To determine whether platelet-rich fibrin lysate (PRF-L) could restore the function of chronically ultraviolet-A (UVA)-irradiated human dermal fibroblasts (HDFs), we isolated and sub-cultured HDFs from six different human foreskins. HDFs were divided into two groups: those that received chronic UVA irradiation (total dosages of 10 J cm-2) and those that were not irradiated. We compared the proliferation rates, collagen deposition, and migration rates between the groups and between chronically UVA-irradiated HDFs in control and PRF-L-treated media. Our experiment showed that chronic UVA irradiation significantly decreased (p<0.05) the proliferation rates, migration rates, and collagen deposition of HDFs, compared to controls. Compared to control media, chronically UVA-irradiated HDFs in 50% PRF-L had significantly increased proliferation rates, migration rates, and collagen deposition (p<0.05), and the migration rates and collagen deposition of chronically UVA-irradiated HDFs in 50% PRF-L were equal to those of normal fibroblasts. Based on this experiment, we concluded that PRF-L is a good candidate material for treating UVA-induced photoaging of skin, although the best method for its clinical application remains to be determined.
Blood Platelets/*cytology/*metabolism ; Cell Movement/radiation effects ; Cell Proliferation/radiation effects ; Cells, Cultured ; Collagen/metabolism ; Fibrin/*metabolism ; Fibroblasts/*cytology/metabolism/*radiation effects ; Humans ; Skin/*cytology ; Time Factors ; Ultraviolet Rays/*adverse effects

OBJECTIVETo observe the effect of solar infrared ray (IR) radiation on the expressions of c-Jun and collagens I and III in cultured human skin fibroblasts (HSFs) and explore the molecular mechanism by which IR radiation causes aging of the skin.

METHODSPrimarily cultured HSFs exposed to IR radiation were examined for changes of the cell viability with MTT assay. The mRNA and protein expressions of c-Jun and collagens I and III was detected with real-time quantitative PCR and immunocytochemistry.

RESULTSMTT assay showed that IR irradiation caused inhibition of cell proliferation compared with the control cells. The mRNA and protein expression of collagen I was decreased significantly by IR irradiation with the increase of the irradiation dose (P<0.01). HSFs irradiated by IR for 12 h showed a dose-dependent reduction of the expression of collagen type III mRNA and protein (P<0.05, P<0.01), but the expression increased dose-dependently in response to IR exposure for 24 h (P<0.05 or 0.01). IR irradiation enhanced the mRNA and protein expression of c-Jun in a dose-dependence manner (P<0.05 or 0.01).

CONCLUSIONSIR irradiation can increase the expression of c-Jun, inhibit the expression of collagen I, and cause disturbance in collagen III expression in human skin fibroblasts, which may be one of the mechanism of IR radiation to initiate and promote skin photoaging.

Cell Proliferation ; Cell Survival ; Cells, Cultured ; Collagen Type I ; metabolism ; Collagen Type III ; metabolism ; Fibroblasts ; metabolism ; radiation effects ; Humans ; Infrared Rays ; Proto-Oncogene Proteins c-jun ; metabolism ; RNA, Messenger ; metabolism ; Skin ; cytology ; Skin Aging ; Ultraviolet Rays

OBJECTIVEThe aim of this review is to comprehensively and unbiasedly summarize the improvements in the techniques for classical corneal collagen cross-linking (CXL) by covering the reasons for this improvement, measure, and effect to approach the future direction of the CXL.

DATA SOURCESAll articles used in this review were mainly retrieved from the PubMed database.

STUDY SELECTIONOriginal articles and reviews were selected if they were related to the improvement in the technique of classical CXL. Data were mainly extracted from 94 articles, which are listed in the reference section of this review.

RESULTSThis innovative research involves every step such as instrument preparation, epithelial management, riboflavin instillation, and UVA irradiation. These clinical and experimental results seem promising.

CONCLUSIONSCXL treatment is the only recent promising method for preventing the progress of keratoconus. The limitations and potential complications that accompany classical CXL such as corneal thickness limitations, ultraviolet-A (UVA) light injury, and the impact of de-epithelialization encourage people to research new improvements in techniques. While this research needs to be further investigated, we hope our review can help related researchers and patients.

