Early Osteogenic Differentiation Research Articles

Osteogenic differentiation of mesenchymal stem cells modulated by a chemically modified super-hydrophilic titanium implant surface.

This study investigated the osteogenic functionality of multipotent mesenchymal stem cells (MSCs) modulated by a chemically modified super-hydrophilic titanium (Ti) bone implant surface to elucidate the biological mechanism underlying the bone healing capacity of this modified Ti surface. A microstructured Ti surface incorporating bioactive ions (in this study, phosphate (P) ions) was prepared by wet chemical treatment. The results showed that the hydrothermally obtained crystalline P-incorporated Ti surface (P surface) displayed long-term super-hydrophilicity (water contact angles <5°) during a 36-week observation period. The hydrophilic P surface enhanced early cellular functions and osteogenic differentiation of multipotent MSCs derived from mouse bone marrow and human adipose tissue. The expression of critical integrins affecting subsequent osteoblast function and osteoblast phenotype genes was notably upregulated in multipotent MSCs grown on the P surface compared with the commercially available grit-blasted microrough clinical oral implant surface. The P surface supported better cell spreading, focal adhesion and ALP activity of MSCs. These results indicate that a super-hydrophilic P-incorporated Ti surface accelerates implant bone healing by enhancing the early osteogenesis functions of multipotent MSCs.

Synergistic Effects of Beta Tri-Calcium Phosphate and Porcine-Derived Decellularized Bone Extracellular Matrix in 3D-Printed Polycaprolactone Scaffold on Bone Regeneration.

Bone-derived extracellular matrix (ECM) is widely used in studies on bone regeneration because of its ability to provide a microenvironment of native bone tissue. However, a hydrogel, which is a main type of ECM application, is limited to use for bone graft substitutes due to relative lack of mechanical properties. The present study aims to fabricate a scaffold for guiding effective bone regeneration. A polycaprolactone (PCL)/beta-tricalcium phosphate (β-TCP)/bone decellularized extracellular matrix (dECM) scaffold capable of providing physical and physiological environment are fabricated using 3D printing technology and decoration method. PCL/β-TCP/bone dECM scaffolds exhibit excellent cell seeding efficiency, proliferation, and early and late osteogenic differentiation capacity in vitro. In addition, outstanding results of bone regeneration are observed in PCL/β-TCP/bone dECM scaffold group in the rabbit calvarial defect model in vivo. These results indicate that PCL/β-TCP/bone dECM scaffolds have an outstanding potential as bone graft substitutes for effective bone regeneration.

ECM Decorated Electrospun Nanofiber for Improving Bone Tissue Regeneration.

Optimization of nanofiber surface properties can lead to enhanced tissue regeneration outcomes in the context of bone tissue engineering. Herein, we developed a facile strategy to decorate elctrospun nanofibers using extracellular matrix (ECM) in order to improve their performance for bone tissue engineering. Electrospun PLLA nanofibers (PLLA NF) were seeded with MC3T3-E1 cells and allowed to grow for two weeks in order to harvest a layer of ECM on nanofiber surface. After decellularization, we found that ECM was successfully preserved on nanofiber surface while maintaining the nanostructure of electrospun fibers. ECM decorated on PLLA NF is biologically active, as evidenced by its ability to enhance mouse bone marrow stromal cells (mBMSCs) adhesion, support cell proliferation and promote early stage osteogenic differentiation of mBMSCs. Compared to PLLA NF without ECM, mBMSCs grown on ECM/PLLA NF exhibited a healthier morphology, faster proliferation profile, and more robust osteogenic differentiation. Therefore, our study suggests that ECM decoration on electrospun nanofibers could serve as an efficient approach to improving their performance for bone tissue engineering.

Bioactive glass induced osteogenic differentiation of human adipose stem cells is dependent on cell attachment mechanism and mitogen-activated protein kinases.

