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Representative histochemical ALP staining, Alizarin Red staining and indirect immunofluorescence images of ahMSC cultured in the presence of 1.5 wt.% C 2 S-TCP material. (A) ahMSCs were positive for alkaline phosphatase staining, acquiring a reddish tint (200×); (B) mineral deposits are stained bright red by the Alizarin Red Solution (100×) (inserted image: 200×); (C) indirect immunofluorescent microscopy of ahMSC. Cells were labeled with rabbit anti-Collagen type I antibody, followed by incubation with Alexa 488-conjugated anti-rabbit secondary antibody (green) (120×); (D) cells were labeled with rabbit anti-osteocalcin antibody, followed by incubation with Alexa 488-conjugated anti-rabbit secondary antibody (green) (120×). (E) Cells were labeled with rabbit anti-osteopontin antibody, followed by incubation with Alexa 488-conjugated anti-rabbit secondary antibody (green) (120×). (F) Cells were labeled with rat anti heparan sulfate antibody, followed by incubation with Alexa 488-conjugated anti-rat secondary antibody (green) (120×). (G) Cells were labeled with mouse anti-β-tubulin, then Alexa 488-conjugated anti-mouse secondary antibody (green) (200×). Scale bar: 300 μm.
Source publication
Bioceramic samples with osteogenic properties, suitable for use in the regeneration of hard tissue, were synthesized. The materials consisting of α-tricalcium phosphate (αTCP) and also αTCP doped with either 1.5wt.% or 3.0wt.% of dicalcium silicate (C2S) in the system Dicalcium Silicate-Tricalcium Phosphate (C2S-TCP) were obtained by solid state re...
Contexts in source publication
Context 1
... skin-derived fibroblast cul- tured in the presence of different active biomaterials for the same time periods. In these samples the presence of the ceramics in the culture me- dium did not induce the extracellular matrix mineralization, confirmed by the negative staining using Alizarin Red S (data not shown). At the end of the study (30 days) (Fig. 6) the cells were positive for alkaline phosphatase staining. Col-I positive groups of cells augmented in the control and also in the samples incubated in the presence of the bioma- terials; but the general aspect of the control cells was always less positive than the ones relative to biomaterials. The HS expression was very high in all ...
Context 2
... of constitu- ent proteins of the ECM (OP, HS-proteoglycans, etc.) and finally the deposition of calcium salts. At the end of the study (30 days) all of the osteoblastic markers studied (ALP, Col I and OC) likewise confirmed the expression of osteoblast phenotype and the expression of ECM pro- teins (OP, HS-proteoglycans) was also detected (Fig. 6). These findings showed that the ahMSCs have carried out a process of differentiation, showing both an osteoblastic phenotype and ability to synthesize min- eralized ECM. The effect of TCPss materials on ahMSC differentiation into osteoblasts was favored by the Si in solid solution in the TCP ceramics as well as the release of Si, Ca ...
Citations
... Calcium silicate ceramics are used as a biomaterial owing to their excellent characteristics such as biocompatibility, bioactivity and biodegradability. [1][2][3] Calcium silicate ceramics are mostly used in dentistry, orthopaedics, drug delivery systems, and tissue engineering. [3][4][5][6] They are also utilized in cosmetic formulations as absorbent, opacifying agent, and bulking agent. ...
... 11,12 They induce cellular adhesion, promote division and differentiation of bone marrow mesenchymal stem cells. 1,2 Also Si +4 ions contribute upregulation of angiogenic gene expression. Upon contact with water and environmental fluids, calcium silicate materials undergo a reaction that leads to the formation of calcium hydroxide. ...
In the current study, a biowaste, chicken eggshell, was used with commercial silica powder as a raw material resource for synthesizing porous bioceramic, larnite, granules with a solid-state reaction method. This study was carried out in two primary stages. In the first step, spherical structures were obtained using a facile spherical salt bead production technique developed by Jinnapat and Kennedy. Powdered chicken eggshells and commercial silica powder were used as the inorganic starting materials to produce the spherical structures. After the shaping procedure, it was determined that spherical granules with a broad range of granule sizes, ranging from less than 2 mm to the micrometer level, were achieved. Then, in the second step, a high-temperature heat treatment was carried out to produce porous larnite granules. Performed XRD analysis revealed that the phase purity of the larnite phase was achieved at the high-temperature heat treatment done at 1250°C for 2 hours. However, it was observed that after sintering, the size of the ceramic granules slightly increased, losing their general spherical shape by sticking to each other during the sintering process. Microstructure investigations of the larnite granules revealed a fine, homogenous microstructure with a bimodal porosity distribution. The coarse porosity between the fine larnite particle clusters was determined to reach up to 15-20 mm.
