Bone-like Nodule Formation Research Articles

3D osteogenic differentiation of human iPSCs reveals the role of TGFβ signal in the transition from progenitors to osteoblasts and osteoblasts to osteocytes

Although the formation of bone-like nodules is regarded as the differentiation process from stem cells to osteogenic cells, including osteoblasts and osteocytes, the precise biological events during nodule formation are unknown. Here we performed the osteogenic induction of human induced pluripotent stem cells using a three-dimensional (3D) culture system using type I collagen gel and a rapid induction method with retinoic acid. Confocal and time-lapse imaging revealed the osteogenic differentiation was initiated with vigorous focal proliferation followed by aggregation, from which cells invaded the gel. Invading cells changed their morphology and expressed osteocyte marker genes, suggesting the transition from osteoblasts to osteocytes. Single-cell RNA sequencing analysis revealed that 3D culture-induced cells with features of periosteal skeletal stem cells, some of which expressed TGFβ-regulated osteoblast-related molecules. The role of TGFβ signal was further analyzed in the transition from osteoblasts to osteocytes, which revealed that modulation of the TGFβ signal changed the morphology and motility of cells isolated from the 3D culture, suggesting that the TGFβ signal maintains the osteoblastic phenotype and the transition into osteocytes requires down-regulation of the TGFβ signal.

2,3-Diphospho-D-glyceric acid inhibits calciprotein particle growth and calcification in MOVAS cells but not in MC3T3-E1 cells

Anionic polyphosphate compounds are well-known to inhibit the formation of hydroxyapatite crystals. Vascular calcification (VC) is associated with an increase in crystalline calciprotein particles (CPPs) in the bloodstream. 2,3-Diphospho-D-glyceric acid (2,3-DPG) is a highly anionic Poly-P compound found in the concave center of erythrocytes where it binds to hemoglobin, thereby reducing oxygen affinity. Since 2.3-DPG exists in plasma, we hypothesized that 2,3-DPG may act as an endogenous inhibitor of VC. In comparison to widely recognized calcification inhibitors such as myo-inositol hexakisphosphate, 2,3-DPG significantly delayed the formation of crystalline CPPs. Additionally, 2,3-DPG inhibited calcification in a mouse vascular smooth muscle cell line (MOVAS) without cytotoxic effects. Taken together with the result that 2,3-DPG did not impact bone-like nodule formation in mouse osteoblast-like MC3T3-E1 cells, these results suggest that 2,3-DPG may inhibit VC selectively.

Effect of MgO addition on the sinterability, mechanical properties and biological cell activities of sintered silicon-substituted hydroxyapatite

The effect of 1 wt% magnesium oxide (MgO) as sintering additive on the sintering behaviour, microstructure, mechanical properties and cell activities of 0.4 and 1.6 wt% silicon-substituted hydroxyapatite (MgO-0.4Si-HA and MgO-1.6Si-HA) was investigated. The powders were synthesized by the wet-chemical neutralization method and the compacts were pressureless sintered in air between 1150 and 1250 °C for 2 h. The results showed that MgO-0.4Si-HA promoted the formation of the secondary phase of β-TCP when sintered at 1250 °C, whilst the stability of the hydroxyapatite phase was not disrupted for the MgO-1.6Si-HA. The presence of MgO was found to be beneficial in suppressing grain growth of Si-HA when sintered at 1250 °C; however, it did not promote densification at lower temperatures (1150 and 1200 °C). It was observed that despite the lower densification, Vickers hardness and compressive strength of the MgO-Si-HA were higher than those of the undoped Si-HA samples. The beneficial effect of MgO in enhancing the biocompatibility of Si-HA has been revealed. It was found that the MgO-doped samples dramatically enhanced the osteogenic differentiation of MC3T3-E1 cells as shown from the higher cell proliferation, alkaline phosphatase activity and bone-like nodule formation when compared to pristine HA.