Bioactive glasses (BaGs) are widely utilised in bone tissue engineering (TE) but the molecular response of cells to BaGs is poorly understood. To elucidate the mechanisms of cell attachment to BaGs and BaG-induced early osteogenic differentiation, we cultured human adipose stem cells (hASCs) on discs of two silica-based BaGs S53P4 (23.0 Na2O - 20.0 CaO - 4.0 P2O5 - 53.0 SiO2 (wt-%)) and 1-06 (5.9 Na2O - 12.0 K2O - 5.3 MgO - 22.6 CaO - 4.0 P2O5 - 0.2 B2O3 - 50.0 SiO2) in the absence of osteogenic supplements. Both BaGs induced early osteogenic differentiation by increasing alkaline phosphatase activity (ALP) and the expression of osteogenic marker genes RUNX2a and OSTERIX. Based on ALP activity, the slower reacting 1-06 glass was a stronger osteoinducer. Regarding the cell attachment, cells cultured on BaGs had enhanced integrinβ1 and vinculin production, and mature focal adhesions were smaller but more dispersed than on cell culture plastic (polystyrene). Focal adhesion kinase (FAK), extracellular signal-regulated kinase (ERK1/2) and c-Jun N-terminal kinase (JNK)-induced c-Jun phosphorylations were upregulated by glass contact. Moreover, the BaG-stimulated osteoinduction was significantly reduced by FAK and mitogen-activated protein kinase (MAPK) inhibitors, indicating an important role for FAK and MAPKs in the BaG-induced early osteogenic commitment of hASCs. Upon indirect insert culture, the ions released from the BaG discs could not reproduce the observed cellular changes, which highlighted the role of direct cell-BaG interactions in the osteopotential of BaGs. These findings gave valuable insight into the mechanism of BaG-induced osteogenic differentiation and therefore provided knowledge to aid the future design of new functional biomaterials to meet the increasing demand for clinical bone TE treatments.

Repositioning Titanium: An In Vitro Evaluation of Laser-Generated Microporous, Microrough Titanium Templates As a Potential Bridging Interface for Enhanced Osseointegration and Durability of Implants.

Although titanium alloys remain the preferred biomaterials for the manufacture of biomedical implants today, such devices can fail within 15 years of implantation due to inadequate osseointegration. Furthermore, wear debris toxicity due to alloy metal ion release has been found to cause side-effects including neurotoxicity and chronic inflammation. Titanium, with its known biocompatibility, corrosion resistance, and high elastic modulus, could if harnessed in the form of a superficial scaffold or bridging device, resolve such issues. A novel three-dimensional culture approach was used to investigate the potential osteoinductive and osseointegrative capabilities of a laser-generated microporous, microrough medical grade IV titanium template on human skeletal stem cells (SSCs). Human SSCs seeded on a rough 90-µm pore surface of ethylene oxide-sterilized templates were observed to be strongly adherent, and to display early osteogenic differentiation, despite their inverted culture in basal conditions over 21 days. Limited cellular migration across the template surface highlighted the importance of high surface wettability in maximizing cell adhesion, spreading and cell-biomaterial interaction, while restricted cell ingrowth within the conical-shaped pores underlined the crucial role of pore geometry and size in determining the extent of osseointegration of an implant device. The overall findings indicate that titanium only devices, with appropriate optimizations to porosity and surface wettability, could yet play a major role in improving the long-term efficacy, durability, and safety of future implant technology.

