Direct Reprogramming of Human Suspension Cells into Mesodermal Cell Lineages via Combined Magnetic Targeting and Photothermal Stimulation by Magnetic Graphene Oxide Complexes.

Suspension cells can provide a source of cells for cellular reprogramming, but they are difficult to transfect by nonviral vectors. An efficient and safe nonviral vector (GO-Fe3 O4 -PEI complexes) based on iron oxide nanoparticle (Fe3 O4 )-decorated graphene oxide (GO) complexed with polyethylenimine (PEI) for the first time is developed for delivering three individual episomal plasmids (pCXLE-hOCT3/4-shp53, pCXLE-hSK, and pCXLE-hUL) encoding pluripotent-related factors of Oct3/4, shRNA against p53, Sox2, Klf4, L-Myc, and Lin28 into human peripheral blood mononuclear cells (PBMCs) simultaneously. The combined treatment of magnetic stirring and near-infrared (NIR)-laser irradiation, which can promote contact between the complexes and floating cells and increase the cell membrane permeability, respectively, is used to conduct multiple physical stimulations for suspension PBMCs transfection. The PCR analysis shows that the combinatorial effect of magnetic targeting and photothermal stimulation obviously promoted the transfection efficiency of suspension cells. The transfected cells show positive expression of the pluripotency markers, including Nanog, Oct4, and Sox2, and have potential to differentiate into mesoderm and ectoderm cells. The results demonstrate that the GO-Fe3 O4 -PEI complex provides a safe, convenient, and efficient tool for reprogramming PBMCs into partially induced pluripotent stem cells, which are able to rapidly transdifferentiate into mesodermal lineages without full reprogramming.

Enhanced anti-cancer activity by curcumin-loaded hydrogel nanoparticle derived aggregates on A549 lung adenocarcinoma cells.

To investigate the anti-cancer activity of curcumin-loaded hydrogel nanoparticle derived aggregates on A549 lung adenocarcinoma cells. Curcumin was incorporated with biopolymeric chitosan, gelatin, and hyaluronan nanoparticles using an electrostatic field system. Characteristics of curcumin-loaded aggregates were examined including size and morphology, incorporation efficiency, stability and in vitro release. Treatment effect on A549 cells were assessed with cell viability assay, apoptosis assay, cell cycle analysis, reactive oxygen species detection, and Western blot. Observation from transmission electron microscopy show that the prepared biopolymeric nanoparticles were approximately 3-4 nm in diameter and that the size of the aggregates increased to approximately 26-55 nm after the incorporation of curcumin with the nanoparticles. The incorporation efficiency of curcumin into the chitosan, gelatin, and hyaluronan nanoparticles was 81, 67, and 78 % respectively. The formation of hyaluronan/curcumin and gelatin/curcumin aggregates seems to improve the stability of curcumin drug. The chitosan/curcumin aggregate has a faster release of curcumin than gelatin/curcumin and hyaluronan/curcumin aggregates. Treatment with chitosan/curcumin, gelatin/curcumin and hyaluronan/curcumin aggregates resulted in higher apoptosis rates of 45, 40 and 32 %, respectively, as compared to pure curcumin (less than 20 %) via Annexin V-FITC/PI analysis. Chitosan/curcumin aggregates induce the highest apoptosis effect (indicated by sub-G1 phase). In summary, chitosan/curcumin, gelatin/curcumin, and hyaluronan/curcumin aggregates represent higher anticancer proliferation properties in A549 cells than curcumin alone that exhibit great potential enhancement by either using fewer drugs or a decreased duration.

Repair of chondral defects with allogenous chondrocyte-seeded hyaluronan/collagen II microspheres in a rabbit model.

