Polyphenols from grape pomace induce osteogenic differentiation in mesenchymal stem cells.

Polyphenols are increasingly investigated for the treatment of periodontitis and research on their use in dental biomaterials is currently being conducted. Grape pomace extracts are a rich source of polyphenols. In the present study, the polyphenols of two different types of grape pomace were characterized and identified by high­performance liquid chromatography­diode array detector, and the effect of polyphenol­rich grape pomace extracts on mesenchymal stem cell (MSC) osteogenic differentiation was investigated. Solid­liquid extraction was used to recover polyphenols from red and white grape pomace. The two extracts have been characterized through the phenolic content and antioxidant power. Human MSCs (hMSCs) from the bone marrow were cultured both with and without given amounts (10 or 20 µg/ml) of the obtained pomace extracts. Their effects on cell differentiation were evaluated by reverse transcription­quantitative polymerase chain reaction, compared with relevant controls. Results showed that both pomace extracts, albeit different in phenolic composition and concentration, induced multiple effects on hMSC gene expression, such as a decreased receptor activator of nuclear factor κ­Β ligand/osteoprotegerin ratio and an enhanced expression of genes involved in osteoblast differentiation, thus suggesting a shift of hMSCs towards osteoblast differentiation. The obtained results provided data in favor of the exploitation of polyphenol properties from grape pomace extracts as complementary active molecules for dental materials and devices for bone regeneration in periodontal defects.

Cloning and Expression Analysis of Human Amelogenin in Nicotiana benthamiana Plants by Means of a Transient Expression System.

Enamel is the covering tissue of teeth, made of regularly arranged hydroxyapatite crystals deposited on an organic matrix composed of 90% amelogenin that is completely degraded at the end of the enamel formation process. Amelogenin has a biomineralizing activity, forming nanoparticles or nanoribbons that guide hydroxyapatite deposit, and regenerative functions in bone and vascular tissue and in wound healing. Biotechnological products containing amelogenin seem to facilitate these processes. Here, we describe the production of human amelogenin in plants by transient transformation of Nicotiana benthamiana with constructs carrying synthetic genes with optimized human or plant codons. Both genes yielded approximately 500 µg of total amelogenin per gram of fresh leaf tissue. Two purification procedures based on affinity chromatography or on intrinsic solubility properties of the protein were followed, yielding from 12 to 150 µg of amelogenin per gram of fresh leaf tissue, respectively, at different purity. The identity of the plant-made human amelogenin was confirmed by MALDI-TOF-MS analysis of peptides generated following chymotrypsin digestion. Using dynamic light scattering, we showed that plant extracts made in acetic acid containing human amelogenin have a bimodal distribution of agglomerates, with hydrodynamic diameters of 22.8 ± 3.8 and 389.5 ± 86.6 nm. To the best of our knowledge, this is the first report of expression of human amelogenin in plants, offering the possibility to use this plant-made protein for nanotechnological applications.

Novel bioceramic-reinforced hydrogel for alveolar bone regeneration.

