Development of an Injectable Biphasic Hyaluronic Acid-Based Hydrogel With Stress Relaxation Properties for Cartilage Regeneration.

Biomimetic stress-relaxing hydrogels with reversible crosslinks attract significant attention for stem cell tissue regeneration compared with elastic hydrogels. However, stress-relaxing hyaluronic acid (HA)-based hydrogels fabricated using conventional technologies lack stability, biocompatibility, and mechanical tunability. Here, it is aimed to address these challenges by incorporating calcium or phosphate components into the HA backbone, which allows reversible crosslinking of HA with alginate to form interpenetrating networks, offering stability and mechanical tunability for mimicking cartilage. Diverse stress-relaxing hydrogels (τ1/2; SR50, 60-2000 s) are successfully prepared at ≈3 kPa stiffness with self-healing and shear-thinning abilities, favoring hydrogel injection. In vitro cell experiments with RNA sequencing analysis demonstrate that hydrogels tune chondrogenesis in a biphasic manner (hyaline or calcified) depending on the stress-relaxation properties and phosphate components. In vivo studies confirm the potential for biphasic chondrogenesis. These results indicate that the proposed stress-relaxing HA-based hydrogel with biphasic chondrogenesis (hyaline or calcified) is a promising material for cartilage regeneration.

Mechanism of Antidepressant Action of (2R,6R)-6-Hydroxynorketamine (HNK) and Its Compounds: Insights from Proteomic Analysis.

The effects of HNK, I5, and I6 on the expression of protein in hippocampus of depressed mice were studied by isobaric tags for relative and absolute quantitation (iTRAQ) to explore the mechanism of their antidepressant action. HNK, I5, and I6 were administered intragastric administration once a day in the morning for 7 days. The drug was subsequently discontinued for 7 days (without any treatment). On the 15th day, mice in each group were given the drug (1.0, 10.0, 30.0 mg/kg) intragastric stimulation and mouse hippocampal tissues were taken to perform iTRAQ to identify differentially expressed proteins, and bioinformatics was used to analyze the functional enrichment of the differentially expressed proteins. Compared with Ctr group, the number of differentially expressed proteins in HNK, I5, and I6 treatment groups was 158, 88, and 105, respectively. The three groups shared 29 differentially expressed proteins. In addition, compared with HNK group, the number of differentially expressed proteins in I5 and I6 groups was 201 and 203, respectively. A total of 47 and 56 differentially expressed proteins were co-expressed in I5 and I6 groups. Bioinformatics analysis showed that these differentially expressed proteins mainly had the functions of binding, biocatalysis, and transport, and mainly participated in cellular process, biological regulation process, biological metabolism process, and stress reaction process. GO and KEGG pathway analysis found that these differentially expressed proteins were involved long-term potentiation, G13 pathway, platelet activation pathway, and MAPK signaling pathway. HNK, I5, and I6 antidepressants are closely related to sudden stress sensitivity, stress resistance, neurotransmitter, and metabolic pathways. This study provides a scientific basis to further elucidate the mechanism and clinical application of HNK, I5, and I6 antidepressants.

Research Models of the Nanoparticle-Mediated Drug Delivery across the Blood-Brain Barrier.

Brain diseases and damages come in many forms such as neurodegenerative diseases, tumors, and stroke. Millions of people currently suffer from neurological diseases worldwide. While Challenges of current diagnosis and treatment for neurological diseases are the drug delivery to the central nervous system. The Blood-Brain Barrier (BBB) limits the drug from reaching the targeted site thus showing poor effects. Nanoparticles that have advantage of the assembly at the nanoscale of available biomaterials can provide a delivery platform with potential to raising brain levels of either imaging therapeutic drugs or imaging. Therefore, successful modeling of the BBB is another crucial factor for the development of nanodrugs. In this review, we analyze the in vitro and in vivo findings achieved in various models, and outlook future development of nanodrugs for the successful treatment of brain diseases and damages.

Therapeutic tissue regenerative nanohybrids self-assembled from bioactive inorganic core / chitosan shell nanounits.

