Sam L. Helgerson

Controlled Release of Bioactive Transforming Growth Factor Beta-1 from Fibrin Gels In Vitro

This study analyzed the ability of fibrin gels to deliver added recombinant transforming growth factor beta-1 (TGF-beta1) in a controlled manner and biologically active form. First, the effects of the amount of TGF-beta1 on the release kinetics were analyzed using a single fibrin gel formulation (fibrinogen complex (FC) at 25 mg/mL, thrombin at 2 IU/mL). Then, the effects of FC and thrombin concentrations were analyzed. Finally, to test the biological activity of the released TGF-beta1 from the gels, medium supernatants taken from gels at day 3 were used as culture medium for human mesenchymal stem cell (HMSC) monolayers. Cell proliferation was analyzed after staining with calcein dye, and changes in cell morphology were observed under fluorescence microscopy at days 1, 4, and 7. At day 7, HMSC chondrogenic differentiation was assessed by Alcian Blue staining and osteogenic differentiation by alkaline phosphatase activity and Alizarin Red staining. Results showed that TGF-beta1 added to fibrin gels was gradually released from the gels and increased with the amount of TGF-beta1 initially seeded, with a total of approximately 50% of the initial amount released by day 10 (with gels containing 25 mg/mL of FC and 2 IU/mL of thrombin). The release was lower with increasing FC concentrations, suggesting a binding affinity of TGF-beta1 with the FC component. Varying the thrombin concentration had a lesser effect. HMSC monolayers cultured with medium supernatants collected from gels at day 3 and containing released TGF-beta1 showed a change in morphology (squared to polygonal), lower cell proliferation, positive Alcian Blue staining but low levels of osteogenic differentiation markers. These results demonstrated that released TGF-beta1 was still bioactive and tended to induce mainly chondrogenic differentiation of the HMSC. Overall, the present study demonstrated that fibrin gels could be used as a carrier matrix for controlled release of bioactive TGF-beta1 by adjusting the concentrations of FC and thrombin in the gels.

Sustained (rh)VEGF165 release from a sprayed fibrin biomatrix induces angiogenesis, up-regulation of endogenous VEGF-R2, and reduces ischemic flap necrosis

This study investigated (1) the release of recombinant human vascular endothelial growth factor ([rh]VEGF165) from an in vitro fibrin matrix, (2) the effects of (rh)VEGF165 released from an in vivo fibrin matrix on ischemic flap necrosis in the rat dorsal skin flap model, and (3) the effects of (rh)VEGF165 released from an in vivo fibrin matrix on VEGF‐R2 expression in transgenic VEGF‐R2/luc mice. In vitro fibrin matrices were spiked with (rh)VEGF165 and demonstrated (rh)VEGF165 release over 88 hours with 66% recovery. Ischemic dorsal flaps were treated with a fibrin sealant (FS), FS spiked with (rh)VEGF165, or left untreated. Flaps treated with FS spiked with (rh)VEGF165 showed greater viability than controls as measured by planimetric analysis. Immunohistochemical analyses revealed stronger neovascularization than that exhibited by controls. Transgenic mice implanted with FS spiked with (rh)VEGF165 had significant increases in VEGF‐R2 expression relative to controls at days 5–13 after implantation. Conclusions drawn from this work are that (1) (rh)VEGF165 is released from an in vitro fibrin matrix at clinically appropriate times, (2) (rh)VEGF165 increases the viability of tissue flaps in vivo, and (3) (rh)VEGF165 induces the expression of VEGF‐R2 expression. This work demonstrates the clinical ability of sprayed FS to locally deliver growth factors to ischemic tissue of patients.

Fibrin as a delivery vehicle for active macrophage activator lipoprotein-2 peptide : in vitro studies

Fibrin sealants have been used in hemostasis and tissue sealing for over 25 years and recent studies have shown them to be an ideal delivery vehicle for cells and bioactive substances. We examined the use of fibrin as a delivery vehicle for the macrophage activator lipoprotein peptide (MALP)‐2. MALP‐2, secreted by mycoplasma, plays an important role in an early influx of leukocytes and infiltration by monocytes and their subsequent activation into macrophages as detected by their secretion of cytokines and chemoattractants. We first showed that MALP‐2 activated several monocytic cell lines by increasing the expression of cytokines and chemoattractants in these cells. Furthermore, using a reverse transcription‐polymerase chain reaction approach, we found that MALP‐2 affected the gene expression of its own receptors: TLR2 and TLR4 in various cell types including fibroblasts, keratinocytes, and endothelial cells. Furthermore, the conditioned medium, containing secreted cytokines and chemoattractants, collected from monocytes treated with MALP‐2 enhanced fibroblast migration using a standard wound culture assay. Next, we examined MALP‐2s effect on the human monocyte cell line when it is mixed with fibrin. Monocytes seeded on three‐dimensional fibrin containing MALP‐2 secreted more cytokines such as interleukin‐6, tumor necrosis factor‐α, and chemoattractants such as macrophage inflammatory protein 1 α and monocyte chemoattractant protein 1 when compared with monocytes seeded on three‐dimensional fibrin in the absence of MALP‐2. This study supports the use of fibrin to deliver MALP‐2, and possibly other peptides, in an active form that might enhance wound healing.

Physical and Epitope Analysis of a Recombinant Human T-Cell Receptor Vα/Vβ Construct Support the Similarity to Immunoglobulin

The genetic organization and protein structure of T-cell receptors (TCR) and immunoglobulins (Ig) are remarkably similar. Through recombinant, physical, and peptide-based immunological studies we demonstrated that rabbit antisera generated against a recombinant single-chain TCR (scTCR) react with defined peptide epitopes of their constituent TCR α and β chains. These antisera cross-react with the λ light-chain Mcg as well as with peptides duplicating its covalent structure. Conversely, rabbit antisera generated to human λ light chains cross-reacted with the recombinant scTCR. Rabbit anti-λ antibodies purified on an scTCR affinity column bound to T-cell lines and to T and B lymphocytes from peripheral blood. Circular dichroism analysis demonstrated plots characteristic of β-sheets for both Mcg and recombinant scTCR. Antisera directed against TCR α-chain synthetic peptides reacted with scTCR, Mcg λ light-chain protein, synthetic peptides from regions of sequence homology in β-chains, and Mcg. Based upon this homology and the serological cross-reactions which reflect conformational determinants, we suggest that the Vα/Vβ antigen-binding domain of this particular monoclonal scTCR construct is substantially similar to the conformational structure of λ light chains.