Jin San Choi

THE INFLUENCE OF ELECTROSPUN ALIGNED POLY(EPSILON-CAPROLACTONE)/COLLAGEN NANOFIBER MESHES ON THE FORMATION OF SELF-ALIGNED SKELETAL MUSCLE MYOTUBES

Current treatment options for restoring large skeletal muscle tissue defects due to trauma or tumor ablation are limited by the host muscle tissue availability and donor site morbidity of muscle flap implantation. Creation of implantable functional muscle tissue that could restore muscle defects may bea possible solution. To engineer functional muscle tissue for reconstruction, scaffolds that mimic native fibers need to be developed. In this study we examined the feasibility of using poly(epsilon-caprolactone) (PCL)/collagen based nanofibers using electrospinning as a scaffold system for implantable engineered muscle. We investigated whether electrospun nanofibers could guide morphogenesis of skeletal muscle cells and enhance cellular organization. Nanofibers with different fiber orientations were fabricated by electrospinning with a blend of PCL and collagen. Human skeletal muscle cells (hSkMCs) were seeded onto the electrospun PCL/collagen nanofiber meshes and analyzed for cell adhesion, proliferation and organization. Our results show that unidirectionally oriented nanofibers significantly induced muscle cell alignment and myotube formation as compared to randomly oriented nanofibers. The aligned composite nanofiber scaffolds seeded with skeletal muscle cells may provide implantable functional muscle tissues for patients with large muscle defects.

Bilayered scaffold for engineering cellularized blood vessels.

Vascular scaffolds fabricated by electrospinning poly(epsilon-caprolactone) (PCL) and collagen have been designed to provide adequate structural support as well as a favorable adhesion substrate for vascular cells. However, the presence of small-sized pores limits the efficacy of smooth muscle cells (SMC) seeding, as these cells could not adequately infiltrate into the scaffolds. To overcome this challenge, we developed a bilayered scaffolding system that provides different pore sizes to facilitate adequate cellular interactions. Based on the fact that pore size increases with the increase in fiber diameter, four different fiber diameters (ranging 0.27-4.45 mum) were fabricated by electrospinning with controlled parameters. The fabricated scaffolds were examined by evaluating cellular interactions, and the mechanical properties were measured. Endothelial cells (EC) seeded on nanoscaled fibers showed enhanced cellular orientation and focal adhesion. Conversely, fabrication of a larger fiber diameter improved SMC infiltration into the scaffolds. To incorporate both of these properties into a scaffold, bilayered vascular scaffolds were produced. The inner layer yielded small diameter fibers and the outer layer provided large diameter fibers. We show that the bilayered scaffolds permit EC adhesion on the lumen and SMC infiltration into the outer layer. This study suggests that the use of bilayered scaffolds may lead to improved vessel formation.

Bioengineering endothelialized neo-corneas using donor-derived corneal endothelial cells and decellularized corneal stroma.

Corneal transplantation is a common transplant procedure performed to improve visual acuity by replacing the opaque or distorted host tissue by clear healthy donor tissue. However, its clinical utility is limited due to a lack of high quality donor corneas. Bioengineered neo-corneas, created using an expandable population of human donor-derived corneal endothelial cells (HCEC), could address this current shortage. The objectives of this study were to establish HCEC isolation and culture protocols and to investigate the feasibility of bioengineering corneal tissue constructs by seeding the cells on decellularized human corneal stroma. HCECs were removed from the discarded corneas of eye donors by enzymatic digestion. Cells were expanded and evaluated for their expression of Na(+)/K(+)-ATPase and zona occludens-1 (ZO-1). Donor corneal stromas were cut to 120-200 microm thickness slices using a microtome and then decellularized. Extracellular matrix components and mechanical properties of the scaffolds were measured after decellularization. To engineer neo-corneas, 130 HCEC/mm(2) were seeded on decellularized human corneal stromas. The resulting constructs were placed in growth medium for 14 days and then analyzed using scanning electron microscopy (SEM), histology, and immunocytochemistry. Seeded cells retain expression of the functional markers Na(+)/K(+)-ATPase and ZO-1 and constructs have biomechanical properties similar to those of normal corneas. These results indicate that construction of neo-corneas, using HCECs derived from discarded donor corneas and decellularized thin-layer corneal stromas, may create a new source of high quality corneal tissue for transplantation.

Factors affecting successful isolation of human corneal endothelial cells for clinical use.

Corneal transplantation is a common transplant procedure used to improve visual acuity by replacing the opaque or distorted host tissue with clear healthy donor tissue. However, its clinical utility is limited due to a lack of donor supply of high-quality corneas. Bioengineered neocorneas, created using an expandable population of human donor-derived corneal endothelial cells (HCECs), could address this shortage. Thus, the objective of this study was to evaluate HCEC sourcing with various isolation methods, including enzymatic digestion, culture medium components, and adhesive proteins. HCECs were obtained from corneas obtained from various aged donors after endothelial keratoplasty. Under a dissection microscope, the Descemets membrane, including the attached corneal endothelium, was stripped from the stroma, and the cells were isolated and expanded by explant culture or by enzymatic digestion with enzymes such as collagenase II, dispase, or trypsin. In order to improve the initial cell attachment, tissue culture plates were coated with collagen IV, fibronectin, or fibronectin–collagen combination coating mix (FNC) before cell plating. We were able to successfully obtain HCECs from 32% (86/269) of donor corneas. Donor age and isolation method influenced the characteristics of the resulting in vitro HCEC culture. Under all conditions tested, FNC-coated plates showed higher quality cultures than the other coatings tested. These results suggest that donor age and HCEC isolation methodology are the two factors that most directly affect the quality of the resulting HCEC culture in vitro. These factors should guide the methodological development for the clinical expansion of HCECs for the generation of bioengineered neocorneas.

In Situ Tissue Regeneration of Renal Tissue Induced by Collagen Hydrogel Injection

Host stem/progenitor cells can be mobilized and recruited to a target location using biomaterials, and these cells may be used for in situ tissue regeneration. The objective of this study was to investigate whether host biologic resources could be used to regenerate renal tissue in situ. Collagen hydrogel was injected into the kidneys of normal mice, and rat kidneys that had sustained ischemia/reperfusion injury. After injection, the kidneys of both animal models were examined up to 4 weeks for host tissue response. The infiltrating host cells present within the injection regions expressed renal stem/progenitor cell markers, PAX‐2, CD24, and CD133, as well as mesenchymal stem cell marker, CD44. The regenerated renal structures were identified by immunohistochemistry for renal cell specific markers, including synaptopodin and CD31 for glomeruli and cytokeratin and neprilysin for tubules. Quantitatively, the number of glomeruli found in the injected regions was significantly higher when compared to normal regions of renal cortex. This phenomenon occurred in normal and ischemic injured kidneys. Furthermore, the renal function after ischemia/reperfusion injury was recovered after collagen hydrogel injection. These results demonstrate that introduction of biomaterials into the kidney is able to facilitate the regeneration of glomerular and tubular structures in normal and injured kidneys. Such an approach has the potential to become a simple and effective treatment for patients with renal failure. Stem Cells Translational Medicine 2018;7:241–250