Chitosan 3D scaffolds with resolvin D1 for vertebral arthrodesis: a pilot study.

PURPOSE: Over the last years, the number of vertebral arthrodesis has been steadily increasing. The use of iliac crest bone autograft remains the "gold standard" for bone graft substitute in these procedures. However, this solution has some side effects, such as the problem of donor site morbidity indicating that there is a real need for adequate alternatives. This pilot study aimed to evaluate the usefulness of chitosan (Ch) porous 3D scaffolds incorporated with resolvin D1 (RvD1) as an alternative implant to iliac bone autograft. METHODS: We have performed bilateral posterolateral lumbar vertebral arthrodesis in a rat animal model. Three experimental groups were used: (i) non-operated animals; (ii) animals implanted with Ch scaffolds incorporated with RvD1 and (iii) animals implanted with iliac bone autograft. RESULTS: The collagenous fibrous capsule formed around the Ch scaffolds with RvD1 is less dense when compared with the iliac bone autograft, suggesting an important anti-inflammatory effect of RvD1. Additionally, new bone formation was observed in the Ch scaffolds with RvD1. CONCLUSION: These results demonstrate the potential of these scaffolds for bone tissue repair applications.

The inflammasome in biomaterial-driven immunomodulation.

Inflammasomes are intracellular structures formed upon the assembly of several proteins that have a considerable size and are very important in innate immune responses being key players in host defense. They are assembled after the perception of pathogens or danger signals. The activation of the inflammasome pathway induces the production of high levels of the pro-inflammatory cytokines Interleukin (IL)-1ß and IL-18 through the caspase activation. The procedure for the implantation of a biomaterial causes tissue injury, and the injured cells will secrete danger signals recognized by the inflammasome. There is growing evidence that the inflammasome participates in a number of inflammatory processes, including pathogen clearance, chronic inflammation and tissue repair. Therefore, the control of the inflammasome activity is a promising target in the development of capable approaches to be applied in regenerative medicine. In this review, we revisit current knowledge of the inflammasome in the inflammatory response to biomaterials and point to the yet underexplored potential of the inflammasome in the context of immunomodulation.

Immunomodulatory biomaterial-based wound dressings advance the healing of chronic wounds via regulating macrophage behavior.

Successful wound healing is a process that has three overlying phases: inflammatory, proliferative and remodeling. Chronic wounds are characterized by a perpetuated inflammation that inhibits the proliferative and remodeling phases and impairs the wound healing. Macrophages are key modulators of the wound healing process. Initially, they are responsible for the wound cleaning and for the phagocytosis of pathogens and afterwards they lead to the resolution of the inflammatory response and they express growth factors important for angiogenesis and cytokines and growth factors needed for cell proliferation and deposition of extracellular matrix. The phenotype of the macrophage changes gradually throughout the healing process from the initial M1 pro-inflammatory phenotype characteristic of the acute response to the M2 pro-regenerative phenotype that allows an accurate tissue repair. In chronic wounds, M1 pro-inflammatory macrophages persist and impair tissue repair. As such, immunomodulatory biomaterials arise as promising solutions to accelerate the wound healing process. In this review, we discuss the importance of macrophages and their polarization throughout the different phases of wound healing; macrophage dysfunction in chronic wounds and the use of immunomodulatory biomaterials to overcome the critical problem of chronic wounds-the continued inflammatory phase that impairs healing.

3D chitosan scaffolds impair NLRP3 inflammasome response in macrophages.

