Human Fetal Skin Fibroblasts Research Articles

Senescence-Associated Heterochromatin Foci Suppress γ-H2AX Focus Formation Induced by Radiation Exposure.

DNA damage is induced by both endogenous and exogenous factors. Repair of DNA double-strand break (DSB), a serious damage that threatens genome stability, decreases with senescence. However, the molecular mechanisms underlying the decline in DNA repair capacity during senescence remain unclear. We performed immunofluorescence staining for phosphorylated histone H2AX (γ-H2AX) in normal human fetal lung fibroblasts and human skin fibroblasts of different ages after chronic irradiation (total dose, 1 Gy; dose rate, 1 Gy/day) to investigate the effect of cellular senescence and organismal aging on DSB repair. Accumulation of DSBs was observed with cellular senescence and organismal aging, probably caused by delayed DSB repair. Importantly, the formation of γ-H2AX foci, an early event in DSB repair, is delayed with cellular senescence and organismal aging. These results suggest that the delay in γ-H2AX focus formation might delay the overall DSB repair. Interestingly, immediate γ-H2AX foci formation was suppressed in cells with senescence-associated heterochromatin foci (SAHF). To investigate the relationship between the γ-H2AX focus formation and SAHF, we used LiCl to relax the SAHFs, followed by irradiation. We demonstrated that LiCl rescued the delayed γ-H2AX foci formation associated with cellular senescence. This indicates that SAHF interferes with γ-H2AX focus formation and inhibits DSB repair in radiation-induced DSB. Our results suggest that therapeutic targeting of SAHFs have potential to resolve DSB repair dysfunction associated with cellular senescence.

Human fetal dermal fibroblast-myeloid cell diversity is characterized by dominance of pro-healing Annexin1-FPR1 signaling

Fetal skin achieves scarless wound repair. Dermal fibroblasts play a central role in extracellular matrix deposition and scarring outcomes. Both fetal and gingival wound repair share minimal scarring outcomes. We tested the hypothesis that compared to adult skin fibroblasts, human fetal skin fibroblast diversity is unique and partly overlaps with gingival skin fibroblasts. Human fetal skin (FS, n= 3), gingiva (HGG, n= 13), and mature skin (MS, n= 13) were compared at single-cell resolution. Dermal fibroblasts, the most abundant cluster, were examined to establish a connectome with other skin cells. Annexin1-FPR1 signaling pathway was dominant in both FS as well as HGG fibroblasts and related myeloid cells while scanty in MS fibroblasts. Myeloid-specific FPR1-ORF delivered in murine wound edge using tissue nanotransfection (TNT) technology significantly enhanced the quality of healing. Pseudotime analyses identified the co-existence of an HGG fibroblast subset with FPR1high myeloid cells of fetal origin indicating common underlying biological processes.

Effects of exosomes derived from fibroblast cells on skin wound healing in Wistar rats

