Fibroblastic Shape Research Articles

A natural nanocomposite based on gelatin-collagen hydrogel and silk fibroin embedded with ZnFe LDH for biological applications

In this research, a novel hydrogel nanobiocomposite was developed by combining natural Collagen (Col) and Gelatin (Ge) biopolymers with Glutaraldehyde (GA), forming a cross-linked Col-Ge hydrogel. This hydrogel was further enhanced by adding Silk fibroin (SF) protein and ZnFe layered double hydroxide (LDH) for improved mechanical properties and antibacterial functionality. Various analytical techniques were employed to comprehensively characterize the structural features of the designed Col-Ge hydrogel/SF/ZnFe LDH nanocomposite. Biological tests, including MTT and hemolytic assays, demonstrated 95.25 % and 96.51 % cell viability for human skin fibroblast cells (Hu02) after 2 and 3 days. Also, in addition to non-toxicity, the fibroblast shape of Hu02 cells was entirely maintained. The nanocomposite exhibited only 4.83 %hemolytic activity on human RBCs, indicating excellent hemocompatibility. In inhibiting bacterial growth, it showed 27.51 % inhibition for E. coli and 16.8 % for S. aureus. These findings suggest that the Col-Ge hydrogel/SF/ZnFe LDH nanobiocomposite holds promise for biomedical applications.

Histological Evaluation of A unilateral Critical-Sized Mandibular Defect Reconstruction using human dental pulp stem cells by Light Microscope and Real-Time Quantitative PCR

Objective: To evaluate the osteogenic differentiation potential of human dental pulp stem cells (DPSCs) isolated from the dental pulp of third molar teeth in vitro cultures, and to evaluate the bone regenerative capacity of human dental pulp stem cells (DPSCs) when transplanted into a unilateral critical-sized bone defect in the mandibular bone in vivo after receiving a hydroxyapatite matrix and polylactic-polyglycolic acid (HA/PLGA) scaffold. Material and methods: A total of 18 mandibular defects were made, and three groups (each n = 6) were created. The first group: the transplanted DPSCs implanted in the critical-sized bone defect after receiving (HA/PLGA) scaffold. The second group received only (HA/PLGA) scaffold. The third group, which served as the control, had a critical-sized defect left empty. After characterization, Von Kossa [VK] and Alizarin red staining were employed to identify differentiated osteoblasts at the 14th and 21st days, and histological analyses, as well as polymerase chain reactions (PCR), were also used. Results: It showed that DPSCs had high proliferation potential and typical fibroblastic shape. Additionally, osteogenic differentiation of DPSCs was validated by morphological alterations, histological examination, and the expression of lineage-specific genes confirmed osteogenic differentiation of DPSCs. Conclusion: High proliferation potential and the capacity to differentiate into osteoblasts are two characteristics of DPSCs taken from impacted third molars.

Characterization of Epithelial Stem Cells and Mesenchymal Stem Cells from Human Hair Follicle

Hair follicle mesenchymal stem cells (HF-MSCs) and epithelial stem cells (HF-EpSCs) play a key role in hair growth and regeneration. The isolation, culture and characterization of HF-MSCs and HF-EpSCs will provide clues for in vitro tissue models and regenerative medicine applications. In this study, HF-MSCs and HF-EpSCs from the hair bulge were harvested using the tissue culture method. HF-MSCs exhibited a fibroblast shape and expressed highly the mesenchymal markers CD44, CD73, CD90 and CD105 and a lack of hematopoietic markers CD45, CD34, CD11b, CD19 and HLA-DR. They showed the potential for differentiation into adipogenic, osteogenic and chondrogenic lineages. HF-EpSCs possessed the keratinocyte shape and expressed typical markers for epithelial stem cells such as CD200, CK15 and CK19. The stemness marker CD49f was preferentially expressed in HF-EpSCs over HF-MSCs. Moreover, the expression of pluripotent factors including c-myc, oct-4, sox-2, klf-4 and nanog at the mRNA level was higher in HF-EpSCs compared to the expression in HF-MSCs. In conclusion, we successfully isolated mesenchymal and epithelial stem cells via culturing the hair follicle bulge tissues and preliminarily evaluated the stemness and plasticity of these two populations. This information will provide clues for further studies on hair regeneration in vitro and in vivo.

