Fibroblastic Morphology Research Articles

Effect of tricalcium silicate cements in gene expression of COL1A1, MAPK's, and NF-kB, and cell adhesion in primary teeth' pulp fibroblasts.

Tricalcium silicate cements (TSCs) regulate gene expression and cell responses from dental tissues surrounding the repair site. The study aimed to evaluate the gene expression levels of Collagen Type I Alpha 1 Chain (COL1A1), Mitogen-Activated Protein Kinases (MAPK's), Nuclear Factor-Kappa B (NF-κB), cell adhesion, and morphology of human dental pulp fibroblasts (hDPFs) from primary teeth treated with eluates obtained from Mineral Trioxide Aggregate (MTA) and Biodentine. hDPFs were treated with eluates from Biodentine and MTA (2.5 mg/mL in culture medium). The control group was a culture without the eluates. Gene expressions of COL1A1, MAPK's, and NF-κB were evaluated using Polymerase Chain Reaction (PCR) and cell adhesion by immunocytochemistry for Vinculin and Integrin β1 expression. Gene expression of MAPK's and NF-κB in hDPFs with the eluates from MTA and Biodentine showed no significant difference versus the control group (p > 0.05), but COL1A1 exhibited a significant difference (p < 0.05). The expression of COL1A1, MAPK's, and NF-κB was lower in cultures with MTA and Biodentine eluates regarding the control group, with no significant difference between MTA and Biodentine (p > 0.05). After 72 h of incubation, the hDPFs cultured with MTA and Biodentine eluates showed an elongated morphology; after 7 d, a loss or/and reduction of the cytoplasmic processes, and smaller nuclei were observed. Vinculin and Integrin β1 were expressed in hDPFs treated with MTA and Biodentine eluates. MTA and Biodentine did not inhibit or generate a significant difference in the expression levels of COL1A1, MAPK's, and NF-κB in hDPFs.

Dipotassium Glycyrrhizinate Against Fibrotic Effects of Histamine Depletes Intracellular Glutathione to Discomfort Mitochondrial, Inducing Cell Apoptosis/ Ferroptosis of Over-Activated Lung Fibroblast

Pulmonary fibrosis (PF) is the common outcome of a variety of chronic lung diseases and systemic diseases, and the consequence of Coronavirus Disease 2019 (COVID-19). There is currently a lack of effective treatments to reverse this disease or prevent its development. The purpose of this study is to investigate the effect of dipotassium glycyrrhizinate (DG) on lung fibroblasts and the possible mechanism of alleviating pulmonary fibrosis. Extract primary lung fibroblasts, establish cell models, and use time-lapse photography to continuously monitor the effects of dipotassium glycyrrhizinate and histamine on the morphology of lung fibroblasts. The content of glutathione in cells was detected by the fluorescence method. Cell mitochondrial membrane potential was examined, protein expressions of P53, Caspase-3, Bax and Bal-2 of lung fibroblasts were evaluated by western blot and immune cytochemistry. Our data demonstrated that dipotassium glycyrrhizinate inhibited histamine-induced cell growth/proliferation, glutathione content, mitochondrial membrane potential; also down-regulated P53 and up-regulated the ratio of Bax/Bcl-2, leading to the ferritosis/apoptosis of activated lung fibroblasts, against the fibrotic effects of histamine. Present study provided a new pharmaceutics mechanism of dipotassium glycyrrhizinate for the treatment of lung fibrosis that dipotassium glycyrrhizinate against fibrotic effects of histamine depletes intracellular glutathione to discomfort mitochondrial, inducing cell apoptosis/ferroptosis of over-activated lung fibroblast.

Effects of the Washing Time and Washing Solution on the Biocompatibility and Mechanical Properties of 3D Printed Dental Resin Materials.