Application of whey protein isolate in bone regeneration: Effects on growth and osteogenic differentiation of bone-forming cells

Recently, milk-derived proteins have attracted attention for applications in the biomedical field such as tissue regeneration. Whey protein isolate (WPI), especially its main component β-lactoglobulin, can modulate immunity and acts as an antioxidant, antitumor, antiviral, and antibacterial agent. There are very few reports of the application of WPI in tissue engineering, especially in bone tissue engineering. In this study, we tested the influence of different concentrations of WPI on behavior of human osteoblast-like Saos-2 cells, human adipose tissue-derived stem cells (ASC), and human neonatal dermal fibroblasts (FIB). The positive effect on growth was apparent for Saos-2 cells and FIB but not for ASC. However, the expression of markers characteristic for early osteogenic cell differentiation [type-I collagen (COL1) and alkaline phosphatase (ALP)] as well as ALP activity, increased dose-dependently in ASC. Importantly, Saos-2 cells were able to deposit calcium in the presence of WPI, even in a proliferation medium without other supplements that support osteogenic cell differentiation. The results indicate that, depending on the cell type, WPI can act as an enhancer of cell proliferation and osteogenic differentiation. Therefore, enrichment of biomaterials for bone regeneration with WPI seems a promising approach, especially due to the low cost of WPI.

RUNX3 plays an important role in mediating the BMP9-induced osteogenic differentiation of mesenchymal stem cells.

Although bone morphogenetic protein9(BMP9) is highly capable of promoting the osteogenic differentiation of mesenchymal stem cells(MSCs) both invitro and invivo, the molecular mechanisms involved remain to be fully elucidated. Runt-related transcription factor(RUNX)3 is an essential regulator of osteoblast/chondrocyte maturation. However, the exact role of RUNX3 in BMP9 osteoinductive activity is unknown. In this study, we sought to investigate the functional role of RUNX3 in the BMP9-induced osteogenic differentiation of MSCs. We found that BMP9 upregulated the endogenous expression of RUNX3 in MSCs. The overexpression or/and knockdown of RUNX3 both increased the levels of alkaline phosphatase(ALP) a marker of BMP9-induced early osteogenic differentiation. Nevertheless, matrix mineralization, a marker of BMP9-induced late osteogenic differentiation was enhanced by the overexpression of RUNX3, whereas it was inhibited by the knockdown of RUNX3. The BMP9-induced expression of osteogenic pivotal transcription factors[inhibitor of differentiation(Id)3, distal-less homeobox5(DLX5) and RUNX2)] was further increased by the overexpression of RUNX3; however, it was reduced by the knockdown of RUNX3. However, the expression levels of Id1 and Id2 were both enhanced by the overexpression or/and knockdown of RUNX3. The BMP9-induced phosphorylation of Smad1/5/8 was increased with the overexpression of RUNX3, and yet was decreased with the knockdown of RUNX3. Collectively, our findings suggest that RUNX3 is an essential modulator of the BMP9-induced osteoblast lineage differentiation of MSCs.

SPIO-Au core-shell nanoparticles for promoting osteogenic differentiation of MC3T3-E1 cells: Concentration-dependence study.

This work aims to explore the concentration-dependence of SPIO-Au core-shell nanoscale particles (NPs) (17.3 ± 1.2 nm in diameter) on biocompatibility and osteogenic differentiation of preosteoblast MC3T3-E1 cells. The stability of NPs was first investigated by UV-vis absorption spectra and zeta potential measurement. Then concentration effects of NPs (1-80 μg/mL) were evaluated on viability, morphology, proliferation, cellular uptake, and alkaline phosphate (ALP) activity levels. Results have shown strong stability and no acute toxicity (viability > 93%) or morphological difference at all concentration levels of NPs. The proliferation results indicated that the concentration of NPs below 40 μg/mL does not affect the cell proliferation for 7 days of incubation. Transmission electron microscopy images revealed the successful internalization of NPs into MC3T3-E1 cells and the dose-dependent accumulation of NPs inside the cytoplasm. The ALP level of MC3T3-E1 cells was improved by 49% (of control) after treated with NPs at 10 μg/mL for 10 days, indicating their positive effect on early osteogenic differentiation. This study confirmed the excellent biocompatibility of SPIO-Au NPs and their great potential for promoting osteogenic differentiation and promised the future application for these NPs in bone engineering including drug delivery, cell labeling, and activity tracking within scaffolds. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3350-3359, 2017.