Natural inorganic/organic nanohybrids are a fascinating model in biomaterials design due to their ultra-microstructure and extraordinary properties. Here, we report unique-structured nanohybrids through self-assembly of biomedical inorganic/organic nanounits, composed of bioactive inorganic nanoparticle core (hydroxyapatite, bioactive glass, or mesoporous silica) and chitosan shell - namely Chit@IOC. The inorganic core thin-shelled with chitosan could constitute as high as 90%, strikingly contrasted with the conventional composites. The Chit@IOC nanohybrids were highly resilient under cyclic load and resisted external stress almost an order of magnitude effectively than the conventional composites. The nanohybrids, with the nano-roughened surface topography, could accelerate the cellular responses through stimulated integrin-mediated focal adhesions. The nanohybrids were also able to load multiple therapeutic molecules in the core and shell compartment and then release sequentially, demonstrating controlled delivery systems. The nanohybrids compartmentally-loaded with therapeutic molecules (dexamethasone, fibroblast growth factor 2, and phenamil) were shown to stimulate the anti-inflammatory, pro-angiogenic and osteogenic events of relevant cells. When implanted in the in vivo calvarium defect model with 3D-printed scaffold forms, the therapeutic nanohybrids were proven to accelerate new bone formation. Overall, the nanohybrids self-assembled from Chit@IOC nanounits, with their unique properties (ultrahigh inorganic content, nano-topography, high resilience, multiple-therapeutics delivery, and cellular activation), can be considered as promising 3D tissue regenerative platforms.

Enhanced growth and myogenic differentiation of spheroid-derived C2C12 cells.

Among many factors of controlling stem cell differentiation, the key transcription factor upregulation via physical force is a good strategy on the lineage-specific differentiation of stem cells. The study aimed to compare growth and myogenic potentials between the parental cells (PCs) and the 1-day-old C2C12 spheroid-derived cells (SDCs) in two-dimensional (2D) and three-dimensional (3D) culture conditions through examination of the cell proliferation and the expression of myogenic genes. The data showed that 1-day-old spheroids had more intense expression of MyoD gene with respect to the PCs. The proliferation of the SDCs is significantly higher than the PCs in a time-dependent manner. The SDCs had also significantly higher myogenic potential than the PCs in 2D and 3D culture conditions. The results suggest that MyoD gene upregulation through cell-cell contacts is the good approach for preparation of seed cells in muscle tissue engineering.

Current Nanoparticle-Based Technologies for Osteoarthritis Therapy.

Osteoarthritis (OA) is a common chronic joint disease that is characterized by joint pain and stiffness, and limitation of motion and the major cause of disability, which reduces life quality of patients and brings a large economic burden to the family and society. Current clinical treatment is mostly limited to symptomatic treatment aimed at pain alleviation and functional improvement, rather than suppressing the progression of OA. Nanotechnology is a promising strategy for the treatment of OA. In this review, we summarize the current experimental progress that focuses on technologies such as liposomes, micelles, dendrimers, polymeric nanoparticles (PNPs), exosomes, and inorganic nanoparticles (NPs) for their potential treatment of OA.

3D culture technologies of cancer stem cells: promising ex vivo tumor models.

Cancer stem cells have been shown to be important in tumorigenesis processes, such as tumor growth, metastasis, and recurrence. As such, many three-dimensional models have been developed to establish an ex vivo microenvironment that cancer stem cells experience under in vivo conditions. Cancer stem cells propagating in three-dimensional culture systems show physiologically related signaling pathway profiles, gene expression, cell-matrix and cell-cell interactions, and drug resistance that reflect at least some of the tumor properties seen in vivo. Herein, we discussed the presently available Cancer stem cell three-dimensional culture models that use biomaterials and engineering tools and the biological implications of these models compared to the conventional ones.

Targeting with nanoparticles for the therapeutic treatment of brain diseases.

Brain diseases including neurodegenerative disorders and tumours are among the most serious health problems, degrading the quality of life and causing massive economic cost. Nanoparticles that load and deliver drugs and genes have been intensively studied for the treatment of brain diseases, and have demonstrated some biological effects in various animal models. Among other efforts taken in the nanoparticle development, targeting of blood brain barrier, specific cell type or local intra-/extra-cellular space is an important strategy to enhance the therapeutic efficacy of the nanoparticle delivery systems. This review underlies the targeting issue in the nanoparticle development for the treatment of brain diseases, taking key exemplar studies carried out in various in vivo models.

