Carbohydrate/protein hydrogels as responsive scaffolds in controlling inorganic crystallization

<p>The strategies that both invertebrate and vertebrate organism use to produce organic-inorganic composite materials for different purposes such as mechanical support and protection for the body are fascinating. While extensive research has been done on understanding the basic principles of biomineral formation, mimicking the critical principles of the mechanisms of biomineralization in vitro and fully capturing the structural information and characteristics remain challenging issues for scientists. Calcium is an essential element in biological systems. It plays a central role in the mineralization and maintenance of the skeleton as well as in fundamental physiological processes including growth and development in vertebrates. Within a biological organism calcium ions are stored, delivered, or released in the presence of different anions such as phosphate, carbonate and citrate. Competition between the different anions which interact with calcium ions in different hydrogel matrices leads to manipulation of the various composite materials produced such as bone and nacre. Soluble anionic acidic macromolecules associated with biominerals play a vital role in modulating the mineral morphology and hierarchy of the organized composite. Understanding the interaction between the constituent ions and the organic matrix is crucial if we are to make synthetic materials, the structure and properties of which replicate those of native biominerals, or materials that have the storage and/or release characteristics of foods, for example. Carbohydrate-based hydrogels versus protein-based hydrogels are used here as scaffolds for the synthesis of calcium carbonate and calcium phosphate biominerals. Water soluble acidic additives are used to modulate the nucleation and growth of the minerals. In particular chitosan and gelatin hydrogel templates were used as the mineralization scaffolds. Three different mineralization methods were used: the Kitano, alternate soaking and McGrath methods. Monomeric vs. polymeric additives (acrylic acid, glutamic acid, aspartic acid and their corresponding polymers) were introduced into all systems in order to control the nucleation and growth of the so-formed minerals. The morphology, crystallinity, polymorphism and composition of the synthesized organic-inorganic composites were investigated. Analyses were carried out using a number of techniques including Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), X-ray Diffraction (XRD), micro-Raman spectroscopy and solid-sate NMR. In the case of calcium carbonate the morphology and crystallinity were more affected by the use of polymeric additives compared with their monomeric equivalents. In particular the calcium carbonate preferentially grew laterally within and on the periphery of the chitosan or gelatin hydrogel scaffold. This results from the formation of a polyelectrolyte complex between the chitosan or gelatin hydrogels and the added polymer. The presence of the polyelectrolyte complex modifies the nucleation of the mineral. Nanoparticles are preferentially formed which then aggregate together maintaining a lateral perspective with the scaffold. In the case of calcium phosphate mostly spherical and platelet-shaped morphologies composed of amorphous calcium phosphate and poorly crystalline hydroxylapatite respectively were always formed within both chitosan and gelatin hydrogel scaffolds. pH was also found to be a key factor in controlling which polymorph of calcium phosphate precipitates. The crystallinity is influenced by the presence of additives for chitosan scaffold. For systems with added aspartic or polyaspartic acid platelet-shaped CaP forms. These crystals are more highly crystalline compared to those where predominately the porous spherical calcium phosphate morphology is observed which form when L-glutamic acid is added. In the presence of polymeric additives nanoparticles form which then aggregate to yield larger crystals. Such aggregation was preferentially observed for gelatin scaffolds. Citrate anions are particularly important in calcium phosphate precipitation in bone. Biomimetic hydroxylapatite-chitosan and hydroxylapatite-gelatin nanocomposite were synthesized where citrate ions were used to control the size and crystallinity of the hydroxylapatite crystals. TEM data show that the size of the hydroxylapatite crystals decreases upon introducing citrate ions into the systems. Solid-state NMR dipolar dephasing data indicate the hydroxylapatite precipitation can be stabilized with 2.5 wt% sodium citrate with respect to the chitosan and gelatin mass. The data included within this thesis illustrate that both gelatin and chitosan hydrogel scaffolds display similar ability in modulating calcium carbonate or calcium phosphate crystallization in the absence and presence of additives. The role of soluble acidic additives is significant in the formation of biominerals. These results reveal therefore the possibility that carbohydrate-based systems, which have many advantages over protein-based systems, could be used to provide more options for fabricating new implantable materials for humans and animals. The results from the combination of techniques used including XRD, SSNMR and TEM showed the possibility of in vitro synthesis of a bio-nanocomposite material in the presence of citrate similar to that of natural bone (in terms of composition and morphology). The achievement of this work demonstrates that new advanced materials with various composite structures and morphologies can be synthesized through a biomimetic biomineralization mechanism under ambient conditions similar to natural materials such as bone and nacre. These advancements have potential application in biomedical research and more specifically in fabrication of implantable materials.</p>