Collagen ; metabolism ; Cornea ; metabolism ; radiation effects ; Humans ; Ultraviolet Rays

Mesenchymal stem cells (MSCs) are multipotent stem cells that differentiate into a variety of cell types. Low frequency pulsed electromagnetic fields (LFPEMFs) therapy can causes biochemical changes at the cellular level to accelerate tissue repair in mammals. So, we tested the hypothesis that LFPEMFs can promote chondrogenic differentiation of rat bone marrow-derived mesenchymal stem cells (rBMSCs) in vitro. The rBMSCs were isolated by adherence method and the third-generation of the rBMSCs were randomly divided into LFPEMFs groups, chondrocyte-induced group and control group. LFPEMFs groups with complete medium were exposed to 50Hz, 1mT PEMFs for 30 min every day, lasting for 10, 15 and 20 d, respectively. Chondrocyte-induced group were treated with chondrogenic media, while control groups were only cultured with complete medium. The mRNA expressions of type II-collagen (Col II) and aggrecan were determined by Real-time fluorescent quantitation PCR. The protein expression of Col II and aggrecan were detected with toluidine blue stain or immunocytochemical stain, respectively. The result showed that the mRNA and protein expression level of Col-II and aggrecan were significantly higher in the LFPEMFs group or chondrocyte-induced group, compared to the control group. It suggest that LFPEMFs could contribute to rBMSCs to differentiate into chondrogenic differentiation in vitro.
Animals ; Bone Marrow Cells ; cytology ; Cell Differentiation ; radiation effects ; Cells, Cultured ; Chondrocytes ; cytology ; Collagen Type II ; genetics ; metabolism ; Electromagnetic Fields ; Male ; Mesenchymal Stromal Cells ; cytology ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo analyze the potential mechanism of preventive and therapeutic effects of (90)Sr on hypertrophic scar, and to observe its clinical effect.

METHODSFibroblasts isolated from human hypertrophic scar were cultured in vitro and radiated by (90)Sr with the dose varying from 0 Gy (control group) to 5 Gy (LD group), 10 Gy (MD group), and 15 Gy (HD group). The cell cycle and apoptosis rate were determined by flow cytometry at post radiation hour (PRH) 24, 48, and 72. The concentration of type I collagen in cell supernatant was detected by enzyme-linked immunosorbent assay (ELISA). Therapeutic effects of (90)Sr radiation were evaluated among 348 patients with hypertrophic scars, 40 patients with keloids, and 114 patients for scar prevention after surgical operation. The number of fibroblasts after HE staining was compared among normal skin tissue, hypertrophic scar, and hypertrophic scar treated with (90)Sr radiation. Data were processed with one-way analysis of variance and q test.

RESULTS(1) Apoptotic rates in MD and HD groups at PRH 48 were higher than those at PRH 24, and the apoptotic rate was similar between MD group and HD group at PRH 72. Apoptotic rate in LD group at PRH 48 was significantly higher than that at PRH 24, but it decreased rapidly at PRH 72, which was significantly lower than those in MD and HD groups (with F values all equal to 916.711, P values all below 0.01). (2) At PRH 24, cell ratios of each phase in LD and HD groups were similar, and cell ratio of S phase in HD group [(48.1 ± 1.0)%] was higher than those in the other three groups (with F values all equal to 200.277, P values all below 0.01). At PRH 72, cell ratio of S phase in MD and HD groups was respectively (85.7 ± 5.2)%, (73.0 ± 8.4)%, implying that cells were blocked in S phase, and the values were all higher than those in control and LD groups (with F values all equal to 111.105, P values all below 0.01). (3) At the same time point, the concentration of type I collagen decreased along with the increase of radiation dose (with F values from 5044.449 to 8234.432, P values all below 0.01). With the same radiation dose, the concentration of type I collagen increased along with prolongation of time (with F values from 333.395 to 2973.730, P values all below 0.01). (4) Clinical observation showed the (obvious) effective rate of radiation for pathological scars and that for scar prevention after surgical operation added up to 88.45%. The number of fibroblasts per 200 times visual field in patients after (90)Sr radiation (86 ± 20) was less than that in patients without treatment [(198 ± 65), F = 208.405, P < 0.05].

CONCLUSIONSThe effect of (90)Sr radiation on fibroblasts and extracellular matrix can contribute to inhibition of scar formation, and the clinical effect is significant.

