Complexos polieletrólitos de quitosana e xantana carregados com ativos naturais e sintéticos: estudo físico/químico e microbiológico para aplicação de drug delivery / Polyelectrolyte complexes of chitosan and xanthan loaded with natural and synthetic actives: physicochemical and microbiological study for drug delivery application

A eficácia dos implantes osseointegrados é amplamente reconhecida na literatura científica. Contudo, infiltrações bacterianas na junção implante-pilar podem desencadear inflamação nos tecidos circundantes, contribuindo para a evolução de condições mais sérias, como a peri-implantite. O objetivo desse estudo foi produzir complexos polieletrólitos (PECs) de quitosana (Q) e xantana (X) em forma de membranas, carregá-las com ativos naturais e sintéticos antimicrobianos, caracterizálas estruturalmente e avaliá-las frente a degradação enzimática, cinética de liberação e ações antimicrobianas com finalidade de aplicação para drug delivery. Membranas de QX a 1% (m/v) foram produzidas em três proporções, totalizando doze grupos experimentais: QX (1:1); QX (1:2), QX (2:1), QX-P (com própolis) (1:1); QX-P (1:2); QX-P (2:1); QX-C (com canela) (1:1); QX-C (1:2); QX-C (2:1) e CLX (com clorexidina 0,2%) (1:1); CLX (1:2); CLX (2:1). Para os estudos de caracterização foram feitas análises da espessura em estado seco; análises morfológicas superficial e transversal em Microscopia Eletrônica de Varredura (MEV); análise estrutural de espectroscopia de infravermelho por transformada de Fourier (FTIR); análise de degradação por perda de massa sob ação da enzima lisozima; e análise da cinética de liberação dos ativos em saliva artificial. Para os testes microbiológicos, análises de verificação de halo de inibição e ação antibiofilme foram feitas contra cepas de Staphylococcus aureus (S. aureus) e Escherichia coli (E. coli). Os resultados demonstraram que a espessura das membranas variou conforme a proporção, sendo que o grupo QX (1:2) apresentou a maior média de 1,022 mm ± 0,2, seguida respectivamente do QX (1:1) com 0,641 mm ± 0,1 e QX (2:1) com 0,249 mm ± 0,1. Nas imagens de MEV é possível observar uma maior presença de fibras, rugosidade e porosidade nos grupos QX (1:2) e QX (1:1) respectivamente, e, no QX (2:1) uma superfície mais lisa, uniforme e fina. No FTIR foram confirmados os picos característicos dos materiais isoladamente, além de observar as ligações iônicas que ocorreram para formação dos PECs. Na análise de degradação, os grupos com ativos naturais adicionados tiveram melhores taxas de sobrevida do que os grupos QX. No teste de liberação, os grupos QX-P tiveram uma cinética mais lenta que os QX-C, cuja liberação acumulada de 100% foi feita em 24 h. Já nos testes do halo inibitório, somente os grupos CLX tiveram ação sobre as duas cepas, e os QX-P tiveram sobre S. aureus. Nas análises antibiofilme, os grupos CLX apresentaram as maiores taxas de redução metabólica nas duas cepas (± 79%); os grupos QX-P apresentaram taxas de redução similares em ambas as cepas, porém com percentual um pouco maior para E. coli (60- 80%) e os grupos QX-C tiveram grande discrepância entre as duas cepas: de 35 a 70% para S. aureus e 14 a 19% para E. coli. Pode-se concluir que, frente as análises feitas, o comportamento do material foi afetado diretamente pelos ativos adicionados a matriz polimérica. As proporções de Q ou X afetaram somente a espessura final. Quanto a aplicação proposta de drug delivery, os dispositivos apresentaram grande potencial, principalmente os grupos CLX e QX-P. (AU)

The effectiveness of osseointegrated implants is widely recognized in scientific literature. However, bacterial infiltrations at the implant-abutment interface may trigger inflammation in surrounding tissues, contributing to the development of more serious conditions, such as peri-implantitis. The aim of this study was to produce chitosan (Q) and xanthan (X) polyelectrolyte complexes (PECs) in the form of membranes, load and evaluate them for enzymatic degradation, release kinetics, and antimicrobial actions for drug delivery applications. QX membranes at 1% (w/v) were produced in three proportions, totaling twelve experimental groups: QX (1:1), QX (1:2), QX (2:1), QX-P (with propolis) (1:1), QX-P (1:2), QX-P (2:1), QX-C (with cinnamon) (1:1), QX-C (1:2), QX-C (2:1), and CLX (with 0.2% chlorhexidine) (1:1), CLX (1:2), CLX (2:1). Characterization studies included analyses of dry state thickness, surface and crosssectional morphology using Scanning Electron Microscopy (SEM), structural analysis by Fourier Transform Infrared (FTIR) spectroscopy, mass loss degradation analysis under lysozyme action, and active release kinetics analysis in artificial saliva. Microbiological tests included verification analyses of inhibition halos and antibiofilm action against strains of Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). Results showed that membrane thickness varied according to proportion, with group QX (1:2) presenting the highest average of 1.022 mm ± 0.2, followed by QX (1:1) with 0.641 mm ± 0.1, and QX (2:1) with 0.249 mm ± 0.1. SEM images showed greater presence of fibers, roughness, and porosity in groups QX (1:2) and QX (1:1) respectively, while QX (2:1) exhibited a smoother, more uniform, and thinner surface. FTIR confirmed characteristic peaks of the materials individually, besides showing ionic bonds formed for PECs. Degradation analysis revealed that groups with added natural actives had better survival rates than QX groups. In release tests, QX-P groups exhibited slower kinetics than QX-C, with 100% cumulative release achieved in 24 h. inhibitory halo tests, only CLX groups exhibited action against both strains, while QX-P acted against S. aureus. Antibiofilm analyses showed CLX groups with the highest metabolic reduction rates in both strains (± 79%); QX-P groups showed similar reduction rates in both strains, slightly higher for E. coli (60-80%), and QX-C groups had a significant discrepancy between strains: 35-70% for S. aureus and 14-19% for E. coli. In conclusion, material behavior was directly affected by added actives to the polymeric matrix. Proportions of Q or X only affected final thickness. Regarding proposed drug delivery applications, the devices showed great potential, especially CLX and QX-P groups.(AU)

Study on injectable chitosan hydrogel with tendon-derived stem cells for enhancing rotator cuff tendon-to-bone healing / 中国修复重建外科杂志

