adipose tissue-derived stromal vascular fraction (SVF), which is known to be inherently composed of a heterogeneous cell population, consisting of the key cell types (as mesenchymal and endothelial/mural progenitor cells), capable of driving important paracrine-mediated cellular processes (e.g. angiogenesis, inflammation) [20-22]. The use of bioactive or inert hydrogel to deliver cells might totally change their paracrine effects and capability to directly interact with the host tissue. Among the recently developed hydrogels, those based on Elastin-Like Recombinamers (ELRs) stand out for their non-toxicity, non-immunogenicity, mechanical stiffness in the range of 1-10 kPa , and the functionalization possibility , offering a versatile cell- delivery platform for multiple tissue regeneration purposes. Moreover, the elastin integration in engineered tissues (e.g. heart valves, myocardium, skin, hyaline cartilage) is relevant for their regeneration, since it is one of the key ECM components and its production usually decreases during adulthood . In this work, we focus on investigating how the ELR-based hydrogels, in their inert or bioactivated form, could modulate the in vitro and in vivo angiogenic potential of human SVF cells. In particular, we hypothesized that the ELRs bioactivation, through the inclusion of functional RGD and REDV sequences, would promote celladhesion and enhance the SVF response in terms of in vitro angiogenic factor release and endothelial cell organization, also improving the in vivo angiogenic potential and integration with the host tissue.
Syndecan-4 and integrins are involved in the cell migration and adhesion processes in several cell types. Syndecan-4, a transmembrane heparan sulfate proteoglycan, is associated to focal adhesions in adherent cells and has been described as a marker of satellite cells in skeletal muscle. In this tissue, β1 integrin forms heterodimers with α5 and α6 during myoblast differentiation and with α7 in adult muscle. Here, we show that the levels of these two cell surface membrane molecules are regulated by spontaneous electrical activity during the differentiation of rat primary myoblasts. Syndecan-4 and β1 integrin protein levels decrease after the inhibition of electrical activity using tetrodotoxin (TTX). Syndecan-4 also decreases substantially in denervated rat tibialis anterior muscle. Indirect immuno ﬂ uorescence analysis shows that syndecan-4 and β 1 integrin co-localize with vinculin, a molecular marker of costameres in skeletal muscle myoﬁbers. Co- localization is lost in inactive myotubes adopting a diffuse pattern, suggesting that the costameric organization is disrupted in TTX-treated myotubes. Moreover, the inhibition of spontaneous electrical activity decreases myotube celladhesion. In summary, this work shows that syndecan-4 and β1 integrin protein levels and their localization in costameric structures are regulated by electrical activity and suggests that this regulatory mechanism inﬂuences the adhesion properties of skeletal myotubes during differentiation.
Background. VCAM-1 (soluble vascular celladhesion molecule-1) plays a role in liver angiogenesis. Hepato- cellular carcinoma (HCC) has important angiogenic activity, so expression of VCAM-1 may be pathogenic. Aim. To assess the association between serum VCAM-1 (sVCAM-1) levels and features of tumour and liver disease in patients with and without HCC, and to study the influence of HCC treatment on sVCAM-1 levels. Material and methods. Concentrations in peripheral (sVCAM-1-P) and hepatic (sVCAM-1-H) veins were analysed using ELISA in 134 consecutive patients with chronic liver disease between May 2004 and February 2006, who underwent a splanchnic haemodynamic study. Of these patients, 58 had HCC. Results. sVCAM-1-P and sVCAM-1-H were well correlated in both groups. No association was found between sVCAM-1-H and tumour features. No differences were observed in sVCAM-1-H between HCC and non-HCC cirrhotic patients. There was a significant linear association between Child-Pugh stage and sVCAM-1-H in HCC-patients (Child-Pugh A [2,485 ± 1,294 ng/mL] vs. Child-Pugh B [3,408 ± 1,338 ng/mL] vs. Child-Pugh C [4,096 ± 862 ng/ mL]; p = 0.007). Seven non-cirrhotic HCC patients had a significantly lower sVCAM-1-H than cirrhotic HCC patients. Treatment of HCC leads to an increase in sVCAM-1-H levels although this was not associated with the necrosis response to treatment. Conclusions. sVCAM-1 levels are more closely associated with the se- verity of underlying liver disease than with the presence of HCC. sVCAM-1 levels are not associated with tumour features or invasiveness; therefore, sVCAM-1 does not seem to play an important role in the angio- genic processes of HCC.