Chitosan (Ch) is used in different biomedical applications to promote tissue repair. However, tissue injury caused by biomaterial implantation lead to the release of danger signals that engage different inflammatory pathways on the host. Different implanted materials activate the inflammasome leading to the modulation of the immune response. Here we have studied how macroscopic biomaterials, Ch scaffolds with different chemical composition: 4% or 15% degree of acetylation (DA) modulate the activation of the NLRP3 inflammasome in vitro. For that, we assessed the NLRP3 inflammasome in bone marrow derived mouse macrophages (BMDM) and human macrophages cultured within 3D Ch scaffolds. We found that both Ch scaffolds did not trigger the NLRP3 inflammasome activation in macrophages. Furthermore, BMDMs and human macrophages cultured in both Ch scaffolds presented a reduction in the number of apoptosis-associated speck-like protein containing a caspase activating recruitment domain (ASC) specks and in IL-1ß release upon classical NLRP3 inflammasome stimulation. We also found a decrease in proIL-1ß in BMDMs after priming with LPS when cultured in Ch scaffolds with DA 4% DA after priming with LPS when compared to Ch scaffolds with 15% DA or to macrophages cultured in cell-culture plates. Our results shows that 3D Ch scaffolds with different DA impair NLRP3 inflammasome priming and activation. STATEMENT OF SIGNIFICANCE: In this research work we have assessed the role of the NLRP3 inflammasome in the macrophage response to 3D chitosan scaffolds with different degrees of acetylation (DA). To our knowledge this is the first work that demonstrates the modulatory capacity of 3D porous chitosan scaffolds in the NLRP3 inflammasome activation, because our results show that Ch scaffolds impair NLRP3 inflammasome assembly in macrophages. Interestingly, our results are in contrast with studies reported in the literature that indicate that chitosan is a powerful activator of the NLRP3 inflammasome in nanoscale chitosan products. Our studies that were performed in large scale chitosan scaffolds, stress out that the process of phagocytosis is pivotal in inflammasome assembly and activation, are rather important since they clearly illustrate the different role of the inflammasome in the biological response to large scale and nanoscale biomaterials.

The inflammasome in host response to biomaterials: Bridging inflammation and tissue regeneration.

The development of new biomaterials to be used in tissue engineering applications is creating new solutions for a range of healthcare problems. The trend in biomaterials research has shifted from biocompatible "immune-evasive" biomaterials to "immune-interactive" materials that modulate the inflammatory response supporting implant integration as well as improving healing and tissue regeneration. Inflammasomes are large intracellular multiprotein complexes that are key players in host defence during innate immune responses and assemble after recognition of pathogens or danger signals. The process of biomaterial implantation causes injury to tissues that will consequently release danger signals that could be sensed by the inflammasome. There are increasing evidences that the inflammasome has a role in several inflammatory processes, from pathogen clearance to chronic inflammation or tissue repair. Thus, modulation of the inflammasome activity appears as an important target in the development of effective approaches in regenerative medicine. In this review, we discuss the main points of the current understanding on the host response to implanted biomaterials and how the paradigm of "immune-evasive" biomaterials has shifted over the last years; the significance of the inflammasome in the inflammatory response to biomaterials; and the growing idea that the immune system is of key importance in an effective tissue repair and regeneration. STATEMENT OF SIGNIFICANCE: We herein discuss the main points of the current understanding on the host response to implanted biomaterials and how the paradigm of "immune-evasive" biomaterials has shifted to "immune-interactive" over the last years; the significance of the inflammasome in the inflammatory response to biomaterials; and the growing idea that the immune system is of key importance in an effective tissue repair and regeneration, supporting the emerging concept of Regenerative Immunology. The inflammasome is a recent and central concept in immunology research. Since the beginning of this century the inflammasome is viewed as key platform of the innate immune response. We believe that, successful modulation of the inflammasome activity will become a milestone in the fields of tissue engineering and regenerative medicine.

Chitosan porous 3D scaffolds embedded with resolvin D1 to improve in vivo bone healing.

The aim of this study was to investigate the effect chitosan (Ch) porous 3D scaffolds embedded with resolvin D1 (RvD1), an endogenous pro-resolving lipid mediator, on bone tissue healing. These scaffolds previous developed by us have demonstrated to have immunomodulatory properties namely in the modulation of the macrophage inflammatory phenotypic profile in an in vivo model of inflammation. Herein, results obtained in an in vivo rat femoral defect model demonstrated that two months after Ch + RvD1 scaffolds implantation, an increase in new bone formation, in bone trabecular thickness, and in collagen type I and Coll I/Coll III ratio were observed. These results suggest that Ch scaffolds embedded with RvD1 were able to lead to the formation of new bone with improvement of trabecular thickness. This study shows that the presence of RvD1 in the acute phase of the inflammatory response to the implanted biomaterial had a positive role in the subsequent bone tissue repair, thus demonstrating the importance of innovative approaches for the control of immune responses to biomedical implants in the design of advanced strategies for regenerative medicine. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1626-1633, 2018.