OBJECTIVE@#To explore the effect of chitosan (CS) hydrogel loaded with tendon-derived stem cells (TDSCs; hereinafter referred to as TDSCs/CS hydrogel) on tendon-to-bone healing after rotator cuff repair in rabbits.@*METHODS@#TDSCs were isolated from the rotator cuff tissue of 3 adult New Zealand white rabbits by Henderson step-by-step enzymatic digestion method and identified by multidirectional differentiation and flow cytometry. The 3rd generation TDSCs were encapsulated in CS to construct TDSCs/CS hydrogel. The cell counting kit 8 (CCK-8) assay was used to detect the proliferation of TDSCs in the hydrogel after 1-5 days of culture in vitro, and cell compatibility of TDSCs/CS hydrogel was evaluated by using TDSCs alone as control. Another 36 adult New Zealand white rabbits were randomly divided into 3 groups ( n=12): rotator cuff repair group (control group), rotator cuff repair+CS hydrogel injection group (CS group), and rotator cuff repair+TDSCs/CS hydrogel injection group (TDSCs/CS group). After establishing the rotator cuff repair models, the corresponding hydrogel was injected into the tendon-to-bone interface in the CS group and TDSCs/CS group, and no other treatment was performed in the control group. The general condition of the animals was observed after operation. At 4 and 8 weeks, real-time quantitative PCR (qPCR) was used to detect the relative expressions of tendon forming related genes (tenomodulin, scleraxis), chondrogenesis related genes (aggrecan, sex determining region Y-related high mobility group-box gene 9), and osteogenesis related genes (alkaline phosphatase, Runt-related transcription factor 2) at the tendon-to-bone interface. At 8 weeks, HE and Masson staining were used to observe the histological changes, and the biomechanical test was used to evaluate the ultimate load and the failure site of the repaired rotator cuff to evaluate the tendon-to-bone healing and biomechanical properties.@*RESULTS@#CCK-8 assay showed that the CS hydrogel could promote the proliferation of TDSCs ( P<0.05). qPCR results showed that the expressions of tendon-to-bone interface related genes were significantly higher in the TDSCs/CS group than in the CS group and control group at 4 and 8 weeks after operation ( P<0.05). Moreover, the expressions of tendon-to-bone interface related genes at 8 weeks after operation were significantly higher than those at 4 weeks after operation in the TDSCs/CS group ( P<0.05). Histological staining showed the clear cartilage tissue and dense and orderly collagen formation at the tendon-to-bone interface in the TDSCs/CS group. The results of semi-quantitative analysis showed that compared with the control group, the number of cells, the proportion of collagen fiber orientation, and the histological score in the TDSCs/CS group increased, the vascularity decreased, showing significant differences ( P<0.05); compared with the CS group, the proportion of collagen fiber orientation and the histological score in the TDSCs/CS group significantly increased ( P<0.05), while there was no significant difference in the number of cells and vascularity ( P>0.05). All samples in biomechanical testing failed at the repair site during the testing process. The ultimate load of the TDSCs/CS group was significantly higher than that of the control group ( P<0.05), but there was no significant difference compared to the CS group ( P>0.05).@*CONCLUSION@#TDSCs/CS hydrogel can induce cartilage regeneration to promote rotator cuff tendon-to-bone healing.

Development of chitosan based microencapsulated spray dried powder of tuna fish oil: oil load impact and oxidative stability / Desenvolvimento de pó microencapsulado por pulverização de óleo de atum à base de quitosana: impacto da carga de óleo e estabilidade oxidativa

Abstract The impact of fish oil concentration on the oxidative stability of microcapsules through the spray drying process using chitosan and maltodextrin as wall material was studied. Emulsions were prepared with different Tuna fish oil (TFO) content (TFO-10%, TFO20%, TF030% TF0-40%) while wall material concentration was kept constant. Microencapsulated powder resulting from emulsion prepared with high fish oil load have high moisture content, wettability, total oil and low encapsulation efficiency, hygroscopicity and bulk tapped density. Oxidative stability was evaluated periodically by placing microcapsules at room temperature. Microcapsules prepared with TFO-10% presented high oxidative stability in terms of peroxide value (2.94±0.04) and anisidine value (1.54±0.02) after 30 days of storage. It was concluded that optimal amounts of fish oil for microencapsulation are 10% and 20% using chitosan and maltodextrin that extended its shelf life during study period.

Resumo Foi estudado o impacto da concentração de óleo de peixe na estabilidade oxidativa de microcápsulas por meio do processo de secagem por atomização, utilizando quitosana e maltodextrina como material de parede. As emulsões foram preparadas com diferentes teores de óleo de atum (TFO) (TFO-10%, TFO20%, TF030% TF0-40%), enquanto a concentração de material de parede foi mantida constante. O pó microencapsulado resultante da emulsão preparada com alta carga de óleo de peixe tem alto teor de umidade, molhabilidade e óleo total e baixa eficiência de encapsulação, higroscopicidade e densidade extraída a granel. A estabilidade oxidativa foi avaliada periodicamente colocando microcápsulas à temperatura ambiente. As microcápsulas preparadas com TFO-10% apresentaram alta estabilidade oxidativa em termos de valor de peróxido (2,94 ± 0,04) e valor de anisidina (1,54 ± 0,02) após 30 dias de armazenamento. Concluiu-se que as quantidades ideais de óleo de peixe para microencapsulação são de 10% e 20% usando quitosana e maltodextrina que prolongaram sua vida útil durante o período de estudo.

