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 Use of Specialized Pro-Resolving Mediators in Biomaterial-Based Immunomodulation.

The implantation of a biomaterial will lead to the immediate onset of an acute inflammatory response, which is of key importance in shaping the quality of the repair process. However, the return to homeostasis is critical to prevent a chronic inflammatory response that may impair the healing process. The resolution of the inflammatory response is now recognized as an active and highly regulated process, being described as specialized immunoresolvents that have a fundamental role in the termination of the acute inflammatory response. These mediators collectively coined as specialized pro-resolving mediators (SPMs) are a family of endogenous molecules that include lipoxins (Lx), resolvins (Rv), protectins (PD), maresins (Mar), Cysteinyl-SPMs (Cys-SPMs) and n-3 docosapentaenoic acid-derived SPMs (n-3 DPA-derived SPMs). SPMs have important anti-inflammatory and pro-resolutive actions such as decreasing the recruitment of polymorphonuclear leukocytes (PMNs), inducing the recruitment of anti-inflammatory macrophages, and increasing macrophage clearance of apoptotic cells through a process known as efferocytosis. Over the last years, the trend in biomaterials research has shifted towards the engineering of materials that are able to modulate the inflammatory response and thus stimulate appropriate immune responses, the so-called immunomodulatory biomaterials. These materials should be able to modulate the host immune response with the aim of creating a pro-regenerative microenvironment. In this review, we explore the potential of using of SPMs in the development of new immunomodulatory biomaterials and we propose insights for future research in this field.

The Role of Neutrophils in Biomaterial-Based Tissue Repair-Shifting Paradigms.

Tissue engineering and regenerative medicine are pursuing clinical valid solutions to repair and restore function of damaged tissues or organs. This can be achieved in different ways, either by promoting endogenous tissue repair or by using biomaterials or medical devices to replace damaged tissues. The understanding of the interactions of the immune system with biomaterials and how immune cells participate in the process of wound healing are critical for the development of successful solutions. Until recently, it was thought that neutrophils participate only in the initial steps of an acute inflammatory response with the role of eliminating pathogenic agents. However, the appreciation that upon activation the longevity of neutrophils is highly increased and the fact that neutrophils are highly plastic cells and can polarize into different phenotypes led to the discovery of new and important actions of neutrophils. In this review, we focus on the roles of neutrophils in the resolution of the inflammatory response, in biomaterial-tissue integration and in the subsequent tissue repair/regeneration. We also discuss the potential of neutrophils for biomaterial-based immunomodulation.

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.

Platelet and leukocyte adhesion to albumin binding self-assembled monolayers.

This study reports the use of tetraethylene glycol-terminated self-assembled monolayers (EG(4) SAMs) as a background non-fouling surface to study the effect of an 18 carbon ligand (C18) on albumin selective and reversible adsorption and subsequent platelet and leukocyte adhesion. Surface characterization techniques revealed an efficient immobilization of different levels of C18 ligand on EG(4) SAMs and an increase of surface thickness and hydrophobicity with the increase of C18 ligands. Albumin adsorption increased as the percentage of C18 ligands on the surface increased, but only 2.5%C18 SAMs adsorbed albumin in a selective and reversible way. Adherent platelets also increased with the amount of immobilized C18. Pre-immersion of samples in albumin before contact with platelets demonstrated an 80% decrease in platelet adhesion. Pre-immersion in plasma was only relevant for 2.5%C18 SAMs since this was the only surface to have less platelet adhesion compared to buffer pre-immersion. EG(4) SAMs adhered negligible amounts of leukocytes, but surfaces with C18 ligands have some adherent leukocytes. Except for 10%C18 SAMs, which increased leukocyte adhesion after albumin pre-adhesion, protein pre-immersion did not influence leukocyte adhesion. It has been shown that a surface with a specific surface concentration of albumin-binding ligands (2.5%C18 SAMs) can recruit albumin selectively and reversibly and minimize the adhesion of platelets, despite still adhering some leukocytes.

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.

Osteogenic, anti-osteoclastogenic and immunomodulatory properties of a strontium-releasing hybrid scaffold for bone repair.

