PIEZO1 mechanically regulates the antitumour cytotoxicity of T lymphocytes.

The killing function of cytotoxic T cells can be enhanced biochemically. Here we show that blocking the mechanical sensor PIEZO1 in T cells strengthens their traction forces and augments their cytotoxicity against tumour cells. By leveraging cytotoxic T cells collected from tumour models in mice and from patients with cancers, we show that PIEZO1 upregulates the transcriptional factor GRHL3, which in turn induces the expression of the E3 ubiquitin ligase RNF114. RNF114 binds to filamentous actin, causing its downregulation and rearrangement, which depresses traction forces in the T cells. In mice with tumours, the injection of cytotoxic T cells collected from the animals and treated with a PIEZO1 antagonist promoted their infiltration into the tumour and attenuated tumour growth. As an immunomechanical regulator, PIEZO1 could be targeted to enhance the outcomes of cancer immunotherapies.

Activin Receptor-Like Kinase 3 Directly Couples Gαq (Guanine Nucleotide-Binding Protein Subunit αq)/ Gαq (Guanine Nucleotide-Binding Protein Subunit α11) to Regulate Vascular Contractility.

BACKGROUND: Vascular smooth muscle cell (VSMC) contractility is critical for blood pressure regulation and vascular homeostasis. Identifying the key molecule that maintains VSMC contractility may provide a novel therapeutic target for vascular remodeling. ALK3 (activin receptor-like kinase 3) is a serine/threonine kinase receptor, and deletion of ALK3 causes embryonic lethality. However, little is known about the role of ALK3 in postnatal arterial function and homeostasis. METHODS: We conducted in vivo studies in a tamoxifen-induced postnatal VSMC-specific ALK3 deletion mice suitable for analysis of blood pressure and vascular contractility. Additionally, the role of ALK3 on VSMC was determined using Western blot, collagen-based contraction assay and traction force microscopy. Furthermore, interactome analysis were performed to identify the ALK3-associated proteins and bioluminescence resonance energy transfer assay was used to characterize Gαq activation. RESULTS: ALK3 deficiency in VSMC led to spontaneous hypotension and impaired response to angiotensin II in mice. In vivo and in vitro data revealed that ALK3 deficiency impaired contraction force generation by VSMCs, repressed the expression of contractile proteins, and inhibited the phosphorylation of myosin light chain. Mechanistically, Smad1/5/8 signaling mediated the ALK3-modulated contractile protein expressions but not myosin light chain phosphorylation. Furthermore, interactome analysis revealed that ALK3 directly interacted with and activated Gαq (guanine nucleotide-binding protein subunit αq)/Gα11 (guanine nucleotide-binding protein subunit α11), thereby stimulating myosin light chain phosphorylation and VSMC contraction. CONCLUSIONS: Our study revealed that in addition to canonical Smad1/5/8 signaling, ALK3 modulates VSMC contractility through direct interaction with Gαq/Gα11, and therefore, might serve as a potential target for modulating aortic wall homeostasis.

Purse-string contraction guides mechanical gradient-dictated heterogeneous migration of epithelial monolayer.

