Differentiation Of Pre-osteoblastic Cells Research Articles

Vancomycin-Loaded Silk Fibroin/Calcium Phosphate/Methylcellulose-Based In Situ Thermosensitive Hydrogel: A Potential Function for Bone Regeneration

This study explores the efficacy of a vancomycin-loaded silk fibroin/calcium phosphate/methylcellulose-based in situ thermosensitive hydrogel (VM-SF/CaP/MC) in promoting the osteogenic differentiation of preosteoblast cells. Three VM-SF/CaP/MC formulations with varying low (L) and high (H) concentrations of silk fibroin (SF) and calcium phosphate (CaP) were prepared: VM-HSF/LCaP/MC, VM-LSF/HCaP/MC, and VM-HSF/HCaP/MC. These hydrogels significantly enhanced MC3T3-E1 cell migration and proliferation in a dose- and time-dependent manner, achieving complete cell migration within 48 h. In addition, they significantly promoted alkaline phosphatase activity, collagen content, and mineralization in MC3T3-E1 cells, indicating their potential for osteogenesis. Among the hydrogel formulations, the VM-HSF/HCaP/MC hydrogel, with high SF and CaP content, demonstrated superior potential in promoting the osteogenic differentiation of MC3T3-E1 cells. It exhibited the highest ALP activity (11.13 ± 0.91 U/mg protein) over 14 days, along with increased collagen content (54.00 ± 1.71 µg/mg protein) and mineralization (15.79 ± 1.48 mM) over 35 days. Therefore, this formulation showed a promising candidate for clinical application in localized bone regeneration, particularly in treating osteomyelitis.

Role of in-situ electrical stimulation on early-stage mineralization and in-vitro osteogenesis of electroactive bioactive glass composites

Bioactive glass (BAG) has emerged as an effective bone graft substitute due to its diverse qualities of biocompatibility, bioactivity, osteoblast adhesion and enhanced revascularization. However, inferior osteogenic capacity of BAG compared to autologous bone grafts continues to limiting it's wide-spread clinical applications towards repairing of bone fractures and healing. In this study, we have fabricated BAG composites with 0.5 to 2 wt% bismuth ferrite (BF, a multiferroic material) with an aim to generate in-situ electrical charges pertinent to early-stage bone regeneration thus mimicking nature bone, which is a piezoelectric material. The fabricated BAG composites were characterised in terms of microstructures, phase analysis, remanent polarization, wettability and subsequently evaluated for in vitro cell proliferation and osteogenesis with and without magnetic field exposure (200 mT, 30 min./day). Pre-osteoblast cells from mice (MC3T3-E1) seeded on these composites exhibited excellent cell growth without any cytotoxicity, which is further supported by FITC/DAPI staining and a live/dead assay. The results of Alizarin Red S assay and increased levels of Alkaline Phosphatase (ALP) activity, at 21 days of culture, suggest that the BAG-BF composites promote in vitro osteogenic differentiation of pre-osteoblast cells. The enhanced osteogenesis of BAG-BF composites was also confirmed through qRT-PCR analysis, which showed rapid upregulation of osteoblastogenic specific genes namely RunX-2, Collagen-1, Bone Sialo Protein, and ALP after 21 days. Additionally, the osteogenic differentiation was assessed by the Western Blot technique, which revealed significantly higher band intensity of osteogenic markers in BAG-1.5 BF and BAG-2 BF composites than pure BAG. These findings clearly demonstrate that in-situ electrical stimulation and osteoconductive capacity of BF reinforced BAG composites have positive impact on osteoblast cell development, bone formation, and healing.

Fabrication of micropatterned PCL-collagen nanofibrous scaffold for cellular confinement induced early osteogenesis

