The structure of materials is known to play an important role in material function. Nowadays, flowerlike structures have gained attention for studies not only in analytical chemistry, but also in biomaterial design. In this study, flowerlike structures were applied in bone regeneration in the form of calcium phosphate microflowers. The material was synthesized by a simple and environmentally friendly method. We characterized the structure and properties of the microflower using various methods. Cytotoxicity and osteogenesis-related gene regulations of the microflower were investigated in vitro. Cell uptake was observed by immunofluorescence. Rat calvarial critical-size defect models were successfully established to further confirm the enhanced bone regeneration ability of this material. We expect that this novel study will be of practical importance for the extended application of flowerlike materials and will provide new insights into the optimization of the morphology of calcium phosphate materials.Keywords: bone regeneration; calcium phosphate; microflowers; osteogenesis; rat calvarial defect model;

Tetrahedral DNA nanostructures (TDNs) are considered promising drug delivery carriers because they are able to permeate cellular membrane and are biocompatible and biodegradable. Furthermore, they can be modified by functional groups. To improve the drug-delivering ability of TDNs, we chose anticancer aptamer AS1411 to modify TDNs for tumor-targeted drug delivery. AS1411 can specifically bind to nucleolin, which is overexpressed on the cell membrane of tumor cells. Furthermore, AS1411 can inhibit NF-κB signaling and reduce the expression of bcl-2. In this study, we compared the intracellular localization of AS1411-modified TDNs (Apt-TDNs) with that of TDNs in different cells under hypoxic condition. Furthermore, we compared the effects of Apt-TDNs and TDNs on cell growth and cell cycle under hypoxic condition. A substantial amount of Apt-TDNs entered and accumulated in the nucleus of MCF-7 cells; however, the amount of Apt-TDNs that entered L929 cells was comparatively less. TDNs entered in much lower quantity in MCF-7 cells than Apt-TDNs. Moreover, there was little difference in the amount of TDNs that entered L929 cells and MCF-7 cells. Apt-TDNs can inhibit MCF-7 cell growth and promote L929 cell growth, while TDNs can promote both MCF-7 and L929 cell growth. Thus, the results indicate that Apt-TDNs are more effective tumor-targeted drug delivery vehicles than TDNs, with the ability to specifically inhibit tumor cell growth.Keywords: anticancer; AS1411; drug delivery; tetrahedral DNA nanostructures; tumor-targeted;

•The differentiation of bone marrow-derived EPCs can be regulated by substrate stiffness.•Venous lineages are obtained when EPCs are cultured on soft substrates.•Growth on Petri dishes strongly induces arterial phenotype of EPCs.•The Ras/Mek pathway plays a significant role in mediating the effects of substrate stiffness on the differentiation of EPCs.Cells sense and respond to the biophysical properties of their surrounding environment by interacting with the extracellular matrix (ECM). Therefore, the optimization of these cell–matrix interactions is critical in tissue engineering. The vascular system is adapted to specific functions in diverse tissues and organs. Appropriate arterial-venous differentiation is vital for the establishment of functional vasculature in angiogenesis. Here, we have developed a polydimethylsiloxane (PDMS)-based substrate capable of simulating the physiologically relevant stiffness of both venous (7 kPa) and arterial (128 kPa) tissues. This substrate was utilized to investigate the effects of changes in substrate stiffness on the differentiation of endothelial progenitor cells (EPCs). As EPCs derived from mouse bone marrow were cultured on substrates of increasing stiffness, the mRNA and protein levels of the specific arterial endothelial cell marker ephrinB2 were found to increase, while the expression of the venous marker EphB4 decreased. Further experiments were performed to identify the mechanotransduction pathway involved in this process. The results indicated that substrate stiffness regulates the arterial and venous differentiation of EPCs via the Ras/Mek pathway. This work shows that modification of substrate stiffness may represent a method for regulating arterial-venous differentiation for the fulfilment of diverse functions of the vasculature.Download high-res image (443KB)Download full-size image

