Skin lesions are traumatic events that lead to increased fluid loss, infections, scars formation and the appearance of immunocompromised regions. The loss of skin integrity can result in significant physiological imbalances and disability or even death. So far, different wound dressings have been developed. Among them, hydrogels are the most applied. Herein, a thermoresponsive hydrogel was produced using chitosan and agarose formed at body temperature (37°C), in order to verify their applicability in the treatment of wounds. Hydrogel bactericidal activity, biocompatibility, morphology, porosity and wettability were characterized by confocal microscopy, MTS assay and SEM. The attained results revealed that hydrogel has a pore size (90–400 µm) compatible with cellular internalization and proliferation. A bactericidal activity was observed for hydrogels containing more than 188 µg/mL of chitosan. The results obtained demonstrated that this 3D network has the suitable properties for improving the healing process of cutaneous wounds.

In the area of tissue engineering different approaches have been studied, so far, for promoting regeneration or replacement of damaged tissues. Among the different materials developed, hydrogels, due to their biocompatibility and similarities with the native extracellular matrix, have emerged as suitable candidates for being used for different therapeutic purposes. Herein, photocrosslinkable hydrogels, composed by chitosan methacrylamide (ChMA) and gelatin methacrylamide (GelMA) were crosslinked by ultraviolet (UV) light, using Irgacue 2959 as photoinitiator. The morphological, physicochemical and biological properties of the hydrogels were characterized by scanning electron microscopy, Fourier transform infrared spectroscopy and nuclear magnetic resonance. The obtained results demonstrated that the developed hydrogels possess suitable properties for being used as 3D constructs on several areas of tissue engineering. Furthermore, these properties may allow their future application as space filling agents or as delivery vehicles of bioactive molecules and cells.

Wound healing is a complex process involving an integrated response by many different cell types and growth factors in order to achieve rapid restoration of skin architecture and function. The present study evaluated the applicability of a chitosan hydrogel (CH) as a wound dressing. Scanning electron microscopy analysis was used to characterize CH morphology. Fibroblast cells isolated from rat skin were used to assess the cytotoxicity of the hydrogel. CH was able to promote cell adhesion and proliferation. Cell viability studies showed that the hydrogel and its degradation by-products are noncytotoxic. The evaluation of the applicability of CH in the treatment of dermal burns in Wistar rats was performed by induction of full-thickness transcutaneous dermal wounds. Wound healing was monitored through macroscopic and histological analysis. From macroscopic analysis, the wound beds of the animals treated with CH were considerably smaller than those of the controls. Histological analysis revealed lack of a reactive or a granulomatous inflammatory reaction in skin lesions with CH and the absence of pathological abnormalities in the organs obtained by necropsy, which supported the local and systemic histocompatibility of the biomaterial. The present results suggest that this biomaterial may aid the re-establishment of skin architecture.

Healing enhancement and pain control are critical issues on wound management. So far, different wound dressings have been developed. Among them, hydrogels are the most applied. Herein, a thermoresponsive hydrogel was produced using chitosan (deacetylation degree 95%) and agarose. Hydrogel bactericidal activity, biocompatibility, morphology, porosity and wettability were characterized by confocal microscopy, MTS assay and SEM. The performance of the hydrogel in the wound healing process was evaluated through in vivo assays, during 21 days. The attained results revealed that hydrogel has a pore size (90–400 μm) compatible with cellular internalization and proliferation. A bactericidal activity was observed for hydrogels containing more than 188 μg/mL of chitosan. The improved healing and the lack of a reactive or a granulomatous inflammatory reaction in skin lesions treated with hydrogel demonstrate its suitability to be used in a near future as a wound dressing.

