Preparation,properties and in vitro cellular response of multi-walled carbon nanotubes/bioactive glass/poly(etheretherketone) biocomposite for bone tissue engineering

Desired bone repair biomaterial must have good biocompatibility and suitable mechanical properties that are equivalent to those of human bones. In this study, multi-walled carbon nanotubes (MWCNTS) was designed to incorporate into bioactive glass/poly(etheretherketone) to fabricate a composite of multi-walled carbon nanotubes/bioactive glass/poly(etheretherketone) (MWCNTS/BG/PEEK) through a compounding and injection-molding process. The microstructures, mechanical properties, thermal stability and bioactivity of the ternary biocomposite, as well as preliminary cell responses of MC3T3-E1 osteoblast cells to this biomaterial, were investigated. The mechanical performance of ternary MWCNTS/BG/PEEK composite was vastly superior to binary BG/PEEK composite. More importantly, cell culture tests showed that cell adhesion, viability and differentiation of MC3T3-E1 cells were significantly promoted on the MWCNTS/BG/PEEK composite. Moreover, it was found that MWCNTS in composite further promoted cell metabolic vitality and osteogenic differentiation of osteoblast cells. Hence, this MWCNTS/BG/PEEK biomaterial may be used as a promising bone graft scaffold in dental and orthopedic applications.     

Effect of the micro/nanostructured topography of polyetheretherketone on the behavior of MC3T3-E1 preosteoblasts

Polyetheretherketone (PEEK) is of interest because of its excellent mechanical properties. However, the bioinert nature of PEEK limits its use in clinical applications. In this study, a series of micro/nanostructures combining nano-, submicron-, and microscale on PEEK were fabricated with 0.5, 1, 4, and 6 min of sulfonation time (S1, S2, S3, and S4). Compared to the flat surface on PEEK, the micro/nanostructure of different sizes all significantly promote cell adhesion, proliferation, and osteogenic differentiation of MC3T3-E1 cells. It is shown that micro/nano-porous structures with smaller size and lower roughness of S1 enabled faster cell propagation. The results of alkaline phosphatase staining, alizarin red staining, and quantitative real-time PCR reveal that the osteogenic activity of MC3T3-E1 cells gradually decreases with the increasement of pore size, indicating that the micro/nanostructured topography of S1 generated substantially increased matrix mineralization and bone-like nodule formation, compared to the 3D network surface. This work provides an effective strategy for designing biomaterials with potential clinical applications.     

Enhanced osteogenic activities of polyetheretherketone surface modified by poly(sodium p-styrene sulfonate) via ultraviolet-induced polymerization

Polyetheretherketone (PEEK) is a promising bone and dental tissue engineering material with excellent mechanical properties and biocompatibility, but its biological inertness deficiency limits its clinical applications. In this study, poly(sodium p-styrene sulfonate) (pNaSS) was grafted onto PEEK surface by ultraviolet (UV) induced polymerization to enhance its osteogenic activities. Attenuated total reflectance Fourier transformed Infrared (ATR-FTIR) spectroscopy, scanning electron microscopy (SEM), and contact angle (CA) analysis were carried out to prove the success of grafting polymerization. Toluidine blue O (TBO) colorimetric assay was utilized to quantify the graft amounts of pNaSS. Results showed that the amounts of grafted pNaSS on the PEEK surface could be well-controlled from 0.59 ± 0.07 mmol/cm² to 5.08 ± 0.20 mmol/cm², through adjusting the UV irradiation time and monomer concentration. The hydrophilicity of PEEK surface was increased and the in vitro mineralization ability was promoted with the introduction of pNaSS. Besides, this surface modification method did not influence the intrinsic mechanical properties of PEEK. in vitro biological studies revealed that cell adhesion, proliferation, and osteogenic differentiation of MC3T3-E1 cells were enhanced with the increase of graft amounts.     

