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SEM image of the ultrasonic culture, bar equal to 30 μm. During the culture period, the physical stimulus caused a wide-ranging coat of the internal surface of the biomaterial: several osteoblasts proliferated and the biomaterial was tending to be hidden by cell-matrix layers (asterisks).
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Bone graft substitutes and cancellous biomaterials have been widely used to heal critical-size long bone defects due to trauma, tumor resection, and tissue degeneration. In particular, porous hydroxyapatite is widely used in reconstructive bone surgery owing to its biocompatibility. In addition, the in vitro modification of cancellous hydroxyapatit...
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Citations
... For instance, a fluid shear stress [9,38,47] or ultrasounds [17] or biomaterial features [12,19,42] lead to the remodeling of bone matrix in vitro. ...
In a murine ventricular cardiac tissue in vitro, via an image processing analysis, we have studied the ergotropic effect (contraction energy) after electromagnetic stimulation (frequency, 75 Hz), isoproterenol administration (10 μM), and their combination. We have found that the electromagnetic stimulation is able to counteract the β-adrenergic action of isoproterenol.
... For example, a fluid shear stress [1][2][3] or ultrasounds [4] or biomaterial features [5] lead to the remodeling of bone matrix in vitro. In addition, the mechanical forces may also change the transcription more rapidly when they are transmitted directly into the nucleus via the cytoskeleton linked to nuclear envelop proteins [6]. ...
In a model of murine ventricular cardiac tissue in vitro , we have studied the inotropic effects of electromagnetic stimulation (frequency, 75 Hz), isoproterenol administration (10 μ M), and their combination. In particular, we have performed an image processing analysis to evaluate the kinematics and the dynamics of beating cardiac syncytia starting from the video registration of their contraction movement. We have found that the electromagnetic stimulation is able to counteract the β -adrenergic effect of isoproterenol and to elicit an antihypertrophic response.
... For these methods, the cells that were seeded on the surface were retained in the pores and on the top surface. However, this aspect cannot be ensured in an in vivo system, in which the body fluids surrounding the biomaterial could drag the cells from the desired site on porous scaffolds is the use of ultrasonic stimulation, which requires sophisticated equipment but promotes the expression of osteoblastic markers in in vitro assays 5 . The use of continuous cultures requires bioreactors, which guarantee a homogeneous distribution of nutrients in the culture medium; however, a significant percentage of the cultured cells is lost when replacing the culture medium due to the flow rate that is used in this technique and the medium adhering to the walls of the equipment. ...
Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs bovine bone matrix Nukbone (NKB) as a scaffold. Thus, the application of MSCs in repair and tissue regeneration processes depends principally on the efficient implementation of the techniques for placing these cells in a host tissue. For this reason, the design of biomaterials and cellular scaffolds has gained importance in recent years because the topographical characteristics of the selected scaffold must ensure adhesion, proliferation and differentiation into the desired cell lineage in the microenvironment of the injured tissue. This option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) employs bovine bone matrix as a cellular scaffold and is an efficient culture technique because the cells respond to the topographic characteristics of the bovine bone matrix Nukbone (NKB), i.e., spreading on the surface, macroporous covering and colonizing the depth of the biomaterial, after the cell isolation process. We present the procedure for isolating and culturing MSCs on a bovine matrix.
... Within the field of cell mechanics, ultrasound devices have been used to investigate the role of acoustic stimulation on cell proliferation [333][334][335][336][337], genetic activity [333,335,337], cell-cell interactions [338], and ECM organization [337], to induce the differentiation of various different cell types [337,339], as well as they have been used for cell manipulation and transport [330]. ...
The interplay between the mechanical properties of cells and the forces that they produce
internally or that are externally applied to them play an important role in maintaining
the normal function of cells. These forces also have a significant effect on the progression
of mechanically related diseases. To study the mechanics of cells, a wide variety of tools
have been adapted from the physical sciences. These tools have helped to elucidate the
mechanical properties of cells, the nature of cellular forces, and mechanoresponses that
cells have to external forces, i.e., mechanotransduction. Information gained from these
studies has been utilized in computational models that address cell mechanics as a collection
of biomechanical and biochemical processes. These models have been advantageous
in explaining experimental observations by providing a framework of underlying cellular
mechanisms. They have also enabled predictive, in silico studies, which would otherwise
be difficult or impossible to perform with current experimental approaches. In this
review, we discuss these novel, experimental approaches and accompanying computational
models. We also outline future directions to advance the field of cell mechanics. In
particular, we devote our attention to the use of microposts for experiments with cells
and a bio-chemical-mechanical model for capturing their unique mechanobiological
properties.
