Cladophora glomerata enriched by biosorption with Mn(II) ions alleviates lipopolysaccharide‐induced osteomyelitis‐like model in MC3T3‐E1, and 4B12 osteoclastogenesis

Abstract Chronic osteomyelitis, a bone infectious disease, is characterized by dysregulation of bone homeostasis, which results in excessive bone resorption. Lipopolysaccharide (LPS) which is a gram‐negative endotoxin was shown to inhibit osteoblast differentiation and to induce apoptosis and osteoclasts formation in vitro. While effective therapy against bacteria‐induced bone destruction is quite limited, the investigation of potential drugs that restore down‐regulated osteoblast function remains a major goal in the prevention of bone destruction in infective bone diseases. This investigation aimed to rescue LPS‐induced MC3T3‐E1 pre‐osteoblastic cell line using the methanolic extract of Cladophora glomerata enriched with Mn(II) ions by biosorption. LPS‐induced MC3T3‐E1 cultures supplemented with C. glomerata methanolic extract were tested for expression of the main genes and microRNAs involved in the osteogenesis pathway using RT‐PCR. Moreover, osteoclastogenesis of 4B12 cells was also investigated by tartrate‐resistant acid phosphatase (TRAP) assay. Treatment with algal extract significantly restored LPS‐suppressed bone mineralization and the mRNA expression levels of osteoblast‐specific genes such as runt‐related transcription factor 2 (Runx2), alkaline phosphatase (ALP) and osteocalcin (OCN), osteopontin (OPN), miR‐27a and miR‐29b. The extract also inhibited osteoblast apoptosis, significantly restored the down‐regulated expression of Bcl‐2, and decreased the loss of MMP and reactive oxygen spices (ROS) production in MC3T3‐E1 cells induced by LPS. Furthermore, pre‐treatment with algal extract strongly decreased the activation of osteoclast in MC3T3‐E1‐4B12 coculture system stimulated by LPS. Our findings suggest that C. glomerata enriched with Mn(II) ions may be a potential raw material for the development of drug for preventing abnormal bone loss induced by LPS in bacteria‐induced bone osteomyelitis.


