Macrophage polarization in response to collagen scaffold stiffness is

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Bio-interactions and Biocompatibility

Macrophage polarization in response to collagen scaffold stiffness is dependent on crosslinking agent used to modulate the stiffness Rukmani Sridharan, Emily J Ryan, Cathal J Kearney, Daniel J. Kelly, and Fergal J O'Brien ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.8b00910 • Publication Date (Web): 01 Dec 2018 Downloaded from http://pubs.acs.org on December 3, 2018

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ACS Biomaterials Science & Engineering

Macrophage polarization in response to collagen scaffold stiffness is dependent on crosslinking agent used to modulate the stiffness Rukmani Sridharan1,2,3; Emily J. Ryan1, 3; Cathal J. Kearney1,2,3; Daniel J. Kelly1,2,3 ;Fergal J. O'Brien1,2,3,* 1 Tissue Engineering Research Group, Department of Anatomy, Royal College of Surgeons in Ireland, Dublin 2, D02 YN77, Ireland 2 Trinity Centre for Bioengineering, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin 2, D02 PN40, Ireland 3 Advanced Materials Bio-Engineering Research Centre (AMBER), Trinity College Dublin, Dublin 2, D02 PN40, Ireland *Corresponding Author E-mail: [email protected]. Phone: +353-1-402 2149. Fax: +353-1-402 2355 Keywords: Macrophage polarization, collagen scaffolds, stiffness, crosslinking

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Abstract Macrophages are the first responders to biomaterial implantation, and determine the success or failure of an implant through their polarization into pro- (M1) and anti- (M2) inflammatory states. It is known that material properties such as stiffness can influence this response, with these properties typically modulated using a crosslinking agent. However, the cellular response comparing different crosslinking agents is often not analyzed. In this study, collagen scaffolds were crosslinked with one physical (DHT) and two chemical crosslinking methods (EDAC and genipin) in order to independently modulate the stiffness of scaffolds. The physical and structural properties of the scaffolds were thoroughly characterized to ensure that macrophage behavior to scaffold stiffness and crosslinking agent employed could be evaluated independent of each other. Through gene expression and protein secretion analysis of THP1 cultures, we demonstrate that the macrophage response to collagen scaffold stiffness is dependent on the crosslinking agent used. Macrophages respond similarly to scaffolds of increasing stiffness generated using the same crosslinking agent. However, when exposed to scaffolds of similar bulk modulus and degradation characteristics crosslinked using different crosslinkers, the cells responded to the crosslinking agent used rather than to the bulk modulus of the scaffolds. Moreover, while genipin crosslinking suppressed both pro- and anti-inflammatory responses from macrophages, EDAC crosslinking promoted a robust pro- and anti-inflammatory response to M1 and M2 factors respectively. The results demonstrate the potential of using tailored individual crosslinking treatments depending on the clinical indication. Taken together, the results from this study highlight the importance of understanding the macrophage response to both chemical and physical properties of scaffolds in order to promote positive remodeling outcomes after biomaterial implantation.


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Introduction Right from early development, where the mechanical forces encountered by a fetus determine its musculoskeletal development 1, to the constant physiological shear stresses that are presented to cells lining blood vessels 2; the mechanical environment of tissues plays an important role in both form and function 3. Accordingly, the importance of biophysical cues in directing cellular responses at the physiological and pathological level is now widely appreciated. For instance, the behavior of several cell types including fibroblasts 4,

mesenchymal stem cells 5-6, endothelial cells 7, macrophages 8 and tumor cells 9 are shown

to be dependent on their mechanical environment. However, these findings are often described in a 2D setting that does not recapitulate the complexity of the 3D microenvironment that cells sense in vivo. 3D collagen-based biomaterials are widely popular in several applications due to their excellent biocompatibility, resemblance to native tissues and their favorable mechanical properties

10-12.

They are often cross-linked with chemical or physical agents in order to

improve their mechanical properties and tailor their degradation rate. One of the most common chemical crosslinkers is 1-ethyl-3-3-dimethyl aminopropyl carbodiimide (EDAC), which forms zero length crosslinks (crosslinking agent is not present in the final crosslinked product) between amine and carboxyl groups – which are commonly found on proteins – with by-products that are easily washed away

13.