Vestibuloplastia con láser de Er Cr:YSGG y nanotransportador biomolécula EPX: un nuevo cicatrizante periodontal / Vestibuloplasty with Er Cr:YSGG laser and EPX biomolecule nanocarrier: a new periodontal healing agent

La resorción ósea alveolar suele dar lugar a que las inserciones de la mucosa interfieran para la construcción, estabilidad y retención de una prótesis removible, una opción que permite modificar este tejido se obtiene por medio de una vestibuloplastia. Actualmente se puede favorecer la cicatrización de heridas utilizando láser de alta potencia aplicado a procedimientos quirúrgicos orales. Se realiza reporte de caso en paciente femenino a la que se realizó procedimiento de vestibuloplastia con láser de Er,Cr:YSGG, utilizando de forma postoperatoria gel de quitosano en nanotransportador biomolécula EPX. Se observa una cicatrización rápida y favorable al combinar ambas terapéuticas, además al utilizar productos con quitosano se disminuye el riesgo de la necrosis de fibroblastos gingivales humanos como recientemente se reportó en el uso de colutorios de clorhexidina (AU)

Alveolar bone resorption often results in mucosal insertions interfering with the construction, stability and retention of a removable prosthesis, an option to modify this tissue is obtained by means of vestibuloplasty. Currently, wound healing can be promoted by using high power laser applied to oral surgical procedures. A case report of a female patient who underwent a vestibuloplasty procedure with laser Er,Cr:YSGG, using chitosan gel with EPX biomolecule nanocarriers postoperatively. A fast and favorable healing is observed when combining both therapeutics, besides, when using products with chitosan, the risk of necrosis of human gingival fibroblasts is reduced, as recently reported in the use of chlorhexidine mouthwashes (AU)

Estudio de biocompatibilidad de matrices poliméricas combinadas, responsivas al pH, con aplicación en Ingeniería de Tejido Óseo / Biocompatibility study of combined pH-responsive polymeric matrices with application in Bone Tissue Engineering

El presente trabajo muestra la obtención de un material a partir de un polímero sintético (TerP) y otro natural, mediante entrecruzamiento físico y su caracterización fisicoquímica y biológica, con el fin de emplearlos para regeneración de tejido óseo. Las membranas fueron obtenidas por la técnica de evaporación del solvente y caracterizadas por espectroscopia FTIR, ensayos de hinchamiento, medidas de ángulo de contacto y microscopia electrónica de barrido (SEM). Se encontró que la compatibilidad entre los polímeros que la constituyen es estable a pH fisiológico y que, al incorporar mayor cantidad del TerP a la matriz, esta se vuelve más hidrofóbica y porosa. Además, teniendo en cuenta la aplicación prevista para dichos materiales, se realizaron estudios de biocompatibilidad y citotoxicidad con células progenitoras de médula ósea (CPMO) y células RAW264.7, respectivamente. Se evaluó la proliferación celular, la producción y liberación de óxido nítrico (NO) al medio de cultivo durante 24 y 48 horas y la expresión de citoquinas proinflamatorias IL-1ß y TNF-α de las células crecidas sobre los biomateriales variando la cantidad del polímero sintético. Se encontró mayor proliferación celular y menor producción de NO sobre las matrices que contienen menos proporción del TerP, además de poseer una mejor biocompatibilidad. Los resultados de este estudio muestran que el terpolímero obtenido y su combinación con un polímero natural es una estrategia muy interesante para obtener un biomaterial con posibles aplicaciones en medicina regenerativa y que podría extenderse a otros sistemas estructuralmente relacionados. (AU)

In the present work, the preparation of a biomaterial from a synthetic terpolymer (TerP) and a natural polymer, physically crosslinked, is shown. In order to evaluate the new material for bone tissue regeneration, physicochemical and biological characterizations were performed. The membranes were obtained by solvent casting and characterized using FTIR spectroscopy, swelling tests, contact angle measurements, and scanning electron microscopy (SEM). It was found that the compatibility between the polymers is stable at physiological pH and the incorporation of a higher amount of TerP into the matrix increases hydrophobicity and porosity.Furthermore, considering the intended application of these materials, studies of biocompatibility and cytotoxicity were conducted with Bone Marrow Progenitor Cells (BMPCs) and RAW264.7 cells, respectively. Cell proliferation, NO production and release into the culture medium for 24 and 48 hours, and proinflammatory cytokine expression of IL-1ß and TNF-α from cells grown on the biomaterials while varying the amount of the synthetic polymer were evaluated. Greater cell proliferation and lower NO production were found on matrices containing a lower proportion of TerP, in addition to better biocompatibility. The results of this study demonstrate that the obtained terpolymer and its combination with a natural polymer is a highly interesting strategy for biomaterial preparation with potential applications in regenerative medicine. This approach could be extended to other structurally related systems. (AU)

Longitudinal analyses of composite resin restoration on erosive lesions: effect of dentin treatment with a chitosan nanoformulation containing green tea

Aim To evaluate the influence of the biomodification of erosive lesions with a chitosan nanoformulation containing green tea (NanoCsQ) on the clinical performance of a composite resin. Methods The study was performed in a split-mouth, randomized and double-blinded model with 20 patients with 40 erosive lesions. The patient's teeth were randomized into two groups (n=20) according to the surface treatment: 1) Without biomodification (control), and 2) Biomodification with NanoCsQ solution (experimental). The lesions were restored with adhesive (Tetric N-bond, Ivoclar) and composite resin (IPS Empress Direct, Ivoclar). The restorations were polished and 7 days (baseline), 6 months, and 12 months later were evaluated according to the United States Public Health Service (USPHS) modified criteria, using clinical exam and photographics. Data were analyzed by Friedman's and Wilcoxon signed-rank tests. Results No significant differences were found between the control and experimental groups (p=0.423), and also among the follow-up periods (baseline, six months, and 12 months) (p=0.50). Regarding the retention criteria, 90% of the restoration had an alpha score in the control group. Only 10% of the restorations without biomodification (control) had a score charlie at the 12-month follow-up. None of the patients reported post-operatory sensitivity. Conclusion The NanoCsQ solution did not negatively affect the performance of the composite resin restorations after 12 months.

Effects of Chitosan Nanoparticles with Long Synthetic siRNAs Targeting VEGF in Triple-Negative Breast Cancer Cells

Abstract Vascular endothelial growth factor (VEGF) is an essential angiogenic factor in breast cancer development and metastasis. Small interfering RNAs (siRNAs) can specifically silence genes via the RNA interference pathway, therefore were investigated as cancer therapeutics. In this study, we investigated the effects of siRNAs longer than 30 base pairs (bp) loaded into chitosan nanoparticles in triple-negative breast cancer cells, compared with conventional siRNAs. 35 bp long synthetic siRNAs inhibited VEGF gene expression by 51.2% and increased apoptosis level by 1.75-fold in MDA-MB-231 cell lines. Furthermore, blank and siRNA-loaded chitosan nanoparticles induced expression of IFN-γ in breast cancer cells. These results suggest that long synthetic siRNAs can be as effective as conventional siRNAs, when introduced into cells with chitosan nanoparticles