BackgroundThe role of exosomes in areas, such as skin wound healing, have been of consideratble interest recently. However, the effects of exosomes derived mainly from fibroblast cells on wound healing have yet to be documented well. The study aimed to evaluate the effects of exosomes derived from fibroblast cells on wound healing in Wistar rats. MethodsHuman fetal skin was isolated afterward centrifuge, and trypsin 0.1% was added to the cells after removing DPBS from the Falcon tube, and the trypsin was removed. The cells were moved to culture flasks. Then, the secondary culture of Human Fetal Skin Fibroblast was done. The pellets containing exosomes were suspended in PBS, and to achieve purified exosomes, the suspended Exosome were passed through a 0.22 µm filter. The exosome solution was kept at − 20 ºC. In the in vivo phase, 48 male Wistar rats were divided into four groups. Group I, low-dose exosome (LDE) solution (150 μl/day), group II high-dose exosome (HDE) solution (300 μl/day), group III commercially available ointment (positive control (PC)) was topically applied on wounds and group VI without treatment (negative control (NC)). A skin biopsy was taken for histopathological analysis. Wound area, depth of ulcer, degree of granulation, and inflammation were assessed. For histopathological assessment, re-epithelialization, inflammatory cells, granulation tissue, crust formation, and collagen maturation (fibrosis) parameters were evaluated. ResultsForty-eight male Wistar rats were included. The HDE group's showed accelerated healing compared to the NC and PC groups at 9 and 12 days. Inflammation and granulation were higher in the HDE, LDE, and PC groups than in the NC group (p < 0.05). The onset of re-epithelialization and collagen deposition was higher in the LDE, HDE, and PC groups, then on nine and 12-day, gradually maturing and extending through the ulcer (p < 0.05). On day 12, in almost all parameters, the LDE and HDE groups showed improved results compared to NC cases (p < 0.05). ConclusionsThe results showed that the utilization of fibroblast-Exo significantly promoted cutaneous wound healing in a rat full-thickness skin ulcer model. This is a potential innovation for cell-free therapy from fibroblast-Exo as a closed structure similar to human cells.

Human Fetal Skin Fibroblast Isolation and Expansion for Clinical Application.

Cell therapy is one of the most hopeful technologies of regenerative medicine approaches. Among various cells, human skin fibroblasts have been progressively used for wound healing as cell-based therapy purposes. By increasing the age, the number of skin fibroblasts' abilities including cell migration, growth, collagen production, etc. decreases. Hence, use of the fetal source is more beneficent. In this respect, this chapter covers the manufacturing of human fetal skin-derived fibroblasts for clinical application.

2-Phenyl-4-quinolone (YT-1) induces G2/M phase arrest and an intrinsic apoptotic mechanism in human leukemia cells

The present study aimed to investigate the biological effects of the new compound 2‑phenyl‑4‑quinolone (YT‑1) on human leukemia cells. Cell viability was determined by propidium iodide (PI) exclusion method followed by flow cytometry. Our results showed that YT‑1 inhibited the cell viability and resulted in morphologic changes to the U937, HL‑60 and K562 cells, respectively. Among them, U937 cells were the most sensitive cell line. On the contrary, YT‑1 had no cytotoxic effects on human fetal skin fibroblast WS1 cells. Flow cytometric analysis indicated that YT‑1 induced G2/M phase arrest and apoptosis (sub‑G1 population) in U937 cells. The presence of apoptotic bodies evidenced by DAPI staining and DNA fragmentation detected by agarose gel electrophoresis further supported the induction of apoptosis in the YT‑1‑treated U937 cells. AnnexinV/PI staining of U937 cells confirmed that the early apoptotic event occurred after YT‑1 exposure. YT‑1 disrupted the mitochondrial membrane potential (ΔΨm) in a time‑dependent manner. YT‑1 increased the protein levels of Bax and Bak but decreased Bcl‑2 and Bid protein levels in U937 cells in a time‑dependent manner. In addition, YT‑1 stimulated the expression of cytochromec and proteolytic activation of caspase‑3 and caspase‑9 after exposure to YT‑1 in U937 cells. In summary, YT‑1 suppressed the viability of U937 leukemia cells through the intrinsic apoptosis pathway. YT‑1 is a potential chemotherapeutic candidate for the treatment of leukemia.

Treatment with hESC-Derived Myocardial Precursors Improves Cardiac Function after a Myocardial Infarction.

