Three-dimensional Collagen Gel Research Articles

Deletion of Tgm2 suppresses BMP-mediated hepatocyte-to-cholangiocyte metaplasia in ductular reaction.

Transglutaminase 2 (Tgm2) plays an essential role in hepatic repair following prolonged toxic injury. During cholestatic liver injury, the intrahepatic cholangiocytes undergo dynamic tissue expansion and remodelling, referred to as ductular reaction (DR), which is crucial for liver regeneration. However, the molecular mechanisms governing the dynamics of active cells in DR are still largely unclear. Here, we generated Tgm2-knockout mice (Tgm2-/-) and Tgm2-CreERT2-Rosa26-mTmG flox/flox (Tgm2CreERT2-R26T/Gf/f) mice and performed a three-dimensional (3D) collagen gel culture of mouse hepatocytes to demonstrate how Tgm2 signalling is involved in DR to remodel intrahepatic cholangiocytes. Our results showed that the deletion of Tgm2 adversely affected the functionality and maturity of the proliferative cholangiocytes in DR, thus leading to more severe cholestasis during DDC-induced liver injury. Additionally, Tgm2 hepatocytes played a crucial role in the regulation of DR through metaplasia. We unveiled that Tgm2 regulated H3K4me3Q5ser via serotonin to promote BMP signalling activation to participate in DR. Besides, we revealed that the activation or inhibition of BMP signalling could promote or suppress the development and maturation of cholangiocytes in DDC-induced DR. Furthermore, our 3D collagen gel culture assay indicated that Tgm2 was vital for the development of cholangiocytes in vitro. Our results uncovered a considerable role of BMP signalling in controlling metaplasia of Tgm2 hepatocytes in DR and revealed the phenotypic plasticity of mature hepatocytes.

Exogenous interleukin-1 beta stimulation regulates equine tenocyte function and gene expression in three-dimensional culture which can be rescued by pharmacological inhibition of interleukin 1 receptor, but not nuclear factor kappa B, signaling

We investigated how Interleukin 1 beta (IL-1β) impacts equine tenocyte function and global gene expression in vitro and determined if these effects could be rescued by pharmacologically inhibiting nuclear factor-κB (NF-KB) or interleukin 1 signalling. Equine superficial digital flexor tenocytes were cultured in three-dimensional (3D) collagen gels and stimulated with IL-1β for two-weeks, with gel contraction and interleukin 6 (IL6) measured throughout and transcriptomic analysis performed at day 14. The impact of three NF-KB inhibitors on gel contraction and IL6 secretion were measured in 3D culture, with NF-KB-P65 nuclear translocation by immunofluorescence and gene expression by qPCR measured in two-dimensional (2D) monolayer culture. In addition, daily 3D gel contraction and transcriptomic analysis was performed on interleukin 1 receptor antagonist-treated 3D gels at day 14. IL-1β increased NF-KB-P65 nuclear translocation in 2D culture and IL6 secretion in 3D culture, but reduced daily tenocyte 3D gel contraction and impacted > 2500 genes at day 14, with enrichment for NF-KB signaling. Administering direct pharmacological inhibitors of NF-KB did reduce NF-KB-P65 nuclear translocation, but had no effect on 3D gel contraction or IL6 secretion in the presence of IL-1β. However, IL1Ra restored 3D gel contraction and partially rescued global gene expression. Tenocyte 3D gel contraction and gene expression is adversely impacted by IL-1β which can only be rescued by blockade of interleukin 1 receptor, but not NF-KB, signalling.

Improvement of the cell viability of hepatocytes cultured in three-dimensional collagen gels using pump-free perfusion driven by water level difference

Cell-containing collagen gels are one of the materials employed in tissue engineering and drug testing. A collagen gel is a useful three-dimensional (3D) scaffold that improves various cell functions compared to traditional two-dimensional plastic substrates. However, owing to poor nutrient availability, cells are not viable in thick collagen gels. Perfusion is an effective method for supplying nutrients to the gel. In this study, we maintained hepatocytes embedded in a 3D collagen gel using a simple pump-free perfusion cell culture system with ordinary cell culture products. Flow was generated by the difference in water level in the culture medium. Hepatocytes were found to be viable in a collagen gel of thickness 3.26 (± 0.16 S.E.)-mm for 3 days. In addition, hepatocytes had improved proliferation and gene expression related to liver function in a 3D collagen gel compared to a 2D culture dish. These findings indicate that our perfusion method is useful for investigating the cellular functions of 3D hydrogels.