Nano-graphene oxide/polyurethane nanofibers: mechanically flexible and myogenic stimulating matrix for skeletal tissue engineering.

For skeletal muscle engineering, scaffolds that can stimulate myogenic differentiation of cells while possessing suitable mechanical properties (e.g. flexibility) are required. In particular, the elastic property of scaffolds is of importance which helps to resist and support the dynamic conditions of muscle tissue environment. Here, we developed highly flexible nanocomposite nanofibrous scaffolds made of polycarbonate diol and isosorbide-based polyurethane and hydrophilic nano-graphene oxide added at concentrations up to 8%. The nano-graphene oxide incorporation increased the hydrophilicity, elasticity, and stress relaxation capacity of the polyurethane-derived nanofibrous scaffolds. When cultured with C2C12 cells, the polyurethane-nano-graphene oxide nanofibers enhanced the initial adhesion and spreading of cells and further the proliferation. Furthermore, the polyurethane-nano-graphene oxide scaffolds significantly up-regulated the myogenic mRNA levels and myosin heavy chain expression. Of note, the cells on the flexible polyurethane-nano-graphene oxide nanofibrous scaffolds could be mechanically stretched to experience dynamic tensional force. Under the dynamic force condition, the cells expressed significantly higher myogenic differentiation markers at both gene and protein levels and exhibited more aligned myotubular formation. The currently developed polyurethane-nano-graphene oxide nanofibrous scaffolds, due to their nanofibrous morphology and high mechanical flexibility, along with the stimulating capacity for myogenic differentiation, are considered to be a potential matrix for future skeletal muscle engineering.

Differential chondro- and osteo-stimulation in three-dimensional porous scaffolds with different topological surfaces provides a design strategy for biphasic osteochondral engineering.

Bone/cartilage interfacial tissue engineering needs to satisfy the differential properties and architectures of the osteochondral region. Therefore, biphasic or multiphasic scaffolds that aim to mimic the gradient hierarchy are widely used. Here, we find that two differently structured (topographically) three-dimensional scaffolds, namely, "dense" and "nanofibrous" surfaces, show differential stimulation in osteo- and chondro-responses of cells. While the nanofibrous scaffolds accelerate the osteogenesis of mesenchymal stem cells, the dense scaffolds are better in preserving the phenotypes of chondrocytes. Two types of porous scaffolds, generated by a salt-leaching method combined with a phase-separation process using the poly(lactic acid) composition, had a similar level of porosity (~90%) and pore size (~150 µm). The major difference in the surface nanostructure led to substantial changes in the surface area and water hydrophilicity (nanofibrous ≫ dense); as a result, the nanofibrous scaffolds increased the cell-to-matrix adhesion of mesenchymal stem cells significantly while decreasing the cell-to-cell contracts. Importantly, the chondrocytes, when cultured on nanofibrous scaffolds, were prone to lose their phenotype, including reduced chondrogenic expressions (SOX-9, collagen type II, and Aggrecan) and glycosaminoglycan content, which was ascribed to the enhanced cell-matrix adhesion with reduced cell-cell contacts. On the contrary, the osteogenesis of mesenchymal stem cells was significantly accelerated by the improved cell-to-matrix adhesion, as evidenced in the enhanced osteogenic expressions (RUNX2, bone sialoprotein, and osteopontin) and cellular mineralization. Based on these findings, we consider that the dense scaffold is preferentially used for the chondral-part, whereas the nanofibrous structure is suitable for osteo-part, to provide an optimal biphasic matrix environment for osteochondral tissue engineering.

Efficacy of collagen and alginate hydrogels for the prevention of rat chondrocyte dedifferentiation.