BackgroundWe previously reported the generation of a reporter line of human embryonic stem cells (hESCs) with enhanced green fluorescent protein (eGFP) expression driven by the α-myosin heavy chain (αMHC) promoter. The GFP+/αMHC+ cells derived from this cell line behave as multipotent, human myocardial precursors (hMPs) in vitro. In this study, we evaluated the therapeutic effects of GFP+/αMHC+ cells isolated from the reporter line in a mouse model of myocardial infarction (MI).MethodsMI was generated in immunodeficient mice. hMPs were injected into murine infarcted hearts under ultrasound guidance at 3 days post-MI. Human fetal skin fibroblasts (hFFs) were injected as control. Cardiac function was evaluated by echocardiography. Infarct size, angiogenesis, apoptosis, cell fate, and teratoma formation were analyzed by immunohistochemical staining.ResultsCompared with control, hMPs resulted in improvement of cardiac function post-MI with smaller infarct size, induced endogenous angiogenesis, and reduced apoptosis of host cardiomyocytes at the peri-infarct zone at 28 days post-MI.ConclusionIntramyocardial injection of hMPs improved cardiac function post-MI. The engraftment rate of these cells in the myocardium post-MI was low, suggesting that the majority of effect occurs via paracrine mechanisms.

Human fetal skin fibroblasts: Extremely potent and allogenic candidates for treatment of diabetic wounds

The number of patients with diabetes has been expected around 300 million by 2025 and 366 million by 2030 by WHO. On the other hand, diabetic wounds as one of the common complications of diabetes represent major health challenges. Recently, wound care biological products have been proposed for treatment of chronic wounds such as the diabetic wound. Accordingly, tissue-engineered skin substitutes have demonstrated promising effects. Some of these products have used adult skin and neonatal foreskin fibroblasts to produce a tissue-engineered skin substitute. Although adult skin and neonatal foreskin fibroblasts have demonstrated promising effects, but fetal skin fibroblasts and keratinocytes have depicted some unique and considerable properties over adult and neonatal skin cells for instance, skin regeneration with no inflammation and scar formation, low immunogenicity, more VEGF-A secretion than their adult counterparts, immunomodulatory effect by the expression of Indoleamine 2,3 dioxygenase, more resistance to oxidative and physical stresses, etc. On the other hand fetal dermal cells with intrinsic IDO-dependent immunosuppressive activity have introduced them as an allogeneic alternative for treatment of chronic wounds. Therefore, based on the mentioned advantages they are ideal skin substitutes. Accordingly, we suggest that using these cells alone or in combination with biocompatible scaffolds for treatment of different types of ulcers such as diabetic wounds.

Reprint of "Biological characteristics of human fetal skin fibroblasts and MG-63 human osteosarcoma cells on tantalum-doped carbon films"

Tantalum is considered a highly biocompatible metal with high corrosion resistance. Metal-doped amorphous carbon films are gaining interest as an effective surface modifier for medical devices. In this study, tantalum (Ta), TaC/amorphous carbon (a-C), and Ta-containing amorphous hydrogenated carbon films (Ta-C:H) were synthesized using a twin-gun magnetron sputtering system to improve biological properties and explore potential applications for surgical implants or devices. Reactive C2H2, activated by the tantalum plasma in the magnetron sputtering process, was used to deposit the Ta-C:H coatings. The deposited TaC/a-C coatings, in which TaC was embedded in the amorphous carbon matrix as a nanocomposite film, exhibited favorable cell viability of the human osteosarcoma cell line (MG-63), similar to uncoated titanium. Among the examined materials, Ta-C:H coatings exhibited the highest biocompatibility for WS1 human fetal skin fibroblast cells derived from soft tissues and demonstrated the highest biological performance.

Preparation of a thermo- and pH-sensitive nanofibrous scaffold with embedded chitosan-based nanoparticles and its evaluation as a drug carrier