Self-assembly of gelatin and collagen in the polyvinyl alcohol substrate and its influence on cell adhesion, proliferation, shape, spreading and differentiation.

Culture substrates display profound influence on biological and developmental characteristic of cells cultured in vitro. This study investigates the influence of polyvinyl alcohol (PVA) substrates blended with different concentration of collagen or/and gelatin on the cell adhesion, proliferation, shape, spreading, and differentiation of stem cells. The collagen/gelatin blended PVA substrates were prepared by air drying. During drying, blended collagen or/and gelatin can self-assemble into macro-scale nucleated particles or branched fibrils in the PVA substrates that can be observed under the optical microscope. These collagen/gelatin blended substrates revealed different surface topography, z-average, roughness, surface adhesion and Young's modulus as examined by the atomic force microscope (AFM). The results of Fourier transform infrared spectroscopy (FTIR) analysis indicated that the absorption of amide I (1,600-1,700cm-1) and amide II (1,500-1,600cm-1) groups increased with increasing collagen and gelatin concentration blended and the potential of fibril formation. These collagen or/and gelatin blended PVA substrates showed enhanced NIH-3T3 fibroblast adhesion as comparing with the pure PVA, control tissue culture polystyrene, conventional collagen-coated and gelatin-coated wells. These highly adhesive PVA substrates also exhibit inhibited cell spreading and proliferation. It is also found that the shape of NIH-3T3 fibroblasts can be switched between oval, spindle and flattened shapes depending on the concentration of collagen or/and gelatin blended. For inductive differentiation of stem cells, it is found that number and ration of neural differentiation of rat cerebral cortical neural stem cells increase with the decreasing collagen concentration in the collagen-blended PVA substrates. Moreover, the PVA substrates blended with collagen or collagen and gelatin can efficiently support and conduct human pluripotent stem cells to differentiate into Oil-Red-O- and UCP-1-positive brown-adipocyte-like cells via ectodermal lineage without the addition of mitogenic factors. These results provide a useful and alternative platform for controlling cell behavior in vitro and may be helpful for future application in the field of regenerative medicine and tissue engineering.

COPA3 peptide supplementation alleviates the heat stress of chicken fibroblasts.

Heat stress inhibits cellular proliferation and differentiation through the production of reactive oxygen species. Under stress conditions, antioxidant drugs promote stable cellular function by reducing the stress level. We sought to demonstrate 9-mer disulfide dimer peptide (COPA3) supplementation stabilizes fibroblast proliferation and differentiation even under heat stress conditions. In our study, fibroblasts were assigned to two different groups based on the temperature, like 38°C group presented as Control - and 43°C group presented as Heat Stress-. Each group was subdivided into two groups depending upon COPA3 treatment, like 38°C + COPA3 group symbolized Control+ and the 43°C + COPA3 group symbolized as Heat Stress+. Heat stress was observed to decrease the fibroblast viability and function and resulted in alterations in the fibroblast shape and cytoskeleton structure. In contrast, COPA3 stabilized the fibroblast viability, shape, and function. Moreover, heat stress and COPA3 were found to have opposite actions with respect to energy production, which facilitates the stabilization of cellular functions by increasing the heat tolerance capacity. The gene expression levels of antioxidant and heat shock proteins were higher after heat stress. Additionally, heat stress promotes the mitogen-activated protein kinase/ extracellular signal-regulated kinase-nuclear factor erythroid 2-related factor 2 (MAPK/ERK-Nrf2). COPA3 maintained the MAPK/ERK-Nrf2 gene expressions that promote stable fibroblast proliferation, and differentiation as well as suppress apoptosis. These findings suggest that COPA3 supplementation increases the heat tolerance capacity, viability, and functional activity of fibroblasts.

Inflammatory and differentiation cellular response to calcium silicate cements: An in vitro study.