MiR-139-5p Represses BMSC Osteogenesis via Targeting Wnt/β-Catenin Signaling Pathway.

Osteogenesis of mesenchymal stem cells (MSCs) has played a necessary role in the repair of bone. According to some reports, microRNAs participate in different physiological activity of the cells, including cell differentiation. This study investigated the function that miR-139-5p plays in the osteogenic differentiation of human bone marrow MSCs (hBMSCs). In addition to miR-139-5p, the effects of alkaline phosphatase (ALP), a membrane-bound metalloenzyme that is considered an early osteogenic differentiation marker, have also been investigated. Calcium-rich deposit (mineralization) is also a typical osteogenic differentiation marker that could be visualized by alizarin red S (ARS) staining. Inhibiting miR-139-5p notably promotes the hBMSC osteoblast differentiation, which, however, will be reduced by overexpressed miR-139-5p. This result has been made based on the alternations of ALP activity, ARS staining, as well as expression of osteogenic genes, including runt-related gene-2 (Runx2), collagen I (Col-1), and osteocalcin (OCN). miR-139-5p exerts its role in BMSC osteogenesis most probably through the Wnt/β-catenin pathway, by direct targeting CTNNB1 and frizzled 4 (FZD4), essential factors of Wnt/β-catenin pathway. In conclusion, according to the present study, inhibiting miR-139-5p could be a promising strategy in hBMSC osteogenesis.

ToF-SIMS study of differentiation of human bone-derived stromal cells: new insights into osteoporosis

Lipids have numerous important functions in the human body, as they form the cells' plasma membranes and play a key role in many disease states, presumably also in osteoporosis. Here, the fatty acid composition of the outer plasma membranes of cells differentiated into the osteogenic and adipogenic direction is studied with surface-sensitive time-of-flight secondary ion mass spectrometry (ToF-SIMS). For data evaluation, principal component analysis (PCA) is applied. Human (bone-derived) mesenchymal stromal cells (hMSCs) from an osteoporotic donor and a control donor are compared to reveal differences in the fatty acid composition of the membranes. The chemical information is correlated to staining and real-time quantitative polymerase chain reaction (rt-qPCR) results to provide insight into the gene expression of several differentiation markers on the RNA level. Adipogenic differentiation of hMSCs from a non-osteoporotic donor correlates with increased relative intensities of all fatty acids under investigation. After osteogenic differentiation of non-osteoporotic cells, the relative mass signal intensities of unsaturated fatty acids such as oleic and linoleic acids are increased. However, the osteoporotic cells show increased levels of palmitic acid in the plasma membrane after exposure to osteogenic differentiation conditions, which correlates to an immature differentiation state relative to non-osteoporotic osteogenic cells. This immature differentiation state is confirmed by increased early osteogenic differentiation factor Runx2 on RNA level and by less calcium mineralization spots seen in von Kossa staining and ToF-SIMS images. Graphical abstract Time-of-flight secondary ion mass spectrometry is applied to analyze the fatty acid composition of the outer plasma membranes of cells differentiated into the adipogenic and osteogenic direction. Cells from an osteoporotic and a control donor are compared to reveal differences due to differentiation and disease stage of the cells.

TRPV4 functions in flow shear stress induced early osteogenic differentiation of human bone marrow mesenchymal stem cells.

Mechanical cues have been shown to induce osteogenic differentiation of bone marrow stromal cells (MSCs). The TRPV4 channel, a Ca2+-permeable membrane ion channel, is implicated in the transduction of external mechanical stimulation into specific intracellular responses in a wide variety of bone cells. However, the role of TRPV4 in transducing and regulating the differentiation of human MSCs in response to flow shear stress (FSS) is unclear. In this study, using FSS and calcium imaging, we demonstrated that FSS activated early osteogenic differentiation, as shown by the early osteogenic differentiation marker osterix (Osx) and alkaline phosphatase (ALP) staining. Increases in intracellular Ca2+ and in the percentage of responding cells were induced by FSS. However, the late osteogenic differentiation marker Ocn and in vitro mineralization were unchanged after FSS stimulation. TRPV4 channels mediated the FSS-induced Ca2+ influx and osteogenic differentiation of MSCs, which were inhibited by a selective TRPV4 blocker HC-067047 and specific Trpv4 siRNA. Ca2+ influx through TRPV4 promoted NFATc1 nuclear localization. These results identify an essential role of TRPV4 in FSS-induced early osteogenic differentiation of human MSCs.