... Because they are mainly composed of tricalcium silicate and dicalcium silicate, CSBSs are considered bioactive bioceramic materials. They release Si and Ca ions, which have an important role in stimulating cell proliferation and differentiation [9]. Tricalcium silicate is the most important component and interacts quickly with water to create a variety of polymorphic crystalline phases depending on temperature, composition, and impurities [10,11]. ...
Introduction:
This study aimed to evaluate the efficacy of a formulation of premixed calcium silicate-based sealer (CSBS) with monocalcium silicate (Mono-CS) as the main component. Its properties were compared with those of a control group (iRoot SP) according to ISO 6876/2012 standards for root canal sealers.
Materials and methods:
The CSBS formulation consisted of two components (powder and liquid). The powder was a mixture of Mono-CS, a radiopacifier, and a thickening agent, and the liquid components were nonaqueous liquid agent and setting accelerator. Three formulation groups with different powder-liquid ratios were prepared: group A, 2 : 1; group B, 3 : 1; and group C, 2 : 1, which also contained calcium chloride as a setting accelerator. The setting time, flow rate, film thickness, and radiopacity of the three CSBS groups and the control group were evaluated and compared. Each test was repeated five times for each group.
Results:
The minimum values of setting time (i.e., working time, initial setting time, and final setting time) were ranked in order of significance as group B, the control group, group C, and group A. The control group had the lowest film thickness at 20 μm, with a nonsignificant difference to group C. The flow rates in group A, group C, and the control group were >20 mm. Furthermore, the experimental groups showed a similar amount of radiopacity as the control group (p > 0.05).
Conclusion:
Mono-CS and calcium chloride can be used in the formulation of root canal sealers, and their properties, including working time, initial setting time, final setting time, flow rate, film thickness, and radiopacity, are consistent with those of iRoot SP and ISO 6876/2012 standards.
... Multipotent ah-BM-MSCs were isolated from bone marrow as previously described (additional information related to the applied methodology, cell isolation, culture, and expansion can be found in previous publications [42,43]). Briefly, bone marrow was collected from the iliac crest of three volunteer patients by direct puncture. ...
... The isolated ah-BM-MSCs were characterized by means of flow cytometry (Beckman Dickinson & Co., Franklin Lakes, NJ, USA; Navios Software v1.2) for mesenchymal (CD90, CD73, 105) and hematopoietic (CD34, CD45) markers as previously described [42,44,45]. Before performing the in vitro assays, single-cell suspensions obtained using culture trypsinization were labeled with fluorochrome-conjugated antibodies: CD73-PE, CD90-APC, CD105-FITC, CD34-APC, and CD45-FITC (Human MSC Phenotyping Cocktail, Miltenyi Biotec, Bergisch Gladbach, Germany) in order to verify the purity of ah-BM-MSCs populations. ...
Acute and chronic bone infections, especially those caused by methicillin-resistant Staphylococcus aureus (MRSA), remains a major complication and therapeutic challenge. It is documented that local administration of vancomycin offers better results than the usual routes of administration (e.g., intravenous) when ischemic areas are present. In this work, we evaluate the antimicrobial efficacy against S. aureus and S. epidermidis of a novel hybrid 3D-printed scaffold based on polycaprolactone (PCL) and a chitosan (CS) hydrogel loaded with different vancomycin (Van) concentrations (1, 5, 10, 20%). Two cold plasma treatments were used to improve the adhesion of CS hydrogels to the PCL scaffolds by decreasing PCL hydrophobicity. Vancomycin release was measured by means of HPLC, and the biological response of ah-BM-MSCs growing in the presence of the scaffolds was evaluated in terms of cytotoxicity, proliferation, and osteogenic differentiation. The PCL/CS/Van scaffolds tested were found to be biocompatible, bioactive, and bactericide, as demonstrated by no cytotoxicity (LDH activity) or functional alteration (ALP activity, alizarin red staining) of the cultured cells and by bacterial inhibition. Our results suggest that the scaffolds developed would be excellent candidates for use in a wide range of biomedical fields such as drug delivery systems or tissue engineering applications.
... All tests were performed in triplicate. Multipotent ah-BM-MSCs were isolated from bone marrow as previously described (additional information related to the applied methodology, cell isolation, culture, and expansion of ah-BM-MSCs can be found in previous publications [40,41]). Briefly, bone marrow was collected from the iliac crest of three volunteer patients by direct puncture. ...
