Surface modification of PHBV nanofiber mats for rapid cell cultivation and harvesting.

To maintain the original function of a specific tissue for therapeutic tissue engineering, an advanced cell culture surface for repeat cell proliferation is necessary. We designed a novel cell proliferation and rapid harvesting surface by combining nonwoven nanofiber mat and a thermo-responsive polymer. Nanofibrous poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) mats were fabricated by the electrospinning technique. A poly(N-isopropylacrylamide) (PNIPAM) thermo-responsive layer was grafted on the PHBV nanofiber mat by electron beam irradiation. The average diameter of the PNIPAM-grafted PHBV nanofibers was determined by SEM. ATR-FTIR and ESCA were used to confirm the grafting of PNIPAM onto the PHBV nanofiber surface. Water contact angles on the mats were measured in response to temperature changes. Human adipose-derived stem cells (ADSCs) were cultured on the PNIPAM-grafted PHBV nanofiber mat to investigate cell proliferation, harvesting, and functionality during repeat subculture. Detached ADSCs from each surface by low temperature treatment and trypsin-EDTA were compared by a fluorescence-activated cell sorter (FACS) using expression of stem cell membrane-specific markers such as CD-13 PE, CD-29 PE, and CD-90 FITC. The mass cultivation and intact harvesting of stem cells by low temperature treatment using a thermo-responsive PHBV nanofiber mat is a promising technique for use in regenerative medicine and stem cell therapy.

Nanofiber mats composed of a chitosan-poly(d,l-lactic-co-glycolic acid)-poly(ethylene oxide) blend as a postoperative anti-adhesion agent.

Postoperative tissue adhesion causes serious complications and suffering in 90% of patients after peritoneum surgery, while commercial anti-adhesion agents cannot completely prevent postoperative peritoneal adhesions. This study demonstrates electrospining of a blended solution of chitosan, poly(d,l-lactic-co-glycolic acid) (PLGA), and poly(ethylene oxide) (PEO) to fabricate a chitosan-based nanofibrous mat as a postoperative anti-adhesion agent. Rheological studies combined with scanning electron microscopy reveal that the spinnability of the chitosan-PLGA solution could be controlled by adjusting the blend ratio and concentration with average fiber diameter from 634 to 913 nm. Biodegradation of the nanofiber specimens showed accelerated hydrolysis by chitosan. Proliferation of fibroblasts and antimicrobial activity of nanofibers containing chitosan was analyzed. Abdominal defects with cecum adhesion in rats demonstrated that the blend nanofiber mats were effective in preventing tissue adhesion as a barrier (4 weeks after abdominal surgery) by coverage of exfoliated peritoneum and insufficient wound sites at the beginning of the wound healing process. Chitosan-PLGA-PEO blend nanofiber mats will provide a promising key as a postoperative anti-adhesion agent. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1906-1915, 2017.

Injectable pullulan hydrogel for the prevention of postoperative tissue adhesion.

Methods for reducing and preventing postoperative abdominal adhesions have been researched for decades; however, despite these efforts, the formation of postoperative peritoneal adhesions is continuously reported. Adhesions cause serious complications such as postoperative pain, intestinal obstruction, and infertility. Tissue adhesion barriers have been developed as films, membranes, knits, sprays, and hydrogels. Hydrogels have several advantages when used as adhesion barriers, including flexibility, low tissue adhesiveness, biodegradability, and non-toxic degraded products. Furthermore, compared with preformed hydrogels, injectable hydrogels can fill and cover spaces of any shape and do not require a surgical procedure for implantation. In this study, pullulan was modified through reaction with 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) to introduce carboxyl and phenyl groups as crosslinking sites. The grafting of tyramine on pullulan allows crosslinking branches on pullulan backbone. We successfully fabricated pullulan hydrogel with an enzymatic reaction using horseradish peroxidase (HRP) and hydrogen peroxide (H2O2). The chemical structure of modified pullulan was analyzed with ATR-FTIR and (1)H NMR spectroscopies. Rheological properties were tested by measuring storage modulus with varying H2O2, HRP, polymer solution concentrations and tyramine substitution rates. Cell viability and animal tests were performed. The modified pullulan hydrogel is an invaluable advance in anti-adhesion agents.

Fabrication and characterization of thermoresponsive polystyrene nanofibrous mats for cultured cell recovery.

Rapid cell growth and rapid recovery of intact cultured cells are an invaluable technique to maintain the biological functions and viability of cells. To achieve this goal, thermoresponsive polystyrene (PS) nanofibrous mat was fabricated by electrospinning of PS solution, followed by the graft polymerization of thermoresponsive poly(N-isopropylacrylamide)(PIPAAm) on PS nanofibrous mats. Image analysis of the PS nanofiber revealed a unimodal distribution pattern with 400 nm average fiber diameter. Graft polymerization of PIPAAm on PS nanofibrous mats was confirmed by spectroscopic methods such as ATR-FTIR, ESCA, and AFM. Human fibroblasts were cultured on four different surfaces, PIPAAm-grafted and ungrafted PS dishes and PIPAAm-grafted and ungrafted PS nanofibrous mats, respectively. Cells on PIPAAm-grafted PS nanofibrous mats were well attached, spread, and proliferated significantly much more than those on other surfaces. Cultured cells were easily detached from the PIPAAm-grafted surfaces by decreasing culture temperature to 20 °C, while negligible cells were detached from ungrafted surfaces. Moreover, cells on PIPAAm-grafted PS nanofibrous mats were detached more rapidly than those on PIPAAm-grafted PS dishes. These results suggest that thermoresponsive nanofibrous mats are attractive cell culture substrates which enable rapid cell growth and recovery from the culture surface for application to tissue engineering and regenerative medicine.

