The creation of a living heart valve is a much-wanted alternative for current valve prostheses that suffer from limited durability and thromboembolic complications. Current strategies to create such valves, however, require the use of cells for in vitro culture, or decellularized human- or animal-derived donor tissue for in situ engineering. Here, we propose and demonstrate proof-of-concept of in situ heart valve tissue engineering using a synthetic approach, in which a cell-free, slow degrading elastomeric valvular implant is populated by endogenous cells to form new valvular tissue inside the heart. We designed a fibrous valvular scaffold, fabricated from a novel supramolecular elastomer, that enables endogenous cells to enter and produce matrix. Orthotopic implantations as pulmonary valve in sheep demonstrated sustained functionality up to 12 months, while the implant was gradually replaced by a layered collagen and elastic matrix in pace with cell-driven polymer resorption. Our results offer new perspectives for endogenous heart valve replacement starting from a readily-available synthetic graft that is compatible with surgical and transcatheter implantation procedures.

In an in-situ approach towards tissue engineered cardiovascular replacement grafts, cell-free scaffolds are implanted that engage in endogenous tissue formation. Bioactive molecules can be incorporated into such grafts to facilitate cellular recruitment. Stromal cell derived factor 1α (SDF1α) is a powerful chemoattractant of lymphocytes, monocytes and progenitor cells and plays an important role in cellular signaling and tissue repair. Short SDF1α-peptides derived from its receptor-activating domain are capable of activating the SDF1α-specific receptor CXCR4. Here, we show that SDF1α-derived peptides can be chemically modified with a supramolecular four-fold hydrogen bonding ureido-pyrimidinone (UPy) moiety, that allows for the convenient incorporation of the UPy-SDF1α-derived peptides into a UPy-modified polymer scaffold. We hypothesized that a UPy-modified material bioactivated with these UPy-SDF1α-derived peptides can retain and stimulate circulating cells in an anti-inflammatory, pro-tissue formation signaling environment. First, the early recruitment of human peripheral blood mononuclear cells to the scaffolds was analyzed in vitro in a custom-made mesofluidic device applying physiological pulsatile fluid flow. Preferential adhesion of lymphocytes with reduced expression of inflammatory factors TNFα, MCP1 and lymphocyte activation marker CD25 was found in the bioactivated scaffolds, indicating a reduction in inflammatory signaling. As a proof of concept, in-vivo implantation of the bioactivated scaffolds as rat abdominal aorta interposition grafts showed increased cellularity by CD68+ cells after 7 days. These results indicate that a completely synthetic, cell-free biomaterial can attract and stimulate specific leukocyte populations through supramolecular incorporation of short bioactive SDF1α derived peptides.

Inflammation is a natural phase of the wound healing response, which can be harnessed for the in situ tissue engineering of small-diameter blood vessels using instructive, bioresorbable synthetic grafts. This process is dependent on colonization of the graft by host circulating cells and subsequent matrix formation. Typically, vascular regeneration in small animals is governed by transanastomotic cell ingrowth. However, this process is very rare in humans and hence less relevant for clinical translation. Therefore, a novel rat model was developed, in which cell ingrowth from the adjacent tissue is inhibited using Gore-Tex sheathing. Using this model, our aim here was to prove that functional blood vessels can be formed in situ through the host inflammatory response, specifically by blood-borne cells. The model was validated by implanting sex-mismatched aortic segments on either anastomoses of an electrospun poly(ɛ-caprolactone) (PCL) graft, filled with fibrin gel, into the rat abdominal aorta. Fluorescent in situ hybridization analysis revealed that after 1 and 3 months in vivo, over 90% of infiltrating cells originated from the bloodstream, confirming the effective shielding of transanastomotic cell ingrowth. Using the validated model, PCL/fibrin grafts were implanted, either or not loaded with monocyte chemotactic protein-1 (MCP-1), and cell infiltration and tissue development were investigated at various key time points in the healing cascade. A phased healing response was observed, initiated by a rapid influx of inflammatory cells, mediated by the local release of MCP-1. After 3 months in vivo, the grafts consisted of a medial layer with smooth muscle cells in an oriented collagen matrix, an intimal layer with elastin fibers, and confluent endothelium. This study proves the regenerative potential of cells in the circulatory system in the setting of in situ vascular tissue engineering.