Novo sistema de liberação de drogas ­ filme de fibroína de seda carregado ou não com insulina para reparo de feridas em mucosa palatina: estudo in vitro e clínico randomizado / A new drug delivery system ­ silk fibroin film loaded or not with insulin on palatal mucosa wound healing: in vitro study and a randomized clinical trial

Several types of periodontal and peri-implant soft tissue defects require surgical treatment to reestablish function and aesthetics. However, local, and systemic factors can jeopardize tissue repair leading to unexpected outcomes and postoperative discomfort. In order to overcome this problem, new devices have been developed to improve surgical procedures outcomes and patient experience. The aim of the present study was to develop a new silk fibroin (SF)/chitosan (CH) film loaded with insulin as a drug delivery system to improve palatal donor area healing after free gingival graft harvesting for ridge preservation. For this, biomaterial development, characterization and in vitro assessment were performed to evaluate the new delivery system. In addition, 3- months outcomes from palatal wound healing following the use of the proposed delivery system were assessed through clinical, patient centered parameters, immunological, microbiological, and histological evaluations. Sixty-nine patients with indication of tooth extraction were enrolled into 3 groups: Control Group (C) (n=23): open wound on palatal mucosa followed by spontaneous healing; SF/CH Film (F) (n=23): open wound on palatal mucosa and silk fibroin film as dressing; Insulin-loaded SF/CH film (IF) (n=23): open wound on palatal mucosa and an insulin- loaded silk fibroin film as a delivery system. : It was verified some characteristics that are favorable to the oral environment, such as mechanical properties, swelling and permeability to water vapor. The biomaterial presented a standard of a controlled release system through diffusion with delivery stability in human saliva, along with an excellent biocompatibility with the absence of cytotoxicity and genotoxicity increasing cell viability in lineage cells (HaCat). F and IF promoted accelerated palatal wound closure on day 7 and 14 after surgery, as well as an early epithelialization, compared to the C group. Both films were capable to reduce pro-inflammatory cytokines (IL-6, TNF-α, IL-1ß) and modulate biomarkers correlated to tissue degradation/remodeling. Spontaneous healing microbiome reported higher genus/species with pathogenic role in the oral mucosa with reduction in health species following this profile until de end of the follow-up. A tendency of eubiosis was observed in F and IF groups throughout healing process. It seems that this new device has a promising application in oral cavity and positively influence wound healing. (AU)

Diversos tipos de defeitos mucogengivais requerem abordagem cirúrgica para o reestabelecimento funcional e estético. Porém, alterações locais e sistêmicas podem prejudicar o processo de reparo gerando resultados inesperados e desconforto ao paciente. Biomateriais vem sendo desenvolvidos para melhorar os resultados dos procedimentos cirúrgicos e a experiência clínica do paciente. O objetivo do presente estudo foi desenvolver um filme de fibroína de seda (FS) e quitosana (QT) carregado com insulina (INS), atuando como um sistema de liberação, para acelerar a cicatrização de feridas na área doadora palatina após procedimento de preservação de rebordo com uso de enxerto gengival livre. Para isso, foi executado o desenvolvimento, caracterização e avaliação in vitro do biomaterial. Ademais, o resultado de 3 meses do reparo das feridas palatinas foi verificado por meio de avaliações clínicas, imunológica, microbiológica, histológica, bem como parâmetros centrados no paciente. Sessenta e nove pacientes foram alocados aleatoriamente nos grupos Controle (C) (n=23): ferida aberta em palato seguido de cicatrização espontânea; Filme de FS/QT (F) (n=23): ferida aberta em palato associada ao filme na área doadora; Filme de FS/QT carregado com INS (IF) (n=23): ferida aberta em palato associada ao filme carregado com INS na área doadora. Verificou-se propriedades mecânicas, bem como de entumecimento e permeabilidade ao vapor de água, favoráveis ao meio bucal sem nenhuma alteração com a inclusão da INS. O dispositivo apresentou liberação controlada por meio de difusão com estabilidade em saliva humana. Excelente biocompatibilidade com ausência de cito e genotoxicidade foi observada em diversos tipos celulares aumentando a viabilidade celular em células de linhagem (HaCat). F e IF favoreceram um fechamento acelerado da ferida palatina aos 7 e 14 dias pós-injuria, assim como uma epitelização precoce destes comparado ao grupo C. F e IF reduziram citocinas pró-inflamatórias (IL6, TNF-α, IL-1ß) além de apresentarem função modulatória na quantificação de biomarcadores relacionados a degradação tecidual. O Grupo C apresentou gênero/espécies com potencial patogênico e redução de microrganismos relacionados a saúde mantendo este perfil aos 14 e 30 dias. Enquanto isso, uma tendência a eubiose foi observado em F e IF ao longo do processo de cicatrização. Deste modo, verifica-se a aplicação promissora do novo dispositivo na cavidade oral bem como capacidade de influenciar positivamente o reparo da mucosa oral. (AU)

Applicatoin of chitosan-based hydrogel in oral tissue engineering / 中南大学学报(医学版)

Pulpitis, periodontitis, jaw bone defect, and temporomandibular joint damage are common oral and maxillofacial diseases in clinic, but traditional treatments are unable to restore the structure and function of the injured tissues. Due to their good biocompatibility, biodegradability, antioxidant effect, anti-inflammatory activity, and broad-spectrum antimicrobial property, chitosan-based hydrogels have shown broad applicable prospects in the field of oral tissue engineering. Quaternization, carboxymethylation, and sulfonation are common chemical modification strategies to improve the physicochemical properties and biological functions of chitosan-based hydrogels, while the construction of hydrogel composite systems via carrying porous microspheres or nanoparticles can achieve local sequential delivery of diverse drugs or bioactive factors, laying a solid foundation for the well-organized regeneration of defective tissues. Chemical cross-linking is commonly employed to fabricate irreversible permanent chitosan gels, and physical cross-linking enables the formation of reversible gel networks. Representing suitable scaffold biomaterials, several chitosan-based hydrogels transplanted with stem cells, growth factors or exosomes have been used in an attempt to regenerate oral soft and hard tissues. Currently, remarkable advances have been made in promoting the regeneration of pulp-dentin complex, cementum-periodontium-alveolar bone complex, jaw bone, and cartilage. However, the clinical translation of chitosan-based hydrogels still encounters multiple challenges. In future, more in vivo clinical exploration under the conditions of oral complex microenvironments should be performed, and the combined application of chitosan-based hydrogels and a variety of bioactive factors, biomaterials, and state-of-the-art biotechnologies can be pursued in order to realize multifaceted complete regeneration of oral tissue.

