Novel method to improve vascularization of tissue engineered constructs with biodegradable fibers.

Tissue engineered grafts lack adequate vascularization and suffer from poor perfusion in vivo curtailing clinical application. Improving vascularization in any tissue implants would hence increase their survivability and treatment efficacy. Many prevascularization strategies established to date involves the angiogenic induction of endothelial progenitor cells in thick tissue engineered scaffolds to obtain vascularization. These 3D scaffolds typically require a dynamic cell culturing system involving/needful of bioreactors to obtain vascularization in thick tissue engineered scaffolds. Herein, we developed a novel method to engineer a vessel network without bioreactor, where 3D blood vessels could be simply obtained in a 2D static cell culturing system. This network could be used to augment the prevascularization of tissue engineered grafts. Endothelial cells (HUVECs) were confluently cultured on resorbable electrospun poly (D, L-lactide-co-glycolide) microfibers of capillary dimensions. These cell encapsulated capillary fibers were further embedded in collagen with HUVECs and vascular endothelial growth factor. Green fluorescent protein and red fluorescent protein expressing HUVECs were used to label cells on fiber and in collagen respectively for visualization and monitoring of capillary network formation. Seeded HUVECs in the hybrid construct were subsequently cultured for 30 days before implantation. Vessel density was measured by the total tubule length per unit area at different time points. In vitro results indicated that the fibers provide contact guidance to form primary networks to direct more vessels branching of HUVECs in hybrid constructs and the vessel integrity of microvasculature was retained after fiber degradation. In addition, these preformed engineered capillaries could capably inosculate with de novo capillaries in collagen when combined, giving rise to a hybrid pre-vascularized scaffold of more extensive vessel network and interconnections, thereby markedly improved prevascularization. When implanted onto the dorsal skin of immune-deficient mice, vessels of hybrid pre-vascularized scaffold also rapidly anastomosed with mice vasculature within a day as confirmed with the immunostaining of endothelial cell markers CD31 and von Willebrand factor. This proof of concept study showed that artificial capillaries formed through contact guidance of endothelial cells on resorbable capillary sized microfibers can significantly enhance prevascularization in tissue engineered constructs intended for surgical implantation.

A 3D biomimetic model of tissue stiffness interface for cancer drug testing.

Contrary to oversimplified preclinical drug screens that derive treatment responses of cancer cells grown on plastic cell culturing surfaces, the actual in vivo scenario for cancer cell invasion is confronted with a diversity of tissue stiffness. After all, the packing of organs and tissues in the body translates to the abundant presence of tissue stiffness interfaces. The invasive dissemination of cancer cells in vivo might be encouraged by favorable tissue stiffness gradients, likely explaining the preferential spread of cancer cells which is subjective to the cancer type and origin of the primary site. Yet these critical tumor microenvironmental influences cannot be recapitulated in 2D preclinical drug screens, hence omitting potentially invaluable in vivo patterns of drug responses that may support safer clinical dosage implementation of cancer drugs. Current attempts to study stiffness implications on cancer cells are largely confined to 2D surfaces of tunable stiffness. While these studies collectively show that cancer cells migrate better on a stiffer matrix, the generation of a biomimetic 3D tissue stiffness interface for cancer cell migration would clearly give a more definitive understanding on the probable push and pull influences of the 3D ECM. Herein, we developed a biomimetic platform which enables the precise placement of spheroids at tissue stiffness interfaces constructed with natural ECM collagen type I. This enables a standardized comparison of spheroid invasion under a 3D stiffness gradient influence. We found that cancer cells in 3D infiltrated more extensively into a softer matrix of 300 Pa while showing significantly reduced invasion into stiffer matrix of 1200 and 6000 Pa. These biomimetic spheroid cultures postinvasion were suitably subjected to paclitaxel treatment and subsequent daily live quantification of apoptotic cells to evaluate the implications of tissue stiffness on chemotherapeutic treatment. We importantly found that cancer cells which more extensively infiltrated the 300 Pa matrix also succumbed to paclitaxel induced apoptosis earlier than cells in stiffer matrices of 1200 and 6000 Pa respectively. This suggests that reduced invasion of cancer cells attributed to increased tissue stiffness barriers may favor their reduced apoptotic susceptibility to chemotherapeutic treatment.

Induction of myogenic differentiation of human mesenchymal stem cells cultured on Notch agonist (Jagged-1) modified biodegradable scaffold surface.

Engineered scaffold surface provides stem cells with vital cues that could determine the eventual fate of stem cells. In this work, biodegradable poly(L-lactide-co-ε-caprolactone) (PLCL) scaffold conjugated with Notch agonist-Jagged-1(JAG) peptide (2.1 kDa) was prepared to initiate myogenic differentiation of human mesenchymal stem cells (hMSCs). The scaffold surface was activated with oxygen plasma and acrylic acid was engrafted via UV polymerization to form a surface bearing carboxylic groups. JAG peptide was subsequently immobilized onto the carboxylated scaffold surface. Surface chemistry and topography were examined using attenuated total reflection Fourier transform infrared, X-ray photoelectron spectroscopy, and atomic force microscopy. Quantitative real time polymerase chain reaction analysis revealed activation of the Notch pathway; furthermore, several specific markers associated with myogenic but not osteogenic differentiation were shown to be up-regulated in hMSCs cultured on the engineered surface. The pro-myocardial effect of surface bound JAG peptide was further affirmed via immunodetection of the distinct myocardial marker, cardiac troponin T. Collectively, our results suggest that PLCL conjugated JAG peptide is a viable strategy to enhance the functional potential of scaffolds to be used as a bioengineered cardiac patch in myocardial infarction repair.