Although EDAC crosslinking is widely

utilized due to its biocompatibility, natural crosslinkers such as genipin have gathered increased interest in recent years due to its non-cytotoxic effects and its potential ability to suppress inflammation 14-17. Genipin crosslinks free amine groups (e.g., lysine, hydroxylysine, and arginine) and forms intramolecular cross-links with collagen

18.

However, unlike EDAC,

the genipin molecule remains in the crosslinked end-product. Biomaterials with a range of chemical, physical and biological properties are increasingly being used to repair and replace damaged tissues

19.

Although historically designed to be

inert to prevent interactions with the immune system, it is now accepted that the biophysical and biochemical properties of biomaterials can be tuned to promote favorable immune responses that will accelerate tissue repair

20-22.

Cells of the immune system (e.g.

neutrophils and macrophages) are the first to respond to biomaterial implantation in the body, with the structure and composition of the implant determining further cellular 3 ACS Paragon Plus Environment


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responses 22. Macrophages are key mediators of the foreign body response 23-24 and polarize into different phenotypes (known as polarization states) depending on changes in their microenvironment

25-28.

The adaptive polarization states of macrophages into M1 pro-

inflammatory and M2 anti-inflammatory states facilitates their role in several processes involved in the wound healing cascade; right from the initial stages of inflammation which is dominated by M1 macrophages to scar formation and matrix deposition to finally the wound resolution stages facilitated by M2 macrophages 27. Accordingly, the macrophage response to the properties of the biomaterial can have a lasting impact on the tissue repair process, with mechanical properties playing an important role in directing this response 22. It has been demonstrated that PEG hydrogels of increasing stiffness (from 130-840 kPa) show an increased recruitment of inflammatory cells, with a thicker foreign body capsule leading to impaired repair 8. In contrast, a recent study utilizing 3D collagen networks crosslinked with 1-ethyl-3-3-dimethyl aminopropyl carbodiimide (EDAC) to modulate the stiffness properties (one crosslinker concentration and a range of 27-100 Pa) showed that the stiffer materials resulted in a shift to an anti-inflammatory phenotype

29.

These contrasting results are likely due to the difference in the range of

stiffness studied (Pa vs. kPa), composition (PEG vs. collagen) and architecture (hydrogel with no pores vs. porous 3D networks) used in addition to species differences (mouse vs. human) of the cells

30.

Moreover, by modulating the stiffness of the materials with chemical

crosslinkers such as EDAC, an additional variable in terms of crosslinking agent used is introduced into the system that is often not accounted for. Hence, it is not possible to delineate the response of macrophages to the increasing stiffness of a material from the crosslinking reagent used, especially when a range of stiffness properties generated with the same crosslinker is not studied. Additionally, while these studies illuminate the important role of mechanical properties on the macrophage response

8, 29,

they often utilize only

limited number of markers to assess the phenotype of macrophages, which often leads to insufficient characterization of the cell phenotype and a poor understanding of their polarization profile To date, a number of studies have evaluated the role of mechanical properties of biomaterials on the macrophage and foreign body response

8, 29, 31.

However, these have

often been studied in 2D materials or in 3D scaffolds utilizing one crosslinking technique to 4 ACS Paragon Plus Environment

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modify their mechanical properties, making it difficult to conclude if the effects observed were due to the mechanical properties or the crosslinking method employed. Our lab and others have shown that the method of crosslinking influences the cellular response to collagen scaffolds

13, 18, 32.

We have also previously shown that increasing the mechanical

properties of collagen-based scaffolds with EDAC crosslinking led to a corresponding increase in the osteogenesis capability of mesenchymal stromal cells (MSCs), validating this system for studying the effect of mechanical properties on cellular behavior

33.

Therefore,

the overall objective of the research presented in this study was to elucidate both the role of collagen scaffold stiffness, and the crosslinking agent used, in directing macrophage polarization. Towards this, the first aim of this study was to develop and characterize collagen scaffolds with a range of mechanical properties through the use of both physical and chemical crosslinking methods. Secondly, the attachment of macrophages to the different scaffolds and their response to the byproducts of scaffold crosslinking was analyzed. Finally, macrophage polarization in response to scaffold properties was assessed through gene expression and protein secretion analysis.