Titanium nanotopography induces osteocyte lacunar-canalicular networks to strengthen osseointegration

Osteocyte network architecture is closely associated with bone turnover. The cellular mechanosensing system regulates osteocyte dendrite formation by enhancing focal adhesion. Therefore, titanium surface nanotopography might affect osteocyte network architecture and improve the peri-implant bone tissue quality, leading to strengthened osseointegration of bone-anchored implants. We aimed to investigate the effects of titanium nanosurfaces on the development of osteocyte lacunar-canalicular networks and osseointegration of dental implants. Alkaline etching created titanium nanosurfaces with anisotropically patterned dense nanospikes, superhydrophilicity, and hydroxyl groups. MLO-Y4 mouse osteocyte-like cells cultured on titanium nanosurfaces developed neuron-like dendrites with increased focal adhesion assembly and gap junctions. Maturation was promoted in osteocytes cultured on titanium nanosurfaces compared to cells cultured on machined or acid-etched micro-roughened titanium surfaces. Osteocytes cultured in type I three-dimensional collagen gels for seven days on nano-roughened titanium surfaces displayed well-developed interconnectivity with highly developed dendrites and gap junctions compared to the poor interconnectivity observed on the other titanium surfaces. Even if superhydrophilicity and hydroxyl groups were maintained, the loss of anisotropy-patterned nanospikes reduced expression of gap junction in osteocytes cultured on alkaline-etched titanium nanosurfaces. Four weeks after placing the titanium nanosurface implants in the upper jawbone of wild-type rats, osteocytes with numerous dendrites were found directly attached to the implant surface, forming well-developed lacunar-canalicular networks around the nano-roughened titanium implants. The osseointegration strength of the nano-roughened titanium implants was significantly higher than that of the micro-roughened titanium implants. These data indicate that titanium nanosurfaces promote osteocyte lacunar-canalicular network development via nanotopographical cues and strengthen osseointegration. Statement of significanceThe clinical stability of bone-anchoring implant devices is influenced by the bone quality. The osteocyte network potentially affects bone quality and is established by the three-dimensional (3D) connection of neuron-like dendrites of well-matured osteocytes within the bone matrix. No biomaterials are known to regulate formation of the osteocyte network. The present study provides the first demonstration that titanium nanosurfaces with nanospikes created by alkali-etching treatment enhance the 3D formation of osteocyte networks by promoting osteocyte dendrite formation and maturation by nanotopographic cues, leading to strengthened osseointegration of titanium implants. Osteocytes attached to the titanium nanosurfaces via numerous cellular projections. The success of osteocyte regulation by nanotechnology paves the way for development of epoch-making technologies to control bone quality.

Disulfiram Exerts Antifibrotic and Anti-Inflammatory Therapeutic Effects on Perimysial Orbital Fibroblasts in Graves' Orbitopathy.

Fibrosis of extraocular muscles (EOMs) is a marker of end-stage in Graves’ orbitopathy (GO). To determine the antifibrotic and anti-inflammatory therapeutic effects and the underlying molecular mechanisms of disulfiram (DSF) on perimysial orbital fibroblasts (pOFs) in a GO model in vitro, primary cultures of pOFs from eight patients with GO and six subjects without GO (NG) were established. CCK-8 and EdU assays, IF, qPCR, WB, three-dimensional collagen gel contraction assays, cell scratch experiments, and ELISAs were performed. After TGF-β1 stimulation of pOFs, the proliferation rate of the GO group but not the NG group increased significantly. DSF dose-dependently inhibited the proliferation, contraction, and migration of pOFs in the GO group. Additionally, DSF dose-dependently inhibited fibrosis and extracellular matrix production markers (FN1, COL1A1, α-SMA, CTGF) at the mRNA and protein levels. Furthermore, DSF mediates antifibrotic effects on GO pOFs partially through the ERK-Snail signaling pathway. In addition, DSF attenuated HA production and suppressed inflammatory chemokine molecule expression induced by TGF-β1 in GO pOFs. In this in vitro study, we demonstrate the inhibitory effect of DSF on pOFs fibrosis in GO, HA production, and inflammation. DSF may be a potential drug candidate for preventing and treating tissue fibrosis in GO.