... Flow cytometry was used to characterize the isolated ah-BM-MSCs (Beckman Dickinson & Co., Franklin Lakes, NJ, USA; Software Navios) for mesenchymal (CD90, CD73, 105) and hematopoietic (CD34, CD45) markers as previously described [40,42,43]. Before performing the in vitro assays, single-cell suspensions obtained by culture trypsinization were labeled with fluorochrome-conjugated antibodies: CD73-PE, CD90-APC, CD105-FITC, CD34-APC, and CD45-FITC (Human MSC Phenotyping Cocktail, Miltenyi Biotec, Bergisch Gladbach, Germany) in order to verify the purity of ah-BM-MSCs populations (data not shown). ...
Acute and chronic bone infections, especially those caused by methicillin-resistant Staphylococcus aureus (MRSA), remains a major complication and therapeutic challenge. It is documented that local administration of vancomycin offers better results than the usual routes of administration (e.g. intravenous) when ischemic areas are present. In this work, we evaluate the antimicrobial efficacy against S. aureus and S. epidermidis and analyzed the potential cytotoxic effect of a novel hybrid 3D-printed scaffold based on poly(caprolactone) and chitosan loaded with different vancomycin concentrations (1, 5, 10 and 20%). For this purpose, vancomycin release was measured by means of HPLC, and the biological response of ah-BM-MSCs in the presence of the scaffolds was evaluated in terms of cytotoxicity (LDH activity), proliferation (AlamarBlue Ⓡ) and osteogenic differentiation (ALP activity, Alizarin Red staining). In addition, two cold plasma treatments were evaluated to improve the adhesion of hydrophobic polymers to hydrogels. The hybrid PCL/CS/Van scaffolds tested were found to be biocompatible, bioactive, and bactericide, as demonstrated by no cytotoxicity or functional alteration and by bacterial inhibition. Our results suggest that the scaffolds developed would be excellent candidates to be used in a wide range of biomedical fields such as drug delivery systems or tissue engineering applications.
... Si promotes neovascularization, through direct or indirect induction, of the release of angiogenic factors by fibroblasts, which activate their receptors in endothelial cells and initiate the cascade of chemical reactions involved in the angiogenesis mechanism (Deng et al., 2017;Nair et al., 2009;Li et al., 2018). Therefore, although Si, documented, promotes cell adhesion, proliferation, and differentiation, the relationship between this ion concentration and the cellular response is not yet fully clarified (De Aza et al., 2013). Moreover, very high Si concentrations seem to cause cell death (Messenguer-Olmo et al., 2012;Lin et al., 2015). ...
In recent decades, researchers in bone tissue bioengineering have focused on developing and improving bioceramics efficient in presenting physical-chemical characteristics similar to bone tissue, aiming to mimic cellular events and mechanisms involved in osteogenesis. Among the materials used, wollastonite (W) has stood out in recent years, mainly due to its bioactivity. Besides, tricalcium phosphate (TCP) is also used primarily due to its osteoinductivity and osteoconductivity. Given their ionic compositions and the physical-chemical properties of W and TCP, scientists have associated these two materials during the synthesis of bioceramics that unite the characteristics of each material into a single biomaterial, called composite. This design enables a variety of association that allows improvements in the biological behavior of these materials. Therefore, W/TCP composites have shown excellent performance, in vitro and in vivo, as they start to exhibit fundamental properties for bone regeneration. These characteristics indicate the use of these new biomaterials in future clinical applications, especially in cases of extensive bone losses, which remain a significant challenge for scientists and biomedical professionals. Nevertheless, despite the advances achieved, many questions must be clarified, and essential to comprehend the mechanisms involved in osteogenesis after implantation. Thus, this study aimed to contextualize the use of W/TCP composites for bone regeneration, to support further studies necessary to identify the biological behavior of these bioceramics and ensure use in clinical practice.
... The ah-BM-MSCs were isolated and cultured as previously described [90,91] and characterized in accordance with the criteria established by the International Society for Cell Therapy (ISCT) [92] (data not shown). Briefly, three healthy patients scheduled for elective orthopedic surgery were recruited for this study. ...
