Physical properties imparted by genipin to chitosan for tissue regeneration with human stem cells: A review.

Genipin is a fully assessed non-cytotoxic crosslinking compound. The chitosan|genipin physical properties such as morphology, roughness, porosity, hydrophilicity, ζ-potential, surface area and surface energy exert control over cell adhesion, migration, phenotype maintenance and intracellular signaling in vitro, and cell recruitment at the tissue-scaffold interface in vivo. For example a therapy using fucose|chitosan|genipin nanoparticles encapsulating amoxicillin, based on the recognition of fucose by H. pylori, leads to sharply improved clinical results. A bioactive scaffold sensitive to environmental stimuli provides an alternative approach for inducing adipose stem cell chondrogenesis: the expression of specific genes, the accumulation of cartilage-related macromolecules and the mechanical properties are comparable to the original cartilage-derived matrix (CDM), thus making the CDM|genipin a contraction-free biomaterial suitable for cartilage tissue engineering. For the regeneration of the cartilage, chitosan|genipin permits to modulate matrix synthesis and proliferation of chondrocytes by dynamic compression; chondrocytes cultured on the composite substrate produce much more collagen-II and sulfated GAG. The main advantages gained in the bone regeneration area with chitosan|genipin are: acceleration of mineral deposition; enhancement of adhesion, proliferation and differentiation of osteoblasts; promotion of the expression of osteogenic differentiation markers; greatly improved viability of human adipose stem cells.

Genipin-Crosslinked Chitosan Gels and Scaffolds for Tissue Engineering and Regeneration of Cartilage and Bone.

The present review article intends to direct attention to the technological advances made since 2009 in the area of genipin-crosslinked chitosan (GEN-chitosan) hydrogels. After a concise introduction on the well recognized characteristics of medical grade chitosan and food grade genipin, the properties of GEN-chitosan obtained with a safe, spontaneous and irreversible chemical reaction, and the quality assessment of the gels are reviewed. The antibacterial activity of GEN-chitosan has been well assessed in the treatment of gastric infections supported by Helicobacter pylori. Therapies based on chitosan alginate crosslinked with genipin include stem cell transplantation, and development of contraction free biomaterials suitable for cartilage engineering. Collagen, gelatin and other proteins have been associated to said hydrogels in view of the regeneration of the cartilage. Viability and proliferation of fibroblasts were impressively enhanced upon addition of poly-l-lysine. The modulation of the osteocytes has been achieved in various ways by applying advanced technologies such as 3D-plotting and electrospinning of biomimetic scaffolds, with optional addition of nano hydroxyapatite to the formulations. A wealth of biotechnological advances and know-how has permitted reaching outstanding results in crucial areas such as cranio-facial surgery, orthopedics and dentistry. It is mandatory to use scaffolds fully characterized in terms of porosity, pore size, swelling, wettability, compressive strength, and degree of acetylation, if the osteogenic differentiation of human mesenchymal stem cells is sought: in fact, the novel characteristics imparted by GEN-chitosan must be simultaneously of physico-chemical and cytological nature. Owing to their high standard, the scientific publications dated 2010-2015 have met the expectations of an interdisciplinary audience.

Emerging biomedical applications of nano-chitins and nano-chitosans obtained via advanced eco-friendly technologies from marine resources.

The present review article is intended to direct attention to the technological advances made in the 2010-2014 quinquennium for the isolation and manufacture of nanofibrillar chitin and chitosan. Otherwise called nanocrystals or whiskers, n-chitin and n-chitosan are obtained either by mechanical chitin disassembly and fibrillation optionally assisted by sonication, or by e-spinning of solutions of polysaccharides often accompanied by poly(ethylene oxide) or poly(caprolactone). The biomedical areas where n-chitin may find applications include hemostasis and wound healing, regeneration of tissues such as joints and bones, cell culture, antimicrobial agents, and dermal protection. The biomedical applications of n-chitosan include epithelial tissue regeneration, bone and dental tissue regeneration, as well as protection against bacteria, fungi and viruses. It has been found that the nano size enhances the performances of chitins and chitosans in all cases considered, with no exceptions. Biotechnological approaches will boost the applications of the said safe, eco-friendly and benign nanomaterials not only in these fields, but also for biosensors and in targeted drug delivery areas.

