Structure and the Anticancer Activity of Vitamin D Receptor Agonists.

Vitamin D is a group of seco-steroidal fat-soluble compounds. The two basic forms, vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol), do not have biological activity. They are converted in the body by a two-step enzymatic hydroxylation into biologically active forms, 1α,25-dihydroxyvitamin D2 [ercalcitriol, 1,25(OH)2D2] and 1α,25-dihydroxyvitamin D3 [calcitriol, 1,25(OH)2D3], which act as classical steroid hormones. 1,25(OH)2D3 exerts most of its physiological functions by binding to the nuclear vitamin D receptor (VDR), which is present in most body tissues to provide support to a broad range of physiological processes. Vitamin D-liganded VDR controls the expression of many genes. High levels of 1,25(OH)2D3 cause an increase in calcium in the blood, which can lead to harmful hypercalcemia. Several analogs of 1,25(OH)2D3 and 1,25(OH)2D2 have been designed and synthesized with the aim of developing compounds that have a specific therapeutic function, for example, with potent anticancer activity and a reduced toxic calcemic effect. Particular structural modifications to vitamin D analogs have led to increased anticancer activity and reduced calcemic action with the prospect of extending work to provide future innovative therapies.

Development of a film-forming oleogel with increased substantivity for the treatment of psoriasis.

The aim of this work was the development of a film-forming formulation (FFF) for the topical treatment of psoriasis that shows an increased substantivity compared to conventional semi-solid dosage forms. The developed formulation is an oleogel. It is based on a combination of castor oil and medium chain triglycerides, and the oil-soluble film former MP-30 (Croda GmbH, Nettetal, Germany), a polyamide that upon mixing with a polar oil entraps the oil und thus substantially increases the viscosity of the formulation up to a semisolid state. Betamethasone dipropionate (BDP) and calcipotriole (CA) were used as active pharmaceutical ingredients (APIs). Oleogels of different compositions were evaluated regarding substantivity, rheological properties, ex-vivo penetration into the skin and ex-vivo permeation through the skin. Marketed products were used as controls. It was found that the amount of betamethasone dipropionate penetrating and permeating into and through the skin from the film-forming formulation is at an intermediate value compared to the marketed products. The substantivity of the developed formulation is described by an amount of 57.7 % formulation that remains on the skin surface and is thus significantly higher compared to the marketed products. In the film forming formulation, the proportion of API penetrating the skin remains the same when the skin repetitively brought in contact with a piece of textile during the penetration experiment. In contrast with the in-market formulations tested, this proportion was reduced by up to 97 %. As a result, the developed formulations can lead to an increased patient compliance.

Design, synthesis, and biological activity of D-bishomo-1α,25-dihydroxyvitamin D3 analogs and their crystal structures with the vitamin D nuclear receptor.

The biologically active metabolite of vitamin D3 - calcitriol - is a hormone involved in the regulation of calcium-phosphate homeostasis, immunological processes and cell differentiation, being therefore essential for the proper functioning of the human body. This suggests many applications of this steroid in the treatment of diseases such as rickets, psoriasis and some cancers. Unfortunately, using therapeutic doses of calcitriol is associated with high concentrations of this compound which causes hypercalcemia. For this reason, new calcitriol analogs are constantly sought, devoid of calcemic effects but maintaining its beneficial properties. In this study, we present the synthesis of vitamin D derivatives characterized by an enlarged (seven-membered) ring D. Preparation of the designed vitamin D compounds required separate syntheses of crucial building blocks (C/D-rings fragments with side chain and rings A) which were combined by different methods, including Wittig-Horner reaction and Suzuki coupling. Biological activities of the target vitamin D analogs were assessed both in vitro and in vivo, demonstrating their significant potency compared to the natural hormone. Furthermore, the successful crystallization of these compounds with the vitamin D receptor (VDR) enabled us to investigate additional molecular interactions with this protein.

Efficiency of Gemini surfactant containing semi-rigid spacer as microbial corrosion inhibitor for carbon steel in simulated seawater.

SRB is one of the main bacteria causing marine microbial corrosion. In order to reduce the loss of microbial corrosion, a Gemini surfactant (12-B-12) containing semi-rigid spacer was used to investigate the anti-bacterial and anti-corrosion performances of carbon steel in simulated seawater by weight-loss test, electrochemical method and surface morphology analysis. The results showed that the inhibition efficiency of 0.01 mM 12-B-12 was as high as 98.3% after 30 days of incubation in simulated seawater with SRB, and the planktonic and sessile SRB on the carbon steel surface can be reduced to undetectable level. Quantum chemical calculation and molecular dynamics simulation were used to study the structure-activity relationship.