Environmentally sensitive poly(N-isopropylacrylamide) (PNIPAAm) nanofibrous scaffolds loaded with a hydrophilic drug were fabricated via an electrospinning process. First, thermally crosslinkable poly(NIPAAm-co-N-methylolacrylamide) (PNN) was synthesized by redox polymerization below the phase transition temperature of PNIPAAm. The phase transition temperature of the PNN copolymer could be altered from 34 to 40 °C by changing the ratio of N-methylolacrylamide (NMA) to NIPAAm. Subsequently, PNN/chitosan nanofibers were electrospun using ethanol/acetic acid/water as a cosolvent. The PNN/chitosan nanofibers were sensitive to both pH and temperature. The fibrous structure of the soaked PNN/chitosan nanofibers was successfully preserved by the crosslinking of NMA. Furthermore, the chitosan-based nanoparticles (NPs) were introduced into the PNN nanofibers (PNN/NPs) to achieve prolonged drug release. The nanoparticles were observed in the PNN nanofibers by transmission electron microscopy. All of the scaffolds examined had high tensile strengths (1.45 MPa or above) and exhibited no significant cytotoxicity toward human fetal skin fibroblasts. Finally, doxycycline hyclate was used as a model drug. The results illustrated that PNN/NPs nanofibrous scaffolds exhibited continuous drug release behavior for up to 1 week, depending on the pH and temperature.

The differential proliferative response of fetal and adult human skin fibroblasts to TGF-β is retained when cultured in the presence of fibronectin or collagen

BackgroundTransforming growth factor-β is a multifunctional and pleiotropic factor with decisive role in tissue repair. In this context, we have shown previously that TGF-β inhibits the proliferation of fetal human skin fibroblasts but stimulates that of adult ones. Given the dynamic reciprocity between fibroblasts, growth factors and extracellular matrix (ECM) in tissue homeostasis, the present study aims to investigate the role of fibronectin and collagen in the proliferative effects of TGF-β on fetal and adult cells. MethodsHuman fetal and adult skin fibroblasts were grown either on plastic surfaces or on surfaces coated with fibronectin or collagen type-I, as well as, on top or within three-dimensional matrices of polymerized collagen. Their proliferative response to TGF-β was studied using tritiated thymidine incorporation, while the signaling pathways involved were investigated by Western analysis and using specific kinase inhibitors. ResultsFetal skin fibroblast-proliferation was inhibited by TGF-β, while that of adult cells was stimulated by this factor, irrespective of the presence of fibronectin or collagen. Both inhibitory and stimulatory activities of TGF-β on the proliferation of fetal and adult fibroblasts, respectively, were abrogated when the Smad pathway was blocked. Moreover, inhibition of fetal fibroblasts was mediated by PKA activation, while stimulation of adult ones was effected through the autocrine activation of FGF receptor and the MEK–ERK pathway. ConclusionsFetal and adult human skin fibroblasts retain their differential proliferative response to TGF-β when cultured in the presence of fibronectin and unpolymerized or polymerized collagen. General significanceThe interplay between TGF-β and ECM supports the pleiotropic nature of this growth factor, in concordance with the different repair strategies between fetuses and adults. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.

Biological characteristics of human fetal skin fibroblasts and MG-63 human osteosarcoma cells on tantalum-doped carbon films

Tantalum is considered a highly biocompatible metal with high corrosion resistance. Metal-doped amorphous carbon films are gaining interest as an effective surface modifier for medical devices. In this study, tantalum (Ta), TaC/amorphous carbon (a-C), and Ta-containing amorphous hydrogenated carbon films (Ta-C:H) were synthesized using a twin-gun magnetron sputtering system to improve biological properties and explore potential applications for surgical implants or devices. Reactive C2H2, activated by the tantalum plasma in the magnetron sputtering process, was used to deposit the Ta-C:H coatings. The deposited TaC/a-C coatings, in which TaC was embedded in the amorphous carbon matrix as a nanocomposite film, exhibited favorable cell viability of the human osteosarcoma cell line (MG-63), similar to uncoated titanium. Among the examined materials, Ta-C:H coatings exhibited the highest biocompatibility for WS1 human fetal skin fibroblast cells derived from soft tissues and demonstrated the highest biological performance.

