A new biphasic microgel as lipoxin delivery system for tissue engineering – BILIPOX

Task 1.1 will be dedicated to synthesis of the microgels of Si-HPMC as LXA4 delivery system.
Si-HPMC will be obtained by the unique condensation reaction of alkoxysilane functions in physiological conditions of temperature and pH. An emulsion templating process will be applied using only the interfacial properties of HPMC without the use of any surfactant. The choice of oily core of BPMGs will be achieved as function of solubility and stability of lipoxin in vegetable injectable oils. The formulation and the conditions of the manufacture of BPMGs will be optimized in term of ratio oil/water and in term of pH to respectively optimize the lipoxin loading and to prevent the risk of its oxydation particularly in acidic pH. Stability of LXA4 may be improved by addition of antioxidants in lipophilic oily phase (a tocopherol).
Task 1.2 will evaluate chemical-physical characterizations and mechanical properties.
The surface and interfacial tension will be measured with a Krüss tensiometer K100 using the Wilhelmy plate method. The dynamic viscosity of the continuous phase will be measured with a coaxial cylinder rheometer. Study of the nanomechanical properties and the scanning topographically of surface will be done by atomic force microscope (AFM).
Task 1.3 will evaluate release profile and absence of degradation of LXA4.
The dosage of lipoxin will be achieved by liquid chromatography tandem mass spectrometry.
Task 2.1 will analyze response of macrophage polarization to LXA4-BPMGs of Si-HPMC in vitro.
To obtain Human macrophages from monocytes, we will benefit of the collaboration with Christophe BLANQUART CRCINA, INSERM UMR 1232. Human monocytes will be incubated with LXA4-BPMGs of Si-HPMC for three days during their differentiation in the presence of GM-CSF (M1-like macrophages) or M-CSF (M2-like macrophages). Characterization of macrophages will be assessed by flow cytometry using CD14, CD163, HLA-DR and CD16. IL-6, IL-10, IL-12, IL-1ß, TNFa, IL-23, IP-10, IL-1RA and TARC will be assessed by RT-PCR and multiplex ELISA using our collaboration with CRCINA, INSERM UMR 1232. TGFß secretion will be studied using commercial reporter cell line. Mix leukocyte reaction (MLR) experiments will be performed to confirm the phenotype of macrophage. To complete the study, differentiated M1 and M2 macrophages will be incubated with LXA4/BMP2-BPMGs of Si-HPMC during 3 days.
WP3 will be dedicated to evaluation of the impact of LXA4-BPMGs of Si-HPMC in two complementary and preclinical models of inflammation (dental pulp inflammation in rat) and (alveolar bone loss in minipig). The first in vivo model (rat) will target the resolution phase of inflammation whereas the second one will focus on the regenerative events following resolution of inflammation.

- We have developed a method to measure active products such as specialized pro-resolving mediator in oily components of BMPG
- We have developed optimized formulation of our BMPG compatible with human clinical use
- We have two different sorts of BPMG that could fit with different clinical needs
- We have developed 2D, 3D, and co-culture model of acute inflammation phase to test our BPMG
- We have developed a platform of macrophage polarization protocol that could be used to test other proposal and to consider other therapeutic proposal

This project will open perspectives for translation to human therapeutic for treatment of periodontitis in an original injectable drug delivery device. BILIPOX project will help to better understand the microgels/macrophage interaction and the impact of inflammation on oral stem cells. Using our original and established preclinical models of inflammation (pulpitis) and established model of alveolar bone loss, we will be able to generate new data about immunoregulation and tissue regeneration in the field of periodontology and endodontics.
Advantages of microgels over other delivery system for API are, first, the absence of species and/or the formation of bypass products (methacrylate derivatives, glutaraldehyde, carbodiimides, or di-halo species) during the cross linking reaction. Indeed, these products generated with other physical or chemical cross-linked hydrogels could interact with biological environment and disturb the cell. Our proposal may also be advantageous as compared with other drug delivery systems (such as synthetic nanoparticle) that have potential adverse immunotoxic effects. Then, methods for preparation and the functionalization of microgels are easily reproducible by our consortium. Si-HPMC are obtained by the unique condensation reaction of alkoxysilane functions in physiological conditions of temperature and pH. An emulsion templating process is applied using only the interfacial properties of HPMC without the use of any surfactant. Stability and release of oil droplet was be released after incubation in physiological conditions for 17 days.
Relevance of new therapeutic strategies using LXA4 over currently available anti-inflammatory drugs, such as anti–TNFa is that they would be able to dampen excessive inflammation without compromising host defence. Therapeutics that enhance resolution might have a better benefit/risk ratio.
Moreover, the strategy with this BPMG is modular. It has been demonstrated that it is possible to co- encapsulate both hydrophilic and hydrophobic drugs. Thus, in future experiment, we may combine co- encapsulation of LXA4 with other hydrophilic compound such Bone morphogenic protein 2 (BMP2).