Using a recently established method to prepare hyaluronan/collagen II (HA/Col II) microspheres for a novel biomaterial to couple with living cells/tissues, this animal model study evaluated the effects on a 4-week healing process of chondral defects by the implantation of allogenous chondrocyte-seeded HA/Col II microspheres that had been cultured in vitro for 7 days prior to implantation compared with unseeded HA/Col II microspheres or an untreated wound. Four weeks postsurgery, the untreated group's defect was filled with translucent soft tissue. At the same time, the edges and demarcation lines of the healing defects that were implanted with either HA/Col II microspheres or chondrocyte-seeded HA/Col II microspheres were infused yet recognizable. Furthermore, the new tissues were well integrated into the surrounding articular cartilage. Less glycosaminoglycan (GAG) staining was observed in the defects implanted with HA/Col II microspheres, which indicated that most of the repair tissues were derived from fibrocartilage formation. Conversely, more GAG staining appeared in the defect implanted with chondrocyte-seeded HA/Col II microspheres, which demonstrated a higher level of hyaline cartilage regeneration. Due to the short healing period assigned to this study, the repaired cartilage showed limited incorporation into the surrounding host cartilage and some loose connection to the subchondral bone.

Protein-based soft actuator with high photo-response and easy modulation for anisotropic cell alignment and proliferation in a liquid environment.

Cell alignment and elongation, which are critical factors correlated with differentiation and maturation in cell biology and tissue engineering, have been widely studied in organisms. Several strategies such as external mechanical strain, geometric topography, micropatterning approaches, and microfabricated substrates have been developed to guide cell alignment, but these methodologies cannot be used for easily denatured natural proteins to modulate the cell behaviour. Herein, for the first time, a novel biocompatible light-controlled protein-based bilayer soft actuator composed of elastin-like polypeptides (ELPs), silk fibroin (SF), graphene oxide (GO), and reduced graphene oxide (rGO), named ESGRG, is developed for efficiently driving cellular orientation and elongation with anisotropic features on soft actuator via remote NIR laser exposure. The actuation of ESGRG could be manipulated by modulating the intensity of NIR and the relative ratio of GO to rGO for promoting myoblasts alignment and nucleus elongation to generate different motions. The results indicate that the YAP and MHC protein expression of C2C12 skeletal muscle cells on ESGRG can be rapidly induced and enhanced by controlling the relative ratio of rGO/GO = 1/4 at a multiple-cycle stimulation with a very low power intensity of 1.2 W cm-2 in friendly liquid environments. This study demonstrates that the ESGRG hydrogel actuator system can modulate the cell-level behaviors via light-driven cyclic bending-motions and can be utilized in applications of soft robotic and tissue engineering such as artificial muscle and maturation of cardiomyocytes.

A smart injectable composite hydrogel with magnetic navigation and controlled glutathione release for promoting in situ chondrocyte array and self-healing in damaged cartilage tissue.

Injectable cell-based hydrogels allow surgical operation in a minimally invasive way for articular cartilage lesions but the chondrocytes in the injectable hydrogels are difficultly arrayed and fixed at the site of interest to repair the cartilage tissue. In this study, an injectable hyaluronic acid-polyacrylic acid (HA-pAA) hydrogel was first synthesized using hyaluronic acid-cyclodextrin (HA-CD) and polyacrylic acid-ferrocene (pAA-Fc) to provide cell-delivery and self-healing. To promote the cell fixation and alignment, porous poly(lactic-co-glycolic acid) (PLGA) magnetic microcapsules (PPMMs) with glutathione (GSH) loaded and iron oxide nanoparticles (IO) located in the shell were designed. The GSH-loaded PPMMs with layer-by-layer (LbL) assembly of hyaluronic acid (HA) and GSH (LbL-PPMMs) can provide a two-stage rapid and slow release of GSH to modulate the self-healing of the HA-pAA hydrogel at the injured site. Furthermore, the chondrocytes embedded in the HA-pAA hydrogel could be delivered through CD44 receptors on the HA polymer chains of LbL-PPMMs toward the surface of the damaged site by an internal magnetic force. The composite hydrogel system of chondrocytes/LbL-PPMMs/HA-pAA can provide the damaged cartilage with a more even and smooth surface than other groups in a rabbit model after 8 weeks of implantation. In addition, the chondrocytes in the deep zone tissue exhibit a columnar array, similar to the cell arrangement in normal cartilage tissue. Together with the cell navigation behavior and GSH release from the LbL-PPMM/HA-pAA hydrogel, a full closure of lesions on the cartilage tissue can be achieved. Our results demonstrate the highly promising potential of the injectable LbL-PPMM/HA-pAA system in cartilage tissue repair.

