# A novel calcium alginate hydrogel formulation to enhance monocyte/macrophage anti-inflammatory activity

Nell Hirt, Mansour Alkobtawi, Enzo Manchon, Richard Proust, Emeline Villain, Stéphane Lack, Emmanuelle Bourrat, Céline Des Courtils, Jean-David Bouaziz, Reem Al-Daccak

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Nell Hirt, Mansour Alkobtawi, Enzo Manchon, Richard Proust, Emeline Villain, et al. A novel calcium alginate hydrogel formulation to enhance monocyte/macrophage anti-inflammatory activity. International Journal of Biological Macromolecules, 2025, 306 (Part 1), pp.141251. 10.1016/j.ijbiomac.2025.141251. hal-05110168

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Submitted on 12 Jun 2025

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# A novel calcium alginate hydrogel formulation to enhance monocyte/macrophage anti-inflammatory activity

Nell Hirt<sup>a,b</sup>, Mansour Alkobtawi<sup>b</sup>, Enzo Manchon<sup>a</sup>, Richard Proust<sup>b</sup>, Emeline Villain<sup>b</sup>, Stéphane Lack<sup>b</sup>, Emmanuelle Bourrat<sup>c</sup>, Céline des Courtils<sup>b</sup>, Jean-David Bouaziz<sup>a,c</sup>, Reem Al-Daccak<sup>a,*</sup>

## ARTICLE INFO

**Keywords:**

Calcium alginate

Hydrogel

Monocytes

Macrophages

Inflammation

Recessive dystrophic epidermolysis bullosa

## ABSTRACT

Alginate hydrogels are biocompatible and present tunable properties making them ideal for biomedical applications. We designed a novel Ca²⁺-Alginate hydrogel and investigated its bioactivity on key component of the immune inflammatory process, the monocytes/macrophages. Our results demonstrate that the developed Ca²⁺-Alginate hydrogel downregulated the expression of inflammation-related markers CD36 and CD64, in both classical and intermediate monocyte subsets. Additionally, the hydrogel upregulated the expression of the anti-inflammatory marker CD206 in both subsets and reduced their capacity to produce TNFα. In macrophages, the hydrogel modulated the pro-inflammatory M1 towards an anti-inflammatory profile, as evidenced by an increased population of CD163⁺CD206⁺ macrophages, typically associated with anti-inflammatory/ immunoregulatory activity, and a decreased production of TNFα. The hydrogel also affected mitochondrial function in M1-macrophages, increasing mitochondrial mass and reducing reactive oxygen species production. The translational potential of the hydrogel was evaluated on circulating monocytes from patients suffering from the severe skin disease recessive dystrophic epidermolysis bullosa. The hydrogel increased the anti-inflammatory classical monocyte subset at the expense of the intermediate inflammatory subset. It also reduced CD36 and CD64, and downregulated TNFα production. Collectively, our findings provide evidence of the anti-inflammatory potential of a Ca²⁺-Alginate hydrogel, suggesting its promising therapeutic application to modulate inflammation.

## 1. Introduction

Natural polymers have gained considerable interest in biomedical applications due to their low toxicity and favorable physicochemical properties. Among these, alginate, a polysaccharide derived from brown algae, stands out for its excellent biocompatibility and gelation ability. It is widely used in various therapeutic strategies for wound healing, vascular and bone regeneration, either alone or as a scaffold for cells and their derivatives. Alginate is a heteropolymer composed of β-D-mannuronic acid (M) and α-L-guluronic acid monomers (G), arranged in MM, GG, or MG blocks [1]. The ratio of these monomers, known as the mannuronic/guluronic (M/G) ratio, varies depending on the algal species, the part of the plant, and environmental factors such as seasonality. This variation influences alginate's physical properties, particularly its

ability to chelate cations, as GG blocks bind cations more strongly than MM blocks. When exposed to divalent cations such as calcium (Ca²⁺), alginate undergoes crosslinking to form ionic hydrogels [2,3]. As Ca²⁺ is a key coagulation factor and a potent cell activator, Ca²⁺ alginate (Ca²⁺-Alg) has attracted considerable attention across a range of biomedical applications [4–7]. Notably, it is endowed with pro-inflammatory properties, stimulates immune response promoting the activation of macrophages and the secretion of pro-inflammatory cytokines (e.g., IL-1β, TNFα) as well as enhancing Natural Killer (NK) cell activity [8–10].

Inflammation is a complex defensive response initiated by the release of inflammatory mediators in response to cell and/or tissue damage caused by pathogens, chemicals, or physical agents. Stress signals released by these damaging stimuli activate resident innate myeloid immune cells, which, in turn, recruit additional immune cells to

<footnote><sup>a</sup> National Institute of Health and Medical Research (INSERM) UMRS-976 HPI, Paris University, Saint-Louis Hospital, 75010 Paris, France</footnote>

<footnote><sup>b</sup> Laboratoires Brothier, Nanterre, France</footnote>

<footnote><sup>c</sup> Dermatology Department, AP-HP, Saint-Louis Hospital, 75010 Paris, France</footnote>

<footnote>* Corresponding author at: Hôpital Saint Louis, Inserm U976, Bâtiment Bazin (porte 2) 1, Avenue Claude Vellefaux, 75010 Paris, France.
E-mail address: reem.al-daccak@inserm.fr (R. Al-Daccak).</footnote>
eliminate pathogens, clear cell debris, repair damaged tissue, and restore homeostasis [11]. However, when inflammation becomes persistent, it can turn chronic and contribute to a variety of pathological conditions, including delayed wound healing, cancer, autoimmune diseases, and degenerative disorders [12]. Therefore, inflammation is a tightly regulated process that requires precise coordination between pro-inflammatory and anti-inflammatory cells and mediators to ensure effective resolution and healing.