... The mononuclear cells were then separated from the bone marrow and washed using a SEPAX ® S-100 device (Biosafe, Eysins, Switzerland). For more details related to the methodology and procedure applied, cell isolation, culture, and expansion of ah-BM-MSCs, please refer to previous publications [90,91]. After determining nucleated cells viability with trypan blue solution (#T8154; Sigma-Aldrich, Saint Louis, MO, USA) for initial expansion, the harvested cells were seeded at a density of 3.75 × 10 5 mL in a 75 cm 2 tissue culture flask (Biofil ® ) with 10 mL of basic growth culture medium (GM) consisting of Dulbecco's Minimal Essential Medium (DMEM) (#31885-023; Gibco, Bleiswijk, the Netherlands) incorporating 10% (v/v) inactivated fetal bovine serum (FBS) (#F7524, Sigma-Aldrich, Saint Louis, MO, USA), 100 U/mL of penicillin, and 100 μg/mL of streptomycin (#P4333, Sigma-Aldrich, Saint Louis, MO, USA), and then incubated at 37 °C in a 5% CO2 atmosphere with 95% of relative humidity. ...
In this work, we evaluated the influence of a novel hybrid 3D-printed porous composite scaffold based on poly(ε-caprolactone) (PCL) and β-tricalcium phosphate (β-TCP) microparticles in the process of adhesion, proliferation, and osteoblastic differentiation of multipotent adult human bone marrow mesenchymal stem cells (ah-BM-MSCs) cultured under basal and osteogenic conditions. The in vitro biological response of ah-BM-MSCs seeded on the scaffolds was evaluated in terms of cytotoxicity, adhesion, and proliferation (AlamarBlue Assay ®) after 1, 3, 7, and 14 days of culture. The osteogenic differentiation was assessed by alkaline phosphatase (ALP) activity, mineralization (Alizarin Red Solution, ARS), expression of surface markers (CD73, CD90, and CD105), and reverse transcription-quantitative polymerase chain reaction (qRT-PCR) after 7 and 14 days of culture. The scaffolds tested were found to be bioactive and biocompatible, as demonstrated by their effects on cytotoxicity (viability) and extracellular matrix production. The mineralization and ALP assays revealed that osteogenic differentiation increased in the presence of PCL/β-TCP scaffolds. The latter was also confirmed by the gene expression levels of the proteins involved in the ossification process. Our results suggest that similar bio-inspired hybrid composite materials would be excellent candidates for osteoinductive and osteogenic medical-grade scaffolds to support cell proliferation and differentiation for tissue engineering, which warrants future in vivo research.
... Another factor that can explain this difference is the ionic concentration of calcium (Ca 2+ ) and silicon (Si) at the implantation site [15]. However, this interrelation is still unclear, which limits to affirm the influence of Si on cellular behavior [47]. ...
... In the W-80 group, which had a lower percentage of β-TCP 20% (wt.), biodegradation occurred more intensively. This can be attributed to the presence of Si associated with TCP, which decreases the temperature of the phase transition from β-TCP to α-TCP, stabilizing the α-TCP phase at room temperature [47][48][49][50][51]. Another factor that may justify these findings is the presence of the newly formed osteoid matrix on the surface of the W-20 scaffolds, which reduced the exposure of the biomaterial to body fluids and multinucleated cells, making biodegradation difficult due to the action of body fluids. ...
In order to provide favorable conditions for bone regeneration, a lot of biomaterials have been developed and evaluated, worldwide. Composite biomaterials have gained notoriety, as they combine desirable properties of each isolated material. Thus, in this research, bone repair capacity of three developed formulations of ceramic scaffolds were evaluated histomorphometrically, after implantation. Scaffolds were based on wollastonite (W) and β-tricalcium phosphate (β-TCP) composites in three different ratios (wt. %). Thirty Wistar rats were randomly assigned to three experimental groups: W-20 (20W/80β-TCP wt. %), W-60 (60W/40β-TCP wt. %), and W-80 (80W/20β-TCP wt. %), evaluated by optical microscopy at biological tests after 15 and 45 days of implantation. Throughout the study, the histological results evidenced that the scaffolds remained at the implantation site, were biocompatible and presented osteogenic potential. The percentage of neoformed mineralized tissue was more evident in the W-20 group (51%), at 45 days. The composite of the W-80 group showed more evident biodegradation than the biomaterials of the W-20 and W-60 groups. Thus, it is concluded that the scaffold containing 20W/80β-TCP (wt. %) promoted more evident bone formation, but all composites evaluated in this study showed notorious bioactivity and promising characteristics for clinical application.
... Ah-MSCs were characterized according to the minimal standard criteria established by Dominici et al. [18]. The methods followed for the culture and expansion of ah-MSCs are found in a previous work [19]. Expanded cells, passage 3 (P3), were collected for the in vitro tests. ...
... Cells were seeded at a density of 5 × 10 3 cells/cm 2 on scaffold surfaces in 48-well plates. Incubation was performed at 37 • C, 5% CO 2 , and 95% relative humidity with 800 µL of GM for 7, 14, 21, and 28 days according to previous works [17,19]. The cells grown in plastic, in the absence of scaffolds, were used as a positive control. ...