The intricate interaction of the scaffold's architecture/geometry and with the cells is essential for tissue engineering and regenerative medicine. Cells sense their surrounding dynamic cues such as biophysical, biomechanical, and biochemical, and respond to them differently. Numerous studies have recently explored and reported the effect of contact guidance by culturing various types of cells on different types of micropatterned substrates such as microgrooves, geometric (square and triangle) micropattern, microstrips, micropatterned nanofibers. Amongst all of these micropatterned polymeric substrates; electrospun nanofibers have been regarded as a suitable substrate as it mimics the native ECM architectures. Therefore, in the present study; stencil-assisted electrospun Grid-lined micropatterned PCL-Collagen nanofibers (GLMPCnfs) were fabricated and its influence on the alignment and differentiation of pre-osteoblast cells (MC3T3-E1) was investigated. The randomly orientated Non-patterned PCL-Collagen nanofibers (NPPCnfs) were used as control. The patterns were characterized for their geometrical features such as area and thickness of deposition using surface profiler and scanning electron microscopy. A 61 % decrease in the overall area of GLMPCnfs as compared to the stencil area demonstrated the potential of electrofocusing phenomenon in the process of patterning electrospun nanofibers into various micron-scale structures. The MC3T3-E1 cells were confined and aligned in the direction of GLMPCnfs as confirmed by a high cellular aspect ratio (AR = 5.41), lower cellular shape index (CSI = 0.243), and cytoskeletal reorganization assessed through the F-actin filament immunocytochemistry (ICC) imaging. The aligned cells along the GLMPCnfs exhibited elevated alkaline phosphatase activity and enhanced mineralization. Furthermore, the gene expression profiling revealed upregulation of key osteogenic markers, such as ALP, OCN, OPN, COL1A1, and osteocyte markers DMP1, and SOST. Consequently, the research highlights the impact of GLMPCnfs on the cellular behaviour that results to the pre-osteoblast differentiation and the potential for stimulant-free early osteogenesis. These results offer an extensive understanding and mechanistic insight into how scaffold topography can be modified to influence cellular responses for effective bone regeneration strategies.

A biomimetic calcium phosphate nanowire coating on titanium surface enhances osteoimmunomodulation and osteointegration

The appropriate immune microenvironment plays crucial roles in bone regeneration. Surface structure and chemistry are key factors affecting immune cells behavior and subsequently regulating the activity of bone-related cells. To achieve rapid osteointegration, this study prepared a biomimetic calcium phosphate (CaP) coating on Ti-based substrates (THCaP) with the help of oleic acid under hydrothermal condition. The coating was composed of hydroxyapatite nanowires and further self-assembled into micropores. Also, the effect of the biomimetic CaP coating on the immune response of macrophages and the impact on angiogenesis and osteogenesis were investigated. In vitro cell culture results showed that compared with pristine Ti and similar titanate nanowire structure (TH), THCaP not only stimulated the polarization of macrophages towards a pro-healing M2 phenotype, but also enhanced the angiogenic and osteogenic differentiation of endothelial cells and pre-osteoblastic cells, respectively. Particularly, macrophages on THCaP induced a favorable immune microenvironment that facilitated osteogenic differentiation of pre-osteoblastic cells. In vivo tests confirmed these findings, showing that the implanted THCaP could suppress the inflammatory response and facilitate new bone formation when compared with pristine Ti and TH implants. These results indicate that preparation of biomimetic CaP network structure is a promising approach to surface design of Ti-based substrates, which is beneficial to generate a favorable osteoimmunomodulatory microenvironment for enhancing osteointegration.

Effect of calcium phosphate/bovine serum albumin coated Al2O3-Ti biocomposites on osteoblast response.

The biological performance of aluminum oxide-titanium (Al2O3-Ti) composites requires special attention to achieve improved osteoblastic differentiation, and subsequent osseointegration/strong anchorage with the surrounding bone. Therefore, the aim of this study was to improve them by providing calcium phosphate (Ca-P)/bovine serum albumin (BSA) coating on their surfaces. Ca-P/BSA coatings were prepared on the surfaces of 75vol.%Ti composites (75Ti-BSA) and pure Ti (100Ti-BSA as a control). The surface characteristics, phase analysis, micro-hardness, BSA release profile and biological responses including cytotoxicity, cell viability, differentiation, mineralization, and cell adhesion were evaluated. The results showed that lower cytotoxicity% and higher mitochondrial activity or viability % were associated with the samples with Ca-P/BSA coatings (particularly 75Ti-BSA having 21.3% cytotoxicity, 111.4% and 288.6% viability at day 1 and 7, respectively). Furthermore, the Ca-P/BSA coating could highly enhance the differentiation of pre-osteoblast cells into osteoblasts in 75Ti-BSA group (ALP concentration of 4.8 ng/ml). However, its influence on cell differentiation in 100Ti-BSA group was negligible. Similar results were also obtained from mineralization assay. The results on cell adhesion revealed that the Ca-P/BSA coated samples differently interacted with MC3T3-E1 cells; enlarged flat cells on 75Ti-BSA vs more spindle-shaped cells on 100Ti-BSA. Ca-P/BSA coated Al2O3-Ti provided promising biological performance, superior to that of uncoated composites. Therefore, they have the potential to improve implant osseointegration.