Directing cell migration in continuous microchannels by topographical amplification of natural directional persistence.

Discrete micropatterns on biomaterial surfaces can be used to guide the direction of mammalian cell movement by orienting cell morphology. However, guiding cell assembly in three-dimensional scaffolds remains a challenge. Here we demonstrate that the random motions of motile cells can be rectified within continuous microchannels without chemotactic gradients or fluid flow. Our results show that uniform width microchannels with an overhanging zigzag design can induce polarization of NIH3T3 fibroblasts and human umbilical vein endothelial cells by expanding the cell front at each turn. These continuous zigzag microchannels can guide the direction of cell movement even for cells with altered intracellular signals that promote random movement. This approach for directing cell migration within microchannels has important potential implications in the design of scaffolds for tissue engineering.

Culture of bovine articular chondrocytes in funnel-like collagen-PLGA hybrid sponges.

Three-dimensional porous scaffolds play an important role in tissue engineering and regenerative medicine. Structurally, these porous scaffolds should have an open and interconnected porous architecture to facilitate a homogeneous cell distribution. Moreover, the scaffolds should be mechanically strong to support new tissue formation. We developed a novel type of funnel-like collagen sponge using embossing ice particulates as a template. The funnel-like collagen sponges could promote the homogeneous cell distribution, ECM production and chondrogenesis. However, the funnel-like collagen sponges deformed during cell culture due to their weak mechanical strength. To solve this problem, we reinforced the funnel-like collagen sponges with a knitted poly(D,L-lactic-co-glycolic acid) (PLGA) mesh by hybridizing these two types of materials. The hybrid scaffolds were used to culture bovine articular chondrocytes. The cell adhesion, distribution, proliferation and chondrogenesis were investigated. The funnel-like structure promoted the even cell distribution and homogeneous ECM production. The PLGA knitted mesh protected the scaffold from deformation during cell culture. Histological and immunohistochemical staining and cartilaginous gene expression analyses revealed the cartilage-like properties of the cell/scaffold constructs after in vivo implantation. The hybrid scaffold, composed of a funnel-like collagen sponge and PLGA mesh, would be a useful tool for cartilage tissue engineering.

Preparation of Open Porous Hyaluronic Acid Scaffolds for Tissue Engineering Using the Ice Particulate Template Method.

A novel method to fabricate highly interconnected porous hyaluronic acid (HA) scaffolds with open surface pore structures was developed by using embossed ice particulates as a template. HA sponges were cross-linked by water-soluble carbodiimide (WSC) and the optimal cross-linking condition was analyzed by infrared spectroscopy. Cross-linking with 50 mM WSC in a 90% (v/v) ethanol/water solvent mixture assured the highest degree of cross-linking and most stable structure and, therefore, was used to cross-link the HA sponges. Observation with a scanning electron microscope showed that the HA scaffolds had funnel-like porous structures. There were large, open pores on the top surfaces and inner bulk pores under the top surface of the funnel-like HA sponges. The inner bulk pores were interconnected with the large, top surface pores and extended into the whole sponge. The pore morphology and density of the large, top surface pores were dependent on the dimension and density of the ice particulates. The size of the inner bulk pores was dependent on the freezing temperature. The funnel-like pore structures of the HA sponges facilitated cell penetration into the inner pores of the sponges and resulted in homogenous cell distribution in the sponges.

Cartilage tissue engineering using funnel-like collagen sponges prepared with embossing ice particulate templates.

Three-dimensional porous scaffolds of collagen have been widely used for tissue engineering and regenerative medicine. In this study, we fabricated funnel-like collagen sponges with open surface pore structures by a freeze-drying method that used embossing ice particulates as a template. By controlling the size of the ice particulates and the temperature of freezing, collagen sponges with different pore structures were prepared. To investigate the effects of different pore structures on cartilage regeneration, the funnel-like collagen sponges were used to culture bovine articular chondrocytes. Scaffolds that were prepared with 400microm ice particulate templates and a freezing temperature of -3 degrees C resulted in the best cell distribution, ECM production, and chondrogenesis. Although funnel-like collagen sponges prepared with 400microm ice particulate templates and a freezing temperature of -1 degrees C and 720microm ice particulates and a freezing temperature of -3 degrees C, showed even cell distribution, the cell seeding efficiencies and sGAG amount per cell were low. However, the scaffolds prepared with 400microm ice particulate templates and a freezing temperature of -5 degrees C or -10 degrees C showed a limited effect on the improvement of cell distribution and chondrogenesis. Control collagen sponges without ice particulates failed to support the formation of homogenous cartilage-like tissue. These results indicate that funnel-like collagen sponges were superior to control collagen sponges and that scaffolds prepared with 400microm ice particulate templates at -3 degrees C were optimal for cartilage tissue engineering.

Electrospinning of microbial polyester for cell culture.

Biodegradable and biocompatible poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a copolymer of microbial polyester, was fabricated as a nanofibrous mat by electrospinning. The specific surface area and the porosity of electrospun PHBV nanofibrous mat were determined. When the mechanical properties of flat film and electrospun PHBV nanofibrous mats were investigated, both the tensile modulus and strength of electrospun PHBV were less than those of cast PHBV film. However, the elongation ratio of nanofiber mat was higher than that of the cast film. The structure of electrospun nanofibers using PHBV-trifluoroethanol solutions depended on the solution concentrations. When x-ray diffraction patterns of bulk PHBV before and after electrospinning were compared, the crystallinity of PHBV was not significantly affected by the electrospinning process. Chondrocytes adhered and grew on the electrospun PHBV nanofibrous mat better than on the cast PHBV film. Therefore, the electrospun PHBV was considered to be suitable for cell culture.