Inflammatory-regenerative cell interaction is believed to mediate hard tissue formation. This study aimed to investigate the interaction between human inflammatory monocytes with human regenerative fibroblasts after exposure to different calcium silicate materials. Human monocytes were cultured on three materials, polystyrene (PS), mineral trioxide aggregates (MTA) and biodentine (BD), in the presence or absence of lipopolysaccharide (LPS). Half of the monocyte-conditioned media (MoCM) of each group was used to analyse inflammatory cytokine secretion, namely TNF-α, IL-1β, IL-1RA and IL-6. The remaining MoCM was used to culture recipient fibroblasts, measuring the cell number (proliferation) and levels of alkaline phosphatase (differentiation) and lactic acid dehydrogenase (cytotoxicity). In absence of LPS, MTA was associated with higher secretion of TNF-α and lower secretion of IL-1β, while BD triggered higher secretions of both cytokines when both materials were compared to control (PS). When LPS was added, higher levels of all analysed cytokines were observed in the PS and BD groups, whereas for the MTA group, only TNF-α and IL-6 were increased. Fibroblasts responded differently to the MoCM from the different groups, revealing significant increases in proliferation and differentiation capacities, particularly when cultured in CM from monocytes exposed to MTA. The morphological evaluation revealed different patterns of fibroblast shape and spread in the different MoCM groups. Calcium silicate materials modulate the monocyte inflammatory response, which subsequently induce differential effects on the recipient fibroblasts. MTA appears to promote the secretion of prodifferentiation signals from the monocytes, which are received by fibroblasts, promoting their proliferation and differentiation. The model represents a promising tool to evaluate the interaction of different cells in response to different materials.

The EGF Motif With CXDXXXXYXCXC Sequence Suppresses Fibrosis in a Mouse Skin Wound Model.

Fibrosis is an essential process for wound healing, but excessive fibrosis, such as keloids and hypertrophic scars, can cause cosmetic and functional problems. These lesions are caused by abnormal deposition and shrinkage of collagen fibers. The light chain of FIX, a plasma protein essential for hemostasis, has the amino acid sequence CXDXXXXYXCXC in the EGF domain. Peptides containing this sequence inhibited stromal growth in a mouse transplant tumor model. In this study, the effect of the FIX light chain on wound healing was studied. A full-layer wound was made on the back of each mouse, and cDNA encoding the light chain of mouse FIX (F9-LC) in an expression vector was injected locally once each week using a non-viral vector. Histochemical analysis of the wound was then performed to assess the effects on wound healing. Moreover, the effect of F9-LC on fibroblasts was studied in vitro. Macroscopic observation showed that wounds with forced expression of F9-LC appeared flatter and had fewer wrinkles than control wounds. Tissue collagen staining and immunostaining revealed that administration of F9-LC suppressed collagen 1 and 3 deposition and decreased α-smooth muscle actin expression. Electron microscopy revealed sparse and disorganized collagen fibers in the F9-LC-treated mice. In experiments using fibroblasts, addition of a recombinant protein of the FIX light chain disrupted the typical spindle shape and alignment of fibroblasts. F9-LC is a new candidate for use in treatments to regulate excessive fibrosis and contraction in wound healing.

Regulation of cellular contractile force, shape and migration of fibroblasts by oncogenes and Histone deacetylase 6.

The capacity of cells to adhere to, exert forces upon and migrate through their surrounding environment governs tissue regeneration and cancer metastasis. The role of the physical contractile forces that cells exert in this process, and the underlying molecular mechanisms are not fully understood. We, therefore, aimed to clarify if the extracellular forces that cells exert on their environment and/or the intracellular forces that deform the cell nucleus, and the link between these forces, are defective in transformed and invasive fibroblasts, and to indicate the underlying molecular mechanism of control. Confocal, Epifluorescence and Traction force microscopy, followed by computational analysis, showed an increased maximum contractile force that cells apply on their environment and a decreased intracellular force on the cell nucleus in the invasive fibroblasts, as compared to normal control cells. Loss of HDAC6 activity by tubacin-treatment and siRNA-mediated HDAC6 knockdown also reversed the reduced size and more circular shape and defective migration of the transformed and invasive cells to normal. However, only tubacin-mediated, and not siRNA knockdown reversed the increased force of the invasive cells on their surrounding environment to normal, with no effects on nuclear forces. We observed that the forces on the environment and the nucleus were weakly positively correlated, with the exception of HDAC6 siRNA-treated cells, in which the correlation was weakly negative. The transformed and invasive fibroblasts showed an increased number and smaller cell-matrix adhesions than control, and neither tubacin-treatment, nor HDAC6 knockdown reversed this phenotype to normal, but instead increased it further. This highlights the possibility that the control of contractile force requires separate functions of HDAC6, than the control of cell adhesions, spreading and shape. These data are consistent with the possibility that defective force-transduction from the extracellular environment to the nucleus contributes to metastasis, via a mechanism that depends upon HDAC6. To our knowledge, our findings present the first correlation between the cellular forces that deforms the surrounding environment and the nucleus in fibroblasts, and it expands our understanding of how cells generate contractile forces that contribute to cell invasion and metastasis.

