Bioinspired gelatin/bioceramic composites loaded with bone morphogenetic protein-2 (BMP-2) promote osteoporotic bone repair.

There are currently several commercialized products approved by the Food and Drug Administration and the European Medicines Agency based on the use of recombinant human BMP-2 for the treatment of non-unions long fractures and spinal fusion. However, the adverse effects recorded with the use of BMPs suggest the need for drug delivery carriers that allow reducing the required doses and improve their cost-effectiveness. Herein, we have developed a new osteoconductive scaffold that reduces the required doses of BMP-2 for promoting bone regeneration in an osteoporotic defect model. The composite is, in brief, a gelatin-based 3D scaffold reinforced with either calcium sulfate or hydroxyapatite as an inorganic osteoconductive biomaterial. To this end, the organic/inorganic composite systems showed high hydration capacity and good in vitro degradability. The incorporation of 7.5% (m/v) ceramic compounds resulted in scaffolds with stiffer Young modulus (179 and 75 kPa for CaSO4_7 and HA_7, respectively) than bare gelatin hydrogels (48 kPa). Studies with human bone-marrow derived mesenchymal stem cells (hBM-MSCs) revealed that the 3D scaffolds promote cell adhesion and proliferation along with osteogenic differentiation capabilities. Specifically, downregulation of stemness (Nanog, Oct4) genes and upregulation of osteogenic markers (ALP, Col1a1, Fmod) by two fold were observed over 10 days under basal culture conditions. Promisingly, the sustained in vitro release of BMP-2 observed from the porous reinforced scaffolds allowed us to address the critical-sized osteoporotic mice calvarial defects with a relatively low growth factor doses (600 ng BMP-2/scaffold) compared to conventional doses at 2-15 micrograms. Overall, this study demonstrates the promising potential of osteoconductive gelatin/calcium bioceramics composites as osteogenic growth factors delivery carriers for bone-regeneration via ultra-low growth factor doses.

Cell shape and spreading of stromal (mesenchymal) stem cells cultured on fibronectin coated gold and hydroxyapatite surfaces.

In order to identify the cellular mechanisms leading to the biocompatibility of hydroxyapatite implants, we studied the interaction of human bone marrow derived stromal (mesenchymal) stem cells (hMSCs) with fibronectin-coated gold (Au) and hydroxyapatite (HA) surfaces. The adsorption of fibronectin was monitored by Quartz Crystal Microbalance with Dissipation (QCM-D) at two different concentrations, 20 µg/ml and 200 µg/ml, and the fibronectin adsorption experiments were complemented with antibody measurements. The QCM-D results show that the surface mass uptake is largest on the Au surfaces, while the number of polyclonal and monoclonal antibodies directed against the cell-binding domain (CB-domain) on the fibronectin (Fn) is significantly larger on the (HA) surfaces. Moreover, a higher number of antibodies bound to the fibronectin coatings formed from the highest bulk fibronection concentration. In subsequent cell studies with hMSC's we studied the cell spreading, cytoskeletal organization and cell morphology on the respective surfaces. When the cells were adsorbed on the uncoated substrates, a diffuse cell actin cytoskeleton was revealed, and the cells had a highly elongated shape. On the fibronectin coated surfaces the cells adapted to a more polygonal shape with a well-defined actin cytoskeleton, while a larger cell area and roundness values were observed for cells cultured on the coated surfaces. Among the coated surfaces a slightly larger cell area and roundness values was observed on HA as compared to Au. Moreover, the results revealed that the morphology of cells cultured on fibronectin coated HA surfaces were less irregular. In summary we find that fibronectin adsorbs in a more activated state on the HA surfaces, resulting in a slightly different cellular response as compared to the fibronectin coated Au surfaces.

Fibronectin adsorption, cell adhesion, and proliferation on nanostructured tantalum surfaces.

The interaction between dental pulp derived mesenchymal stem cells (DP-MSCs) and three different tantalum nanotopographies with and without a fibronectin coating is examined: sputter-coated tantalum surfaces with low surface roughness <0.2 nm, hut-nanostructured surfaces with a height of 2.9 +/- 0.6 nm and a width of 35 +/- 8 nm, and dome structures with a height of 13 +/- 2 nm and a width of 52 +/- 14 nm. Using ellipsometry, the adsorption and the availability of fibronectin cell-binding domains on the tantalum surfaces were examined, as well as cellular attachment, proliferation, and vinculin focal adhesion spot assembly on the respective surfaces. The results showed the highest fibronectin mass uptake on the hut structures, with a slightly higher availability of cell-binding domains and the most pronounced formation of vinculin focal adhesion spots as compared to the other surfaces. The proliferation of DP-MSCs was found to be significantly higher on dome and hut surfaces coated with fibronectin compared to the uncoated flat tantalum surfaces. Consequently, the results presented in this study indicate that fibronectin-coated nanotopographies with a vertical dimension of less than 5 nm influence cell adhesion. This rather interesting behavior is argued to originate from the more available fibronectin cell-binding domains observed on the hut structures.