Results Generation of scaffolds with similar bulk modulus and degradation characteristics using different crosslinking agents In order to independently evaluate the role of both scaffold crosslinking and the resultant stiffness on macrophage behavior, we treated collagen scaffolds with one physical crosslinking (dehydrothermal (DHT)) treatment and two chemical crosslinking treatments



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(EDAC and genipin (Gen)). Table 1 lists the nomenclature of the different groups generated and the target compressive modulus. Table 1: Collagen scaffolds crosslinked with DHT, EDAC and genipin to produce a range of bulk moduli Physical

Chemical

Target bulk

crosslinking

crosslinking

modulus (kPa)

DHT

DHT

-

0.5

0.1% Gen

DHT

0.1% Genipin

1

0.3% Gen

DHT

0.3% Genipin

1.5

0.05% EDAC

DHT

0.05% EDAC

1

0.1% EDAC

DHT

0.1% EDAC

1.5

Group name

Mechanical testing revealed that the compressive modulus of the scaffolds increased with increasing EDAC and genipin concentration (Figure 1A). While scaffolds treated with DHT crosslinking alone had a compressive modulus of 0.42 kPa, this increased to 0.93 kPa and 1.6 kPa when additionally treated with 0.1% Gen and 0.3% Gen, representing a two-fold and three-fold increase in compressive modulus, respectively. Upon treatment with 0.05% and 0.1% EDAC, compressive modulus of DHT crosslinked scaffolds again increased two-fold and three-fold to 0.82 kPa and 1.5 kPa respectively. The compressive modulus of scaffolds treated with 0.1% Gen and 0.05% EDAC were similar to each other, and the compressive modulus of scaffolds treated with 0.3% Gen and 0.1% EDAC were also similar, giving us the opportunity to independently evaluate the effect of bulk scaffold stiffness and crosslinking of collagen scaffolds on macrophage behavior.


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Figure 1: The effect of crosslinking on collagen scaffold mechanical and structural properties. (A)

Compressive modulus of scaffolds significantly increased upon crosslinking with genipin and EDAC. 0.1% Gen and 0.05% EDAC crosslinking doubled the elastic modulus of DHT scaffolds (p≤0.001) and (p≤0.05), while 0.3% Gen and 0.1% EDAC crosslinking tripled the compressive modulus of DHT scaffolds (p≤0.0001). (B) A ninhydrin test was performed to determine the amount of free amine groups in crosslinked scaffolds, and plotted as the percent of crosslinked amine groups of uncrosslinked scaffolds. The amount of crosslinked amine groups significantly increased with increasing concentration of crosslinkers (p≤0.0001). (C) Scaffolds degraded to different extents upon treatment with collagenase for 2 hours, as assessed by the remaining dry weight upon freeze drying and the (D) HYP content of degraded scaffolds. Results are represented as mean±stdev for three independent experiments. A one-way ANOVA with Tukey’s Post-test was used for statistical analysis. * (p≤0.05),** (p≤0.01) and **** (p≤0.0001).

In order to characterize the degree of crosslinking in the scaffolds, the percentage of free amines in each group was determined using a ninhydrin assay. The inverse of free amine content (which represents the degree of crosslinking) of each crosslinked group was presented as a percentage of DHT scaffolds (Figure 1B). As expected, a significantly higher degree of crosslinking was observed when the scaffolds were treated with increasing amounts of EDAC or genipin compared to DHT scaffolds, with no difference observed between 0.1% Gen and 0.05% EDAC or 0.3% Gen and 0.1% EDAC scaffolds. This indicates that upon crosslinking, there are less free amines available as binding sites for cellular attachment. On analyzing the ability of crosslinked scaffolds to withstand degradation to collagenase through measuring the remaining dry weight (Figure 1C) and hydroxyproline (HYP) content (which corresponds to the amount of remaining collagen) (Figure 1D) after degradation, we observed that DHT scaffolds were almost completely degraded (4% dry weight and 0.89% 7 ACS Paragon Plus Environment


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HYP) upon exposure to collagenase. The ability of scaffolds to resist degradation significantly increased (p≤0.0001) upon crosslinking with 0.1% Gen (51% dry weight and 57% HYP) and 0.05% EDAC (77% dry weight and 85% HYP), with no significant difference between the two groups. 0.3% Gen and 0.1% EDAC scaffolds did not degrade when treated with collagenase for 2 hours. This again demonstrates the differential degrees of crosslinking obtained when using increasing concentration of crosslinking agents. The compressive modulus, crosslinking degree and degradation properties of scaffolds treated with 0.1% Gen and 0.05% EDAC were similar to each other, and different from scaffolds treated with 0.3% Gen and 0.1% EDAC, which were similar to each other. Taken together, these results demonstrate that we were able to successfully fabricate collagen scaffolds with three different compressive moduli or stiffness properties (~0.5 kPa, ~1 kPa and ~1.5 kPa) that had different degradation properties and three different crosslinking methods (DHT, EDAC and genipin), giving us the opportunity to independently evaluate the effect of stiffness and crosslinking of collagen scaffolds on macrophage behavior. Crosslinking agents do not adversely affect macrophage attachment and protein production

Figure 2: The effect of scaffold stiffness and crosslinking on macrophage attachment.