Oral chondroprotection with nutraceuticals made of chondroitin sulphate plus glucosamine sulphate in osteoarthritis.

Oral supplementation of chondroitin sulphate plus glucosamine helps repair the articular surface in osteoarthritis. Chondroitin-S reduces the concentration of the pro-inflammatory cytokines and transcription factor involved in inflammation. GlcN.S enhances cartilage specific matrix components and prevents collagen degeneration in chondrocytes by inhibiting hydrolytic enzymes, and preventing the oxidation of lipids and proteins. Chondroitin-S plus GlcN.S are slow-acting drugs that alleviate pain and partly restore joint function in OA patients. Orally administered pharmaceutical-grade chondroitin-S plus GlcN.S stabilize the joint space narrowing and significantly decrease the number of patients with new erosive OA. They are safe and no adverse events have ever been reported; they are recommended by EULAR and OARSI. The cost/effectiveness of the oral chondroitin-S plus GlcN.S therapy derives from the reduction of costs for physiotherapy, and for gastroprotective and non-steroidal drugs. The synergistic association of these two world-widely preferred nutraceuticals is a step forward in the management of OA.

Chitosan stabilizes platelet growth factors and modulates stem cell differentiation toward tissue regeneration.

The idea of using chitosan as a functional delivery aid to support simultaneously PRP, stem cells and growth factors (GF) is associated with the intention to use morphogenic biomaterials to modulate the natural healing sequence in bone and other tissues. For example, chitosan-chondroitin sulfate loaded with platelet lysate was included in a poly(D,L-lactate) foam that was then seeded with human adipose-derived stem cells and cultured in vitro under osteogenic stimulus: the platelet lysate provided to the bone tissue the most suitable assortment of GF which induces the osteogenic differentiation of the mesenchymal stem cells. PDGF, FGF, IGF and TGF-ß were protagonists in the repair of callus fractures. The release of GF from the composites of chitosan-PRP and either nano-hydroxyapatite or tricalcium phosphate was highly beneficial for enhancing MSC proliferation and differentiation, thus qualifying chitosan as an excellent vehicle. A number of biochemical characteristics of chitosan exert synergism with stem cells in the regeneration of soft tissues.

Chitosan, hyaluronan and chondroitin sulfate in tissue engineering for cartilage regeneration: a review.

Injection of hyaluronan into osteoarthritic joints restores the viscoelasticity, augments the flow of joint fluid, normalizes endogenous hyaluronan synthesis, and improves joint function. Chitosan easily forms polyelectrolyte complexes with hyaluronan and chondroitin sulfate. Synergy of chitosan with hyaluronan develops enhanced performances in regenerating hyaline cartilage, typical results being structural integrity of the hyaline-like neocartilage, and reconstitution of the subchondral bone, with positive cartilage staining for collagen-II and GAG in the treated sites. Chitosan qualifies for the preparation of scaffolds intended for the regeneration of cartilage: it yields mesoporous cryogels; it provides a friendly environment for chondrocytes to propagate, produce typical ECM, and assume the convenient phenotype; it is a good carrier for growth factors; it inactivates metalloproteinases thus preventing collagen degradation; it is suitable for the induction of the chondrogenic differentiation of mesenchymal stem cells; it is a potent means for hemostasis and platelet delivery.

Biomedical exploitation of chitin and chitosan via mechano-chemical disassembly, electrospinning, dissolution in imidazolium ionic liquids, and supercritical drying.