Advances in the structure and function of chitosanase / 生物工程学报

Chitosanases represent a class of glycoside hydrolases with high catalytic activity on chitosan but nearly no activity on chitin. Chitosanases can convert high molecular weight chitosan into functional chitooligosaccharides with low molecular weight. In recent years, remarkable progress has been made in the research on chitosanases. This review summarizes and discusses its biochemical properties, crystal structures, catalytic mechanisms, and protein engineering, highlighting the preparation of pure chitooligosaccharides by enzymatic hydrolysis. This review may advance the understandings on the mechanism of chitosanases and promote its industrial applications.

Preparation of chitosan hydrochloride stabilized emulsion and its immunostimulatory effect / 生物工程学报

In order to increase the ability of oil-emulsion adjuvant to stimulate cellular immunity, chitosan hydrochloride with positive charge was selected to stabilize oil-in-water emulsion (CHE). In this paper, model antigen ovalbumin was selected to prepare vaccines with emulsion adjuvant, commercial adjuvant or no adjuvant. The emulsion was characterized by measuring the particle size, electric potential and antigen adsorption rate. BALB/c mice were immunized by intramuscular injection. Serum antibody levels, the numbers of IL-4-secreting cells in splenocytes, cytotoxic T lymphocyte (CTL) response, and the expression of central memory T cells were measured to evaluate the immunostimulatory effect. The results showed that chitosan hydrochloride can effectively stabilize the emulsion. The emulsion size is about 600 nm, and the antigen adsorption rate is more than 90%. After immunization, CHE could increase serum antibodies levels and increase IL-4 secretion. Expression of CTL surface activation molecules was also increased to stimulate CTL response further and to increase the CD44+CD62L+ in T cells proportion. CHE as adjuvant can stimulate humoral and cellular immunity more efficiently, and is expected to extend the duration of protection.

Preparation, characterization and activity evaluation of Spirulina-chitooligosaccharides capable of inhibiting biofilms / 生物工程学报

The biofilms formed by pathogenic microorganisms seriously threaten human health and significantly enhance drug resistance, which urgently call for developing drugs specifically targeting on biofilms. Chitooligosaccharides extracted from shrimp and crab shells are natural alkaline oligosaccharides with excellent antibacterial effects. Nevertheless, their inhibition efficacy on biofilms still needs to be improved. Spirulina (SP) is a microalga with negatively charged surface, and its spiral structure facilitates colonization in the depth of the biofilm. Therefore, the complex of Spirulina and chitooligosaccharides may play a synergistic role in killing pathogens in the depth of biofilm. This research first screened chitooligosaccharides with significant bactericidal effects. Subsequently, Spirulina@Chitooligosaccharides (SP@COS complex was prepared by combining chitooligosaccharides with Spirulina through electrostatic adsorption. The binding of the complex was characterized by zeta potential, z-average size, and fluorescence labeling. Ultraviolet-visible spectroscopy (UV-Vis) showed the encapsulation efficiency and the drug loading efficiency reached up to 90% and 16%, respectively. The prepared SP@COS2 exhibited a profound synergistic inhibition effect on bacterial and fungal biofilms, which was mainly achieved by destroying the cell structure of the biofilm. These results demonstrate the potential of Spirulina-chitooligosaccharides complex as a biofilm inhibitor and provide a new idea for addressing the harm of pathogenic microorganisms.

Baicalin, silver titanate, Bletilla striata polysaccharide and carboxymethyl chitosan in a porous sponge dressing for burn wound healing / 中西医结合学报

OBJECTIVE@#This study tests the efficacy of Bletilla striata polysaccharide (BSP), carboxymethyl chitosan (CMC), baicalin (BA) and silver titanate (ST) in a wound dressings to fight infection, promote healing and provide superior biocompatibility.@*METHODS@#The antibacterial activity of BA and ST was evaluated in vitro using the inhibition zone method. BA/ST/BSP/CMC porous sponge dressings were prepared and characterized. The biocompatibility of BA/ST/BSP/CMC was assessed using the cell counting kit-8 assay. The therapeutic effect of BA/ST/BSP/CMC was further investigated using the dorsal skin burn model in Sprague-Dawley rats.@*RESULTS@#The wound dressing had good antibacterial activity against Escherichia coli and Staphylococcus aureus through BA and ST, while the combination of BSP and CMC played an important role in promoting wound healing. The BA/ST/BSP/CMC porous sponge dressings were prepared using a freeze-drying method with the concentrations of BA and ST at 20 and 0.83 mg/mL, respectively, and the optimal ratio of 5% BSP to 4% CMC was 1:3. The average porosity, water absorption and air permeability of BA/ST/BSP/CMC porous sponge dressings were measured to be 90.43%, 746.1% and 66.60%, respectively. After treatment for 3 and 7 days, the healing rates of the BA/ST/BSP/CMC group and BA/BSP/CMC group were significantly higher than those of the normal saline (NS) group and silver sulfadiazine (SSD) group (P < 0.05). Interleukin-1β expression in the BA/ST/BSP/CMC group at 1 and 3 days was significantly lower than that in the other three groups (P < 0.05). After being treated for 3 days, vascular endothelial growth factor expression in the BA/BSP/CMC group and BA/ST/BSP/CMC group was significantly higher than that in the NS group and SSD group (P < 0.05). Inspection of histological sections showed that the BA/ST/BSP/CMC group and BA/BSP/CMC group began to develop scabbing and peeling of damaged skin after 3 days of treatment, indicating accelerated healing relative to the NS group and SSD group.@*CONCLUSION@#The optimized concentration of BA/ST/BSP/CMC dressing was as follows: 6 mg BSP, 14.4 mg CMC, 0.5 mg ST and 12 mg BA. The BA/ST/BSP/CMC dressing, containing antibacterial constituents, was non-cytotoxic and effective in accelerating the healing of burn wounds, making it a promising candidate for wound healing. Please cite this article as: Gong YR, Zhang C, Xiang X, Wang ZB, Wang YQ, Su YH, Zhang HQ. Baicalin, silver titanate, Bletilla striata polysaccharide and carboxymethyl chitosan in a porous sponge dressing for burn wound healing. J Integr Med. 2023; 21(5): 487-495.