Mechanical heterogeneity has been recognized as an important role in mediating collective cell migration, yet the related mechanism has not been elucidated. Herein, we fabricate heterogeneous stiffness gradients by leveraging microelastically-patterned hydrogels with varying periodic distance. We observe that a decrease in the periodic distance of the mechanical heterogeneity is accompanied by an overall increase in the velocity and directionality of the migrating monolayer. Moreover, inhibition of ROCK- and myosin ⅡA- but not Rac1-mediated contraction reduces monolayer migration on the mechanically heterogeneous substrates. Furthermore, we find that F-actin and myosin ⅡA form purse-string at the leading edge on the mechanically heterogeneous substrates. Together, these findings not only show that the orientational cell-cell contraction promotes collective cell migration under the mechanical heterogeneity, but also demonstrate that the mechanosensation arising from large-scale cell-cell interactions through purse-string formation mediated cell-cell orientational contraction can feed back to regulate the reorganization of epithelial tissues. STATEMENT OF SIGNIFICANCE: By detecting the links between heterogenous rigidity and collective cell migration behavior at the molecular level, we reveal that collective cell migration in the mechanical heterogeneity is driven by ROCK- and myosin-ⅡA-dependent cytoskeletal tension. We confirm that cytoskeletal tension across the epithelial tissue is holistically linked through F-actin and myosin-ⅡA, which cooperate to form purse-string structures for modulating collective tissue behavior on the exogenous matrix with mechanical heterogeneity. Mechanical heterogeneity initiates tissue growth, remodelling, and morphogenesis by orientating cell contractility. Therefore, tensional homeostasis across large-scale cell interactions appears to be necessary and sufficient to trigger collective tissue behavior. Overall, these findings shed light on the role of mechanical heterogeneity in tissue microenvironment for reorganization and morphogenesis.

Super-resolution traction force microscopy with enhanced tracer density enables capturing molecular scale traction.

Cell traction mediates the biochemical and mechanical interactions between the cell and its extracellular matrix (ECM). Traction force microscopy (TFM) is a powerful technique for quantitative cellular scale traction analysis. However, it is challenging to characterize macromolecular scale traction events with current TFM due to the limited sampling density and algorithmic precision. In this article, we introduce a super-resolution TFM by utilizing a novel substrate surface modification method. Our TFM technique achieved a spatial resolution comparable to fluorescence microscopy and precision comparable to the rupture force of an integrin-ligand bond. Correlated imaging of TFM with fluorescence microscopy demonstrated that the residing paxillin highly correlated with traction while α5 integrin was located differently. Time-lapse TFM imaging captured a transient traction variation as the adhesion protein passed by. Thus, the novel super-resolution TFM benefits the studies on cellular biochemical and mechanical interactions.

Mechanical stretching boosts expansion and regeneration of intestinal organoids through fueling stem cell self-renewal.

Intestinal organoids, derived from intestinal stem cell self-organization, recapitulate the tissue structures and behaviors of the intestinal epithelium, which hold great potential for the study of developmental biology, disease modeling, and regenerative medicine. The intestinal epithelium is exposed to dynamic mechanical forces which exert profound effects on gut development. However, the conventional intestinal organoid culture system neglects the key role of mechanical microenvironments but relies solely on biological factors. Here, we show that adding cyclic stretch to intestinal organoid cultures remarkably up-regulates the signature gene expression and proliferation of intestinal stem cells. Furthermore, mechanical stretching stimulates the expansion of SOX9+ progenitors by activating the Wnt/ß-Catenin signaling. These data demonstrate that the incorporation of mechanical stretch boosts the stemness of intestinal stem cells, thus benefiting organoid growth. Our findings have provided a way to optimize an organoid generation system through understanding cross-talk between biological and mechanical factors, paving the way for the application of mechanical forces in organoid-based models.

Collagen gel contraction assays: From modelling wound healing to quantifying cellular interactions with three-dimensional extracellular matrices.

Cells respond to and actively remodel the extracellular matrix (ECM). The dynamic and bidirectional interaction between cells and ECM, especially their mechanical interactions, has been found to play an essential role in triggering a series of complex biochemical and biomechanical signal pathways and in regulating cellular functions and behaviours. The collagen gel contraction assay (CGCA) is a widely used method to investigate cell-ECM interactions in 3D environments and provides a mechanically associated readout reflecting 3D cellular contractility. In this review, we summarize various versions of CGCA, with an emphasis on recent high-throughput and low-consumption CGCA techniques. More importantly, we focus on the technique of force monitoring during the contraction of collagen gel, which provides a quantitative characterization of the overall forces generated by all the resident cells in the collagen hydrogel. Accordingly, we present recent biological applications of the CGCA, which have expanded from the initial wound healing model to other studies concerning cell-ECM interactions, including fibrosis, cancer, tissue repair and the preparation of biomimetic microtissues.