Monocytes and macrophages are key players in the innate immune response and the inflammatory process [13]. These myeloid cells are highly heterogeneous with monocytes being classified as CD14<sup>+</sup>CD16<sup>-</sup> classical, CD14<sup>+</sup>CD16<sup>+</sup> intermediate, and CD14<sup>-</sup>CD16<sup>+</sup> non-classical monocytes. Classical monocytes are the most abundant subset in circulation and are characterized by robust phagocytic and antimicrobial activity. Intermediate monocytes are more pro-inflammatory specialized in antigen-presentation, as well as the production of cytokines and reactive oxygen species (ROS). Non-classical monocytes act as patrolling cells, playing crucial roles in antiviral responses and tissue repair [14,15]. Upon activation, monocytes can differentiate into either pro-inflammatory M1-macrophages or anti-inflammatory M2-macrophages, depending on the microenvironment. M1-macrophages defend the host against pathogens, primarily through ROS production, and secrete pro-inflammatory cytokines, such as IL-1β and TNFα. M2-macrophages are rather involved in resolving inflammation, promoting tissue repair and secreting immune regulatory cytokines, such as IL-10 [16-18].

In this study, we formulated a novel Ca²⁺-Alg hydrogel with the aim of exploring its potential for biomedical applications. Ca²⁺ is a critical second messenger in immune cell signaling, and its release from alginate hydrogels is often immunostimulatory. Intriguingly, our results demonstrated that the bioactive Ca²⁺-Alg hydrogel finetunes the behavior of primary human monocytes and macrophages from healthy donors towards anti-inflammatory behavior. Within our translational perspective, the hydrogel also effectively modulates diseased circulating monocytes from patients suffering from the severe skin disease recessive dystrophic epidermolysis bullosa (RDEB), marked by a pronounced inflammatory dimension. Immune dysregulation of the inflammatory process is often implicated in chronic diseases and impaired tissue repair. Within this notion, this novel Ca²⁺ hydrogel may thus offer a promising approach to inflammatory disease management strategies.

## 2. Methods

### 2.1. Ca²⁺-Alg hydrogel preparation

Sodium alginate extracted from brown algae of the genus *Laminaria* (M/G = 1.5, pharmaceutical grade) was mixed at 2% with 0.9% NaCl in water. The resulting solution was sprayed through a nozzle into a solution of 1% calcium chloride (CaCl₂) and 0.6% NaCl in water. The CaCl₂ solution provides Ca²⁺ ions that displace the Na⁺ ions from the alginate chains and replace them via interaction with the alginate carboxylate groups. The interaction of Ca²⁺ ions with alginate results in the ionic crosslinking of alginate chains, leading to the formation of a stable calcium alginate hydrogel. The spraying process is carried out in a continuously stirred tank using magnetic stirrers within a suitable enclosure. Operating conditions include a pressure of approximately 1.8 bars in the tank and 2.5 bars in the enclosure. The process lasts about 90 min and ensures ionic exchange, alginate crosslinking and calcium alginate hydrogel formation. The resulting mixture is subjected to three rounds of centrifugation at 2500 rpm for 2 min each. After each centrifugation step, the pellet containing the calcium alginate hydrogel is resuspended in demineralized water and filtered under vacuum using a 0.1 µm filter to remove residual salts. Finally, the purified hydrogel is sterilized by ultraviolet (UV) treatment.

### 2.2. Ca²⁺-Alg hydrogel viscosity

The viscosity of the Ca²⁺-Alg hydrogel is evaluated with a rheometer (TA Instrument, model HR10), using a steel cone and plate geometry (4 cm, 2°, 59,000 µm truncation gap). For this measure, a shear stress continuous ramp is used (10 to 150 Pa, 2 min) at 25 °C (Peltier plate).

### 2.3. Characterization of Ca²⁺-Alg hydrogel

Characterization of Ca²⁺-Alg hydrogel was proceeded through light microscopy and laser granulometric analysis. For light microscopy, samples of Ca²⁺-Alg hydrogel were placed between cavity slide (diameter 15-18 mm, depth 0,6-0,8, Marienfeld, ref. 1,320,000) and coverslip and observed with a reverse optical microscope (Leica, model DMi8). Images were obtained at various magnification (x5, x10, x20, x40 and x100) and treated using Leica LAS X software (version 3.0.0.15697). Laser granulometry was performed by FILAB laboratory, France using a Malvern Mastersizer 3000. This includes a Hydro MV liquid analysis module and an Aero MV dry analysis module. Briefly, six measurements were performed for each Ca²⁺-Alg hydrogel sample to check the repeatability of the results according to the criteria of ISO 13320:2020. The resulting analysis was reported as the relative distribution of the volume (%) of Ca²⁺-Alg hydrogel particles by size (µm). The dv₁₀ value represents the size of the particles below which 10% of the sample lies, dv₉₀ is the size of the particles below which 90% of the sample lies, and dv₅₀ is the median of the particle size distribution.