Dedifferentiation of chondrocytes remains a major problem in cartilage tissue engineering. The development of hydrogels that can preserve chondrogenic phenotype and prevent chondrocyte dedifferentiation is a meaningful strategy to solve dedifferentiation problem of chondrocytes. In the present study, three gels were prepared (alginate gel (Alg gel), type I collagen gel (Col gel), and their combination gel (Alg/Col gel)), and the in vitro efficacy of chondrocytes culture while preserving their phenotypes was investigated. While Col gel became substantially contracted with time, the cells encapsulated in Alg gel preserved the shape over the culture period of 14 days. The mechanical and cell-associated contraction behaviors of Alg/Col gel were similar to those of Alg. The cells in Alg and Alg/Col gels exhibited round morphology, whereas those in Col gel became elongated (i.e. fibroblast-like) during cultures. The cells proliferated with time in all gels with the highest proliferation being attained in Col gel. The expression of chondrogenic genes, including SOX9, type II collagen, and aggrecan, was significantly up-regulated in Alg/Col gel and Col gel, particularly in Col gel. However, the chondrocyte dedifferentiation markers, type I collagen and alkaline phosphatase (ALP), were also expressed at significant levels in Col gel, which being contrasted with the events in Alg and Alg/Col gels. The current results suggest the cells cultured in hydrogels can express chondrocyte dedifferentiation markers as well as chondrocyte markers, which draws attention to choose proper hydrogels for chondrocyte-based cartilage tissue engineering.

Intra-articular biomaterials-assisted delivery to treat temporomandibular joint disorders.

The temporomandibular joint disorder, also known as myofascial pain syndrome, is considered one of the prevalent chronic pain diseases caused by muscle inflammation and cartilage degradation in head and neck, and thus influences even biopsychosocial conditions in a lifetime. There are several current treatment methodologies relieving inflammation and preventing degradation of the joint complex. One of the promising non-surgical treatment methods is an intra-articular injection of drugs such as corticosteroids, analgesics, and anti-depressants. However, the side effects of drugs due to frequent injections and over-doses, including dizziness, dry mouth, and possible drug dependency are considered limitations. Thus, the delivery of therapeutic molecules through the use of nano/microparticles is currently considered as a promising strategy primarily due to the controlled release. This review highlights the nano/microparticle systems for effective intra-articular therapeutics delivery to prevent cartilage degradation and protect subchondral bone in a temporomandibular joint.

Chondrogenic Potential of Dedifferentiated Rat Chondrocytes Reevaluated in Two- and Three-Dimensional Culture Conditions.

For the cartilage repair, the cell sources currently adopted are primarily chondrocytes or mesenchymal stem cells (MSCs). Due to the fact that chondrocytes dedifferentiate during 2-dimensional (2D) expansion, MSCs are generally more studied and considered to have higher potential for cartilage repair purposes. Here we question if the dedifferentiated chondrocytes can regain the chondrogenic potential, to find potential applications in cartilage repair. For this we chose chondrocytes at passage 12 (considered to have sufficiently dedifferentiated) and the expression of chondrogenic phenotypes and matrix syntheses were examined over 14 days. In particular, the chondrogenic potential of MSCs was also compared. Results showed that the dedifferentiated chondrocytes proliferated actively over 14 days with almost 2.5-fold increase relative to MSCs. Moreover, the chondrogenic ability of chondrocytes was significantly higher than that of MSCs, as confirmed by the expression of a series of mRNA levels and the production of cartilage extracellular matrix molecules in 2D-monolayer and 3-dimensional (3D)-spheroid cultures. Of note, the significance was higher in 3D-culture than in 2D-culture. Although more studies are needed such as the use of different cell passages and human cell source, and the chondrogenic confirmation under in vivo conditions, this study showing that the dedifferentiated chondrocytes can also be a suitable cell source for the cell-based cartilage repair, as a counterpart of MSCs, will encourage further studies regarding this issue.

Promoting angiogenesis with mesoporous microcarriers through a synergistic action of delivered silicon ion and VEGF.