The Osteogenesis Effect and Underlying Mechanisms of Local Delivery of gAPN in Extraction Sockets of Beagle Dogs

A plastic and biodegradable bone substitute consists of poly (l-lactic-co-glycolic) acid and 30 wt % β-tricalcium phosphate has been previously fabricated, but its osteogenic capability required further improvement. We investigated the use of globular adiponectin (gAPN) as an anabolic agent for tissue-engineered bone using this scaffold. A qualitative analysis of the bone regeneration process was carried out using μCT and histological analysis 12 weeks after implantation. CBCT (Cone Beam Computed Tomography) superimposition was used to characterise the effect of the different treatments on bone formation. In this study, we also explored adiponectin’s (APN) influence on primary cultured human jaw bone marrow mesenchymal stem cells gene expressions involved in the osteogenesis. We found that composite scaffolds loaded with gAPN or bone morphogenetic protein 2 (BMP2) exhibited significantly increased bone formation and mineralisation following 12 weeks in the extraction sockets of beagle dogs, as well as enhanced expression of osteogenic markers. In vitro investigation revealed that APN also promoted osteoblast differentiation of primary cultured human jaw bone marrow mesenchymal stem cells (h-JBMMSCs), accompanied by increased activity of alkaline phosphatase, greater mineralisation, and production of the osteoblast-differentiated genes osteocalcin, bone sialoprotein and collagen type I, which was reversed by APPL1 siRNA. Therefore, the composite scaffold loaded with APN exhibited superior activity for guided bone regeneration compared with blank control or Bio-Oss^® (a commercially available product). The composite scaffold with APN has significant potential for clinical applications in bone tissue engineering.     

Superior Osteo-Inductive and Osteo-Conductive Properties of Trabecular Titanium vs. PEEK Scaffolds on Human Mesenchymal Stem Cells: A Proof of Concept for the Use of Fusion Cages

Fusion cages composed of titanium and its alloys are emerging as valuable alternative to standard polyetheretherketone (PEEK) ones routinely used in cervical and lumbar spine surgery. Aim of this study was to evaluate osteo-inductive and osteo-conductive ability of an innovative trabecular titanium (T-Ti) scaffold on human mesenchymal stem cells (hMSCs), in both absence and presence of biochemical osteogenic stimuli. Same abilities were assessed on PEEK and standard 2D plastic surface, the latter meant as gold-standard for in vitro differentiation studies. hMSCs adhered and colonized both T-Ti and PEEK scaffolds. In absence of osteogenic factors, T-Ti triggered osteogenic induction of MSCs, as demonstrated by alkaline phosphatase activity and calcium deposition increments, while PEEK and standard 2D did not. Addition of osteogenic stimuli reinforced osteogenic differentiation of hMSCs cultured on T-Ti in a significantly higher manner with respect to standard 2D plastic culture surfaces, whereas PEEK almost completely abolished the process. T-Ti driven differentiation towards osteoblasts was confirmed by gene and marker expression analyses, even in absence of osteogenic stimuli. These results clearly indicate superior in vitro osteo-inductive and osteo-conductive capacity of T-Ti compared to PEEK, and make ground for further studies supporting the use of T-Ti cages to improve bone fusion.     

Exosome: A Novel Approach to Stimulate Bone Regeneration through Regulation of Osteogenesis and Angiogenesis

The clinical need for effective bone regeneration therapy remains in huge demands. However, the current “gold standard” treatments of autologous and allogeneic bone grafts may result in various complications. Furthermore, safety considerations of biomaterials and cell-based treatment require further clarification. Therefore, developing new therapies with stronger osteogenic potential and a lower incidence of complications is worthwhile. Recently, exosomes, small vesicles of endocytic origin, have attracted attention in bone regeneration field. The vesicles travel between cells and deliver functional cargoes, such as proteins and RNAs, thereby regulating targeted cells differentiation, commitment, function, and proliferation. Much evidence has demonstrated the important roles of exosomes in osteogenesis both in vitro and in vivo. In this review, we summarize the properties, origins and biogenesis of exosomes, and the recent reports using exosomes to regulate osteogenesis and promote bone regeneration.     