Promotion of conjunctival fibroblast-mediated collagen gel contraction by mast cells through up-regulation of matrix metalloproteinase release and activation

Mast cells and conjunctival fibroblasts contribute to conjunctival wound healing and allergic ocular inflammation. The number of mast cells in the conjunctiva is increased in individuals with cicatricial fibrosis-causing ocular surface diseases and after glaucoma filtering surgery, suggesting that these cells may contribute to the scarring observed after such surgery. We studied the potential mechanism of fibroblast–mast cell interaction in the healing of conjunctival wounds using a three-dimensional collagen gel culture system. We found that mast cells derived from the bone marrow of mice embedded in a collagen gel did not induce gel contraction. However, an increase in mast cells was associated with increased collagen gel contraction mediated by mouse conjunctival fibroblasts. The extent of collagen degradation was not affected by the co-culture of mast cells and conjunctival fibroblasts. Gelatin zymography disclosed that mast cells increased the amounts of both the pro form of matrix metalloproteinase (MMP)–9 and the active form of MMP-2 in supernatants of conjunctival fibroblast cultures. Furthermore, the potentiating effect of mast cells on contraction of the collagen gel through conjunctival fibroblasts was attenuated by the addition of a synthetic MMP inhibitor. Thus, current results suggest that mast cells accelerate the conjunctival fibroblast-dependent contraction of collagen gel by increasing the release as well as activation of MMPs. Therefore, the interaction between mast cells and conjunctival fibroblasts may contribute to conjunctival scar formation after glaucoma filtering surgery.

Detecting long-range interactions between migrating cells

Chemotaxis enables cells to systematically approach distant targets that emit a diffusible guiding substance. However, the visual observation of an encounter between a cell and a target does not necessarily indicate the presence of a chemotactic approach mechanism, as even a blindly migrating cell can come across a target by chance. To distinguish between the chemotactic approach and blind migration, we present an objective method that is based on the analysis of time-lapse recorded cell migration trajectories: For each movement step of a cell relative to the position of a potential target, we compute a p value that quantifies the likelihood of the movement direction under the null-hypothesis of blind migration. The resulting distribution of p values, pooled over all recorded cell trajectories, is then compared to an ensemble of reference distributions in which the positions of targets are randomized. First, we validate our method with simulated data, demonstrating that it reliably detects the presence or absence of remote cell-cell interactions. In a second step, we apply the method to data from three-dimensional collagen gels, interspersed with highly migratory natural killer (NK) cells that were derived from two different human donors. We find for one of the donors an attractive interaction between the NK cells, pointing to a cooperative behavior of these immune cells. When adding nearly stationary K562 tumor cells to the system, we find a repulsive interaction between K562 and NK cells for one of the donors. By contrast, we find attractive interactions between NK cells and an IL-15-secreting variant of K562 tumor cells. We therefore speculate that NK cells find wild-type tumor cells only by chance, but are programmed to leave a target quickly after a close encounter. We provide a freely available Python implementation of our p value method that can serve as a general tool for detecting long-range interactions in collective systems of self-driven agents.