MECHANICAL STIMULATION OF PIEZOELECTRIC SCAFFOLDS PROMOTES CELL OSTEOGENIC DIFFERENTIATION

Bone is a dynamic tissue that undergoes continuous mechanical forces. Mechanical stimuli applied on scaffolds resembling a part of the human bone tissue affects the osteogenesis [1]. Poly(3,4-ethylenedioxythiophene) (PEDOT) is a piezoelectric material that responds to mechanical stimulation producing an electrical signal, which in turn promotes the osteogenic differentiation of bone-forming cells by opening voltage-gated calcium channels [2]. In this study we examined the biological behavior of pre-osteoblastic cells seeded onto lyophilized piezoelectric PEDOT-containing scaffolds applying uniaxial compression.Two different concentrations of PEDOT (0.10 and 0.15% w/v) were combined with a 5% w/v poly(vinyl alcohol) (PVA) and 5% w/v gelatin, casted into wells, freeze dried and crosslinked with 2% v/v (3-glycidyloxypropyl)trimethoxysilane and 0.025% w/v glutaraldehyde. The scaffolds were physicochemically characterized by FTIR, measurement of the elastic modulus, swelling ratio and degradation rate. The cell-loaded scaffolds were subjected to uniaxial compression with a frequency of 1 Hz and a strain of 10% for 1 h every second day for 21 days. The loading parameters were selected to resemble the in vivo loading situation [3]. Cell viability and morphology on the PEDOT/PVA/gelatin scaffolds was determined. The alkaline phosphatase (ALP) activity, the collagen and calcium production were determined.The elastic modulus of PEDOT/PVA/gelatin scaffolds ranged between 1 and 5 MPa. The degradation rate indicates a mass loss of 15% after 21 days. The cell viability assessment displays excellent biocompatibility, while SEM images display well-spread cells. The ALP activity at days 3, 7 and 18 as well as the calcium production are higher in the dynamic culture compared to the static one. Moreover, energy dispersive spectroscopy analysis revealed the presence of calcium phosphate in the extracellular matrix after 14 days. The results demonstrate that PEDOT/PVA/gelatin scaffolds promote the adhesion, proliferation, and osteogenic differentiation of pre-osteoblastic cells under mechanical stimulation, thus favoring bone regeneration.

A Doubly Fmoc-Protected Aspartic Acid Self-Assembles into Hydrogels Suitable for Bone Tissue Engineering.

Hydrogels have been used as scaffolds for biomineralization in tissue engineering and regenerative medicine for the repair and treatment of many tissue types. In the present work, we studied an amino acid-based material that is attached to protecting groups and self-assembles into biocompatible and stable nanostructures that are suitable for tissue engineering applications. Specifically, the doubly protected aspartic residue (Asp) with fluorenyl methoxycarbonyl (Fmoc) protecting groups have been shown to lead to the formation of well-ordered fibrous structures. Many amino acids and small peptides which are modified with protecting groups display relatively fast self-assembly and exhibit remarkable physicochemical properties leading to three-dimensional (3D) networks, the trapping of solvent molecules, and forming hydrogels. In this study, the self-assembling fibrous structures are targeted toward calcium binding and act as nucleation points for the binding of the available phosphate groups. The cell viability, proliferation, and osteogenic differentiation of pre-osteoblastic cells cultured on the formed hydrogel under various conditions demonstrate that hydrogel formation in CaCl2 and CaCl2-Na2HPO4 solutions lead to calcium ion binding onto the hydrogels and enrichment with phosphate groups, respectively, rendering these mechanically stable hydrogels osteoinductive scaffolds for bone tissue engineering.