Mechanical transmission enables EMT cancer cells to drive epithelial cancer cell migration to guide tumor spheroid disaggregation.

Cell phenotype heterogeneity within tumor tissue, especially which due to the emergence of epithelial-mesenchymal transition (EMT) in cancer cells, is associated with cancer invasion and metastasis. However, our understanding of the cellular mechanism(s) underlying the cooperation between EMT cell and epithelial cancer cell migration remains incomplete. Herein, heterotypic tumor spheroids containing both epithelial and EMT cancer cells were generated in vitro. We observed that EMT cells dominated the peripheral region of the self-organized heterotypic tumor spheroid. Furthermore, our results demonstrated that EMT cells could serve as leader cells to improve the collective migration efficiency of epithelial cancer cells and promote dispersion and invasion of the tumor spheroids, which was regulated by the force transition between EMT cells and epithelial cancer cells. Mechanistically, our data further suggest that force transmission is mediated by heterophilic N-cadherin/E-cadherin adhesion complexes between EMT and epithelial cancer cells. Impairment of N-cadherin/E-cadherin adhesion complex formation abrogated the ability of EMT cells to guide epithelial cancer cell migration and blocked the dispersion of tumor spheroids. Together, our data provide new insight into the mechanical interaction between epithelial and EMT cancer cells through heterophilic cadherin adhesion, which enables cooperative tumor cell migration, highlighting the role of EMT cells in tumor invasion.

Bioinspired porous microspheres for sustained hypoxic exosomes release and vascularized bone regeneration.

Exosomes derived from mesenchymal stem cells (MSCs) have demonstrated regenerative potential for cell-free bone tissue engineering, nevertheless, certain challenges, including the confined therapeutic potency of exosomes and ineffective delivery method, are still persisted. Here, we confirmed that hypoxic precondition could induce enhanced secretion of exosomes from stem cells from human exfoliated deciduous teeth (SHEDs) via comprehensive proteomics analysis, and the corresponding hypoxic exosomes (H-Exo) exhibited superior potential in promoting cellular angiogenesis and osteogenesis via the significant up-regulation in focal adhesion, VEGF signaling pathway, and thyroid hormone synthesis. Then, we developed a platform technology enabling the effective delivery of hypoxic exosomes with sustained release kinetics to irregular-shaped bone defects via injection. This platform is based on a simple adsorbing technique, where exosomes are adsorbed onto the surface of injectable porous poly(lactide-co-glycolide) (PLGA) microspheres with bioinspired polydopamine (PDA) coating (PMS-PDA microspheres). The PMS-PDA microspheres could effectively adsorb exosomes, show sustained release of H-Exo for 21 days with high bioactivity, and induce vascularized bone regeneration in 5-mm rat calvarial defect. These findings indicate that the hypoxic precondition and PMS-PDA porous microsphere-based exosome delivery are efficient in inducing tissue regeneration, hence facilitating the clinical translation of exosome-based therapy.

Vascularized pulp regeneration via injecting simvastatin functionalized GelMA cryogel microspheres loaded with stem cells from human exfoliated deciduous teeth.

Dental pulp necrosis are serious pathologic entities that causes tooth nutrition deficiency and abnormal root development, while regeneration of functional pulp tissue is of paramount importance to regain tooth vitality. However, existing clinical treatments, which focus on replacing the necrotic pulp tissue with inactive filling materials, fail to restore pulp vitality and functions, thus resulting in a devitalized and weakened tooth. Currently, dental pulp regeneration via stem cell-based therapy for pulpless teeth has raised enormous attention to restore the functional pulp. Here, a novel design of injectable simvastatin (SIM) functionalized gelatin methacrylate (GelMA) cryogel microspheres (SMS) loaded with stem cells from human exfoliated deciduous teeth (SHEDs) was established to refine SHEDs biological behaviors and promote in vivo vascularized pulp-like tissue regeneration. In this system, SIM encapsulated poly (lactide-co-glycolide) (PLGA) nanoparticles were incorporated into GelMA cryogel microspheres via cryogelation and O1/W/O2 emulsion method. SMS with sustained release of SIM promoted SHEDs adhesion, proliferation and exhibited cell protection properties during the injection process. Furthermore, SMS enhanced SHEDs odontogenic differentiation and angiogenic potential, and SHEDs loaded SMS (SHEDs/SMS) are beneficial for human umbilical vein endothelial cells (HUVECs) migration and angiogenesis, demonstrating their potential for use in promoting vascularized tissue regeneration. SHEDs/SMS complexes were injected into cleaned human tooth root segments for subcutaneous implantation in nude mice. Our results demonstrated that SHEDs/SMS could induce vessel-rich pulp-like tissue regeneration in vivo and that such an injectable nano-in-micro multistage system for the controlled delivery of bioactive reagents would be suitable for clinical application in endodontic regenerative dentistry.