RGD bicycles suggest that these bicycles provide an interesting alternative to promote fast celladhesion on 2D biomaterial surfaces compared with well-known linear or monocyclic RGD peptides. However, our initial hypothesis that high-affinity integrin-binding RGD bicycles, as determined in solid-phase immunoassays , should improve integrin-mediated celladhesion and proliferation to a significantly greater extent than monocyclic and linear RGD was only partly confirmed. Furthermore, we have shown that covalent RGD-functionalization of ELRs via copper-free click reaction is more efficient for inducing integrin-mediated celladhesion and proliferation than the recombinant synthesis of ELRs comprising RGD as part of their backbone. This strategy could be used to ensure correct exposure of the bioactive sequence, thereby guaranteeing an optimal cell-material interaction. Finally, we believe that ELRs functionalized with integrin-selective RGD-bicycles represent an attractive and efficient way to design integrin-selective polymer surfaces with the potential to evaluate cell-adhesion behavior and tailor high integrin peptides for specific biomedical applications.
(A) Table of proteomics results of TRPC6 binding partners from wild type (WT) human podocytes overexpressing TRPC6-GFP. TRPC6 was seen to bind to TRPC3, TRPC7, and PLCy2, interactions that have been described previously. Novel interactions with calpain 2 and caldesmon-1 were identified. Podocytes expressing the GFP protein only were used as a control. (B) Interactions reported by proteomics were confirmed in TRPC6 KO cells expressing WT TRPC6-GFP (T6K + WT) by co- immunoprecipitation (TRAP lane). Control agarose beads were used to demonstrate immunoprecipitation was specific to TRPC6 (control lane). Additional interactions with calpain 1 and ERK 1/2 were also identified. (C) Based on the proteomics results the phosphorylation and/or cleavage state of focal adhesion kinase (FAK), talin-1, caldesmon-1 and ERK 1/2 was ascertained through Western blotting. T6K had increased FAK phosphorylation at Tyr 397 and decreased ERK phosphorylation compared to control and T6K + WT cells. Cleavage of FAK, talin-1 and caldesmon-1 was decreased in T6K cells compared to controls. For densitometry see supplementary figure 3.
However, the final list of genes comprised 86 probe sets (74 genes) commonly yielded by the two methodologies which were differentially expressed in LGMD2A compared to unaffected muscle biopsies. Of these 74 genes, 53 were overexpressed and 21 had a reduced expression in the LGMD2A patients and all the genes were clustered into functional groups (Table 2). Transcripts were classified according to different biological processes, as obtained from LocusLink (www.ncbi.nlm.nih.gov/LocusLink/): extracellular matrix proteins/phosphate transport, celladhesion, muscle development, transcription factors, signaling pathways, metabolic process, transport, ubiquitin cycle, and other functions. The additional supporting process, the Correlation-based Feature Subset selection (CFS)  highlighted a set of 21 genes. Of these 21 genes, 7 corresponded to the previously determined group of 74 genes. In turn, Symmetrical Uncertainty Ranking returned correlation coefficients higher than 0.5 for 24 genes within the list. Note that the highest correlation was 0.816 when the coefficient was constrained between 1 (maximum) and 0 (minimum). The average coefficient for the whole gene list was 0.36, with a standard deviation of 0.177.
lenalidomide (50 mg/kg, daily) or vehicle. As shown on ﬁgure 2e, after 3 weeks of treatment, tumors from lenalidomide-treated mice had a signiﬁcant (P ¼ 0.04) lower volume than tumors from the vehicle group. The immunohistochemistry analysis of repre- sentative tumor sections revealed that lenalidomide therapy efﬁciently reduced the hallmarks of bortezomib resistance and aggressiveness in REC-1 tumors, such as phospho-histone H3, IRF4, CD38, Blimp-1 and CCL3, and restored the expression of PAX5 (Figure 2f and Supplementary Figure S1B). Western blot analysis of representative whole tumor specimens, indicated that IRF4 and its target gene MYC experimented a 35% and a 85% protein decrease after lenalidomide therapy, respectively (Supplementary Figure S1E). In addition, the interferon-regulated genes CXCL10 and CXCL11 were the most downregulated genes in lenalidomide-receiving tumors among a panel of 90 genes related to immune regulation (data not shown), in agreement with previous ﬁndings in ABC-DLBCL cells. 20 Finally, lenalidomide was also able to reduce the length and number of intramural tumor blood vessels, as shown by a reduction in platelet endothelial celladhesion molecule-1 staining (Figure 2f and Supplementary Figure S1B). Thus, lenalidomide activity appeared to be stronger in MCL cell lines and tumors resistant to bortezomib, mediated by the downregulation of IRF4 and plasma-cell-related antigens and cytokines, and impaired angiogenesis.
