Hugo Schmoekel

Synthetic matrix metalloproteinase-sensitive hydrogels for the conduction of tissue regeneration: Engineering cell-invasion characteristics

Synthetic hydrogels have been molecularly engineered to mimic the invasive characteristics of native provisional extracellular matrices: a combination of integrin-binding sites and substrates for matrix metalloproteinases (MMP) was required to render the networks degradable and invasive by cells via cell-secreted MMPs. Degradation of gels was engineered starting from a characterization of the degradation kinetics (kcat and Km) of synthetic MMP substrates in the soluble form and after crosslinking into a 3D hydrogel network. Primary human fibroblasts were demonstrated to proteolytically invade these networks, a process that depended on MMP substrate activity, adhesion ligand concentration, and network crosslinking density. Gels used to deliver recombinant human bone morphogenetic protein-2 to the site of critical defects in rat cranium were completely infiltrated by cells and remodeled into bony tissue within 4 wk at a dose of 5 μg per defect. Bone regeneration was also shown to depend on the proteolytic sensitivity of the matrices. These hydrogels may be useful in tissue engineering and cell biology as alternatives for naturally occurring extracellular matrix-derived materials such as fibrin or collagen.

Repair of bone defects using synthetic mimetics of collagenous extracellular matrices

We have engineered synthetic poly(ethylene glycol) (PEG)–based hydrogels as cell-ingrowth matrices for in situ bone regeneration. These networks contain a combination of pendant oligopeptide ligands for cell adhesion (RGDSP) and substrates for matrix metalloproteinase (MMP) as linkers between PEG chains. Primary human fibroblasts were shown to migrate within these matrices by integrin- and MMP-dependent mechanisms. Gels used to deliver recombinant human bone morphogenetic protein-2 (rhBMP-2) to the site of critical- sized defects in rat crania were completely infiltrated by cells and were remodeled into bony tissue within five weeks. Bone regeneration was dependent on the proteolytic sensitivity of the matrices and their architecture. The cell-mediated proteolytic invasiveness of the gels and entrapment of rhBMP-2 resulted in efficient and highly localized bone regeneration.

Bone healing in the rat and dog with nonglycosylated BMP-2 demonstrating low solubility in fibrin matrices.

A novel form of recombinant human bone morphogenetic protein‐2 (BMP‐2) was explored for effective incorporation and long‐term retention into fibrin ingrowth matrices. The solubility of native BMP‐2 is greatly dependent on its glycosylation. To enhance retention of BMP‐2 in fibrin matrices, a nonglycosylated form (nglBMP‐2), which is less soluble than the native glycosylated protein, was produced recombinantly and evaluated in critical‐size defects in the rat calvarium (group n = 6). When 1 or 20 μg nglBMP‐2 was incorporated by precipitation within the matrix, 74 ± 4% and 98 ± 2% healing was observed in the rat calvarium, respectively, as judged radiographically by closure of the defect at 3 weeks. More soluble forms of BMP‐2, used as controls, induced less healing, demonstrating a positive correlation between low solubility, retention in vitro, and healing in vivo. Subsequently, the utility of nglBMP‐2 was explored in a prospective veterinary clinical trial for inter‐carpal fusion in dogs, replacing the standard‐of‐care, namely autologous cancellous autograft, with nglBMP‐2 in fibrin. In a study of 10 sequential canine patients, fibrin with 600 μg/ml nglBMP‐2 performed better than autograft in the first weeks of bone healing and comparably thereafter. Furthermore, a greater fraction of animals treated with nglBMP‐2 in fibrin demonstrated bone bridging across each of the treated joints at both 12 and 17 weeks than in animals treated with autograft. These results suggest that evaluation in a human clinical setting of nonglycosylated BMP‐2 in fibrin matrices might be fruitful.

Functionalization of polysulfide nanoparticles and their performance as circulating carriers

We here present an evaluation of the carrier performance of nanoparticles that are biofunctional, i.e. derivatized to provide a controlled biological activity, and environmentally responsive, since they respond to the presence of oxidants. In particular, we focus on the possibilities (a) to make the nanoparticles detectable and (b) to control their uptake in phagocytic cells, which determines their lifetime in vivo. We first describe techniques for labeling selectively the nanoparticle surface or bulk with imaging moieties (fluorophores or gold). We then show how surface composition and size, which are both controlled through the use of PEG derivatives, influence uptake by macrophages in vitro and blood circulation in vivo: for example, in vitro uptake is negligible for small (40 nm) particles but not for larger (100 nm) ones and, correspondingly, in vivo blood circulation half-life time decreases from 6.0 to 2.9 h. However, upon decoration with RGD peptides also the small particles can be significantly internalized.