Strontium (Sr) is known to stimulate osteogenesis, while inhibiting osteoclastogenesis, thus encouraging research on its application as a therapeutic agent for bone repair/regeneration. It has been suggested that it may possess immunomodulatory properties, which might act synergistically in bone repair/regeneration processes. To further explore this hypothesis we have designed a Sr-hybrid system composed of an in situ forming Sr-crosslinked RGD-alginate hydrogel reinforced with Sr-doped hydroxyapatite (HAp) microspheres and studied its in vitro osteoinductive behaviour and in vivo inflammatory response. The Sr-hybrid scaffold acts as a dual Sr2+ delivery system, showing a cumulative Sr2+ release of ca. 0.3 mM after 15 days. In vitro studies using Sr2+concentrations within this range (0 to 3 mM Sr2+) confirmed its ability to induce osteogenic differentiation of mesenchymal stem/stromal cells (MSC), as well as to reduce osteoclastogenesis and osteoclasts (OC) functionality. In comparison with a similar Sr-free system, the Sr-hybrid system stimulated osteogenic differentiation of MSC, while inhibiting the formation of OC. Implantation in an in vivo model of inflammation, revealed an increase in F4/80+/CD206+ cells, highlighting its ability to modulate the inflammatory response as a pro-resolution mediator, through M2 macrophage polarization. Therefore, the Sr-hybrid system is potentially an appealing biomaterial for future clinical applications.

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.

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.

The influence of functional groups of self-assembled monolayers on fibrous capsule formation and cell recruitment.

The contribution of the surface chemistry of an implant to the thickness of the fibrous capsule formed after implantation was herein investigated. For that, self-assembled monolayers (SAMs) of alkanethiols on gold with different terminal functional groups (COOH, OH, and CH(3)) were used. These surfaces were implanted in subcutaneous air pouches of BALB/c mice and the ensuing fibrous capsules were evaluated and compared with the initial inflammatory response caused by the implant. The thickness of the fibrous capsules that are under organization around the implant was measured 1 week after implantation by histology. Inflammatory exudates were collected from the air pouches 24 h after the implantation of SAMs and were analyzed by flow cytometry. A significant increase in the thickness of fibrous capsules was seen around implanted CH(3)-terminated SAMs, and also in gold surfaces, in comparison with the air pouch wall of sham-operated mice and of COOH- and OH-covered SAMs. The CH(3)-coated implants also recruited higher numbers of inflammatory cells; this enhancement involved a significant number of Mac-1(+) cells. Our data indicate that implant surfaces coated with CH(3) induce thick fibrous capsules and this may be the result of the stronger inflammatory effect of CH(3) in comparison with COOH or OH chemical groups.

The attraction of Mac-1+ phagocytes during acute inflammation by methyl-coated self-assembled monolayers.

We have used self-assembled monolayers (SAMs) of alkanethiolates on gold to study the contribution of methyl terminal functional groups in implant-triggered inflammation. The CH3-coated biomaterials were inserted in an air-pouch cavity of the BALB/c mouse and the in situ inflammatory response was monitored 4, 24, 48 and 72 h later. Flow cytometry was applied to define surface expression of the adhesion receptor Mac-1 (CD11b/CD18), a marker of activated leukocytes, and also of CD3 and B220 antigens (T and B lymphocytes). The CH3-coated surfaces caused a significant enhancement in the number of Mac-1+ cells in the implant. The only significant change in T and B lymphocytes was a transient increase in T cells detected 48 h after the implantation. Peak numbers of Mac-1+ phagocytes were observed 24 h after implantation. We conclude that if CH3 is present at the surface of implants, this chemical group will trigger a significant enhancement of activated phagocytes involved in the inflammatory reaction, and this phenomenon may extend the local phlogistic event.

Inflammatory cell recruitment and adhesion to methyl-terminated self-assembled monolayers: effect of implantation time.

The contribution of methyl groups in implant-triggered inflammation was investigated in vivo using self-assembled monolayers (SAMs) of alkanethiols on gold. The CH(3)-coated implants were inserted in an air-pouch cavity induced in BALB/c mice. The in situ inflammatory response was monitored 24, 48, and 72 hours later. Inflammatory cells recovered from the air pouches were counted and observed by light microscopy. The cellularity of the implant surfaces was defined by scanning electron microscopy (SEM). In comparison with gold implants, the CH(3)-coated SAMs recruited a significantly higher number of inflammatory cells. Polymorphonuclear leukocytes (PMN) were more numerous than mononuclear cells (Mo) in the exudates recovered from the air pouches with CH(3)-coated SAMs. The opposite PMN/Mo proportion was observed in air pouches of the two control groups (mice receiving gold implants or sham-operated animals). A low density of adherent cells was seen on CH(3)-coated implants, with no significant quantitative differences during the time course of the study. In contrast, the gold-coated surfaces were covered with numerous cells during all of the 3 days of the inflammation. In conclusion, implants with CH(3) surfaces are likely to induce PMN-dominated local acute inflammation but these surfaces are not associated with a significant adherence of leukocytes to the implant.

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.

Inflammatory responses and cell adhesion to self-assembled monolayers of alkanethiolates on gold.