Dual growth factor loaded nonmulberry silk fibroin/carbon nanofiber composite 3D scaffolds for invitro and invivo bone regeneration.

In recent years the potential application of nanocomposite biomaterials in tissue engineering field is gaining importance because of the combined features of all the individual components. A bottom-up approach is acquired in this study to recreate the bone microenvironment. The regenerated silk protein fibroin obtained from nonmulberry tropical tasar Antheraea mylitta species is reinforced with functionalized carbon nano fiber (CNF) and the composite sponges are fabricated using facile green aqueous based method. Biophysical investigations show that the matrices are porous and simultaneously bioactive when incubated in simulated body fluid. The reinforcement of CNF influences the mechanical property of the matrices by increasing the compressive modulus up to 46.54MPa (∼4.3 times of the control fibroin sponge) in hydrated state, which is higher than the minimum required human trabecular bone modulus (10MPa). The composite matrices are found to be non-hemolytic as well as cytocompatible. The growth factors (BMP-2 and TGF-β1) loaded composites show sustained release kinetics and an early attachment, growth, proliferation, and osteogenic differentiation of the osteoblasts and mesenchymal stem cells. The matrices are immunocompatible as evidenced by minimal release of pro-inflammatory cytokines both invitro and invivo. In order to support the invitro study, invivo analysis of new bone formation within the implants is performed through radiological, μ-CT, fluorochrome labeling and histological analysis, which show statistically better bone formation on growth factor loaded composite scaffolds. The study clearly shows the potential attributes of these composite matrices as an extra cellular matrix for supporting successful osseointegration process.

Electroactive BaTiO3 nanoparticle-functionalized fibrous scaffolds enhance osteogenic differentiation of mesenchymal stem cells.

It has been proven that the surface topographic cues of fiber arrangement can induce osteogenic differentiation of mesenchymal stem cells. However, this effect alone is weak and insufficient to meet the needs of regenerative medicine. In this work, electroactivity concept was introduced to enhance the osteoinductivity of fibrous scaffolds. The randomly oriented and aligned electroactive fibrous scaffolds of poly-(l-lactic acid) (PLLA) with incorporation of ferroelectric ceramic BaTiO3 (BTO) nanoparticles (NPs) were fabricated by electrospinning. Physicochemical properties, including fiber morphology, microstructure, composition, thermal stability, surface roughness, and surface wettability, of these fibrous scaffolds were studied. The dielectric properties of the scaffolds were evaluated. The results showed that the randomly oriented BTO/PLLA composite fibrous scaffolds had the highest dielectric permittivity of 1.19, which is of the same order of magnitude as the natural bone. The combined effects of fiber orientation and electrical activity on the osteogenic responses of bone marrow mesenchymal stem cells (BM-MSCs) were specifically investigated. Randomly oriented composite fibrous scaffolds significantly promoted polygonal spreading and encouraged early osteogenic differentiation in BM-MSCs, whereas aligned composite fibrous scaffolds promoted cell elongation and discouraged osteogenic differentiation. These results evidenced that randomly fiber orientation and biomimetic electric activity have combining effects on osteogenic differentiation of BM-MSCs. Our findings indicate that coupling effects of multi-physical properties should be paid more attention to mimic the microenvironment for enhancing osteogenic differentiation of BM-MSCs.