Three-dimensional (3D) printing technology is highly regarded in the field of dentistry. Three-dimensional printed resin restorations must undergo a washing process to remove residual resin on the surface after they have been manufactured. However, the effect of the use of different washing solutions and washing times on the biocompatibility of the resulting resin restorations is unclear. Therefore, we prepared 3D-printed denture teeth and crown and bridge resin, and then washed them with two washing solutions (isopropyl alcohol and tripropylene glycol monomethyl ether) using different time points (3, 5, 10, 15, 30, 60, and 90 min). After this, the cell viability, cytotoxicity, and status of human gingival fibroblasts were evaluated using confocal laser scanning. We also analyzed the flexural strength, flexural modulus, and surface SEM imaging. Increasing the washing time increased the cell viability and decreased the cytotoxicity (p < 0.001). Confocal laser scanning showed distinct differences in the morphology and number of fibroblasts. Increasing the washing time did not significantly affect the flexural strength and surface, but the flexural modulus of the 90 min washing group was 1.01 ± 0.21 GPa (mean ± standard deviation), which was lower than that of all the other groups and decreased as the washing time increased. This study confirmed that the washing time affected the biocompatibility and mechanical properties of 3D printed dental resins.

Silver sulfadiazine loaded core-shell airbrushed nanofibers for burn wound healing application

Ideal dressing materials for complex and large asymmetric burns should have the dual properties of anti-bacterial and regenerative with advanced applicability of direct deposit on the wound at the patient bedside. In this study, core-shell nanofibers (polycaprolactone; PCL and polyethylene oxide; PEO) with different percent of silver sulfadiazine (SSD) loading (2–10%) were prepared by the airbrushing method using a custom build device. Results indicate a sustained release profile of silver sulfadiazine (SSD) up to 28 days and concentration-dependent anti-bacterial activity. The morphology and proliferation of human dermal fibroblast (HDF) cells and human dental follicle stem cells (HDFSC) on the silver sulfadiazine loaded nanofibers confirm the biocompatibility of airbrushed nanofibers. Moreover, upregulation of extracellular matrix (ECM) proteins (Col I, Col III, and elastin) support the differentiation and regenerative properties of silver sulfadiazine nanofiber mats. This was further confirmed by the complete recovery of rabbit burn wound models within 7 days of silver sulfadiazine loaded nanofiber dressing. Histopathology data show silver sulfadiazine loaded core-shell nanofibers' anti-inflammatory and proliferative activity without any adverse response on the tissue. Overall data display that the airbrushed silver sulfadiazine-loaded core-shell nanofibers are effective dressing material with the possibility of direct fiber deposition on the wound to cover, heal, and regenerate large asymmetric burn wounds.

Growth kinetics and phenotypic markers expression in human dermal stem cells

Human dermal stem cells (DSCs) have generated significant interest in the field of regenerative medicine due to their prospects of autologous transplantation. The present study evaluated the growth kinetics and phenotypic markers expression in human DSCs. The primary cultures of DSCs (n=3) were established by explant culture and characterization of the cells was carried out by assessing morphology, viability, proliferation rate, population doubling time (PDT), cell cycle status and the expression of cell surface markers such as CD29, CD73, CD90 and CD166. The cells released from tissue explants showed spindleshaped fibroblast morphology with the mean percentage viability varying between 93.43% and 100% from passages 1 to 4. DSCs displayed a strong and steady proliferative potential with an average PDT of 42.55 hrs. Cell cycle profile of DSCs demonstrated the majority of cells (59.80% to 76.29%) at G0/G1 phase. Further, the phenotypic profile of markers confirmed the stromal origin of DSCs by exhibiting positivity for CD29, CD73, CD90 and CD166. In conclusion, the growth kinetics and expression of phenotypic markers are consistent with the notion that skin dermis contains a population of stem cells and can serve as a potential autologous source for therapeutic applications.