Radiation-induced mast cell mediators differentially modulate chemokine release from dermal fibroblasts

Ionizing radiation has been demonstrated to result in degranulation of dermal mast cells. Chemokines are thought to play a crucial role in the early phase of the cutaneous radiation reaction. In human skin, mast cells are located in close proximity to dermal fibroblasts, which thus are a potential target for the action of mast cell mediators. In this study, we evaluated the effects of mast cell-derived histamine, serotonin, tumour necrosis factor (TNF)-α and tryptase on chemokine release from dermal fibroblasts. Human mast cells (HMC-1) were investigated for histamine release and cytokine production after ionizing radiation using enzyme-linked immunosorbent assay (ELISA) and flow cytometry. Receptor expression on human fetal foreskin fibroblasts (HFFF2) and human adult skin fibroblasts (HDFa) was examined by flow cytometry. Chemokine mRNA and protein expression were analyzed by gene array and ELISA, respectively. Ionizing radiation significantly increased histamine release and cytokine expression by HMC-1 cells. Receptors for histamine, serotonin, TNF-α and tryptase were detected both in HFFF2 and in HDFa cells. Dermal fibroblasts constitutively expressed distinct sets of chemokine mRNA. Mast cell mediators differentially affected the release of chemokines CCL8, CCL13, CXCL4 and CXCL6 by fibroblasts. Our data suggest that radiation-induced mast cell mediators have a tremendous impact on inflammatory cell recruitment into irradiated skin. We postulate the activation of mast cells to be an initial key event in the cutaneous radiation reaction, which might offer promising targets for treatment of both normal tissue side effects in radiation therapy and radiation injuries.

Human amniotic fluid stimulates the proliferation of human fetal and adult skin fibroblasts: The roles of bFGF and PDGF and of the ERK and Akt signaling pathways

Fetuses and adults follow different repair strategies for the healing of skin wounds. Experimental evidence indicates that this most probably reflects the intrinsic characteristics of fetal tissue, although environmental factors may also contribute to this phenomenon. Accordingly, the aim of the present study was to investigate the effect of the in utero environment, i.e., amniotic fluid, on one of the major parameters of wound healing, namely cell proliferation, and especially its effect on cultures of both human fetal and adult skin fibroblasts. We found that second trimester human amniotic fluid is a potent stimulant of DNA synthesis and proliferation of cells from both developmental stages. This effect is due to the presence of growth factors, especially basic fibroblast growth factor and platelet-derived growth factor, because inhibitors of their respective receptor kinases and specific neutralizing antibodies can significantly inhibit cell proliferation. Furthermore, we found that this mitogenic effect is mediated through the activation of the MEK/ERK and the PI3K/Akt signaling pathways. Interestingly, we have not observed any significant differences between fetal and adult fibroblasts in their response to amniotic fluid, indicating that cells from both developmental stages respond equally to this complex mixture of regulatory molecules.

Derivation of Induced Pluripotent Stem Cells from Fetal Human Skin Fibroblasts

The isolation and study of autologous human stem cells remain among the most urgent problems in cell biology and biomedicine to date. Induced pluripotent stem cells can be derived from human somatic cells by the overexpression of a number of genes. In this study we reprogrammed fetal human skin fibroblasts by transduction with retroviral vectors carrying murine Oct4 , Sox2 , Klf4 , and c-Myc cDNAs. As a result, cells with the protein expression and gene transcription pattern characteristic of human embryonic stem cells were derived. These induced pluripotent cells are capable of differentiation in vitro into the ectoderm, mesoderm, and endoderm derivatives.