Tranexamic Acid Promotes Murine Bone Marrow-Derived Osteoblast Proliferation and Inhibits Osteoclast Formation In Vitro

Despite modern surgical trauma care, bleeding contributes to one-third of trauma-related death. A significant improvement was obtained through the introduction of tranexamic acid (TXA), which today is widely used in emergency and elective orthopedic surgery to control bleeding. However, concerns remain regarding potential adverse effects on bone turnover and regeneration. Therefore, we employed standardized cell culture systems including primary osteoblasts, osteoclasts, and macrophages to evaluate potential effects of TXA on murine bone cells. While osteoblasts derived from calvarial digestion were not affected, TXA increased cell proliferation and matrix mineralization in bone marrow-derived osteoblasts. Short-term TXA treatment (6 h) failed to alter the expression of osteoblast markers; however, long-term TXA stimulation (10 days) was associated with the increased expression of genes involved in osteoblast differentiation and extracellular matrix synthesis. Similarly, whereas short-term TXA treatment did not affect gene expression in terminally differentiated osteoclasts, long-term TXA stimulation resulted in the potent inhibition of osteoclastogenesis. Finally, in bone marrow-derived macrophages activated with LPS, simultaneous TXA treatment led to a reduced expression of inflammatory cytokines and chemokines. Collectively, our study demonstrates a differential action of TXA on bone cells including osteoanabolic, anti-resorptive, and anti-inflammatory effects in vitro which suggests novel treatment applications.     

Herbally derived polymeric nanofibrous scaffolds for bone tissue regeneration

Hydroxyapatite (HA), the bone mineral and Cissus quadrangularis (CQ), a medicinal plant with osteogenic activity, are attaining increasing interest as a potential therapeutic agent for enhanced bone tissue regeneration. In the present study a synergistic effect of these two agents were analyzed by fabricating PCL‐CQ‐HA nanofibrous scaffolds by electrospinning and compared with PCL‐CQ and PCL (control) nanofibrous scaffolds. Morphology, composition, hydrophilicity, and mechanical properties of the electrospun PCL, PCL‐CQ, PCL‐CQ‐HA nanofibrous scaffolds were examined by Field emission scanning electron microscopy (FESEM), Fourier transform infrared spectroscopy (FTIR), Contact angle and Tensile tests, respectively. The response of human foetal osteoblast cells on these scaffolds were evaluated using MTS assay, alkaline phosphatase activity, alizarin red staining, and osteocalcin expression for bone tissue regeneration. While the observed cellular response to both groups of scaffolds was better than for the control PCL scaffold, the PCL‐CQ‐HA nanofibrous scaffolds provided the most favorable substrate for cell proliferation and mineralization. The results showed that PCL‐CQ‐HA nanofibrous scaffolds had appropriate surface roughness for the osteoblast adhesion, proliferation, and mineralization comparing with other scaffolds. The observed investigation of physicochemical and biological properties suggests that the CQ‐HA loaded PCL nanofibrous scaffolds serve as a potential biocomposite material for bone tissue engineering. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 39835.     

Biological Characteristics and Osteogenic Differentiation of Ovine Bone Marrow Derived Mesenchymal Stem Cells Stimulated with FGF-2 and BMP-2

Cell-based therapies using mesenchymal stem cells (MSCs) are a promising tool in bone tissue engineering. Bone regeneration with MSCs involves a series of molecular processes leading to the activation of the osteoinductive cascade supported by bioactive factors, including fibroblast growth factor-2 (FGF-2) and bone morphogenetic protein-2 (BMP-2). In this study, we examined the biological characteristics and osteogenic differentiation potential of sheep bone marrow MSCs (BM-MSCs) treated with 20 ng/mL of FGF-2 and 100 ng/mL BMP-2 in vitro. The biological properties of osteogenic-induced BM-MSCs were investigated by assessing their morphology, proliferation, phenotype, and cytokine secretory profile. The osteogenic differentiation was characterized by Alizarin Red S staining, immunofluorescent staining of osteocalcin and collagen type I, and expression levels of genetic markers of osteogenesis. The results demonstrated that BM-MSCs treated with FGF-2 and BMP-2 maintained their primary MSC properties and improved their osteogenic differentiation capacity, as confirmed by increased expression of osteocalcin and collagen type I and upregulation of osteogenic-related gene markers BMP-2, Runx2, osterix, collagen type I, osteocalcin, and osteopontin. Furthermore, sheep BM-MSCs produced a variety of bioactive factors involved in osteogenesis, and supplementation of the culture medium with FGF-2 and BMP-2 affected the secretome profile of the cells. The results suggest that sheep osteogenic-induced BM-MSCs may be used as a cellular therapy to study bone repair in the preclinical large animal model.     