... The manufacturing process adheres to the strict control measures and clinical documentation in accordance with a quality assurance system based on international standards (ISO 13485 and ISO 9001). The data of safety concerning the use in humans of the bone substitute of bovine origins, were provided by the manufacturing company at registration site of the product; they are also well documented by the scientific literature available on Orthoss® (5)(6)(7)(8)(9)(10). Chemically speaking, Orthoss® is a matrix made of natural hydroxyapatite, the highly osteoconductive natural matrix possesses a topography which is very similar to the human bone. ...
... Chemically speaking, Orthoss® is a matrix made of natural hydroxyapatite, the highly osteoconductive natural matrix possesses a topography which is very similar to the human bone. Orthoss® facilitates angiogenesis and migration of osteoblasts throughout the matrix, and when it is implanted, the matrix is structurally integrated into the surrounding bone and it is incorporated into the physiological remodeling process (8)(9)(10). ...
Tridimensional scaffolds can promote bone regeneration as a framework supporting the migration of cells from the surrounding tissue into the damaged tissue and as delivery systems for the controlled or prolonged release of cells, genes, and growth factors. The goal of the work was to obtain an advanced medical device for bone regeneration through coating a decellularized and deproteinized bone matrix of bovine origin with a biodegradable, biocompatible polymer, to improve the cell engraftment on the bone graft. The coating protocol was studied and set up to obtain a continuous and homogeneous polylactide-co-glycolide (PLGA) coating on the deproteinized bone matrix Orthoss® block without occluding pores and decreasing the scaffold porosity. The PLGA-coated scaffolds were characterized for their morphology and porosity. The effects of PLGA polymer coating on cell viability were assessed with the 3-(4,5-dimethyl-2-thiazolyl)-2,5 diphenyl-2H-tetrazolium assay. The polymer solution concentration and the number of polymeric layers were the main variables affecting coating efficiency and porosity of the original decellularized bone matrix. The designed polymer coating protocol did not affect the trabecular structure of the original decellularized bone matrix. The PLGA-coated decellularized bone matrix maintained the structural features, and it improved the ability in stimulating fibroblasts attachment and proliferation.
... The AM-hMSC obtained, were cultured in H-DMEM under standard cultured conditions. All non-adherent cells were removed with the medium change after one week, while adherent cells were reached 90% confluence and recovered by trypsinization, then, seeded into 24-well cell culture plates at a density of 5 x10 5 cells on pre-moistened NKB as well as polystyrene disks and incubated overnight according to Fassina L, et al. [9]. Later, disks were placed in a new 24-well cell culture plate (time zero) in three conditions: 1) positive condition (+OS), AM-hMSC seeded on polystyrene disk with H-DMEM supplemented with 82 µg/mL ascorbic acid, 100 nM dexamethasone, and 10 mM β-glycerophosphate (H-DMEM-OS); 2) negative condition (-NKB), AM-hMSC cultured on polystyrene disk in H-DMEM and, 3) AM-hMSC with H-DMEM seeded on NKB disk as experimental condition (+NKB). ...
... In this context, NKB exhibits the required properties for being used as a bone regeneration biomaterial; contains mineral and collagen components, optimal pore sizes (200-2000 µm) and porous interconnectivity that, as physiologic human cancellous bone, represents an optimum structure for distribution of vessels and cell proliferation [13,14]. These are properties that create a suitable topography and environment for cell adhesion, proliferation and differentiation necessaries for osteoinductive activity [9, 15]. Also, NKB topography offers a suitable environment for cell adhesion, as showed by spreading cells seen at SEM images offigure 1A , where AM-hMSCs cultured on NKB adapt its morphological characteristics in response to surface topography (roughness, porosity, interconnectivity and chemical composition) as have been reported for bone scaffolds [16, 17]. ...
Bovine bone matrix Nukbone® (NKB) is an osseous tissue-engineering biomaterial that retains its mineral and organic phases and its natural bone topography and has been used as a xenoimplant for bone regeneration in clinics. There are not studies regarding its influence of the NKB in the behaviour of cells during the repairing processes. The aim of this research is to demonstrate that NKB has an osteoinductive effect in human mesenchymal stem cells from amniotic membrane (AM-hMSCs). Results indicated that NKB favours the AM-hMSCs adhesion and proliferation up to 7 days in culture as showed by the scanning electron microscopy and proliferation measures using an alamarBlue assay. Furthermore, as demonstrated by reverse transcriptase polymerase chain reaction, it was detected that two gene expression markers of osteoblastic differentiation: the core binding factor and osteocalcin were higher for AM-hMSCs co-cultured with NKB in comparison with cultivated cells in absence of the biomaterial. As the results indicate, NKB possess the capability for inducing successfully the osteoblastic differentiation of AM-hMSC, so that, NKB is an excellent xenoimplant option for repairing bone tissue defects.
