Young Min Ju

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.

Combined systemic and local delivery of stem cell inducing/recruiting factors for in situ tissue regeneration

Whereas the conventional tissue engineering strategy involves the use of scaffolds combined with appropriate cell types to restore normal functions, the concept of in situ tissue regeneration uses host responses to a target‐specific scaffold to mobilize host cells to a site of injury without the need for cell seeding. For this purpose, local delivery of bioactive molecules from scaffolds has been generally used. However, this approach has limited stem cell recruitment into the implants. Thus, we developed a combination of systemic delivery of substance P (SP) and local release of stromal‐derived factor‐1α (SDF‐1α) from an implant. In this study, we examined whether this combined system would significantly enhance recruitment of host stem cells into the implants. Flow cytometry and immunohistochemistry for CD29/CD45, CD146/α‐smooth muscle actin, and c‐kit demonstrated that this system significantly increased the number of stem cell‐like cells within the implants when compared with other systems. In vitro culture of the cells that had infiltrated into the scaffolds from the combined system confirmed that host stem cells were recruited into these implants and indicated that they were capable of differentiation into multiple lineages. These results indicate that this combined system may lead to more efficient tissue regeneration.—Ko, I. K., Ju, Y. M., Chen, T., Atala, A., Yoo, J. J., Lee, S.J. Combined systemic and local delivery of stem cell inducing/recruiting factors for in situ tissue regeneration. FASEB J. 26, 158–168 (2012). www.fasebj.org

Engineered small diameter vascular grafts by combining cell sheet engineering and electrospinning technology.

Tissue engineering offers an attractive approach to creating functional small-diameter (<5mm) blood vessels by combining autologous cells with a natural and/or synthetic scaffold under suitable culture conditions, which results in a tubular construct that can be implanted in vivo. We have previously developed a vascular scaffold fabricated by electrospinning poly(ε-caprolactone) (PCL) and type I collagen that mimics the structural and biomechanical properties of native vessels. In this study, we investigated whether a smooth muscle cell (SMC) sheet could be combined with the electrospun vascular scaffolds to produce a more mature smooth muscle layer as compared to the conventional cell seeding method. The pre-fabricated SMC sheet, wrapped around the vascular scaffold, provided high cell seeding efficiency (approx. 100%) and a mature smooth muscle layer that expressed strong cell-to-cell junction, connexin 43 (CX43), and contractile proteins, α smooth muscle actin (α-SMA) and myosin light chain kinase (MLCK). Moreover, bioreactor-associated preconditioning of the SMC sheet-combined vascular scaffold maintained high cell viability (95.9 ± 2.7%) and phenotypes and improved cellular infiltration and mechanical properties (35.7% of tensile strength, 47.5% of elasticity, and 113.2% of elongation at break).

End-to-side neurorrhaphy using an electrospun PCL/collagen nerve conduit for complex peripheral motor nerve regeneration.

In cases of complex neuromuscular defects, finding the proximal stump of a transected nerve in order to restore innervation to damaged muscle is often impossible. In this study we investigated whether a neighboring uninjured nerve could serve as a source of innervation of denervated damaged muscle through a biomaterial-based nerve conduit while preserving the uninjured nerve function. Tubular nerve conduits were fabricated by electrospinning a polymer blend consisting of poly(ε-caprolactone) (PCL) and type I collagen. Using a rat model of common peroneal injury, the proximal end of the nerve conduit was connected to the side of the adjacent uninjured tibial branch (TB) of the sciatic nerve after partial axotomy, and the distal end of the conduit was connected to the distal stump of the common peroneal nerve (CPN). The axonal continuity recovered through the nerve conduit at 8 weeks after surgery. Recovery of denervated muscle function was achieved, and simultaneously, the donor muscle, which was innervated by the axotomized TB also recovered at 20 weeks after surgery. Therefore, this end-to-side neurorrhaphy (ETS) technique using the electrospun PCL/collagen conduit appears to be clinically feasible and would be a useful alternative in instances where autologous nerve grafts or an adequate proximal nerve stump is unavailable.

A novel tissue-engineered trachea with a mechanical behavior similar to native trachea

A novel tissue-engineered trachea was developed with appropriate mechanical behavior and substantial regeneration of tracheal cartilage. We designed hollow bellows scaffold as a framework of a tissue-engineered trachea and demonstrated a reliable method for three-dimensional (3D) printing of monolithic bellows scaffold. We also functionalized gelatin sponge to allow sustained release of TGF-β1 for stimulating tracheal cartilage regeneration and confirmed that functionalized gelatin sponge induces cartilaginous tissue formation in vitro. A tissue-engineered trachea was then created by assembling chondrocytes-seeded functionalized gelatin sponges into the grooves of bellows scaffold and it showed very similar mechanical behavior to that of native trachea along with substantial regeneration of tracheal cartilage in vivo. The tissue-engineered trachea developed here represents a novel concept of tracheal substitute with appropriate mechanical behavior similar to native trachea for use in reconstruction of tracheal stenosis.

In situ regeneration of skeletal muscle tissue through host cell recruitment.

