Youngmee Jung

Manufacture of elastic biodegradable PLCL scaffolds for mechano-active vascular tissue engineering

A soft and very elastic poly(lactide-co-ε-caprolactone) (PLCL)(50:50, M n 185 × 103) was synthesized. Tubular scaffolds were prepared by an extrusion-particulate leaching method for mechano-active vascular tissue engineering. The copolymer was very flexible but completely rubber-like elastic. Even the high porous PLCL scaffolds (90% salt wt) exhibited 200% elongation, but recovery over 85% in a tensile test. Moreover, the PLCL scaffolds maintained their high elasticity also in culture media under cyclic mechanical strain conditions. The highly porous scaffold (90% salt wt) withstood for an initial 1 week without any deformation and sustained for 2 weeks in culture media under cyclic stress of 10% amplitude and at 1 Hz frequency which are similar to the natural vascular conditions. Vascular smooth muscle cells (VSMCs) were seeded on to the PLCL scaffolds. The cell adhesion and proliferation on the scaffolds of various pore-size were increased with increasing pore size. For the pore sizes of 50-100 μm, 100-150 μm, 150-200 μm and 200-250 μm, the ratios of cell numbers were about 1:1.2:1.9:2.2, respectively, at both 12 h and 5 days. Similarly, the higher porous scaffolds exhibited more cell adhesion and proliferation compared to lower porous one, where the effect was more pronounced in the longer proliferation period. SMC-seeded scaffolds were implanted subcutaneously in athymic nude mice to confirm the biocompatibility. Such a high elastic property and proper biocompatibility to SMCs of PLCL scaffolds prepared in this study will be very useful to engineer SM-containing tissues such as blood vessels under mechanically dynamic environments (mechano-active tissue engineering).

Rapid formation of multicellular spheroids in double-emulsion droplets with controllable microenvironment

An attractive option for tissue engineering is to use of multicellular spheroids as microtissues, particularly with stem cell spheroids. Conventional approaches of fabricating spheroids suffer from low throughput and polydispersity in size, and fail to supplement cues from extracellular matrix (ECM) for enhanced differentiation. In this study, we report the application of microfluidics-generated water-in-oil-in-water (w/o/w) double-emulsion (DE) droplets as pico-liter sized bioreactor for rapid cell assembly and well-controlled microenvironment for spheroid culture. Cells aggregated to form size-controllable (30–80 μm) spheroids in DE droplets within 150 min and could be retrieved via a droplet-releasing agent. Moreover, precursor hydrogel solution can be adopted as the inner phase to produce spheroid-encapsulated microgels after spheroid formation. As an example, the encapsulation of human mesenchymal stem cells (hMSC) spheroids in alginate and alginate-arginine-glycine-aspartic acid (-RGD) microgel was demonstrated, with enhanced osteogenic differentiation further exhibited in the latter case.

The enhancement of mature vessel formation and cardiac function in infarcted hearts using dual growth factor delivery with self-assembling peptides

For successful treatment of myocardial infarction (MI), it is important to prevent cardiac fibrosis and maintain cardiac function by protecting cardiomyocytes and inducing angiogenesis. To establish functional and stable vessels, various growth factors, ones stimulating both endothelial cells (EC) and vascular smooth muscle cells (VSMC), are required. Self-assembling peptides form fibers (<10 nm) and provide 3-dimensional microenvironments that can recruit EC and VSMC to promote vascularization and long-term delivery of growth factors. Here we demonstrate myocardial protection of infarcted heart using dual growth factor delivery with self-assembling peptides. After coronary artery ligation in rats, growth factors (PDGF-BB and FGF-2) with self-assembling peptides were injected. There were 6 rats in each group. Hearts were harvested at 4 and 8 weeks for functional and histological analysis. Infarct size and cardiomyocyte apoptosis in dual growth factors along with self-assembling peptides group were dramatically reduced compared to sham. The capillary and arterial density of this group recovered with angiogenic synergism and cardiac functions had almost recovered. In conclusion, dual growth factors along with self-assembling peptides lead to myocardial protection, stable vessel formation, and improvement in cardiac function.