Near-infrared excited graphene oxide/silver nitrate/chitosan coating for improving antibacterial properties of titanium implants / 中国修复重建外科杂志

OBJECTIVE@#To design and construct a graphene oxide (GO)/silver nitrate (Ag3PO4)/chitosan (CS) composite coating for rapidly killing bacteria and preventing postoperative infection in implant surgery.@*METHODS@#GO/Ag3PO4 composites were prepared by ion exchange method, and CS and GO/Ag3PO4 composites were deposited on medical titanium (Ti) sheets successively. The morphology, physical image, photothermal and photocatalytic ability, antibacterial ability, and adhesion to the matrix of the materials were characterized.@*RESULTS@#The GO/Ag3PO4 composites were successfully prepared by ion exchange method and the heterogeneous structure of GO/Ag3PO4 was proved by morphology phase test. The heterogeneous structure formed by Ag3PO4 and GO reduced the band gap from 1.79 eV to 1.39 eV which could be excited by 808 nm near-infrared light. The photothermal and photocatalytic experiments proved that the GO/Ag3PO4/CS coating had excellent photothermal and photodynamic properties. In vitro antibacterial experiments showed that the antibacterial rate of the GO/Ag3PO4/CS composite coating against Staphylococcus aureus reached 99.81% after 20 minutes irradiation with 808 nm near-infrared light. At the same time, the composite coating had excellent light stability, which could provide stable and sustained antibacterial effect.@*CONCLUSION@#GO/Ag3PO4/CS coating can be excited by 808 nm near infrared light to produce reactive oxygen species, which has excellent antibacterial activity under light.

Nanosilver alleviates foreign body reaction and facilitates wound repair by regulating macrophage polarization / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Foreign body reactions induced by macrophages often cause delay or failure of wound healing in the application of tissue engineering scaffolds. This study explores the application of nanosilver (NAg) to reduce foreign body reactions during scaffold transplantation. An NAg hybrid collagen-chitosan scaffold (NAg-CCS) was prepared using the freeze-drying method. The NAg-CCS was implanted on the back of rats to evaluate the effects on foreign body reactions. Skin tissue samples were collected for histological and immunological evaluation at variable intervals. Miniature pigs were used to assess the effects of NAg on skin wound healing. The wounds were photographed, and tissue samples were collected for molecular biological analysis at different time points post-transplantation. NAg-CCS has a porous structure and the results showed that it could release NAg constantly for two weeks. The NAg-CCS group rarely developed a foreign body reaction, while the blank-CCS group showed granulomas or necrosis in the subcutaneous grafting experiment. Both matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) were reduced significantly in the NAg-CCS group. The NAg-CCS group had higher interleukin (IL)-10 and lower IL-6 than the blank CCS group. In the wound healing study, M1 macrophage activation and inflammatory-related proteins (inducible nitric oxide synthase (iNOS), IL-6, and interferon-‍γ (IFN-‍γ)) were inhibited by NAg. In contrast, M2 macrophage activation and proinflammatory proteins (arginase-1, major histocompatibility complex-II (MHC-II), and found in inflammatory zone-1 (FIZZ-1)) were promoted, and this was responsible for suppressing the foreign body responses and accelerating wound healing. In conclusion, dermal scaffolds containing NAg suppressed the foreign body reaction by regulating macrophages and the expression of inflammatory cytokines, thereby promoting wound healing.

Research Advances in Medical Materials and Products for Soft Tissue Repairs / 中国医疗器械杂志

Soft tissue is an indispensable tissue in human body. It plays an important role in protecting the body from external physical, chemical or biological factors. Mild soft tissue injuries can self-heal, while severe soft tissue injuries may require related treatment. Natural polymers (such as chitosan, hyaluronic acid, and collagen) and synthetic polymers (such as polyethylene glycol and polylactic acid) exhibit good biocompatibility, biodegradability and low toxicity. It can be used for soft tissue repairs for antibacterial, hemostatic and wound healing purposes. Their related properties can be enhanced through modification or preparation of composite materials. Commonly used soft tissue repairs include wound dressings, biological patches, medical tissue adhesives, and tissue engineering scaffolds. This study introduces the properties, mechanisms of action and applications of various soft tissue repair medical materials, including chitosan, hyaluronic acid, collagen, polyethylene glycol and polylactic acid, and provides an outlook on the application prospects of soft tissue repair medical materials and products.

Research Progress of Carboxymethyl Chitosan-Based Haemostatic Materials and Their Haemostatic Mechanism --Review / 中国实验血液学杂志

Effective haemostatic materials can quickly control bleeding and achieve the purpose of saving patients' lives. In recent years, chitosan-based haemostatic materials have shown good haemostatic effects, but their application is limited because chitosan is almost insoluble in water. Carboxymethyl chitosan-based haemostatic materials can promote hemostasis by activating red blood cells and aggregating platelets. In addition, carboxymethyl chitosan can bind with Ca2+ to activate platelets and coagulation factors, and start endogenous coagulation pathways, which can adsorb fibrinogen in plasma to promote haemostasis. In this paper, the latest research progress of carboxymethyl chitosan-based haemostatic materials and their haemostatic mechanism were reviewed, in order to further strengthen the understanding of the haemostatic mechanism of carboxymethyl chitosan-based haemostatic materials, and provide new idea for the research and clinical application of carboxymethyl chitosan-based haemostatic materials.

Efeito biológico do laser de baixa intensidade em osteoblastos cultivados na superfície de arcabouços poliméricos tridimensionais / Biological effect of low-intensity laser on osteoblasts cultured on the surface of three-dimensional polymeric scaffolds