### 2.4. Cultures of peripheral blood mononuclear cells within Ca²⁺-Alg hydrogel

Peripheral blood mononuclear cells (PBMC) were prepared from blood samples of healthy donors by Ficoll-Hypaque density gradient. Donors signed an informed consent following human ethics committee "Comité consultatif pour la protection des personnes dans les recherches biomédicales" - Saint Louis Hospital, Paris, France", and the study has been approved by the institution. Briefly, 0.5 × 10⁶ PBMC/mL were seeded in 24-well plates in complete RPMI medium supplemented with 10% human serum, 1% PenStrep and 1% L-glutamine for 3 days, in the absence or presence of 10% Ca²⁺-Alg at 37 °C in 5% CO₂. At the end of culture, cells were recovered from the hydrogel using VitroGel® Cell Recovery Solution according to manufacturer's procedures. Cells were then treated with FcR blocking reagent for 20 min at 4 °C to prevent unspecific bindings, stained with antibodies against lineage specific markers (Table 1). Cell viability was then evaluated by 7-aminoactinomycin D [7-AAD] staining and flow cytometry. Cells were acquired on a BD Fortessa flow cytometer and analyzed using FlowJo software (10.8.1). Unsupervised clustering was performed on the expression values of the markers using the FlowSOM algorithm (OMIQ, www.omiq.ai).

### 2.5. Monocyte/macrophage cultures with Ca²⁺-Alg hydrogel

Circulating CD14⁺ monocytes were isolated from whole blood

<table><caption>**Table 1**
List of antibodies used for PBMC staining.</caption><thead><tr><td>Antibody</td><td>Clone</td><td>Source</td><td>Identifier</td></tr></thead><tbody><tr><td>CD3-BUV805</td><td>UCTH1</td><td>BD Bioscience</td><td>Cat#612895</td></tr><tr><td>CD4-APCH7</td><td>RPA-T4</td><td>BD Bioscience</td><td>Cat#560158</td></tr><tr><td>CD8-BV395</td><td>RPA-T8</td><td>BD Bioscience</td><td>Cat#563795</td></tr><tr><td>CD45RA-BV785</td><td>HI100</td><td>BD Bioscience</td><td>Cat#563870</td></tr><tr><td>CD16-BV711</td><td>3G8</td><td>BD Bioscience</td><td>Cat#563127</td></tr><tr><td>CD19-BV650</td><td>SJ25C1</td><td>BD Bioscience</td><td>Cat#563226</td></tr><tr><td>CCR7-PECY7</td><td>3D12</td><td>BD Bioscience</td><td>Cat#557648</td></tr><tr><td>CD14-PE</td><td>REA599</td><td>Miltenyi Biotec</td><td>Cat#130-110-577</td></tr><tr><td>CD56-APC</td><td>REA196</td><td>Miltenyi Biotec</td><td>Cat#130-113-310</td></tr></tbody></table>
samples from healthy donors or RDEB patients using StraightFrom® Whole Blood CD14 MicroBeads (130-090-879, Miltenyi Biotech). The clinical-biological cohort of RDEB patients is followed and managed at the Dermatology Department - French national reference center for rare diseases of the skin and mucous membranes of genetic origin (MAGEC) - Saint-Louis Hospital. All patients signed informed consent following human ethics committee "Comité consultatif pour la protection des personnes dans les recherches biomédicales" (Direction de la Recherche Clinique, de l'Innovation des relations avec les universités et les organismes de recherche (DRCI), AP-HP, Saint-Louis Hospital). Briefly, 0.4 × 10⁶ monocytes/mL were seeded in 24 well-plates for 3 days in the absence or presence of 10 % Ca²⁺-Alg at 37 °C in 5 % CO₂. For macrophage experiments, cells were polarized from CD14⁺CD16⁻ classical monocytes. Monocytes were negatively selected from PBMC using magnetic beads (130-117-337, Miltenyi Biotech) following manufacturer's instruction, then seeded (0.4 × 10⁶ monocytes/mL) in a 24-well plate in complete medium and polarized towards pro-inflammatory M1-like or anti-inflammatory M2-like macrophages. For M1-like polarization CD14⁺CD16⁻ monocytes were cultured in the presence of Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) (50 ng/mL) for 6 days then treated for another 24 h with a combination of IFN-γ (50 ng/mL) and lipopolysaccharide (LPS) (50 ng/mL). For M2-like polarization CD14⁺CD16⁻ monocytes were cultured in human macrophage colony-stimulating factor (M-CSF) (50 ng/mL) for 6 days then treated for another 24 h with IL-4 (20 ng/mL) and IL-13 (20 ng/mL). Polarized macrophages were then cultured in the absence or presence of 10 % of Ca²⁺-Alg hydrogel for an additional 3 days.

At the end of monocytes or macrophage cultures, cells were recovered from hydrogel using VitroGel® Cell Recovery Solution according to manufacturer's procedures and their phenotype was determined by flow cytometry. After recovery, cells were treated with FcR blocking reagent for 20 min at 4 °C to prevent unspecific bindings and then labelled with specific antibodies against myeloid markers (Table 2). Cells were then acquired on BD Fortessa flow cytometer and analyzed using FlowJo software (10.8.1). Unsupervised clustering was performed on the expression values of the markers using the FlowSOM algorithm (OMIQ, www.omiq.ai).