Traditional Japanese apricot (Prunus mume) induces osteocalcin in osteoblasts.

The fruit of Prunus mume (ume, also known as Japanese apricot) has been used as a functional food in Japan since ancient times. We previously reported that ume stimulates the differentiation of preosteoblastic cells. Osteocalcin (OCN) is secreted by osteoblasts, and there is known association with glucolipid metabolism and cognitive function. This study sought to clarify the relationship between ume extracts and OCN production both in vitro and in vivo. Alkaline phosphatase activity and OCN level in the ethyl acetate extracts of ume-treated extracts were significantly increased in preosteoblast MC3T3-E1 cells compared with the control group. In human study, serum OCN level was significantly higher in the high ume intake group than in the low intake group in community-dwelling participants over 60 years old. These results suggest that ume has the potential to upregulated OCN production both in vitro and in vivo.

Composite Scaffolds from Gelatin and Bone Meal Powder for Tissue Engineering.

Bone tissue engineering offers versatile solutions to broaden clinical options for treating skeletal injuries. However, the variety of robust bone implants and substitutes remains largely uninvestigated. The advancements in hydrogel scaffolds composed of natural polymeric materials and osteoinductive microparticles have shown to be promising solutions in this field. In this study, gelatin methacrylate (GelMA) hydrogels containing bone meal powder (BP) particles were investigated for their osteoinductive capacity. As natural source of the bone mineral, we expect that BP improves the scaffold’s ability to induce mineralization. We characterized the physical properties of GelMA hydrogels containing various BP concentrations (0, 0.5, 5, and 50 mg/mL). The in vitro cellular studies revealed enhanced mechanical performance and the potential to promote the differentiation of pre-osteoblast cells. The in vivo studies demonstrated both promising biocompatibility and biodegradation properties. Overall, the biological and physical properties of this biomaterial is tunable based on BP concentration in GelMA scaffolds. The findings of this study offer a new composite scaffold for bone tissue engineering.

Comparative gene expression analysis for pre-osteoblast MC3T3-E1 cells under non-adhesive culture toward osteocyte differentiation

Osteocytes play an important role to modulate the bone remodeling and are also known as terminally differentiated cells originated from the osteoblast precursor cells, but its differentiation mechanism remains unclear. Since an efficient invitro method to evoke the osteocyte differentiation from the osteoblast precursor cells has not been established, we conducted the comparative gene expression analysis for mouse pre-osteoblast MC3T3-E1 cells in order to elucidate the key factors to induce the osteocyte differentiation from the pre-osteoblast cells. In this study, we prepared four different culture environments by modulating their cell-substrate interaction and cell-cell interaction; (i) low and (ii) high cell density on the adhesive culture models, and (iii) low and (iv) high cell density on the non-adhesive floating culture models. By comparing these conditions in terms of cell-substrate and cell-cell interaction, we showed that the elimination of cell-substrate interaction under non-adhesive floating culture greatly up-regulated the gene expression of osteocyte markers in the pre-osteoblast cells. Moreover, the presence of moderate cell-cell interaction in the non-adhesive spheroid culture further enhanced the up-regulation of osteocyte markers for the pre-osteoblast cells. The results altogether suggest the most appropriate culture environment to induce the invitro osteocyte differentiation of pre-osteoblast cells.

Impact of Hydrolyzed Collagen from Defatted Sea Bass Skin on Proliferation and Differentiation of Preosteoblast MC3T3-E1 Cells.

Osteoporosis is a serious problem affecting health of the elderly. Drugs (bisphosphonates) applied for treatment are often accompanied by adverse side effects. Thus, fish byproduct-derived peptides, particularly hydrolyzed collagen (HC) from defatted sea bass skin, could be a safe source of anti-osteoporosis agents. This study aimed to examine the effects of HC on proliferation and differentiation of preosteoblast cells. HC prepared using papain before Alcalase hydrolysis was determined for molecular weight (MW) distribution. Thereafter, the resulting HC (50–800 µg/mL) was added to the cell. Proliferation, alkaline phosphatase activity (AP-A) and mineralization of cells were investigated. Moreover, the expression of runt-related transcription factor 2 (RUNX2) and the p-Akt/Akt pathway were also determined using Western blot. The results showed that HC had an MW < 3 kDa. HC (50–200 µg/mL) could promote cell proliferation. Nevertheless, HC at 100 µg/mL (HC-100) had enhanced AP-A and increased mineralization during the first 7 days of culture. Moreover, HC-treated cells had higher calcium depositions than the control (p < 0.05). Additionally, cells treated with HC-100 had higher levels of RUNX2 and p-Akt expressions than control (p < 0.05). Therefore, HC could be a promising functional ingredient to promote osteoblast proliferation and differentiation, which could enhance bone strength.