Cell attachment was quantified by measuring DNA content of cells seeded on DHT, 0.1% Gen, 0.3% Gen, 0.05% EDAC and 0.1% EDAC scaffolds one day after seeding. No significant difference was observed between groups. A one-way ANOVA with Tukey’s Post-test was used for statistical analysis. Results are represented as mean ± stdev from three independent experiments. ** (p≤0.01).

On assessing macrophage attachment to different scaffolds, we found that scaffolds crosslinked with either EDAC or genipin led to better cellular attachment compared to DHT scaffolds (p≤0.001) irrespective of crosslinker concentration, with no difference between the various EDAC or genipin concentrations (Figure 3). It has been suggested that macrophages are capable of reacting to the residues associated with the crosslinking process left behind after crosslinking biomaterials 8 ACS Paragon Plus Environment

34.

We used ‘pre-

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conditioned medium’ which was produced by incubating crosslinked scaffolds in THP1 growth medium for 24 hours, in order to analyse the macrophage response to these residues, measured through the analysis of cellular metabolic activity (Figure 3A), release of pro- (MIP1α, IL6) and anti-inflammatory (IL10) cytokines (Figure 3B-D). No differences were observed in the macrophage response to the pre-conditioned medium from DHT, EDAC and genipin scaffolds. This confirms that all residues associated with the crosslinking process were eliminated during the washing steps, allowing us to next study the response of macrophages to the scaffold physical properties without byproduct affecting the results.

Figure 3: Response of macrophages to pre-conditioned medium from crosslinked scaffolds.

Macrophages seeded on plastic were treated with growth medium exposed to scaffolds for 24 hours (preconditioned medium) to ensure that the crosslinking by-products did not adversely alter macrophage response. (A) Cell metabolic activity , (B) anti-inflammatory factor production (IL10) , and pro-inflammatory factor production, (C) macrophage inflammatory protein (MIP1α) and (D) IL6 were not altered by treatment with preconditioned medium. Results are representative of two independent experiments. A one-way ANOVA with Tukey’s Post-test was used for statistical analysis. ns= not significant.

Macrophage response to collagen scaffold stiffness depends on the crosslinking agent used to modulate the stiffness In order to analyze the gene expression and protein secretion of macrophages cultured on different scaffolds, cells were cultured in basal medium (M0), to observe if scaffold properties induced changes in baseline gene and protein expression; and in M1 (IFN + LPS) and M2 (IL4 + IL13) induction medium, to observe how scaffold properties influence polarization into different phenotypes (Figure 4). In M0 medium, EDAC crosslinking significantly increased the secretion of pro-inflammatory factor IL6 (p≤0.01) and anti-inflammatory factor IL1Ra (p≤0.05 9 ACS Paragon Plus Environment


Figure 4: Characterization of macrophage gene expression and protein secretion in response to scaffold stiffness, crosslinking and the presence of induction factors.

Sandwich ELISA and qRT-PCR for markers of the M1 (left panel: A, B, C, D) and M2 (right panel: E, F, G, H) phenotypes shown as protein production (pg/ml) for ELISA or fold change over 18S for gene expression upon M1(IFN + LPS) or M2(IL4 + IL13) induction. In M0 medium, EDAC crosslinking significantly increased IL6 and IL1Ra production compared to DHT scaffolds. On M1(IFN + LPS) induction, EDAC significantly increased TNFα, IL6 and MIP1α production, while genipin significantly decreased IL6 production and increased MIP1α production compared to DHT scaffolds. On M2(IL4 + IL13) induction, EDAC significantly increased IL10 and IL1Ra secretion and CCL13 and CCL22 expression compared to DHT scaffolds. Genipin led either to a decrease or no change in IL10 and IL1Ra production and CCL13 expression. A two-way ANOVA with Bonferroni’s post-test was used for statistical analysis. Data are representative of four independent experiments for protein analysis (A, B, C, E, F) and three independent experiments for gene expression analysis (D, G, H) and are presented as mean ± stdev. * (p≤0.05), ** (p≤0.01),***(p≤0.001) and **** (p≤0.0001) represent significant difference compared to DHT scaffolds unless otherwise indicated. ns=not significant.