## 2.6. Cytokines assays

Monocytes or macrophages were cultured as previously described for 3 days in the absence or presence of Ca²⁺-Alg hydrogel. LPS (50 ng/mL) was added 24 h before the end of the culture, the supernatants were collected. The level of TNF-α and IL-10 in supernatants was then determined using MACSPlex Mix Cytotoxic basic kit (130-125-767, 130-125-806, 130-125-838, Miltenyi Biotech) according to manufacturer's procedures. Samples were acquired using a Canto II (BD) and analyzed using FlowJo (10.8.1) software.

<table><caption>**Table 2**
List of antibodies used for monocytes/macrophages staining.</caption><thead><tr><td>Antibody</td><td>Clone</td><td>Source</td><td>Identifier</td></tr></thead><tbody><tr><td>CD14-V500</td><td>M5e2</td><td>BD Bioscience</td><td>Cat#561391</td></tr><tr><td>CD163-BV421</td><td>GH161</td><td>BD Bioscience</td><td>Cat#562643</td></tr><tr><td>CD16-FITC</td><td>3G8</td><td>BD Bioscience</td><td>Cat#555406</td></tr><tr><td>CD86-PECF594</td><td>2331</td><td>BD Bioscience</td><td>Cat#562390</td></tr><tr><td>CD80-APCH7</td><td>L307.4</td><td>BD Bioscience</td><td>Cat#561134</td></tr><tr><td>CD206-APC</td><td>19.2</td><td>BD Bioscience</td><td>Cat#561763</td></tr><tr><td>HLA-DR-PE</td><td>L243</td><td>BD Bioscience</td><td>Cat#e0615</td></tr><tr><td>CD36-BV395</td><td>CB38</td><td>BD Bioscience</td><td>Cat#565422</td></tr><tr><td>CD16-BV711</td><td>3G8</td><td>BD Bioscience</td><td>Cat#563127</td></tr><tr><td>CD64-PECY7</td><td>REA978</td><td>Miltenyi Biotec</td><td>Cat#130-116-301</td></tr><tr><td>CD14-PE</td><td>REA599</td><td>Miltenyi Biotec</td><td>Cat#130-110-577</td></tr><tr><td>CD11b-BV510</td><td>REA713</td><td>Miltenyi Biotec</td><td>Cat#130-110-617</td></tr><tr><td>HLA-DR-BV421</td><td>G46-6</td><td>BD Bioscience</td><td>Cat#562804</td></tr><tr><td>CD11c-FITC</td><td>BHy6</td><td>BD Bioscience</td><td>Cat#561355</td></tr><tr><td>CCR2-APC</td><td>K036C2</td><td>Biolegend</td><td>Cat#357208</td></tr></tbody></table>

## 2.7. Assessment of mitochondrial mass and reactive oxygen species production

Mitochondrial mass in macrophages were determined using the MitoTracker Green FM (M7514, Invitrogen) according to the manufacturer's protocol. Briefly, cells were incubated in PBS containing 100 nM MitoTracker Green FM for 30 min in the dark at 37 °C, then washed twice. Levels of reactive oxygen species (ROS) in macrophages were determined using the DCFDA/H2DCFDA-Cellular ROS Assay Kit (ab113851, Abcam, Cambridge, UK) according to the manufacturer's protocol. Briefly, cells were incubated with 25 µM DCFDA for 30 min at 37 °C. At the end of labelling procedures, cells were directly acquired with Canto II (BD) flow cytometer and analyzed using FlowJo (10.8.1) software.

## 2.8. Quantification and statistical analysis

Statistical analysis was performed using GraphPad Prism 9.5.0. For 2 groups, comparisons were performed using unpaired t-test assuming Gaussian distribution with parametric test Anderson-Darling (A2*)/ D'Agostino-Pearson omnibus (K2)/Shapiro-Wilk (W)/Kolmogorov-Smirnov (distance), or with Mann Whitney test. For 3 groups, comparisons were performed using One-way ANOVA assuming Gaussian distribution or with nonparametric test. P-values ≤0.05 were considered statistically significant.

# 3. Results

## 3.1. Production and physico-chemical characterization of the Ca²⁺-Alg hydrogel

The Ca²⁺-Alg hydrogel production steps are outlined in the left panel of Fig. 1A. First, a Na⁺-Alg solution with a mannuronic-to-guluronic (M/G) ratio of 1.5, was sprayed into a calcium-chloride (CaCl₂) solution. This process facilitated an ionic exchange between Na⁺ from the alginate and Ca²⁺ from the CaCl₂ solution, leading to the conversion of Na⁺-Alg into Ca²⁺-Alg and the ionic crosslinking of alginate chains to form Ca²⁺-Alg particles. Particles were subjected to a triple washing process to ensure purity (Fig. 1A, left panel). The resulting Ca²⁺-alginate hydrogel was a translucent, slightly yellowish formulation with a fluid consistency and remained stable for over two years (Fig. 1A, right panel). Viscosity profiles exhibited a non-newtonian behavior, with higher shear rate inducing viscosity decrease, and remained stable after sterilization (Fig. 1B). Microscopic analysis revealed spherical particles (Fig. 1C), and laser granulometry analysis showed an average particle size (Dv {4:3}) of 116 µm (Fig. 1D). This procedure converted Na⁺-Alg into Ca²⁺-Alg hydrogel of specific formulation, M/G ratio and particle size, which together will influence the hydrogel's mechanical properties.