Fabrication and biological assessment of halloysite-doped micro/nano structures on titanium surface

Titanium (Ti) is widely used as a biomaterial for dental implants, but its insufficient angiogenic and osteogenic properties prolong the restoration period. In this study, halloysite nanotubes (HNTs) were embedded on the surface of Ti via micro arc oxidation (MAO) treatment to enhance its early osseointegration. The surface physiochemical properties were examined, and the results confirmed that HNTs were successfully incorporated into the MAO coating. It was also discovered that the surface wettability and negative charge of the corresponding samples increased. In cytocompatibility tests involving human umbilical vein endothelial cells (HUVECs) and MC3T3-E1 cells, the HNT group exhibited no cytotoxicity compared with the MAO group and the best performance for cell adhesion and spreading among the three groups (Ti, MAO and HNT). Regarding angiogenesis, the HNT group outperformed the Ti and MAO groups in cell migration, tube formation, and angiogenic gene expression of HUVECs. Furthermore, with regard to osteogenesis, the HNT group exhibited the highest levels of alkaline phosphatase activity, collagen secretion, mineralized calcium nodules, and osteogenic gene expression of MC3T3-E1 cells among the three groups with significant differences. These findings indicated that the HNT specimens could significantly promote angiogenesis as well as osteogenesis at both the cellular and molecular levels.     

89Sr-doped hydroxyapatite nanoparticles as a potential therapeutic agent for bone tumors

Hydroxyapatite (HA) nanoparticles have been studied due to their high biocompatibility, similarity with bone tissue, and their capacity for bone regeneration since these nanoparticles can easily adhere on osteosarcoma and osteoblast cells, promoting osteoblast growth and osteosarcoma cell uptake. These materials may still accumulate spontaneously and selectively in regions of bone tumors through the enhanced permeability and retention effect. HA also allows the incorporation of strontium in your network. Strontium as a biochemical analog of calcium can maintain the osteogenesis characteristics of HA allowing the production of the radioactive isotopes strontium-89 and phosphorus-32 through neutron irradiation. These radioisotopes are beta emitters that enable the treatment of bone tumors, while the affected region is regenerated. In this work, we investigated the synthesis of strontium-doped HA nanorods through the hydrothermal coprecipitation method as a potential therapeutic agent for bone tumors. All materials were successfully obtained and demonstrated high cell viability, maintaining the osteogenic capacity, making these materials promising agents for the specific treatment of bone tumors. The results indicate that the Sr provides an increase in therapeutic potential due to its beta emission.     

3D Printed SiOC(N) Ceramic Scaffolds for Bone Tissue Regeneration: Improved Osteogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells

Bone tissue engineering has developed significantly in recent years as there has been increasing demand for bone substitutes due to trauma, cancer, arthritis, and infections. The scaffolds for bone regeneration need to be mechanically stable and have a 3D architecture with interconnected pores. With the advances in additive manufacturing technology, these requirements can be fulfilled by 3D printing scaffolds with controlled geometry and porosity using a low-cost multistep process. The scaffolds, however, must also be bioactive to promote the environment for the cells to regenerate into bone tissue. To determine if a low-cost 3D printing method for bespoke SiOC(N) porous structures can regenerate bone, these structures were tested for osteointegration potential by using human mesenchymal stem cells (hMSCs). This includes checking the general biocompatibilities under the osteogenic differentiation environment (cell proliferation and metabolism). Moreover, cell morphology was observed by confocal microscopy, and gene expressions on typical osteogenic markers at different stages for bone formation were determined by real-time PCR. The results of the study showed the pore size of the scaffolds had a significant impact on differentiation. A certain range of pore size could stimulate osteogenic differentiation, thus promoting bone regrowth and regeneration.     

