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.

The Neuroimmune Interplay in Joint Pain: The Role of Macrophages.

Chronic pain associated with joint disorders, such as rheumatoid arthritis (RA), osteoarthritis (OA) and implant aseptic loosening (AL), is a highly debilitating symptom that impacts mobility and quality of life in affected patients. The neuroimmune crosstalk has been demonstrated to play a critical role in the onset and establishment of chronic pain conditions. Immune cells release cytokines and immune mediators that can activate and sensitize nociceptors evoking pain, through interaction with receptors in the sensory nerve terminals. On the other hand, sensory and sympathetic nerve fibers release neurotransmitters that bind to their specific receptor expressed on surface of immune cells, initiating an immunomodulatory role. Macrophages have been shown to be key players in the neuroimmune crosstalk. Moreover, macrophages constitute the dominant immune cell population in RA, OA and AL. Importantly, the targeting of macrophages can result in anti-nociceptive effects in chronic pain conditions. Therefore, the aim of this review is to discuss the nature and impact of the interaction between the inflammatory response and nerve fibers in these joint disorders regarding the genesis and maintenance of pain. The role of macrophages is highlighted. The alteration in the joint innervation pattern and the inflammatory response are also described. Additionally, the immunomodulatory role of sensory and sympathetic neurotransmitters is revised.

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.

NAP-2 Secreted by Human NK Cells Can Stimulate Mesenchymal Stem/Stromal Cell Recruitment.

Strategies for improved homing of mesenchymal stem cells (MSCs) to a place of injury are being sought and it has been shown that natural killer (NK) cells can stimulate MSC recruitment. Here, we studied the chemokines behind this recruitment. Assays were performed with bone marrow human MSCs and NK cells freshly isolated from healthy donor buffy coats. Supernatants from MSC-NK cell co-cultures can induce MSC recruitment but not to the same extent as when NK cells are present. Antibody arrays and ELISA assays confirmed that NK cells secrete RANTES (CCL5) and revealed that human NK cells secrete NAP-2 (CXCL7), a chemokine that can induce MSC migration. Inhibition with specific antagonists of CXCR2, a receptor that recognizes NAP-2, abolished NK cell-mediated MSC recruitment. This capacity of NK cells to produce chemokines that stimulate MSC recruitment points toward a role for this immune cell population in regulating tissue repair/regeneration.

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.

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.

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.

Enhanced mesenchymal stromal cell recruitment via natural killer cells by incorporation of inflammatory signals in biomaterials.

An exacerbated inflammatory response questions biomaterial biocompatibility, but on the other hand, inflammation has a central role in the regulation of tissue regeneration. Therefore, it may be argued that an 'ideal' inflammatory response is crucial to achieve efficient tissue repair/regeneration. Natural killer (NK) cells, being one of the first populations arriving at an injury site, can have an important role in regulating bone repair/regeneration, particularly through interactions with mesenchymal stem/stromal cells (MSCs). Here, we studied how biomaterials designed to incorporate inflammatory signals affected NK cell behaviour and NK cell-MSC interactions. Adsorption of the pro-inflammatory molecule fibrinogen (Fg) to chitosan films led to a 1.5-fold increase in adhesion of peripheral blood human NK cells, without an increase in cytokine secretion. Most importantly, it was found that NK cells are capable of stimulating a threefold increase in human bone marrow MSC invasion, a key event taking place in tissue repair, but did not affect the expression of the differentiation marker alkaline phosphatase (ALP). Of significant importance, this NK cell-mediated MSC recruitment was modulated by Fg adsorption. Designing novel biomaterials leading to rational modulation of the inflammatory response is proposed as an alternative to current bone regeneration strategies.