Cartilage regeneration with highly-elastic three-dimensional scaffolds prepared from biodegradable poly(l-lactide-co-ɛ-caprolactone)

Compressive mechanical stimuli are crucial in regenerating cartilage with tissue engineering, which creates a need for scaffolds that can maintain their mechanical integrity while delivering mechanical signals to adherent cells during strain applications. With these goals in mind, the aim of this study was to develop a mechano-active scaffold that facilitated effective cartilaginous tissue formation under dynamic physiological environments. Using a gel-pressing method, we fabricated a biodegradable and highly-elastic scaffold from poly(L-lactide-co-epsilon-caprolactone) (PLCL; 5:5), with 85% porosity and a 300-500-microm pore size, and we compared it to control scaffolds made of rigid polylactide (PLA) or poly(lactide-co-glycolide) (PLGA). After tensile mechanical tests and recovery tests confirmed the elasticity of the PLCL scaffolds, we seeded them with rabbit chondrocytes, cultured them in vitro, and subcutaneously implanted them into nude mice for up to eight weeks. The PLCL scaffolds possessed a completely rubber-like elasticity, were easily twisted and bent, and exhibited an almost complete (over 97%) recovery from applied strain (up to 500%); the control PLA scaffolds showed little recovery. In vitro and in vivo accumulations of extracellular matrix on the cell-PLCL constructs demonstrated that they could not only sustain but also significantly enhance chondrogenic differentiation. Moreover, the mechanical stimulation of the dynamic in vivo environment promoted deposition of the chondral extracellular matrix onto the PLCL. In contrast, on the PLA scaffolds, most of the chondrocytes had de-differentiated and formed fibrous tissues. In a rabbit defect model, the groups treated with PLCL scaffolds exhibited significantly enhanced cartilage regeneration compared to groups harboring an empty control or PLGA scaffolds. These results indicated that the mechano-active PLCL scaffolds effectively delivered mechanical signals associated with biological environments to adherent chondrocytes, suggesting that these elastic PLCL scaffolds could successfully be used for cartilage regeneration.

In situ chondrogenic differentiation of human adipose tissue-derived stem cells in a TGF-β1 loaded fibrin–poly(lactide-caprolactone) nanoparticulate complex

When conducting cartilage tissue engineering with stem cells, it is well known that chemical and physical stimulations are very important for the induction and maintenance of chondrogenesis. In this study, we induced chondrogenic differentiation of human adipose tissue-derived stem cells (hASCs) in situ by effective stimulation via the continuous controlled release of TGF-beta1 from a heparin-functionalized nanoparticle-fibrin-poly(lactide-co-caprolactone) (PLCL) complex. PLCL scaffolds were fabricated with 85% porosity and 300-500 microm pore size by a gel-pressing method. Heparin-functionalized nanoparticles were prepared by a solvent-diffusion method, composed of poly(lactide-co-glycolide) (PLGA), Pluronic F-127, and heparin, and then TGF-beta1 was loaded to the nanoparticles. A mixture of hASCs, fibrin gels and TGF-beta1 loaded nanoparticles was then seeded onto PLCL scaffolds and cultured in vitro, after which they were subcutaneously implanted into nude mice for up to five weeks. The results of in vitro and in vivo studies revealed that chondrogenic differentiation of the hASCs on the complex was induced and sustained by continuous stimulation by TGF-beta1 from the heparin-functionalized nanoparticles. In addition, there was no significant difference between the predifferentiation condition prior to incubation in chondrogenic medium and the proliferation condition, which suggests that in situ chondrogenic differentiation of hASCs was induced by the TGF-beta1 loaded nanoparticles. Consequently, the hybridization of fibrin and PLCL scaffolds for three-dimensional spatial organization of cells and the effective delivery of TGF-beta1 using heparin-functionalized nanoparticles can induce hASCs to differentiate to a chondrogenic lineage and maintain their phenotypes.