Bioactivation Treatment with Mixed Acid and Heat on Titanium Implants Fabricated by Selective Laser Melting Enhances Preosteoblast Cell Differentiation.

Selective laser melting (SLM) is a promising technology capable of producing individual characteristics with a high degree of surface roughness for implants. These surfaces can be modified so as to increase their osseointegration, bone generation and biocompatibility, features which are critical to their clinical success. In this study, we evaluated the effects on preosteoblast proliferation and differentiation of titanium metal (Ti) with a high degree of roughness (Ra = 5.4266 ± 1.282 µm) prepared by SLM (SLM-Ti) that was also subjected to surface bioactive treatment by mixed acid and heat (MAH). The results showed that the MAH treatment further increased the surface roughness, wettability and apatite formation capacity of SLM-Ti, features which are useful for cell attachment and bone bonding. Quantitative measurement of osteogenic-related gene expression by RT-PCR indicated that the MC3T3-E1 cells on the SLM-Ti MAH surface presented a stronger tendency towards osteogenic differentiation at the genetic level through significantly increased expression of Alp, Ocn, Runx2 and Opn. We conclude that bio-activated SLM-Ti enhanced preosteoblast differentiation. These findings suggest that the mixed acid and heat treatment on SLM-Ti is promising method for preparing the next generation of orthopedic and dental implants because of its apatite formation and cell differentiation capability.

3D-printed biodegradable composite scaffolds with significantly enhanced mechanical properties via the combination of binder jetting and capillary rise infiltration process

For hard tissue engineering applications, biodegradable composite scaffolds have been extensively investigated because of their satisfactory mechanical properties and biocompatibility. Recently, 3D printing processes have received substantial attention in the tissue engineering field because of their ability to be customized for tissues that have suffered different types of loss or damage for each patient. However, previous studies on material extrusion-based techniques lack flexibility in the filler loading amount and cannot fulfill requirements that aim to enhance mechanical properties and biocompatibility. Herein, we propose a biodegradable polymer-based composite scaffolds with high ceramic loadings fabricated using the binder jetting (BJ) technique conjugated with capillary rise infiltration. A calcium sulfate hemihydrate (CSH) scaffold was fabricated using BJ-based 3D printing. Thereafter, CSH was transformed into biphasic calcium phosphate (BCP) using hydrothermal treatment, followed by heat treatment. Melted polycaprolactone (PCL) was infiltrated in the resulting BCP scaffold. BCP was then completely dispersed in the PCL matrix, and the calculated PCL loading in the BCP matrix exceeded 40 vol%. The PCL/BCP composite scaffold demonstrated the highest compressive strength, moduli, and toughness with the fracture mode shifted from brittle to less brittle. Moreover, a stable PCL/BCP surface promotes initial cell responses and shows sufficient proliferation and differentiation of pre-osteoblast cells.

Improved osseointegration of 3D printed Ti-6Al-4V implant with a hierarchical micro/nano surface topography: An in vitro and in vivo study