## 3.2. Ca²⁺-Alg hydrogel preserves PBMC viability

Because the overall bioactivity of hydrogels, is governed by their mechanical properties, we investigated the bioactivity of the developed Ca²⁺-Alg hydrogel. Our first objective was to assess its biocompatibility with immune cells. Peripheral blood mononuclear cells (PBMC) were selected as a model to evaluate potential cytotoxicity, given their role as critical circulating immune cells involved in host defense. PBMC from healthy donors were cultured in the presence or absence of Ca²⁺-Alg hydrogel, and the viability of various immune cell populations —T cells (CD3⁺), B cells (CD19⁺), NK cells (CD56⁻), and monocytes (CD14⁺)— was determined by flow cytometry (Fig. 2A). After 3 days of culture, PBMC in the presence of Ca²⁺-Alg showed preserved viability in CD3⁺ T cells, CD56⁺ NK cells, and CD19⁺ B cells, with a notable increase in the viability of CD14⁺ monocytes (Fig. 2B). These results collectively suggest that the Ca²⁺-Alg hydrogel does not induce cytotoxicity in these circulating immune cell populations.
<figure><img src="image_4.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>D</figcaption></figure>

<table><thead><tr><th rowspan="2">Test</th><th colspan="4">Laser particle size distribution (µm)</th></tr><tr><th>dV<sub>10</sub></th><th>dV<sub>50</sub></th><th>dV<sub>90</sub></th><th>Dv(4:3)</th></tr></thead><tbody><tr><td>1</td><td>43.0</td><td>93.2</td><td>208</td><td>114</td></tr><tr><td>2</td><td>43.1</td><td>94.1</td><td>213</td><td>116</td></tr><tr><td>3</td><td>43.0</td><td>93.6</td><td>213</td><td>118</td></tr><tr><td>4</td><td>43.1</td><td>93.6</td><td>215</td><td>117</td></tr><tr><td>5</td><td>43.1</td><td>93.6</td><td>211</td><td>114</td></tr><tr><td>6</td><td>43.2</td><td>93.6</td><td>215</td><td>117</td></tr><tr><td>Mean</td><td><strong>43.1</strong></td><td><strong>93.7</strong></td><td><strong>212</strong></td><td><strong>116</strong></td></tr><tr><td>Standard deviation</td><td>0.0766</td><td>0.306</td><td>2.71</td><td>1.7</td></tr></tbody></table>

<figure><img src="image_7.png" /><figcaption>**Fig. 1.** Production and characterization of the Ca²⁺-Alg hydrogel (A, left panel) Key steps for the production process of the Ca²⁺-Alg hydrogel. (A, right panel) Hydrogel visual characteristics (B) Viscosity as a function of shear stress before (grey curve) and after (orange curve) sterilization. (C) Light microscopic observation of Ca²⁺-Alg particles. (D left panel) Particle size distribution by laser diffraction granulometry. dV₁₀: the portion of particles with diameters smaller than this value is 10 %, dV₅₀: the portion of particles with diameters smaller and larger than this value are 50 %, dV₉₀: the portion of particles with diameters smaller than this value is 90 %, and Dv {4:3} is the average particle size relative to volume. (D right panel) Average particle size distribution curve with volume density (%) representing the highest volume percentage of the particle size distribution displayed in the y-axis and the particle size in the x-axis.</figcaption></figure>

To further evaluate the effect of our hydrogel, we applied t-distributed stochastic neighbor embedding (t-SNE) and the FlowSOM unbiased clustering algorithm. Using lineage-specific markers, we identified distinct subsets within investigated immune populations: CD3, CD4, CD8, CCR7, and CD45RA for T cells; CD19 for B cells; CD56 and CD16 for NK cells; and CD14 for monocytes (Fig. 2C upper left panel). CD4⁺ and CD8⁺ T cells, CD56⁺ NK cells and CD19⁺ B cells showed similar abundance in the presence of Ca²⁺-Alg hydrogel, while the abundance of CD14⁺ monocytes increased (Fig. 2C lower panel). EdgeR volcano plot analysis further confirmed these results highlighting a marked increase in monocytes abundance (from 1.53 % to 6.51 %) (Fig. 2D). These results suggest that the hydrogel preserves the overall viability of circulating immune cells, while selectively enhancing the abundance of CD14⁺ monocytes.