Carbohydrate/protein hydrogels as responsive scaffolds in controlling inorganic crystallization

<p>The strategies that both invertebrate and vertebrate organism use to produce organic-inorganic composite materials for different purposes such as mechanical support and protection for the body are fascinating. While extensive research has been done on understanding the basic principles of biomineral formation, mimicking the critical principles of the mechanisms of biomineralization in vitro and fully capturing the structural information and characteristics remain challenging issues for scientists. Calcium is an essential element in biological systems. It plays a central role in the mineralization and maintenance of the skeleton as well as in fundamental physiological processes including growth and development in vertebrates. Within a biological organism calcium ions are stored, delivered, or released in the presence of different anions such as phosphate, carbonate and citrate. Competition between the different anions which interact with calcium ions in different hydrogel matrices leads to manipulation of the various composite materials produced such as bone and nacre. Soluble anionic acidic macromolecules associated with biominerals play a vital role in modulating the mineral morphology and hierarchy of the organized composite. Understanding the interaction between the constituent ions and the organic matrix is crucial if we are to make synthetic materials, the structure and properties of which replicate those of native biominerals, or materials that have the storage and/or release characteristics of foods, for example. Carbohydrate-based hydrogels versus protein-based hydrogels are used here as scaffolds for the synthesis of calcium carbonate and calcium phosphate biominerals. Water soluble acidic additives are used to modulate the nucleation and growth of the minerals. In particular chitosan and gelatin hydrogel templates were used as the mineralization scaffolds. Three different mineralization methods were used: the Kitano, alternate soaking and McGrath methods. Monomeric vs. polymeric additives (acrylic acid, glutamic acid, aspartic acid and their corresponding polymers) were introduced into all systems in order to control the nucleation and growth of the so-formed minerals. The morphology, crystallinity, polymorphism and composition of the synthesized organic-inorganic composites were investigated. Analyses were carried out using a number of techniques including Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), X-ray Diffraction (XRD), micro-Raman spectroscopy and solid-sate NMR. In the case of calcium carbonate the morphology and crystallinity were more affected by the use of polymeric additives compared with their monomeric equivalents. In particular the calcium carbonate preferentially grew laterally within and on the periphery of the chitosan or gelatin hydrogel scaffold. This results from the formation of a polyelectrolyte complex between the chitosan or gelatin hydrogels and the added polymer. The presence of the polyelectrolyte complex modifies the nucleation of the mineral. Nanoparticles are preferentially formed which then aggregate together maintaining a lateral perspective with the scaffold. In the case of calcium phosphate mostly spherical and platelet-shaped morphologies composed of amorphous calcium phosphate and poorly crystalline hydroxylapatite respectively were always formed within both chitosan and gelatin hydrogel scaffolds. pH was also found to be a key factor in controlling which polymorph of calcium phosphate precipitates. The crystallinity is influenced by the presence of additives for chitosan scaffold. For systems with added aspartic or polyaspartic acid platelet-shaped CaP forms. These crystals are more highly crystalline compared to those where predominately the porous spherical calcium phosphate morphology is observed which form when L-glutamic acid is added. In the presence of polymeric additives nanoparticles form which then aggregate to yield larger crystals. Such aggregation was preferentially observed for gelatin scaffolds. Citrate anions are particularly important in calcium phosphate precipitation in bone. Biomimetic hydroxylapatite-chitosan and hydroxylapatite-gelatin nanocomposite were synthesized where citrate ions were used to control the size and crystallinity of the hydroxylapatite crystals. TEM data show that the size of the hydroxylapatite crystals decreases upon introducing citrate ions into the systems. Solid-state NMR dipolar dephasing data indicate the hydroxylapatite precipitation can be stabilized with 2.5 wt% sodium citrate with respect to the chitosan and gelatin mass. The data included within this thesis illustrate that both gelatin and chitosan hydrogel scaffolds display similar ability in modulating calcium carbonate or calcium phosphate crystallization in the absence and presence of additives. The role of soluble acidic additives is significant in the formation of biominerals. These results reveal therefore the possibility that carbohydrate-based systems, which have many advantages over protein-based systems, could be used to provide more options for fabricating new implantable materials for humans and animals. The results from the combination of techniques used including XRD, SSNMR and TEM showed the possibility of in vitro synthesis of a bio-nanocomposite material in the presence of citrate similar to that of natural bone (in terms of composition and morphology). The achievement of this work demonstrates that new advanced materials with various composite structures and morphologies can be synthesized through a biomimetic biomineralization mechanism under ambient conditions similar to natural materials such as bone and nacre. These advancements have potential application in biomedical research and more specifically in fabrication of implantable materials.</p>