Calcium aluminate cement-based blends for application to fill in bone defects

The need to treat bone defects is increasing dramatically as the population grows old. Calcium aluminate cement, CAC, has been applied to fill in bone defects. CAC has been used in orthopedics, as it can avoid some problems of commercial products, polymethylmethacrylate, PMMA, regarding its too low consistency and strong odor and exothermic reactions which can damage the surrounding tissue. Besides that, CAC can be molded in situ to provide intimate fitting to the defect rims. The injectability of cements is an important aspect when minimally invasive surgical techniques are used and for applications with limited accessibility and narrow cavities. At present work, CAC was initially analyzed via zeta potential and viscosity when mixed with different dispersant additives. Then, CAC blends containing different materials, alumina, zirconia, zinc oxide, hydroxyapatite, tricalcium phosphate, chitosan, collagen, or zinc oxide-bismuth oxide, were evaluated regarding their injectability and by means of in vitro mineralization and ex vivo uniaxial cold crushing strength tests. The best injectability conditions, 76 wt% solids, hypodermic needle, were used for the ex vivo tests. In the latter, the compositions selected by in vitro tests were injected to repair 3-mm defects on mouse femurs cut into pieces of 10 mm, whereas collagen and PMMA were inserted with a spatula. After setting the materials for 24 h at 37 °C in body fluid solution, the pieces were submitted to uniaxial cold crushing strength tests. The blends alumina, collagen, and chitosan resulted in greater reddish-stained areas indicating enhanced mineralized matrix formation for these groups, which also presented higher values of calcium deposits indicating a successful in vitro bone-like nodule formation. The filling of defects with CACH and its blends increased the resistance of bones when compared to bones with the added defect, with lower effect verified in the presence of chitosan and collagen.

Biological responses of MC3T3-E1 on calcium carbonate coatings fabricated by hydrothermal reaction on titanium

Titainum (Ti) implants have been successfully used in orthopaedic and dental surgery. However, poor early bone tissue integration is still a common cause of implant failure. This could be modulated by improving the material bonding or adhesion directly to the bone by surface roughening and/or a bioresorbable and osteoconductive coating. In this study, we report on the biological behaviours of the Ti substrate with modified surface roughness and/or a calcium carbonate (CaCO3) coating. The roughened Ti surface was prepared using an acid etching reaction, and the CaCO3 coating on the substrates was synthesized by the hydrothermal treatment of Ti in calcium citrate complexes. This study demonstrates that surface roughening of Ti alone does not improve the biological response of the MC3T3-E1 cells, but a CaCO3 coating on the smooth Ti surface increases cell responses, and these effects are further enhanced by the combination of coating a roughened Ti surface with CaCO3. The larger the cell area, the greater the cell proliferation and increased bone-like nodule formation were observed on the CaCO3 coating of the roughened Ti surface. This observation was also supported by a higher ALP value. The cell behaviours found in the current study further support the development of CaCO3 coatings towards clinical application.

Stereocomplex poly(lactic acid)-based composite nanofiber membranes with highly dispersed hydroxyapatite for potential bone tissue engineering

The development of materials with excellent mechanical properties and stem cell affinity for bone repair is a topic that has received significant interest in the field of bone tissue engineering. In this study, stereocomplex poly(lactic acid)-based composite nanofiber membranes that are compatible with bone marrow stem cells (BMSCs) were prepared with poly(d-lactic acid)-grafted hydroxyapatite and enantiomeric poly(lactic acid)s. The mechanical property tests showed that, compared with those from a heated poly(l-lactic acid) (PLLA) nanofiber membrane, the tensile strength and Young's modulus of the most strengthened composite nanofiber membrane herein increased by 30.16% and 34.56%, respectively. The X-ray diffraction results indicated that the stereocomplex crystallite (SC) of poly(lactic acid) formed in heated composite nanofiber membranes. The differential scanning calorimetry results showed that the melting temperature of the SC of heated composite nanofiber membranes was approximately 221 °C. Live/dead staining and scanning electron microscopy were used to observe the proliferation and adhesion of BMSCs cultured on the nanofiber membranes. Compared with the PLLA nanofiber membrane, increased type I collagen expression and improved formation of bone-like nodules can be observed after induction for nine days on the composite nanofiber membrane, indicating the potential application of the composite nanofiber membrane for bone tissue engineering.