Recently developed technology permits to optimize simultaneously surface area, porosity, density, rigidity and surface morphology of chitin-derived materials of biomedical interest. Safe and ecofriendly disassembly of chitin has superseded the dangerous acid hydrolysis and provides higher yields and scaling-up possibilities: the chitosan nanofibrils are finding applications in reinforced bone scaffolds and composite dressings for dermal wounds. Electrospun chitosan nanofibers, in the form of biocompatible thin mats and non-wovens, are being actively studied: composites of gelatin + chitosan + polyurethane have been proposed for cardiac valves and for nerve conduits; fibers are also manufactured from electrospun particles that self-assemble during subsequent freeze-drying. Ionic liquids (salts of alkylated imidazolium) are suitable as non-aqueous solvents that permit desirable reactions to occur for drug delivery purposes. Gel drying with supercritical CO(2) leads to structures most similar to the extracellular matrix, even when the chitosan is crosslinked, or in combination with metal oxides of interest in orthopedics.

Chitins and chitosans as immunoadjuvants and non-allergenic drug carriers.

Due to the fact that some individuals are allergic to crustaceans, the presumed relationship between allergy and the presence of chitin in crustaceans has been investigated. In vivo, chitin is part of complex structures with other organic and inorganic compounds: in arthropods chitin is covalently linked to proteins and tanned by quinones, in fungi it is covalently linked to glucans, while in bacteria chitin is diversely combined according to Gram(+/-) classification. On the other hand, isolated, purified chitin is a plain polysaccharide that, at the nano level, presents itself as a highly associated structure, recently refined in terms of regularity, nature of bonds, crystallinity degree and unusual colloidal behavior. Chitins and modified chitins exert a number of beneficial actions, i.e., (i) they stimulate macrophages by interacting with receptors on the macrophage surface that mediate the internalization of chitin particles to be degraded by lysozyme and N-acetyl-beta-glucosaminidase (such as Nod-like, Toll-like, lectin, Dectin-1, leukotriene 134 and mannose receptors); (ii) the macrophages produce cytokines and other compounds that confer non-specific host resistance against bacterial and viral infections, and anti-tumor activity; (iii) chitin is a strong Th1 adjuvant that up-regulates Th1 immunity induced by heat-killed Mycobacterium bovis, while down- regulating Th2 immunity induced by mycobacterial protein; (iv) direct intranasal application of chitin microparticles into the lung was also able to significantly down-regulate allergic response to Dermatophagoids pteronyssinus and Aspergillus fumigatus in a murine model of allergy; (v) chitin microparticles had a beneficial effect in preventing and treating histopathologic changes in the airways of asthmatic mice; (vi) authors support the fact that chitin depresses the development of adaptive type 2 allergic responses. Since the expression of chitinases, chitrotriosidase and chitinase-like proteins is greatly amplified during many infections and diseases, the common feature of chitinase-like proteins and chitinase activity in all organisms appears to be the biochemical defense of the host. Unfortunately, conceptual and methodological errors are present in certain recent articles dealing with chitin and allergy, i.e., (1) omitted consideration of mammalian chitinase and/or chitotriosidase secretion, accompanied by inactive chitinase-like proteins, as an ancestral defensive means against invasion, capable to prevent the insurgence of allergy; (2) omitted consideration of the fact that the mammalian organism recognizes more promptly the secreted water soluble chitinase produced by a pathogen, rather than the insoluble and well protected chitin within the pathogen itself; (3) superficial and incomplete reports and investigations on chitin as an allergen, without mentioning the potent allergen from crustacean flesh, tropomyosine; (4) limited perception of the importance of the chemical/biochemical characteristics of the isolated chitin or chitosan for the replication of experiments and optimization of results; and (5) lack of interdisciplinarity. There is quite a large body of knowledge today on the use of chitosans as biomaterials, and more specifically as drug carriers for a variety of applications: the delivery routes being the same as those adopted for the immunological studies. Said articles, that devote attention to the safety and biocompatibility aspects, never reported intolerance or allergy in individuals and animals, even when the quantities of chitosan used in single experiments were quite large. Therefore, it is concluded that crab, shrimp, prawn and lobster chitins, as well as chitosans of all grades, once purified, should not be considered as "crustacean derivatives", because the isolation procedures have removed proteins, fats and other contaminants to such an extent as to allow them to be classified as chemicals regardless of their origin.

Chitin deacetylases: properties and applications.

