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.

Bioactive zinc-doped sol-gel coating modulates protein adsorption patterns and in vitro cell responses.

Zinc is an essential element with an important role in stimulating the osteogenesis and mineralization and suppressing osteoclast differentiation. In this study, new bioactive ZnCl2-doped sol-gel materials were designed to be applied as coatings onto titanium. The biomaterials were physicochemically characterized and the cellular responses evaluated in vitro using MC3T3-E1 osteoblasts and RAW264.7 macrophages. The effect of Zn on the adsorption of human serum proteins onto the material surface was evaluated through nLC-MS/MS. The incorporation of Zn did not affect the crosslinking of the sol-gel network. A controlled Zn2+ release was obtained, reaching values below 10 ppm after 21 days. The materials were no cytotoxic and lead to increased gene expression of ALP, TGF-ß, and RUNX2 in the osteoblasts. In macrophages, an increase of IL-1ß, TGF-ß, and IL-4 gene expression was accompanied by a reduced TNF-α liberation. Proteomic results showed changes in the adsorption patterns of proteins associated with immunological, coagulative, and regenerative functions, in a Zn dose-dependent manner. The variations in protein adsorption might lead to the downregulation of the NF-κB pathway, thus explain the observed biological effects of Zn incorporation into biomaterials. Overall, these coatings demonstrated their potential to promote bone tissue regeneration.

Proteomic analysis of calcium-enriched sol-gel biomaterials.

Calcium is an element widely used in the development of biomaterials for bone tissue engineering as it plays important roles in bone metabolism and blood coagulation. The Ca ions can condition the microenvironment at the tissue-material interface, affecting the protein deposition process and cell responses. The aim of this study was to analyze the changes in the patterns of protein adsorption on the silica hybrid biomaterials supplemented with different amounts of CaCl2, which can function as release vehicles. This characterization was carried out by incubating the Ca-biomaterials with human serum. LC-MS/MS analysis was used to characterize the adsorbed protein layers and compile a list of proteins whose affinity for the surfaces might depend on the CaCl2 content. The attachment of pro- and anti-clotting proteins, such as THRB, ANT3, and PROC, increased significantly on the Ca-materials. Similarly, VTNC and APOE, proteins directly involved on osteogenic processes, attached preferentially to these surfaces. To assess correlations with the proteomic data, these formulations were tested in vitro regarding their osteogenic and inflammatory potential, employing MC3T3-E1 and RAW 264.7 cell lines, respectively. The results confirmed a Ca dose-dependent osteogenic and inflammatory behavior of the materials employed, in accordance with the protein attachment patterns.

The effect of strontium incorporation into sol-gel biomaterials on their protein adsorption and cell interactions.

It is known strontium can both inhibit the osteoclast formation and stimulate the osteoblast maturation, so biomaterials containing this element can favour bone structure stabilisation. The addition of Sr to biomaterials could affect their interactions with proteins and cells. Here, a silica-hybrid sol-gel network doped with different amounts of SrCl2 and applied as coatings on titanium discs was examined. in vitro analysis was performed to determine the potential effect of Sr in the coatings, showing enhanced gene expression of osteogenic markers (alkaline phosphatase and transforming growth factor-ß) in MC3T3-E1 incubated with Sr-doped biomaterials. The examination of inflammatory markers (tumour necrosis factor-α and interleukin 10) in RAW 264.7 macrophages revealed an anti-inflammatory potential of these materials. Proteins adsorbed onto the coatings incubated with human serum (3 h at 37 °C) were also analysed; mass spectrometry was used to characterise the proteins adhering to materials with different Sr content. Adding Sr to the coatings increased their affinity to APOE and VTNC proteins (associated with anti-inflammatory and osteogenic functions). Moreover, the proteins involved in coagulation processes, such as prothrombin, were more abundant on the coatings containing Sr than on the base sol-gel surfaces. Correlations between gene expression and proteomic results were also examined.

Proteomic analysis of silica hybrid sol-gel coatings: a potential tool for predicting the biocompatibility of implants in vivo.

The interactions of implanted biomaterials with the host organism determine the success or failure of an implantation. Normally, their biocompatibility is assessed using in vitro tests. Unfortunately, in vitro and in vivo results are not always concordant; new, effective methods of biomaterial characterisation are urgently needed to predict the in vivo outcome. As the first layer of proteins adsorbed onto the biomaterial surfaces might condition the host response, mass spectrometry analysis was performed to characterise these proteins. Four distinct hybrid sol-gel biomaterials were tested. The in vitro results were similar for all the materials examined here. However, in vivo, the materials behaved differently. Six of the 171 adsorbed proteins were significantly more abundant on the materials with weak biocompatibility; these proteins are associated with the complement pathway. Thus, protein analysis might be a suitable tool to predict the in vivo outcomes of implantations using newly formulated biomaterials.