Angiogenic capacity of biomaterials is a key asset to drive vascular ingrowth during tissue repair and regeneration. Here we design a unique angiogenic microcarrier based on sol-gel derived mesoporous silica. The microspheres offer a potential angiogenic stimulator, Si ion, 'intrinsically' within the chemical structure. Furthermore, the highly mesoporous nature allows the loading and release of angiogenic growth factor 'extrinsically'. The Si ion is released from the microcarriers at therapeutic ranges (over a few ppm per day), which indeed up-regulates the expression of hypoxia inducing factor 1α (HIF1α) and stabilizes it by blocking HIF-prolyl hydroxylase 2 (PHD2) in HUVECs. This in turn activates the expression of a series of proangiogenic molecules, including bFGF, VEGF, and eNOS. VEGF is incorporated effectively within the mesopores of microcarriers and is then released continuously over a couple of weeks. The Si ion and VEGF released from the microcarriers synergistically stimulate endothelial cell functions, such as cell migration, chemotactic homing, and tubular networking. Furthermore, in vivo neo-blood vessel sprouting in chicken chorioallantoic membrane model is significantly promoted by the Si/VEGF releasing microcarriers. The current study demonstrates the synergized effects of Si ion and angiogenic growth factor through a biocompatible mesoporous microsphere delivery platform, and the concept provided here may open the door to a new co-delivery system of utilizing ions with growth factors for tissue repair and regeneration.

Effects of Type I Collagen Concentration in Hydrogel on the Growth and Phenotypic Expression of Rat Chondrocytes.

It is controversial whether type I collagen itself can maintain and improve chondrogenic phenotype of chondrocytes in a three-dimensional (3D) environment. In this study, we examined the effect of type I collagen concentration in hydrogel (0.5, 1, and 2 mg/ml) on the growth and phenotype expression of rat chondrocytes in vitro. All collagen hydrogels showed substantial contractions during culture, in a concentration-dependent manner, which was due to the cell proliferation. The cell viability was shown to be the highest in 2 mg/ml collagen gel. The mRNA expression of chondrogenic phenotypes, including SOX9, type II collagen, and aggrecan, was significantly up-regulated, particularly in 1 mg/ml collagen gel. Furthermore, the production of type II collagen and glycosaminoglycan (GAG) content was also enhanced. The results suggest that type I collagen hydrogel is not detrimental to, but may be useful for, the chondrocyte culture for cartilage tissue engineering.

Co-culture of Human Dental Pulp Stem Cells and Endothelial Cells Using Porous Biopolymer Microcarriers: A Feasibility Study for Bone Tissue Engineering.

Delivery of stem cells with osteogenesis while enabling angiogenesis is important for vascularized bone tissue engineering. Here a three-dimensional (3D) co-culture system of dental pulp stem cells (DPSCs) and endothelial cells (ECs) was designed using porous microcarriers, and the feasibility of applying to bone tissue engineering was investigated in vitro. Highly porous spherical microcarriers made of degradable biopolymers were prepared with sizes of hundreds of micrometers. The microcarriers loaded with DPSCs were co-cultured with ECs embedded in a hydrogel of type I collagen. An optimal co-culture medium that preserves the viability of ECs while stimulating the osteogenic differentiation of DPSCs was found to be a 10:1 of osteogenic medium:endothelial medium. The co-cultured constructs of DPSCs/ECs showed significantly higher level of alkaline phosphatase activity than the mono-cultured cells. Moreover, the expressions of genes related with osteogenesis and angiogenesis were significantly up-regulated by the co-cultures with respect to the mono-cultures. Results imply the interplay between ECs and DPSCs through the designed 3D co-culture models. The microcarrier-enabled co-cultured cell system is considered to be useful as an alternative tool for future vascularized bone tissue engineering.

Delivery of dexamethasone from bioactive nanofiber matrices stimulates odontogenesis of human dental pulp cells through integrin/BMP/mTOR signaling pathways.

