Engineering fibrin-binding TGF-β1 for sustained signaling and contractile function of MSC based vascular constructs

Abstract

We present a strategy to conjugate TGF-β1 into fibrin hydrogels to mimic the in vivo presentation of the growth factor in a 3D context. To this end, we engineered fusion proteins between TGF-β1 and a bi-functional peptide composed of a Factor XIII domain and a plasmin cleavage site. In another version the protease cleavage site was omitted to examine whether the growth factor that could not be released from the scaffold by cells had different effects on tissue constructs. The optimal insertion site which yielded correctly processed, functional protein was found between the latency associated peptide and mature TGF-β1 domains. In solution the fusion proteins exhibited similar biological activity as native TGF-β1 as evidenced by inhibition of cell proliferation and promoter activity assays. Immunoprecipitation experiments demonstrated that the fusion TGF-β1 protein bound to fibrinogen in a Factor XIII dependent manner and could be released from the peptide by the action of plasmin. In contrast to bolus delivery, immobilized TGF-β1 induced sustained signaling in fibrin-embedded cells for several days as evidenced by Smad2 phosphorylation. Prolonged pathway activation correlated with enhanced contractile function of vascular constructs prepared from hair follicle mesenchymal stem cells or bone marrow derived smooth muscle cells. Our results suggest that fibrin-immobilized TGF-β1 may be used to enhance the local microenvironment and improve the function of engineered tissues in vitro and potentially also after implantation in vivo where growth factor delivery faces overwhelming challenges.

Hybrid Biomaterial with Conjugated Growth Factors and Mesenchymal Stem Cells for Ectopic Bone Formation

“…Previous work in our laboratory has shown that fibrin immobilized TGF-b1-promoted MSC differentiation to SMCs uniformly throughout fibrin rings, eliminating growth factor diffusion limitations. 33,42 VEGF has been used to promote angiogenesis in many previous studies, however, neovessels formed only in the presence of VEGF have been shown to be unstable and degrade rapidly in vitro, and at a slower extent in vivo. This is likely due to the lack of pericytes and SMCs, which typically enwrap neovessels within the body.…”

“…Fibrin has been extensively studied as a scaffold and a number of studies have shown that fibrin is a growth factor carrier for tissue regeneration. 33,42 In addition, fibrin has also been used to deliver stem cells for bone tissue engineering. 43,44 Ideally, incorporation of a vascularized network into engineered bone substitutes to mimic the structure of natural bone tissue is necessary to promote rapid bone regeneration.…”

“…32 In addition, immobilization of TGF-b1 into fibrin led to prolonged phosphorylation of Smad2, which was accompanied by increased function that is, contractility of SMCs embedded within the hydrogels. 33 In this study, we capitalized on previous developments in our laboratories to develop a hybrid biomaterial comprising nCS and fibrin to deliver BMP2-producing MSCs and angiogenic growth factors, VEGF and FGF9, to induce robust angiogenesis and enhance bone regeneration.…”

Differential Effects of Culture Senescence and Mechanical Stimulation on the Proliferation and Leiomyogenic Differentiation of MSC from Different Sources: Implications for Engineering Vascular Grafts

“…8,25,[27][28][29] In the current study, the same protocol was used to prepare TEV constructs by polymerizing fibrin hydrogels around the silastic tubing on the 24-PP, which measures 4.75 mm in diameter. 10 6 cells of each cell isolation were suspended in 800 mL of fibrinogen (Enzyme Research Laboratories) and mixed with 200 mL of thrombin (Sigma) with final concentrations of 2.5 mg and 2.5 U, respectively.…”

Hair Follicle: A Novel Source of Multipotent Stem Cells for Tissue Engineering and Regenerative Medicine

“…77 Recent studies showed that biomaterials can be decorated with various biomolecules to provide appropriate growth or differentiation signals as necessary. [78][79][80][81][82][83] It might be interesting to examine whether this approach could be applied in the context of HFs to overcome the current limitations and facilitate studies of human HF biology.…”