The Biological Effects of Ozone Gas on Soft and Hard Dental Tissues and the Impact on Human Gingival Fibroblasts and Gingival Keratinocytes

Ozone is an allotropic form of oxygen, so in the medical field ozone therapy has special effects. Starting from the premise that bio-oxidative ozone therapy reduces the number of bacteria, in the present study two approaches were proposed: to evaluate the biological effects of ozone gas on the tooth enamel remineralization process and to demonstrate its impact on the morphology and confluence of human primary gingival cells, namely keratinocytes (PGK) and fibroblasts (HGF). The ozone produced by HealOzone was applied in vivo to 68 M1s (first permanent molars), both maxillary and mandibular, on the occlusal surfaces at pit and fissure. The molars included in the study recorded values between 13 and 24 according to the DIAGNOdent Pen 2190 scale, this being the main inclusion/exclusion criterion for the investigated molars. Because the gas can make contact with primary gingival cells during the ozonation process, both human gingival fibroblasts and keratinocytes were exposed to different doses of ozone (20 s, 40 s, 60 s), and its effects were observed with the Olympus IX73 inverted microscope. The contact of ozone with the human primary gingival cells demonstrates cell sensitivity to the action of ozone, this being higher in fibroblasts compared to keratinocytes, but it is not considered toxic because all the changes are reversible at 48 h after exposure.

In Vitro Biocompatibility of CPP-ACP and Fluoride-containing Desensitizers on Human Gingival Cells.

To analyze the biocompatibility of different desensitizers containing casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) and fluoride in their composition: MI Varnish (MV), Clinpro White Varnish (3M Oral Care), Profluorid Varnish (VOCO), Duraphat (Colgate) and Embrace Varnish (Pulpdent) on human gingival fibroblast cells (hGF). Human gingival fibroblast (hGF) cells were exposed to several desensitizer extracts at different concentrations (0.1%, 1%, and 4% eluates). Then, in vitro biocompatibility was studied by analyzing the IC50 value, cell proliferation (MTT assay and cell cycle), cell migration (wound healing assay), cell morphology and F-actin content (immunocytofluorescence), and induction of apoptosis/necrosis (flow cytometry). Data were analyzed by one-way analysis of variance (ANOVA) followed by Tukey test. The lowest cell viability and IC50 were observed in all concentrations of Embrace Varnish-treated hGFs (p<0.001), whereas the highest were exhibited by those treated with Clinpro White Varnish. Similar effects were evidenced when induction of apoptosis/necrosis and cell migration assays were assessed. Finally, MI Varnish, Profluorid Varnish, Duraphat, and Embrace Varnish extracts showed lower numbers of attached cells, some of them with an unusual fibroblastic morphology when cultured with 4% concentration of the varnishes, while Clinpro White Varnish exhibited a similar number of cells with an evident actin cytoskeleton compared to the control group. The results obtained in this study indicate that hGFs show better in vitro biocompatibility after exposure to Clinpro White Varnish, even at the highest concentration employed, making it the most eligible for topical applications. In contrast, Embrace Varnish exhibited a high cytotoxicity towards hGFs that could potentially delay the healing process and regeneration of the oral mucosa, although more studies are needed to confirm this hypothesis.

Evaluating Mouse Fibroblast Interaction with Implant Surfaces in a 3D Microenvironment

Previous studies assessing fibroblast interactions with implants have mainly relied on measurements such as cell migration, gene expression, and cell adhesion. For these studies, testing cellular behavior at the implant surface was done by imaging the cell-implant interface using standard microscopy techniques in 2D tissue culture dishes. The true behavior of cells relative to the implant can best be assessed in a more physiologic 3D microenvironment. The embedding of the implant disks in 3D collagen gels was standardized with labeled fibroblasts to allow the imaging of fibroblast morphology and behavior when proximal to or binding to the implant disks. This allowed comparison of the behavior of laser-microgrooved and machined implant disk surfaces quantitatively in an in vitro 3D microenvironment. This in vitro imaging assay revealed for the first time in a 3D microenvironment setting the statistically significant impact laser-microgrooved disk surfaces have on both cell adherence and recruitment of cells in proximity to the disk. It also allowed visualization of membrane protrusivity and cytoskeletal organization in cells adherent to the implant disk. This assay provides a simple and effective way of observing cell behavior on and around the implant disk surface in a more physiologic 3D setting. Within the limits of this study, it revealed that the laser-microgrooved implant surface demonstrates significant superiority in fibroblast recruitment and binding in a 3D microenvironment.