Adolescent ; Adult ; Apoptosis ; radiation effects ; Cell Cycle ; radiation effects ; Cells, Cultured ; Child ; Child, Preschool ; Cicatrix, Hypertrophic ; metabolism ; pathology ; radiotherapy ; Collagen Type I ; metabolism ; Female ; Fibroblasts ; radiation effects ; Humans ; Male ; Strontium Radioisotopes ; therapeutic use ; Young Adult

PURPOSE: The purpose of this study is to investigate the mechanism of cellular proliferation of electromagnetic field (EMF) on human intervertebral disc (IVD) cells. MATERIALS AND METHODS: Human IVD cells were cultured three-dimensionally in alginate beads. EMF was exposed to IVD cells with 650Omega, 1.8 millitesla magnetic flux density, 60 Hz sinusoidal wave. Cultures were divided into a control and EMF group. Cytotoxicity, DNA synthesis and proteoglycan synthesis were measured by MTT assay, [3H]-thymidine, and [35S]-sulfate incorporation. To detect phenotypical expression, reverse transcription-polymerase chain reactions (RT-PCR) were performed for aggrecan, collagen type I, and type II mRNA expression. To assess action mechanism of EMF, IVD cells were exposed to EMF with NG-Monomethyl-L-arginine (NMMA) and acetylsalicylic acid (ASA). RESULTS: There was no cytotoxicity in IVD cells with the EMF group in MTT assay. Cellular proliferation was observed in the EMF group (p < 0.05). There was no difference in newly synthesized proteoglycan normalized by DNA synthesis between the EMF group and the control. Cultures with EMF showed no significant change in the expression of aggrecan, type I, and type II collagen mRNA compared to the control group. Cultures with NMMA (blocker of nitric oxide) or ASA (blocker of prostaglandin E2) exposed to EMF demonstrated decreased DNA synthesis compared to control cultures without NMMA or ASA (p < 0.05). CONCLUSION: EMF stimulated DNA synthesis in human IVD cells while no significant effect on proteoglycan synthesis and chondrogenic phenotype expressions. DNA synthesis was partially mediated by nitric oxide and prostaglandin E2. EMF can be utilized to stimulate proliferation of IVD cells, which may provide efficient cell amplification in cell therapy to degenerative disc disease.
Adult ; Aspirin/pharmacology ; Cell Proliferation/*radiation effects ; Collagen/metabolism ; Dinoprostone/metabolism ; *Electromagnetic Fields ; Enzyme Inhibitors/pharmacology ; Female ; Humans ; Intervertebral Disk/*pathology/radiation effects ; Male ; Middle Aged ; Nitric Oxide/metabolism ; Tetrazolium Salts/pharmacology ; Thiazoles/pharmacology ; omega-N-Methylarginine/pharmacology

OBJECTIVETo investigate the effects of preemptive freezing with different temperature and cross-linking methods on the ultrastructure of collagen membrane and its influence on human fibroblast proliferation.

METHODSBovine collagen type I solution in concentration of 10 g/L was preliminarily frozen at -20 degrees C or - 80 degrees C for 12 hours, and lyophilized at -70 degrees C for 48 hours. The diameter of apertures in collagen membranes prepared with two different preliminary temperatures were observed by scanning electron microscope (SEM) and compared. The preliminary freezing temperature of - 80 degrees C was used for the following study. The apertures of collagen membrane performed with cross-linking glutaraldehyde and ultraviolet (UV) radiation cross-linking with glutaraldehyde (double cross - linking) after preliminary freezing were also compared. The proliferation of human fibroblasts inoculated in above cross-linking collagens were assessed by MTT assay, in terms of absorption value.

RESULTSThe mean diameter of apertures of collagen membrane pre-frozen at -20 degrees C was (172 +/- 374 microm, while that at -80 degrees C was (99 +/- 24) microm. The apertures of collagen membrane were reduced in size after glutaraldehyde cross-linking, while those of double cross-linking showed no change in size. There was obvious difference in absorption value of fibroblasts 8 days after seeding between above two cross-linking methods (1.534 +/- 0.013 for glutaraldehyde cross-linking, 3.778 +/- 0.010 for double cross-linking, P < 0.05).

CONCLUSIONThe collagen membrane after preliminary freezing at - 80 degrees C and double cross-linking with UV radiation and glutaraldehyde may be used as a dermal skeleton substitute.

Animals ; Cattle ; Cells, Cultured ; Coculture Techniques ; Collagen ; ultrastructure ; Cross-Linking Reagents ; chemistry ; Dermis ; cytology ; Fibroblasts ; cytology ; drug effects ; radiation effects ; Freezing ; Glutaral ; chemistry ; Humans ; Skin, Artificial ; Tissue Scaffolds ; Ultraviolet Rays

BACKGROUNDConnective tissue growth factor (CTGF) is a potent fibrogenic cytokine which has been associated with progressive glomerulosclerosis and tubulointerstitial fibrosis. We investigated the role of CTGF on the progression of a rat model of radiation nephropathy.