Targeting hepatocyte growth factor in epithelial\u2013stromal interactions in an in vitro experimental model of human periodontitis

Periodontitis is a chronic inflammatory disease leading to progressive connective tissue degradation and loss of the tooth-supporting bone. Clinical and experimental studies suggest that hepatocyte growth factor (HGF) is involved in the dysregulated fibroblast–epithelial cell interactions in periodontitis. The aim of this study was to explore effects of HGF to impact fibroblast-induced collagen degradation. A patient-derived experimental cell culture model of periodontitis was applied. Primary human epithelial cells and fibroblasts isolated from periodontitis-affected gingiva were co-cultured in a three-dimensional collagen gel. The effects of HGF neutralizing antibody on collagen gel degradation were tested and transcriptome analyses were performed. HGF neutralizing antibody attenuated collagen degradation and elicited expression changes of genes related to extracellular matrix (ECM) and cell adhesion, indicating that HGF signaling inhibition leads to extensive impact on cell–cell and cell–ECM interactions. Our study highlights a potential role of HGF in periodontitis. Antagonizing HGF signaling by a neutralizing antibody may represent a novel approach for periodontitis treatment.

Autocrine Hyaluronan Influences Sprouting and Lumen Formation During HUVEC Tubulogenesis In Vitro.

Although many studies have focused on a role for hyaluronan (HA) of interstitial extracellular matrix (presumably produced by non-vascular "stromal" cells) in regulating vascular growth, we herein examine the influence of "autocrine HA" produced by vascular endothelial cells themselves on tubulogenesis, using human umbilical vein endothelial cells (HUVECs) in angiogenic and vasculogenic three-dimensional collagen gel cultures. Relative to unstimulated controls, tubulogenic HUVECs upregulated HAS2 mRNA and increased the synthesis of cell-associated HA (but not HA secreted into media). Confocal microscopy/immunofluorescence on cultures fixed with neutral-buffered 10% formalin (NBF) revealed cytoplasmic HAS2 in HUVEC cords and tubes. Cultures fixed with NBF (with cetylpyridinium chloride added to retain HA), stained for HA using "affinity fluorescence" (biotinylated HA-binding protein with streptavidin-fluor), and viewed by confocal microscopy showed HA throughout tube lumens, but little/no HA on the abluminal sides of the tubes or in the surrounding collagen gel. Lumen formation in angiogenic and vasculogenic cultures was strongly suppressed by metabolic inhibitors of HA synthesis (mannose and 4-methylumbelliferone). Hyaluronidase strongly inhibited lumen formation in angiogenic cultures, but not in vasculogenic cultures (where developing lumens are not open to culture medium). Collectively, our results point to a role for autocrine, luminal HA in microvascular sprouting and lumen development. (J Histochem Cytochem 69: 415-428, 2021).

Sustained Delivery of SB-431542, a Type I Transforming Growth Factor Beta-1 Receptor Inhibitor, to Prevent Arthrofibrosis.

Fibrosis of the knee is a common disorder resulting from an aberrant wound healing response and is characterized by extracellular matrix deposition, joint contraction, and scar tissue formation. The principal regulator of the fibrotic cascade is transforming growth factor beta-1 (TGF-β1), a factor that induces rapid proliferation and differentiation of resident fibroblasts. In this study, we demonstrate successful inhibition of TGF-β1-driven myofibroblastic differentiation in human fibroblast-like synoviocytes using a small molecule TGF-β1 receptor inhibitor, SB-431542. We also demonstrate successful encapsulation of SB-431542 in poly(D,L-lactide-co-glycolide) (PLGA) as a potential prophylactic treatment for arthrofibrosis and characterize drug release and bioactivity in a three-dimensional collagen gel contraction assay. We assessed the effects of TGF-β1 and SB-431542 on cell proliferation and viability in monolayer cultures. Opposing dose-dependent trends were observed in cell proliferation, which increased in TGF-β1-treated cultures and decreased in SB-431542-treated cultures relative to control (p < 0.05). SB-431542 was not cytotoxic at the concentrations studied (0-50 μM) and inhibited TGF-β1-induced collagen gel contraction in a dose-dependent manner. Specifically, TGF-β1-treated gels contracted to 18% ± 1% of their initial surface area, while gels treated with TGF-β1 and ≥10 μM SB-431542 showed no evidence of contraction (p < 0.0001). Upon removal of the compound, all gels contracted to control levels after 44 h in culture, necessitating sustained delivery for prolonged inhibition. To this end, SB-431542 was encapsulated in PLGA microspheres (SBMS) that had an average diameter of 87.5 ± 24 μm and a loading capacity of 4.3 μg SB-431542 per milligram of SBMS. Functional assessment of SBMS revealed sustained inhibition of TGF-β1-induced gel contraction as well as hallmark features of myofibroblastic differentiation, including α-smooth muscle actin expression and connective tissue growth factor production. These results suggest that SB-431542 may be used to counter TGF-β1-driven events in the fibrotic cascade in the knee cartilage. Impact statement Arthrofibrosis is the most prevalent comorbidity resulting from orthopedic procedures such as total knee arthroplasty that is characterized by excess deposition and accumulation of extracellular matrix. Despite its prevalence, treatments are generally palliative, and there is no effective prophylactic therapy. We report that the small molecule transforming growth factor beta-1 (TGF-β1) receptor inhibitor, SB-431542, can inhibit the TGF-β1-driven myofibroblastic differentiation of fibroblast-like synoviocytes. To provide sustained inhibition, we explored the use of SB-laden microspheres as a prophylactic therapy in a three-dimensional contraction model of fibrosis and propose that such therapies will have the potential to improve the standard of care for arthrofibrosis.