Characterization and evaluation of ascorbic acid-induced cell sheet formation in human periodontal ligament stem cells: An in vitro study

ObjectivesPeriodontal ligament-derived stem cells (PDLSCs) are regarded as a viable option for periodontal regeneration using cell sheet technology. The objective of the present in vitro study was to characterize human PDLSCs based on their phenotypic and biological properties and to evaluate the ascorbic acid (AA or vitamin C)-induced cell sheet by analyzing the molecular markers. MethodsPDLSCs were established from premolars, and their morphology, viability, proliferation, phenotypic marker expression, and ability to differentiate into osteocytes and adipocytes were analyzed. PDLSCs were then induced to form cell sheets using 100 μM AA, and gene expression was examined by real-time polymerase chain reaction. ResultsPDLSCs showed fibroblastic morphology with >95% viability. The cells were highly proliferative and positive for surface antigens CD29, CD73, and CD90 but negative for CD34 and CD45. They were capable of differentiating into osteocytes and adipocytes. Induction with 100 μM AA transformed PDLSCs into two-to three-layered cell sheets. There was no significant upregulation in ALP and RUNX2 expression in the AA-induced cell sheet. However, the expression levels of late osteoblast differentiation marker (bone gamma-carboxy glutamate protein); cementogenic markers (cementum attachment protein and CP23), and genes encoding extracellular matrix (ECM) proteins [collagen type 1 alpha 1 and integrin beta 1) were higher in AA-induced cell sheets by PDLSCs. ConclusionsThe stimulating effect of AA on cell sheet formation by PDLSCs was confirmed by the expression of typical markers involved in osteogenesis/cementogenesis and ECM secretion, which makes this procedure a prospective option for periodontal tissue regeneration applications.

Dye-Mediated Photo-Oxidation Biomaterial Fixation: Analysis of Bioinductivity and Mechanical Properties of Bovine Pericardium for Use in Cardiac Surgery.

Extracellular matrix bioscaffolds can influence the cardiac microenvironment and modulate endogenous cellular mechanisms. These materials can optimize cardiac surgery for repair and reconstruction. We investigated the biocompatibility and bioinductivity of bovine pericardium fixed via dye-mediated photo-oxidation on human cardiac fibroblast activity. We compared a dye-mediated photo-oxidation fixed bioscaffold to glutaraldehyde-fixed and non-fixed bioscaffolds reported in contemporary literature in cardiac surgery. Human cardiac fibroblasts from consenting patients were seeded on to bioscaffold materials to assess the biocompatibility and bioinductivity. Human cardiac fibroblast gene expression, secretome, morphology and viability were studied. Dye-mediated photo-oxidation fixed acellular bovine pericardium preserves human cardiac fibroblast phenotype and viability; and potentiates a pro-vasculogenic paracrine response. Material tensile properties were compared with biomechanical testing. Dye-mediated photo-oxidation fixed acellular bovine pericardium had higher compliance compared to glutaraldehyde-fixed bioscaffold in response to tensile force. The biocompatibility, bioinductivity, and biomechanical properties of dye-mediated photo-oxidation fixed bovine pericardium demonstrate its feasibility as a bioscaffold for use in cardiac surgery. As a fixed yet bioinductive solution, this bioscaffold demonstrates enhanced compliance and retains bioinductive properties that may leverage endogenous reparative pathways. Dye-mediated photo-oxidation fixed bioscaffold warrants further investigation as a viable tool for cardiac repair and reconstruction.

Methacrylated Silk Fibroin Hydrogels: pH as a Tool to Control Functionality.