Adipose-derived stem cells (ADSCs) are considered to be ideal stem cell sources for bone regeneration owing to their ability to differentiate into osteo-like cells. Therefore, they have attracted increasing attention in recent years. Tetrahedral DNA nanostructures (TDNs), a new type of DNA-based biomaterials, have shown great potential for biomedical applications. In the present work, we aimed to investigate the role played by TDNs in osteogenic differentiation and proliferation of ADSCs and tried to explore if the canonical Wnt signal pathway could be the vital biological mechanism driving these cellular responses. Upon exposure to TDNs, ADSCs proliferation and osteogenic differentiation were significantly enhanced, accompanied by the up-regulation of genes correlated with the Wnt/β-catenin pathway. In conclusion, our results indicate that TDNs are crucial regulators of the increase in osteogenic potential and ADSCs proliferation, and this noteworthy discovery could provide a promising novel approach toward ADSCs-based bone defect regeneration.Tetrahedral DNA nanostructures (TDNs) have been considered as a promising material in biomedical fields. Here, we provide an insight into the ADSCs cellular response to TDNs from the view of cell growth and osteogenic differentiation. We demonstrated that TDNs can promote the osteogenic differentiation and proliferation of ADSCs by means of improving osteogenic-specific genes and proteins expression, ALP activity and calcium deposit and activation of classical Wnt/β-catenin pathway was the main mechanism involved in these cellular responses to TDNs.Download high-res image (309KB)Download full-size image

Nowadays, DNA nanostructures are extensively researched for their biocompatibility, editable functionality, and structural stability. Tetrahedral DNA nanostructures (TDNs), widely known for their membrane permeability, are regarded as potential candidates for drug delivery. However, the stability and membrane permeability of TDNs call for further enhancement if in vivo usage is ascribed. To overcome the drawbacks of TDNs, ethylene imine (PEI, 25 kDa, branched)—a classic cationic polymer in the field of gene delivery—was applied. Via a facile one-pot synthesis method, a PEI/TDNs complex was formed. Subsequently, a DNase protection assay, a cytotoxicity assay, endocytosis-related experiments, and lysosome staining were performed to examine the potential of PEI/TDNs as a delivery vehicle. The combination of PEI and TDNs not only overcame the drawbacks of each substance but also retained their individual merits. Traditionally, drug-delivery vehicles that enable enhanced cell entry and lysosome escape are often compromised by their toxicity and poor multifunctionality. We believe this novel PEI/TDNs complex with enhanced systemic stability, biocompatibility, cell-entry ability, and lysosome-escape ability and unsurpassed editable functionality could be a powerful tool as a multi-functional delivery vehicle in targeted drug delivery, in vivo imaging, and other related fields.

•Carbon dots (CDs) were fabricated by the green hydrothermolysis of milk.•The CDs were complexed with doxorubicin (DOX) with a view to targeted drug delivery.•The CD-DOX complexes displayed pH-dependent DOX release behavior.•Compared to free DOX, CD-DOX complexes were significantly more deadly to cancer cells.•Increased efficacy was based on localized drug release in the nuclei of cancer cells.Chemotherapy is widely applied against various kinds of carcinoma. Generally, chemotherapeutic agents, such as Doxorubicin (DOX), Paclitaxel (PTX), 5-Fluorouracil (5-FU), Methotrexate (MTX), and Vinblastine (VLB) are combined with a view to maximizing their efficacy. Unfortunately, chemotherapeutics are indiscriminate and also kill normal healthy cells, resulting in serious side effects. This non-productive and destructive distribution of chemotherapeutics is regarded as one of the largest problems associated with chemotherapy. Recently, the application of carbon dots (CDs) in cancer therapy has attracted considerable attention due to their attractive properties, such as biocompatibility and low toxicity. We report herein on the fabrication of CD-DOX antitumor drug complexes, from the combination of CDs and DOX, with a view to providing a novel and efficient strategy for cancer treatment. CDs were synthesized by hydrothermal treatment of milk, a simple and environmentally friendly synthetic process. DOX was conjugated to the CDs through electrostatic interactions via the multiple surface CD functional groups. The CD-DOX complexes exhibited pH-dependent DOX release behavior. A cytotoxicity study demonstrated that the CDs were non-cytotoxic in the range of concentrations used. Compared to free DOX, the CD-DOX complexes were significantly more destructive to the adenoid cystic carcinoma cell line (ACC-2), but exhibited lower toxicity to a mouse fibroblast cell line (L929). Confocal microscopy and flow cytometry confirmed that CD-DOX complexes increased cancer therapy efficiency through the localization of a much higher quantity of drugs in the nuclei of tumor cells and induced a higher rate of apoptosis in ACC-2 cells, compared to DOX alone.Download high-res image (105KB)Download full-size image