Combined heterogeneity in cell size and deformability promotes cancer invasiveness.

Phenotypic heterogeneity is increasingly acknowledged to confer several advantages to cancer progression and drug resistance. Here, we probe the collective importance of heterogeneity in cell size and deformability in breast cancer invasion. A computational model of invasion of a heterogeneous cell aggregate predicts that combined heterogeneity in cell size and deformability enhances invasiveness of the whole population, with maximum invasiveness at intermediate cell-cell adhesion. We then show that small cells of varying deformability, a subpopulation predicted to be enriched at the invasive front, exhibit considerable overlap with the biophysical properties of cancer stem cells (CSCs). In MDA-MB-231 cells, these include CD44 hi CD24- mesenchymal CSCs, which are small and soft, and CD44 hi CD24+ hybrid CSCs, which exhibit a wide range of size and deformability. We validate our predictions by tracking the pattern of cell invasion from spheroids implanted in three-dimensional collagen gels, wherein we show temporal enrichment of CD44 hi cells at the invasive front. Collectively, our results illustrate the advantages imparted by biophysical heterogeneity in enhancing cancer invasiveness.This article has an associated First Person interview with the first author of the paper.

A conserved PI(4,5)P2-binding domain is critical for immune regulatory function of DOCK8.

DOCK8 is a Cdc42-specific guanine-nucleotide exchange factor that is essential for development and functions of various subsets of leukocytes in innate and acquired immune responses. Although DOCK8 plays a critical role in spatial control of Cdc42 activity during interstitial leukocyte migration, the mechanism remains unclear. We show that the DOCK homology region (DHR)-1 domain of DOCK8 binds specifically to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and is required for its recruitment to the plasma membrane. Structural and biochemical analyses reveal that DOCK8 DHR-1 domain consists of a C2 domain-like core with loops creating the upper surface pocket, where three basic residues are located for stereospecific recognition of phosphoinositides. Substitution of the two basic residues, K576 and R581, with alanine abolished PI(4,5)P2 binding in vitro, ablated the ability of DOCK8 to activate Cdc42 and support leukocyte migration in three-dimensional collagen gels. Dendritic cells carrying the mutation exhibited defective interstitial migration in vivo. Thus, our study uncovers a critical role of DOCK8 in coupling PI(4,5)P2 signaling with Cdc42 activation for immune regulation.

Peripheral sensory neurons promote angiogenesis in neurovascular models derived from hESCs.