Fetal skin fibroblasts: A cell model for studying the retinoid pathway in congenital diaphragmatic hernia

Although there is strong evidence that genetic factors play a pathogenic role in congenital diaphragmatic hernia (CDH), few causal genes have been identified in humans. A number of studies, essentially in animal models, have suggested that disruption of the retinoid signaling pathway plays a major role in the pathogenesis of CDH. Our hypothesis is that human fetal skin fibroblasts express some metabolic and molecular actors of the retinoid pathway and that they offer convenient cellular material for investigating the molecular retinoid pathway defects associated with CDH. We first established the expression of receptors, enzymes and binding proteins involved in the retinoic acid (RA) pathway in non-CDH fetal skin fibroblasts using RT-PCR and immunocytochemistry approaches. We then studied the expression of these genes in skin fibroblasts from seven fetuses with isolated and nonisolated CDH. Fetal skin fibroblasts expressed enzymes involved in RA metabolism as well as nuclear receptors for signal transduction. Basal levels of retinoic acid receptor, retinaldehyde dehydrogenase 2, and CYP26 (cytochrome P450 RAI) expression were altered in two of seven fetuses. Interestingly, these genes were previously described as abnormally expressed in CDH physiopathology. Our results suggest that human fetal skin fibroblasts could be useful for studying retinoid signaling pathway disruption in the context of CDH. Our proposal is strengthened by the fact that we identified CDH fetuses for which molecular and metabolic actors of the retinoid pathway were not detected.

Amphiphilic copolymers for liquid bandage application studies

This research pertains to a new class of liquid bandage polymers which are promising for assisting advanced wound healing by serving as substrates to promote cell viability and proliferation. Amphiphilic nitrogen-containing polymer poly (3-methacryloyloxypropyltris (trimethylsiloxy)silane-co-N-isopropylacrylamide) (poly (TRIS-co-NIPAM)) was synthesized and investigated with further comparison to several different wound care polymers including commercialized 3M Nexcare No Sting Liquid Bandage. Cell viability on different polymers was tested on fetal human skin fibroblasts (HSFs) and neonatal human epidermal keratinocytes (HEKs). Test results were quantified by Sulforhodamine B (SRB) in vitro cytotoxicity assay. It was demonstrated that both HSFs and HEKs survive better on the poly (TRIS-co-NIPAM) film as the cell seeding substrate compared to other candidate polymer formulations, as well as to the commercial 3M No Sting Liquid Bandage polymer. Thus we conclude that wound healing could be accelerated by this new class of liquid bandage polymer, particularly for early-stage wounds..

Cytotoxicity, Antimicrobial Activity and Composition of Essential Oil from Tanacetum balsamita L. Subsp. Balsamita

The aerial parts of Tanacetum balsamita subsp. balsamita were collected at full flowering stage from Tabriz, Iran. The essential oil was isolated by hydrodistillation and analyzed by combination of capillary GC and GC-MS. Twenty seven components were identified, representing 99.2% of the total oil. Carvone (51.0%), beta-thujone (20.8%), 1,8-cineole (4.4%) and alpha-thujone (3.2%) were the main components. The in vitro antimicrobial activity of the essential oil of T. balsamita subsp. balsamita was studied against eight Gram-positive and Gram-negative bacteria (Bacillus subtilis, B. pumulis, Enterococcus faecalis, Staphylococcus aureus, S. epidermidis, Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae) and three fungi (Candida albicans, Saccharomyces cerevisiae and Aspergillus niger). The results of the antimicrobial activity of the essential oil according to the disc diffusion method and MIC values indicated that the oil exhibited moderate to high antimicrobial activity. The in vitro toxicological study based on the MTT cytotoxicity assay on human fetal skin fibroblast (HFSF) and Monkey kidney (Vero) cell lines showed IC50 values of 2500 and 1250 microg/mL, respectively.