Enhanced Osseointegration Capability of Poly(ether ether ketone) via Combined Phosphate and Calcium Surface-Functionalization.

Biomedical applications of poly(ether ether ketone) (PEEK) are hindered by its inherent bioinertness and lack of osseointegration capability. In the present study, to enhance osteogenic activity and, hence, the osseointegration capability of PEEK, we proposed a strategy of combined phosphate and calcium surface-functionalization, in which ozone-gas treatment and wet chemistry were used for introduction of hydroxyl groups and modification of phosphate and/or calcium, respectively. Surface functionalization significantly elevated the surface hydrophilicity without changing the surface roughness or topography. The cell study demonstrated that immobilization of phosphate or calcium increased the osteogenesis of rat mesenchymal stem cells compared with bare PEEK, including cell proliferation, alkaline phosphatase activity, and bone-like nodule formation. Interestingly, further enhancement was observed for samples co-immobilized with phosphate and calcium. Furthermore, in the animal study, phosphate and calcium co-functionalized PEEK demonstrated significantly enhanced osseointegration, as revealed by a greater direct bone-to-implant contact ratio and bond strength between the bone and implant than unfunctionalized and phosphate-functionalized PEEK, which paves the way for the orthopedic and dental application of PEEK.

Ozone-gas-mediated surface hydrophilization enhances the cell responses to titanium

Surface wettability is attracting increasing attention as an important factor that affects the cell/tissue responses of implant materials. Time-dependent hydrocarbon adsorption on the titanium (Ti) implant surface has been reported to decrease the surface hydrophilicity by adsorption of hydrocarbon contaminants from the atmosphere and dramatically decrease the osseointegration over time. In this study, we found that ozone-gas-mediated removal of hydrocarbon contaminants and introduction of –OH groups significantly increased the surface hydrophilicity of acid-etched Ti without changing the original surface topography. Ozone-gas-treated Ti dramatically enhanced osteogenic differentiation of mesenchymal stem cells, as evidenced by greater cell proliferation, alkaline phosphatase activity, and bone-like nodule formation compared with original Ti.

In vitro bone-like nodules generated from patient-derived iPSCs recapitulate pathological bone phenotypes.

The recapitulation of bone formation via the in vitro generation of bone-like nodules is frequently used to understand bone development. However, current bone-induction techniques are slow and difficult to reproduce. Here, we report the formation of bone-like nodules within ten days, via the use of retinoic acid (RA) to induce the osteogenic differentiation of human induced pluripotent stem cells (hiPSCs) into osteoblast-like and osteocyte-like cells that create human bone tissue when implanted in calvarial defects in mice. We also show that the induction of bone formation depends on cell signalling through the RA receptors RARα and RARβ, which simultaneously activate the BMP (bone morphogenetic protein) and Wnt signalling pathways. Moreover, by using patient-derived hiPSCs, the bone-like nodules recapitulated the osteogenesis-imperfecta phenotype, which was rescued via the correction of disease-causing mutations and partially by an mTOR (mechanistic target of rapamycin) inhibitor. The method of inducing bone nodules may serve as a fast and reproducible model for the study of the formation of both healthy and pathological bone.

Scaffolds’ production based on calcium aluminate blends and their biological properties

Scaffolds are artificial structures that provide a temporary template to withstand mechanical loads in vivo during the process of bone regeneration. They should present a highly porous three-dimensional structure with an interconnected network of pores for the tissue growth and the transport of nutrients and metabolic residues. Additionally, scaffolds must be easily manufactured in different shapes and sizes. A 4 wt% of additives (alumina, zirconia, zinc oxide, hydroxyapatite, or tricalcium phosphate) was added to CAC (calcium aluminate cement), and these blends were used for scaffolds preparation via polymeric sponge replica. Aqueous suspensions were prepared with different solid contents. Sponges impregnated with 55 wt% solid were evaluated for the decomposition temperature by thermogravimetric analysis. The scaffolds microstructures were characterized by scanning electron microscopy and mercury intrusion porosimetry. The relative density was also calculated considering the theoretical density. The scaffolds (60 wt%) were characterized by cytotoxicity assay, total protein content, alkaline phosphatase activity, mineralized bone-like nodule formation, and cell adhesion. The blends prepared with 60 and 55 wt% solid were most suitable for sponge impregnation, resulting in scaffolds with higher fraction of open porosity. The tricalcium phosphate blends presented the best results regarding mineralized nodules. The MTT tests and osteoblast cells adhered to the scaffolds provided predictive evidence of biocompatibility of the material which, associated with the satisfactory results obtained in the cell differentiation tests (ALP), suggested that the scaffolds developed are promising materials. Additionally, all scaffolds allowed the mineralized matrix formation which is a key issue for osteogenesis.

