputed tomography and fractal analysis.

The macrophysical properties of granular biomaterials used to fill bone defects have rarely been considered. Granules of a given biomaterial occupy three-dimensional (3-D) space when packed together and create a macroporosity suitable for the invasion of vascular and bone cells. Granules of ß-tricalcium phosphate were prepared using polyurethane foam technology and increasing the amount of material powder in the slurry (10, 11, 15, 18, 21 and 25 g). After sintering, granules of 1000-2000 µm were prepared by sieving. They were analyzed morphologically by scanning electron microscopy and placed in polyethylene test tubes to produce 3-D scaffolds. Microcomputed tomography (microCT) was used to image the scaffolds and to determine porosity and fractal dimension in three dimensions. Two-dimensional sections of the microCT models were binarized and used to compute classical morphometric parameters describing porosity (interconnectivity index, strut analysis and star volumes) and fractal dimensions. In addition, two newly important fractal parameters (lacunarity and succolarity) were measured. Compression analysis of the stacks of granules was done. Porosity decreased as the amount of material in the slurry increased but non-linear relationships were observed between microarchitectural parameters describing the pores and porosity. Lacunarity increased in the series of granules but succolarity (reflecting the penetration of a fluid) was maximal in the 15-18 g groups and decreased noticeably in the 25 g group. The 3-D arrangement of biomaterial granules studied by these new fractal techniques allows the optimal formulation to be derived based on the lowest amount of material, suitable mechanical resistance during crushing and the creation of large interconnected pores.