UNLABELLED: The osseointegration of dental implants and their consequent long-term success is guaranteed by the presence, in the extraction site, of healthy and sufficient alveolar bone. Bone deficiencies may be the result of extraction traumas, periodontal disease and infection. In these cases, placement of titanium implants is contraindicated until a vertical bone augmentation is obtained. This goal is achieved using bone graft materials, which should simulate extracellular matrix (ECM), in order to promote osteoblast proliferation and fill the void, maintaining the space without collapsing until the new bone is formed. In this work, we design, develop and characterize a novel, moldable chitosan-pectin hydrogel reinforced by biphasic calcium phosphate particles with size in the range of 100-300µm. The polysaccharide nature of the hydrogel mimics the ECM of natural bone, and the ceramic particles promote high osteoblast proliferation, assessed by Scanning Electron Microscopy analysis. Swelling properties allow significant adsorption of water solution (up to 200% of solution content) so that the bone defect space can be filled by the material in an in vivo scenario. The incorporation of ceramic particles makes the material stable at different pH and increases the compressive elastic modulus, toughness and ultimate tensile strength. Furthermore, cell studies with SAOS-2 human osteoblastic cell line show high cell proliferation and adhesion already after 72h, and the presence of ceramic particles increases the expression of alkaline phosphatase activity after 1week. These results suggest a great potential of the developed moldable biomaterials for the regeneration of the alveolar bone. STATEMENT OF SIGNIFICANCE: The positive fate of a surgical procedure involving the insertion of a titanium screw still depends on the quality and quantity of alveolar bone which is present in the extraction site. Available materials are basically hard scaffold materials with un-predictable behavior in different condition and difficult shaping properties. In this work we developed a novel pectin-chitosan hydrogel reinforced with ceramic particles. Polysaccharides simulate the extracellular matrix of natural bone and the extensive in vitro cells culture study allows to assess that the incorporation of the ceramic particles promote a pro-osteogenic response. Shape control, easy adaption of the extraction site, predictable behavior in different environment condition, swelling properties and an anti-inflammatory response are the significant characteristics of the developed biomaterial.

Engineered porous scaffolds for periprosthetic infection prevention.

Periprosthetic infection is a consequence of implant insertion procedures and strategies for its prevention involve either an increase in the rate of new bone formation or the release of antibiotics such as vancomycin. In this work we combined both strategies and developed a novel, multifunctional three-dimensional porous scaffold that was produced using hydroxyapatite (HA) and ß-tricalcium phosphate (ß-TCP), coupled with a pectin (PEC)-chitosan (CHIT) polyelectrolyte (PEI), and loaded with vancomycin (VCA). By this approach, a controlled vancomycin release was achieved and serial bacterial dilution test demonstrated that, after 1week, the engineered construct still inhibits the bacterial growth. Degradation tests show an excellent behavior in a physiological and acidic environment (<10% of mass loss). Furthermore, the PEI coating shows an anti-inflammatory response, and good cell proliferation and migration were demonstrated in vitro using osteoblast SAOS-2 cell line. This new engineered construct exhibits excellent properties both as an antibacterial material and as a stimulator of bone formation, which makes it a good candidate to contrast periprosthetic infection.

Collagen type I coating stimulates bone regeneration and osteointegration of titanium implants in the osteopenic rat.

PURPOSE: To investigate the effects of titanium implants functionalised with collagen type I (TiColl) on bone regeneration and osteointegration in a healthy and osteopenic rat animal model. METHOD: TiColl screws were implanted into the femoral condyles of healthy and osteopenic rats and compared with acid-etched titanium (Ti) screws. The osteointegration process was evaluated by a complementary approach combining microtomographic, histological, histomorphometric and biomechanical investigations at four and 12 weeks. RESULTS: The TiColl screw also ensured a greater mechanical stability; the push-out values for TiColl screws increased from four to 12 weeks (+28 %). The energy necessary to detach the bone from the screw was significantly higher for TiColl-functionalised screws in comparison to Ti screws (+23 %) at 12 weeks. Histomorphometric investigation revealed that total bone-to-implant contact was higher in TiColl screws in comparison to Ti screws (P < 0.05) and at epiphyseal level, increased bone-to-implant contact was found with TiColl screws in comparison to Ti screws (P < 0.05) in an ovariectomy (OVX) condition. A significant increase in the measured total bone ingrowth from four to 12 weeks was detected for both materials, but more significant for the TiColl material (P < 0.0005). Finally, bone ingrowth in the TiColl group was significantly higher (P < 0.005) in comparison to that of Ti screws in the SHAM condition at metaphyseal level at 12 weeks. CONCLUSION: The present results showed that TiColl is effective in promoting implant osteointegration even in compromised bone.

Affecting osteoblastic responses with in vivo engineered potato pectin fragments.