Multifunctional Immunoliposomes Enhance the Immunotherapeutic Effects of PD-L1 Antibodies against Melanoma by Reprogramming Immunosuppressive Tumor Microenvironment.

The immunosuppressive tumor microenvironment (TME) can significantly limit the immunotherapeutic effects of the PD-L1 antibody (aPDL1) by inhibiting the infiltration of CD8+ cytotoxic T cells (CTLs) into the tumor tissues. However, how to reprogram the immunosuppressive TME and promote the infiltration of CTLs remains a huge challenge for aPDL1 to achieve the maximum benefits. Herein, the authors design a multifunctional immunoliposome that encapsulates the adrenergic receptor blocker carvedilol (CAR) and connects the "don't eat me" signal antibody (aCD47) and aPDL1 in series via a reactive oxygen species (ROS)-sensitive linker on the surface. In ROS-enriched immunosuppressive TME, the multifunctional immunoliposome (CAR@aCD47/aPDL1-SSL) can first release the outer aCD47 to block the "do not eat me" pathway, promote the phagocytosis of tumor cells by phagocytic cells, and activate CTLs. Then, the aPDL1 on the liposome surface is exposed to block the PD-1/PD-L1 signaling pathway, thereby inducing CTLs to kill tumor cells. CAR encapsulated in CAR@aCD47/aPDL1-SSL can block the adrenergic nerves in the tumor tissues and reduce their densities, thereby inhibiting angiogenesis in the tumor tissues and reprogramming the immunosuppressive TME. According to the results, CAR@aCD47/aPDL1-SSL holds an effective way to reprogram the immunosuppressive TME and significantly enhance immunotherapeutic efficiency of aPDL1 against the primary cancer and metastasis.

Integration of electrotaxis and durotaxis in cancer cells: Subtle nonlinear responses to electromechanical coupling cues.

Cells in living organisms live in multiphysics-coupled environments. There is growing evidence indicating that both exogenous electric field (EEF) and extracellular stiffness gradient (ESG) can regulate directional movement of cells, which are known as electrotaxis and durotaxis, respectively. How single cells respond to the ubiquitous electromechanical coupling cues, however, remains mysterious. Using microfluidic chip-based methodology and finite element-based electromechanical coupling design strategies, we develope an electromechanical coupling microchip system, enabling us to quantitatively investigate polarization and directional migration governed by EEF and ESG at the single cell level. It is revealed that both of electrotaxis and durotaxis nonlinearly depend on the physiological EEF and ESG, respectively. Specific combinations of EEF and ESG can subtly modify the polarization states of single cells and thus induce hyperpolarization and depolarization. Cells can integrate electrotaxis and durotaxis in response to multi-cue microenvironments via subtle mechanisms involving cooperation and competition during cellular electrosensing and mechanosensing. The work offers a platform for quantifying migration and polarization of cells driven by electromechanical cues, which is essential not only for elucidating physiological and pathological processes like embryo development, and invasion and metastasis of cancer cells, but for manipulating cell behaviors in a controllable and programmable fashion.

Injectable GelMA Cryogel Microspheres for Modularized Cell Delivery and Potential Vascularized Bone Regeneration.