Recently, much attention has been paid to DNA again due to the successful synthesis of DNA-based nanostructures that can enter cells via endocytosis and thus have great potential in biomedical fields. However, the impacts of DNA nanostructures on life activities of a living cell are unknown. Herein, the promotion effect of tetrahedral DNA nanostructure (TDN) on cell growth and the underlying molecular mechanisms are reported. Upon exposure to TDN, cell proliferation is significantly enhanced, accompanied by up-regulation of cyclin-dependent kinase like-1 gene, changes in cell cycle distribution, and up-regulation of the Wnt/β-catenin signaling-related proteins (β-catenin, Lef 1 and cyclin D). In contrast, single-stranded DNA (ssDNA) shows no such functions. Furthermore, TDN is able to reverse the inhibition effect of DKK1, a specific inhibitor for Wnt/β-catenin pathway. Hence, the Wnt/β-catenin pathway is the target for TDN to promote cell proliferation. The findings allow TDN to be a novel functional nanomaterial that has great potential in tissue repair and regeneration medicine.

Due to its evascular, aneural, and alymphatic conditions, articular cartilage shows extremely poor regenerative ability. Thus, directing chondrocyte toward a desired location and function by utilizing the mechanical cues of biomaterials is a promising approach for effective tissue regeneration. However, chondrocytes cultured on Petri dish will lose their typical phenotype which may lead to compromised results. Therefore, we fabricated polydimethylsiloxane (PDMS) materials with various stiffness as culture substrates. Cell morphology and focal adhesion of chondrocytes displayed significant changes. The cytoskeletal tension of the adherent cells observed by average myosin IIA fluorescent intensity increased as stiffness of the underlying substrates decreased, consistent with the alteration of chondrocyte phenotype in our study. Immunofluorescent images and q-PCR results revealed that chondrocyte cultured on soft substrates showed better chondrocyte functionalization by more type II collagen and aggrecan expression, related to the lowest mRNA level of Rac-1, RhoA, ROCK-1, and ROCK-2. Taken together, this work not only points out that matrix elasticity can regulate chondrocyte functionalization via RhoA/ROCK pathway, but also provides new prospect for biomechanical control of cell behavior in cell-based cartilage regeneration.Keywords: chondrocytes; cytoskeleton; elastic substrates; phenotype; RhoA/ROCK pathway

Self-assembled tetrahedral DNA nanostructures (TDNs) with precise sizes have been extensively applied in various fields owing to their exceptional mechanical rigidity, structural stability, and modification versatility. In addition, TDNs can be internalized by mammalian cells and remain mainly intact within the cytoplasm by escaping degradation by nucleases. Here, we studied the effects of TDNs on cell migration and the underlying molecular mechanisms. TDNs remarkably enhanced the migration of rat adipose-derived stem cells and down-regulated the long noncoding RNA (lncRNA) XLOC 010623 to activate the mRNA expression of Tiam1 and Rac1. Furthermore, TDNs highly up-regulated the mRNA and protein expression of RHOA, ROCK2, and VCL. These results indicate that TDNs suppressed the transcription of lncRNA XLOC 010623 and activated the TIAM1/RAC1 and RHOA/ROCK2 signaling pathways to promote cell migration. On the basis of these findings, TDNs show a high potential for application in tissue repair and regenerative medicine as a functional three-dimensional DNA nanomaterial.Keywords: ASCs; cell migration; mechanical rigidity; self-assembled; structural stability; TDNs