A primeira parte do trabalho avaliou, através de uma revisão sistemática de estudos in vitro, a aplicabilidade da fotobiomodulação como uma ferramenta auxiliar na engenharia de tecidos. De 8373 estudos inicialmente identificados a partir das estratégias de busca, dez artigos atingiram os critérios de inclusão para análise. Os dados obtidos na maioria dos estudos revisados indicaram que a laserterapia de baixa intensidade (LBI) pode aumentar a proliferação e diferenciação de células cultivadas na superfície dos biomateriais. Na segunda parte do trabalho foi avaliado o efeito da LBI na dose de 4 J/cm2 na proliferação de osteoblastos (OFCOL II) cultivados na superfície de arcabouços poliméricos tridimensionais (3D) de ácido polilático (PLA) e de PLA associado a quitosana (PLA/Q) produzidos pela técnica de fiação por sopro em solução. O ensaio do Alamar Blue demonstrou que as células OFCOL II cultivadas sobre os arcabouços 3D de PLA e irradiadas apresentaram uma maior atividade proliferativa quando comparadas aos grupos não irradiados no intervalo de 72 h. Além disso, as células OFCOL II cultivadas sobre arcabouços de PLA/Q também apresentaram uma maior atividade proliferativa em 24 h. A análise pela microscopia eletrônica de varredura (MEV) mostrou que os osteoblastos se encontravam ancorados em concavidades das fibras nos arcabouços examinados. Concluiu-se que o modelo proposto apresentou um potencial para estudos na área da engenharia tecidual óssea. Na terceira parte do trabalho foi avaliada a influência da LBI infravermelha (IV) e vermelha (V) em diferentes dosagens (1 J/cm², 4 J/cm² e 6 J/cm²) na proliferação e viabilidade das células OFCOL II. O ensaio do Alamar Blue mostrou diferenças significativas (p<0,05) na atividade mitocondrial do grupo IV utilizando a dose de 1 J/cm2 e 4 J/cm2, nos intervalos de 24 e 48 h. Já o ensaio do Live/Dead evidenciou que a LBI induziu aumento da viabilidade celular no grupo IV na dose de 4 J/cm2, quando comparada com os demais grupos. Em conjunto, os resultados sugerem que a LBI pode promover bioestimulação in vitro de osteoblastos, inclusive quando cultivados na superfície de arcabouços poliméricos 3D, representando assim uma ferramenta promissora nas técnicas de engenharia tecidual óssea (AU).

The first part of the work evaluated, through a systematic review of in vitro studies, the applicability of photobiomodulation as an auxiliary tool in tissue engineering. Of 8373 studies initially identified from the search strategies, ten articles met the inclusion criteria for analysis. Data obtained from most of the reviewed studies indicated that low-intensity laser therapy (LLLT) could increase the proliferation and differentiation of cells cultured on the surface of biomaterials. The second part of the work evaluated the effect of LLLT at a dose of 4 J/cm² on the proliferation of osteoblasts (OFCOL II) cultivated on the surface of threedimensional (3D) polymer scaffolds of polylactic acid (PLA) and PLA associated with chitosan (PLA/Q) produced by the solution blow spinning technique. The Alamar Blue assay demonstrated that OFCOL II cells cultured on 3D PLA scaffolds and irradiated showed more significant proliferative activity when compared to non-irradiated groups within 72 h. Furthermore, OFCOL II cells cultured on PLA/Q scaffolds showed higher proliferative activity at 24 h. Analysis by scanning electron microscopy (SEM) showed that the osteoblasts were anchored in the concavities of the fibers of the examined scaffolds. It was concluded that the proposed model showed potential for studies in the field of bone tissue engineering. The third part of the work evaluated the influence of infrared (IR) and red (R) laser therapy at different dosages (1 J/cm², 4 J/cm², and 6 J/cm²) on the proliferation and viability of OFCOL II cells. The Alamar Blue assay showed significant differences (p<0.05) in the mitochondrial activity of group IR using the dose of 1 J/cm² and 4 J/cm² at 24 and 48 h. The Live/Dead assay showed that LLLT induced an increase in cell viability in the IR group at a dose of 4 J/cm² compared to the other groups. Taken together, the results suggest that LLLT can promote in vitro biostimulation of osteoblasts, even when cultivated on the surface of 3D polymeric scaffolds, thus representing a promising tool in bone tissue engineering techniques (AU).

Biocomponents Based on Hydroxiapatite: Influence of Sterilization on the Mechanical Resistance / Biocomponentes à base de hidroxiapatita: Influência da esterilização na resistência mecânica

Abstract Objective This study aimed to evaluate the influence of sterilization on the compressive and flexural mechanical strength of hydroxyapatite-based biocomponents obtained through freeze-dried bovine bone, and its association with chitosan. Methods Freeze-dried bovine bone was processed into 100 μm particles and mixed with 50% of its weight in chitosan. The mixture was packed in metallic molds for preparing the specimens, and sterilized at 127°C using an autoclave for subsequent experimentation. The specimens were subjected to compression and flexion tests following norm 5833 of the International Organization for Standardization (ISO), with 6 × 12 mm cylindrical blocks (for compression tests) and 75 × 10 × 3.3 mm plates (for flexion tests) as samples. The samples were divided into four groups of 20 specimens each, with 10 for compression and 10 for flexion tests. Three groups were sterilized (autoclave, gamma rays, and ethylene oxide), whereas the fourth group (control) was not. The mechanical tests obtained from the different sterilization processes were compared using analysis of variance (ANOVA, p< 0.05), followed by the Tukey multiple comparison test of means, with a 95% confidence interval. Results The specimens presented mean compressive strengths of 10.25 MPa for the control group and 3.67 MPa, 9.65 MPa, and 9.16 MPa after ethylene oxide, gamma ray, and autoclave sterilization, respectively. Flexion test results showed an average resistance of 0.40 MPa in the control group, and 0.15 MPa, 0.17 MPa, and 0.30 MPa after ethylene oxide, gamma ray, and autoclave sterilization, respectively. There were statistically significant differences observed in the maximum compression of the ethylene oxide-sterilized group compared with that of the control group (p= 0.0002), gamma ray-sterilized (p= 0.0003), and the autoclaved (p= 0.0006) groups. There was a statistically significant difference in maximum flexion of the specimens sterilized by gamma rays when compared with the control group (p= 0.0245). However, low flexural strengths were observed in all specimens. Conclusion The autoclave sterilization group did not result in statistically significant differences in either compression or flexion strength tests. Thus, the autoclave proved to be the best sterilization option for the hydroxyapatite-based biocomponents in this study.