Graphene-oxide-modified β-tricalcium phosphate bioceramics stimulate in vitro and in vivo osteogenesis

Graphene oxide (GO) has attracted much interest for applications in bone tissue engineering; however, until now, the interaction between GO and stem cells, and the in vivo bone-forming ability of GO have not been explored. The aim of this study was to produce GO-modified β-tricalcium phosphate (β-TCP-GRA) bioceramics and then explore the material’s osteogenic capacity in vitro and in vivo, as well as unravel some of the molecular mechanisms behind this. β-TCP-GRA disks and scaffolds were successfully prepared by a simple GO/water suspension soaking method in combination with heat treatment. These scaffolds were found to significantly enhance the proliferation, alkaline phosphatase activity, and osteogenic gene expression of human bone marrow stromal cells (hBMSCs), when compared with β-TCP without GO modification (controls). Activation of the Wnt/β-catenin signaling pathway in hBMSCs appears to be the mechanism behind this osteogenic induction by β-TCP-GRA. β-TCP-GRA scaffolds led to an increased rate of in vivo new bone formation compared to β-TCP controls, indicative of the stimulatory effect of GO on in vivo osteogenesis, making GO modification of β-TCP a very promising method for applications in bone tissue engineering, in particular for the regeneration of large bone defects.     

Vinculin focal adhesion of osteoblast‐like cells on PEEK coated with ultra‐thin polymer nano films

PEEK is the polymer of choice to replace metal encapsulants and other parts in active medical implants fixated into bone. The current challenge is to improve its biocompatibility with bone tissue to ultimately achieve osseointegration. PEEK sheets surfaces coated with plasma deposited nano thin polymer films using CH₄, (CH₄ + O₂) and (CH₄ + N₂) gases. PEEK samples plasma treated with nonpolymerizing gases (O₂) were also used for comparison. The adhesion performance of osteoblast like cells on the plasma‐treated PEEK surfaces and the presence of Vinculin in these cells were evaluated after long culturing period (12 days). X‐ray photoelectron spectroscopy and Auger spectroscopy were used to provide surface molecular information, surface hardness and molecular density. All plasma‐treated surfaces retained functionality after the sterilization process. PEEK surfaces with high number of oxygen functional groups and particularly oxygen rich thin polymer coating (plasma deposition using CH₄+O₂ gas mixture) resulted in strong cellular adhesion strength and large Vinculin amount. Further, osteoblast‐like cells responded better to surfaces with lower molecular density acting like another signal for cell adhesion. The osteoblast‐like cells response was weaker for surfaces with both thin films with nitrogen functional groups and nonfunctional (nonpolar) films. Furthermore, thin films rich in nitrogen functional groups repelled the cells, showed abnormal cells shape, smaller Vinculin amount and induced thicker cellular clusters with poor spread. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015 , 132, 42181.     

Characterization of Osteogenesis and Chondrogenesis of Human Decellularized Allogeneic Bone with Mesenchymal Stem Cells Derived from Bone Marrow,Adipose Tissue,and Wharton’s Jelly