Standard reconstructive procedures for restoring normal function after skeletal muscle defects involve the use of existing host tissues such as muscular flaps. In many instances, this approach is not feasible and delays the rehabilitation process and restoration of tissue function. Currently, cell-based tissue engineering strategies have been used for reconstruction; however, donor tissue biopsy and ex vivo cell manipulation are required prior to implantation. The present study aimed to overcome these limitations by demonstrating mobilization of muscle cells into a target-specific site for in situ muscle regeneration. First, we investigated whether host muscle cells could be mobilized into an implanted scaffold. Poly(l-lactic acid) (PLLA) scaffolds were implanted in the tibialis anterior (TA) muscle of rats, and the retrieved scaffolds were characterized by examining host cell infiltration in the scaffolds. The host cell infiltrates, including Pax7+ cells, gradually increased with time. Second, we demonstrated that host muscle cells could be enriched by a myogenic factor released from the scaffolds. Gelatin-based scaffolds containing a myogenic factor were implanted in the TA muscle of rats, and the Pax7+ cell infiltration and newly formed muscle fibers were examined. By the second week after implantation, the Pax7+ cell infiltrates and muscle formation were significantly accelerated within the scaffolds containing insulin-like growth factor 1 (IGF-1). Our data suggest an ability of host stem cells to be recruited into the scaffolds with the capability of differentiating to muscle cells. In addition, the myogenic factor effectively promoted host cell recruitment, which resulted in accelerating muscle regeneration in situ.

Diaphragmatic muscle reconstruction with an aligned electrospun poly(ε-caprolactone)/collagen hybrid scaffold.

Large diaphragmatic muscle defects in congenital diaphragmatic hernia (CDH) are reconstructed by prosthetic materials or autologous grafts, which are associated with high complications and reherniation. In this study we examined the feasibility of using aligned electrospun poly(ε-caprolactone) (PCL)/collagen hybrid scaffolds for diaphragmatic muscle reconstruction. The hybrid scaffolds were implanted into a central left hemi-diaphragmatic defect (approximately 70% of the diaphragmatic tissue on the left side) in rats. Radiographic and magnetic resonance imaging (MRI) analyses showed no evidence of herniation or retraction up to 6 months after implantation. Histological and immunohistochemical evaluations revealed ingrowth of muscle tissue into the scaffolds. The mechanical properties of the retrieved diaphragmatic scaffolds were similar to those of normal diaphragm at the designated time points. Our results show that the aligned electrospun hybrid scaffolds allowed muscle cell migration and tissue formation. The aligned scaffolds may provide implantable functional muscle tissues for patients with diaphragmatic muscle defects.

CD133 antibody conjugation to decellularized human heart valves intended for circulating cell capture

The long term efficacy of tissue based heart valve grafts may be limited by progressive degeneration characterized by immune mediated inflammation and calcification. To avoid this degeneration, decellularized heart valves with functionalized surfaces capable of rapid in vivo endothelialization have been developed. The aim of this study is to examine the capacity of CD133 antibody-conjugated valve tissue to capture circulating endothelial progenitor cells (EPCs). Decellularized human pulmonary valve tissue was conjugated with CD133 antibody at varying concentrations and exposed to CD133 expressing NTERA-2 cl.D1 (NT2) cells in a microflow chamber. The amount of CD133 antibody conjugated on the valve tissue surface and the number of NT2 cells captured in the presence of shear stress was measured. Both the amount of CD133 antibody conjugated to the valve leaflet surface and the number of adherent NT2 cells increased as the concentration of CD133 antibody present in the surface immobilization procedure increased. The data presented in this study support the hypothesis that the rate of CD133(+) cell adhesion in the presence of shear stress to decellularized heart valve tissue functionalized by CD133 antibody conjugation increases as the quantity of CD133 antibody conjugated to the tissue surface increases.

Functional recovery of denervated muscle by neurotization using nerve guidance channels

Tissue‐engineered muscle has been proposed as a means of repairing volumetric muscle defects to restore anatomical and functional recovery. We have previously demonstrated that denervated muscle, which is analogous to engineered muscle construct, can be reinnervated by direct transplantation of host nerve (neurotization) in a rat model. However, the use of this approach is not possible if the length of host nerve is inadequate and cannot be mobilized to the insertion site of the engineered muscle. In this study we investigated whether neurotization coupled with nerve guidance channels would increase the regeneration of neuromuscular junctions (NMJs) in completely denervated muscle and encourage neurofunctional recovery. Seventy‐two Lewis rats were evaluated in three groups, a normal control group (n = 8), a denervated group (n = 32) and a neurotization coupled with nerve guidance group (n = 32). Neurofunctional behaviour and histological evaluations were performed at 4, 8, 12 and 20 weeks postoperatively. Extensor postural thrust (EPT) and compound muscle action potential (CMAP) amplitude were significantly improved in the nerve guidance group when compared with the denervated group, even though these values were different from those of the normal control group at 20 weeks postoperation. Regeneration of axons and NMJs was demonstrated histologically in the nerve guidance group. Neurotization coupled with nerve guidance channels leads to regeneration of axons and NMJs in completely denervated muscle. To our knowledge, this is the first report to show that nerve guidance can allow re‐innervation in denervated muscle containing long‐gap nerve injuries. Copyright

PD33-12 IN VIVO EVALUATION OF FUNCTIONALIZED MUSCLE SCAFFOLDS FOR RECONSTRUCTION

INTRODUCTION: Every tissue in the body contains some type of stem or progenitor cells. These cells are believed to be part of underlying regenerative machinery that is responsible for daily maintenance and repair of injured tissue. In this study, we aimed to regenerate muscle functionality following volumetric muscle loss through the use of a target-specific scaffolding system. The objectives of this study were to evaluate the functionalized decellularized tissue scaffolds on the host muscle cell migration and muscle function recovery in vivo.