Resumo Objetivo O objetivo deste estudo foi avaliar a influência da esterilização na resistência mecânica à compressão e flexão de biocomponentes à base de hidroxiapatita obtida a partir de osso bovino liofilizado e sua associação com quitosana. Métodos O osso bovino liofilizado foi processado em partículas de 100 μm e misturado à quitosana em proporção de 50% de seu peso. A mistura foi acondicionada em moldes metálicos para preparo dos espécimes e esterilizada a 127°C em autoclave para posterior experimentação. Os espécimes foram submetidos a ensaios de compressão e flexão seguindo a norma 5833 da International Organization for Standardization (ISO); os espécimes eram blocos cilíndricos de 6 × 12 mm (para ensaios de compressão) e placas de 75 × 10 × 3,3 mm (para ensaios de flexão). As amostras foram divididas em quatro grupos de 20 espécimes cada, sendo 10 para ensaios de compressão e 10 para ensaios de flexão. Três grupos foram esterilizados (por autoclavagem, raios gama e óxido de etileno), enquanto o quarto grupo (controle) não foi. Os testes mecânicos obtidos nos diferentes processos de esterilização foram comparados por análise de variância (ANOVA, p< 0,05) seguido pelo teste de comparação múltipla de médias de Tukey, com intervalo de confiança de 95%. Resultados Os espécimes apresentaram resistências médias à compressão de 10,25 MPa para o grupo de controle e 3,67 MPa, 9,65 MPa e 9,16 MPa após esterilização com óxido de etileno, raios gama e autoclavagem, respectivamente. Os resultados do teste de flexão mostraram uma resistência média de 0,40 MPa no grupo de controle, e 0,15 MPa, 0,17 MPa e 0,30 MPa após esterilização com óxido de etileno, raios gama e autoclavagem, respectivamente. A compressão máxima observada no grupo esterilizado com óxido de etileno foi estatisticamente diferente à obtida no grupo de controle (p= 0,0002), esterilizado com raios gama (p= 0,0003) e autoclavado (p= 0,0006). A flexão máxima dos espécimes esterilizados com raios gama foi estatisticamente diferente à observada no grupo de controle (p= 0,0245). No entanto, a resistência à flexão foi baixa em todos os espécimes. Conclusão A esterilização em autoclave não foi associada a diferenças estatisticamente significativas nos testes de compressão ou flexão. Assim, a autoclave foi a melhor opção de esterilização para os biocomponentes à base de hidroxiapatita neste estudo.

Uso de quitina e quitosana como adsorventes de amônia de efluentes aquícolas: revisão de literatura / Use of chitin and quitosan as adsorvents of ammonia of aquaculture effluents: a literature review / Uso de quitina y quitosana como adsorbentes de amonia de efluentes acuícolas: revisión de literatura

Os resíduos provenientes da aquicultura são derivados da ração e da excreção dos peixes e podem estar sedimentados, suspensos ou dissolvidos, ocasionando elevados valores de DBO, DQO, nitrogênio e fósforo. A produção de camarões no Brasil tem gerado elevadas quantidades de resíduos sólidos, tendo em vista que os exoesqueletos dos camarões correspondem a cerca de 40% do seu peso total, resultando num forte impacto ambiental. Diversas pesquisas envolvendo a quitina estão sendo desenvolvidas na área de tratamento de água, devido principalmente a sua capacidade de formar filme, sendo utilizada em sistemas filtrantes. Este polissacarídeo também pode ser utilizado como agente floculante no tratamento de efluentes, como adsorvente na clarificação de óleos, e principalmente na produção de quitosana. Atualmente a quitosana possui aplicações multidimensionais, desde áreas como a nutrição humana, biotecnologia, ciência dos materiais, indústria farmacêutica, agricultura, terapia genética e proteção ambiental. A quitosana é muito eficiente na remoção de poluentes em diferentes concentrações. Apresenta alta capacidade e grande velocidade de adsorção, boa eficiência e seletividade tanto em soluções que possuem altas ou baixas concentrações. O uso da biotecnologia, através do processo de adsorção utilizando adsorventes naturais e baratos, como a quitina e quitosana, minimiza os impactos ambientais da aquicultura tanto em relação aos provocados pelo lançamento de efluentes no meio ambiente quanto aos causados pelo descarte inadequado dos resíduos do processamento de camarões.(AU)

Aquaculture residues are derived from fish feed and excretion and may be sedimented, suspended or dissolved, resulting in high BOD, COD, nitrogen and phosphorus values. Shrimp production in Brazil has generated high amounts of solid waste, since shrimp exoskeletons account for about 40% of their total weight, resulting in a strong environmental impact. Several researches involving chitin are being developed in the area of water treatment, mainly due to its ability to form film, being used in filter systems. This polysaccharide can also be used as a flocculating agent in the treatment of effluents, as an adsorbent in the clarification of oils, and especially in the production of chitosan. Currently, chitosan has multidimensional applications, from areas such as human nutrition, biotechnology, materials science, pharmaceutical industry, agriculture, gene therapy and environmental protection. Chitosan is very efficient in the removal of pollutants at different concentrations. It presents high capacity and high adsorption velocity, good efficiency and selectivity both in solutions that have high or low concentrations. The use of biotechnology, through the adsorption process using natural and cheap adsorbents such as chitin and chitosan, minimizes the environmental impacts of aquaculture both in relation to those caused by the release of effluents into the environment and those caused by the inappropriate disposal of processing residues of shrimps.(AU)

Los residuos procedentes de la acuicultura se derivan de la ración y de la excreción de los peces y pueden estar sedimentados, suspendidos o disueltos, ocasionando elevados valores de DBO, DQO, nitrógeno y fósforo. La producción de camarones en Brasil ha generado grandes cantidades de residuos sólidos, teniendo en cuenta que los exoesqueletos de los camarones corresponden a cerca del 40% de su peso total, resultando en un fuerte impacto ambiental. Varias investigaciones involucrando la quitina se están desarrollando en el área de tratamiento de agua, debido principalmente a su capacidad de formar película, siendo utilizada en sistemas filtrantes. Este polisacárido también puede ser utilizado como agente floculante en el tratamiento de efluentes, como adsorbente en la clarificación de aceites, y principalmente en la producción de quitosana. Actualmente la quitosana posee aplicaciones multidimensionales, desde áreas como la nutrición humana, biotecnología, ciencia de los materiales, industria farmacéutica, agricultura, terapia genética y protección ambiental. La quitosana es muy eficiente en la eliminación de contaminantes en diferentes concentraciones. Presenta alta capacidad y gran velocidad de adsorción, buena eficiencia y selectividad tanto en soluciones que poseen altas o bajas concentraciones. El uso de la biotecnología, a través del proceso de adsorción utilizando adsorbentes naturales y baratos, como la quitina y quitosana, minimiza los impactos ambientales de la acuicultura tanto en relación a los provocados por el lanzamiento de efluentes en el medio ambiente en cuanto a los causados por el descarte inadecuado de los residuos del procesamiento de camarones.(AU)