EFFICACY OF INTRALESIONAL VERAPAMIL HYDROCHLORIDE VERSUS INTRALESIONAL TRIAMCINOLONE ACETONIDE IN HYPERTROPHIC SCARS AND KELOIDS

Background: Keloids and hypertrophic scars are dermal fibro-proliferative disorders lead to disfigurement, pain and pruritus. Their management is still challenging as there is no universally accepted treatment regimen. Corticosteroid injections, most commonly triamcinolone acetonide, continue to play a major role in the management of keloids. Verapamil is a phenylalkylamine calcium channel blocker antiarrhythmic agent that has antifibrotic effect. Objective: To compare efficacy of intralesional verapamil hydrochloride and triamcinolone acetonide in hypertrophic scars and keloids. Patients and methods: Forty Egyptian patients with keloids or hypertrophic scars were divided into two equal groups. The patients were recruited from the Dermatology outpatient clinics of Al-Zahraa University Hospital during the period from May 2016 to May 2017. Informed written consents were obtained from all patients. Group A: Intralesional Verapamil Hydrochloride. Group B: Intralesional Triamcinolone Acetonide. The efficacy of treatment was evaluated by Digital photography and Vancouver scar scale before and after treatment. Two- fine millimeter punch biopsies were taken from 5 patients of the verapamil group before and after treatment to demonstrate the histopathological changes induced by verapamil. Results: Both drugs improved total Vancouver scar scale, vascularity score and pliability score of keloid or hypertrophic scar nearly equally with no statistical significant difference. Both drugs improved height of keloid or hypertrophic scar significantly, but triamcinolone showed better improvement. Verapamil highly improved pigmentation score of keloids and hypertrophic scars, but triamcinolone showed non- significant improvement. Side effects were reported in 4 patients of triamcinolone group with no side effects in verapamil group. The histopathological examination after treatment with verapamil injection showed marked reduction of collagen deposition and alteration of the fibroblast shape (from elongated to spherical), and these changes are similar to histopathological changes which occur after corticosteroids injection. Conclusion: Verapamil was among the several therapeutic modalities, have an option for keloids and hypertrophic scars with an extremely low cost and fewer adverse effects.

Heterogeneity and Remodeling of Ion Currents in Cultured Right Atrial Fibroblasts From Patients With Sinus Rhythm or Atrial Fibrillation.

Cardiac fibroblasts express multiple voltage-dependent ion channels. Even though fibroblasts do not generate action potentials, they may influence cardiac electrophysiology by electrical coupling via gap junctions with cardiomyocytes, and through fibrosis. Here, we investigate the electrophysiological phenotype of cultured fibroblasts from right atrial appendage tissue of patients with sinus rhythm (SR) or atrial fibrillation (AF). Using the patch-clamp technique in whole-cell mode, we observed steady-state outward currents exhibiting either no rectification or inward and/or outward rectification. The distributions of current patterns between fibroblasts from SR and AF patients were not significantly different. In response to depolarizing voltage pulses, we measured transient outward currents with fast and slow activation kinetics, an outward background current, and an inward current with a potential-dependence resembling that of L-type Ca2+ channels. In cell-attached patch-clamp mode, large amplitude, paxilline-sensitive single channel openings were found in ≈65% of SR and ∼38% of AF fibroblasts, suggesting the presence of “big conductance Ca2+-activated K+ (BKCa)” channels. The open probability of BKCa was significantly lower in AF than in SR fibroblasts. When cultured in the presence of paxilline, the shape of fibroblasts became wider and less spindle-like. Our data confirm previous findings on cardiac fibroblast electrophysiology and extend them by illustrating differential channel expression in human atrial fibroblasts from SR and AF tissue.