Cell-based therapies for regenerative medicine have been characterized by the low retention and integration of injected cells into host structures. Cell immobilization in hydrogels for target cell delivery has been developed to circumvent this issue. In this work mesenchymal stem cells isolated from Wistar rats bone marrow (rMSCs) were immobilized in alginate beads fabricated using an innovative approach involving the gellification of the liquid precursor droplets onto biomimetic superhydrophobic surfaces without the need of any precipitation bath. The process occurred in mild conditions preventing the loss of cell viability. Furthermore, fibronectin (FN) was also immobilized inside alginate beads with high efficiency in order to mimic the composition of the extracellular matrix. This process occurred in a very fast way (around 5 min), at room temperature, without aggressive mechanical strengths or particle aggregation. The methodology employed allowed the production of alginate beads exhibiting a homogenous rMSCs and FN distribution. Encapsulated rMSCs remained viable and were released from the alginate for more than 20 days. In vivo assays were also performed, by implanting these particles in a calvarial bone defect to evaluate their potential for bone tissue regeneration. Microcomputed tomography and histological analysis results showed that this hybrid system accelerated bone regeneration process. The methodology employed had a dual role by preventing cell and FN loss and avoiding any contamination of the beads or exchange of molecules with the surrounding environment. In principle, the method used for cell encapsulation could be extended to other systems aimed to be used in tissue regeneration strategies.

Hydrogels and lyophilisates were obtained by chemical crosslinking of gelatin using N-hydroxysuccinimide and N, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride. The systems were characterized with respect to the degree of crosslinking, morphology, water uptake, in vitro drug release and biocompatibility studies. Pilocarpine hydrochloride, a drug for the treatment of glaucoma, was loaded by soaking in an aqueous solution containing the drug. In vitro, the released drug percentage varied between 29.2% and 99.2% in 8 h of study. The release data were fitted to the Korsmeyer–Peppas equation to calculate the release exponent, which indicated anomalous transport for the release of pilocarpine. The corneal endothelial cell culture tests indicated that the prepared biomaterials are not cytotoxic.

The integrity of skin, as the largest organ of the human body, must be preserved in order to play its role as a protective barrier, in the maintenance of fluid homeostasis and temperature regulation. Skin lesions can occur due to different causes, being burns those responsible for extensive skin loss, infection, electrolyte imbalances and respiratory failure. In order to decrease the effects of skin damages, new skin substitutes have been developed to accelerate the healing process and thus restore the native structure of skin. Among the existent materials, hydrogels own the most desirable characteristics of an “ideal dressing”, such as biocompatibility and biodegradability. Currently, new photocrosslinkable hydrogels have been developed for tissue engineering purposes. Taking into account the intrinsic properties of hydrogels, the work plan developed during this master thesis allowed the production of photocrosslinkable hydrogels, composed of chitosan and gelatine that present some of the properties required for wound regeneration. Initially, methacrylate groups were added to the chitosan and gelatine primary amine groups, leading to the synthesis of methacrylamide chitosan (MAC) and methacrylamide gelatine (MAG). The chemical modification of the polymers was confirmed by proton nuclear magnetic resonance (1H NMR). Then, MAC and MAG hydrogels were produced using ultraviolet (UV) light in the presence of a photoinitiator (Irgacue 2959). The hydrogels were subsequently characterized by scanning electron microscopy and Fourier transform infrared spectroscopy. Porosity and swelling properties were also analyzed and revealed that the hydrogels with a higher content of chitosan had higher porosity and swelling capacity. The cytotoxic profile of the hydrogels was evaluated through an MTS assay, using human fibroblast cells. Cell adhesion on the surface of the hydrogels was visualized through scanning electron microscopy. The results obtained demonstrated that the hydrogels developed herein possess suitable properties for being used as wound dressings. Therefore, in the future in vivo studies will be performed to evaluate the histocompatibility of the hydrogels and their capacity to improve the wound healing process. In addition, growth factors and antimicrobial agents can be incorporated in the hydrogels, in order to improve wound repair and prevent bacterial infections, respectively.