Effects of 3-dimensional Bioprinting Alginate/Gelatin Hydrogel Scaffold Extract on Proliferation and Differentiation of Human Dental Pulp Stem Cells

IntroductionAlginate/gelatin hydrogel (Alg-Gel) scaffold has been applied in tissue engineering, but the research on its application in dental tissues regeneration is still lacking. We investigated the effect of this scaffold on human dental pulp stem cells (hDPSCs). MethodshDPSCs were cultured in both Alg-Gel and 3D-printed Alg-Gel scaffolds. Cell growth and adhesion were compared using fluorescein isothiocyanate–phalloidin staining and scanning electron microscopic micrographs. Changes in the proliferation in hDPSCs cultured in the complete culture medium containing aqueous extracts of the Alg-Gel or 3D-printed Alg-Gel scaffolds were examined using Cell Counting Kit-8 assay and flow cytometry analysis. Cells were cultured in the mineralization medium containing aqueous extracts of the Alg-Gel or 3D-printed Alg-Gel scaffolds for 7 or 14 days, and the differentiation of cells was shown by alizarin red S staining and alkaline phosphatase staining. The messenger RNA and protein expression of mineralization-related genes were detected with real-time polymerase chain reaction and Western blotting. Elemental analysis was used to test the material extract composition. ResultsMore cells were grown and adhered to the 3D-printed Alg-Gel scaffolds than the Alg-Gel scaffolds. The aqueous extracts of 3D-printed scaffolds can promote cell proliferation, and compared with Alg-Gel scaffolds, the extracts of 3D-printed scaffolds were more effective. Compared with the negative control group, 3D-printed Alg-Gel scaffold and Alg-Gel scaffold aqueous extracts promoted osteogenic/odontoblastic differentiation of hDPSCs with the enhanced formation of bone-like nodules and the alkaline phosphatase staining. The expression of mineralization-related genes was also up-regulated. 3D-printed scaffold aqueous extract contained more calcium and phosphorus ions than the Alg-Gel scaffold. ConclusionsThese findings suggest that compared with the Alg-Gel scaffold, 3D-printed Alg-Gel is more suitable for the growth of hDPSCs, and the scaffold extracts can better promote cell proliferation and differentiation.

Fibre Laser Treatment of Beta TNZT Titanium Alloys for Load-Bearing Implant Applications: Effects of Surface Physical and Chemical Features on Mesenchymal Stem Cell Response and Staphylococcus aureus Bacterial Attachment

A mismatch in bone and implant elastic modulus can lead to aseptic loosening and ultimately implant failure. Selective elemental composition of titanium (Ti) alloys coupled with surface treatment can be used to improve osseointegration and reduce bacterial adhesion. The biocompatibility and antibacterial properties of Ti-35Nb-7Zr-6Ta (TNZT) using fibre laser surface treatment were assessed in this work, due to its excellent material properties (low Young’s modulus and non-toxicity) and the promising attributes of fibre laser treatment (very fast, non-contact, clean and only causes changes in surface without altering the bulk composition/microstructure). The TNZT surfaces in this study were treated in a high speed regime, specifically 100 and 200 mm/s, (or 6 and 12 m/min). Surface roughness and topography (WLI and SEM), chemical composition (SEM-EDX), microstructure (XRD) and chemistry (XPS) were investigated. The biocompatibility of the laser treated surfaces was evaluated using mesenchymal stem cells (MSCs) cultured in vitro at various time points to assess cell attachment (6, 24 and 48 h), proliferation (3, 7 and 14 days) and differentiation (7, 14 and 21 days). Antibacterial performance was also evaluated using Staphylococcus aureus (S. aureus) and Live/Dead staining. Sample groups included untreated base metal (BM), laser treated at 100 mm/s (LT100) and 200 mm/s (LT200). The results demonstrated that laser surface treatment creates a rougher (Ra value of BM is 199 nm, LT100 is 256 nm and LT200 is 232 nm), spiky surface (Rsk > 0 and Rku > 3) with homogenous elemental distribution and decreasing peak-to-peak distance between ripples (0.63 to 0.315 µm) as the scanning speed increases (p < 0.05), generating a surface with distinct micron and nano scale features. The improvement in cell spreading, formation of bone-like nodules (only seen on the laser treated samples) and subsequent four-fold reduction in bacterial attachment (p < 0.001) can be attributed to the features created through fibre laser treatment, making it an excellent choice for load bearing implant applications. Last but not least, the presence of TiN in the outermost surface oxide might also account for the improved biocompatibility and antibacterial performances of TNZT.