The effect of equiaxial stretching on the osteogenic differentiation and mechanical properties of human adipose stem cells

Although mechanical cues are known to affect stem cell fate and mechanobiology, the significance of such stimuli on the osteogenic differentiation of human adipose stem cells (hASCs) remains unclear. In this study, we investigated the effect of long-term mechanical stimulation on the attachment, osteogenic differentiation and mechanical properties of hASCs. Tailor-made, pneumatic cell stretching devices were used to expose hASCs to cyclic equiaxial stretching in osteogenic medium. Cell attachment and focal adhesions were visualised using immunocytochemical vinculin staining on days 3 and 6, and the proliferation and alkaline phosphatase activity, as a sign of early osteogenic differentiation, were analysed on days 0, 6 and 10. Furthermore, the mechanical properties of hASCs, in terms of apparent Young’s modulus and normalised contractility, were obtained using a combination of atomic force microscopy based indentation and computational approaches. Our results indicated that cyclic equiaxial stretching delayed proliferation and promoted osteogenic differentiation of hASCs. Stretching also reduced cell size and intensified focal adhesions and actin cytoskeleton. Moreover, cell stiffening was observed during osteogenic differentiation and especially under mechanical stimulation. These results suggest that cyclic equiaxial stretching modifies cell morphology, focal adhesion formation and mechanical properties of hASCs. This could be exploited to enhance osteogenic differentiation.

Effects of Hydroxyapatite and Hypoxia on Chondrogenesis and Hypertrophy in 3D Bioprinted ADMSC Laden Constructs.

Hydrogel-based cartilage tissue engineering strategies require the induction and long-term maintenance of adipose derived mesenchymal stem cells (ADMSC) into a stable chondrogenic phenotype. However, ADMSC exhibit the tendency to undergo hypertrophic differentiation, rather than forming permanent hyaline cartilage phenotype changes. This may hinder their implementation in articular cartilage regeneration, but may allow the possibility for bone and bone to soft tissue interface repair. In this study, we examined the effects of hydroxyapatite (HAp) on the chondrogenesis and hypertrophy of ADMSC within bioprinted hyaluronic acid (HA)-based hydrogels. We found that a small amount of HAp (∼10% of polymer concentration) promoted both chondrogenic and hypertrophic differentiation of ADMSC. Increased HAp contents promoted hypertrophic conversion and early osteogenic differentiation of encapsulated ADMSC. Subsequently, ADMSC-laden, stratified constructs with nonmineralized and mineralized layers (i.e., HA based and HA-HAp based) were 3D bioprinted. The constructs were conditioned in chondrogenic medium in either a normoxic or hypoxic environment for 8 weeks to assess the effects of oxygen tension on ADMSC differentiation and interface integration. We further implanted the bioprinted constructs subcutaneously into nude mice for 4 weeks. It was found that hypoxia partially inhibited hypertrophic differentiation by significantly down-regulating the expression of COL10A1, ALP, and MMP13. In addition, hypoxia also suppressed spontaneous calcification of ADMSC and promoted interface integration. This study demonstrates that both HAp content and hypoxia are important to mediate chondrogenesis, hypertrophy, and endochondral ossification of ADMSC. An optimized recipe and condition will allow for 3D bioprinting of multizonal grafts with integrated hard tissue and soft tissue interfaces for the treatment of complex orthopedic defects.

Fabrication of human hair keratin/jellyfish collagen/eggshell-derived hydroxyapatite osteoinductive biocomposite scaffolds for bone tissue engineering: From waste to regenerative medicine products