The last decade has witnessed significant progress in the development of photosensitive polymers for in situ polymerization and 3D printing applications. Light-mediated sol-gel transitions have immense potential for tissue engineering applications as cell-laden materials can be crosslinked within minutes under mild environmental conditions. Silk fibroin (SF) is extensively explored in regenerative medicine applications due to its ease of modification and exceptional mechanical properties along with cytocompatibility. To efficiently design SF materials, the in vivo assembly of SF proteins must be considered. During SF biosynthesis, changes in pH, water content, and metal ion concentrations throughout the silkworm gland divisions drive the transition from liquid silk to its fiber form. Herein, we study the effect of the glycidyl-methacrylate-modified SF (SilkMA) solution pH on the properties and secondary structure of SilkMA hydrogels by testing formulations prepared at pH 5, 7, and 8. Our results demonstrate an influence of the prepolymer solution pH on the hydrogel rheological properties, compressive modulus, optical transmittance, and network swellability. The hydrogel pH did not affect the in vitro viability and morphology of human dermal fibroblasts. This work demonstrates the utility of the solution pH to tailor the SilkMA conformational structure development toward utility and function and shows the need to strictly control the pH to reduce batch-to-batch variability and ensure reproducibility.

Effects of Sex and 17 β-Estradiol on Cardiac Fibroblast Morphology and Signaling Activities In Vitro.

Several studies have demonstrated estrogen’s cardioprotective abilities in decreasing the fibrotic response of cardiac fibroblasts (CFs). However, the majority of these studies are not sex-specific, and those at the cellular level utilize tissue culture plastic, a substrate with a much higher stiffness than physiological conditions. Understanding the intrinsic differences between male and female CFs under more physiologically “healthy” conditions will help to elucidate the divergences in their complex signaling networks. We aimed to do this by conducting a sex-disaggregated analysis of changes in cellular morphology and relative levels of profibrotic signaling proteins in CFs cultured on 8 kPa stiffness plates with and without 17 β-estradiol (E2). Cyclic immunofluorescent analysis indicated that there was a negligible change in cellular morphology due to sex and E2 treatment and that the differences between male and female CFs occur at a biochemical rather than structural level. Several proteins corresponding to profibrotic activity had various sex-specific responses with and without E2 treatment. Single-cell correlation analysis exhibited varied protein–protein interaction across experimental conditions. These findings demonstrate the need for further research into the dimorphisms of male and female CFs to develop better tailored sex-informed prevention and treatment interventions of cardiac fibrosis.

Cell Properties of Lung Tissue-Resident Macrophages Propagated by Co-Culture with Lung Fibroblastic Cells from C57BL/6 and BALB/c Mice.

Tissue-resident macrophages (Mø) originating from foetal precursors are maintained by self-renewal under tissue/organ-specific microenvironments (niches). We recently developed a simple propagation method applicable to tissue-resident Mø by co-culturing. Here, we examined the properties of lung tissue-resident Mø propagated by co-culturing with lung interstitial cells. The intracardially and intratracheally perfused lung from BALB/c and C57BL/6 mice could minimise the contamination of alveolar Mø and lung monocytes. Lung tissue-resident Mø could be largely propagated under standard culture media along with the propagation of lung interstitial cells demonstrating a fibroblastic morphology. Propagated lung Mø showed characteristic expression properties for Mø/monocyte markers: high expressions of CD11b, CD64 and CD206; substantial expressions of Mertk; and negative expressions of Ly6C, MHC II and Siglec-F. These properties fit with those of lung interstitial Mø of a certain population that can undergo self-renewal. Propagated fibroblastic cells by co-culturing with lung Mø possessed niche properties such as Csf1 and Tgfb1 expression. Propagated lung Mø from both the mouse types were polarised to an M2 phenotype highly expressing arginase 1 without M2 inducer treatment, whereas the M1 inducers significantly increased the iNOS-positive cell percentages in C57BL/6 mice relative to those in BALB/c mice. This is the first study to demonstrate fundamental properties of lung tissue-resident Mø propagated by co-culturing. Propagated lung Mø showing features of lung interstitial Mø can serve as an indispensable tool for investigating SARS-CoV-2 diseases, although lung interstitial Mø have gained little attention in terms of their involvement in SARS-CoV-2 disease pathology, in contrast to alveolar and recruited Mø.

