Indong Jun

The stimulation of myoblast differentiation by electrically conductive sub-micron fibers.

Myotubes assemble with bundles of myofibers to form the structural units in skeletal muscle. Therefore, myotube formation plays an important role in restoring muscular functions, and substrates to promote the differentiation of myoblasts to myotubes need to be developed for muscle tissue engineering. In this study, we developed electrically conductive composite fibers of poly(L-lactide-co-epsilon-caprolactone) (PLCL) blended with polyaniline (PANi) using an electrospinning method, and then investigated the effect of these composite fibers on the differentiation of myoblasts. The prepared PLCL/PANi fibers showed no significant difference in fiber diameter or contact angle, regardless of the incorporation of PANi. The fibers containing 30% PANi (PLCL/PANi-30) maintained elastic properties of maximum elongation at break (160+/-14.4%). The composite fibers were cytocompatible, as the DNA content on each fiber was similar for up to 8 days of C2C12 myoblast culture. After 4 days of culture, the number of cells positive for sarcomeric myosin was 3.6-times greater on the electrically conductive fibers (21+/-1 and 19+/-2 for PLCL/PANi-15 and -30 fibers, respectively) than on the PLCL/PANi-0 fibers (6+/-2). Furthermore, the level of myogenin expression detected on day 8 of culture on PLCL/PANi-15 was approximately 1.6-fold greater than the PLCL/PANi-0 fibers. Similar results were observed for the expression of other genes including troponin T (2-fold greater) and the myosin heavy chain gene (3-fold greater). These results indicate that electrically conductive substrates can modulate the induction of myoblasts into myotube formation without additional electrical stimulation, suggesting that these fibers may have potential as a temporary substrate for skeletal tissue engineering.

Mussel-inspired surface modification of poly(L-lactide) electrospun fibers for modulation of osteogenic differentiation of human mesenchymal stem cells.

Development of biomaterials to control the fate of stem cells is important for stem cell based regeneration of bone tissue. The objective of this study is to develop functionalized electrospun fibers using a mussel-inspired surface coating to regulate adhesion, proliferation and differentiation of human mesenchymal stem cells (hMSCs). We prepared poly(L-lactide) (PLLA) fibers coated with polydopamine (PD-PLLA). The morphology, chemical composition, and surface properties of fiber were characterized by SEM, AFM, XPS, Raman spectra and water contact angle measurements. Incubation of fibers in dopamine solution for 1h resulted in formation of polydopamine with only negligible effects on the roughness and hydrophobicity of the fibers. However, PD-PLLA fibers modulated hMSC responses in several aspects. Firstly, adhesion and proliferation of hMSCs cultured on PD-PLLA were significantly enhanced relative to those on PLLA. In addition, the ALP activity of hMSCs cultured on PD-PLLA (1.74±0.14 nmole/DNA/30 min) was significantly higher than on PLLA (0.97±0.07 nmole/DNA/30 min). hMSCs cultured on PD-PLLA showed up-regulation of genes associated with osteogenic differentiation as well as angiogenesis. Furthermore, the calcium deposition from hMSCs cultured on PD-PLLA (41.60±1.74 μg) was significantly greater than that on PLLA (30.15±1.21 μg), which was double-confirmed by alizarin red S staining. Our results suggest that the bio-inspired coating synthetic degradable polymer can be used as a simple technique to render the surface of synthetic biodegradable fibers to be active for directing the specific responses of hMSCs.

Polydopamine-mediated immobilization of multiple bioactive molecules for the development of functional vascular graft materials

In this study, we introduced a simple method for polydopamine-mediated immobilization of dual bioactive factors for the preparation of functionalized vascular graft materials. Polydopamine was deposited on elastic and biodegradable poly(lactic acid-co-ɛ-caprolactone) (PLCL) films, and a cell adhesive RGD-containing peptide and basic fibroblast growth factor were subsequently immobilized by simple dipping. We used an enzyme-linked immunosorbent assay and fluorescamine assay to confirm that we had stably immobilized bioactive molecules on the polydopamine-coated PLCL film in a reaction time-dependent manner. When human umbilical vein endothelial cells (HUVEC) were cultured on the prepared substrates, the number of adherent cells and proliferation of HUVEC for up to 14 days were greatest on the film immobilized with dual factors. On the other hand, the film immobilized with RGD peptide exhibited the highest migration speed compared to the other groups. The expression of cluster of differentiation 31 and von Willebrand factor, which indicates maturation of endothelial cells, was highly stimulated in the dual factor-immobilized group, and passively adsorbed factors showed a negligible effect. The immobilization of bioactive molecules inspired by polydopamine was successful, and adhesion, migration, proliferation and differentiation of HUVEC were synergistically accelerated by the presence of multiple signaling factors. Collectively, our results have demonstrated that a simple coating with polydopamine enables the immobilization of multiple bioactive molecules for preparation of polymeric functionalized vascular graft materials.