Three-dimensional (3D) printing technology is serving as a promising approach of fabricating titanium (Ti) and its alloys used for bone tissue engineering. However, the biological inertness nature of Ti material limits its capability to bind directly with the bone tissue. This paper aims to enhance the bioactivity and osteogenesis of 3D printed Ti-6Al-4V implants by constructing a hierarchical micro/nano-topography on the surface. Ti-6Al-4V implants were prepared by the electron beam melting (EBM) technique. A method combining ultrasonic acid etching with anodic oxidation is proposed for surface modification of EBM Ti-6Al-4V implants in this study. The acid etching step was to remove any existent residual powders on the implant's surface and construct micro-pits and -grooves on the EBM microrough surface. Nanotube arrays with a diameter of 40–50 nm were superimposed on the micro-structured substrate via anodic oxidation. The results of in vitro experiments showed that the hierarchical micro/nano-structured surface on Ti-6Al-4V after acid etching and anodic oxidation (AN) promoted the proliferation and osteogenic differentiation of pre-osteoblast cells (MC3T3-E1) via enhancing the surface hydrophilicity and bioactivity compared with the polished Ti surface (P). Micro-CT and histological analysis were used to assess the in vivo osteogenic properties enhancement. The results 8 weeks after the surgery showed the ratio of bone volume to total volume (BV/TV) of AN implant was 43.4%, which represented 1.5 times that of as-printed implants (AM) without any post-treatment. Considerable increment of bone-to-implant contact area was also detected from the micro-CT reconstructed 3D models in comparison with AM implants and acid etched (AE) EBM implants. In conclusion, the hierarchical micro/nano topography generated on the EBM native surface showed an improvement of bioactivity and osteogenic properties, which is expected to accelerate the application of 3D printed orthopedic and dental implants in clinics. Statement of significanceTraditional titanium implants have the nature of biological inertness, which limits their capability to bind directly with the bone tissue. The failure of implants after couple of years of implantation will cause huge pain to the patients. In this work, a surface modification method for 3D printed implants was developed to construct a hierarchical micro/nano-structure. Through the in vitro and in vivo experiments, we proved that this hierarchical micro/nano-structure induced a better promotion effect on osteoblast proliferation and differentiation comparing with untreated surface or polished surface, and was also capable of bolstering the new bone formation, suggesting a potent strategy to improve the biological properties of 3D printed titanium implants. The work is expected to accelerate the application of 3D printed orthopedic and dental implants in clinics.

Outlining cell interaction and inflammatory cytokines on UV-photofunctionalized mixed-phase TiO2 thin film.

Photofunctionalization mediated by ultraviolet (UV) light seems to be a promising approach to improve the physico-chemical characteristics and the biological response of titanium (Ti) dental implants. Seeing that photofunctionalization is able to remove carbon from the surface, besides to promote reactions on the titanium dioxide (TiO2) layer, coating the Ti with a stable TiO2 film could potentialize the UV effect. Thus, here we determined the impact of UV-photofunctionalized mixed-phase (anatase and rutile) TiO2 films on the physico-chemical properties of Ti substrate and cell biology. Mixed-phase TiO2 films were grown by radiofrequency magnetron sputtering on commercially pure titanium (cpTi) discs, and samples were divided as follow: cpTi (negative control), TiO2 (positive control), cpTi UV, TiO2 UV (experimental). Photofunctionalization was performed using UVA (360nm - 40W) and UVC (250nm - 40W) lamps for 48h. Surfaces were analyzed in terms of morphology, topography, chemical composition, crystalline phase, wettability and surface free energy. Pre-osteoblastic cells (MC3T3E1) were used to assess cell morphology and adhesion, metabolism, mineralization potential and cytokine secretion (IFN-γ, TNF-α, IL-4, IL-6 and IL-17). TiO2-coated surfaces exhibited granular surface morphology and greater roughness. Photofunctionalization increased wettability (p<0.05) and surface free energy (p<0.001) on both surface conditions. TiO2-treated groups featured normal cell morphology and spreading, and greater cellular metabolic activity at 2 and 4days (p<0.05), whereas UV-photofunctionalized surfaces enhanced cell metabolism, cell adhered area, and calcium deposition (day 14) (p<0.05). In general, assessed proteins were found slightly affected by either UV or TiO2 treatments. Altogether, our findings suggest that UV-photofunctionalized TiO2 surface has the potential to improve pre-osteoblastic cell differentiation and the ability of cells to form mineral nodules by modifying Ti physico-chemical properties towards a more stable context. UV-modified surfaces modulate the secretion of key inflammatory markers.

Synthesis and Characterization of Melatonin-Loaded Chitosan Microparticles Promote Differentiation and Mineralization in Preosteoblastic Cells.