Evaluation of bone growth using artificial bone substitute (osteoset) and platelet gel mixtures: a preliminarily study in dogs.

Platelet gels (PG), activated by bovine thrombin (BT), have increasingly been used in orthopedic surgery. However, BT may induce immunological reactions and carry potential viral and prion risks. To avoid these side effects, thrombin derived from human plasma (human thrombin, HT) is becoming the preferred platelet activator to prepare PG. However, limited experience and data on the clinical benefits of HT-generated PG (HTPG) in orthopedic surgery is reported. Consequently, we designed and performed a series of studies in dogs to compare the impacts of promotion of bone growth by an artificial bone substitute (Osteoset) in combination with HTPG or without it in the spinal repair experiments. X-ray observations and histological studies were performed at predetermined periods post-operation. The preliminary results revealed the preparation of HTPG was easy and required less than 30 minutes. HTPG was capable of embedding the artificial bone substitute Osteoset to prepare a sticky and easily manipulated composite for the application into spinal defect. We found HTPG exhibited enhancement of grafting capacity in consolidation of bone mass. After 12 weeks, tissue reconstruction reached approximately 80% of the injury defects when treated by HTPG/Osteoset combination, but only 30 approximately 40% in the absence of HTPG. The physiological activity of artificial bone substitute combined with PG activated by HT may therefore open beneficial prospects for more successful and safer bone formation in spine procedures in the near future.

Development of chondrocyte-seeded electrosprayed nanoparticles for repair of articular cartilage defects in rabbits.

Due to limited self-healing capacity in cartilages, there is a rising demand for an innovative therapy that promotes chondrocyte proliferation while maintaining its biofunctionality for transplantation. Chondrocyte transplantation has received notable attention; however, the tendencies of cell de-differentiation and de-activation of biofunctionality have been major hurdles in its development, delaying this therapy from reaching the clinic. We believe it is due to the non-stimulative environment in the injured cartilage, which is unable to provide sustainable physical and biological supports to the newly grafted chondrocytes. Therefore, we evaluated whether providing an appropriate matrix to the transplanted chondrocytes could manipulate cell fate and recovery outcomes. Here, we proposed the development of electrosprayed nanoparticles composed of cartilage specific proteins, namely collagen type II and hyaluronic acid, for implantation with pre-seeded chondrocytes into articular cartilage defects. The fabricated nanoparticles were pre-cultured with chondrocytes before implantation into injured articular cartilage. The study revealed a significant potential for nanoparticles to support pre-seeded chondrocytes in cartilage repair, serving as a protein delivery system while improving the survival and biofunctionality of transplanted chondrocytes for prolonged period of time.

Guided tissue regeneration for using a chitosan membrane: an experimental study in rats.

Barrier membranes are employed clinically to deflect the growth of gingival tissues away from root surface. They provide an isolated space over the regions with the defective tissues that allow the relatively slow growing periodontal ligament fibroblasts to be repopulated onto the root surface. Several makes of bioabsorbable membranes are now commercially available. In this study, we have employed chitosan as barrier membrane material and evaluated it for a guided tissue regeneration application. Three types of chitosan membranes: Chi-NaOH, Chi-Na(5)P(3)O(10), and Chi-Na(2)SO(3)(each was gelated by NaOH, crosslinked by Na(5)P(3)O(10) and Na(2)SO(3), respectively), were prepared to be evaluated by the following categories: the mechanical strength to create an effective space, the rapid rate to reach hydrolytic equilibrium in phosphate-buffered solution, and the ease of clinical manipulative operations. Consequently, standardized, transosseous and critical sized skull defects were made in adult rats and the defective regions were covered with the specifically prepared chitosan membranes. After 4 weeks of recovering, varying degrees of bone healing were observed beneath the chitosan membranes in comparison to the control group. The chitosan covered regions showed a clear boundary space between connective tissues and bony tissues. Apparently, this process resulted in a good cell occlusion and beneficial osteogenesis effect to the bone. As for the control group, the bone defect was filled with connective tissue, and a destruction of the integrity of newly formed bone was observed. Among the chitosan membranes tested in this study, Chi-NaOH membrane provided a higher percentage of new bone formation than those from the Chi-Na(5)P(3)O(10) and Chi-Na(2)SO(3) families.