Therapeutically relevant design of scaffolds is of special importance in the repair and regeneration of tissues including dentin and pulp. Here we exploit nanofiber matrices that incorporate bioactive glass nanoparticles (BGNs) and deliver the odontogenic drug dexamethasone (DEX) to stimulate the odontogenic differentiation of human dental pulp cells (HDPCs). DEX molecules were first loaded onto the BGN, and then the DEX-BGN complex was incorporated within the biopolymer nanofiber matrix through electrospinning. The release of DEX continued over a month, showing a slow releasing profile. HDPCs cultured on the DEX-releasing BGN matrices were viable, proliferating well up to 14 days. The odontogenic differentiation, as assessed by alkaline phosphatase activity, mRNA expression of genes, and mineralization, was significantly stimulated on the matrices incorporating BGN and further on those releasing DEX. The DEX-releasing BGN matrices highly upregulated the expression of the integrin subsets α1, α5, and ß3 as well as integrin downstream signaling molecules, including focal adhesion kinase (FAK), Paxillin, and RhoA, and activated bone morphogenetic protein mRNA and phosphorylation of Smad1/5/8. Furthermore, the DEX-releasing BGN-matrices stimulated Akt and mammalian target of rapamycin (mTOR), which was proven by the inhibition study. Collectively, the designed therapeutic nanofiber matrices that incorporate BGN and deliver DEX were demonstrated to promote odontogenesis of HDPCs, and the integrins, bone morphogenetic protein, and mTOR signaling pathways are proposed to be the possible molecular mechanisms. While further in vivo studies are still needed, the DEX-releasing bioactive scaffolds are considered as a potential therapeutic nanomatrix for regenerative endodontics and tissue engineering.

Magnetic nanocomposite scaffolds combined with static magnetic field in the stimulation of osteoblastic differentiation and bone formation.

Magnetism has recently been implicated to play significant roles in the regulation of cell responses. Allowing cells to experience a magnetic field applied externally or scaffolding them in a material with intrinsic magnetic properties has been a possible way of utilizing magnetism. Here we aim to investigate the combined effects of the external static magnetic field (SMF) with magnetic nanocomposite scaffold made of polycaprolactone/magnetic nanoparticles on the osteoblastic functions and bone formation. The SMF synergized with the magnetic scaffolds in the osteoblastic differentiation of primary mouse calvarium osteoblasts, including the expression of bone-associated genes (Runx2 and Osterix) and alkaline phosphatase activity. The synergism was demonstrated in the activation of integrin signaling pathways, such as focal adhesion kinase, paxillin, RhoA, mitogen-activated protein kinase, and nuclear factor-kappaB, as well as in the up-regulation of bone morphogenetic protein-2 and phosphorylation of Smad1/5/8. Furthermore, the SMF/magnetic scaffold-stimulated osteoblasts promoted the angiogenic responses of endothelial cells, including the expression of vascular endothelial growth factor and angiogenin-1 genes and the formation of capillary tubes. When the magnetic scaffolds were implanted in mouse calvarium defects, the application of SMF significantly enhanced the new bone formation at 6 weeks, as revealed by the histological and micro-computed tomographic analyses. Current findings suggest that the combinatory application of external (SMF) and internal (scaffold) magnetism can be a promising tool to regenerative engineering of bone.

Porous microcarrier-enabled three-dimensional culture of chondrocytes for cartilage engineering: A feasibility study.

Cartilage repair is substantially intractable due to poor self-healing ability. Porous microspheres can be a fascinating three-dimensional matrix for cell culture and injectable carrier in cartilage engineering. In this study, we assessed the feasible use of porous biopolymer microspheres for chondrocyte carriers. When seeded onto the blended biopolymer microspheres and followed by a dynamic spinner flask culture, the chondrocytes showed robust growth behaviors during the culture period. The gene expressions of SOX9, type II collagen, and aggrecan were significantly upregulated after 2-week of culture. Furthermore, immunolocalization of type II collagen and secretion of glycosaminolglycan became prominent. The results suggest the feasible usefulness of the porous microspheres as the cell culture matrix and the subsequent delivery into cartilage defects.

Isolation and culture of primary rat adipose derived stem cells using porous biopolymer microcarriers.

Adipose-derived stem cells (ADSCs) are an attractive source of material for mesenchymal stem cell research due to the abundance of adipose and relative ease of access compared with bone marrow. A key consideration for research is whether cell isolation methods can be improved, to reduce the process steps needed to isolate and expand cell material. In the current study, we used macroporous biopolymer microcarriers to isolate primary ADSCs. We found that the method was capable of isolating ADSCs that were subsequently capable of being transferred to culture dishes and expanded in vitro. Moreover, flow cytometry revealed that they expressed typical stem cell markers and were capable of undergoing tri-lineage differentiation. In summary, it is feasible to use biopolymer microcarriers for retrieval of viable ADSCs that retain identity markers of stem cell function.