In the adult tissues, blood vessels traverse the body with neurons side by side; and share common signaling molecules. Developmental studies on animal models have shown that peripheral sensory neurons (PSNs) secrete angiogenic factors and endothelial cells (ECs) secrete neurotrophic factors which contribute to their coexistence, thereby forming the peripheral neurovascular (PNV) unit. Despite the large number of studies showing that innervation and vascularization complement each other, the interaction between human PSNs and ECs is still largely unknown. To study this interaction and to evaluate if PSNs affect angiogenesis, we derived both PSNs and ECs from human embryonic stem cells (hESCs) and developed a co-culture system. Seeding the two cell types together showed that PSNs induced endothelial morphogenesis with formation of vessel-like structures (VLSs). The PSN precursors, neural crest stem cells also induced VLS formation in the co-culture system; however, to a lesser extent. This sheds new light on the in vitro angiogenic potential of these cell types. PSNs derived from hESCs are powerful tools for studying development and disease as human PSNs are inaccessible for in vitro assays. Our novel approach, with optimized media condition allowed for integrating hESC-derived PSNs with hESC-derived ECs in three-dimensional (3D) collagen gel for creating a completely humanised PNV model. This preliminary model showed that innervation improves the development of vascularized channels in vitro, and provides insight to the development of innervated 3D models in future.

Acquired contractile ability in human endometrial stromal cells by passive loading of cyclic tensile stretch

The uterus plays an important and unique role during pregnancy and is a dynamic organ subjected to mechanical stimuli. It has been reported that infertility occurs when the peristalsis is prevented, although its mechanisms remain unknown. In this study, we found that mechanical strain mimicking the peristaltic motion of the uterine smooth muscle layer enabled the endometrial stromal cells to acquire contractility. In order to mimic the peristalsis induced by uterine smooth muscle cells, cyclic tensile stretch was applied to human endometrial stromal cells. The results showed that the strained cells exerted greater contractility in three-dimensional collagen gels in the presence of oxytocin, due to up-regulated alpha-smooth muscle actin expression via the cAMP signaling pathway. These in vitro findings underscore the plasticity of the endometrial stromal cell phenotype and suggest the possibility of acquired contractility by these cells in vivo and its potential contribution to uterine contractile activity. This phenomenon may be a typical example of how a tissue passively acquires new contractile functions under mechanical stimulation from a neighboring tissue, enabling it to support the adjacent tissue’s functions.

Requirement for the collagen receptor Endo180 in collagen gel contraction mediated by corneal fibroblasts

The interaction of keratocytes with extracellular matrix components plays an important role in the maintenance of corneal transparency and shape as well as in the healing of corneal wounds. In particular, the interaction of these cells with collagen and cell-mediated collagen contraction contribute to wound closure. Endo180 is a receptor for collagen that mediates its cellular internalization. We have now examined the role of Endo180 in collagen contraction mediated by corneal fibroblasts (activated keratocytes). Antibodies to Endo180 inhibited the contractile activity of mouse corneal fibroblasts embedded in a three-dimensional collagen gel and cultured in the presence of serum, with this effect being both concentration and time dependent and essentially complete at an antibody concentration of 0.2 μg/ml. Whereas corneal fibroblasts cultured in a collagen gel manifested a flattened morphology with prominent stress fibers under control conditions, they showed a spindlelike shape with few stress fibers in the presence of antibodies to Endo180. Antibodies to Endo180 had no effect on the expression of α–smooth muscle actin or the extent of collagen degradation in collagen gel cultures of corneal fibroblasts. Immunohistofluorescence analysis did not detect the expression of Endo180 in the unwounded mouse cornea. However, Endo180 expression was detected in keratocytes migrating into the wound area at 3 days after a corneal incisional injury. Together, our results suggest that Endo180 is required for the contraction of collagen matrix mediated by corneal fibroblasts and that its expression in these cells may contribute to the healing of corneal stromal wounds.

Three-Dimensional Co-culture of Primary Human Osteocytes and Mature Human Osteoclasts in Collagen Gels.