Synergistic effect of surface phosphorylation and micro-roughness on enhanced osseointegration ability of poly(ether ether ketone) in the rabbit tibia

This study was aimed to investigate the osseointegration ability of poly(ether ether ketone) (PEEK) implants with modified surface roughness and/or surface chemistry. The roughened surface was prepared by a sandblast method, and the phosphate groups on the substrates were modified by a two-step chemical reaction. The in vitro osteogenic activity of rat mesenchymal stem cells (MSCs) on the developed substrates was assessed by measuring cell proliferation, alkaline phosphatase activity, osteocalcin expression, and bone-like nodule formation. Surface roughening alone did not improve MSC responses. However, phosphorylation of smooth substrates increased cell responses, which were further elevated in combination with surface roughening. Moreover, in a rabbit tibia implantation model, this combined surface modification significantly enhanced the bone-to-implant contact ratio and corresponding bone-to-implant bonding strength at 4 and 8 weeks post-implantation, whereas modification of surface roughness or surface chemistry alone did not. This study demonstrates that combination of surface roughness and chemical modification on PEEK significantly promotes cell responses and osseointegration ability in a synergistic manner both in vitro and in vivo. Therefore, this is a simple and promising technique for improving the poor osseointegration ability of PEEK-based orthopedic/dental implants.

Surface plasma treatment and phosphorylation enhance the biological performance of poly(ether ether ketone).

Poly(ether ether ketone) (PEEK) has emerged as an alternative endosseous material to metal implants mainly because of its lack of allergic sensitivity and radiolucency, while maintaining similar mechanical properties with bone. However, a disadvantage of PEEK is its weak osseointegration ability compared with metal implants. To overcome this, we prepared a phosphate group-modified PEEK by plasma treatment and subsequent phosphorylation reaction. Plasma treatment and phosphate modification of PEEK changed its hydrophobic surface to a hydrophilic surface while maintaining the original surface topography and roughness. Phosphate modification increased the bioactivity of rat bone marrow stromal cells (BMSCs), including proliferation, alkaline phosphatase activity, and bone-like nodule formation; however, this effect was negligible in plasma-treated PEEK. In addition, phosphate modification attenuated the phenotypic polarization of lipopolysaccharide-primed RAW264.7 macrophages to an inflammatory phenotype, based on the finding that macrophages on phosphate-modified PEEK produced decreased levels of the inflammatory cytokine and increased levels of the anti-inflammatory cytokine. Finally, in an animal study, phosphate-modified PEEK exhibited a doubled pullout force from the femur bone cavity compared with bare PEEK. Thus, we conclude that phosphate modification can significantly improves the implant-bone bonding strength of PEEK by enhancing BMSCs activity and reducing excessive inflammation.