Enhanced osteogenic differentiation of alendronate-conjugated nanodiamonds for potential osteoporosis treatment

BackgroundAlendronate (Alen) is promising material used for bone-targeted drug delivery due to its high bone affinity and therapeutic effects on bone diseases. In addition, Alen can enhance the osteogenic differentiation of osteoblastic cell. Recently, nanodiamonds (NDs) with hardness, non-toxicity, and excellent biocompatibility are employed as promising materials for carrier systems and osteogenic differentiation. Therefore, we prepared Alen-conjugated NDs (Alen-NDs) and evaluated their osteogenic differentiation performances.MethodsAlen-NDs were synthesized using DMTMM as a coupling reagent. Morphological change of Mouse calvaria-derived preosteoblast (MC3T3-E1) treated with Alen-NDs was observed using the confocal microscope. The osteogenic differentiation was confirmed by cell proliferation, alkaline phosphatase (ALP), calcium deposition, and real-time polymerase chain reaction assay.ResultsAlen-NDs were prepared to evaluate their effect on the proliferation and differentiation of osteoblastic MC3T3-E1 cells. The Alen-NDs had a size of about 100 nm, and no cytotoxicity at less than 100 μg/mL of concentration. The treatment of NDs and Alen-NDs reduced the proliferation rate of MC3T3-E1 cells without cell death. Confocal microscopy images confirmed that the treatment of NDs and Alen-NDs changed the cellular morphology from a fibroblastic shape to a cuboidal shape. Flow cytometry, alkaline phosphatase (ALP) activity, calcium deposition, and real-time polymerase chain reaction (RT-PCR) confirmed the higher differentiation of MC3T3-E1 cells treated by Alen-NDs, compared to the groups treated by osteogenic medium and NDs. The higher concentration of Alen-ND treated in MC3T3-E1 resulted in a higher differentiation level.ConclusionsAlen-NDs can be used as potential therapeutic agents for osteoporosis treatment by inducing osteogenic differentiation.

Control of fibroblast shape in sequentially formed 3D hybrid hydrogels regulates cellular responses to microenvironmental cues

Cell shape plays important roles in regulating cell behavior; however, independently controlling cell shape in three dimensions is a challenging undertaking, and how cell shape affects cellular responses to mechanical and biochemical cues in three dimensions remains unclear. Here, we present a hydrogel-based platform to control cell shape in three dimensions by using sequentially formed hybrid hydrogels consisting of collagen and alginate. By adjusting the cross-linking time of the alginate, we fixed the shape of NIH 3T3 fibroblasts at different spreading states. Then, we explored the influence of cell shape on the cell responses to microenvironmental cues by using cardiac fibroblasts (CFs) as model cells. We found that the spreading state of the CFs influences their responses to both mechanical (i.e., matrix stiffness) and biochemical (i.e., transforming growth factor-β1 (TGF-β1)) cues in three dimensions. Additional experiments revealed that integrin β1 in focal adhesions and Smad2/3 are involved in mediating the cell shape-dependent responses of CFs to matrix stiffness and TGF-β1 cues, respectively. This work represents the first step in understanding how cell shape influences cell responses to mechanical and biochemical cues in three dimensions and can be instructive for developing novel approaches to target cell shape regulation for treating fibrosis and other diseases.

Adaptation to chronic acidic extracellular pH elicits a sustained increase in lung cancer cell invasion and metastasis

Acidic extracellular pH (pHe) is an important microenvironment for cancer cells. This study assessed whether adaptation to acidic pHe enhances the metastatic phenotype of tumor cells. The low metastatic variant of Lewis lung carcinoma (LLCm1) cells were subjected to stepwise acidification, establishing acidic pHe-adapted (LLCm1A) cells growing exponentially at pH 6.2. These LLCm1A cells showed increased production of matrix metalloproteinases (MMPs), including MMP-2, -3, -9, and -13, and pulmonary metastasis following injection into mouse tail veins. Although LLCm1A cells exhibited a fibroblastic shape, keratin-5 expression was increased and α-smooth muscle actin expression was reduced. Despite serial passage of these cells at pH 7.4, high invasive activity through Matrigel® was sustained for at least 28 generations. Thus, adaptation to acidic pHe resulted in a more invasive phenotype, which was sustained during passage at pH 7.4, suggesting that an acidic microenvironment at the primary tumor site is important in the acquisition of a metastatic phenotype.