Photoresponsive transformation from spherical to nanotubular assemblies: anticancer drug delivery using macrocyclic cationic gemini amphiphiles.

We developed NIR-light-responsive macrocyclic cationic gemini amphiphiles, one of which displayed various favorable properties of lipids. The NIR-light-mediated cleavage of the strained dioxacycloundecine ring led to the conversion of the spherical to a nanotubular self-assembly in the aqueous medium. This photo-mediated transformation from the spherical to nanotubular self-assembly resulted in the release of encapsulated hydrophobic anticancer drug molecule doxorubicin (Dox) in a controlled manner. The potent cationic gemini amphiphile also displayed lower cytotoxicity and efficient NIR-light-mediated Dox release efficacy to cancerous cells.

Structure-Activity Relationship Study of an Alkynylphosphonate and Vynilphosphonate Analogues of Calcitriol.

BACKGROUND: 1α,25-dihydroxy vitamin D3 (calcitriol) shows potent growth-inhibitory properties on different cancer cell lines, but its hypercalcemic effects have severely hampered its therapeutic application. Therefore, it is important to develop synthetic calcitriol analogues that retain or even increase its antitumoral effects and lack hypercalcemic activity. Based on previous evidence of the potent antitumor effects of the synthetic alkynylphosphonate EM1 analogue, we have now synthesized a derivative called SG. OBJECTIVE: The aim of the present work is to evaluate the calcemic activity and the antitumor effect of SG, comparing these effects with those exerted by calcitriol and with those previously published for EM1. In addition, we propose to analyze by in silico studies, the chemical structure-biological function relationship of these molecules. METHODS: We performed the synthesis of vinylphosphonate SG analogue; in vitro assays on different cancer cell lines; in vivo assays on mice; and in silico assays applying computational molecular modeling. RESULTS: The SG compound lacks hypercalcemic activity, similar to the parent compound EM1. However, the antitumor activity was blunted, as no antiproliferative or anti-migratory effects were observed. By in silico assays, we demonstrated that SG analogue has a lower affinity for the VDRligand- binding domain than the EM1 compound due to lack of interaction with the important residues His305 and His397. CONCLUSION: These results demonstrate that the chemical modification in the lateral side chain of the SG analogue affects the antitumoral activity observed previously for EM1 but does not affect the calcemic activity. These results contribute to the rational design and synthesis of novel calcitriol analogues.

Gemini Curcumin Suppresses Proliferation of Ovarian Cancer OVCAR-3 Cells via Induction of Apoptosis.

BACKGROUND: Ovarian cancer has the highest mortality rate among gynecological malignancies. Despite recent advances in treatment, most patients still suffer from poor prognosis. Curcumin has shown highly cytotoxic effects against different types of cancer. However, its poor bioavailability restricts its clinical application. Gemini Curcumin (Gemini-Cur) has been developed to overcome this limitation. OBJECTIVE: Here, we aimed to unravel the inhibitory effect of Gemini-Cur in ovarian cancer. METHODS: OVCAR-3 cells were treated with free curcumin and Gemni-Cur in a time- and dose-dependent manner. Then, the anticancer activity was investigated by uptake kinetics, cellular viability and apoptotic assays. Furthermore, we evaluated the BAX/Bcl-2 expression ratio by real-time PCR and western blotting. RESULTS: Our data showed that gemini surfactant nanoparticles enhance the cellular uptake of curcumin compared to free curcumin (p<0.01). Regarding the growth inhibitory effect of nano-curcumin, the results demonstrated that Gemini-Cur suppresses the proliferation of OVCAR-3 cells through induction of apoptosis (p<0.001). CONCLUSION: The results illustrate that Gemini-Cur nanoparticles have a great potential for developing novel therapeutics against ovarian cancer.

Immunomodulatory Dual-Sized Microparticle System Conditions Human Antigen Presenting Cells Into a Tolerogenic Phenotype In Vitro and Inhibits Type 1 Diabetes-Specific Autoreactive T Cell Responses.

