Decellularized scaffolds containing hyaluronic acid and EGF for promoting the recovery of skin wounds.

There is no effective therapy for the treatment of deep and large area skin wounds. Decellularized scaffolds can be prepared from animal tissues and represent a promising biomaterial for investigation in tissue regeneration studies. In this study, MTT assay showed that epidermal growth factor (EGF) increased NIH3T3 cell proliferation in a bell-shaped dose response, and the maximum cell proliferation was achieved at a concentration of 25 ng/ml. Decellularized scaffolds were prepared from pig peritoneum by a series of physical and chemical treatments. Hyaluronic acid (HA) increased EGF adsorption to the scaffolds. Decellularized scaffolds containing HA sustained the release of EGF compared to no HA. Rabbits contain relatively large skin surface and are less expensive and easy to be taken care, so that a rabbit wound healing model was use in this study. Four full-thickness skin wounds were created in each rabbit for evaluation of the effects of the scaffolds on the skin regeneration. Wounds covered with scaffolds containing either 1 or 3 µg/ml EGF were significantly smaller than with vaseline oil gauzes or with scaffolds alone, and the wounds covered with scaffolds containing 1 µg/ml EGF recovered best among all four wounds. Hematoxylin-Eosin staining confirmed these results by demonstrating that significantly thicker dermis layers were also observed in the wounds covered by the decellularized scaffolds containing HA and either 1 or 3 µg/ml EGF than with vaseline oil gauzes or with scaffolds alone. In addition, the scaffolds containing HA and 1 µg/ml EGF gave thicker dermis layers than HA and 3 µg/ml EGF and showed the regeneration of skin appendages on day 28 post-transplantation. These results demonstrated that decellularized scaffolds containing HA and EGF could provide a promising way for the treatment of human skin injuries.

Performance of permeable pavement systems on stormwater permeability and pollutant removal.

Permeable pavement is an effective means for stormwater runoff control and pollutant removal. However, relatively few studies have examined the characteristics of permeable brick and corresponding permeable pavement system (PPS). In this work, the permeable pavement systems consisted of surface permeable brick layer (concrete or ceramic) with structural layer (including a cement mortar layer, a permeable concrete layer, and a gravel layers) were selected as typical cases to assess their permeability and runoff pollutant removal performance by laboratory experiments. The results indicated that PPS had obvious outflow hysteresis effect. The PPS with ceramic brick layer reached the saturation flow rate earlier and showed larger outflow rate than that with concrete brick layer. Both types of PPSs had a relatively high efficiency (83.8-95.2%) in removing suspended solids (SS) in stormwater runoff mainly due to the interception and filtration of the surface brick layer, whereas the structural layer of the PPS played a vital role in the removal of total phosphorus (TP). The percentage of total nitrogen (TN) removal efficiency via ceramic brick layer accounted for via corresponding PPS was obviously larger than that of concrete brick layer. The PPS also displayed a certain chemical oxygen demand (COD) removal ability: around 14.0-27.0% for concrete type and 20.9-28.9% for ceramic type. Subsequently, a multi-objective evaluation model was implemented based on the analytic hierarchy process (AHP) method to identify the optimal scheme in relation to four indices: permeability, environmental benefit, compressive strength, and comprehensive economic cost. The results showed, insofar, the ceramic PPS is preferred with a better economic performance. Our study attempts to select optimal designs of PPS and provides insight into the permeable capacity and the efficiency of pollutant removal in PPS.

Cross-linked decellularized porcine corneal graft for treating fungal keratitis.

This study aims to develop a cross-linked decellularized porcine corneal graft (cDPC) as a substitute for lamellar donor corneas and to evaluate the feasibility of using cDPC to treat fungal keratitis. The cDPC was prepared by decellularization, chemical crosslinking and γ-ray irradiation. Transparency, effectiveness of decellularization and biomechanical strength of cDPC were evaluated. The safety and efficacy of using cDPC to treat fungal keratitis were evaluated in the rabbit model. The transparency of cDPC was similar to that of a native porcine cornea (NPC), and no intact cells were observed in cDPC except for an insignificant amount of residual shrinking cellular nucleus. Compared to the NPC, the biomechanical strength of the cDPC was significantly increased. In the rabbit model of lamellar keratoplasty, the implanted cDPC reduced the incidence of corneal perforation, and also maintained transparency in majority. The results of this study suggest that the cDPC is capable of restoring the original transparency of cornea while effectively treating fungal keratitis. The cDPC is a highly promising ideal substitute for the donor human cornea.

Establishment of a Long-Term Chick Forebrain Neuronal Culture on a Microelectrode Array Platform.