High concentration of phosphorus is a distinctive feature of myelin. An X-ray elemental microanalysis study using freeze-fracture scanning electron microscopy of rat sciatic nerve.

We have used rat sciatic nerves submitted to freezing and freeze-fracture to determine the elemental composition of small domains of the peripheral nerve studied at high resolution by scanning electron microscopy. We found that myelin of Schwann cells is unique in its high content in phosphorus (P) that was more than 10 times higher than P measured in any other cells. This high concentration in P makes myelin chemistry suitable of monitoring at the subcellular level using the herein described methodology.

Development of an immunomodulatory biomaterial: using resolvin D1 to modulate inflammation.

In our search for immunomodulatory biomaterials capable of modulating the inflammatory response through M2 macrophage polarization, we report here on a new strategy that resulted from the incorporation of resolvin D1 (RvD1), a pro-resolution lipid mediator in porous 3D chitosan (Ch) scaffolds, followed by its lyophilisation. We have investigated the inflammatory response caused by this biomaterial in vivo using a mouse air-pouch model of inflammation. We found that this developed material caused a decrease in inflammatory cells recruited to the implant site, together with higher numbers of F4/80(+)/CD206(+) cells (M2 macrophages) and lower numbers of F4/80(+)/CCR7(+) cells (M1 macrophages). It also induced a general decrease in pro-inflammatory cytokines, and caused a decrease in the inflammatory cells observed around and within the implanted scaffolds, when compared with Ch alone or Ch not submitted to lyophilisation after RvD1 incorporation. Our results demonstrate that we were able to develop an immunomodulating biomaterial that triggers a shift in the macrophage response towards a M2 reparative response that will be advantageous for the host.

Modulation of the inflammatory response to chitosan through M2 macrophage polarization using pro-resolution mediators.

Tissue engineering and regenerative medicine have created a demand for biomaterials with specific functions such as the ability to modify the host immune response. The objective of this study was to evaluate the effect of two different pro-resolution lipid mediators, lipoxin A4 (LxA4) and resolvin D1 (RvD1), in the modulation of the inflammatory response to biomaterials through M2 macrophage polarization. This was investigated in vivo using a mouse air-pouch model of inflammation. Our results demonstrated that both LxA4 and RvD1 are able to shift the macrophage response to implanted Ch scaffolds to an M2 reparative response. The injection of these pro-resolution mediators caused a decrease in inflammatory cells recruited to the implant site together with higher numbers of F4/80(+)/CD206(+) cells (M2 macrophages) and lower numbers of F4/80(+)/CCR7(+) cells (M1 macrophages); it also induced a general decrease in several pro-inflammatory cytokines; and caused a significant decrease in the thickness and area of the fibrous capsule formed around the implanted scaffolds. In conclusion, the use of either LxA4 or RvD1 allowed the in vivo control of macrophage phenotypic profile and thus may play a significant role in regenerative medicine applications, namely through modulation of the inflammatory response.

Immunosuppression abrogates resistance of young rabbits to Rabbit Haemorrhagic Disease (RHD).

Rabbit Haemorrhagic Disease (RHD) is caused by a calicivirus (RHDV) that kills 90% of infected adult European rabbits within 3 days. Remarkably, young rabbits are resistant to RHD. We induced immunosuppression in young rabbits by treatment with methylprednisolone acetate (MPA) and challenged the animals with RHDV by intramuscular injection. All of these young rabbits died within 3 days of infection due to fulminant hepatitis, presenting a large number of RHDV-positive dead or apoptotic hepatocytes, and a significant seric increase in cytokines, features that are similar to those of naïve adult rabbits infected by RHDV. We conclude that MPA-induced immunosuppression abrogates the resistance of young rabbits to RHD, indicating that there are differences in the innate immune system between young and adult rabbits that contribute to their distinct resistance/susceptibility to RHDV infection.

Macrophage polarization following chitosan implantation.