Cell therapeutics hold tremendous regenerative potential and the therapeutic effect depends on the effective delivery of cells. However, current cell delivery carriers with unsuitable cytocompatibility and topological structure demonstrate poor cell viability during injection. Therefore, porous shape-memory cryogel microspheres (CMS) are prepared from methacrylated gelatin (GelMA) by combining an emulsion technique with gradient-cooling cryogelation. Pore sizes of the CMS are adjusted via the gradient-cooling procedure, with the optimized pore size (15.5 ± 6.0 µm) being achieved on the 30-min gradient-cooled variant (CMS-30). Unlike hydrogel microspheres (HMS), CMS promotes human bone marrow stromal cell (hBMSC) and human umbilical vein endothelial cell (HUVEC) adhesion, proliferated with high levels of stemness for 7 d, and protects cells during the injection process using a 26G syringe needle. Moreover, CMS-30 enhances the osteogenic differentiation of hBMSCs in osteoinductive media. CMS can serve as building blocks for delivering multiple cell types. Here, hBMSC-loaded and HUVEC-loaded CMS-30, mixed at a 1:1 ratio, are injected subcutaneously into nude mice for 2 months. Results show the development of vascularized bone-like tissue with high levels of OCN and CD31. These findings indicate that GelMA CMS of a certain pore size can effectively deliver multiple cells to achieve functional tissue regeneration.

Calcium and TRPV4 promote metastasis by regulating cytoskeleton through the RhoA/ROCK1 pathway in endometrial cancer.

Transient receptor potential vanilloid 4 (TRPV4) is a calcium-permeable cation channel that has been associated with several types of cancer. However, its biological significance, as well as its related mechanism in endometrial cancer (EC) still remains elusive. In this study, we examined the function of calcium in EC, with a specific focus on TRPV4 and its downstream pathway. We reported here on the findings that a high level of serum ionized calcium was significantly correlated with advanced EC progression, and among all the calcium channels, TRPV4 played an essential role, with high levels of TRPV4 expression associated with cancer progression both in vitro and in vivo. Proteomic and bioinformatics analysis revealed that TRPV4 was involved in cytoskeleton regulation and Rho protein pathway, which regulated EC cell migration. Mechanistic investigation demonstrated that TRPV4 and calcium influx acted on the cytoskeleton via the RhoA/ROCK1 pathway, ending with LIMK/cofilin activation, which had an impact on F-actin and paxillin (PXN) levels. Overall, our findings indicated that ionized serum calcium level was significantly associated with poor outcomes and calcium channel TRPV4 should be targeted to improve therapeutic and preventive strategies in EC.

Multifunctional Immunoliposomes Combining Catalase and PD-L1 Antibodies Overcome Tumor Hypoxia and Enhance Immunotherapeutic Effects Against Melanoma.

BACKGROUND: Immune checkpoint blockades (ICBs) are a promising treatment for cancers such as melanoma by blocking important inhibitory pathways that enable tumor cells to evade immune attack. Programmed death ligand 1 monoclonal antibodies (aPDL1s) can be used as an ICB to significantly enhance the effectiveness of tumor immunotherapy by blocking the PD-1/PD-L1 inhibitory pathway. However, the effectiveness of aPDL1s may be limited by low selectivity in vivo and immunosuppressed tumor microenvironment including hypoxia. PURPOSE: To overcome the limitations, we develop a multifunctional immunoliposome, called CAT@aPDL1-SSL, with catalase (CAT) encapsulated inside to overcome tumor hypoxia and aPDL1s modified on the surface to enhance immunotherapeutic effects against melanoma. METHODS: The multifunctional immunoliposomes (CAT@aPDL1-SSLs) are prepared using the film dispersion/post-insertion method. The efficacy of CAT@aPDL1-SSLs is verified by multiple experiments in vivo and in vitro. RESULTS: The results of this study suggest that the multifunctional immunoliposomes preserve and protect the enzyme activity of CAT and ameliorate tumor hypoxia. Moreover, the enhanced cellular uptake of CAT@aPDL1-SSLs in vitro and their in vivo biodistribution suggest that CAT@aPDL1-SSLs have great targeting ability,resulting in improved delivery and accumulation of immunoliposomes in tumor tissue.Finally, by activating and increasing the infiltration of CD8+ T cells at the tumor site, CAT@aPDL1-SSLs inhibit the growth of tumor and prolong survival time of mice,with low systemic toxicity. CONCLUSION: In conclusion, the multifunctional immunoliposomes developed and proposed in this study are a promising candidate for melanoma immunotherapy, and could potentially be combined with other cancer therapies like radiotherapy and chemotherapy to produce positive outcomes.