The biosensor system formed by culturing primary animal neurons on a microelectrode array (MEA) platform is drawing an increasing research interest for its power as a rapid, sensitive, functional neurotoxicity assessment, as well as for many other electrophysiological related research purposes. In this paper, we established a long-term chick forebrain neuron culture (C-FBN-C) on MEAs with a more than 5 month long lifespan and up to 5 month long stability in morphology and physiological function; characterized the C-FBN-C morphologically, functionally, and developmentally; partially compared its functional features with rodent counterpart; and discussed its pros and cons as a novel biosensor system in comparison to rodent counterpart and human induced pluripotent stem cells (hiPSCs). Our results show that C-FBN-C on MEA platform 1) can be used as a biosensor of its own type in a wide spectrum of basic biomedical research; 2) is of value in comparative physiology in cross-species studies; and 3) may have potential to be used as an alternative, cost-effective approach to rodent counterpart within shared common functional domains (such as specific types of ligand-gated ion channel receptors and subtypes expressed in the cortical tissues of both species) in large-scale environmental neurotoxicant screening that would otherwise require millions of animals.

Use of decellularized scaffolds combined with hyaluronic acid and basic fibroblast growth factor for skin tissue engineering.

Skin damage is one of the common clinical skin diseases, and the main cure is the use of skin graft, especially for large area of skin injury or deep-skin damage. However, skin graft demand is far greater than that currently available. In this study, xenogeneic decellularized scaffold was prepared with pig peritoneum by a series of biochemical treatments to retain normal three-dimensional tissue scaffold and remove cells and antigenic components from the tissue. Scaffold was combined with hyaluronic acid (HA) plus two different concentrations of basic fibroblast growth factor (bFGF) and tested for its use for the repair of skin wounds. HA enhanced bFGF to adsorb to the decellularized scaffolds and slowed the release of bFGF from the scaffolds in vitro. A total of 20 rabbits were sacrificed on day 3, 6, 11, or 14 postsurgery. The wound healing rate and the thickness of dermis layer of each wound were determined for analyzing the wound repair. Statistical analysis was performed by the two-tailed Student's t-test. Wounds covered with scaffolds containing 1 µg/mL bFGF had higher wound healing rates of 47.24%, 74.69%, and 87.54%, respectively, for days 6, 11, and 14 postsurgery than scaffolds alone with wound healing rates of 28.17%, 50.31%, and 61.36% and vaseline oil gauze with wound healing rates of 24.84%, 42.75%, and 57.62%. Wounds covered with scaffolds containing 1 µg/mL bFGF showed more dermis regeneration than the other wounds and had dermis layer of 210.60, 374.40, and 774.20 µm, respectively, for days 6, 11, and 14 postsurgery compared with scaffolds alone with dermis layer of 116.60, 200.00, and 455.40 µm and vaseline oil gauze with dermis layer of 82.60, 186.20, and 384.40 µm. There was no significant difference in wound healing rates and thickness of dermis layer between wounds covered with scaffolds containing 1 and 3 µg/mL bFGF on days 3, 6, 11, and 14 postsurgery. The decellularized scaffolds combined with HA and bFGF can be further tested for skin tissue engineering.

Microfluidics-based laser cell-micropatterning system.

The ability to place individual cells into an engineered microenvironment in a cell-culture model is critical for the study of in vivo relevant cell-cell and cell-extracellular matrix interactions. Microfluidics provides a high-throughput modality to inject various cell types into a microenvironment. Laser guided systems provide the high spatial and temporal resolution necessary for single-cell micropatterning. Combining these two techniques, the authors designed, constructed, tested and evaluated (1) a novel removable microfluidics-based cell-delivery biochip and (2) a combined system that uses the novel biochip coupled with a laser guided cell-micropatterning system to place individual cells into both two-dimensional (2D) and three-dimensional (3D) arrays. Cell-suspensions of chick forebrain neurons and glial cells were loaded into their respective inlet reservoirs and traversed the microfluidic channels until reaching the outlet ports. Individual cells were trapped and guided from the outlet of a microfluidic channel to a target site on the cell-culture substrate. At the target site, 2D and 3D pattern arrays were constructed with micron-level accuracy. Single-cell manipulation was accomplished at a rate of 150 µm s(-1) in the radial plane and 50 µm s(-1) in the axial direction of the laser beam. Results demonstrated that a single-cell can typically be patterned in 20-30 s, and that highly accurate and reproducible cellular arrays and systems can be achieved through coupling the microfluidics-based cell-delivery biochip with the laser guided system.

Enhancement of skin wound healing with decellularized scaffolds loaded with hyaluronic acid and epidermal growth factor.