Alternative approach of cell encapsulation by Volvox spheres.

Volvox sphere is a bio-mimicking concept of a biomaterial structure design able to encapsulate chemicals, drugs and/or cells. The aim of this study was to prepare Volvox spheres encapsulating AML12 liver cells and mesenchymal stem cells (MSCs) via a high voltage electrostatic field system. The results demonstrated that AML12 liver cells and MSCs could be successfully encapsulated into the inner spheres and the outer sphere of the Volvox spheres. The improved cell viability of MSCs was achieved by the addition of collagen and polyethylene glycol into the preparation components of the Volvox spheres. Collagen material potentially provides extracellular matrix-like structure for cell adhesion while polyethylene glycol provides a void/loose space for permeability of metabolites. The encapsulated MSCs were able to differentiate into hepatocytes or hepatocyte-like cells and express liver cell markers including albumin, alpha feto-protein and cytokeratin 18. The encapsulated cells secreted albumin to about 140 ng on day 14. Based on these observations, we conclude that Volvox spheres can be used as an alternative approach to encapsulate multiple types of cells, here AML12 hepatocyte cell line and MSCs. Nevertheless, efforts are still needed to improve the viability of the encapsulated cells and increase the differentiation of MSCs into functional liver cells.

Evaluation of the ability of xanthan gum/gellan gum/hyaluronan hydrogel membranes to prevent the adhesion of postrepaired tendons.

After tendon-repair surgery, adhesion between the surgical tendon and the synovial sheath is often presented resulting in poor functional repair of the tendon. This may be prevented using a commercially available mechanical barrier implant, Seprafilm, which is composed of hyaluronan (HA) and carboxymethyl cellulose hydrogels. In a rat model, prepared membranes of various compositions of gellan gum (GG), xanthan gum (XG) and HA as well as Seprafilm were wrapped around repaired tendons and the adhesion of the tendons was examined grossly and histologically after 3 weeks of healing. Certain formulations of the XG/GG/HA hydrogel membranes reduced tendon adhesion with equal efficacy but without reducing the tendon strength compared to Seprafilm. The designed membranes swelled rapidly and blanketed onto the tendon tissue more readily and closely than Seprafilm. Also they degraded slowly, which allowed the membranes to function as barriers for extended periods.

Preparation and evaluation of collagen I/ gellan gum/ß-TCP microspheres as bone graft substitute materials.

Collagen I is the main component of protein in bone and exhibits many excellent applications in biomedical fields. Gellan gum possesses good biocompatible, biodegradable and good mechanical property, and shows great potentials as tissue engineering scaffold or cell culture substrate. Therefore, the aim of this study was to use collagen I, gellan gum and ß-TCP to prepare collagen I/gellan gum/ß-TCP microspheres by emulsion method as bone graft substitute materials. The preliminary results showed that collagen I/gellan gum/ß-TCP microspheres had particle size distribution between 500-1000 µP in diameter and exhibited better mechanical strength. These microspheres also showed good biocompatibility in cell activity test.

Surface plasmon resonance biosensor with high anti-fouling ability for the detection of cardiac marker troponin T.