Silencing of Forkhead Box M1 Reverses Transforming Growth Factor-β1-Induced Invasion and Epithelial-Mesenchymal Transition of Endometriotic Epithelial Cells

Aim: The aim of this study was to investigate the expression of Forkhead box M1 (FoxM1) in endometriosis and determine FoxM1’s possible effects on endometriotic epithelial cells (EECs) invasion and epithelial-mesenchymal transition (EMT). Methods: The expression of FoxM1 and E-cadherin in endometrium and ectopic tissues was analyzed by immunohistochemistry. The transforming growth factor-β1 (TGF-β1) was added to induce EMT of EECs, which were purified from ectopic tissues. The Short hairpin RNA (ShRNA) intervention technique was used to silence FoxM1. The morphological changes of EECs were observed by microscope. The invasion ability of EECs was determined by transwell invasion assay. The expression of FoxM1 and EMT-related gene (E-cadherin, N-cadherin, vimentin, and Snail) in EECs was detected by quantitative reverse transcription-polymerase chain reaction and western blot. Results: FoxM1 expression was significantly increased, while E-cadherin expression was significantly decreased in ectopic tissues than that in endometrium tissues. After TGF-β1 treatment, EECs showed a transformation from an epithelial sheet-like structure to a mesenchymal fibroblastic spindle shape; EECs invasion ability was enhanced; the level changes of EMT-related molecule also indicated an EMT phenotype of EECs. After FoxM1-shRNA intervention, TGF-β1-induced changes of EECs in morphology, invasion ability and EMT-related molecule expressions were partially reversed. Conclusions: Silencing of FoxM1 could reverse TGF-β1-induced invasion and EMT of EECs.

The circadian expression of osteogenic factors in periodontal tissue loading mechanical force: new concepts of the personalized orthodontic care.

The need for orthodontic treatment continues to increase. Strategies that shorten the treatment course and reduce discomfort are most welcome in clinic. Circadian rhythm plays important role in various physiological processes, including bone formation. This study intended to depict a possible circadian releasing property of the osteogenic factors within the periodontal tissue during orthodontic treatment, which may direct a more efficient and satisfactory orthodontic treatment to the patient. Primary periodontal ligament cells (PDLCs) were obtained from the Sprague-Dawley (SD) rats. An equibiaxial strain value of 12% was applied on rat PDLCs (rPDLCs). After 2h stimuli of 10-7M dexamethasone (DX), the osteogenic genes' expressions were detected by real-time polymerase chain reaction (RT-PCR) at Zeitgeber times 0, 4, 8, 12, 16, 20, and 24. An orthodontic appliance was placed on 45 SD rats. Animals were maintained under 12-h light/dark periods and euthanized at 9 time points over the diurnal cycle. The orthodontic sensitive tissues of the mesial root of the maxillary first molar were collected for RT-PCR and immunohistological assay. The rPDLCs displayed typical fibroblastic spindle shape, and subcultured steadily in vitro. Induced by DX, the mRNA expression of Col-1, OPN, and IBSP within the loaded/unloaded rPDLCs oscillated as that of the main clock gene Per-1. The osteogenic genes' expressions as well as the protein releases sustained a circadian oscillation trend in vivo. This study indicates the existence of a circadian rhythm of the osteogenic factors within the orthodontic sensitive tissues, which highlights the importance of precise timing of force loading in further orthodontic treatment. Thus, a periodicity pattern of orthodontic traction at night may prove a more efficient tooth movement while minimizing the treatment window and discomfort complains.