Paxillin was undetectable (Figure 2B). These data sug- gest that cell-substratum adhesions and the formation of focal adhesions are highly dependent on overexpression of Hakai, probably due to the specific downregulation of Paxillin. Although Paxillin primarily functions as a molecular adapter or scaffold protein that provides mul- tiple docking sites at the plasma membrane, it is also important for the transduction of external signals to eli- cit changes in cell motility and modulate gene expres- sion by the various MAP kinase cascades. Paxillin binds to many proteins that are involved in effecting changes in the organization of the actin cytoskeleton, and are thus necessary for cell motility associated with tumor metastasis . Therefore, the complete absence of Pax- illin at focal adhesions may be one of the mechanisms that explain the loss of celladhesion to the substrate.
The possibility of employing ELR-RGD materials to im- prove the regeneration, or induce the differentiation, of endo- thelium , cartilage , bone [48, 54], muscle , pan- creas , or neuronal tissue  has been described. Inter- esting results have also been obtained in the case of epithe- lial oral tissue, for which conventional artificial 3D scaffolds are unable to sustain the potential proliferative capacity of oral epithelial cells during extended culture times. In order to improve celladhesion on the natural structural protein colla- gen, an ELR-RGD/collagen mixture was electrospun and cross-linked to fabricate a suitable highly porous epithelial 3D scaffold . The nanofiber 3D ELR scaffold is initially cultivated with human fibroblasts that are able to proliferate, infiltrate the network and neo-synthesize their own ECM, finally forming a lamina propria equivalent. Co-culture with oral epithelial cells seeded over this lamina equivalent gen- erates a non-keratinized, multilayered oral epithelial mucosal equivalent. The 3D hybrid scaffold is able to sustain in vitro culture for six weeks. Moreover, it was found to express the specific oral epithelial marker and is histologically very similar to the native version. In contrast, the collagen-only electrospun 3D scaffold used as negative control was poorly colonized by the cells and its resulting epithelium was thin- ner . Other types of epithelial cells have also been found to benefit if cultured on ELR-RGD as a substrate for subse- quent subretinal transplantation. Indeed, ELR-RGD supports are able to sustain the growth and maintain the phenotype, and functional characteristics of retinal pigment epithelial cells [58, 59].
increasing morbidity of bone fractures and defects due to changes in the age pyramid, and the limitations in the use of auto-, allo-, and xenografts. In order to assess this hypothesis, an ELR containing RGD (Arg-Gly-Asp) cell-adhesion sequences and another one fused to the osteogenic bone morphogenetic protein-2 (BMP-2) were used to form injectable physically cross-linked hydrogels. Moreover, elastase-sensitive domains were included in both ELR molecules, thereby conferring biodegradation as a result of enzymatic cleavage and avoiding the need for scaffold removal after bone regeneration. Both ELRs and their combination showed excellent in vitro cytocompatibility, and the culture of cells on RGD-containing ELRs resulted in optimal celladhesion. In addition, hydrogels based on a mixture of both ELRs were implanted in a pilot study involving a femoral bone injury model in New Zealand White rabbits, showing complete regeneration in six out of seven cases, with the other showing partial closure of the defect. Moreover, bone neo- formation was confirmed using different techniques, such as radiography, computed tomography and histology. Therefore, this hydrogel system therefore displays significant potential in the regeneration of bone defects, promoting self-regeneration by the surrounding tissue with no involvement of stem cells or osteogenic factors other than BMP-2, which is released in a controlled manner by elastase-mediated cleavage from the ELR backbone.
MB 231 cell line) response to epidermal grow factor gradients (Wang et al., 2004). Thus, the fabricated microfluidic gradient generator system has potential for many applications relating to various biomedical research. This research demonstrate that the system provides a way to conduct study and analysis on cell response to chemical gradients. In order to study celladhesion and cell migration studies have been shown that reflection interference contrast microscopy (RICM) could be used to do single cell observation (Laurent, 2009). Cells dynamics like spreading (Sengupta, 2006) and membrane fluctuations (Zidovska, 2011) had been studied with RICM previously.