### 3.3. Ca²⁺-Alg hydrogel finetunes monocytes towards anti-inflammatory behavior

Given the pronounced effect of the Ca²⁺-Alg hydrogel on monocytes and their crucial role in regulating inflammatory processes, we further investigated the impact of our newly developed hydrogel on circulating CD14⁺ monocytes (Fig. 3A). Dimensional reduction using uniform manifold approximation and projection (UMAP) revealed that culturing CD14⁺ monocytes with Ca²⁺-Alg hydrogel for 3 days led to significant changes in their spatial distribution (Fig. 3B left panel). This shift was

accompanied by alterations in the proportions of monocyte subsets. Under control conditions, CD14⁺CD16⁻ classical monocytes, CD14⁺CD16⁺ intermediate monocytes, and CD14⁻CD16⁺ non-classical monocytes accounted for 47.7 %, 45.7 %, and 1.16 %, respectively. However, culturing in the presence of Ca²⁺-Alg hydrogel resulted in a significant increase in the proportion of CD14⁺CD16⁻ classical monocytes (66.5 %), while the CD14⁺CD16⁺ intermediate monocyte population notably decreased to 6.2 % (Fig. 3B right panel). No major changes were observed in CD14⁻CD16⁺ non-classical monocyte subset (2.23 %). To further interpret these findings, we analyzed the expression of key surface markers associated with the immune inflammatory response. Flow cytometry revealed that the Ca²⁺-Alg hydrogel significantly decreased the expression of inflammation-associated markers CD36 and CD64 in both classical and intermediate monocytes (Fig. 3D, E). In contrast, both monocyte subsets upregulated the expression of CD206, a marker typically associated with anti-inflammatory M2-macrophages, in the presence of the hydrogel (Fig. 3F). These results suggest that the newly developed hydrogel may promote monocyte polarization towards an anti-inflammatory phenotype. To further corroborate these findings, we measured the production of the pro-inflammatory cytokine TNFα by circulating monocytes upon LPS stimulation in the presence or absence of Ca²⁺-Alg hydrogel. The presence of the hydrogel drastically decreased TNFα production by LPS-stimulated monocytes (Fig. 3G). Together, these results underscore that the newly developed Ca²⁺-Alg hydrogel system can finetune monocytes towards an anti-inflammatory
<figure><img src="image_8.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>**Fig. 2.** Ca²⁺Alg hydrogel effect on PBMC viability and population distribution. (A) Schematic representation of the experimental design to assess the effect of Ca²⁺-Alg on human PBMC. (B) Bar graphs comparing the percentage of PBMC living cells. (C upper panel) Heatmap illustrating the expression of 9 markers based on FlowSOM clustering. (C lower panel) tsne illustrating the abundance of major immune cell populations of 12 PBMC samples from a total of 6 PBMC samples in control medium or Ca²⁺-Alg. (D left panel) EdgeR-based Volcano presenting differentially increased (left) and decreased (right) immune cell populations in PBMC in Ca²⁺-Alg vs control medium. The horizontal dashed line indicates the significant threshold of *p*-value = 0.05. (D right panel) Bar graph comparing the percentage of CD14⁺ monocytes cultured or not with Ca²⁺-Alg. Paired *t*-test, \* *p* < 0.05.</figcaption></figure>
<figure><img src="image_12.png" /><figcaption>**Fig. 3.** Ca²⁺-Alg hydrogel finetunes monocytes behavior. (A) Schematic representation of the experimental design to assess the activity of Ca²⁺-Alg hydrogel on circulating monocytes. (B left panel) UMAP comparing the distribution of circulating CD14⁺ monocytes between control medium and Ca²⁺-Alg (n = 3). Colors vary according to cell density. (B right panel) Bar graph comparing the percentage of monocyte subsets in control medium and Ca²⁺-Alg. (D, E, F) Bar graphs depicting the fold change of CD36, CD64, and CD206 expression in monocyte subsets normalized on control medium, respectively. (G) Quantification of TNFα concentration in monocyte culture supernatant after LPS stimulation. Paired t-test or two-way ANOVA, \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001, \*\*\*\*p < 0.0001.</figcaption></figure>

behavior.

### 3.4. Ca²⁺-Alg hydrogel promotes anti-inflammatory-like macrophages

Upon activation, monocytes differentiate into either pro- or anti-inflammatory macrophages depending on their microenvironment. Given the observed effect of Ca²⁺-Alg on CD14⁺ monocytes, we further investigated its impact on in vitro polarized pro-inflammatory M1-like macrophages (Fig. 4A). To generate M1-like macrophages, CD14⁺CD16⁻ classical monocytes were cultured with GM-CSF for 6 days, followed by 24 h treatment with IFNγ and LPS. Dimensional reduction using UMAP analysis revealed that culturing pro-inflammatory M1-like macrophages with Ca²⁺-Alg hydrogel altered their spatial distribution (Fig. 4B) and upregulated the expression of two markers of macrophage activation: the co-stimulatory CD80 molecule (Fig. 4C) and the antigen-presenting human leukocyte antigen HLA-DR (Fig. 4D). Concomitantly with this upregulation, we observed a