Regulating Fibroblast Shape and Mechanics through Photoresponsive Surfaces with Concentric Circular Topographic Patterns

AbstractMaterial signals in the form of surface topographies, proved to be potent regulators of cell functions and fate, through mechanotransduction pathways. While a wealth of data is related to regular topographic patterns, i.e., lines, pit, or protrusions, there are comparatively few studies addressing the effects of circular, concentric patterns. Yet, curvatures affecting cell shape dramatically alter cell contractility and behavior. Additionally, the vast majority of patterned surfaces are static in nature and this prevents to understand how cells perceive and respond to the topographic patterns. Here, a technique is exploited for dynamically embossing micrometerscale circular pattern on azopolymeric substrates using a confocal laser microscope and it is analyzed how NIH‐3T3 reacts to the underlying topography in terms of changes in shape and mechanical properties. A characteristic pattern arrangement is found which most effectively alters cell morphology and orientation. Cells perceive the concentric pattern and reconfigure as fast as 2 h after pattern inscription. The changes in morphology also reflect dramatic changes in cell mechanics and cytoskeletal arrangements. The reported method is useful to manipulate cell shape and mechanics in a facile and cost‐effective manner and most importantly enables investigate mechanotransduction events dynamically.

The human umbilical cord stem cells improve the viability of OA degenerated chondrocytes.

Osteoarthritis (OA) affects a large number of patients; however, human umbilical cord stem cells exhibit therapeutic potential for treating OA. The aim of the present study was to explore the interaction between human umbilical cord stem cells and degenerated chondrocytes, and the therapeutic potential of human umbilical cord stem cells on degenerated chondrocytes. Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) were harvested from human umbilical cords, and flow cytometry was used to analyze the surface antigen markers, in addition, chondrogenic, osteogenic and adipogenic differentiation on the cells was investigated. OA cells at P3 were cocultured with hUC-MSCs in a separated co-culture system, and reverse transcription-polymerase chain reaction and western blot were used to evaluate the mRNA, and protein expression of collagen type II (Col2), SRY-box 9 (sox-9) and aggrecan. The level of inflammatory cytokines, tumor necrosis factor-α, interleukin (IL)-1β, IL-6, IL-10, were analyzed by ELISA in the supernatant. hUC-MSCs grow in a fibroblastic shape with stable proliferation. hUC-MSCs expressed cluster of differentiation 44 (CD44), CD73, CD90, CD105; while did not express CD34, CD45, CD106, CD133. After multi-induction, hUC-MSCs were able to differatiate into adipogenic, osteogenic and chondrogenic lineage. hUC-MSCs inhibited the expression of matrix metalloproteinase-13, collagen type X α1 chain and cyclooxygenase-2 in OA chondrocytes, and enhanced the proliferation of OA chondrocytes, while OA chondrocytes stimulated the production of Col2, sox-9 and aggrecan and promoted hUC-MSCs differentiate into chondrocytes. Flow cytometry analysis demonstrated hUC-MSCs have a predominant expression of stem cell markers, while the hematopoietic and endothelial markers were absent. Osteogenic, chondrogenic and adipogenic differentiation was observed in certain induction conditions. hUC-MSCs improved the proliferation of OA chondrocytes and downregulated the expression of inflammatory cytokines, while OA chondrocytes promoted MSCs to differentiate into chondrocytes. Taken together, the co-culture of hUC-MSCs and OA chondrocytes may provide a therapeutic potential in OA treatment.

Pdlim7 Regulates Arf6-Dependent Actin Dynamics and Is Required for Platelet-Mediated Thrombosis in Mice.