for 0.05% EDAC and p≤0.0001 for 0.1% EDAC) compared to DHT scaffolds. MIP1α was also significantly increased in EDAC scaffolds compared to genipin crosslinked scaffolds. This 10 ACS Paragon Plus Environment

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suggests that even without induction factors, the scaffold properties are capable of directly modulating the macrophage response, with EDAC crosslinking increasing the secretion of both pro- and anti-inflammatory factors irrespective of stiffness. Upon addition of M1(IFN+LPS) medium, 0.05% EDAC scaffolds significantly increased secretion of TNFα from macrophages compared to all other groups (p≤0.001). Interestingly, IL6 production was significantly decreased in genipin crosslinked groups compared to DHT and EDAC groups, suggesting that genipin is capable of downregulating the pro-inflammatory response. However, the pro-inflammatory marker MIP1α was significantly increased in both genipin and EDAC scaffolds compared to DHT, with a further increase in MIP1α secretion on 0.1% EDAC scaffolds compared to 0.05% EDAC scaffolds (24±5 ng/ml and 18±1 ng/ml) (p≤0.05). Interestingly, pro-inflammatory gene CXCL11 was significantly downregulated in both EDAC and genipin groups compared to DHT controls (Figure 4D). Together, the data suggests that EDAC crosslinking promoted an enhanced expression of pro-inflammatory factors upon addition of M1 factors, while genipin crosslinking generally suppressed proinflammatory factor expression in macrophages. Upon M2(IL4

+ IL13)

induction, EDAC crosslinking led to significantly increased production of

anti-inflammatory proteins (IL10 and IL1Ra) and genes (CCL13 and CCL22) irrespective of stiffness (Figure 4E,F,G,H). Interestingly, genipin crosslinking did not lead to significant increases in the secretion of IL10 or IL1Ra compared to DHT scaffolds. Instead, macrophages on 0.3% Gen scaffolds produced significantly decreased levels of both IL10 (p≤0.01) and IL1Ra (p≤0.05) compared to DHT scaffolds. The data suggests that EDAC crosslinked scaffolds led to a robust increase in anti-inflammatory factor production compared to DHT scaffolds, while 0.3% Genipin crosslinking suppressed anti-inflammatory responses. Taken together, the results from this study illustrate that although macrophages cultured on EDAC scaffolds promote an increase in pro-inflammatory factor expression in M0 medium compared to DHT scaffolds, they respond in a robust manner to both M1 and M2 stimuli by increasing the expression and secretion of pro- and anti-inflammatory factors respectively. We report that genipin crosslinked scaffolds not only suppress a pro-inflammatory response from macrophages, but also dampen a robust anti-inflammatory response to M2 factors (with no increase in IL10, IL1Ra production between M0 and M2 induction).



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Discussion It is now well understood that cellular behavior is directed by the chemical and physical properties of the materials they interact with 22. As one of the first responders to biomaterial implantation, macrophages play an important role in determining the outcome of biomaterial implantation

23.

This study aimed to investigate the role of collagen scaffold

stiffness – as determined by different crosslinking methods – on macrophage polarization. By using one physical and two chemical crosslinking methods, we developed a system to study the role of both collagen scaffold stiffness and the specific crosslinking agent used independent of each other. We show that macrophage response to increases in substrate stiffness of collagen scaffolds is dependent on the crosslinking agent employed to modulate the stiffness properties. While genipin crosslinking suppressed both pro- and antiinflammatory responses from macrophages, EDAC crosslinking promoted a robust proinflammatory response to M1 factors and anti-inflammatory response to M2 factors. Together, this study highlights the effect of different crosslinking treatments on the regenerative response and illustrates the importance of understanding the complex interactions of macrophages with both the physical and chemical properties of scaffolds. Collagen-based scaffolds developed in our laboratory 11, 35 were used to evaluate the role of 3D scaffold stiffness as controlled by different crosslinking methods on macrophage polarization. Although the lyophilized collagen scaffolds have a low bulk modulus (

Macrophage polarization in response to collagen scaffold stiffness is

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