Bone Sialoprotein Immobilized in Collagen Type I Enhances Bone Regeneration In vitro and In vivo.

The use of bioactive molecules is a promising approach to enhance the bone healing properties of biomaterials. The aim of this study was to define the role of bone sialoprotein (BSP) immobilized in collagen type I in various settings. In vitro studies with human primary osteoblasts in mono- or in co-culture with endothelial cells demonstrated a slightly increased gene expression of osteogenic markers as well as an increased proliferation rate in osteoblasts after application of BSP immobilized in collagen type I. Two critical size bone defect models were used to analyze bone regeneration. BSP incorporated in collagen type I increased bone regeneration only marginally at one concentration in a calvarial defect model. To induce the mechanical stability, three-dimensional printing was used to produce a stable porous cylinder of polylactide. The cylinder was filled with collagen type I and immobilized BSP and implanted into a femoral defect of critical size in rats. This hybrid material was able to significantly induce bone regeneration. Our study clearly shows the osteogenic effect of BSP when combined with collagen type I as carrier and thereby offers various approaches and options for its use as bioactive molecule in bone substitute materials.

Improvement of wound healing by the development of ECM-inspired biomaterial coatings and controlled protein release.

Implant design has evolved from biochemically inert substrates, minimizing cell and protein interaction, towards sophisticated bioactive substrates, modulating the host response and supporting the regeneration of the injured tissue. Important aspects to consider are the control of cell adhesion, the discrimination of bacteria and non-local cells from the desired tissue cell type, and the stimulation of implant integration and wound healing. Here, the extracellular matrix acts as a role model providing us with inspiration for sophisticated designs. Within this scope, small bioactive peptides have proven to be miscellaneously deployable for the mediation of surface, cell and matrix interactions. Combinations of adhesion ligands, proteoglycans, and modulatory proteins should guide multiple aspects of the regeneration process and cooperativity between the different extracellular matrix components, which bears the chance to maximize the therapeutic efficiency and simultaneously lower the doses. Hence, efforts to include multiple of these factors in biomaterial design are well worth. In the following, multifunctional implant coatings based on bioactive peptides are reviewed and concepts to implement strong surface anchoring for stable cell adhesion and a dynamic delivery of modulator proteins are discussed.

Extracellular Matrix Components Regulate Bone Sialoprotein Expression in MDA-MB-231 Breast Cancer Cells.

Bone sialoprotein (BSP) has become a target in breast cancer research as it is associated with tumor progression and metastasis. The mechanisms underlying the regulation of BSP expression have been largely elusive. Given that BSP is involved in the homing of cancer cells in bone metastatic niches, we addressed regulatory effects of proteolytic cleavage and extracellular matrix components on BSP expression and distribution in cell culture models. Therefore, MDA-MB-231 human breast cancer cells were kept in 2D and 3D spheroid cultures and exposed to basement membrane extract in the presence or absence of matrix metalloproteinase 9 or the non-polar protease, dispase. Confocal imaging of immunofluorescence samples stained with different antibodies against human BSP demonstrated a strong inducing effect of basement membrane extract on anti-BSP immunofluorescence. Similarly, protease incubation led to acute upregulation of anti-BSP immunofluorescence signals, which was blocked by cycloheximide, suggesting de novo formation of BSP. In summary, our data show that extracellular matrix components play an important function in regulating BSP expression and hint at mechanisms for the formation of bone-associated metastasis in breast cancer that might involve local control of BSP levels by extracellular matrix degradation and release of growth factors.

Peptide-mediated surface coatings for the release of wound-healing cytokines.

Supporting the wound healing process by sending the appropriate cytokine signals can shorten healing time and overcome chronic inflammation syndromes. Even though adhesion peptides consisting of Arg-Gly-Asp (RGD) are commonly used to enhance cell-surface interactions, peptide-mediated cytokine delivery has not been widely exploited so far. Cytokines interact with high affinity with their cognitive receptors but also with sulfated glycosaminoglycans (GAGs), both of which form a base for incorporation of cytokines into functional biomaterials. Here, we report on a mussel-derived surface coating as a prospective cytokine delivery system using covalently bound heparin mimetics, receptor-derived chemokine-binding peptides, and heparin-binding peptides (HBP). The latter enabled non-covalent immobilization of heparin on the surface followed by chemokine binding and release, whereas the former allowed direct non-covalent chemokine immobilization. The peptide displayed excellent binding to custom-made polystyrene 96-well plates, enabling convenient testing of several compounds. Released chemokine successfully induced migration in Jurkat cells, especially for the non-covalent heparin immobilization approach using HBPs as evaluated in a transwell assay. In comparison, heparin-mimetic coatings, comprised of sulfated peptides and GAG derivatives, proved less efficient with respect to amount of immobilized chemokine and migratory response. Thus, our study provides a roadmap for further rational optimization and translation into clinics.

Multifunctional coatings combining bioactive peptides and affinity-based cytokine delivery for enhanced integration of degradable vascular grafts.

Insufficient endothelialization of cardiovascular devices is a high-risk factor for implant failure. Presentation of extracellular matrix (ECM)-derived coatings is a well-known strategy to improve implant integration. However, the complexity of the system is challenging and strategies for applying multifunctionality are required. Here, we engineered mussel-derived surface-binding peptides equipped with integrin (c[RGDfK]) and proteoglycan binding sites (FHRRIKA) for enhanced endothelialization. Surface-binding properties of the platform containing l-3,4-dihydroxyphenylalanine (DOPA) residues were confirmed for hydrophilized polycaprolactone-co-lactide scaffolds as well as for glass and polystyrene. Further, heparin and the heparin-binding angiogenic factors VEGF, FGF-2 and CXCL12 were immobilized onto the peptide in a modular assembly. Presentation of bioactive peptides greatly enhanced human umbilical vein endothelial cell (HUVEC) adhesion and survival under static and fluidic conditions. In subsequent investigations, peptide-heparin-complexes loaded with CXCL12 or VEGF had an additional increasing effect on cell viability, differentiation and migration. Finally, hemocompatibility of the coatings was ensured. This study demonstrates that coatings combining adhesion peptides, glycosaminoglycans and modulators are a versatile tool to convey ECM-inspired multifunctionality to biomaterials and efficiently promote their integration.

Endothelialization of Titanium Surfaces by Bioinspired Cell Adhesion Peptide Coatings.

Common interventional therapies for cardiovascular occlusive diseases, such as the implantation of stents, are at risk of complications like thrombosis or restenosis. Drug-eluting stents have improved patency but simultaneously worsen the endothelialization of the implant. Here, we present a novel peptide coating derived from three proteins of the extracellular matrix named fibronectin, laminin, and elastin. Their active sequences RGD, SIKVAV, and VGVAPG were immobilized onto titanium surfaces by a carrier peptide containing l-3,4-dihydroxyphenylalanine (DOPA). Simultaneous functionalization of the carrier peptide with cyclic c[RGDfK] and SIKVAV had the most potent influence on adhesion, proliferation, viability, and angiogenesis of endothelial cells. By presentation of two adhesion peptides in one molecule, a synergistic enhancement of cell-surface interactions was achieved. Overall, this work clearly demonstrates the advantages of spatially defined peptide coatings for the endothelialization of titanium and thus describes a promising approach for the coating of stents.