A great variety of products are available to improve the moisture damage resistance of the mixtures. These products include the use of mineral fillers (mineral powders with physical size passing the 0.063 mm sieve) which have a decisive influence on the adhesion between the aggregate and the binder for two reasons . The first reason is that the mineral filler fill the voids in the bituminous mixtures, thereby preventing water entry. The second reason is that several mineral fillers exhibit a better chemical affinity with the bitumen than with the aggregate. In this regard, some authors indicate that the use of Portland cement as filler leads to an improvement in the water resistance of the mixture [8, 16]. Other authors stated that using fly ash as mineral filler also improves the water resistance of HMA [5, 16]. Nevertheless, hydrated lime is the most used anti-stripping agent, generally in percentages of 1% to 2% by dry weight of the aggregate [3, 5, 16-17].
Additional file 1: Figure S1. VHL, HIF, HDAC1 and related gene expression in renal tumor cell lines. Gene expression analysis of renal tumor cell lines using the Broad-Novartis cancer cell line encyclopedia show the levels of players in the VHL-HIF axis. The top row indicates the gene analyzed in different renal tumor cell lines (in the left most column). Red color indicates gene upregulation and blue color indicates downregulation of genes in the renal tumor cell lines. Figure S2. Hypoxia induces HDAC 1 expression in clear cell renal tumor cell line. a) Parental VHL null cells C2 and 786 – 0 were compared to cells with wt-VHL introduced for HIF-1 α , HIF-2 α and HDAC 1 protein expression. The left panel measures protein expression in C2 isogeneic cell lines and the right panel measures protein expression in 786 – 0 isogenic cell lines. The numbers below the bands represent densitometry performed by Image J analysis on representative immunoblots relative to their respective isogeneic VHL null cells with GAPDH serving as a loading control. b) HDAC 1 expression was compared between 786 – 0 and 786-0VHL cells under normoxic and hypoxic conditions (mimicked by the use of 100 μ M cobalt chloride) after overnight serum starvation. The numbers below the bands represent densitometry performed by Image J analysis on representative immunoblots relative to 786 – 0 bands in normoxic conditions with total GAPDH serving as loading control. c-d) HDAC 1 protein expression was quantitatively measured by flow cytometry under normoxic and hypoxic conditions. * p < 0.05 indicates statistically different HDAC 1 expression in wt-VHL cells as compared to VHL null cell lines. The error bars represent standard errors from biological triplicate experiments with technical replicates within each experiment. e) The figure is a representative image obtained from flow cytometry. The Y-axis represents HDAC 1 expression measured by FITC and Y-axis represents cell cycle measured by propidium idodide. Figure S3. Panobinostat and tamoxifen treatment increases acetylated α -tubulin in renal tumor cell lines. a-d) C2 and 786 – 0 cells were treated with 10 μ M hydroxy tamoxifen and/or 50nM panobinostat for 4 h. Representative immunofluorescence images of acetylated α -tubulin (in red), HDAC 6 (in green) and ER- α (in green) are shown. Fig. 7 Schema of HDACs, hypoxia inducible factors and ER- α in clear cell
• Background and Aims Cultured cell suspensions have been the preferred model to study the apoplast as well as to monitor metabolic and cell cycle-related changes. Previous work showed that methyl jasmonate (MeJA) inhibits leaf growth in a CORONATINE INSENSITIVE 1 (COI1)-dependent manner, with COI1 being the jasmonate (JA) receptor. Here, the effect of COI1 overexpression on the growth of stably transformed arabidopsis cell cultures is described. • Methods Time-course experiments were carried out to analyse gene expression, and protein and metabolite levels. • Key Results Both MeJA treatment and the overexpression of COI1 modify growth, by altering cell prolifer- ation and expansion. DNA content as well as transcript patterns of cell cycle and cell wall remodelling markers were altered. COI1 overexpression also increases the protein levels of OLIGOGALACTURONIDE OXIDASE 1, BETA-GLUCOSIDASE/ENDOGLUCANASES and POLYGALACTURONASE INHIBITING PROTEIN2, reinforcing the role of COI1 in mediating defence responses and highlighting a link between cell wall loosening and growth regulation. Moreover, changes in the levels of the primary metabolites alanine, serine and succinic acid of MeJA-treated Arabidopsis cell cultures were observed. In addition, COI1 overexpression positively affects the availability of metabolites such as β-alanine, threonic acid, putrescine, glucose and myo-inositol, thereby provid- ing a connection between JA-inhibited growth and stress responses.