significant increase in the anti-inflammatory/immunoregulatory CD163⁺CD206⁺ macrophage population (Fig. 4E). To further explore these phenotypic changes, we analyzed cytokine secretion by M1-macrophages cultured with Ca²⁺-Alg hydrogel, comparing them to M1 and anti-inflammatory M2-macrophages derived from M-CSF/IL-4/IL-13-stimulated CD14⁺CD16⁻ classical monocytes. Upon stimulation, M1-macrophages—whether cultured with or without Ca²⁺-Alg hydrogel—secreted both pro-inflammatory TNFα and anti-inflammatory IL-10 cytokines. Despite variability between donors, the TNFα to IL-10 ratio in M1-macrophages was lower in the presence of Ca²⁺-Alg hydrogel, bringing it closer to the ratio observed in anti-inflammatory M2-like macrophages (Fig. 4F). This result, coupled with the increased CD163⁺CD206⁺ macrophage population, further supports the anti-inflammatory potential of our hydrogel. The production of ROS is frequently elevated in response to stress or under chronic inflammation and is a hallmark of M1-macrophages [19]. We therefore investigated the effect of the hydrogel on mitochondrial function. As depicted in
<figure><img src="image_13.png" /><figcaption>**Fig. 4.** Ca²⁺-Alg hydrogel promotes anti-inflammatory-like macrophages. (A) Schematic representation of the experimental design to investigate the activity of Ca²⁺-Alg hydrogel on M1-like macrophages. (B) UMAP comparing the distribution of M1-like macrophages between control medium and Ca²⁺-Alg (n = 7). Colors vary according to cell density. (C, D) Bar graph comparing the fold change of CD80 and HLA-DR expression in monocyte subsets normalized on M1 control medium, respectively. (E) Bar graph comparing the percentage of CD163/CD206 in M1-like macrophages in control medium and Ca²⁺-Alg. (F) TNFα/IL-10 ratio of produced cytokines in culture supernatant of M1-like macrophages in control medium and Ca²⁺-Alg compared to M2-like macrophages. (G) Bar graph deciphering the mitochondrial mass represented as MitoTracker Green geomean. (H) ROS production assessed by the percentage of positive cells for the DCFDA probe. Paired t-test or one-way ANOVA, \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001.</figcaption></figure>

**Fig. 4G, M1-like macrophages cultured with Ca²⁺-Alg hydrogel showed increased mitochondrial mass, which may indicate a shift towards a more energy-demanding M2 phenotype. In addition, Ca²⁺-Alg hydrogel significantly reduced ROS production by M1-like macrophages (Fig. 4H), bringing their ROS levels closer to those typically observed in M2-like macrophages. These changes in macrophage behavior suggest**

that the hydrogel may influence intrinsic intracellular mechanisms, potentially reprogramming pro-inflammatory macrophages towards an anti-inflammatory phenotype.
### 3.5. Ca²⁺-Alg hydrogel impacts RDEB diseased monocytes

Under physiological conditions, CD14⁺CD16⁻ classical monocytes are the dominant subset of peripheral monocytes. The transition from CD14⁺CD16⁻ classical monocytes to pro-inflammatory CD14⁺CD16⁺ intermediate monocytes, key producers of inflammatory cytokines, is a marked feature of many inflammatory diseases [20]. Within our findings, we investigated the translational potential of the Ca²⁺-Alg hydrogel on monocytes isolated from patients suffering from RDEB as an ex-vivo model of diseased monocytes. We isolated circulating CD14⁺ monocytes from RDEB and cultured them for 3 days with or without Ca²⁺-Alg hydrogel (Fig. 5A). In the presence of the hydrogel, the proportion of CD14⁺CD16⁻ classical monocytes in RDEB samples increased from 15.5% to 64%, while the proportion of CD14⁺CD16⁺ intermediate monocytes decreased from 73.5% to 15.5% (Fig. 5B and C). No alterations were observed in non-classical monocytes. Concomitantly, Ca²⁺-Alg hydrogel significantly reduced the expression of inflammation-associated markers CD36 and CD64 on both classical and intermediate RDEB monocytes (Fig. 5D and E), suggesting a potential modulation of their inflammatory phenotype. The presence of Ca²⁺-Alg hydrogel also reduced the production of TNFα by LPS-stimulated RDEB monocytes (Fig. 5F), further supporting its potential to modulate the inflammatory response. Together, these findings provide the first evidence for the translational potential of our newly developed Ca²⁺-Alg hydrogel as inflammation-lessening agent.

### 4. Discussion

In this study, we demonstrated the bioactivity of a novel Ca²⁺-Alg hydrogel and revealed its unexpected anti-inflammatory properties,

challenging the conventional understanding of Ca²⁺-Alg as a pro-inflammatory material. Ca²⁺-Alg hydrogels are made by ionic cross-linking of Alg with Ca²⁺. The release of Ca²⁺ ions from these hydrogels is often immunostimulatory, promoting inflammatory responses both in vitro and in vivo [9]. The Alg molecule itself, at high M/G ratio (>1), has been reported to drive the immunostimulatory/inflammatory bioactivity of Alg hydrogels [21,22]. On macrophages, this inflammatory stimulation has been reported to involve TLR2 and 4 activation [23]. However, our results demonstrated that the developed Ca²⁺-hydrogel exhibits anti-inflammatory bioactivity despite having an M/G ratio of 1.5. This observed divergence could arise from cation type and the specific physicochemical properties of the hydrogel beyond the M/G ratio, such as alginate molecular weight (oligosaccharides vs polysaccharides), purity, and gelation (visco-elasticity). These physicochemical properties depend on, and influence, the concentration and type of cations that are bound and released. Our hydrogel is primarily Ca²⁺-Alg, whereas in the literature some are Na⁺-Alg or other cations. A combination of all these elements could explain the anti-inflammatory results with our hydrogel.

Our Ca²⁺-Alg hydrogel formulation selectively influenced the balance between classical and intermediate monocyte subsets, favoring the classical subset, and modulated macrophages from an M1 to an M2 phenotype. We also demonstrated that this hydrogel increased the mitochondrial mass of M1-macrophages decreasing their ROS production.