In the present study, we aimed at fabricating an osteoinductive biocomposite scaffold using keratin obtained from human hair, jellyfish collagen and eggshell-derived nano-sized spherical hydroxyapatite (nHA) for bone tissue engineering applications. Keratin, collagen and nHA were characterized with the modified Lowry method, free-sulfhydryl groups and hydroxyproline content analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), attenuated total reflectance-fourier transform infrared spectroscopy (ATR-FTIR) and thermal gravimetric analysis (TGA) which confirmed the success of the extraction and/or isolation processes. Human adipose mesenchymal stem cells (hAMSCs) were isolated and the cell surface markers were characterized via flow cytometry analysis in addition to multilineage differentiation capacity. The undifferentiated hAMSCs were highly positive for CD29, CD44, CD73, CD90 and CD105, but were not seen to express hematopoietic cell surface markers such as CD14, CD34 and CD45. The cells were successfully directed towards osteogenic, chondrogenic and adipogenic lineages in vitro. The microarchitecture of the scaffolds and cell attachment were evaluated using scanning electron microscopy (SEM). The cell viability on the scaffolds was assessed by the MTT assay which revealed no evidence of cytotoxicity. The osteogenic differentiation of hAMSCs on the scaffolds was determined histologically using alizarin red S, osteopontin and osteonectin stainings. Early osteogenic differentiation markers of hAMSCs were significantly expressed on the collagen-keratin-nHA scaffolds. In conclusion, it is believed that collagen-keratin-nHA osteoinductive biocomposite scaffolds have the potential of being used in bone tissue engineering.

Influences of vibration on rapid osteogenic response of osteoblasts

This study investigated the rapid response of osteoblasts, which were derived from low-magnitude high-frequency vibration (LMHFV). Refractory period-derived memory response was also observed. MC3T3-E1 cells were incubated and received LMHFV stimulation (0.49 g, 40 Hz) for 30 min. After application of LMHFV, mRNA levels of earlier osteogenic differentiation markers Runt-related transcription factor 2 (Runx2), collagen typeⅠ(Col-Ⅰ), and alkaline phosphatase (ALP) were immediately detected by real-time fluorescence quantitative polymerase chain reaction in the absence or presence of antioxidant. Simultaneously, concentrations of mitochondrial reactive oxygen species (ROS) and average mitochondrial length were also measured. Osteoblasts in the vibration group showed decreased gene expressions of Runx2, Col-Ⅰ, and ALP (P<0.01) and increased levels of mitochondrial ROS (P<0.01) and shortened mitochondria (P<0.01), whereas antioxidant treatment resulted in recovery from changes in the above indicators (P<0.01). LMHFV can downregulate mRNA levels of early osteogenic differentiation markers, promote ROS generation, and mitochondrial fission. .

In vitro effects of hyaluronic acid on human periodontal ligament cells

BackgroundHyaluronic acid (HA) has been reported to have a positive effect on periodontal wound healing following nonsurgical and surgical therapy. However, to date, a few basic in vitro studies have been reported to investigating the potential of HA on human periodontal ligament (PDL) cell regeneration. Therefore, the aim of this study was to investigate the effect of HA on PDL cell compatibility, proliferation, and differentiation in vitro.MethodsEither non-cross-linked (HA_ncl) or cross-linked (HA_cl) HA was investigated. Human PDL cells were seeded in 7 conditions as follows (1) Control tissue culture plastic (TCP) (2) dilution of HA_ncl (1:100), (3) dilution of HA_ncl (1:10), 4) HA_ncl directly coated onto TCP, (5) dilution of HA_cl (1:100), 6) dilution of HA_cl (1:10) and (7) HA_cl directly coated onto TCP. Samples were then investigated for cell viability using a live/dead assay, an inflammatory reaction using real-time PCR and ELISA for MMP2, IL-1 and cell proliferation via an MTS assay. Furthermore, the osteogenic potential of PDL cells was assessed by alkaline phosphatase(ALP) activity, collagen1(COL1) and osteocalcin(OCN) immunostaining, alizarin red staining, and real-time PCR for genes encoding Runx2, COL1, ALP, and OCN.ResultsBoth HA_ncl and HA_cl showed high PDL cell viability (greater than 90%) irrespective of the culturing conditions. Furthermore, no significant difference in both mRNA and protein levels of proinflammatory cytokines, including MMP2 and IL-1 expression was observed. Both diluted HA_ncl and HA_cl significantly increased cell numbers compared to the controlled TCP samples at 3 and 5 days. HA_ncl and HA_cl in standard cell growth media significantly decreased ALP staining, COL1 immunostaining and down-regulated early osteogenic differentiation, including Runx2, COL1, and OCN mRNA levels when compared to control samples. When osteogenic differentiation medium (ODM) was added, interestingly, the expression of early osteogenic markers increased by demonstrating higher levels of COL1 and ALP expression; especially in HA 1:10 diluted condition. Late stage osteogenic markers remained inhibited.ConclusionsBoth non-cross-linked and cross-linked HA maintained high PDL cell viability, increased proliferation, and early osteogenic differentiation. However, HA was consistently associated with a significant decrease in late osteogenic differentiation of primary human PDL cells. Future in vitro and animal research is necessary to further characterize the effect of HA on periodontal regeneration.