Upon vessel injury, platelets become activated and rapidly reorganize their actin cytoskeleton to adhere to the site of endothelial damage, triggering the formation of a fibrin-rich plug to prevent further blood loss. Inactivation of Pdlim7 provides the new perspective that regulation of actin cytoskeletal changes in platelets is dependent on the encoded PDZ-LIM protein. Loss-of-function of Pdlim7 triggers hypercoagulopathy and causes significant perinatal lethality in mice. Our in vivo and in vitro studies reveal that Pdlim7 is dynamically distributed along actin fibers, and lack of Pdlim7 leads to a marked inability to rearrange the actin cytoskeleton. Specifically, the absence of Pdlim7 prevents platelets from bundling actin fibers into a concentric ring that defines the round spread shape of activated platelets. Similarly, in mouse embryonic fibroblasts, loss of Pdlim7 abolishes the formation of stress fibers needed to adopt the typical elongated fibroblast shape. In addition to revealing a fundamental cell biological role in actin cytoskeletal organization, we also demonstrate a function of Pdlim7 in regulating the cycling between the GTP/GDP-bound states of Arf6. The small GTPase Arf6 is an essential factor required for actin dynamics, cytoskeletal rearrangements, and platelet activation. Consistent with our findings of significantly elevated initial F-actin ratios and subsequent morphological aberrations, loss of Pdlim7 causes a shift in balance towards an increased Arf6-GTP level in resting platelets. These findings identify a new Pdlim7-Arf6 axis controlling actin dynamics and implicate Pdlim7 as a primary endogenous regulator of platelet-dependent hemostasis.

Generation of functional hepatocyte-like cells from human deciduous periodontal ligament stem cells.

Human deciduous periodontal ligament stem cells have been introduced for as an easily accessible source of stem cells from dental origin. Although recent studies have revealed the ability of these stem cells in multipotential attribute, their efficiency of hepatic lineage differentiation has not been addressed so far. The aim of this study is to investigate hepatic lineage fate competence of periodontal ligament stem cells through direct media induction. Differentiation of periodontal ligament stem cells into hepatocyte-like cells was conducted by the exposure of two phase media induction. First phase was performed in the presence of hepatocyte growth factors to induce a definitive endoderm formation. In the subsequent phase, the cells were treated with oncostatin M and dexamethosone followed by insulin and transferrin to generate hepatocyte-like cells. Hepatic-related characters of the generated hepatocyte-like cells were determined at both mRNA and protein level followed by functional assays. Foremost changes observed in the generation of hepatocyte-like cells were the morphological features in which these cells were transformed from fibroblastic shape to polygonal shape. Temporal expression of hepatic markers ranging from early endodermal up to late markers were detected in the hepatocyte-like cells. Crucial hepatic markers such as glycogen storage, albumin, and urea secretion were also shown. These findings exhibited the ability of periodontal ligament stem cells of dental origin to be directed into hepatic lineage fate. These cells can be regarded as an alternative autologous source in the usage of stem cell-based treatment for liver diseases.

Fetal Cartilage-Derived Cells Have Stem Cell Properties and Are a Highly Potent Cell Source for Cartilage Regeneration.

Current strategies for cartilage cell therapy are mostly based on the use of autologous chondrocytes or mesenchymal stem cells (MSCs). However, these cells have limitations of a small number of cells available and of low chondrogenic ability, respectively. Many studies now suggest that fetal stem cells are more plastic than adult stem cells and can therefore more efficiently differentiate into target tissues. However, the characteristics and the potential of progenitor cells from fetal tissue remain poorly defined. In this study, we examined cells from human fetal cartilage at 12 weeks after gestation in comparison with bone marrow-derived MSCs or cartilage chondrocytes from young donors (8-25 years old). The fetal cartilage-derived progenitor cells (FCPCs) showed higher yields by approximately 24 times than that of chondrocytes from young cartilage. The morphology of the FCPCs was polygonal at passage 0, being similar to that of the young chondrocytes, but it changed later at passage 5, assuming a fibroblastic shape more akin to that of MSCs. As the passages advanced, the FCPCs showed a much greater proliferation ability than the young chondrocytes and MSCs, with the doubling times ranging from 2∼4 days until passage 15. The surface marker profile of the FCPCs at passage 2 was quite similar to that of the MSCs, showing high expressions of CD29, CD90, CD105, and Stro-1. When compared to the young chondrocytes, the FCPCs showed much less staining of SA-β-gal, a senescence indicator, at passage 10 and no decrease in SOX9 expression until passage 5. They also showed a much greater chondrogenic potential than the young chondrocytes and the MSCs in a three-dimensional pellet culture in vitro and in polyglycolic acid (PGA) scaffolds in vivo. In addition, they could differentiate into adipogenic and osteogenic lineages as efficiently as MSCs in vitro. These results suggest that FCPCs have stem cell properties to some extent and that they are more active in terms of proliferation and chondrogenic differentiation than young chondrocytes or MSCs.