Current monotherapeutic agents fail to restore tolerance to self-antigens in autoimmune individuals without systemic immunosuppression. We hypothesized that a combinatorial drug formulation delivered by a poly-lactic-co-glycolic acid (PLGA) dual-sized microparticle (dMP) system would facilitate tunable drug delivery to elicit immune tolerance. Specifically, we utilized 30 µm MPs to provide local sustained release of granulocyte-macrophage colony-stimulating factor (GM-CSF) and transforming growth factor ß1 (TGF-ß1) along with 1 µm MPs to facilitate phagocytic uptake of encapsulated antigen and 1α,25(OH)2 Vitamin D3 (VD3) followed by tolerogenic antigen presentation. We previously demonstrated the dMP system ameliorated type 1 diabetes (T1D) and experimental autoimmune encephalomyelitis (EAE) in murine models. Here, we investigated the system's capacity to impact human cell activity in vitro to advance clinical translation. dMP treatment directly reduced T cell proliferation and inflammatory cytokine production. dMP delivery to monocytes and monocyte-derived dendritic cells (DCs) increased their expression of surface and intracellular anti-inflammatory mediators. In co-culture, dMP-treated DCs (dMP-DCs) reduced allogeneic T cell receptor (TCR) signaling and proliferation, while increasing PD-1 expression, IL-10 production, and regulatory T cell (Treg) frequency. To model antigen-specific activation and downstream function, we co-cultured TCR-engineered autoreactive T cell "avatars," with dMP-DCs or control DCs followed by ß-cell line (ßlox5) target cells. For G6PC2-specific CD8+ avatars (clone 32), dMP-DC exposure reduced Granzyme B and dampened cytotoxicity. GAD65-reactive CD4+ avatars (clone 4.13) exhibited an anergic/exhausted phenotype with dMP-DC presence. Collectively, these data suggest this dMP formulation conditions human antigen presenting cells toward a tolerogenic phenotype, inducing regulatory and suppressive T cell responses.

Protection from Ultraviolet Damage and Photocarcinogenesis by Vitamin D Compounds.

Exposure of skin cells to UV radiation results in DNA damage, which if inadequately repaired, may cause mutations. UV-induced DNA damage and reactive oxygen and nitrogen species also cause local and systemic suppression of the adaptive immune system. Together, these changes underpin the development of skin tumours. The hormone derived from vitamin D, calcitriol (1,25-dihydroxyvitamin D3) and other related compounds, working via the vitamin D receptor and at least in part through endoplasmic reticulum protein 57 (ERp57), reduce cyclobutane pyrimidine dimers and oxidative DNA damage in keratinocytes and other skin cell types after UV. Calcitriol and related compounds enhance DNA repair in keratinocytes, in part through decreased reactive oxygen species, increased p53 expression and/or activation, increased repair proteins and increased energy availability in the cell when calcitriol is present after UV exposure. There is mitochondrial damage in keratinocytes after UV. In the presence of calcitriol, but not vehicle, glycolysis is increased after UV, along with increased energy-conserving autophagy and changes consistent with enhanced mitophagy. Reduced DNA damage and reduced ROS/RNS should help reduce UV-induced immune suppression. Reduced UV immune suppression is observed after topical treatment with calcitriol and related compounds in hairless mice. These protective effects of calcitriol and related compounds presumably contribute to the observed reduction in skin tumour formation in mice after chronic exposure to UV followed by topical post-irradiation treatment with calcitriol and some, though not all, related compounds.

Differential Impact of Calcitriol and Its Analogs on Tumor Stroma in Young and Aged Ovariectomized Mice Bearing 4T1 Mammary Gland Cancer.

(1) Background: Vitamin D compounds (VDC) are extensively studied in the field of anticancer properties, including breast cancer. Previously, we showed that calcitriol and its analogs (PRI-2191 and PRI-2205) stimulate metastasis in 4T1 murine mammary gland cancer models in young mice, whereas the reverse effect was observed in aged ovariectomized (OVX) mice; (2) Methods: We determined the phenotype of monocytes/macrophages using FACS and examined the expression of selected genes and proteins by Real-Time PCR and ELISA; (3) Results: Activities of VDC are accompanied by an increase in the percentage of Ly6Clow anti-inflammatory monocytes in the spleen of young and a decrease in aged OVX mice. Treatment of young mice with VDC resulted in an increase of CCL2 plasma and tumor concentration and Arg1 in tumor. In later stage of tumor progression the expression of genes related to metastasis in lung tissue was decreased or increased, in old OVX or young mice, respectively; (4) Conclusions: Pro- or anti-metastatic effects of calcitriol and its analogs in young or aged OVX mice, respectively, can be attributed to the differences in the effects of VDC on the tumor microenvironment, as a consequence of differences in the immunity status of young and aged mice.