Anisotropic stiffness gradient-regulated mechanical guidance drives directional migration of cancer cells.

Interfacial interactions between cancer cells and surrounding microenvironment involve complex mechanotransduction mechanisms that are directly associated with tumor invasion and metastasis. Matrix remodeling triggers heterogeneity of stiffness in tumor microenvironment and thus generates anisotropic stiffness gradient (ASG). The migration of cancer cells mediated by ASG, however, still remains elusive. Based on a multi-layer polymerization method of microstructured hydrogels with surface topology, we develop an in vitro experimental platform for mechanical interactions of cancer cells with ASG matrix microenvironment. We show that mechanical guidance of mesenchymal cells is essentially modulated by ASG, leading to a spontaneous directional migration along the orientation parallel to the maximum stiffness although there is no stiffness gradient in the direction. The ASG-regulated mechanical guidance presents an alternative way of cancer cell directional migration. Further, our findings indicate that the mechanical guidance occurs only in mesenchymal cancer cells, but not in epithelial cancer cells, implying that cell contractility may contribute to ASG-regulated migration of cells. This work is not only helpful for elucidating the role of matrix remodeling in mediating tumor cell invasion and metastasis, but has potential implications for developing specific cancer treatments. STATEMENT OF SIGNIFICANCE: Local extracellular matrix (ECM) stiffening triggers mechanical heterogeneity in tumor microenvironment, which can exert a crucial impact on interfacial interactions between tumor cells and surrounding ECM. The underlying mechanobiological mechanism that tumor cells are modulated by mechanically heterogeneous ECM, however, still remains mysterious to a great extent. Through our established in vitro platform and analysis, we have demonstrated that anisotropic stiffness gradient (ASG) has the ability to elicit directional migration of cells, essentially depending on local stiffness gradients and the corresponding absolute stiffness values. This study is not only crucial for revealing the role of matrix remodeling in regulating tumor invasion and metastasis, but also offers a valuable guidance for developing anti-tumor therapies from the biomechanical perspective.

Thickness Tunable Wedding-Cake-like MoS2 Flakes for High-Performance Optoelectronics.

Atomically thin transition-metal dichalcogenides (TMDCs) have received substantial interest due to their typical thickness-dependent optical and electronic properties and related applications in optoelectronics. However, the large-scale, thickness-tunable growth of such materials is still challenging. Herein, we report a fast growth of thickness-tunable wedding-cake-like MoS2 flakes on 6-in. soda-lime glass by using NaCl-coated Mo foils as metal precursors. The MoS2 thicknesses are tuned from one layer (1L) to >20L by controlling the concentrations of NaCl promoter. To attest to the ultrahigh crystal quality, related devices based on 1L-multilayer MoS2 lateral junctions have been constructed and display a relatively high rectification ratio (∼103) and extra high photoresponsitivity (∼104 A/W). Thanks to the scalable sizes, uniform distributions of the flakes and homogeneous optical properties, the applications in ultraviolet (UV) irradiation filtering eyewear are also demonstrated. Our work should hereby propel the scalable production of layer-controlled TMDC materials as well as their optical and optoelectrical applications.

Biophysical phenotypes and determinants of anterior vs. posterior primitive streak cells derived from human pluripotent stem cells.