| INTRODUC TI ON
Bone is considered as being a highly dynamic tissue undergoing continuous remodelling cycles, consisting of bone neoformation trough osteoblast differentiation and osteoclast-induced resorption. Bone remodelling is a fundamental process for the reconstruction of fractured bones, adaptation of the skeleton to mechanical solicitations and calcium homeostasis. Bone replacement and neoformation basically respond to complex mechanisms that can be grouped into three main stages, with bone resorption initiation by osteoclasts, bone resorption transitioning to proper bone formation, and finally bone formation after osteoblasts differentiation. Excessive pathological bone loss often occurs when an imbalance between formation and bone resorption appears, leading to the development of many severe inflammatory conditions such as osteomyelitis, bacterial arthritis and infected orthopaedic implants. 1,2 Musculoskeletal infections, such as osteomyelitis, are among the most common and most challenging degenerative inflammatory diseases in clinical medicine. These are generally defined as an inflammatory process that could be limited to the bone and the joint, or can propagate to the bone marrow, the periosteum and the surrounding soft tissues, arising from the inoculation of one or more infectious organisms, and thus inducing significant destruction and necrosis of the local bone tissue, sequestrum formation and apposition of new bone tissue. 3,4 Bone infections are often accompanied by the formation of biofilms responsible for pathological bone resorption and reactive bone formation. These biofilms typically comprise a set of various soluble factors such as proteins, lipids, lipopolysaccharides (LPS) and DNA that are produced and secreted by the bacteria in order to form a protective matrix that will counter antibiotic and immune treatments, thus making the therapeutic management rather hazardous. 5 LPS, an endotoxin found in the outer membrane of all gram-negative bacteria, was one of the first bacterial components to be widely implicated in the process of bone resorption observed during infection. 6 LPS is known to trigger the release of many pro-inflammatory cytokines from neutrophils and macrophages such as interleukin-1α (IL-1α) and tumour necrosis factor-α (TNF-α), which subsequently initiate the inflammatory cascades that causes tissue damage and destruction. It has been proposed that released LPS from infected root canals could modulate the secretion of IL-1α and TNF-α from macrophages, and thus stimulate metalloproteinase-1 (MMP-1) synthesis that will in turn initiate dramatic bone resorption. 7 In addition, LPS application to bone marrow cell cultures has been shown to result in the generation of osteoclasts; furthermore, local injection of LPS in the femur resulted in a rapid and massive formation of osteoclasts as well as the expansion of eroded bone areas.
Osteoclast formation is a key step in the bone resorption process, as only osteoclasts have the ability to actively resorb while having a limited life span of two weeks at most. 8 The differentiation of osteoclasts initiated from a cluster of hematopoietic monocyte/macrophage-like stem cells responds to several systemic and local stimuli, such as hormones, cytokines, growth factors and eicosanoids. 9 The differentiation of osteoclasts takes place in several stages and is essentially characterized by the emergence of tartrate-resistant acid phosphatase-positive cells (TRAP), fusion into multinucleate cells, initiation of bone resorption and induction of spontaneous apoptosis. 10 The cytokine RANKL is an essential factor in the initiation and formation of osteoclasts. 11 Binding of RANKL to its receptor triggers the recruitment of the various adaptation factors associated with the TNF receptor (TRAF) and activates subsequently several downstream signal pathways, in particular those involving MAPKs, NF-κBs and PI3K/Akt. Then, activation of transcription factors such as activator protein 1 (AP-1) and activated T-cell cytoplasmic nuclear factor 1 (Nuclear Factor Of Activated T Cells 1) occurs. The recruitment of these downstream factors initiates the differentiation and functioning of osteoclasts by inducing the expression of specific genes, including TRAP, cathepsin K (CTSK) and matrix metalloproteinase 9 (MMP-9), thus resulting in the final formation of mature osteoclasts. 12,13 Manganese (Mn), an essential ubiquitous trace element, is known to be required for normal growth, development and cellular homeostasis, and is thus one of the most important minerals to bone tissue.
Physiologically, Mn is involved in protein metabolism and regeneration of the connective tissue. Moreover, it is closely associated with certain enzymatic activities such as superoxide dismutase (SOD) and Arginase, as well as metallothionein. Its main function is to activate various enzymes that control the metabolism of carbohydrates, proteins and lipids (including cholesterol) and nitrogen metabolism even in maintaining the normalization of the synthesis and secretion of insulin as well. 14,15 In the skeleton, manganese positively modulates RANKL/OPG ratio during bone formation, determining thus thickness of trabecular bone area and increasing trabecular number. 16 It has been reported that manganese deficiencies are at the origin of various bone malformations, stunted growth and impaired motor coordination, leading in the long term to osteoporosis development and to the occurrence of congenital disorders of the skeletal system, such as chondrodystrophy; the use of trace element supplementation such as manganese seems to be a plausible strategy for the management of bone homeostasis disorders. 17 The phyla of macroalgae have been widely recognized to bring a novelty and a diversity of chemical and pharmacological functional ingredients. In addition, algae are considered as real rich sources of various highly bioactive compounds found in marine resources such as polyphenols, pigments, vitamins, carbohydrates, proteins, lipids and minerals. 18 As one of the most common and important filamentous green algae in freshwater, Cladophora glomerata or commonly known as 'cotton-mat' or 'blanket weed' is attracting more and more attention not only because of its high nutritional value, but also because of its richness in various secondary metabolites giving it a high potential in therapeutics for the treatment of among others inflammation, oxidative stress and infectious diseases. 19,20 Recently, the concept of mutual potentiation of the therapeutic and physiological effects of natural substrates and trace elements has been introduced and is part of the new innovative perspectives in therapeutics. Thus, biosorption process can be applied in order to improve the bioavailability of the microelements for the animals while combining the nutritional and curative properties of the used biomasses. 21,22 In that context, the methanolic extract obtained from C. glomerata enriched with Mn (II) ions via biosorption process was applied to a model of   LPS-induced osteomyelitis on MC3T3 pre-osteoblasts cell line, as   well as on the osteoclastogenesis process induced on 4B12 cells in   the present investigation, with the goal of combining the beneficial   effects of manganese on bone metabolism, and the anti-inflamma-tory, antiapoptotic and antioxidant effects of C. glomerata extract.