The development of genipin-crosslinked poly(caprolactone) (PCL)/gelatin nanofibers for tissue engineering applications.

Composite nanofibers of poly(caprolactone) (PCL) and gelatin crosslinked with genipin are prepared. The contact angles and mechanical properties of crosslinked PCL-gelatin nanofibers decrease as the gelatin content increases. The proliferation of myoblasts is higher in the crosslinked PCL-gelatin nanofibers than in the PCL nanofibers, and the formation of myotubes is only observed on the crosslinked PCL-gelatin nanofibers. The expression level of myogenin, myosin heavy chain, and troponin T genes is increased as the gelatin content is increased. The results suggest that PCL-gelatin nanofibers crosslinked with genipin can be used as a substrate to modulate proliferation and differentiation of myoblasts, presenting potential applications in muscle tissue engineering.

In situ hydrogelation and RGD conjugation of tyramine-conjugated 4-arm PPO–PEO block copolymer for injectable bio-mimetic scaffolds

RGD-conjugated Tetronic–tyramine (RGD–Tet–TA) hydrogels were formed in situ, and cell attachment and spreading on the hydrogels were evaluated for an injectable tissue-regenerative scaffold. The hydrogels were prepared by simple mixing GRGDGGGGGY (RGD–Y) in a tyramine-conjugated Tetronic (Tet–TA) polymer solution in the presence of horseradish peroxidase (HRP) and hydrogen peroxide (H2O2). The physico-chemical properties such as mechanical properties and swelling ratio were evaluated and the conjugated RGD concentration and distribution were measured using fluoresamine assay and fluorescence observation. An in vitro cell study was investigated using osteoblasts (MC3T3–E1) on the hydrogel surfaces for 24 h, in which the viability and morphology of the adhered cells were observed using live/dead and F-actin assays. The results demonstrate that the in situ conjugation of RGD peptides to tyrosine residues enhanced the attachment and spreading of osteoblasts in a surface density-dependent manner. Therefore, this study suggests that in situ conjugation of the RGD peptide to injectable hydrogels via an enzymatic reaction may be an efficient tool to prepare in situ forming bio-mimetic hydrogels for tissue regenerative medicine.

Electroactive Electrospun Polyaniline/Poly[(L-lactide)-co-(ε-caprolactone)] Fibers for Control of Neural Cell Function

Blends of PAni and PLCL are electrospun to prepare uniform fibers for the development of electrically conductive, engineered nerve grafts. PC12 cell viability is significantly higher on RPACL fibers than on PLCL-only fibers, and the electrical conductivity of the fibers affects the differentiation of PC12 cells; the number of cells positively-stained and their expression level are significantly higher on RPACL fibers. PC12 cell bodies display an oriented morphology with outgrowing neurites. On RPACL fibers, the expression level of paxillin, cdc-42, and rac is positively affected and proteins including RhoA and ERK exist as more activated state. These results suggest that electroactive fibers may hold promise as a guidance scaffold for neuronal tissue engineering.

Creating Hierarchical Topographies on Fibrous Platforms Using Femtosecond Laser Ablation for Directing Myoblasts Behavior.

Developing an artificial extracellular matrix that closely mimics the native tissue microenvironment is important for use as both a cell culture platform for controlling cell fate and an in vitro model system for investigating the role of the cellular microenvironment. Electrospinning, one of the methods for fabricating structures that mimic the native ECM, is a promising technique for creating fibrous platforms. It is well-known that align or randomly distributed electrospun fibers provide cellular contact guidance in a single pattern. However, native tissues have hierarchical structures, i.e., topographies on the micro- and nanoscales, rather than a single structure. Thus, we fabricated randomly distributed nanofibrous (720 ± 80 nm in diameter) platforms via a conventional electrospinning process, and then we generated microscale grooves using a femtosecond laser ablation process to develop engineered fibrous platforms with patterned hierarchical topographies. The engineered fibrous platforms can regulate cellular adhesive morphology, proliferation, and distinct distribution of focal adhesion proteins. Furthermore, confluent myoblasts cultured on the engineered fibrous platforms revealed that the direction of myotube assembly can be controlled. These results indicate that our engineered fibrous platforms may be useful tools in investigating the roles of nano- and microscale topographies in the communication between cells and ECM.