Allogeneic bone grafts are a promising material for bone implantation due to reduced operative trauma, reduced blood loss, and no donor-site morbidity. Although human decellularized allogeneic bone (hDCB) can be used to fill bone defects, the research of revitalizing hDCB blocks with human mesenchymal stem cells (hMSCs) for osteochondral regeneration is missing. The hMSCs derived from bone marrow, adipose tissue, and Wharton’s jelly (BMMSCs, ADMSCs, and UMSCs, respectively) are potential candidates for bone regeneration. This study characterized the potential of hDCB as a scaffold for osteogenesis and chondrogenesis of BMMSCs, ADMSCs, and UMSCs. The pore sizes and mechanical strength of hDCB were characterized. Cell survival and adhesion of hMSCs were investigated using MTT assay and F-actin staining. Alizarin Red S and Safranin O staining were conducted to demonstrate calcium deposition and proteoglycan production of hMSCs after osteogenic and chondrogenic differentiation, respectively. A RT-qPCR was performed to analyze the expression levels of osteogenic and chondrogenic markers in hMSCs. Results indicated that BMMSCs and ADMSCs exhibited higher osteogenic potential than UMSCs. Furthermore, ADMSCs and UMSCs had higher chondrogenic potential than BMMSCs. This study demonstrated that chondrogenic ADMSCs- or UMSCs-seeded hDCB might be potential osteochondral constructs for osteochondral regeneration.     

Systemic Administration of G-CSF Accelerates Bone Regeneration and Modulates Mobilization of Progenitor Cells in a Rat Model of Distraction Osteogenesis

Granulocyte colony-stimulating factor (G-CSF) was shown to promote bone regeneration and mobilization of vascular and osteogenic progenitor cells. In this study, we investigated the effects of a systemic low dose of G-CSF on both bone consolidation and mobilization of hematopoietic stem/progenitor cells (HSPCs), endothelial progenitor cells (EPCs) and mesenchymal stromal cells (MSCs) in a rat model of distraction osteogenesis (DO). Neovascularization and mineralization were longitudinally monitored using positron emission tomography and planar scintigraphy. Histological analysis was performed and the number of circulating HSPCs, EPCs and MSCs was studied by flow cytometry. Contrary to control group, in the early phase of consolidation, a bony bridge with lower osteoclast activity and a trend of an increase in osteoblast activity were observed in the distracted callus in the G-CSF group, whereas, at the late phase of consolidation, a significantly lower neovascularization was observed. While no difference was observed in the number of circulating EPCs between control and G-CSF groups, the number of MSCs was significantly lower at the end of the latency phase and that of HSPCs was significantly higher 4 days after the bone lengthening. Our results indicate that G-CSF accelerates bone regeneration and modulates mobilization of progenitor cells during DO.     

Hesperidin Promotes Osteogenesis and Modulates Collagen Matrix Organization and Mineralization In Vitro and In Vivo

This study evaluated the direct effect of a phytochemical, hesperidin, on pre-osteoblast cell function as well as osteogenesis and collagen matrix quality, as there is little known about hesperidin’s influence in mineralized tissue formation and regeneration. Hesperidin was added to a culture of MC3T3-E1 cells at various concentrations. Cell proliferation, viability, osteogenic gene expression and deposited collagen matrix analyses were performed. Treatment with hesperidin showed significant upregulation of osteogenic markers, particularly with lower doses. Mature and compact collagen fibrils in hesperidin-treated cultures were observed by picrosirius red staining (PSR), although a thinner matrix layer was present for the higher dose of hesperidin compared to osteogenic media alone. Fourier-transform infrared spectroscopy indicated a better mineral-to-matrix ratio and matrix distribution in cultures exposed to hesperidin and confirmed less collagen deposited with the 100-µM dose of hesperidin. In vivo, hesperidin combined with a suboptimal dose of bone morphogenetic protein 2 (BMP2) (dose unable to promote healing of a rat mandible critical-sized bone defect) in a collagenous scaffold promoted a well-controlled (not ectopic) pattern of bone formation as compared to a large dose of BMP2 (previously defined as optimal in healing the critical-sized defect, although of ectopic nature). PSR staining of newly formed bone demonstrated that hesperidin can promote maturation of bone organic matrix. Our findings show, for the first time, that hesperidin has a modulatory role in mineralized tissue formation via not only osteoblast cell differentiation but also matrix organization and matrix-to-mineral ratio and could be a potential adjunct in regenerative bone therapies.