Macrophages are a key cell in the host response to implants and can be polarized into different phenotypes capable of inducing both detrimental and beneficial outcomes in tissue repair and remodeling, being important in tissue engineering and regenerative medicine. The objective of this study was to evaluate the macrophage response to 3D porous chitosan (Ch) scaffolds with different degrees of acetylation (DA, 5% and 15%). The M1/M2 phenotypic polarization profile of macrophages was investigated in vivo using a rodent air-pouch model. Our results show that the DA affects the macrophage response. Ch scaffolds with DA 5% induced the adhesion of lower numbers of inflammatory cells, being the M2 the predominant phenotypic profile among the adherent macrophages. In the inflammatory exudates F4/80(+)/CD206(+) cells (M2 macrophages) appeared in higher numbers then F4/80(+)/CCR7(+) cells (M1 macrophages), in addition, lower levels of pro-inflammatory cytokines together with higher levels of anti-inflammatory cytokines were found. Ch scaffolds with DA 15% showed opposite results, since M1 were the predominant macrophages both adherent to the scaffold and in the exudates, together with high levels of pro-inflammatory cytokines. In conclusion, Ch scaffolds with DA 5% induced a benign M2 anti-inflammatory macrophage response, whereas Ch scaffolds with DA 15% caused a macrophage M1 pro-inflammatory response.

Cardiac venous arterialization in acute myocardial infarction: how great is the benefit?

OBJECTIVES: Cardiac venous arterialization has been proposed as an alternative approach for myocardial revascularization in ischaemic heart disease. It is based on using the cardiac venous system to transport arterial blood from a systemic artery to infarcted myocardial areas. Our goal was to evaluate its benefit in reducing acute myocardial infarct size and its effects on cardiac performance. METHODS: In a group of pigs, the left internal mammary artery was anastomosed to the left anterior descending vein; this vein was ligated proximally. The left anterior descending coronary artery was also occluded. Over 5 days, several diagnostic procedures were used to characterize and measure the extent of myocardial infarct, namely ECG, echocardiography, cardiac biomarkers and histopathology. Data were compared with those from a control group of pigs, which were submitted to ligation of only the left anterior descending coronary artery. RESULTS: In the experimental group, echocardiography revealed that the ejection fraction and thickness of the ventricular walls remained unchanged 4 days after surgery, in contrast to the major alterations in the control group. In fact, the ejection fraction in the control group decreased by 21% (P < 0.001), with a reduction of 31% (P < 0.004) in the thickness of the interventricular septum at end systole and enlargement of the left ventricular lumen by 28% (P < 0.001). In the experimental group, the sum for ST segment shift was 50% lower (P = 0.038) and the total ventricular histological lesion size was 50% smaller (P < 0.001). Within this lesion, the area of necrotic tissue was 70% smaller (P < 0.001). Cardiac biomarkers were not different between the two groups (P > 0.2). CONCLUSIONS: This study reveals that selective cardiac venous arterialization can nourish the myocardium and is able to reduce infarct size by more than 50%, while protecting cardiac performance. We believe, therefore, that further investigation should be carried out into this technique in order for it to be considered as an option in coronary surgery.

Exposure to industrial wideband noise increases connective tissue in the rat liver.

Rats were daily exposed (eight hours/day) for a period of four weeks to the same high-intensity wideband noise that was recorded before in a large textile plant. Histologic observation of liver sections of the rats was used to perform quantitative comparison of hepatic connective tissue (dyed by Masson trichromic staining) between the noise-exposed and control animals. For that, we have photographed at random centrolobular areas of stained rat liver sections. We found that noise exposure resulted in significant enhancement in the area of collagen-rich connective tissue present in the centrolobular domain of the rat liver. Our data strengthen previous evidence showing that fibrotic transformation is a systemic effect of chronic exposure of rodents and humans to industrial wideband noise.

Regulatory T cells are decreased in acute RHDV lethal infection of adult rabbits.

Rabbit hemorrhagic disease virus (RHDV) is the etiologic agent of rabbit hemorrhagic disease (RHD), an acute lethal infection that kills 90% of adult rabbits due to severe acute liver inflammation. Interestingly, young rabbits are naturally resistant to RHDV infection. Here, we have compared naturally occurring CD4(+)Foxp3(+) regulatory T cells (Tregs) between young and adult rabbits after infection by RHDV. The number and frequency of Tregs was decreased in the spleen of adult rabbits 24h after the RHDV infection; this was in contrast with the unchanged number and frequency of splenic Tregs found in young rabbits after the same infection. Also, serum levels of IL-10 and TGF-ß were enhanced in the infected adult rabbits whereas no alteration was observed in infected young rabbits. However, this increase is accompanied by a burst of pro-inflammatory cytokines, but seems not able to prevent the death of the animals with severe acute liver inflammation in few days after infection. Since Tregs downregulate inflammation, we conclude that their decrease may contribute to the natural susceptibility of adult rabbits to RHDV infection.