Micro-/nano-structured scaffolds with a weight composition of 46.6% α-tricalcium phosphate (α-TCP)—53.4% silicocarnotite (SC) were synthesized by the polymer replica method. The scanning electron microscopy (SEM) analysis of the scaffolds and natural cancellous bone was performed for comparison purposes. Scaffolds were obtained at three cooling rates via the eutectoid temperature (50 °C/h, 16.5 °C/h, 5.5 °C/h), which allowed the surface nanostructure and mechanical strength to be controlled. Surface nanostructures were characterized by transmission electron microscopy (TEM) and Raman analysis. Both phases α-TCP and SC present in the scaffolds were well-identified, looked compact and dense, and had neither porosities nor cracks. The non-cytotoxic effect was evaluated in vitro by the proliferation ability of adult human mesenchymal stem cells (ah-MSCs) seeded on scaffold surfaces. There was no evidence for cytotoxicity and the number of cells increased with culture time. A dense cell-hydroxyapatite layer formed until 28 days. The SEM analysis suggested cell-mediated extracellular matrix formation. Finally, scaffolds were functionalized with the alkaline phosphatase enzyme (ALP) to achieve biological functionalization. The ALP was successfully grafted onto scaffolds, whose enzymatic activity was maintained. Scaffolds mimicked the micro-/nano-structure and chemical composition of natural cancellous bone by considering cell biology and biomolecule functionalization.
... This reabsorption occurs in parallel with an increase in newly formed bone, a phenomenon known as "creeping substitution" [50,51], which considers that the incorporation of the material into the bone is favored by its fragmentation, being continuously replaced by the newly formed bone. In previous studies [52], we determined that the increase of C 2 S proportion in TCP-C 2 S ceramic tends to decrease the resorption time of the material. On the other hand, an increase in the proportion of C 2 S leads to a major bone tissue formation, probably due to the Si released to the surrounding tissue. ...
Calcium phosphate materials are widely used as bone substitutes due to their bioactive and biodegradable properties. Also, the presence of silicon in their composition seems to improve the bioactivity of the implant and promote bone tissue repair. The aim of this study was to develop a novel ceramic scaffold by partial solid-state sintering method with a composition lying in the field of the Nurse’s A-phase–silicocarnotite, in the tricalcium phosphate–dicalcium silicate (TCP–C2S) binary system. Also, we evaluated its osteogenic and osteoconductive properties after being implanted into tibia defects in New Zealand rabbits. X-ray, microcomputer tomography, and histomorphometry studies demonstrated that this porous ceramic is highly biocompatible and it has excellent osteointegration. The material was being progressively reabsorbed throughout the study and there was no unspecified local or systemic inflammatory response observed. These results suggest that ceramic imitates the physicochemical characteristics of bone substitutes used in bone reconstruction.
... Multipotent hMSCs were isolated from bone marrow as described previously [16]. The study was approved by the Institutional Ethics Committee (Virgen de la Arrixaca University Hospital ID: 101212/1/AEMPS), while all patients signed an informed consent. ...
Background: Cell-Based Therapies (CBT) constitute a valid procedure for increasing the quantity and quality of bone in areas with an inadequate bone volume. However, safety and efficacy should be investigated prior to clinical application. The objective of this study was to evaluate the biodistribution, safety and osteogenic capacity of bone marrow-derived human mesenchymal stem cells (hBMMSCs) pre-seeded into β-tricalcium phosphate (TCP) and implanted into NOD/SCID mice at subcutaneous and intramuscular sites. Methods: hBMMSCs were isolated, characterized and then cultured in vitro on a porous β-TCP scaffold. Cell viability and attachment were analyzed and then hBMMSCs seeded constructs were surgically placed at subcutaneous and intramuscular dorsal sites into NOD/SCID mice. Acute and subchronic toxicity, cell biodistribution and efficacy were investigated. Results: There were no deaths or adverse events in treated mice during the 48-hour observation period, and no toxic response was observed in mice. In the 12-week subchronic toxicity study, no mortalities, abnormal behavioral symptoms or clinical signs were observed in the saline control mice or the hBMMSCs/β-TCP groups. Finally, our results showed the bone-forming capacity of hBMMSCs/β-TCP since immunohistochemical expression of human osteocalcin was detected from week 7. Conclusions: These results show that transplantation of hBMMSCs/β-TCP in NOD/SCID mice are safe and effective, and might be applied to human bone diseases in future clinical trials.