| Chemicals
All chemicals and reagents were obtained from Sigma-Aldrich; cell culture reagents were purchased from Gibco BRL unless otherwise stated.

| Algal biomass
The biomass of freshwater macroalgae-C. glomerata, was collected from the surface of the pond in Tomaszówek, Łódź Province, Poland (51°27′21″N, 20°07′43″E) in October 2016. The study was carried out on private land, and the owner of the land gave permission to conduct the study on this site. No specific permissions were required for this location/activity. These studies did not involve endangered or protected species. Then, the biomass was air-dried and fine milled using grinding mills (Retsch GM 300).

| Biosorption process of Mn(II) ions by algal biomass
The biosorption of Mn(II) ions was carried out according to the procedure described by Michalak and Chojnacka. 23 In brief, the experiments were performed in Erlenmeyer flasks containing 500 mL of Poland SA). The initial pH of the solution was adjusted to 5, according to our previous studies, 23

| Extraction of the enriched with Mn(II) ions algal biomass | 7285
of CgMn or Mn(II) for 72 hours. To evaluate the effect of CgMn on LPSinduced pre-osteoblasts viability, cells were pre-treated with 0.5% and 1% of CgMn for 24 hours and then challenged with 1 µg/mL LPS for additional 24 hours in the absence of FBS. At the end of each related treatment, all media were removed and 100 µL of a 10% resazurin solution was added to each well. Cells were cultured for an additional 2 hours. Afterwards, absorbance was measured at the specific wavelengths: 600 nm for resazurin and 690 nm as a background absorbance using a microplate reader (Spectrostar Nano; BMG Labtech). The effect of the methanolic extract obtained from C. glomerata enriched with Mn(II) ions via biosorption process on metabolic activity of cells was expressed as mean of metabolized resazurin compared with that of blank that consists of resazurin with medium only.

| Evaluation of cell morphology
Changes in cellular morphology were evaluated using confocal micros- Samples were after that rinsed with Hank's balanced saline solution (HBSS); then, cell membranes were permeabilized using 0.1% Triton X-100 solution for 15 minutes at room temperature. Actin filaments were stained using atto-590-labelled phalloidin at dilution 1:800 with HBSS for 40 minutes, in the dark at room temperature. Nuclei were imaged by mean of diamidino-2-phenylindole (DAPI), using the ProLong™ Diamond Antifade Mountant with DAPI (Invitrogen™). Mitochondria were labelled using the Mito Red fluorescence dye diluted at 1:1000 in culture medium and incubated for 30 minutes at 37°C in a CO 2 incubator, prior to PFA fixation. Confocal microscope images were acquired as z-stacks having a z-interval of 15, 20 or 25 µm between two consecutive optical slices at a digital size of 512 × 512 pixels, and captured with a Canon PowerShot camera. Obtained photomicrographs were merged and analysed using ImageJ software.

| Detection of apoptosis by Annexin V labelling
The degree of apoptosis in LPS-induced MC3T3-E1 cell popula-

| Quantification of multicaspase activity
Multicaspases activity was quantified by using the Muse MultiCaspase assay kit (Merck Millipore). MC3T3-E1 cells were pretreated with 0.5% and 1% of methanolic extract from Mn(II)-enriched C. glomerata for 24 hours and exposed to 1 µg/mL of LPS, and multicaspase activity was subsequently assessed according to the manufacturer's instructions, using the Muse Cell Analyzer (Millipore).

| Determination of intracellular reactive oxygen species
Quantitative measurements of intracellular ROS namely Superoxide

| Coculture system of MC3T3-E1 with 4B12 cells
Transwell cell culture inserts (Greiner Bio-One) with 3 μm pore-size filters were placed in individual wells of 24-well plates. Briefly, 4B12 osteoclastogenic precursor cells (5 × 10 4 cells/well) were seeded and grown on the well plates; subsequently, pre-osteoblastic MC3T3-E1 cells (3 × 10 4 cells/mL) were seeded and grown on the transwell inserts on top. Pre-osteoblasts were then supplemented with the two concentrations of extract from Mn(II)-enriched C glomerate and challenged with LPS for osteoclastogenic factors production. Cocultures were thus maintained for 10 days in standard culture conditions. On the 11th day, inserts were discarded, and osteoclastogenic-4B12 differentiated cells were collected for TRAP staining, RT-PCR analysis and Phalloidin labelling as previously described for confocal evaluation. After washing-off the remaining fixing mixture with deionized water, cell nuclei were stained for 2 minutes using haematoxylin solution followed by rinsing in tap water. Stained samples were imaged using an inverted microscope (AxioObserverA1; Zeiss), and pictures were acquired using a Cannon PowerShot digital camera.