Insulin prevents leptin inhibition of RM1 prostate cancer cell growth.

The association between obesity and cancer is controversial: whereas several epidemiological, clinical and research studies using cancer cell lines have supported that high levels of insulin and leptin could favor prostate cancer development and dissemination, other studies have demonstrated opposite effects or even absence of association. The main goal of this study was to evaluate the in vitro proliferation of murine androgen insensitive prostate carcinoma cells RM1 in the presence of leptin and insulin. After assessing and confirming the presence of leptin and insulin receptors in RM1 cells by immunocytochemistry, cells were cultured in the presence of different concentrations of leptin (0, 25, 50, 100 and 200 ng/mL), insulin (0, 50, 100, 150 and 200 nM) or leptin plus insulin ( 25 ng/ml + 50 nM; 50 ng/ml + 100 nM; 100 ng/ml + 150 nM; 200 ng/ml + 200 nM; 25 ng/ml + 150 nM; 100 ng/ml + 50 nM of leptin plus insulin, respectively). Cell proliferation was evaluated by assessing the percentage of resazurin reduction, a surrogate marker of cell metabolic rate. Leptin significantly decreased the percentage of resazurin reduction in all studied concentrations while there was only a slight or non significant difference in RM1cell proliferation in the presence of insulin or insulin combined with leptin when compared with control. These results show that leptin decreases RM1 prostate cancer cell proliferation at the studied concentrations, while insulin is able to antagonize the leptin inhibition of RM1 prostate cancer cell growth in vitro. The difference in cell growth that is modulated by the various hormonal environments may explain the heterogeneous behavior of prostate cancer in the obese human population.

A simple and rapid method for isolation of caliciviruses from liver of infected rabbits.

Rabbit Haemorrhagic Disease Virus (RHDV), a member of the Caliciviridae family, is the etiologic agent of Rabbit Haemorrhagic Disease (RHD); this viral disease is highly contagious and kills more than 90% of infected adult rabbits. Research on experimental calicivirus infection uses inocula obtained from livers of rabbits dying from calicivirus infection. This implies that caliciviruses have to be purified from liver homogenates. Current methods to isolate caliciviruses from rabbit livers are time consuming. We propose here a new procedure for fast purification of rabbit caliciviruses from liver homogenates that uses centrifugation through an iodixanol gradient. This method offers in approximately 2 h a sample with a high degree of calicivirus purity, as shown by its biochemical and immunocytochemistry analysis, which is also able to kill adult rabbits from RHD within 48 h of inoculation.

Surgical porcine myocardial infarction model through permanent coronary occlusion.

Using domestic pigs as an animal model, we here validated a reproducible and standardized myocardial infarction (MI) surgical model, to achieve the largest possible infarct extent with the lowest morbidity and mortality. To this end, we included several anesthetic and perisurgical precautions to minimize surgical complications. Mortality and morbidity rates were compared among groups of pigs that underwent permanent occlusion at different locations of either the left circumflex or left anterior descending artery. In addition, to compare the resulting MI between groups, data were collected by using cardiac biomarkers (including troponin I), electrocardiography, and echocardiography. These data were correlated to the final mean infarct size calculated by microscopic studies. Proximal occlusions lead to high mortality rates, whereas distal occlusions induced rather small MI areas. The optimal occlusion site in terms of morbidity, mortality, and lesion extent was the midpoint of the left anterior descending artery. In this group, only one pig died, and group cardiac data showed a rise in biomarker levels, marked left ventricular dysfunction on electrocardiography and echocardiography, and well-defined transmural MI in both ventricles. Infarct size quantitated through histologic studies revealed an average 15% ventricular lesion. Because interanimal variability in results from this group was negligible, we consider that the induced myocardial injury of this model is reliable.

Early acute depletion of lymphocytes in calicivirus-infected adult rabbits.