The acute inflammatory response and the adhesion of cells to self-assembled monolayers (SAMs) of well-defined surface chemistry was studied in vivo using a rodent air-pouch model of inflammation. SAMs with three different terminal functional groups (OH, COOH and CH3) were implanted in subcutaneous air pouches induced in BALB/c mice. After 24 h, inflammatory cells were recovered from the air pouches and the implants were removed and prepared for observation by scanning electron microscopy (SEM). The implants coated with OH and CH3, were found to cause the highest recruitment of inflammatory cells into the subcutaneous pouches. Polymorphonuclear neutrophils (PMNs) leukocytes predominated over mononuclear cells in inflammatory exudates of SAMs-coated implants, the opposite being found in uncoated implants (controls). CH3-coated implants induced the highest number of inflammatory cells and also the largest percentage of PMNs seen in the subcutaneous pouches. Control and OH-covered implants presented the higher densities of attached inflammatory cells detected by SEM. In contrast, the CH3-coated implants showed a very low density of cells adherent to the implant surface. We conclude that the chemical nature and the degree of hydrophobicity of the surface of implants modulate both the local acute inflammatory reaction and the adhesion of leukocytes.

Adhesion of human leukocytes to biomaterials: an in vitro study using alkanethiolate monolayers with different chemically functionalized surfaces.

The adhesion of human leukocytes to self-assembled monolayers of well-defined surface chemistry was investigated in vitro. Polymorphonuclear (PMN) and mononuclear leukocytes were isolated from human blood by centrifugation techniques. The effect on adhesion of cell activation produced by pre-incubation of leukocytes with phytohemagglutinin (PHA) and phorbol 12-myristate 13-acetate (PMA) was also studied. Gold substrates were modified by treatment with alkanethiols with three different terminal chemical groups: COOH, OH, and CH(3). After incubation with the two subpopulations of leukocytes, the monolayers were washed, treated with fixative, stained with a Giemsa method, and observed by light microscopy to quantify the number of attached leukocytes. Comparative quantification of the density of leukocyte adhesion to the three types of self-assembled monolayers was determined. The hydrophobic surface expressing CH(3) was found to be the one that induced the highest adhesion density of leukocytes, both of PMN and mononuclear cells. In vitro activation of both mononuclear and PMN leukocytes further increased cell adhesion to the chemically defined monolayers that were used. This enhancement was higher for PHA-activated than for PMA-stimulated mononuclear cells, whereas PMA treatment of neutrophils resulted in a higher rate of adhesion of these cells than PHA stimulation.

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.

Interactions of leukocytes and platelets with poly(lysine/leucine) immobilized on tetraethylene glycol-terminated self-assembled monolayers.

Surfaces that bind heparin are important for biomaterials for blood deheparinization. In our recent work it was demonstrated that a polypeptide composed of L-lysine and L-leucine (pKL), after immobilization onto tetra(ethylene glycol) terminated self-assembled monolayers (EG4-SAMs), can bind heparin from blood plasma in a selective, concentration-dependent way. During this work the effect of this peptide on platelet adhesion and activation and leukocyte adhesion was studied. The surface charge of these nanostructured surfaces was evaluated in order to correlate the effect of positively charged amine groups and hydrophobic methyl groups on the behavior of platelets and leukocyte adhesion. The results demonstrated that the presence of pKL decreased leukocyte adhesion to EG4-SAMs at all concentrations used. This effect is even more pronounced when surfaces were pre-immersed in heparinized plasma. In contrast, there is an increase in platelet adhesion and activation with increased percentage immobilized pKL. This effect is enhanced when surfaces were pre-immersed in heparinized plasma. However, adsorbed pKL in very low amounts does not induce platelet adhesion and activation compared with EG4, even when pre-immersed in plasma. Since only low pKL amounts are necessary to induce heparin selectivity, these results are promising for the development of heparin-binding biomaterials for blood deheparinization.

Evaluation of the effect of the degree of acetylation on the inflammatory response to 3D porous chitosan scaffolds.

The effect of the degree of acetylation (DA) of 3D chitosan (Ch) scaffolds on the inflammatory reaction was investigated. Chitosan porous scaffolds with DAs of 4 and 15% were implanted using a subcutaneous air-pouch model of inflammation. The initial acute inflammatory response was evaluated 24 and 48 h after implantation. To characterize the initial response, the recruitment and adhesion of inflammatory cells to the implant site was studied. The fibrous capsule formation and the infiltration of inflammatory cells within the scaffolds were evaluated for longer implantation times (2 and 4 weeks). Chitosan with DA 15% attracted the highest number of leukocytes to the implant site. High numbers of adherent inflammatory cells were also observed in this material. For longer implantation periods Ch scaffolds with a DA of 15% induced the formation of a thick fibrous capsule and a high infiltration of inflammatory cells within the scaffold. Our results indicate that the biological response to implanted Ch scaffolds was influenced by the DA. Chitosan with a DA of 15% induce a more intense inflammatory response when compared with DA 4% Ch. Because inflammation and healing are interrelated, this result may provide clues for the relative importance of acetyl and amine functional groups in tissue repair and regeneration.