In terms of a novel scaffold with well good osteoinductive and osteoconductive capacity, melatonin (Mel) possesses positive effects on chemical linkage in scaffold structures, which may allow osteogenic differentiation. The aim of this study is to fabricate Mel-loaded chitosan (CS) microparticles (MPs) as a novel bone substitute through generating a Mel sustained release system from Mel-loaded CS MPs and evaluating its effect on the osteogenic capacity of MC3T3-E1 in vitro. The physical-chemical characteristics of the prepared CS MPs were examined by both Fourier transform infrared spectroscopy and scanning electron microscopy. The released profile and kinetics of Mel from MPs were quantified, and the bioactivity of the released Mel on preosteoblastic MC3T3-E1 cells was characterized in vitro. An in vitro drug release assay has shown high encapsulation efficiency and sustained release of Mel over the investigation period. In an osteogenesis assay, Mel-loaded CS MPs have significantly enhanced alkaline phosphatase (ALP) mRNA expression and ALP activity compared with the control group. Meanwhile, the osteoblast-specific differentiation genes, including runt related transcription factor 2 (Runx2), bone morphogentic protein-2 (Bmp2), collagen I (Col I), and osteocalcin (Ocn), were also significantly upregulated. Furthermore, quantificational alizarin red-based assay demonstrated that Mel-loaded CS MPs notably enhanced the calcium deposit of MC3T3-E1 compared with controls. In essence, Mel-loaded CS MPs can control the release of Mel for a period of time to accelerate osteogenic differentiation of preosteoblast cells in vitro.

Macrolactin A protects against LPS-induced bone loss by regulation of bone remodeling

An imbalance between bone resorption and bone formation leads to several kinds of bone diseases such as rheumatoid arthritis, osteoporosis and Paget’s disease. The imbalance between bone formations relative to bone resorption is responsible in bone remodeling. Several studies have suggested that macrolactin A (MA) has potent anti-inflammatory, anti-cancer and anti-angiogenic effects in various cell types. We investigate whether macrolactin A (MA) could inhibit bone loss and enhance bone formation. We used bone marrow monocytes/macrophages (BMMs) cells to study osteoclast activity and MC3T3-E1 cells to study osteoblast activity. MA suppressed tartrate resistant acid phosphatase (TRAP) positive multinucleated cells in a concentration-dependent manner, as well as at a specific time point. MA markedly reduced bone resorption activity and F-actin ring formation. Moreover, MA markedly suppressed receptor activator of nuclear factor k-B ligand (RANKL)-induced osteoclastogenic marker genes and transcription factors in-vitro. MA repressed osteoclast differentiation via activation of the phosphoinositide kinase-3/Akt, extracellular signal-regulated kinase 1/2 (ERK 1/2), c-Jun N-terminal kinase (JNK), nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) and c-Fos signaling pathways. MA enhanced pre-osteoblast cell differentiation on mineralization activity, alkaline phosphatase (ALP) activity, and the expression of osteoblastogenic markers including osterix, RUNX-2, SMAD4, BMP-2, and ALP. Importantly, MA repressed lipopolysaccharide (LPS)-induced inflammatory bone loss in mice as shown by TRAP staining of femurs and μCT analysis. Therefore, MA could be a promising candidate for the inhibition and management of osteoporosis, arthritis, and bone lytic diseases.

Effects of the Sintering Process on Nacre-Derived Hydroxyapatite Scaffolds for Bone Engineering.

A hydroxyapatite scaffold is a suitable biomaterial for bone tissue engineering due to its chemical component which mimics native bone. Electronic states which present on the surface of hydroxyapatite have the potential to be used to promote the adsorption or transduction of biomolecules such as protein or DNA. This study aimed to compare the morphology and bioactivity of sinter and nonsinter marine-based hydroxyapatite scaffolds. Field emission scanning electron microscopy (FESEM) and micro-computed tomography (microCT) were used to characterize the morphology of both scaffolds. Scaffolds were co-cultured with 5 × 104/cm2 of MC3T3-E1 preosteoblast cells for 7, 14, and 21 days. FESEM was used to observe the cell morphology, and MTT and alkaline phosphatase (ALP) assays were conducted to determine the cell viability and differentiation capacity of cells on both scaffolds. Real-time polymerase chain reaction (rtPCR) was used to identify the expression of osteoblast markers. The sinter scaffold had a porous microstructure with the presence of interconnected pores as compared with the nonsinter scaffold. This sinter scaffold also significantly supported viability and differentiation of the MC3T3-E1 preosteoblast cells (p < 0.05). The marked expression of Col1α1 and osteocalcin (OCN) osteoblast markers were also observed after 14 days of incubation (p < 0.05). The sinter scaffold supported attachment, viability, and differentiation of preosteoblast cells. Hence, sinter hydroxyapatite scaffold from nacreous layer is a promising biomaterial for bone tissue engineering.