Photocrosslinked gelatin hydrogel improves wound healing and skin flap survival by the sustained release of basic fibroblast growth factor

Biomaterials traditionally used for wound healing can act as a temporary barrier to halt bleeding, prevent infection, and enhance regeneration. Hydrogels are among the best candidates for wound healing owing to their moisture retention and drug-releasing properties. Photo-polymerization using visible light irradiation is a promising method for hydrogel preparation since it can easily control spatiotemporal reaction kinetics and rapidly induce a single-step reaction under mild conditions. In this study, photocrosslinked gelatin hydrogels were imparted with properties namely fast wound adherence, strong wet tissue surface adhesion, greater biocompatibility, long-term bFGF release, and importantly, ease of use through the modification and combination of natural bio-macromolecules. The production of a gelatin hydrogel made of natural gelatin (which is superior to chemically modified gelatin), crosslinked by visible light, which is more desirable than UV light irradiation, will enable its prolonged application to uneven wound surfaces. This is due to its flexible shape, along with the administration of cell growth factors, such as bFGF, for tissue regeneration. Further, the sustained release of bFGF enhances wound healing and skin flap survival. The photocrosslinking gelatin hydrogel designed in this study is a potential candidate to enhance wound healing and better skin flap survival.

Combined therapy of platelet-rich plasma and basic fibroblast growth factor using gelatin-hydrogel sheet for rotator cuff healing in rat models

Introduction Excellent outcomes of arthroscopic rotator cuff repair for small and medium tears have been recently reported. However, re-tears after surgery have been a common complication after surgical repair of large and massive rotator cuff tears and often occur in early postoperative phase. It was previously reported that basic fibroblast growth factor and platelet-rich plasma enhanced rotator cuff tear healing. We hypothesized that this combined therapy could enhance rotator cuff healing after rotator cuff repair in a rat model. This study aimed to evaluate the efficacy of combined therapy of platelet-rich plasma and basic fibroblast growth factor with gelatin-hydrogel sheet. Methods To create a rotator cuff defect, the infraspinatus tendon of Sprague Dawley rat was resected from the greater tuberosity. The infraspinatus tendons were repaired and covered with gelatin-hydrogel sheet impregnated with PBS (control group), basic fibroblast growth factor (bFGF group), platelet-rich plasma (PRP group), or both basic fibroblast growth factor and platelet-rich plasma (combined group). Histological examinations were conducted using hematoxylin and eosin, safranin O, and immunofluorescence staining, such as Isolectin B4, type II collagen at 2 weeks postoperatively. For mechanical analysis, ultimate failure load of the tendon-humeral head complex was evaluated at 6 weeks postoperatively. Results In the hematoxylin and eosin staining, the tendon maturing score of the combined group was higher than that of the control group at postoperative 2 weeks. In the safranin O staining, stronger proteoglycan staining was observed in the combined group compared with the other groups at postoperative 2 weeks. Vascular staining with isolectin B4 in 3 treatment groups was significantly higher than that in the control group. Type II collagen expression in the combined group was significantly higher than those in the other groups. The ultimate failure load of the combined group was significantly higher than that of the control group. Conclusion Combined therapy of basic fibroblast growth factor and platelet-rich plasma promoted angiogenesis, tendon maturing and fibrocartilage regeneration at the enthesis, which could enhance the mechanical strength. It was suggested that combined basic fibroblast growth factor and platelet-rich plasma might enhance both tendon and bone–tendon junction healing, and basic fibroblast growth factor and platelet-rich plasma might be synergistic.

Adhesive and normal stress-dependent dynamic friction of a gelatin hydrogel

Adhesion and friction of soft solids on hard surfaces are the important properties for a variety of practical applications. In the present study, Coulomb's law of friction is used for characterizing adhesive friction as well as normal stress-dependent dynamic friction of a gelatin hydrogel on a fixed glass surface. The experimental data, concerning normal stress-dependent dynamic friction of different shear velocity, are obtained from literature. It is observed that both components of friction increase with shear velocity. More importantly, the scaling law shows that adhesive stress varies almost linearly with corresponding coefficient of friction of the hydrogel. A dynamic friction model is also used to analyze the same experimental data to predict a negative normal stress at which dynamic friction reduces to zero, and this result matches closely with the experimental value.