... Thus, a great challenge consists in finding methods to selectively improve differentiation toward tissue, which would consequently reduce the differentiation period. Several stimulation methods that use different sources of energy have been widely tested to improve the rate of differentiation of primary cell culture to osteoblasts, that is, ultrasound [1,2] and electromagnetic fields [3][4][5]. In particular, following a biomimetic approach, mechanical stress has been demonstrated to be a good stimulus. ...
In order to verify whether differentiation of adult stem cells toward bone tissue is promoted by high-frequency vibration (HFV), bone marrow stromal cells (BMSCs) were mechanically stimulated with HFV (30 Hz) for 45 minutes a day for 21 or 40 days. Cells were seeded in osteogenic medium, which enhances differentiation towards bone tissue. The effects of the mechanical treatment on differentiation were measured by Alizarin Red test, (q) real-time PCR, and protein content of the extracellular matrix. In addition, we analyzed the proliferation rate and apoptosis of BMSC subjected to mechanical stimulation. A strong increase in all parameters characterizing differentiation was observed. Deposition of calcium was almost double in the treated samples; the expression of genes involved in later differentiation was significantly increased and protein content was higher for all osteogenic proteins. Lastly, proliferation results indicated that stimulated BMSCs have a decreased growth rate in comparison with controls, but both treated and untreated cells do not enter the apoptosis process. These findings could reduce the gap between research and clinical application for bone substitutes derived from patient cells by improving the differentiation protocol for autologous cells and a further implant of the bone graft into the patient.
... However, when the ionic effect was combined with the effect of topography in the comparative SLA 10/30/TTCP group, the proliferative ability of E1 cells was shown to be significant lower (P < 0.05) than in the SLA 10/30 group. The calcium and phosphate ions became incorporated into the apatite that formed in an intimate association with the organic component, leading to bone formation [31,32]. Figure 6 showed the promoted E1 bone cell growth was largely due to the topography rather than the ions. The ion effect played a less important role than the roughness with respect to E1 cell proliferation. ...
The major challenge for dental implants is achieving optimal esthetic appearance and a concept to fulfill this criterion is evaluated. The key to an esthetically pleasing appearance lies in the properly manage the soft tissue profile around dental implants. A novel implant restoration technique on the surface was proposed as a way to augment both soft- and hard-tissue profiles at potential implant sites. Different levels of roughness can be attained by sandblasting and acid etching, and a tetracalcium phosphate was used to supply the ions. In particular, the early stage attaching and repopulating abilities of bone cell osteoblasts (MC3T3-E1), fibroblasts (NIH 3T3), and epithelial cells (XB-2) were evaluated. The results showed that XB-2 cell adhesive qualities of a smooth surface were better than those of the roughened surfaces, the proliferative properties were reversed. The effects of roughness on the characteristics of 3T3 cells were opposite to the result for XB-2 cells. E1 proliferative ability did not differ with any statistical significance. These results suggest that a rougher surface which provided calcium and phosphate ions have the ability to enhance the proliferation of osteoblast and the inhibition of fibroblast growth that enhance implant success ratios.
Pulsed electromagnetic field (PEMF) has drawn attention as a potential tool to improve the ability of bone biomaterials to integrate into the surrounding tissue. We investigated the effects of PEMF (frequency, 75 Hz; magnetic induction amplitude, 2 mT; pulse duration, 1.3 ms) on human osteoblast-like cells (SAOS-2) seeded onto wool keratin scaffolds in terms of proliferation, differentiation, and production of the calcified bone extracellular matrix. The wool keratin scaffold offered a 3D porous architecture for cell guesting and nutrient diffusion, suggesting its possible use as a filler to repair bone defects. Here, the combined approach of applying a daily PEMF exposure with additional osteogenic factors stimulated the cells to increase both the deposition of bone-related proteins and calcified matrix onto the wool keratin scaffolds. Also, the presence of SAOS-2 cells, or PEMF, or osteogenic factors did not influence the compression behavior or the resilience of keratin scaffolds in wet conditions. Besides, ageing tests revealed that wool keratin scaffolds were very stable and showed a lower degradation rate compared to commercial collagen sponges. It is for these reasons that this tissue engineering strategy, which improves the osteointegration properties of the wool keratin scaffold, may have a promising application for long term support of bone formation in vivo.
Gelatin-based cryogels have been seeded with human SAOS-2 osteoblasts. In order to overcome the drawbacks associated with in vitro culture systems, such as limited diffusion and inhomogeneous cell–matrix distribution, we have described the application of electromagnetic and ultrasound stimulation to physically enhance the cell culture in vitro. The results indicate that the physical stimulation of cell-seeded gelatin-based cryogels upregulates the bone matrix production.