Stem cell recruitment and angiogenesis of neuropeptide substance P coupled with self-assembling peptide nanofiber in a mouse hind limb ischemia model

For the successful treatment of ischemia, it is important to resupply sufficient blood into ischemic regions by inducing angiogenesis. Many stem cell transplantation studies have been reported to enhance angiogenesis, especially those relating to mesenchymal stem cells (MSCs); however cell transplantation has a number of limitations, such as the low rate of cell survival and donor cell shortage. In this study, we developed bioactive peptides by immobilizing substance P into self-assembling peptides, and their MSCs recruiting ability and therapeutic effects were evaluated by using ischemic hind limb models. Limb ischemia was produced in athymic mice, and 1% (wt/vol) peptides were injected into ischemic sites (n = 6 in each group: ischemia, substance P, RADA16-II, RADA16-II + substance P, and RADA16-II + RADA-SP (bioactive peptides)). The tissues were harvested for histological analysis and tissue perfusion measurement at 1, 3, 7, and 28 days after injection. We observed that bioactive peptides assembled themselves (<10 nm nanofibers) and formed 3-dimensional (3D) microenvironments within ischemic regions. In the animal study, it was observed that by applying bioactive peptides, substance P continued to be released at 28 days, and consequently, MSCs were successfully recruited into ischemic regions. Bioactive peptides could prevent fibrosis, promote neovascularization, enhance tissue perfusion, and prevent limb salvages. Our results demonstrated that bioactive peptides are one of the most powerful tools for the treatment of ischemia, through their recruitment of autologous MSCs and promotion of angiogenesis without cells transplantation.

The correlation between human adipose-derived stem cells differentiation and cell adhesion mechanism.

In recent years, research in the areas of stem cells has dramatically increased, including studies of cellular adhesion to a substrate. We sought to determine the adhesive properties of human adipose-derived stem cells (hASCs) for extracellular matrix proteins. The adhesion of hASCs to collagens and laminin was completely inhibited by a monoclonal antibody, Mab 2253, which binds to the beta1 integrin subunit. These data indicate that hASC adhesion to collagens and laminin was exclusively mediated by an integrin. Cell adhesion on fibronectin (Fn) was inhibited by the heparin-binding peptide (HBP) in the presence of Mab 2253, but not by either Mab 2253 or HBP alone. These results indicate that both the beta1 subunit and the heparan sulfate proteoglycan participated in the cell adhesion to Fn. Microscopic views showed extensive spreading of hASCs cultured on Fn, whereas the cells maintained a round shape when cultured on a heparin-binding domain (HBD) substrate. hASCs differentiated into adipocytes, which stained positive for lipid vacuoles by Oil Red-O analysis, more readily on HBD substrate than on FN substrate. These results suggest that hASCs have an adhesion mechanism for the HBD of Fn and hASC morphology is controlled by the adhesion mechanism and strongly correlated with adipogenic differentiation.

Enhanced regeneration of the ligament-bone interface using a poly(L-lactide-co-ε-caprolactone) scaffold with local delivery of cells/BMP-2 using a heparin-based hydrogel.

Recently, the ligament-bone (LTB) junction has been emphasized for the effective transmission of mechanical force and the reduction in stress concentration between the soft ligament and hard bone tissue. The aim of this study was to regenerate an integrated LTB interface by inoculating LTB-relevant cells, isolated from fibrocartilage (FC) or ligament (LIG), separately into each designated region in a single porous cylindrical PLCL scaffold. An injectable, heparin-based hydrogel that has proved to be effective in the culture of chondrocytes as well as the sustained release of growth factor was employed to locally deliver fibrochondrocytes and osteoinductive bone morphogenetic protein-2 (BMP-2) into the FC region, to promote FC regeneration. In in vitro experiments the hydrogel-combined FC systems produced significantly larger amounts of calcium and glycosaminoglycans (GAGs), but less collagen and DNA than FC samples without the hydrogel and all LIG samples. After in vivo subcutaneous implantation in mice for 8 weeks the secreted calcium and GAG contents of the hydrogel-containing FC samples were superior or similar to those of the in vitro hydrogel-containing FC samples at 6 weeks. As a result of the enhanced production of calcium and GAG, the in vivo hydrogel-containing FC samples produced the highest compressive modulus among all samples. Histological and immunofluorescence analysis as well as elemental analysis also confirmed a denser and more homogeneous distribution of calcium, GAG, osteocalcin and neovascularization marker in the in vitro/in vivo hydrogel-containing FC systems than those without hydrogel. These results also show the beneficial effects of BMP-2 added using the hydrogel. In summary, the use of a heparin-based hydrogel for the local delivery of fibrochondrocytes and BMP-2 could accelerate the maturation and differentiation of LTB-specific FC tissues, and it was also possible to recreate the unique stratification of calcified FC and LIG tissues in a single porous PLCL scaffold in terms of both biochemical and biomechanical properties.