Osteoclasts are pivotal cells for bone remodeling and their activity is coordinated by osteocytes that reside inside the bone matrix. In vitro co-cultures of osteocytes and osteoclasts are therefore advantageous to analyze the crosstalk between these cell species. In this study, primary osteocytes were isolated from human bone in a multistep isolation process and embedded into three-dimensional collagen gels. Mature human osteoclasts were generated by differentiation of human peripheral blood mononuclear cells (PBMCs). Different surfaces were tested for osteoclast formation: suspension dishes, collagen gels, and normal tissue culture polystyrene. After detachment from the surfaces, osteoclasts showed typical morphology and gene expression of osteoclast markers. Osteoclasts that were differentiated on collagen exhibited the highest osteoclast marker expression. Cocultivation of mature osteoclasts with osteocytes was performed in a transwell system, with osteocytes, embedded in collagen gels at the apical side and osteoclasts on the basal side of a porous polyethylen terephtalate membrane, which allowed the separate gene expression analysis for osteocytes and osteoclasts. After 7 days of co-culture both cell species showed their typical morphology, which is multinucleated giant cells for osteoclasts and star-shaped cells with dendritic extensions for osteocytes. Furthermore, osteoclast markers tartrate-resistant acid phosphatase, carbonic anhydrase II, and cathepsin K were detected both on gene expression and protein level in single and co-cultures. Osteocytes showed gene expression of typical osteocyte markers E11, sclerostin, dentin matrix protein 1, osteocalcin, and receptor activator of nuclear factor-κ ligand both in single and co-culture. Impact statement This study is the first to establish an in vitro bone model that contains both primary human osteocytes and primary human osteoclasts. Previous studies applied rodent osteocyte cell lines to examine the influence of osteocytes on osteoclast function. This model mimics the clinical situation better since osteocytes are postmitotic cells whose function might be different in primary state compared with a proliferating cell line. Furthermore, the co-culture model can be the basis for in vitro triple culture models involving osteoblasts as the third bone cell species.

Osteogenic cells form mineralized particles, a few μm in size, in a 3D collagen gel culture.

Osteogenic cells form mineralized matrices in vitro, as well as in vivo. The formation and shape of the mineralized matrices are highly regulated by the cells. In vitro formation of mineralized matrices by osteogenic cells can be a model for in vivo osteogenesis. In this study, using a three-dimensional (3D) collagen gel culture system, we developed a new in vitro model for the formation of mineralized particles, a few µm in size, by the osteogenic cells. Human osteosarcoma (HOS) cells formed spherical mineralized matrices (about 12 µm) at approximately 7 days when cultured with β-glycerophosphate (β-GP)-containing culture media on 2D tissue culture plates. Alternately, when they were cultured in a 3D collagen gel containing β-GP, they formed mineralized particles with about 1.7 µm in the gel at approximately 3 days. Calcium precipitation in the gel was evaluated by measuring the gel turbidity. This type of mineralization of HOS cells, which formed mineralized particles inside the gel, was also observed in a peptide-based hydrogel culture. The mineralized particles were completely diminished by inhibiting the activity of Pit-1, phosphate cotransporter, of the HOS cells. When mouse osteoblast-like MC3T3-E1 cells, which form large and flat mineralized matrices in 2D osteogenic conditions at approximately 3 weeks of culture, were cultured in a 3D collagen gel, they also formed mineralized particles in the gel, similar to those in HOS cells, at approximately 18 days. Thus, osteogenic cells cultured in the 3D collagen gel form mineralized particles over a shorter period, and the mineralization could be easily determined by gel turbidity. This 3D gel culture system of osteogenic cells acts as a useful model for cells forming particle-type mineralized matrices, and we assume that the mineralized particles in the 3D hydrogel are calcospherulites, which are derived from matrix vesicles secreted by osteogenic cells.

Synthesis and secretome release by human bone marrow mesenchymal stem cell spheroids within three-dimensional collagen hydrogels: Integrating experiments and modelling.