Formation of the primitive streak (PS) marks one of the most important developmental milestones in embryonic development. However, our understanding of cellular mechanism(s) underlying cell fate diversification along the anterior-posterior axis of the PS remains incomplete. Furthermore, differences in biophysical phenotypes between anterior and posterior PS cells, which could affect their functions and regulate their fate decisions, remain uncharacterized. Herein, anterior and posterior PS cells were derived using human pluripotent stem cell (hPSC)-based in vitro culture systems. We observed that anterior and posterior PS cells displayed significantly different biophysical phenotypes, including cell morphology, migration, and traction force generation, which was further regulated by different levels of Activin A- and BMP4-mediated developmental signaling. Our data further suggested that intracellular cytoskeletal contraction could mediate anterior and posterior PS differentiation and phenotypic bifurcation through its effect on Activin A- and BMP4-mediated intracellular signaling events. Together, our data provide new information about biophysical phenotypes of anterior and posterior PS cells and reveal an important role of intracellular cytoskeletal contractility in regulating anterior and posterior PS differentiation of hPSCs. STATEMENT OF SIGNIFICANCE: Formation of the primitive streak (PS) marks one of the most important developmental milestones in embryonic development. However, molecular and cellular mechanism(s) underlying functional diversification of embryonic cells along the anterior-posterior axis of the PS remains incompletely understood. This work describes the first study to characterize the biophysical properties of anterior and posterior PS cells derived from human pluripotent stem cells (hPSCs). Importantly, our data showing the important role of cytoskeleton contraction in controlling anterior vs. posterior PS cell phenotypic switch (through its effect on intracellular Smad signaling activities downstream of Activin A and BMP4) should shed new light on biomechanical regulations of the development and anterior-posterior patterning of the PS. Our work will contribute significantly to uncovering new biophysical principles and cellular mechanisms driving cell lineage diversification and patterning during the PS formation.

Effect of Optimized Concentrations of Basic Fibroblast Growth Factor and Epidermal Growth Factor on Proliferation of Fibroblasts and Expression of Collagen: Related to Pelvic Floor Tissue Regeneration.

BACKGROUND: Fibroblasts were the main seed cells in the studies of tissue engineering of the pelvic floor ligament. Basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) were widely studied but at various concentrations. This study aimed to optimize the concentrations of combined bFGF and EGF by evaluating their effects on proliferation and collagen secretion of fibroblasts. METHODS: Fibroblasts were differentiated from rat adipose mesenchymal stem cells (ADSCs). Flow cytometry and immunohistochemistry were used for cell identification. The growth factors were applied at concentrations of 0, 1, 10, and 100 ng/ml as three groups: (1) bFGF alone, (2) EGF alone, and (3) bFGF mixed with EGF. Cell proliferation was evaluated by Cell Counting Kit-8 assays. Expression of Type I and III collagen (Col-I and Col-III) mRNAs was evaluated by real-time quantitative reverse transcription-polymerase chain reaction. Statistical analysis was performed with SPSS software and GraphPad Prism using one-way analysis of variance and multiple t-test. RESULTS: ADSCs were successfully isolated from rat adipose tissue as identified by expression of typical surface markers CD29, CD44, CD90, and CD45 in flow cytometry. Fibroblasts induced from ADSC, compared with ADSCs, were with higher mRNA expression levels of Col I and Col III (F = 1.29, P = 0.0390). bFGF, EGF, and the mixture of bFGF with EGF can enhanced fibroblasts proliferation, and the concentration of 10 ng/ml of the mixture of bFGF with EGF displayed most effectively (all P < 0.05). The expression levels of Col-I and Col-III mRNAs in fibroblasts displayed significant increases in the 10 ng/ml bFGF combined with EGF group (all P < 0.05). CONCLUSIONS: The optimal concentration of both bFGF and EGF to promote cell proliferation and collagen expression in fibroblasts was 10 ng/ml at which fibroblasts grew faster and secreted more Type I and III collagens into the extracellular matrix, which might contribute to the stability of the pelvic floor microenvironment.