Cartilaginous tissue formation using a mechano-active scaffold and dynamic compressive stimulation

It is known that complex loading is involved in the development and maintenance of articular cartilage in the body. It means the compressive mechanical stimulation is a very important factor for formation of articular cartilage using a tissue-engineering technique. The objective of this study is to engineer cartilaginous constructs with mechano-active scaffolds and to evaluate the effect of dynamic compression for regeneration of cartilage. The mechano-active scaffolds were prepared from a very elastic poly(L-lactide-co-ε-caprolactone) (PLCL) with 85% porosity and 300–500 μm pore size using a gel-pressing method. The scaffold was seeded with 2 × 106 chondrocytes and the continuous compressive deformation of 5% strain was applied with 0.1 Hz for 10 days and 24 days, respectively. Then, the chondrocytes-seeded constructs were implanted subcutaneously into nude mice. Mechano-active scaffolds with complete rubber-like elasticity showed almost complete (over 97%) recovery at an applied strain of up to 500%. The amount of chondral extracellular matrix was increased significantly by mechanical stimulation on the highly elastic mechano-active scaffolds. Histological analysis showed the mechanically stimulated implants formed mature and well-developed cartilaginous tissue, as evidenced by the chondrocytes within lacunae and the abundant accumulation of sulfated GAGs. However, unhealthy lacunae shapes and hypertrophy forms were observed in the implants stimulated mechanically for 24 days, compared with those stimulated for 10 days. In conclusion, the proper periodical application of dynamic compression can encourage chondrocytes to maintain their phenotypes and enhance the production of GAGs, which would improve the quality of cartilaginous tissue formed both in vitro and in vivo.

Fabrication of a new tubular fibrous PLCL scaffold for vascular tissue engineering.

Biodegradable macroporous scaffolds have been developed for tissue-engineering applications. We fabricated and characterized a new tubular, macroporous, fibrous scaffold using a very elastic biodegradable co-polymer, poly(L-lactide-co-caprolactone) (PLCL, 5:5) in a gel-spinning process. A viscous PLCL solution was spun as a gel-phase under swirl-flow conditions and was subsequently fabricated to produce a tubular fibrous scaffold on a rotating cylindrical shaft in a methanol solution. The porosity and median pore size of the fibrous PLCL scaffolds were 55–75% and 120–150 μm, respectively, using a 5–10% PLCL solution. The use of a 7.5% (w/v) solution resulted in scaffolds with tensile strength and elastic modulus of 3.39 MPa and 1.22 MPa, respectively. The scaffolds exhibited 500–600% elongation-at-break. The tensile strength and modulus of fibrous PLCL scaffolds were proven to decrease on lowering the concentration of the PLCL spinning solution; however, the tensile strength and modulus of fibrous PLCL scaffolds, produced from 5% solutions, are approximately 4- and 5-times higher than those of extruded PLCL scaffolds. These properties indicated that the fibrous PLCL scaffolds were very elastic and mechanically strong. The scaffolds appeared to be well interconnected between the pores as determined by SEM imaging analysis. In addition, the cell-seeding efficiency was 2-fold higher using gel-spun scaffolds than using extruded scaffolds. These results suggest that the gel-spun fibrous PLCL scaffold is an excellent matrix for vascular tissue-engineering applications.