Effect of micro-roughening of poly(ether ether ketone) on bone marrow derived stem cell and macrophage responses, and osseointegration

Poly(ether ether ketone) (PEEK) has emerged as a candidate to replace metal implants because of its satisfactory mechanical properties, radiolucency, and lack of metal allergy. However, PEEK lacks osseointegration ability limiting its clinical applications. To overcome this problem, we prepared PEEK with a micro-rough surface using the sandblast method to modulate its osseointegration property; the sandblast method is simple, cost-effective, and is already applied to clinical metal implants. The surface roughness of the sandblasted PEEK was about 2.3 μm, whereas that of mirror-polished PEEK was 0.06 μm. Rat bone marrow-derived mesenchymal stem cells (RMSCs) showed higher proliferation, osteocalcin (OC) expression and bone-like nodule formation on micro-roughened PEEK compared with those cultured on mirror-polished PEEK, suggesting that micro-roughening facilitated RMSCs proliferation and differentiation. The micro-roughened surface slightly mitigated secretion of inflammatory C-C motif chemokine 2 (CCL-2) from lipopolysaccharide (LPS)-stimulated macrophages, but not of tumor necrosis factor α (TNFα) and interleukin-6 (IL-6). Finally, to compare osseointegration, specimens were implanted in rat femur bone marrow cavities, and then the pull-out force was measured. The pull-out force of micro-roughened PEEK was about four times higher than that of the mirror-polished PEEK. These results showed that micro-roughening of PEEK using the sandblast method was able to improve osseointegration, partly through elevating proliferation and differentiation of RMSCs.

Cytotoxicity Analysis of Ti-7.5Mo Alloy After Biomimetic Surface Treatment to Use as Dental Materials

Titanium (Ti) and its alloys are widely used for medical and dental fields due to their excellent biocompatibility, high corrosion resistance, high specific strength and excellent mechanical properties. Different methods have been developed to improve the surface properties of titanium-based implant materials, and consequently the bone-bonding ability. The Ti-7.5Mo alloy was activated by an alkaline treatment with 5M NaOH, heat treatment and subsequent immersion in SBFx5 to investigate the in vitro response of osteoblastic-like cells (MG-63) on altered biomimetic surfaces. Sample surfaces were characterized by scanning electron microscopy. Cytotoxicity was assessed by the MTT assay, total protein content, alkaline phosphatase activity (ALP) and mineralized bone-like nodule formation. It was shown the que alkali treatment led to the formation of sodium titanate and immersion in SBFx5 formed a film of calcium phosphate. The alkaline treatment and heat treatment of 7.5 Ti-Mo alloys followed by soaking them in SBFx5 for 24 hours is a suitable technique once the final samples were biocompatible, allowed the attachment of the osteoblastic-like cells (MG-G3), and increased the mineralized like-bone nodules formation by these cells.

Strontium- and cobalt-substituted bioactive glasses seeded with human umbilical cord perivascular cells to promote bone regeneration via enhanced osteogenic and angiogenic activities.

Bone regeneration is considered as an unmet clinical need. The most recent researches focused on incorporation of strontium (Sr2+) and cobalt (Co2+) ions into bioactive glasses structure. Strontium is an alkaline earth metal which is currently used in the treatment of osteoporosis. Also, cobalt is considered as another promising element in the bone regeneration field that may induce hypoxia-mediated angiogenesis. In this study, the osteogenic potential of the strontium- and cobalt-substituted bioactive glasses in granule form seeded with human umbilical cord perivascular cells (HUCPVCs) was evaluated in vitro and in vivo. Indeed, the main goal of this study was to improve the osteogenenic and angiogenic properties of bioactive glasses through the incorporation of strontium and cobalt ions in the glass composition. Although some researches have been conducted on this subject, the influence of the simultaneous use of strontium and cobalt ions on the improvement of bone healing in vivo has been not yet well understood and, therefore, deserves further investigation.

Bioactive Glass Particles in Two-Dimensional and Three-Dimensional Osteogenic Cell Cultures.