Rapid Transfer of Endothelial Cell Sheet Using a Thermosensitive Hydrogel and Its Effect on Therapeutic Angiogenesis

In this study, thermosensitive hydrogels incorporated with multiple cell-interactive factors were developed as a substrate to form monolayer of human umbilical vein endothelial cells (HUVECs) that can be detached and transferrable to target sites as a cell-sheet in response to temperature change. The cell adhesive peptide (RGD) and growth factor (bFGF) covalently incorporated within the hydrogel significantly enhanced adhesion and proliferation of HUVECs, allowing for the formation of their confluent monolayer. Meanwhile, the precisely controllable change in the size of the hydrogels was observed by a repeated increase and decrease in temperature from 37 to 4 °C. By exploiting this unique behavior, the detachment and transfer of HUVEC sheet confluently cultured at 37 °C was rapidly induced within 10 min by expansion of the hydrogels when the temperature was decreased to 4 °C. The transferred cell sheet was highly viable and maintained robust cell-cell junction. Finally, the process of cell sheet transfer was directly applied onto an ischemic injury in the hind limb of mice. The transplanted HUVECs as a sheet retarded tissue necrosis over 14 days in comparison with that of direct injection of the same number of cells. Our results suggest that the developed multifunctional Tetronic-tyramine hydrogels could serve as an ideal substrate to modulate the formation of an endothelial cell layer that could potentially be utilized to treat peripheral arterial disease.

Therapeutic angiogenesis by a myoblast layer harvested by tissue transfer printing from cell-adhesive, thermosensitive hydrogels.

Peripheral arterial disease (PAD) is characterized by the altered structure and function of arteries caused by accumulated plaque. There have been many studies on treating this disease by the direct injection of various types of therapeutic cells, however, the low cell engraftment efficiency and diffusion of the transplanted cells have been major problems. In this study, we developed an approach (transfer printing) to deliver monolayer of cells to the hindlimb ischemic tissue using thermosensitive hydrogels, and investigated its efficacy in long term retention upon transplantation and therapeutic angiogenesis. We first investigated the in vitro maintenance of robust cell-cell contacts and stable expression of the ECM proteins in myoblast layer following transfer printing process. In order to confirm the therapeutic effect of the myoblasts in vivo, we cultured a monolayer of C2C12 myoblasts on thermosensitive hydrogels, which was then transferred to the hindlimb ischemia tissue of athymic mice directly from the hydrogel by conformal contact. The transferred myoblast layer was retained for a longer period of time than an intramuscularly injected cell suspension. In addition, the morphology of the mice and laser Doppler perfusion (28 days after treatment) supported that the myoblast layer enhanced the therapeutic effects on the ischemic tissue. In summary, the transplantation of the C2C12 myoblast layer using a tissue transfer printing method could represent a new approach for the treatment of PAD by therapeutic angiogenesis.

Genetically Engineered Myoblast Sheet for Therapeutic Angiogenesis

Peripheral arterial disease is a common manifestation of systemic atherosclerosis, which results in more serious consequences of ischemic events in peripheral tissues such as the lower extremities. Cell therapy has been tested as a treatment for peripheral ischemia that functions by inducing angiogenesis in the ischemic region. However, the poor survival and engraftment of transplanted cells limit the efficacy of cell therapy. In order to overcome such challenges, we applied genetically engineered cell sheets using a cell-interactive and thermosensitive hydrogel and nonviral polymer nanoparticles. C2C12 myoblast sheets were formed on Tetronic-tyramine (Tet-TA)-RGD hydrogel prepared through a highly efficient and noncytotoxic enzymatic reaction. The myoblast sheets were then transfected with vascular endothelial growth factor (VEGF) plasmids using poly(β-amino ester) nanoparticles to increase the angiogenic potential of the sheets. The transfection increased the VEGF expression and secretion from the C2C12 sheets. The enhanced angiogenic effect of the VEGF-transfected C2C12 sheets was confirmed using an in vitro capillary formation assay. More importantly, the transplantation of the VEGF-transfected C2C12 sheets promoted the formation of capillaries and arterioles in ischemic muscles, attenuated the muscle necrosis and fibrosis progressed by ischemia, and eventually prevented ischemic limb loss. In conclusion, the combination of cell sheet engineering and genetic modification can provide more effective treatment for therapeutic angiogenesis.