Molecular simulation is becoming a very powerful tool for studying dynamic phenomena in materials. The simulation yields information about interaction at length and time scales unattainable by experimental mea- surements and unpredictable by continuum theories. This is especially meaningful when referring to bonding between a polymer and a metal substrate. A very important characteristic of polymers is that their physical properties do not rely on the detailed chemical structure of the molecular chains but only on their flexibility, and accordingly they will be able to adopt different conformations. In this paper, a molecular simulation of the bonding between vinyl ester polymer and steel is presented. Four different polymers with increasing chain lengths have been studied. Atomic co-ordinates are adjusted in order to reduce the molecular energy. Conformational changes in the macromolecules have been followed to obtain the polymer pair correlation function. Radius of gyration and end-to-end distance distributions of the individual chains have been used as a quantitative measurement of their flexibility. There exists a correlation between flexibility of the molecular chains and the energy of adhesion between the polymer and the metal substrate. Close contacts between the two materials are established at certain points but every atom up to a certain distance from the interface contributes to the total value of the adhesion energy of the system.
scribed by a decaying oscillation . The operation in discontinuous conduction mode can there- fore also influence the stability of the control system. This problem can be solved in three ways: Parametrization of the control system according to the critical minimum load , operation only in continuous conduction mode, or replacement of the diode with a transistor to allow bidirectional current flow in the freewheeling path (see chapter 2.2.3). To charge a lithium ion cell from a 12 V battery, the input voltage is 𝑉 1 = 12 𝑉 and 𝑉 2 represents the cell voltage at the output. An ideal buck converter can theoretically reach an efficiency of 100%. In reality, losses are defined by the non-ideal behavior of the components. These are mainly switching and conduction losses of the transistor, the ohmic resistances of the choke and printed circuit board (PCB) traces, equivalent series resistance of the capacitor, switching and conduction losses of the diode, and static losses of the control and feedback circuit. Thus, the reduction of losses is mainly achievable by parameter optimization or reduction of the switching frequency. For an unchanged current ripple, the induct- ance of the choke then must be increased.
The following reagents were purchased from Sigma Chemical Co. (St. Louis, MO, USA): trypan blue, 8-(4-chlorophenylthio)- 2-O-methyladenosine-3,5-cyclic monophosphate (Me-cAMP), N6- phenyladenosine-3,5-cyclic monophosphate (6-Bz-cAMP), antibodies to α -smooth muscle actin and β -tubulin, ERK inhibitor (PD98059), JNK inhibitor (SP600125), p38 inhibitor (SB202190) and ALK5 inhibitor (SB431542). Trypsin/EDTA, prestained molecular weight standard, fetal bovine serum (FBS) and fetal calf serum (FCS) were from GIBCO BRL (Carlsbad, CA, USA). Smad3 inhibitor (SiS3) and all organic and inorgan- ic compounds were purchased from Merck (Darmstadt, Germany). The enhanced chemiluminescence (ECL) reagent was acquired from Perkin Elmer Life Sciences, Inc. (Boston, MA, USA). Sterile plastic material was purchased from Falcon® (NJ, USA). The primary antibodies for EPAC-1, phospho-Smad2, phospho-ERK, phospho-JNK, phospho-p38, total-ERK, total-JNK, total-p38 and total-Smad2 were purchased from Cell Signal- ing Technology (Boston, MA, USA). The rabbit and mouse secondary antibodies were acquired from Calbiochem (Darmstadt, Germany). Accutase®, TGF- β 1 and pro-collagen antibody were purchased to Chemicon (Temecula, USA). Rat tail type I collagen was obtained from Santa Cruz Biotechnology Inc. (California, USA).