Pectins, complex plant-derived polysaccharides, are novel candidates for biomaterial nanocoatings. Pectic rhamnogalacturonan-I regions (RG-I) can be enzymatically treated to so-called modified hairy regions (MHR). We surveyed the growth and differentiation of murine preosteoblastic MC3T3-E1 cells on Petri dishes coated with RG-Is from native or genetically engineered potato tubers. Uncoated tissue culture polystyrene (TCPS) and aminated (AMI) dishes served as controls. MHRPTR_GAL sample was depleted of galactose (9 mol % galactose; 23 mol % arabinose) and MHRPTR_ARA of arabinose (61 mol % galactose; 6 mol % arabinose). Wild-type (modified hairy region from potato pectin (MHRP)_WT) fragment contained default amounts (58 mol % galactose; 13 mol % arabinose) of both sugars. Focal adhesions (FAs) indicating cellular attachment were quantified. Reverse transcriptase polymerase chain reaction (RT-PCR) of alkaline phosphatase and osteocalcin genes indicating osteoblastic differentiation was performed along with staining the produced calcium with tetracycline as an indicator of osteoblastic differentiation. Osteoblasts proliferated on all the samples to some extent. The control surfaces performed better than any of the pectin samples, of which the MHRP_WT seemed to function best. FA length was greater on MHRPTR_GAL than on other pectin samples, otherwise the mutants did not significantly deviate. RT-PCR results indicate that differences between the samples at the gene expression level might be even subtler. However, tetracycline-stained calcium-containing mineral was detected merely only on uncoated TCPS. These results indicate the possibility to affect bone cell growth with in vivo-modified pectin fragments, consecutively providing information on the significance of certain monosaccharides on the biocompatibility of these polysaccharides.

Pectin-coated titanium implants are well-tolerated in vivo.

Multiform coated titanium implants are widely used in orthopedic and dental surgery. In this study, we have investigated the reactivity of pectin-coated titanium samples implanted under the latissimus dorsi-muscle fascia of rats. Samples were coated with two enzyme treated apple pectins; modified hairy regions (MHR-A and MHR-B) that differed in chemical structure. Aminated (AMI) and uncoated titanium (Ti) served as controls. The thicknesses of the peri-implant fibrous tissue capsules formed 1 or 3 weeks after implantation were measured as indicative of possible inflammatory reactions toward the biomaterials. After 1 week, the MHR-B implant was surrounded by a thicker fibrous capsule (42.9 microm) than any of the other sample types: MHR-A (33.2 microm), AMI (32.5 microm), and Ti (32.3 microm), the last one being the only statistically significant difference. After 3 weeks, however, this difference disappeared; the capsule thicknesses around MHR-B and Ti implants had decreased to the values found for AMI and MHR-A. Additionally, the capsule formation represents merely a stromal rather than an inflammatory reaction, as indicated by the absence of activated macrophages or foreign body giant cells in the capsules. These results indicate for the first time the in vivo tolerability of covalently linked pectins, and suggest the feasibility of pectin-coated bone and dental implants for clinical use.

Enzymatically-tailored pectins differentially influence the morphology, adhesion, cell cycle progression and survival of fibroblasts.

Improved biocompatibility and performance of biomedical devices can be achieved through the incorporation of bioactive molecules on device surfaces. Five structurally distinct pectic polysaccharides (modified hairy regions (MHRs)) were obtained by enzymatic liquefaction of apple (MHR-B, MHR-A and MHR-alpha), carrot (MHR-C) and potato (MHR-P) cells. Polystyrene (PS) Petri dishes, aminated by a plasma deposition process, were surface modified by the covalent linking of the MHRs. Results clearly demonstrate that MHR-B induces cell adhesion, proliferation and survival, in contrast to the other MHRs. Moreover, MHR-alpha causes cells to aggregate, decrease proliferation and enter into apoptosis. Cells cultured in standard conditions with 1% soluble MHR-B or MHR-alpha show the opposite behaviour to the one observed on MHR-B and -alpha-grafted PS. Fibronectin was similarly adsorbed onto MHR-B and tissue culture polystyrene (TCPS) control, but poorly on MHR-alpha. The Fn cell binding site (RGD sequence) was more accessible on MHR-B than on TCPS control, but poorly on MHR-alpha. The disintegrin echistatin inhibited fibroblast adhesion and spreading on MHR-B-grafted PS, which suggests that MHRs control fibroblast behaviour via serum-adhesive proteins. This study provides a basis for the design of intelligently-tailored biomaterial coatings able to induce specific cell functions.