1. Aubeux D, Peters OA, Hosseinpour S, Tessier S, Geoffroy V, Pérez F, Gaudin A. Specialized pro-resolving lipid mediators in endodontics: a narrative review. BMC Oral Health. 2021;21:276. doi:10.1186/s12903-021-01619-8
2. Aubeux D, Renard E, Pérez F, Tessier S, Geoffroy V, Gaudin A. Review of Animal Models to Study Pulp Inflammation. Front Dent Med. 2021;2 May:21. doi:10.3389/fdmed.2021.673552
1. Peters OA, Paranjpe A, Gaudin A. Dentine–Pulp Complex Regeneration. Regenerative Approaches in Dentistry 2021 Springer (Ed)
1. Poster presentation, congrès “2nd Resolution Days” LXA4 encapsulated in a new biphasic microgel attenuates in vitro inflammatory cytokine production.
2. Gaudin A. Modulation of the inflammatory response for dental pulp regeneration. Bruxelles 2020

The background of this project is the resolution of inflammation in clinical situations associated with widespread conditions such as pulpitis and periodontitis. Pulpitis (inflammation of the dental pulp tissue) can lead to very acute dental pain, which represents one of the main emergencies in dental practice, the most prevalent form of orofacial pain and may lead to overuse of opioid analgesic. Periodontitis (inflammation of the gums and supporting structures of the teeth) is the sixth-most prevalent disease in the world, the most common bone diseases in human and the primary cause for tooth loss in adults leading to significant economic and medical consequences. Although the tissues involved and the treatments are different, these two diseases share common characteristics. Uncontrolled or excessive inflammation can lead to pulp tissue lesions (pulpitis) and bone defects (periodontitis). Scarring and fibrosis in pulpitis or periodontitis prevent the return to homeostasis. As a matter of fact, healing, regeneration, and reconstruction of diseased tissues are significantly hampered. Extensive work over the past few decades has revealed that the resolution of acute inflammation is crucial to avoid persistent chronic inflammation and support repair or regeneration. Macrophages are considered the primary effector cells in regulating tissue repair. Moreover, the microenvironment of injured sites plays a vital role in the reprogramming of macrophages.
Recently, an emphasis has been placed on immune modulatory roles of lipoxins. Lipoxin A4 (LXA4) is an endogenous anti-inflammatory, pro-resolving molecules heralded that regulate functions of the innate immune system (attenuate monocyte recruitment, and induce a pro-resolving M2 phenotype) and also modulate the adaptive immune system by decreasing memory B-cell responses. The major challenge in using lipoxins or analogs is their hydrophobicity, chemical instability in acid and light along with shorter half-life.
In order to overcome these drawbacks, our proposal aims to co-encapsulate both hydrophilic and hydrophobic drugs in a new family of injectable and biphasic hydrogels of silylated (hydroxypropyl) methyl cellulose (Si-HPMC). Recent studies showed that LXA4 might induce and modulate M1 to M2 transition. However, the underlying mechanisms remain to be elucidated.
This project has a robust exploratory dimension by devising new therapeutics that enhance on one side resolution phase of inflammation and the other side regenerative mechanism. In vitro, and in vivo preclinical results of the BILIPOX project would be an important milestone for hydrogels modular strategy.
To address this issue, we propose a 3-year project divided in 1 organizational work package (WP0), and 3 experimental WPs (WP1 to WP3) with specific aims as described below:
WP 1: To prepare and analyze biphasic microgel of Si-HPMC as LXA4 delivery system in vitro
WP 2: To study the effect of LXA4-biphasic microgels of Si-HPMC on macrophage phenotype and under simulated in vitro inflammatory conditions.
WP 3: To assess the effect of LXA4-biphasic microgels of Si-HPMC in preclinical models of inflammation (pulpitis and periodontitis)
In this context, the BILIPOX project aims to provide and assess an optimized formulation for an innovative injectable biphasic microgel as LXA4 delivery system. The results of BILIPOX will provide a better understanding of microgels/macrophage interaction. This would be an important milestone for hydrogels modular strategy that open a new way to design and to multi-functionalize drug delivery systems. Our pharmacological approach will open perspectives for translation to human therapeutic by administration in dental pulp space or in periodontal bone lesion for treatment of pulpitis and periodontitis.