Tissue factor (TF) is a membrane protein, which, when in contact with blood, initiates the extrinsic pathway of the coagulation cascade by binding to Coagulation Factor VIIa (FVIIa) (Kasthuri et al., 2013; Henriquez et al.,2011). The complex TF-FVIIa promotes the formation of Coagulation Factor Xa (FXa) via proteolytic cleavage and the subsequent initiation of the common pathway of the coagulation cascade (Krupiczoic et al., 2008). TF is expressed in response to pro-inflammatory and pro-apoptotic factor exposure in endothelial cells and in the cell-microenvironment, like macrophages, neutrophils and fibroblasts. Additionally, Tissue Factor has been reported to be overexpressed in different tumor types (and in non-adherent cancer cells), suggesting a contribution to coagulation cascade activation in tumor biology. Moreover, chemotherapeutic drugs can stimulate the activation of the coagulation cascade by increasing TF levels or by decreasing Tissue Factor Protein Inhibitor-1 (TFPI-1), a protein that specifically binds TF acting as an inhibitor of the proteolytic activity of the TF-FVIIa complex (Haddad et al., 2006) Additionally, other mechanisms have been proposed associated to chemotherapy-procoagulant effect, including cellular stress and apoptosis of endothelial cells (Kirwan et al., 2015). Isolation of platelet and endothelial-derived microvesicles from melanoma patients showed a procoagulant potential compared to healthy subjects, suggesting an association of melanoma presence with thrombogenesis (Laresche et al., 2014).
with stroke outcome remained statistically significant The major limitation of the study is that treatment despite the inclusion in the models of powerful negative allocation was not randomly assigned. Therefore, we are outcome predictors such as stroke recurrence and bleeding very cautious when extrapolating the prognostic implica- complications. These findings are in discrepancy with most tions of these results. Nevertheless, we emphasize that both previous antithrombotic trials which consistently failed to treatment groups evidenced similar neurological impair- show a net clinical benefit for early anticoagulation in ment at hospital admission. The time to treatment onset acute ischemic stroke. We argue that the lack of agreement was also similar in both groups and the type of care given could mainly be due to the lower dose of UH selected in to all patients was not influenced by the antithrombotic previous clinical trials, the excessive delay to treatment regime. However, as a result of the unblinded nature of the onset, and the inclusion of a significant proportion of study we cannot totally exclude the possibility that the subjects with lacunar stroke. anticoagulated patients were more carefully monitored. Our results are better understood in light of recent Obviously, the only way to circumvent such a limitation is advances regarding the mechanisms of action of UH. In by performing a new clinical trial. Based on our results, we addition to its well described antithrombotic effects UH believe that such a clinical trial is fully warranted . Its also inhibits leukocyte rolling on the vessel wall , due design has been recently completed under the acronym to its ability to block selectins on leukocyte and platelets RAPID (rapid anticoagulation prevents ischemic damage) . Although the expression of adhesion molecules and, hopefully, it will be carried out in approximately forty seems to be unaffected, UH impairs the cell-adhesive centers from seven European countries. Bringing together interactions between leukocytes and endothelial cells . previous experimental data and the findings discussed in Recently, in vitro studies have also shown than UH this study, patients with lacunar stroke will not be in- attenuates the increase in inducible nitric oxide synthase cluded, and the delay to treatment onset will be minimized (iNOS) and NO release after cytokine activation . to optimize the ‘neuroprotective’ effects of high-dose UH. These findings suggest that UH influences the cytokine–
The resorption is a complex process where osteoclast membrane plays a fundamental role. In the membrane there is an area that attaches to the bone matrix and delimits the area to be resorbed. The bone area to be resorbed is exposed to an acidic environment, and then the matrix components are dissolved and endocytosed by the osteoclast 12–14 . This phase starts with the enrolment and spreading of osteoclast progenitors to bone which interact with osteoblast stromal cells and the result of such fusion are osteoclasts. After fusion, the αvβ3 integrin receptor binds to osteopontin at a tripeptide arginine- glycine-aspartic acid (RGD) recognition site. Therefore, osteoclasts are polarized 13,14,17 and suffer a rearrangement of the cytoskeleton which forms multiple focal adhesions. After this rearrangement, osteoclasts present a sealing zone, a ruffled border and a functional secretory domain. The sealing zone presents many focal adhesions that seal a zone beneath the cell in contact with bone matrix forming the resorption lacunae. After the sealing, the ruffled border secretes vacuolar-type H + -ATPase and cysteine protease cathepsin K. The purpose of the former is to acidify the lacunae to enable bone mineral dissolution and the purpose of the latter is to degrade organic phase.