3D-printed scaffolds with carbon nanotubes for bone tissue engineering: Fast and homogeneous one-step functionalization

Three-dimensional (3D) printing is a promising technology for tissue engineering. However, 3D-printing methods are limited in their ability to produce desired microscale features or electrochemical properties in support of robust cell adhesion, proliferation, and differentiation. This study addresses this deficiency by proposing an integrated, one-step, method to increase the cytocompatibility of 3D-printed scaffolds through functionalization leveraging conductive carbon nanotubes (CNTs). To this end, CNTs were first sonicated with water-soluble single-stranded deoxyribonucleic acid (ssDNA) to generate a negatively charged ssDNA@CNT nano-complex. Concomitantly, 3D-printed poly(propylene fumarate) (PPF) scaffolds were ammonolyzed to introduce free amine groups, which can take on a positive surface charge in water. The ssDNA@CNT nano-complex was then applied to 3D-printed scaffolds through a simple one-step coating utilizing electric-static force. This fast and facile functionalization step resulted in a homogenous and non-toxic coating of CNTs to the surface, which significantly improved the adhesion, proliferation, and differentiation of pre-osteoblast cells. In addition, the CNT based conductive coating layer enabled modulation of cell behavior through electrical stimuli (ES) leading to cellular proliferation and osteogenic gene marker expression, including alkaline phosphatase (ALP), osteocalcin (OCN), and osteopontin (OPN). Collectively, these data provide the foundation for a one-step functionalization method for simple, fast, and effective functionalization of 3D printed scaffolds that support enhanced cell adhesion, proliferation, and differentiation, especially when employed in conjunction with ES. Statement of SignificanceThree-dimensional (3D) printing is a promising technology for tissue engineering. However, 3D-printing methods have limited ability to produce desired features or electrochemical properties in support of robust cell behavior. To address this deficiency, the current study proposed an integrated, one-step method to increase the cytocompatibility of 3D-printed scaffolds through functionalization leveraging conductive carbon nanotubes (CNTs). This fast and facile functionalization resulted in a homogenous and non-toxic coating of CNTs to the surface, which significantly improved the adhesion, proliferation, and differentiation of cells on the 3D-printed scaffolds.

Effect of sodium ascorbyl phosphate on osteoblast viability and differentiation.

Sodium ascorbyl phosphate (SAP) is a hydrophilic and stable L-ascorbic acid derivative, being converted by the cell phosphatases into free ascorbic acid (AA), which allows its sustained release in the medium. AA participates in the maintenance and healing of the periodontium. It presents a regulatory role of the osteoblastic activity, stimulating the deposition of collagen extracellular matrix followed by the induction of genes associated with the osteoblastic phenotype. It also acts in the elimination of reactive oxygen species, abundantly produced by defense cells in periodontal disease. The aim of this study was to evaluate the effect of SAP on osteoblast viability and differentiation. Mouse preosteoblastic cells of the MC3T3-E1 strain were used. Cell viability was assessed by the trypan blue dye exclusion assay and the expression of genes related to osteoblast differentiation by quantitative PCR. Collagen I secretion was evaluated by ELISA, and mineralized matrix formation was assayed by Alizarin red S staining. The results showed that SAP at concentrations from 50 to 500µmol/L does not influence preosteoblast cell viability, but stimulates their differentiation, observed by the induction of RUNX2, COL1A1, and BGLAP2; by the higher secreted levels of collagen I; and also by the increase in the mineralization of the extracellular matrix in cells exposed to this agent at 200 or 400µmol/L, compared with those not exposed. By its stability and capacity to induce preosteoblastic cell differentiation, our results indicate that the incorporation of SAP into local release devices, membranes/scaffolds or biomaterials, could favor bone tissue formation and therefore periodontal healing.