Designing a surface recognition layer with high anti-fouling ability, high affinity, and high specificity is an important issue to produce high sensitivity biosensing transducers. In this study, a self-assembled monolayer (SAM) consisting of a homogeneous mixture of oligo(ethylene glycol) (OEG)-terminated alkanethiolate and mercaptohexadecanoic acid (MHDA) on Au was employed for immobilizing troponin T antibody and applied in detecting cardiac troponin T by using surface plasmon resonance (SPR). The mixed SAM showed no phase segregation and exhibited human serum albumin resistance, particularly with an antibody-immobilized surface. X-ray photoemission spectra revealed that the chemical composition ratio of OEG to the mixed SAM was 69% and the OEG packing density was 82%. The specific binding of troponin T on the designed surface indicated a good linear correlation (R=0.991, P<0.0009) at concentrations lower than 50 µgmL(-1) with the limit of detection of 100 ngmL(-1) using a SPR measuring instrument. It is concluded that the mixed SAM functions as designed since it has high detection capability, high accuracy and reproducibility, as well as shows strong potential to be applied in rapid clinical diagnosis for label-free detection within 2 min.

Preparation and characterization of hyaluronan/collagen II microspheres under an electrostatic field system with disc electrodes.

Collagen II and hyaluronan are the two major components of the native extracellular matrix (ECM). Both biopolymers are responsible for providing the associated tissues with tensile strength, and also serve as a structural scaffold for cell adhesion and growth. Over the years, many researchers have focused on the preparation and evaluation of man-made ECM comprising the two polymers in the form of a membrane for chondrocyte culture applications. Here, a simple and in situ method, involving the injection of the hyaluronan/collagen II (HA/Col II) mixture solution through a pair of hollow-centered parallel disc electrodes (HCPDEs) of a high-voltage electrostatic field system, was developed and employed to prepare HA/Col II microspheres in watery phase. The HA/Col II microspheres were firmed up by a two-step cross-linking treatment (first by FeCl(3) and then by 1-ethyl-3-(3-dimethyl aminopropyl) carbodimide, EDC) to secure the spherical structure shape. Then, at 37 degrees C, reconstitution treatment of the Col II molecules was conducted to further strengthen the microspheres. Depending on treatment conditions, the resulting series of HA/Col II microspheres all exhibited good sphericity in the range of 486+/-43 to 679+/-24microm in diameter. Furthermore, the ratio and amount of HA/Col II in the mixture solutions would affect the morphological structure and basic characteristics, including mechanical strength, thermal properties and water content. In the preliminary study, the HA/Col II microspheres have shown to provide favorable ECM characteristics, with appropriate mechanical strength, and exhibited a 3D inclination.

Influences of hyaluronan on type II collagen fibrillogenesis in vitro.

The effect to the kinetics of type II collagen fibrillogenesis with the addition of hyaluronan (HA), (Mw of 1.8x10(6) Da), at various concentrations of HA (0.01, 0.05 and 0.1 wt.%) for a series of fibril formation systems was examined in this study. Evidences deduced from the turbidity-time curves revealed that the inclusion of HA had minor or no impact to the fibrillogenesis of type II collagen (collagen conc. at 0.2 mg/mL). The apparent rate constants, klag (lag phase) increased slightly but kg (growth phase) decreased not very significantly with addition of HA, as compared to the case of pure collagen. This leads us to believe tentatively that, with the addition of HA to collagen solutions, the nucleation process of the fibril formation might have been sped up slightly whereas the growth process slowed up slightly. However, data from TEM observations on the resulting fibrils indicated that the presence of HA did not significantly affect the diameters and the characteristic D-banding periods of the collagen fiber formed. And, from the statistical analyses, we found only insignificant difference (P>0.05) between the specimens from the various experimental groups. It seems to indicate that the ultimate packing of collagen monomers was probably not interfered or affected significantly by the presence of HA in vitro.

Influence of alginate on type II collagen fibrillogenesis.

Collagen II is the majority of extracellular matrix components in articular cartilage, which with the major functions of preventing expansion of the tissue and distributing the load of body weight. To obtain man-made ECM, the reconstitution of collagen could be conducted in the presence of negatively charged polysaccharide, such as alginate. Alginate is an anionic polysaccharide capable of eversible gelated in calcium ion solution to prepare different shapes of biomaterials. Its well-known biocompatibility makes it an ideal material in biomedical applications. Thus, the aim of this study was to evaluate the effects of alginate on the fibrillogenesis of type II collagen. The preliminary results revealed that inclusion of alginate into soluble type II collagen solution could inhibit the development of turbidity of collagen solution, and the apparent rate constants in lag and growth phases decreased during collagen formation period, both rate constants decreased to about one-third of the original constants, respectively. From TEM observations, the collagen fibrils were significantly thicker in 0.05% and 0.1% alginate as compared with pure collagen solution. Furthermore, the D-periods of collagen fibers kept unchanged significantly under all reconstituted conditions, which meant the packing of collagen monomer was probably not affected by adding these amounts of alginate.