Current therapy for skin wound healing still relies on skin transplantation. Many studies were done to try to find out ways to replace skin transplantation, but there is still no effective alternative therapy. In this study, decellularized scaffolds were prepared from pig peritoneum by a series of physical and chemical treatments, and scaffolds loaded with hyaluronic acid (HA) and epidermal growth factor (EGF) were tested for their effect on wound healing. MTT assay showed that EGF increased NIH3T3 cell viability and confirmed that EGF used in this study was biologically active in vitro. Scanning electron microscope (SEM) showed that HA stably attached to scaffolds even after soaking in PBS for 48 h. ELISA assay showed that HA increased the adsorption of EGF to scaffolds and sustained the release of EGF from scaffolds. Animal study showed that the wounds covered with scaffolds containing HA and EGF recovered best among all 4 groups and had wound healing rates of 49.86%, 70.94% and 87.41% respectively for days 10, 15 and 20 post-surgery compared to scaffolds alone with wound healing rates of 29.26%, 42.80% and 70.14%. In addition, the wounds covered with scaffolds containing EGF alone were smaller than no EGF scaffolds on days 10, 15 and 20 post-surgery. Hematoxylin-Eosin (HE) staining confirmed these results by showing that on days 10, 15 and 20 post-surgery, the thicker epidermis and dermis layers were observed in the wounds covered with scaffolds containing HA and EGF than scaffolds alone. In addition, the thicker epidermis and dermis layers were also observed in the wounds covered with scaffolds containing EGF than scaffolds alone. Skin appendages were observed on day 20 only in the wound covered with scaffolds containing HA and EGF. These results demonstrate that the scaffolds containing HA and EGF can enhance wound healing.

Generation of human epidermis-derived mesenchymal stem cell-like pluripotent cells (hEMSCPCs).

We isolated human epidermis-derived mesenchymal stem cell-like pluripotent cells (hEMSCPCs) and demonstrate efficient harvesting, maintenance in vitro for at least 30 passages, reprogramming into multiple phenotypes in vivo, and integration into adult host tissues after injection into the mouse blastocyst to create chimeras. Cell phenotype was examined by karyotyping, immunostaining, immunofluorescence, and flow cytometry. A nested PCR protocol using primers specific for human SRY genes was designed to detect hEMSCPC-derived cells in female chimeric mice. FISH was used to validate the results of nested PCR. Results indicated that hEMSCPCs were derived from epidermis but were distinct from epidermal cells; they resembled mesenchymal stem cells (MSCs) morphologically and expressed the main markers of MSCs. About half of all female offspring of mice implanted with embryos injected with hEMSCPCs at the blastocyst stage harbored the human Y chromosome and tissue-specific human protein, thereby demonstrating the transdifferentiation of hEMSCPCs.

Transfect bone marrow stromal cells with pcDNA3.1-VEGF to construct tissue engineered bone in defect repair.

BACKGROUND: We previously showed that nano-hydroxyapatite/carboxymethyl chitosan (n-Ha/CMCS) displayed excellent mechanical properties, good degradation rates and exceptional biocompatibility, with negligible toxicity. The aim of this study was to determine the effect of the same composite with vascular endothelial growth factor (VEGF)- transfected bone marrow stromal cells (BMSCs) in a rabbit radial defect model. METHODS: The nano-hydroxyapatite was produced through co-precipitation. The n-HA/CMCS scaffold was produced by particle filtration and lyophilization followed by genipin crosslinking. Total RNA from rabbit bone was reverse-transcribed to synthesize VEGF165-pcDNA3.1 that was transfected into the BMSCs. The composite was implanted into a rabbit radial defect model, and the osteogenic activity examined by gross morphology, X-ray examination and hematoxylin and eosin (HE) staining. RESULTS: The microstructure and mechanical property of the n-HA/CMCS scaffold resembled natural cancellous bone. Compared with glutaric dialdehyde crosslinked scaffolds, the genipin crosslinked scaffold was less toxic, and displayed a higher capacity to promote cell adhesion and proliferation. Spontaneous fluorescence of the composite permitted visualization of the composite-bone interface and the adhesion behavior of cells on the scaffold under laser scanning confocal microscopy. The scaffold with VEGF-transfected BMSCs bridged the bony defect and promoted healing, with most of the implanted material being replaced by natural bone over time with little residual implant. Using X-ray, we noted obvious callus formation and recanalization of the bone marrow cavity. Furthermore, HE stained sections showed new cortical bone formation. CONCLUSIONS: The n-HA/CMCS scaffold composite with VEGF-trasnfected BMSCs is biocompatible, nontoxic, promotes the infiltration and formation of the microcirculation, and stimulates bone defect repair. Furthermore, the degradation rate of the composite matched that of growing bone. Overall, this composite material is potentially useful for bone defect repair.