| Quantification of osteogenic and osteoclastogenic-related genes expression
Total RNA was isolated from MC3T3-E1 or 4B12 cells by using Trizol reagent (Sigma) as preconized by the supplier. RNA purity and con-

| miRNAs expression analysis
Total RNA was used to generate cDNA using a Mir-X miRNA First-

| Statistical analysis
Statistical analysis was performed using GraphPad Prism 5.0.
Statistical significance was determined using one-way analysis of variance (ANOVA) with Dunnett's post hoc multiple comparison test.

| Biosorption process of Mn(II) ions by algal biomass
The multielemental composition of the solution before and after biosorption of Mn(II) ions by C. glomerata is presented in Table 3.
Biosorption capacity (q; mg/g) was evaluated as the difference between the initial concentration (C 0 ; mg/L) and concentration of metal ions in the solution at time t-at equilibrium (C eq ; mg/L) divided per the content of the biomass in the solution (C S ; g/L). For and 20 times, respectively. This indicates that the main mechanism of the biosorption process is the ion exchange between metal ions in the solution (Mn(II) ions) and light metal ions naturally bound with functional groups of molecules constituting the algal cell wall. 23,27 The pH of the solution before biosorption process was 5.035 ± 0.007 and after biosorption process (after sorption equilibrium) 6.537 ± 0.036. This is in agreement with the results presented by Tang et al, 28 who observed an increase in pH (equilibrium pH higher than the initial pH) for the sorption of metal ions (Cu(II), Zn(II), Cd (II) and Pb(II)) by the algae-Oocysis sp and Chlorococcum sp. The increase of pH during the sorption of heavy metals may be explained by the ion-exchange mechanism according to which there is an exchange between the metal ions originally bound to the functional groups on the algal cell wall (Na(I), K(I) and Ca(II)) and weak basic heavy metal ions. This will lead to an increase in pH of the bulk solution from the corresponding shift in the hydrolysis equilibrium of the heavy metal ions.

| Extraction of the enriched with Mn(II) ions algal biomass
In Table 4, the multielemental composition of the methanolic extract obtained from the enriched with Mn(II) ions C. glomerata is presented.

| Effect of the methanolic extract from Mn(II)enriched C. glomerata on cellular metabolism and morphology in LPS-induced MC3T3-E1 Cells
The metabolic activity of MC3T3-E1 cells cultivated as a monoculture in the presence of extract as well as after LPS challenging has been calculated using a Resazurin-based assay ( Figure 1). Cell viability of pre-osteoblasts was not significantly affected in any of the three 0.5%, 1% and 2% groups; the increase in concentrations up to 4% and 8% of the Mn(II)-enriched C. glomerata extract significantly reduced cellular viability rate as compared to the group of untreated cells (P < .05), indicating that the extract probably exerts some cytotoxicity toward the tested cells ( Figure 1A). Therefore, the two 0.5% and 1% concentrations were selected for the further experiments due to the absence of obvious cytotoxic effects on MC3T3-E1 cells. TOX8 assays showed that cell metabolism in MC3T3-E1 cells   with one or more prominent nucleoli; and actin cytoskeleton network was more well defined, dense and homogeneously distributed, while less stress fibres were recorded ( Figure 1C). For further apoptosis assessment, total caspase activity was measured within the cells by flow cytometric-based assay. Figure 2C showed that, in absence of extract, LPS led to a marked activation of total cellular multicaspases (P < .001) of about 74.35 ± 1.58%,