This study aimed to investigate the influence of a three-dimensional cell culture model and bioactive glass (BG) particles on the expression of osteoblastic phenotypes in rat calvaria osteogenic cells culture. Cells were seeded on two-dimensional (2D) and three-dimensional (3D) collagen with BG particles for up to 14 days. Cell viability and alkaline phosphatase (ALP) activity was performed. Cell morphology and immunolabeling of noncollagenous bone matrix proteins were assessed by epifluorescence and confocal microscopy. The expressions of osteogenic markers were analyzed using RT-PCR. Mineralized bone-like nodule formation was visualized by microscopy and calcium content was assessed quantitatively by alizarin red assay. Experimental cultures produced a growing cell viability rate up to 14 days. Although ALP activity at 7 days was higher on BG cultures, cells on 3D and 3D+BG had an activity decrease of ALP at 14 days. Three-dimensional conditions favored the immunolabeling for OPN and BSP and the expression of ALP and COL I mRNAs. BG particles influenced positively the OC and OPN mRNAs expression and calcified nodule formation in vitro. The results indicated that the 3D cultures and BG particles contribute to the expression of osteoblastic phenotype and to differentiated and mineralized matrix formation.

Mesenchymal stromal cell and osteoblast responses to oxidized titanium surfaces pre-treated with λ=808nm GaAlAs diode laser or chlorhexidine: in vitro study.

Preservation of implant biocompatibility following peri-implantitis treatments is a crucial issue in odontostomatological practice, being closely linked to implant re-osseointegration. Our aim was to assess the responses of osteoblast-like Saos2 cells and adult human bone marrow-mesenchymal stromal cells (MSCs) to oxidized titanium surfaces (TiUnite®, TiU) pre-treated with a 808±10nm GaAlAs diode laser operating in non-contact mode, in continuous (2W, 400J/cm2; CW) or pulsed (20kHz, 7μs, 0.44W, 88J/cm2; PW) wave, previously demonstrated to have a strong bactericidal effect and proposed as optional treatment for peri-implantitis. The biocompatibility of TiU surfaces pre-treated with chlorhexidine digluconate (CHX) was also evaluated. In particular, in order to mimic the in vivo approach, TiU surfaces were pre-treated with CHX (0.2%, 5min); CHX and rinse; and CHX, rinse and air drying. In some experiments, the cells were cultured on untreated TiU before being exposed to CHX. Cell viability (MTS assay), proliferation (EdU incorporation assay; Ki67 confocal immunofluorescence analysis), adhesion (morphological analysis of actin cytoskeleton organization), and osteogenic differentiation (osteopontin confocal immunofluorescence analysis; mineralized bone-like nodule formation) analyses were performed. CHX resulted cytotoxic in all experimental conditions. Diode laser irradiation preserved TiU surface biocompatibility. Notably, laser treatment appeared even to improve the known osteoconductive properties of TiU surfaces. Within the limitations of an in vitro experimentation, this study contributes to provide additional experimental basis to support the potential use of 808±10nm GaAlAs diode laser at the indicated irradiation setting, in the treatment of peri-implantitis and to discourage the use of CHX.

The stimulatory effect of silica nanoparticles on osteogenic differentiation of human mesenchymal stem cells

Silica-based materials with favourable biocompatibility are generally considered as excellent candidates for applications in biomedical fields. However, previous researches mainly focused on the safety of silica-based materials, their effects on osteogenic differentiation of human mesenchymal stem cells (hMSCs) still need further investigations. In this study, core–shell fluorescent silica nanoparticles (silica NPs) with three different sizes (S1 ~ 50 nm, S2 ~ 200 nm, S3 ~ 400 nm, respectively) were prepared according to the Stöber method. The silica NPs with different sizes did not affect the cell viability (even up to a concentration of 500 µg ml−1), showing size- and dose-independent cytocompatibility of silica NPs on hMSCs. Uptake of silica NPs significantly enhanced the activity of alkaline phosphatase (ALP) and the formation of bone-like nodules of hMSCs after osteogenic induction. At the concentration of 10 µg ml−1, after treating hMSCs with larger sized silica NPs (S2 and S3), higher ALP activity of hMSCs was measured and larger sized bone-like nodules were formed by hMSCs compared with that treated with smaller sized silica NPs (S1).The enhanced osteogenic potential of hMSCs treated with silica NPs may be attributed to the Si released from silica NPs due to the lysosomal degradation inside hMSCs. These results demonstrate the stimulatory effect of silica NPs on osteogenic differentiation of hMSCs and the application potential of silica NPs in bone tissue engineering.