Myocardial infarction results in loss of cardiac cell types, inflammation, extracellular matrix (ECM) degradation, and fibrotic scar. Transplantation of bone marrow-derived mesenchymal stem cells (BM-MSCs) is being explored as they could differentiate into cardiomyocyte-like cells, integrate into host tissue, and enhance resident cell activity. The ability of these cells to restore lost ECM, remodel the inflammatory scar tissue, and repair the injured myocardium remains unexplored. We here elucidated the synthesis and deposition of ECM (e.g., elastin, sulfated glycosaminoglycans, hyaluronan, collagen type III, laminin, fibrillin, lysyl oxidase, and nitric oxide synthases), matrix metalloproteinases (MMPs) and their inhibitors (TIMPs), and other secretome (cytokines, chemokines, and growth factors) in adult human BM-MSC spheroid cultures within three-dimensional collagen gels. The roles of species-specific type I collagen and 5-azacytadine were assessed over a 28-day period. Results revealed that human collagen (but not rat-derived) suppressed MSC proliferation and survival, and MSCs synthesized and released a variety of ECM proteins and secretome over the 28 days. Matrix deposition is at least an order of magnitude lower than their release levels at every time point, most possibly due to elevated MMP levels and interleukins with a concomitant decrease in TIMPs. Matrix synthesis over the 28-day period was fitted to a competitive inhibition form of Michaelis-Menten kinetics, and the production and decay rates of ECM, MMPs, and TIMPs, along with the kinetic model parameters quantified. Such an integrated experimental and modelling approach would help elucidate the critical roles of various parameters (e.g., cell encapsulation and delivery vehicles) in stem cell-based transplantation therapies.

Depletion of Ras Suppressor-1 (RSU-1) promotes cell invasion of breast cancer cells through a compensatory upregulation of a truncated isoform

Extracellular matrix (ECM)-adhesion proteins and actin cytoskeleton are pivotal in cancer cell invasion. Ras Suppressor-1 (RSU-1), a cell-ECM adhesion protein that interacts with PINCH-1, thus being connected to Integrin Linked Kinase (ILK), alpha-parvin (PARVA), and actin cytoskeleton, is up-regulated in metastatic breast cancer (BC) samples. Apart from the originally-identified gene (RSU-1L), an alternatively-spliced isoform (RSU-1-X1) has been reported. We used non-invasive MCF-7 cells, expressing only RSU-1L, and highly invasive MDA-MB-231-LM2 expressing both isoforms and generated stable shRNA-transduced cells lacking RSU-1L, while the truncated RSU-1-X1 isoform was depleted by siRNA-mediated silencing. RSU-1L depletion in MCF-7 cells resulted in complete abrogation of tumor spheroid invasion in three-dimensional collagen gels, whereas it promoted MDA-MB-231-LM2 invasion, through a compensatory upregulation of RSU-1-X1. When RSU-1-X1 was also eliminated, RSU-1L-depletion-induced migration and invasion were drastically reduced being accompanied by reduced urokinase plasminogen activator expression. Protein expression analysis in 23 human BC samples corroborated our findings showing RSU-1L to be upregulated and RSU-1-X1 downregulated in metastatic samples. We demonstrate for the first time, that both RSU-1 isoforms promote invasion in vitro while RSU-1L elimination induces RSU-1-X1 upregulation to compensate for the loss. Hence, we propose that both isoforms should be blocked to effectively eliminate metastasis.

Depletion of glycosphingolipids induces excessive response of chondrocytes under mechanical stress

Glycosphingolipids (GSLs) are ubiquitous membrane components that play an indispensable role in maintaining chondrocyte homeostasis. To gain better insight into roles of GSLs, we studied the effects of GSL-deletion on the physiological responses of chondrocytes to mechanical stress. Mice lacking Ugcg gene (Ugcg-/-) were genetically generated to obtain GSL-deficient mice, and their chondrocytes from the joints were used for functional analyses in vitro culture experiments. The cells were seeded in a three-dimensional collagen gel and subjected to 5%, 10% or 16% cyclic tensile strain for either 3 or 24 h. The gene expressions of chondrocyte anabolic and catabolic factors, and the induction of Ca2+ signaling were analyzed. Our results revealed that chondrocytes derived from GSL-deficient mice exhibited an elevation in the expression of catabolic factors (ADAMTS-5, MMP-13) following the exposure to strain with amplitudes of 10%. Likewise, applying cyclic tensile strain with these amplitudes resulted in an increased Ca2+ oscillation ratio in chondrocytes from GSL-deficient as compared to the ratio from control mice. These results demonstrated that deletion of GSL stimulated the catabolic responses of chondrocytes to mechanical stress via the augmentation of the sensitivity to mechanical stress that may lead to the cartilage deterioration. These findings suggest that the regulation of the physiological responses of chondrocytes by GSLs could be a potential target in a therapeutic intervention in osteoarthritis.