Responses of fibroblasts and glial cells to nanostructured platinum surfaces.

The chronic performance of implantable neural prostheses is affected by the growth of encapsulation tissue onto the stimulation electrodes. Encapsulation is associated with activation of connective tissue cells at the electrode's metallic contacts, usually made of platinum. Since surface nanotopography can modulate the cellular responses to materials, the aim of the present work was to evaluate the 'in vitro' responses of connective tissue cells to platinum strictly by modulating its surface nanoroughness. Using molecular beam epitaxy combined with sputtering, we produced platinum nanostructured substrates consisting of irregularly distributed nanopyramids and investigated their effect on the proliferation, cytoskeletal organization and cellular morphology of primary fibroblasts and transformed glial cells. Cells were cultured on these substrates and their responses to surface roughness were studied. After one day in culture, the fibroblasts were more elongated and their cytoskeleton less mature when cultured on rough substrates. This effect increased as the roughness of the surface increased and was associated with reduced cell proliferation throughout the observation period (4 days). Morphological changes also occurred in glial cells, but they were triggered by a different roughness scale and did not affect cellular proliferation. In conclusion, surface nanotopography modulates the responses of fibroblasts and glial cells to platinum, which may be an important factor in optimizing the tissue response to implanted neural electrodes.

Enhanced surface activation of fibronectin upon adsorption on hydroxyapatite.

In this study the adsorption characteristics and the structure of fibronectin adsorped on hydroxyapatite (Ha) and a reference gold substrate (Au) is examined by quartz crystal microbalance with dissipation (QCM-D) and atomic force microscopy (AFM) at the following concentrations: 20 microg/mL, 30 microg/mL, 40 microg/mL, 100 microg/mL, 200 microg/mL, and 500 microg/mL. The conformational changes of the fibronectin molecules upon surface binding were examined as well with monoclonal antibody directed against the cell binding-domain (CB domain) of fibronectin. The QCM-D and AFM results show that the fibronectin uptake is larger on Au as compared with Ha regardless of the protein bulk concentration used in the experiment, suggesting that the individual fibronectin molecules in general attach to the surfaces in a more unfolded configuration on Ha. Moreover the dissipation values obtained with QCM-D indicate that the individual fibronectin molecules bind in a more compact and rigid configuration on Au compared to the Ha surface. In particular the monoclonal antibody data show that the CB domain on fibronectin is more available on Ha, where such cell-recognizing abilities are more pronounced at low fibronectin surface coverage. The results demonstrate that the detailed molecular structure of fibronectin and its functional activity depend significantly on both the underlying surface chemistry as well as the fibronectin surface coverage.

The influence of glancing angle deposited nano-rough platinum surfaces on the adsorption of fibrinogen and the proliferation of primary human fibroblasts.

We have used the glancing angle deposition (GLAD) method as a simple and fast method to generate nano-rough surfaces for protein adsorption experiments and cell assays. The surface roughness and the detailed geometrical surface morphology of the thin films were characterized by atomic force microscopy (AFM) and scanning electron microscopy (SEM). As the GLAD deposition angle approaches grazing incidence, sharp and whisker-like columnar protrusions are formed. Smaller and less sharp surface features appear for the thin films synthesized at higher deposition angles. By changing the GLAD deposition angle together with the total amount of mass deposited per area on the respective surfaces, the size of the surface features can be varied on the nanoscale. Using the GLAD topographies as model surfaces, we have investigated the influence of the nano-roughness on fibrinogen adsorption and on the proliferation of primary human fibroblasts. It is found that fibrinogen, an important blood protein, preferentially adheres on the whisker-like nano-rough substrates in comparison to a flat surface. Furthermore, the proliferation of the human fibroblasts is significantly reduced on the nano-rough substrates. These results demonstrate that the GLAD technique can be used to fabricate nano-rough surface morphologies that significantly influence both protein and cellular adhesion to surfaces and are therefore well suited for biological assays.

Bovine serum albumin adsorption on nano-rough platinum surfaces studied by QCM-D.

The adsorption of bovine serum albumin (BSA) on platinum surfaces with a root-mean-square roughness ranging from 1.49nm to 4.62nm was investigated using quartz crystal microbalance with dissipation (QCM-D). Two different BSA concentrations, 50microg/ml and 1mg/ml, were used, and the adsorption studies were complemented by monitoring the antibody interaction with the adsorbed BSA layer. The adsorption process was significantly influenced by the surface nano-roughness, and it was observed that the surface mass density of the adsorbed BSA layer is enhanced in a non-trivial way with the surface roughness. From a close examination of the energy dissipation vs. frequency shift plot obtained by the QCM-D technique, it was additionally observed that the BSA adsorption on the roughest surface is subject to several distinct adsorption phases revealing the presence of structural changes facilitated by the nano-rough surface morphology during the adsorption process. These changes were in particular noticeable for the adsorption at the low (50microg/ml) BSA concentration. The results confirm that the nano-rough surface morphology has a significant influence on both the BSA mass uptake and the functionality of the resulting protein layer.