Design, synthesis and biological evaluation of novel 2-alkylidene 19-norcalcitriol analogs.

Continuing our studies aimed at A-ring modified vitamin D compounds, we designed novel 19-norcalcitriol derivatives bearing at C-2 pegylated chains of different lengths. The terminal fragments of these substituents contain hydroxyls or moieties possessing nitrogen and/or sulfur atoms capable of transition metal ions complexation. Also, two conjugate-type platinum(II) complexes of 19-norcalcitriol were obtained in which l-methionine served as chelating moiety. The convergent synthesis of the target 19-norcalcitriol analogs involved several steps with the crucial one being condensation of A-ring phosphine oxide and the known Grundmann ketone by Wittig-Horner reaction. Further elaboration of the 2-alkylidene substituent provided all final compounds which were then tested to determine their affinity for the vitamin D receptor and cytotoxic activity.

A sensitive "Switch-on" phosphorescent probe for ferrous iron quantification in drug and In vitro imaging of living cells.

Iron plays an important role in various physiological processes. However, the detailed biological functions of iron have not been sufficiently explored because of a lack of effective methods to monitoring iron, especially the labile ferrous ion (Fe2+). In the current study, a novel turn-on phosphorescent probe for Fe2+ quantification and visualization has been proposed based on the hybrid nanocomposite of manganese dioxide and gemini iridium complex (MnO2-GM-Ir). The surfactant-like GM-Ir with positive charges was beneficial to combine with the negatively charged manganese dioxide (MnO2) nanosheets, and thus endowing the MnO2-GM-Ir nanocomposite excellent dispersion ability in the water as well as efficiently avoiding the interference to the detection caused by the agglomeration of nanocomposite. Phosphorescence of GM-Ir was effectively quenched by MnO2 nanosheets through fluorescence resonance energy transfer (FRET) and the inner filter effect (IFE), while the phosphorescence could be significantly recovered in the presence of Fe2+via a selective Fe2+-mediated reduction of MnO2 nanosheets, indicating a highly-specific selectivity towards Fe2+ with a low detection limit (80 nM). The drug test assay and in vitro imaging studies further proved that the MnO2-GM-Ir nanocomposite could be employed as a promising probe for the quantitative detection of exogenous Fe2+ in drug and in vitro imaging of living cells.

Antiproliferative activity of side-chain truncated vitamin D analogs (PRI-1203 and PRI-1204) against human malignant melanoma cell lines.

Vitamin D compounds are versatile molecules widely considered as promising agents in cancer prevention and treatment, including melanoma. Previously we investigated series of double point modified vitamin D2 analogs as well as non-calcemic 20S-hydroxyvitamin D3 and 21-hydroxypregnacalciferol as to their anti-melanoma activity. Surprisingly, short side-chain vitamin D analogs were found to be biologically active compounds. Thus, here we tested novel derivatives of pregnacalciferol with an additional hydroxyl at the end of the truncated side chain, PRI-1203 and PRI-1204, as to their potency against human melanoma A375 and RPMI7951 cell lines. Tested compounds are geometric isomers, with 19-methylene positioned in PRI-1203 like in a calcitriol molecule, but reversed in the PRI-1204 analog to the (5E,7E) geometry (5,6-trans). We noticed a decrease in cells viability exerted by PRI-1203. The antiproliferative effect of PRI-1204 was very low, emphasizing the importance of the natural 19-methylene geometry in the PRI-1203. PRI-1203 was also effective in inhibition of A375 melanoma cells migration. PRI-1203, but not PRI-1204, increased the percentage of A375 and RPMI7951 melanoma cells in the G0/G1 phase of cell cycle, possibly in a p21 and p27 independent manner. Both, analogs have very low effect on the level of CYP24A1 mRNA, in comparison to active form of vitamin D - 1.25(OH)2D3. In addition, both tested compounds failed to elicit VDR translocation to the nucleus. Thus, it could be postulated that side chain shortening strongly affects binding of analogs to VDR and activation of genomic responses, however do not impair their antiproliferative activities.

Photophysical behavior of heme group: Unfolding of hemoglobin and myoglobin in the presence of Gemini surfactants of different molecular architectures.