Chitin deacetylases, occurring in marine bacteria, several fungi and a few insects, catalyze the deacetylation of chitin, a structural biopolymer found in countless forms of marine life, fungal cell and spore walls as well as insect cuticle and peritrophic matrices. The deacetylases recognize a sequence of four GlcNAc units in the substrate, one of which undergoes deacetylation: the resulting chitosan has a more regular deacetylation pattern than a chitosan treated with hot NaOH. Nevertheless plain chitin is a poor substrate, but glycolated, reprecipitated or depolymerized chitins are good ones. The marine Vibrio sp. colonize the chitin particles and decompose the chitin thanks to the concerted action of chitinases and deacetylases, otherwise they could not tolerate chitosan, a recognized antibacterial biopolymer. In fact, chitosan is used to prevent infections in fishes and crustaceans. Considering that chitin deacetylases play very important roles in the biological attack and defense systems, they may find applications for the biological control of fungal plant pathogens or insect pests in agriculture and for the biocontrol of opportunistic fungal human pathogens.

The biocompatibility of dibutyryl chitin in the context of wound dressings.

Dibutyryl chitin (DBC) is a modified chitin carrying butyryl groups at 3 and 6 positions; its peculiarity is that it dissolves promptly in common solvents, while being insoluble in aqueous systems. The high biocompatibility of dibutyryl chitin in the form of films and non-wovens has been demonstrated for human, chick and mouse fibroblasts by the Viability/Cytotoxicity assay, In situ Cell Proliferation assay, Neutral Red Retention assay, Lactate Dehydrogenase Release assay, MTS cytotoxicity assay, and scanning electron microscopy. DBC was hardly degradable by lysozyme, amylase, collagenase, pectinase and cellulase over the observation period of 48 days at room temperature, during which no more than 1.33% by weight of the DBC filaments (0.3 mm diameter) was released to the aqueous medium. DBC non-wovens were incorporated into 5-methylpyrrolidinone chitosan solution and submitted to freeze-drying to produce a reinforced wound dressing material. The latter was tested in vivo in full thickness wounds in rats. The insertion of 4x4 mm pieces did not promote any adverse effect on the healing process, as shown histologically. DBC is therefore suitable for contacting intact and wounded human tissues.

Alkaline chitosan solutions.

Rigid and transparent hydrogels were obtained upon pouring chitosan salt solutions into saturated ammonium hydrogen carbonate. Incubation at 20 degrees C for 5 days yielded chitosan carbamate ammonium salt, Chit-NHCO(2)(-)NH(4)(+) a chemical species that either by hydrolysis or by thermal treatment decomposed to restore chitosan in free amine form. Chitosans of different degrees of acetylation, molecular sizes and origins (squid and crustaceans) were used as hydrochloride, acetate, glycolate, citrate and lactate salts. Their hydrogels obtained in ammonium hydrogen carbonate yielded chitosan solutions at pH values as high as 9.6, from which microspheres of regenerated chitosans were obtained upon spray-drying. These materials had a modest degree of crystallinity depending on the partial acylation that took place at the sprayer temperature (168 degrees C). Citrate could cross-link chitosan and impart insolubility to the microspheres. Chloride on the contrary permitted to prepare microspheres of chitosan in free amine form. By the NH(4)HCO(3) treatment, the cationicity of chitosan could be reversibly masked in view of mixing chitosan with alginate in equimolar ratio without coacervation. The clear and poorly viscous solutions of mixed chitosan carbamate and alginate were spray-dried at 115 degrees C to manufacture chitosan-alginate microspheres having prevailing diameter approx 2 micron.

Natural and artificial chitosan-inorganic composites.

Taking inspiration from many chitin-inorganic composites in nature, a number of recent articles throw light on the manufacture of such composites based on calcium carbonate, calcium phosphate and silica. These novel materials are important in the field of blood-compatible materials, bone substitutes, and cements for bone repair and reconstruction. This approach provides an attractive alternative to the processing of inorganic thin films, especially in applications where substrates cannot be exposed to high temperatures.