Canine dental pulp and umbilical cord-derived mesenchymal stem cells as alternative sources for cell therapy in dogs

The use of regenerative medicine for pets has been growing in recent years, and an increasing number of studies have contributed to the widespread use of cell therapies in clinical veterinary medicine. Mesenchymal stem cells (MSCs) can be isolated from different sources such as dental pulp and umbilical cord. Aiming safety and reproducibility of cell therapy in clinical practice by using sources easily obtained that are usually discarded, this study isolated, characterized, and evaluated the proliferation and colony formation potential of canine dental pulp-derived mesenchymal stem cells (cDPSCs) and canine umbilical cord tissue (cUCSCs). Three samples from each source were collected, isolated, and cultured. MSCs were differentiated into three lineages and quantified by spectrophotometry. For immunophenotypic characterization, antibodies were used to analyze the expression of cell surface markers, and 7-AAD and Annexin-V were used to analyze cell viability and apoptosis, respectively. For the clonogenic assay, cells were cultured, the colonies were stained, and counted. For the proliferation assay, the cells were plated in flasks for three days and added EdU nucleoside. cDPSCs and cUCSCs showed plastic adherence and fibroblastic morphology after cultivation. Both sources showed differentiation potential and showed CD29 and CD44 positivity and CD14, CD45, CD34 and HLA-DR negativity, and low mortality and apoptosis rates. There was no difference in proliferation rates between sources. Overall, although cUCSCs had a higher number of colony-forming units than cDPSCs, both sources presented MSCs characteristics and can be used safely as alternative sources in cell therapy.

Evaluation of the In Vitro Biocompatibility of PEDOT:Nafion Coatings.

Poly(3,4-ethylenedioxythiophene)-Nafion (PEDOT:Nafion) is emerging as a promising alternative to PEDOT-polystyrene sulfonate (PEDOT:PSS) in organic bioelectronics. However, the biocompatibility of PEDOT:Nafion has not been investigated to date, limiting its deployment toward in vivo applications such as neural recording and stimulation. In the present study, the in vitro cytotoxicity of PEDOT:Nafion coatings, obtained by a water-based PEDOT:Nafion formulation, was evaluated using a primary cell culture of rat fibroblasts. The surface of PEDOT:Nafion coating was characterized by Atomic Force Microscopy (AFM) and water contact angle measurements. Fibroblasts adhesion and morphology was investigated by scanning electron microscopy (SEM) and AFM measurements. Cell proliferation was assessed by fluorescence microscopy, while cell viability was quantified by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT), lactate dehydrogenase (LDH) and neutral red assays. The results showed that PEDOT:Nafion coatings obtained by the water dispersion were not cytotoxic, making the latter a reliable alternative to PEDOT:PSS dispersion, especially in terms of chronic in vivo applications.

From Thermogelling Hydrogels toward Functional Bioinks: Controlled Modification and Cytocompatible Crosslinking.

Hydrogels are key components in bioink formulations to ensure printability and stability in biofabrication. In this study, a well-known Diels-Alder two-step post-polymerization modification approach is introduced into thermogelling diblock copolymers, comprising poly(2-methyl-2-oxazoline) and thermoresponsive poly(2-n-propyl-2-oxazine). The diblock copolymers are partially hydrolyzed and subsequently modified by acid/amine coupling with furan and maleimide moieties. While the thermogelling and shear-thinning properties allow excellent printability, trigger-less cell-friendly Diels-Alder click-chemistry yields long-term shape-fidelity. The introduced platform enables easy incorporation of cell-binding moieties (RGD-peptide) for cellular interaction. The hydrogel is functionalized with RGD-peptides using thiol-maleimide chemistry and cell proliferation as well as morphology of fibroblasts seeded on top of the hydrogels confirm the cell adhesion facilitated by the peptides. Finally, bioink formulations are tested for biocompatibility by incorporating fibroblasts homogenously inside the polymer solution pre-printing. After the printing and crosslinking process good cytocompatibility is confirmed. The established bioink system combines a two-step approach by physical precursor gelation followed by an additional chemical stabilization, offering a broad versatility for further biomechanical adaptation or bioresponsive peptide modification.