Fluorescence studies were performed to determine the photophysical behavior of heme group in the presence of cationic Gemini surfactants of different architectures. Both hemoglobin and myoglobin were used to understand the heme group interactions with Gemini surfactants under the influence of temperature variation and were compared with homologous monomeric surfactants. The results were also supplemented from the size and zeta potential measurements of both proteins. Gemini surfactants showed marked effect on the unfolding behavior of hemoglobin that mainly contributed by the stronger hydrophobic interactions of double hydrocarbon chains as well as methylene spacer in the head group region with the hydrophobic domains of hemoglobin. Myoglobin with single polypeptide chain did not show similar unfolding behavior in the presence of Gemini surfactants rather it was readily solubilized in the surfactant solution and that too in the presence of monomeric surfactants rather than Gemini surfactants. The results highlighted the mechanistic aspects by which water soluble globular proteins interact with amphiphilic molecules of different functionalities and thus, helped to predict the interactions of both hemoglobin and myoglobin with the complex biological molecules possessing similar functionalities.

Amphiphilic gemini-iridium (III) complex for rapid and selective detection of picric acid in water and intracellular.

Inspired by the structure and properties of gemini surfactant, a novel amphiphilic gemini-iridium complex (GIC-Ir) has been developed, which can spontaneously form vesicles by self-assembly and exhibit excellent dispersibility and high emission intensity in water. The emission of GIC-Ir can be rapidly and selectively quenched by picric acid (PA) due to the aromatic groups and two long-chain quaternary ammonium (QA) groups with positive charge, which endow GIC-Ir vesicles outstanding capability to capture negatively charged PA, and greatly promote the interaction between GIC-Ir and PA. Theoretical calculations and spectral studied indicated that the photoinduced electron transfer and resonance energy transfer may be responsible to the emission quenching. Furthermore, the real water samples and in vitro studies further prove that GIC-Ir can be used as a promising chemosensor for the detection of PA both in water and intracellular.

Friction and Adsorption Properties of Oleic Acid-Based Gemini Amphiphile at Silica/Ester Oil Interfaces.

We characterized the friction and adsorption properties of an oleic acid-based gemini amphiphile having two carboxylic acid headgroups. We employed silica as a solid material, and diethyl sebacate and bis (2-ethylhexyl) sebacate as polar ester oils. Oleic acid and stearic acid were used as comparative amphiphilic materials. These amphiphiles were soluble in the ester oils, and the solubility of the gemini amphiphile was lower than that of the other two amphiphiles. Quartz crystal microbalance with dissipation monitoring measurements suggested that the gemini amphiphile had greater adsorption capability than the two comparative amphiphiles. The greater adsorption density of the gemini amphiphile resulted in the formation of a rigid interfacial film, as suggested by the normal force curves obtained by atomic force microscopy (AFM). We assessed the friction property of these systems using a ball-on-plate-type friction analyzer and by friction-mode AFM (friction force curve). These measurements confirmed that the gemini amphiphile had a smaller kinetic friction coefficient than that of the other two amphiphiles. These results suggest the potential of the gemini amphiphile as a friction modifier in polar oils.

Novel calcitriol analogue with an oxolane group: In vitro, in vivo, and in silico studies.

The active form of vitamin D3 , calcitriol, is a potent antiproliferative compound. However, when effective antitumor doses of calcitriol are used, hypercalcemic effects are observed, thus blocking its therapeutic application. To overcome this problem, structural analogues have been designed with the aim of retaining or even increasing the antitumor effects while decreasing its calcemic activity. This report aims at gaining insights into the structure-activity relationships of the novel oxolane-containing analogue, AM-27, recently synthesized. We herein demonstrate that this compound has antiproliferative and antimigratory effects in squamous cell carcinoma, glioblastoma, and breast cancer cell lines. Analyses of the mechanisms underlying the AM-27 effects on cell viability revealed induction of apoptosis by the analogue. Importantly, nonmalignant cell lines were little or not affected by the compound. In addition, the analogue did not produce hypercalcemia in mice. Also, in silico studies involving docking and molecular dynamics techniques showed that AM-27 is able to bind to the human vitamin D receptor with a higher affinity than the natural ligand calcitriol, a feature that is mostly derived from an electrostatic interaction pattern. Altogether, the proapoptotic effect observed in cancer cells, the lack of calcemic activity in mice, and the differential effects in normal cells suggest the potential of AM-27 as a therapeutic compound for cancer treatment.

111In-Labeled Glycoprotein Nonmetastatic b (GPNMB) Targeted Gemini Surfactant-Based Nanoparticles against Melanoma: In Vitro Characterization and in Vivo Evaluation in Melanoma Mouse Xenograft Model.