Monovalent DNA–gold nanoparticle (mDNA–AuNP) conjugates hold great promise for widespread applications, especially the construction of well-defined, molecule-like nanosystems. Previously reported methods all rely on the use of thiolated DNA to functionalize AuNPs, resulting in relatively low yields. Here, we report a facile method to rapidly prepare mDNA–AuNPs using a poly-adenine (polyA)-mediated approach. As polyA can selectively bind to AuNPs with high controllability of the surface density of DNA, we can use a DNA strand with a sufficiently long polyA to wrap around the surface of an individual AuNP, preventing further the adsorption of additional strands. Based on this observation, we obtained mDNA–AuNPs with a nearly quantitative yield of ~90% using 80 As, as confirmed by both gel electrophoresis and transmission electron microscope observation. The yields of mDNA–AuNPs were insensitive to the stoichiometric ratio between DNA and AuNPs, suggesting the click chemistry-like nature of this polyA-mediated reaction. mDNA–AuNPs exhibited rapid kinetics and high efficiency for sequence-specific hybridization. More importantly, we demonstrated that AuNPs of fixed valences could form well-defined heterogenous oligomeric nanostructures with precise, atom-like control.

As novel applied nanomaterials, both graphene oxide (GO) and its reduced form (rGO) have attracted global attention, because of their excellent properties. However, the lack of comprehensive understanding of their interactions with biomacromolecules highly limits their biomedical applications. This work aims to initiate a systematic study on the property changes of GO/rGO upon interaction with serum proteins and on how their degree of reduction and exposure concentration affect this interaction, as well as to analyze the possible biomedical impacts of the interaction. We found that the adsorption of proteins on GO/rGO occurred spontaneously and rapidly, leading to significant changes in size, zeta potential, and morphology. Compared to rGO, GO showed a higher ability in quenching intrinsic fluorescence of serum proteins in a concentration-dependent manner. The protein adsorption efficiency and the types of associated proteins varied, depending on the degree of reduction and concentration of graphene. Our findings indicate the importance of evaluating the potential protein adsorption before making use of GO/rGO in drug delivery, because the changed physicochemical properties after protein adsorption will have significant impacts on safety and effectiveness of these delivery systems. On the other hand, this interaction can also be used for the separation, purification, or delivery of certain proteins.Keywords: drug delivery; graphene oxide; nanomaterials; protein adsorption; reduced graphene oxide; safety;

In this study, water-soluble, one-step highly reduced and functionalized graphene oxide was prepared via a facile, environment-friendly method by using tea polyphenol (TP), which acted as both reducing agent and stabilizer. The product obtained, that is, tea polyphenol–reduced graphene oxide (TPG), was used as a reinforcing building block for the modification of a mechanically weak chitosan (CS), TPG/CS. The morphology and physicochemical and mechanical properties of the composite were examined by various characterizations. The tensile strength and elastic modulus of CS were greatly improved by TPG, as compared to the findings for GO incorporation. Additionally, to our knowledge, this study is an in-depth analysis of the osteoblast functions of CS/TPG, including aspects such as cell cytotoxicity, proliferation, and expression of ossification genes, alkaline phosphatase (ALP), and Runt-related transcription factor (Runx2), which showed advantages in favorably modulating cellular activity. It was concluded that TPG/CS showed a higher elastic modulus, better hydrophilicity, and excellent biocompatibility than the pristine chitosan for promoting the proliferation and differentiation of osteoblasts, as well as for accelerating the expression of ALP and Runx2 (as shown by reverse transcription polymerase chain reaction (RT-PCR)). These results may provide new prospects for the use of TPG in the modification of biomaterials and for broadening the application of TPG in biological fields.Keywords: chitosan; graphene oxide; mechanical properties; osteoblast; tea polyphenol

The management of chondral defects is a challenging topic of current interest for scientists and surgeons, which has a crucial impact on human cost. Even after several centuries after its first observation, this problem has still not found a satisfactory and definitive answer. Cartilage tissue engineering, which involves novel natural scaffolds, has emerged as a promising strategy for cartilage regeneration and repair. In this study, bio-plasticpoly-3-hydroxybutyrate-4-hydroxybutyrate (P34HB) film was first fabricated. The characteristics of P34HB film were tested using SEM and AFM. Cell morphologies on P34HB film were obtained using SEM and fluorescence microscopy after cell seeding. The tests of cell adhesion and proliferation on P34HB film were conducted using MTT and CCK-8 assays, respectively. Furthermore, full cartilage defects in rats were created and P34HB films were implanted to evaluate their healing effects within 8 weeks. It was found that P34HB film, as a biomaterial implant, possessed good in vitro properties for cell adhesion, migration, and proliferation. Importantly, in the in vivo experiment, P34HB film exhibited desirable healing outcomes. These results demonstrated that P34HB film was a good scaffold for cartilage tissue engineering for improving cell proliferation and adhesion.