Bone-forming perivascular cells: Cellular heterogeneity and use for tissue repair.

Mesenchymal progenitor cells are broadly distributed across perivascular niches-an observation conserved between species. One common histologic zone with a high frequency of mesenchymal progenitor cells within mammalian tissues is the tunica adventitia, the outer layer of blood vessel walls populated by cells with a fibroblastic morphology. The diversity and functions of (re)generative cells present in this outermost perivascular niche are under intense investigation; we have reviewed herein our current knowledge of adventitial cell potential with a somewhat narrow focus on bone formation. Antigens of interest to functionally segregate adventicytes are discussed, including CD10, CD107a, aldehyde dehydrogenase isoforms, and CD140a, among others. Purified adventicytes (such as CD10+ , CD107alow , and CD140a+ cells) have stronger osteogenic potential and promote bone formation in vivo. Recent bone tissue engineering applications of adventitial cells are also presented. A better understanding of perivascular progenitor cell subsets may represent a beneficial advance for future efforts in tissue repair and bioengineering.

Different Conformations of Fibronectin Fibrils Formed Under Cell-Free Condition in Vitro display Distinct Effects on Cell Adhesion and Spreading

Introduction: Insoluble fibrils are the active form of fibronectin (FN) with exposed functional domains capable of interacting with a variety of biomolecules and cells to support many cellular processes. Recent studies show that the formation of FN fibrils can occur in cell-free systems. Currently, the characteristic of FN fibrils and their potential in wound healing application are not fully understood. Our project aims at (i) using urea to induce the formation of FN fibrils with different conformations and (ii) to characterize their conformation-dependent effect on cellular adhesion and spreading.Method: To induce FN fibrillogenesis, native plasma FN at various initial concentrations (0.25, 0.5, 0.75 and 1 mg/ml) was treated by dialysis in urea at concentrations of 1, 2, and 4 M for 16h at room temperature followed by dialysis in PBS buffer (to remove urea). Morphological analysis of formed FN fibrils was conducted bylight microscopy and scanning electron microscopy (SEM). To assess biological effects of FN fibrils on cells in relationto their morphologies, platelet and fibroblast cell adhesion assays were performed. Specifically, platelet rich plasma (5x107 platelets/ml) or fibroblasts (L929, 104 cells/ml) were incubated on surfaces pre-coated with 0.25 and 0.75 mg/ml native plasma FN or FN fibrils for 30 min at 370C.Results: Our cell-free FN fibrillogenesis experiments in vitro indicated that the optimal concentrations of FN and urea were 1 mg/ml and 2 M, respectively, to induce formation of FN fibrils with various conformations. FN only formed minuscule fibrils at 1 M urea while there was no fibril formation at 4 M urea. In contrast, FN fibrils with various conformations ranging from fibrillar FN to non-fibrillar FN (i.e., nodular FN aggregates) were formed when using 2 M urea. Fibril formation was shown to be dependent on the FN concentration. FN at low concentrations (0.25 or 0.5 mg/ml) formed a few insoluble minuscule fibrils or nodular FN aggregates with variation in size ranging from 10 to 50 µm. FN at higher concentrations (0.75 or 1 mg/ml) formed much more nodular FN and fibrillar FN with diameters mostly ranging from 10 to 30 µm that tended to interlace into the fibrillar matrix. SEM analysis documented morphological variations of fibrils formed under different conditions. For example, at low FN concentration (0.25 mg/ml) small fibrils with a smooth surface were formed. By contrast, larger fibrils with attached nodular FN and a resulting rough surface were formed at high FN concentration (≥ 0.75 mg/ml). Adhesion assays indicated that platelets adhered approximately 6 times more firmly on FN fibrils than on native plasma FN (n=3, p < 0.05).On immobilized native FN, adherent platelets were evenly distributed or formed small clusters, while fibrillar FN induced platelet accumulation on the matrix. Fibroblasts adhered both on non-fibrillar FN and matrix of small fibrils (diameter in range of 10-30 µm) but did not interact to larger fibrillar bundles (diameter > 292 µm). Investigations of FN-cell interaction by SEM revealed a structure-dependent effect of FN on fibroblast morphology after adhesion. Specifically, cells remained in spherical shape and separately adhered on nodular FN while extending their shape in multiple directions on the rough surface of FN fibrils.Conclusion: Treatment of native plasma FN by urea at a concentration of 2 M induces irreversible formation of heterogeneous FN fibrils, displaying a rough surface due to attachment of FN nodules. The conformational and compositional changes of the FN matrix can lead to firm adhesion of platelets and fibroblasts and may thus provide a future tool to improve wound healing upon local application. DisclosuresNo relevant conflicts of interest to declare.