F I G U R E 3
Mitochondrial membrane potential analysis. A, Scattered blots representation of live and dead depolarized cells percentages for one representative experiment. B, Bar charts represent the average percentages ± SD of total depolarization for three repetitions. C, MitoRed stained cells were observed using an inverted epi-fluorescent confocal microscope; scale bar size 20 µm; magnification was set at 60-fold. Asterisk (*) refers to comparison of all treated groups to untreated healthy cells. Hashtag (#) refers to comparison of Cladophora glomerata-treated groups to LPS-induced cells. */ # P < .05, **/ ## P < .01, ***/ ### P < .001

| Influence of Mn(II)-enriched C. glomerata methanolic extract on mitochondrial membrane potential depolarization in LPS-induced MC3T3-E1 cells
Mitochondria are known to play a key role to elicit apoptosis in response to many stresses, and the loss of mitochondrial membrane potential represents a hallmark for an early event in apoptosis induction. 29 In order to investigate whether mitochondrial dysfunctions are involved in LPS-induced apoptosis, a change of the mitochondrial membrane potential was analysed using a flow cytometric-based assay. Results in Figure 3B  were characterized by a significant reduction in the fluorescence intensity emitted by the dye (Figure 3C). was recorded for the group exposed to 0.5% extract, where the number of positive fluorescent cells appeared to be higher than that of the normal cells (P < .001), but still remained less than that of the stressed cells (P < .01). Concerning the group that received the clear Mn(II) ions, similar trend to that of the 0.5% extract group was observed ( Figure 4B).

| Effect of Mn(II)-enriched C glomerate methanolic extract on the mRNA expression of osteoblast-specific genes in LPSinduced MC3T3-E1 cells
To  Figure 5B).

| Effect of methanolic extract of Mn(II)-enriched C. glomerata on LPS-induced MC3T3-E1_4B12 coculture osteoclastogenic differentiation
To study the ability of LPS-induced osteoblasts to support osteoclast formation, MC3T3-E1 cells were induced by LPS to stimulate

| D ISCUSS I ON
Excessive bone resorption during chronic inflammatory pathologies, such as septic arthritis and osteomyelitis, is usually initiated following activation of bacterial-induced inflammatory responses. 30  including equine ASCs cells affected with metabolic syndrome that are known to be prone to significant apoptosis. 19,25 Recent studies have elucidated that normal mitochondrial function is importantly required for osteogenic differentiation, and its dysfunction has already been associated with impaired osteogenesis. [33][34][35] The toxic effect produced by LPS was demonstrated to be closely associated with mitochondrial dysfunction. 36 In this investigation, LPS induced dramatical MMP collapse in MC3T3-E1, as well as reduced  42 Manganese is a trace element specially required for MnSOD enzyme in order to reduce mitochondrial oxidative stress. Additionally, MnSOD is a primary antioxidant that scavenges superoxide formed within the mitochondria and protects against oxidative stress. In this prospect, it can be assumed that the biosorption process that was performed on the C. glomerata biomass, was effective in enhancing the biological properties of the algae, particularly in the improvement of the antioxidant status of the extract as well as the cells themselves. 15,43 The inhibitory effect of LPS on osteoblast differentiation pathway was investigated by evaluating mRNA expression levels of osteoblast marker genes. During the present investigation, LPS application was found to highly stimulate the production of the pro-osteoclastogenic factor RANKL, as well as miR- 16- of Fos proto-oncogene (c-Fos) and Nuclear Factor Of Activated T Cells 1, which is considered as the master regulator of osteoclast formation, that regulates a number of osteoclast specific genes such as TRAP, cathepsin K, calcitonin receptor and osteoclast-associated receptor (OSCAR). 59 TRAP, which is a di-iron-containing metalloenzyme largely expressed in osteoclasts, is subsequently found to be dramatically up-transcribed during osteoclast differentiation following Nuclear Factor Of Activated T Cells 1 recruitment to the TRAP promoter. 60 Although the present research has been able to demonstrate a promising effect of the C. glomerata

| CON CLUS ION
The present investigation showed the significant effectiveness of

CO N FLI C T O F I NTE R E S T S
Not applicable.

CO N S E NT FO R PU B LI C ATI O N
Not applicable.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data sets generated and/or analysed during the current study are presented in the article, the accompanying Source Data or