64 RECONSTRUCTION OF HETEROGENEOUS EMBRYOS BY HUMAN SOMATIC CELLS AND BOVINE ENUCLEATED OOCYTES AND ISOLATION OF PUTATIVE HUMAN EMBRYONIC STEM CELL CLONES

This study was undertaken to reconstruct heterogeneous nuclear-transferred embryos by using human fetal skin fibroblast cells as nuclear donor cells and the enucleated bovine oocytes as recipient cytoplasts for the purpose of investigating the feasibility of enucleated bovine oocyte cytoplasm as a means of reprogramming human somatic cell nuclei in an attempt to generate an accessible, autologous, and potentially unlimited source of totipotent human embryonic stem cells for transplantation medicine. Bovine ovaries were recovered at a local abattoir and oocytes were in vitro-matured and employed as recipient cytoplasts. A human fibroblast cell line was derived from an aborted fetus at 4 months of age, serum-starved, and used as donor somatic cells. The cultured nuclear transfer embryos were visually assessed for the first completion of cleavage at 48 h of culture, and for subsequent developmental stages at 72 to 168 h. The fusion of fibroblast cells into recipient cytoplasm was induced by electroporation. The fused oocytes were activated by ionomycin with 2 m mL–1 6-DMAP. The activated reconstructed embryos were co-cultured with bovine cumulus cells in synthetic oviduct fluid supplemented with amino acid (SOFaa) and 10% (v/v) fetal calf serum (FCS) for 168 h. Morulae and blastocysts were used for isolating embryonic stem cells. The results indicated that human fetal skin fibroblast cells could maintain normal morphology and characteristics in culture conditions. They proliferated constantly and presented a regular growth curve in culture. Of these cells, 83.3% retained normal numbers of chromosomes after over 20 culture passages. The skin fibroblast cells exhibited normal morphology and retained normal numbers of chromosomes (2n = 64) in serum starvation culture after undergoing freezing and thawing treatment. The first completed cleavage of xenonuclear transplantation human embryos occurred between 24 and 48 h after activation, and morula and blastocyst development was completed between 72 and 168 h. The cleavage rate and the percentage of blastocyst development of the reconstructed embryos were 80.0% and 7.5%, respectively. Putative embryonic stem cell clones were observed, with nest-like morphology, after 3–7 days of culture on a fibroblast cell layer. Identifications by alkaline phosphatase (AKP) showed that the clones presented a positive reaction, which demonstrated that the isolated stem cell clones were embryonic stem cells. This study demonstrated that xenonuclear-transferred human embryos can undergo embryonic division and subsequent development to morula and blastocyst stage, and that human fetal fibroblast nuclei can be reprogrammed in bovine enucleated oocytes. Xenonuclear-transferred human embryos can be an alternative for obtaining human embryonic stem cells.

Preparation of cell-specific membranes for bone regeneration by peptide grafting

In the present work, cell-specific peptides were immobilized on chitosan and PLA scaffolds. The amount of immobilization, determined by HPLC, was confirmed to be in the effective range. Results from cultures of human fetal skin fibroblasts, rat osteosarcoma, and periodontal fibroblasts demonstrated that the immobilization of cell-specific peptides could effectively enhance the attachment and mineralization of osteoblastic cells on scaffolds. The results indicate that, with immobilization of osteoblast-specific peptides, scaffolds with specificity to osteoblastic cells can be obtained which are believed to be of practical use for generation of bone-like tissues.

Specific inactivation of cysteine protease-type cathepsin by singlet oxygen generated from naphthalene endoperoxides

Singlet oxygen is a causal factor in light-induced skin photoaging and the cytotoxic process of tumor cells in photodynamic chemotherapy. To develop a better understanding of the functional consequences of protein modification by singlet oxygen, the effects of naphthalene endoperoxide on lysosomal protease, cathepsin, were examined. When the soluble fraction of normal human fetal skin fibroblast cells was treated with the endoperoxide, the activities of cysteine proteases, cathepsins B and L/S, were inhibited, but that of aspartate protease, cathepsin D/E, was not. The reduction of the endoperoxide-treated soluble fractions by treatment with dithiothreitol barely recovered the activities. Cathepsin B, purified from normal human liver, exhibited similar profiles to that in cytosol. These data suggest that singlet oxygen oxidatively modifies an amino acid residue essential for catalysis and consequently results in the irreversible inactivation of cysteine protease-type cathepsin.