Biocompatibility Evaluation of Human and Porcine Acellular Dermal Matrix on Human Primary Gingival Fibroblasts: In Vitro Comparative Study.

Objective Allogeneic and xenogeneic acellular dermal matrix (ADM) grafts have been used to treat periodontal soft tissue defects. The purpose of the current study was to compare the effect of human ADM (AlloDerm) and porcine ADM (Derma) on human primary gingival fibroblasts in vitro regarding the biocompatibility test. Materials and Methods Gingival fibroblasts were obtained from healthy adult gingiva and seeded on AlloDerm or Derma ADM in 96-well plate. The control cells were grown on a surface-treated polystyrene cell-culture plate without matrix. The cells were cultured for 3, 7, and 14 days. The fibroblasts morphology was examined using inverted microscopy, and the cell viability of fibroblasts adherent to the dermal matrix was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay after 3, 7, and 14 days in culture. The data were statistically evaluated by one-way analysis of variance. p -Value of 0.05 was considered significant. Results Gingival fibroblasts adjacent to the AlloDerm and Derma matrices were healthy, attached to the well, and did not exhibit any cytopathic changes similar to control. There were no statistically significant differences in the cell viability between the gingival fibroblasts attached to Derma and AlloDerm on day 3 ( p = 0.841), day 7 ( p = 0.198), and day 14 ( p = 0.788). Conclusion Considering this in vitro study’s limitations, both human and porcine ADM were compatible with the surrounding human primary gingival fibroblasts. No significant differences were observed in the cell viability between the gingival fibroblasts that were attached to Derma and AlloDerm.

Effects of Mechanical Compression on Cell Morphology and Function in Human Corneal Fibroblasts

ABSTRACT Purpose: To explore the effect of mechanical compression (similar to that induced by eye rubbing) on cell morphology, proliferation, apoptosis, and extracellular matrix synthesis and degradation in human corneal fibroblasts. Materials and methods: Human corneal fibroblasts were isolated from corneal lenticule tissue of 10 patients after small incision lenticule extraction surgery. A system was established to mechanically compress corneal fibroblasts with pressure ranging from 0 to 524 Pa. Morphological changes, cell proliferation, apoptosis, and corneal matrix synthesis and degradation were examined using microscopy imaging, bromodeoxyuridine staining, flow cytometry, and qPCR analysis in human corneal fibroblasts after mechanical compression. Results: Human corneal fibroblasts showed short and thick cytoplasmic extensions, as well as a relatively low aspect ratio, suggesting significant morphological alterations caused by high levels of compressive stress. Mechanical compression inhibited cell proliferation and promoted cell apoptosis. Furthermore, compressive stress led to significant elevation in the expression of genes related to extracellular matrix degradation (matrix metalloproteinases MMP1 and MMP9) as early as 6 h after compression and moderate changes in the expression of tissue inhibitors of metalloproteinases. In addition, the mRNA expression levels of COL1A1, lumican, and vimentin were reduced 24 and 48 h after mechanical compression. Conclusions: Mechanical compression alters cell morphology, inhibits proliferation, induces apoptosis, upregulates genes related to extracellular matrix degradation, and downregulates corneal structural genes in human corneal fibroblasts. This study provides evidence that compressive stress significantly influences corneal keratocytes. Our findings suggest that this mechanical effect may be related to keratoconus associated with chronic eye rubbing.