Analysis of root plasma membrane aquaporins from Brassica oleracea: post-translational modifications, de novo sequencing and detection of isoforms by high resolution mass spectrometry.

Plasma membrane Intrinsic Proteins (PIPs), a subfamily of aquaporins, are ubiquitous membrane channel proteins that play a crucial role in water uptake in plants. The use of high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) analysis of peptides has previously shown to be a valuable tool to differentiate among PIP homologues sharing a high sequence homology and also to characterize their post-translational modifications (PTMs). The recent introduction of mass spectrometers able to measure peptide mass with high mass accuracy, together with new alternative ways of peptide fragmentation allows the identification and characterization of proteins from nonsequenced organisms, such as broccoli. In this study, we combined three endoproteases (trypsin, Glu-C and Lys-C) with HPLC-MS/MS analysis and two types of peptide fragmentations, CID (collision induced dissociation) and HCD (higher-energy C-trap dissociation), to identify PIP isoforms and PTMs from broccoli roots. After de novo sequencing analysis, eight peptides showing homology to Arabidopsis thaliana PIPs were identified. Although Arabidopsis nomenclature of PIP isoforms has not been defined for broccoli, our results agree with the occurrence of seven AtPIP isoforms (PIP 1;1, PIP 1;2, PIP 1;3 and PIP2;2, PIP 2;3, PIP2;1 and PIP2;7) in broccoli roots, as compared to the plant model A. thaliana. To our knowledge, these results represent the deepest characterization of the PIPs isolated from the roots of broccoli, a crop with increasing agronomical interest.

Do sequence repeats play an equivalent role in the choline-binding module of pneumococcal LytA amidase?

LytA amidase breaks down the N-acetylmuramoyl-l-alanine bonds in the peptidoglycan backbone of Streptococcus pneumoniae. Its polypeptide chain has two modules: the NH(2)-terminal module, responsible for the catalytic activity, and the COOH-terminal module, constructed by six tandem repeats of 20 or 21 amino acids (p1-p6) and a short COOH-terminal tail. The polypeptide chain must contain at least four repeats to efficiently anchor the autolysin to the choline residues of the cell wall. Nevertheless, the catalytic efficiency decreases by 90% upon deletion of the final tail. The structural implications of deleting step by step the two last (p5 and p6) repeats and the final COOH-tail and their effects on choline-amidase interactions have been examined by comparing four truncated mutants with LytA amidase by means of different techniques. Removal of this region has minor effects on secondary structure content but significantly affects the stability of native conformations. The last 11 amino acids and the p5 repeat stabilize the COOH-terminal module; each increases the module transition temperature by about 6 degrees C. Moreover, the p5 motif also seems to participate, in a choline-dependent way, in the stabilization of the NH(2)-terminal module. The effects of choline binding on the thermal stability profile of the mutant lacking the p5 repeat might reflect a cooperative pathway providing molecular communication between the choline-binding module and the NH(2)-terminal region. The three sequence motives favor the choline-amidase interaction, but the tail is an essential factor in the monomer <--> dimer self-association equilibrium of LytA and its regulation by choline. The final tail is required for preferential interaction of choline with LytA dimers and for the existence of different sets of choline-binding sites. The p6 repeat scarcely affects the amidase stability but could provide the proper three-dimensional orientation of the final tail.

Thermal and pH-induced conformational changes of a beta-sheet protein monitored by infrared spectroscopy.

The stability of a lentil lectin, an all-beta protein, has been perturbed by changes in pH and temperature. In the pH interval 5.0 --> 10.0, the overall secondary structure does not undergo significant changes. However, if the individual components of the infrared amide I band are considered, changes in band components attributed to variations in beta-sheet and beta-turns cross-interactions are detected. The combined effects of pH and temperature reveal that the protein is more compact at pH 7.5 with lower denaturation temperatures at pH 5.0 or 10.0, indicating a less stable protein under those conditions. According to our results, the structural stability of the beta-sheet would depend not only on the intermolecular interactions among the strands but also on the conformation of the segments connecting these strands. The protein infrared band assignment has also been examined since the three-dimensional structure of the lentil lectin protein is known from X-ray diffraction studies. Two of the bands observed are attributed to beta-sheet. The one at 1620 cm-1, not affected if the medium is deuterated, is assigned to hairpins composed by two strands connected by a rigid turn whereas that located at 1633 cm-1 corresponds to strands associated by more flexible segments. The band appearing at 1645 cm-1 in H2O corresponds to the open, flexible loops that are connecting the beta-strands. The simplest assumption of the various secondary structure components having identical IR extinction coefficients is enough to provide IR-derived data that are in good agreement with the structure solved by X-ray diffraction.