Modulating in vitro bone cell and macrophage behavior by immobilized enzymatically tailored pectins.

Previous work has reported the results of a multidisciplinary effort producing a proof-of-concept on the use of pectic polysaccharides in the surface modification of medical devices. This study was designed to learn more about the capability of engineered rhamnogalacturonan-I (RG-I) fractions of apple pectin to control bone cell and macrophage behavior. Thermanox or polystyrene Petri dishes were surface modified with two different modified hairy regions (MHRs) obtained by different enzymatic liquefaction processes of apples differing in relative amounts and lengths of their neutral side chains: (long-haired) MHR-alpha and (short-haired) MHR-B. Bone explants from 14-day-old chick embryos were cultured for 14 days on both pectic substrata. MHR-B promoted cell migration and differentiation, MHR-alpha did not. On MHR-alpha, J774.2 macrophages grew well, their percentage in G1 phase was decreased and in S phase increased, and they did not secrete either proinflammatory-cytokines or nitrites. Contrasting results were gained from macrophages on MHR-B, except for nitrite secretion. Thus, we conclude that coatings from tailored pectins show different biological activities in vitro and are potential innovative candidates for improving the biocompatibility of medical devices in various applications.

Effect of modified pectin molecules on the growth of bone cells.

The aim of this study was to investigate molecular candidates for bone implant nanocoatings, which could improve biocompatibility of implant materials. Primary rat bone cells and murine preosteoblastic MC3T3-E1 cells were cultured on enzymatically modified hairy regions (MHR-A and MHR-B) of apple pectins. MHRs were covalently attached to tissue culture polystyrene (TCPS) or glass. Uncoated substrata or bone slices were used as controls. Cell attachment, proliferation, and differentiation were investigated with fluorescence and confocal microscopy. Bone cells seem to prefer MHR-B coating to MHR-A coating. On MHR-A samples, the overall numbers as well as proportions of active osteoclasts were diminished compared to those on MHR-B, TCPS, or bone. Focal adhesions indicating attachment of the osteoblastic cells were detected on MHR-B and uncoated controls but not on MHR-A. These results demonstrate the possibility to modify surfaces with pectin nanocoatings.

Effects on interfacial properties and cell adhesion of surface modification by pectic hairy regions.

Polystyrene Petri dishes, aminated by a plasma deposition process, were surface modified by the covalent linking of two different enzymatically modified hairy regions (HRs) from pectin containing, for example, rhamnogalacturonan-I and xylogalacturonan structural elements. The two polysaccharide preparations share the same structural elements of apple pectin, but the relative amounts and lengths of the neutral side chains present differ. Surface analysis by X-ray photoelectron spectroscopy, contact angle measurement, and atomic force microscope (AFM) force-separation curves was used to characterize the effects on surface chemistry and interfacial forces of the surface modification process. Cell adhesion experiments using continuous L-929 fibroblasts and primary aortic smooth muscle cells were performed to evaluate the effect of the polysaccharide nature on cell adhesion. Results show that immobilization of the HR affects the interfacial field of forces and the cell behavior: "equilibrium" contact angles, obtained by a recently introduced vibrational approach, decrease after HR immobilization reaching a value close to 20 degrees . AFM force-separation curves show a more extended (or softer) interface in the case of the HR bearing longer side chains. Accordingly, depending on the HR preparation, cells shifted from spread morphology and adhesion behavior quantitatively comparable to that observed on conventional tissue culture polystyrene to rounded morphology and significantly lower adhesion. These data show that engineering of plant pectins can be a valuable tool to prepare novel and finely tuned polysaccharides having different chemico-physical and biological properties, to be used in the surface modification of medical devices and materials.