M2-macrophages that are typically associated with tissue repair and resolution of inflammation, exhibit higher mitochondrial mass compared to M1-like macrophages. This observation may indicate that the Ca²⁺-Alg hydrogel is not only influencing the activation state of M1-like macrophages, as previously observed in regard of both phenotype

<figure><img src="image_14.png" /><figcaption>**Fig. 5.** Ca²⁺-Alg hydrogel impact RDEB inflammatory monocytes. (A) Schematic representation of experimental design to evaluate the activity of Ca²⁺-Alg hydrogel on RDEB circulating monocytes compared to control medium. (B) UMAP visualizing FlowSOM-obtained clusters of RDEB monocytes subsets (n = 6). (C) Percentage of RDEB circulating monocytes subsets. (D, E) Bar graph comparing the expression of CD36 and CD64, respectively within circulating monocytes subsets. (F) Quantification of TNFα concentration in monocyte culture supernatant after LPS stimulation. Paired t-test, \*p < 0.05, \*\*p < 0.01.</figcaption></figure>
and cytokine secretion modulation, but could also promotes a metabolic shift, potentially fine-tuning M1 cells towards M2 macrophages. The reduction in ROS production further supports the notion that our hydrogel may impact on the global mitochondrial fitness of macrophages, possibly promoting a shift towards a more anti-inflammatory, tissue-regenerative phenotype. These findings align with emerging evidence suggesting that other Alg hydrogels, such as sulfated Alg-based hydrogels and melanin nanoparticle (MNP)/Alg hydrogels, also modulate the abundance and function of monocyte/macrophage subsets [24,25].

This study did not investigate the specific mechanisms or signaling pathways underlying the regulatory bioactivity of the newly developed Ca²⁺-Alg hydrogel. It is likely that the unique properties of our formulation—acting either together or separately—could influence key intracellular signaling pathways, such as PI3K/AKT, NF-κB/TLR, and JAK/STAT, which are often implicated in monocyte/macrophage behavior [26–28]. Future studies should employ global screening approaches to identify the key intracellular signaling pathways responsible for its unexpected bioactivity, followed by targeted experimental validation.

We recently demonstrated that adult patients with RDEB exhibit an unbalanced distribution of monocyte subsets, with a shift towards the inflammatory intermediate subset [29]. Herein, we used monocytes from these patients to assess the translational potential of the developed hydrogel. Specifically, we showed that our Ca²⁺-Alg hydrogel can restore a more physiological balance of RDEB monocyte subsets, increasing the classical at the expanse of intermediate subsets. Therefore, the developed hydrogel could be useful in restoring the homeostatic balance between steady-state and activated/inflammatory monocyte subsets in an inflammatory disease context.

This is the first study to report the intrinsic anti-inflammatory properties of Ca²⁺-Alg in its pure form, without the need for additional sulfation or incorporation of bioactive compounds. The viscosity profile of this hydrogel, which exhibits non-Newtonian behavior and decreases at higher shear rates, is typical of topical semisolid formulations, facilitating smoother skin application [30,31], further supporting this notion. Notwithstanding, the versatility of the Ca²⁺-Alg hydrogel also offers exciting opportunities for future development. Supplementing it with bioactive agents, such as ions, peptides, or other molecules, could enhance its therapeutic potential and expand its range of applications. While we acknowledge the limitations of this study, including the lack of direct comparisons with other anti-inflammatory agents and the absence of detailed mechanistic insights, our findings lay a solid foundation for future research into both the bioactivity and clinical applications of this hydrogel.

In conclusion, our findings highlight the anti-inflammatory potential of a novel Ca²⁺-Alg hydrogel, offering new insights into the design of biomaterials for immune modulation and tissue repair. Our study encourages future research into its mechanisms of action and therapeutic applications, advancing its potential for clinical translation in the management of inflammatory disorders.

### CRediT authorship contribution statement

**Nell Hirt:** Writing – original draft, Investigation, Data curation, Conceptualization. **Mansour Alkobtawi:** Validation, Project administration, Funding acquisition, Conceptualization. **Enzo Manchon:** Formal analysis, Data curation. **Richard Proust:** Project administration, Funding acquisition, Conceptualization. **Emeline Villain:** Resources. **Stéphane Lack:** Resources. **Emmanuelle Bourrat:** Resources. **Céline des Courtils:** Validation, Project administration, Funding acquisition, Conceptualization. **Jean-David Bouaziz:** Funding acquisition, Conceptualization. **Reem Al-Daccak:** Writing – review & editing, Writing – original draft, Validation, Supervision, Investigation, Funding acquisition, Formal analysis, Conceptualization.

### Declaration of competing interest

Mansour Alkobtawi, Richard Proust, Céline des Courtils, Emeline Villain and Stéphane Lack are employed by Laboratoires Brothier, all other authors declare no conflict of interest.

### Acknowledgements

Nell Hirt conducted her PhD studies with a CIFRE financial support between Association Nationale de la Recherche et de la Technologie (ANRT) and Laboratoires BROTHIER. The work was supported by Laboratoires Brothier, AO MARS 2020-la Société Française de Dermatologie, AP-RM-22-006-Fondation d'Avenir, and Inserm grants for UMRS976. The authors thank Niclas Setterblad (Technical platform – Institute de Recherche Saint-Louis).

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