Engineering Nanofiber Scaffolds with Biomimetic Cues for Differentiation of Skin-Derived Neural Crest-like Stem Cells to Schwann Cells.

Our laboratory reported the derivation of neural crest stem cell (NCSC)-like cells from the interfollicular epidermis of the neonatal and adult epidermis. These keratinocyte (KC)-derived Neural Crest (NC)-like cells (KC-NC) could differentiate into functional neurons, Schwann cells (SC), melanocytes, and smooth muscle cells in vitro. Most notably, KC-NC migrated along stereotypical pathways and gave rise to multiple NC derivatives upon transplantation into chicken embryos, corroborating their NC phenotype. Here, we present an innovative design concept for developing anisotropically aligned scaffolds with chemically immobilized biological cues to promote differentiation of the KC-NC towards the SC. Specifically, we designed electrospun nanofibers and examined the effect of bioactive cues in guiding KC-NC differentiation into SC. KC-NC attached to nanofibers and adopted a spindle-like morphology, similar to the native extracellular matrix (ECM) microarchitecture of the peripheral nerves. Immobilization of biological cues, especially Neuregulin1 (NRG1) promoted the differentiation of KC-NC into the SC lineage. This study suggests that poly-ε-caprolactone (PCL) nanofibers decorated with topographical and cell-instructive cues may be a potential platform for enhancing KC-NC differentiation toward SC.

Cell-free vascular grafts that grow with the host.

Cell-free small diameter vascular grafts, based on small intestinal submucosa (SIS) functionalized with heparin and vascular endothelial growth factor (VEGF) manufactured and implanted successfully into the arterial system of neonatal lambs, where they remained patent and grew in size with the host to a similar extent and with similar rate as native arteries. Acellular tissue engineered vessels (A-TEV) integrated seamlessly into the native vasculature and developed confluent, functional endothelium that afforded patency. The medial layer was infiltrated by smooth muscle cells, showed no signs of calcification and developed contractile function. The vascular wall underwent remarkable extracellular matrix remodeling exhibiting elastin fibers and even inner elastic lamina within six months. Taken together, our results suggest that VEGF-based A-TEVs may be suitable for treatment of congenital heart disorders to alleviate the need for repeated surgeries, which are currently standard practice.

Implantation of VEGF-functionalized cell-free vascular grafts: regenerative and immunological response.

Recently, our group demonstrated that immobilized VEGF can capture flowing endothelial cells (ECs) from the blood in vitro and promote endothelialization and patency of acellular tissue-engineered vessels (A-TEVs) into the arterial system of an ovine animal model. Here, we demonstrate implantability of submillimeter diameter heparin and VEGF-decorated A-TEVs in a mouse model and discuss the cellular and immunologic response. At 1 mo postimplantation, the graft lumen was fully endothelialized, as shown by expression of EC markers such as CD144, eNOS, CD31, and VEGFR2. Interestingly, the same cells coexpressed leukocyte/macrophage (Mϕ) markers CD14, CD16, VEGFR1, CD38, and EGR2. Notably, there was a stark difference in the cellular makeup between grafts containing VEGF and those containing heparin alone. In VEGF-containing grafts, infiltrating monocytes (MCs) converted into anti-inflammatory M2-Mϕs, and the grafts developed well-demarcated luminal and medial layers resembling those of native arteries. In contrast, in grafts containing only heparin, MCs converted primarily into M1-Mϕs, and the endothelial and smooth muscle layers were not well defined. Our results indicate that VEGF may play an important role in regulating A-TEV patency and regeneration, possibly by regulating the inflammatory response to the implants.-Smith, R. J., Jr., Yi, T., Nasiri, B., Breuer, C. K., Andreadis, S. T. Implantation of VEGF-functionalized cell-free vascular grafts: regenerative and immunological response.

Fabrication of porous scaffolds with decellularized cartilage matrix for tissue engineering application.

Due to the avascular nature of articular cartilage, damaged tissue has little capacity for spontaneous healing. Three-dimensional scaffolds have potential for use in tissue engineering approach for cartilage repair. In this study, bovine cartilage tissue was decellularized and chemically crosslinked hybrid chitosan/extracellular matrix (ECM) scaffolds were fabricated with different ECM weight ratios by simple freeze drying method. Various properties of chitosan/ECM scaffolds such as microstructure, mechanical strength, swelling ratio, and biodegradability rate were investigated to confirm improved structural and biological characteristics of chitosan scaffolds in the presence of ECM. The results indicated that by introducing ECM to chitosan, pore sizes in scaffolds with 1% and 2% ECM decreased and thus the mechanical properties were improved. The presence of ECM in the same scaffolds also improved the swelling ratio and biodegradation rate in the hybrid scaffolds. MTT cytotoxicity assays performed on chondrocyte cells cultured on chitosan/ECM scaffolds having various amounts of ECM showed that the greatest cell attachment belongs to the sample with intermediate ECM content (2% ECM). Overall, it can be concluded from all obtained results that the prepared scaffold with intermediate concentration of ECM could be a proper candidate for use in cartilage tissue engineering.

Monocyte Recruitment for Vascular Tissue Regeneration.

A strategy to recruit monocytes (MCs) from blood to regenerate vascular tissue from unseeded (cell-free) tissue engineered vascular grafts is presented. When immobilized on the surface of vascular grafts, the fusion protein, H2R5 can capture blood-derived MC under static or flow conditions in a shear stress dependent manner. The bound MC turns into macrophages (Mϕ) expressing both M1 and M2 phenotype specific genes. When H2R5 functionalized acellular-tissue engineered vessels (A-TEVs) are implanted into the mouse aorta, they remain patent and form a continuous endothelium expressing both endothelial cell (EC) and MC specific proteins. Underneath the EC layer, multiple cells layers are formed coexpressing both smooth muscle cell (SMC) and MC specific markers. Lineage tracing analysis using a novel CX3CR1-confetti mouse model demonstrates that fluorescently labeled MC populates the graft lumen by two and four weeks postimplantation, providing direct evidence in support of MC/Mϕ recruitment to the graft lumen. Given their abundance in the blood, circulating MCs may be a great source of cells that contribute directly to the endothelialization and vascular wall formation of acellular vascular grafts under the right chemical and biomechanical cues.

Endothelialization of arterial vascular grafts by circulating monocytes.

Recently our group demonstrated that acellular tissue engineered vessels (A-TEVs) comprised of small intestinal submucosa (SIS) immobilized with heparin and vascular endothelial growth factor (VEGF) could be implanted into the arterial system of a pre-clinical ovine animal model, where they endothelialized within one month and remained patent. Here we report that immobilized VEGF captures blood circulating monocytes (MC) with high specificity under a range of shear stresses. Adherent MC differentiate into a mixed endothelial (EC) and macrophage (Mφ) phenotype and further develop into mature EC that align in the direction of flow and produce nitric oxide under high shear stress. In-vivo, newly recruited cells on the vascular lumen express MC markers and at later times they co-express MC and EC-specific proteins and maintain graft patency. This novel finding indicates that the highly prevalent circulating MC contribute directly to the endothelialization of acellular vascular grafts under the right chemical and biomechanical cues.