Melanoma is a devastating form of skin cancer with high tendency to metastasis. This work addresses the development of new targeted nanoparticles that can be used for single-photon emission computed tomography (SPECT) imaging of melanoma. Melanoma-specific glycoprotein nonmetastatic b (GPNMB) antigen targeted and nontargeted gemini nanoparticles were prepared, characterized, and radiolabeled with 111In. 111In-labeled nanoparticles were composed of gemini surfactant grafted with monoclonal antibody Fab fragment that targeted GPNMB. Specific uptake of GPNMB-Fab was studied in six melanoma cell lines using flow cytometry. In vitro cellular uptake and internalization were studied using flow cytometry, confocal laser scanning microscopy, and radiometric techniques. Specific uptake of anti-GPNMB targeted nanoparticles was observed in GPNMB expressing cells, which was higher than low expressing or control cells. In vitro studies showed that conjugation of GPNMB targeted nanoparticles led to enhanced intracellular uptake of the nanodelivery system, which is critical for drug delivery. In vivo distribution of the nanoparticles was studied by microSPECT/CT imaging and ex vivo biodistribution. Tumor uptake was significantly higher ( p < 0.05) in nontargeted nanoparticles (5.47 ± 0.46%IA/cc) compared to GPNMB targeted nanoparticles (1.87 ± 0.27% ID/cc), which might be attributed to the high spleen uptake of the targeted formulation. These findings demonstrated that the radiolabeled gemini nanoparticles are promising for image-guided radiotherapy of melanoma. Formulation optimization is needed to improved tumor uptake and in vivo intracellular delivery for radiotherapeutic applications.

Without 1α-hydroxylation, the gene expression profile of 25(OH)D3 treatment overlaps deeply with that of 1,25(OH)2D3 in prostate cancer cells.

Recently, the antiproliferative action of 1,25(OH)2D3 (1,25D3), an active metabolite of vitamin D3, in the management of prostate cancer has been argued rigorously. In this study, we found that at a physiological concentration, 25(OH)D3 (25D3), the precursor of 1,25D3 and an inactive form of vitamin D because of its much weaker binding activity to the vitamin D receptor (VDR) compared with 1,25D3, had a gene expression profile similar to that of 1,25D3 in prostate cancer LNCaP cells. By immunocytochemistry, western blotting, and CYP27B1 and/or VDR knockdown by small interfering RNAs, we found that 10-7 M 25D3, which is within its uppermost physiological concentration in the bloodstream, induced VDR nuclear import and robustly activated its target genes in the virtual absence of CYP27B1 expression. Comprehensive microarray analyses verified 25D3 bioactivity, and we found that 25D3 target gene profiles largely matched those of 1,25D3, while the presence a small subset of 25D3- or 1,25D3-specific target genes was not excluded. These results indicated that 25D3 shares bioactivity with 1,25D3 without conversion to the latter. Metallothionein 2A was identified as a 1,25D3-specific repressive target gene, which might be a prerequisite for 1,25D3, but not 25D3, to exert its anti-proliferative action in LNCaP cells.

Differentiation of osteoclast precursors on gellan gum-based spongy-like hydrogels for bone tissue engineering.

Bone tissue engineering with cell-scaffold constructs has been attracting a lot of attention, in particular as a tool for the efficient guiding of new tissue formation. However, the majority of the current strategies used to evaluate novel biomaterials focus on osteoblasts and bone formation, while osteoclasts are often overlooked. Consequently, there is limited knowledge on the interaction between osteoclasts and biomaterials. In this study, the ability of spongy-like gellan gum and hydroxyapatite-reinforced gellan gum hydrogels to support osteoclastogenesis was investigated in vitro. First, the spongy-like gellan gum and hydroxyapatite-reinforced gellan gum hydrogels were characterized in terms of microstructure, water uptake and mechanical properties. Then, bone marrow cells isolated from the long bones of mice and cultured in spongy-like hydrogels were treated with 1,25-dihydroxyvitamin D3 to promote osteoclastogenesis. It was shown that the addition of HAp to spongy-like gellan gum hydrogels enables the formation of larger pores and thicker walls, promoting an increase in stiffness. Hydroxyapatite-reinforced spongy-like gellan gum hydrogels support the formation of the aggregates of tartrate-resistant acid phosphatase-stained cells and the expression of genes encoding DC-STAMP and Cathepsin K, suggesting the differentiation of bone marrow cells into pre-osteoclasts. The hydroxyapatite-reinforced spongy-like gellan gum hydrogels developed in this work show promise for future use in bone tissue scaffolding applications.