Rabbit Haemorrhagic Disease (RHD) is a lethal infection caused by calicivirus that kills 90% of the infected adult rabbits within 3 days. The calicivirus replicates in the liver and causes a fulminant hepatitis. Most studies on the pathology of RHD have been focused on the fulminant liver disease. This may not be the only mechanism in the pathogenesis of RHD: calicivirus infection may also induce leukopenia in the infected adult rabbits. We show now by flow cytometry analysis that the calicivirus induces an early decrease in B and T cells, in both spleen and liver. The depletion of B and T cells was associated with apoptosis labelled by annexin V. These changes occurred in rabbits before they showed enzymatic evidence of liver damage and persisted after liver transaminase values were very high. We conclude that depletion of lymphocytes caused by the calicivirus infection precedes or attends liver damage. The relative contribution of this lymphocyte depletion for the pathogenesis of the fatal calicivirus infection of rabbits remains to be investigated.

Prostate cancer cell proliferation and angiogenesis in different obese mice models.

Obesity has been associated with increased incidence and aggressiveness of prostate cancer. Although controversial, several studies suggest that leptin could influence tumour cell growth and proliferation. The main goal of this study was to assess cellular growth of prostate adenocarcinoma cells in obese mice with different endogenous hormonal environments in what relates to leptin circulating levels and sensitivity. Four groups of mice (n = 6/group) were used, namely obese mice with congenital non-functioning leptin receptor OBR (db/db), obese mice with congenital leptin deficiency (ob/ob), mice with diet induced obesity (DIO) and normal weight C57BL/6J mice (control). All groups of mice were injected subcutaneously with 3.0 x 10(5) RM1 cells/500 microl PBS (murine prostate carcinoma androgen insensitive cells) and tumour growth and angiogenesis were evaluated 14 days after inoculation. The tumours induced in ob/ob and DIO mice were significantly larger (P < 0.001) while those induced in db/db mice were significantly smaller (P = 0.047), when compared with controls. Morphometric analysis revealed that mitotic index and Ki-67 positive nuclear density, both cell proliferation markers, were also significantly lower in the tumours of db/db mice (P < 0.001) when compared to controls. An inverse correlation was observed between leptin plasma levels and tumour weight (r = -0.642, P < 0.001), mitotic index (r = -0.646, P < 0.01) and Ki-67 positive nuclear density (r = -0.795, P < 0.001). These results suggest that high leptin concentrations are not favourable to RM1 cell growth and proliferation. On the contrary, high plasma leptin levels were associated with less cellular proliferation and angiogenesis in vivo.

Structure of the rat nasal mucosa after acute and chronic hyperbaric oxygen therapy.

OBJECTIVE: We aimed at the identification of putative morphologic changes induced in the rat nasal mucosa by acute or chronic hyperbaric oxygen (HBO2) treatment. STUDY DESIGN: Nasal samples were obtained from three groups of eight adult Wistar rats. The first group was submitted to 30 daily sessions of 100-minute-long HBO2 treatments at 2.5 ATA, the second group to a single 485-minute-long HBO2 session following the U.S. Navy Table 6 extended twice at 2.8 and 1.9 ATA, and the third group was composed of rats not submitted to any HBO2 therapy. METHODS: Samples of anterior portion of the lower nasal turbinates (concha nasalis ventralis) were collected after sacrifice and head dissection of the animals. The samples were processed for light and electron microscopy and for morphometric analysis. Inflammatory leukocyte infiltration was evaluated by a semiquantitative method. Non-parametric ANOVA was applied to evaluate statistical differences between leukocyte infiltration, and ANOVA one-way was used to evaluate the thickness of epithelium and basement membrane from samples of HBO2-treated rats and untreated rats. RESULTS: Samples of the turbinate mucosa of the rats submitted to chronic HBO2 treatment showed a significant increase in the thickness of the epithelium and a mild enhancement in the number of infiltrating leukocytes when compared with data from samples from untreated rats or from rats submitted to a single HBO2 treatment. CONCLUSIONS: Chronic HBO2 treatment causes only minor changes in the architecture of the nasal mucosa of the rat; they reflect a mild inflammatory response of the respiratory tract to the increase in pressure and in oxygen content induced by HBO2. No significant morphological changes were observed after a single HBO2 treatment.