Initiator-free injectable hydrogels are very interesting for drug and/or cell delivery applications, since they can be administered in a minimally invasive way, and avoid the use of potentially harmful chemical initiators. In the current work, oxidized dextran crosslinked with adipic acid dihydrazide hydrogels were further characterized and tuned to produce formulations, with the aim of producing an injectable formulation for the possible treatment of posterior eye diseases. The gelation rate and the hydrogel dissolution profile were shown to be dependent on the balance between the degree of dextran oxidation, and the concentration of both components. For the in vitro studies, rabbit corneal endothelial cells were seeded on the hydrogels to assess cytotoxicity. Hydrogels prepared with low oxidized dextrans were able to promote cell adhesion and proliferation to confluence in just 24 h, while more highly oxidized samples promoted cell adhesion and proliferation, but without achieving confluence. Cell viability studies were performed using MTS assays to verify the non-cytotoxicity of hydrogels and their degradation byproducts, rendering these formulations attractive for further in vivo studies.

Nesta Dissertação foi investigada a biodegradação de hidrogéis à base de alginato de sódio e quitosana em solo argiloso. O solo foi obtido de uma propriedade privada situada no município de Campos dos Goytacazes, região norte fluminense do Estado do Rio de Janeiro, promissora para a expansão agrícola de soja e milho. Foram realizados ensaios de biodegradação, onde o pH e a umidade do solo foram monitorados. Os hidrogéis degradados foram analisados por espectroscopia na região do infravermelho (FTIR), espectroscopia Raman, termogravimetria (TG), microscopia eletrônica de varredura (SEM) e microscopia confocal de varredura a laser (CLSM). Com o objetivo de se obter mais informações a respeito dos microrganismos responsáveis pela biodegradação dos hidrogéis, foi feito um estudo microbiológico do solo. Os ensaios de biodegradabilidade revelaram que os hidrogéis foram completamente biodegradados em seis semanas. O solo com hidrogel se manteve mais úmido do que o solo sem hidrogel, após sete dias, até oito semanas. Tais resultados indicaram o potencial do material como condicionador de solo. Por meio do FTIR e do Raman, foi possível identificar a quebra das cadeias poliméricas através da ligação glicosídica, mesmo com os polissacarídeos reticulados eletrostaticamente. Análises de SEM e de microscopia confocal evidenciaram a fragmentação da estrutura do hidrogel, alterando assim a sua morfologia. Os ensaios microbiológicos comprovaram que os hidrogéis à base de alginato de sódio e quitosana foram utilizados como fonte de carbono por fungos isolados do solo argiloso utilizado, com indicativos de que os fungos dos gêneros Aspergillus, Penicillium e Cunninghamella, além de leveduras, foram responsáveis, entre outros, por tal consumo.

The healing is a complex and dynamic event aimed at the restoration of tissue integrity. However, some factors, such as diabetes, can harm this physiological event. Chitosan, a derivative of chitin, has been considered as a versatile and effective option for the treatment of wounds. This study aimed to evaluate and compare the effect of chitosan 2% gel and chitosan spray based on secondary intention healing of skin wounds in diabetic rats. 54 rats were used, divided randomly into three groups: control group (CG), chitosan 2% gel group (GG), and Chitosan spray group (GS) with 18 animals each. The rats in GC, GG, and GS had their wounds treated daily with saline solution 0.9%, chitosan 2% gel, and chitosan spray, respectively. The groups were divided into three subgroups (n = 6) according to the times of histological analysis of wound (third, seventh, and 14th day). The induction of diabetes in the rats took place by intraperitoneal application of alloxan (120 mg/kg). After confirming the state of diabetes, the animals were anesthetized and the wounds made with the aid of a punch 9.0mm in the dorsal region. Macroscopic and microscopic analyzes were performed. Macroscopic analysis showed that after 14 days, the chitosan 2% gel and chitosan spray gave a contraction rate of 100% and 99%, respectively. At the histological level, there was the anticipated recruitment of mononuclear cells, fibroblasia, and deposition of dense fibrovascular tissue and marked fibrosis in the wounds of GG and GS, emphasizing the ability of this biopolymer to optimize the healing of diabetics. It can therefore be a therapeutic option for the development of other formulations that provide wound repair in diabetic patients.