Tetracycline-incorporated polymer nanofibers as a potential dental implant surface modifier.

This study investigated the antimicrobial and osteogenic properties of titanium (Ti) disks superficially modified with tetracycline (TCH)-incorporated polymer nanofibers. The experiments were carried out in two phases. The first phase dealt with the synthesis and characterization (i.e., morphology, mechanical strength, drug release, antimicrobial activity, and cytocompatibility) of TCH-incorporated fibers. The second phase was dedicated to evaluating both the antimicrobial and murine-derived osteoprecursor cell (MC3T3-E1) response of Ti-modified with TCH-incorporated fibers. TCH was successfully incorporated into the submicron-sized and cytocompatible fibers. All TCH-incorporated mats presented significant antimicrobial activity against periodontal pathogens. The antimicrobial potential of the TCH-incorporated fibers-modified Ti was influenced by both the TCH concentration and bacteria tested. At days 5 and 7, a significant increase in MC3T3-E1 cell number was observed for TCH-incorporated nanofibers-modified Ti disks when compared to that of TCH-free nanofibers-modified Ti-disks and bare Ti. A significant increase in alkaline phosphatase (ALP) levels on the Ti disks modified with TCH-incorporated nanofiber on days 7 and 14 was seen, suggesting that the proposed surface promotes early osteogenic differentiation. Collectively, the data suggest that TCH-incorporated nanofibers could function as an antimicrobial surface modifier and osteogenic inducer for Ti dental implants. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2085-2092, 2017.

Highly Concentrated Alginate-Gellan Gum Composites for 3D Plotting of Complex Tissue Engineering Scaffolds.

In tissue engineering, additive manufacturing (AM) technologies have brought considerable progress as they allow the fabrication of three-dimensional (3D) structures with defined architecture. 3D plotting is a versatile, extrusion-based AM technology suitable for processing a wide range of biomaterials including hydrogels. In this study, composites of highly concentrated alginate and gellan gum were prepared in order to combine the excellent printing properties of alginate with the favorable gelling characteristics of gellan gum. Mixtures of 16.7 wt % alginate and 2 or 3 wt % gellan gum were found applicable for 3D plotting. Characterization of the resulting composite scaffolds revealed an increased stiffness in the wet state (15%–20% higher Young’s modulus) and significantly lower volume swelling in cell culture medium compared to pure alginate scaffolds (~10% vs. ~23%). Cytocompatibility experiments with human mesenchymal stem cells (hMSC) revealed that cell attachment was improved—the seeding efficiency was ~2.5–3.5 times higher on the composites than on pure alginate. Additionally, the composites were shown to support hMSC proliferation and early osteogenic differentiation. In conclusion, print fidelity of highly concentrated alginate-gellan gum composites was comparable to those of pure alginate; after plotting and crosslinking, the scaffolds possessed improved qualities regarding shape fidelity, mechanical strength, and initial cell attachment making them attractive for tissue engineering applications.