Achieving long circulating delivery of nanoparticles (NPs) is important for efficient drug therapy, but it is difficult due largely to proteins adsorption (opsonization) or/and nonsufficient stability of NPs. In this present work, we aimed to address the above issues by constructing a phospholipid and BSA-based nanocomplex system, namely BSA–phospholipid NPs (BSA-PL-NPs). Combining sodium dodecyl sulfate-polyacrylamide gel electrophoresis, X-ray photoelectron spectroscopy and proteins adsorption property, we confirmed that some BSA molecules were fixed on the inner surface of BSA-PL-NPs via hydrophobic interactions and the others were located in the core area. This special configuration allowed BSA-PL-NPs to not only maintain the antiadsorption and low phagocytosis properties but also have the slow zero-order drug release and the enhanced nanostructure stability. Interestingly, we found that BSA-PL-NPs had no cytotoxicity to mouse L929 fibroblasts but could stimulate the cells’ growth instead. In conclusion, BSA-PL-NPs have a great potential to be developed as a long-circulation drug delivery system, and the ready availability, biocompatibility and nontoxicity of phospholipids and albumin give this system great promise for practical use.Keywords: drug delivery; long circulation; nanoparticles; proteins adsorption; stability; toxicity

Pericytes are essential to vascularization, but the purification and characterization of pericytes remain unclear. Smooth muscle actin alpha (α-SMA) is one maker of pericytes. The aim of this study is to purify the α-SMA positive cells from bone marrow and study the characteristics of these cells and the interaction between α-SMA positive cells and endothelial cells. The bone marrow stromal cells were harvested from α-SMA-GFP transgenic mice, and the α-SMA-GFP positive cells were sorted by FACS. The proliferative characteristics and multilineage differentiation ability of the α-SMA-GFP positive cells were tested. A 3-D culture model was then applied to test their vascularization by loading α-SMA-GFP positive cells and endothelial cells on collagen-fibronectin gel. Results demonstrated that bone marrow stromal cells are mostly α-SMA-GFP positive cells which are pluripotent, and these cells expressed α-SMA during differentiation. The α-SMA-GFP positive cells could stimulate the endothelial cells to form tube-like structures and subsequently robust vascular networks in 3-D culture. In conclusion, the bone marrow derived pluripotent cells are pericytes and can contribute to vascularization.

ObjectiveArticular cartilage is a highly specialized tissue which forms the surfaces in synovial joints. Full-thickness cartilage defects caused by trauma or microfracture surgery heal via the formation of fibrotic tissue characterized by a high content of collagen I (COL I) and subsequent poor mechanical properties. The goal of this study is to investigate the molecular mechanisms underlying fibrosis after joint injury.DesignRat knee joint models were used to mimic cartilage defects after acute injury. Immunohistochemistry was performed to detect proteins related to fibrosis. Human fetal chondrocytes and bone marrow stromal cells (BMSCs) were used to study the influence of the lipid lysophosphatidic acid (LPA) on COL I synthesis. Quantitative PCR, ELISA and immunohistochemistry were performed to evaluate the production of COL I. Chemical inhibitors were used to block LPA signaling both in vitro and in vivo.ResultsAfter full-thickness cartilage injury in rat knee joints, stromal cells migrating to the injury expressed high levels of the LPA-producing enzyme autotaxin (ATX); intact articular cartilage in rat and humans expressed negligible levels of ATX despite expressing the LPA receptors LPAR1 and LPAR2. LPA-induced increases in COL I production by chondrocytes and BMSCs were mediated by the MAP kinase and PI3 Kinase signaling pathways. Inhibition of the ATX/LPA axis significantly reduced COL I-enriched fibrocartilage synthesis in full-thickness cartilage defects in rats in favor of the collagen II-enriched normal state.ConclusionTaken together, these results identify an attractive target for intervention in reducing the progression of post-traumatic fibrosis and osteoarthritis.