Evaluation of chitosan/beta-tricalcium phosphate microspheres as a constituent to PMMA cement.

Two methods, a traditional emulsion technique and a high voltage electrostatically modified encapsulation system, were used to fabricate degradable chitosan/beta -tricalcium phosphate (beta-TCP) microspheres. The two distinct kinds of microspheres both exhibited good sphericity and the beta-TCP was trapped well inside the chitosan gel. The microspheres prepared by high voltage electrostatic system exhibited a rougher outer surface and narrower size distribution. These microspheres were then used as an added constituent to commercially available PMMA bone cement. Four modified cement composites that were prepared with different composition ratios of the two kinds of chitosan/beta-TCP microspheres that were made from emulsion technique (C1P1 and C2P1) and from a process by a high voltage electrostatic system (EC1P1 and EC2P1) were compared with the PMMA cement (Pure P). The characteristics of these materials indicate that with the addition of chitosan/beta-TCP microspheres as a constituent into the PMMA cement significantly decreases the curing peak temperature. Furthermore, the setting time increases from 3.5 min to 9 min, as compared to the PMMA cement. These changes could be beneficial for the handling of the bone cement paste and causing less damage to the surrounding tissues. Understandably, the presence of chitosan/beta-TCP microspheres in the prepared composites reduced the ultimate compressive strength and bending strength. From the degradation test and SEM observations, the modified chitosan/beta -TCP/PMMA composites could be degraded gradually and create rougher surfaces that would be beneficial to cell adherence and growth.

Microencapsulation of parathyroid tissue with photosensitive poly(L-lysine) and short chain alginate-co-MPEG.

Human parathyroid glands were encapsulated using the alginate-PLL system in this study. In order to improve the mechanical strength and the biocompatibility, the microcapsules were fabricated with a three-layer structure that consisted of alginate/photosensitive poly(L-lysine)/short chain alginate-co-MPEG. These modified microcapsules were used for encapsulating human parathyroid tissue. In vitro experiments revealed that microencapsulated parathyroid glands maintained differentiative properties in culture, and the capsular membrane was freely permeable to the human parathyroid hormone. For in vivo experiments, these capsules were transplanted into parathyroidectomized SD-rats. After parathyroidectomy, serum calcium decreased from 2.25 to 1.68 mmol/L and remained in a constantly low concentration until transplantation. Parathyroidectomized SD-rats were normocalcemic after transplant of encapsulated parathyroid tissue. The microcapsules were then explanted at 12 weeks for examination. Histological evaluations of excised transplants revealed that the microcapsules remained intact structurally and were free of cell adhesions. The results demonstrated that human parathyroid tissue microencapsulated by this system retains stability and is functional both in vitro and in vivo. This encapsulating system will have valuable application for endocrine surgery in the future.

Biocompatible microcapsules with enhanced mechanical strength.

A block copolymer, (short-chain alginate)-co-MPEG, was synthesized and used for coating the capsular membranes of the photosensitive microcapsules. The resulted microcapsules exhibited an excellent mechanical strength. The permeability test results revealed that the capsular membrane was freely permeable to cytochrome C and myoglobin, less permeable to serum albumin, and almost impermeable to IgG. In the cell attachment test, the results showed that the surface formed by (short-chain alginate)-co-MPEG copolymer could effectively reduce cell adhesion as compared to poly(L-lysine) and alginate. The microcapsules were evaluated by intraperitoneal implantation experiment of mice. The results demonstrated that microcapsules coated with (short-chain alginate)-co-MPEG were more biocompatible than the conventional alginate/PLL/alginate microcapsules.