METHODSThe model of radiation nephropathy in rats was established as follows: control group (n = 12), underwent only laparotomy; irradiated group (n = 20), underwent a laparotomy, then the rats were subjected to a single dose 25 Gy X-ray to the kidneys. The rats were followed up one, three, six and nine months after renal exposure to radiation.

RESULTSRenal dysfunction was noted early in irradiated rats. Histological analysis showed focal glomerular sclerotic lesions at an early stage after irradiation. Radiation-induced glomerular and tubulointerstitial injuries were particularly severe the sixth month after irradiation as compared to the control group (P < 0.01). By immunohistochemistry, increased expression of CTGF was noted in the irradiated kidneys, which began to increase from the first month after irradiation, and remained significantly higher at the sixth and ninth month after irradiation (P < 0.01). Upon Western blot analysis CTGF protein expression showed an increase in the radiation treated kidneys compared with the control rats. The expression of CTGF closely correlated with the progression of radiation nephropathy. The expression of alpha-smooth muscle actin, vimentin, type III collagen and type IV collagen was also high in the irradiated kidney as compared to control rat kidneys (P < 0.05), and was most severe at the sixth and ninth month after irradiation (P < 0.01). By double immunostaining, CTGF expressing cells were found to be alpha-SMA-positive myofibroblasts and vimentin-positive tubular epithelial cells. Glomerular expression of CTGF closely correlated with the glomerular expression of alpha-SMA (r = 0.628, P < 0.01), vimentin (r = 0.462, P < 0.05) and accumulation of type IV collagen (r = 0.584, P < 0.01) in the irradiated kidney. Similarly, the expression of CTGF was positively correlated with the expression of alpha-SMA (r = 0.613, P < 0.01), vimentin (r = 0.629, P < 0.01), deposition of type III collagen (r = 0.741, P < 0.001) and type IV collagen (r = 0.799, P < 0.0001) in the tubulointerstitium of the irradiated kidney. Finally the expression of CTGF after the irradiation of the kidney positively correlated with the levels of blood urea nitrogen and serum creatinine.

CONCLUSIONOverexpression of CTGF may play an important role in the development and progression of glomerulosclerosis and tubulointerstitial fibrosis in radiation nephropathy.

Actins ; analysis ; Animals ; Collagen Type III ; analysis ; Collagen Type IV ; analysis ; Connective Tissue Growth Factor ; analysis ; physiology ; Kidney ; radiation effects ; Kidney Diseases ; etiology ; Male ; Radiation Injuries, Experimental ; etiology ; Rats ; Rats, Wistar ; Vimentin ; analysis

PURPOSE: To determine the levels of bone and cartilage turnover markers in men with ankylosing spondylitis (AS) and to investigate their associations with disease activity, bone mineral density, and radiographic damage of the spine. PATIENTS AND METHODS: This cross-sectional study enrolled 35 men with newly diagnosed AS. The bone mineral densities (BMD) of their lumbar spines and proximal femurs, Bath AS Disease Activity Index (BASDAI), and Bath AS Radiographic Index (BASRI) were evaluated. Urinary C-terminal telopeptide fragments of type I collagen (CTX-I) and type II collagen (CTX-II) levels were determined by enzyme-linked immunosorbent assay, and serum levels of bone-specific alkaline phosphatase (BALP) and osteocalcin were determined by an enzyme immunoassay. Levels of biochemical markers were compared with those of 70 age-matched healthy men. RESULTS: Patients with AS had significantly higher mean urinary CTX-I and CTX-II levels than control subjects (p < 0.05). Elevated urinary CTX-I levels correlated well with BASDAI, femoral BMD, and femoral T score (p < 0.05), and elevated urinary CTX-II levels correlated well with spinal BASRI (p < 0.05) in patients with AS. Mean serum BALP and osteocalcin levels did not differ between patients and controls and did not show any significant correlations with BMD, BASDAI, or BASRI in men with AS. Conclusions: Elevated CTX-I reflects disease activity and loss of femoral BMD while elevated CTX-II levels correlate well with radiographic damage of the spine, suggesting the usefulness of these markers for monitoring disease activity, loss of BMD, and radiographic damage in men with AS.
Adolescent ; Adult ; Alkaline Phosphatase/blood ; Biological Markers/*analysis/blood/urine ; *Bone Density ; Bone and Bones/*metabolism/radiation effects ; Cartilage/metabolism/radiation effects ; Collagen Type I/urine ; Collagen Type II/urine ; Cross-Sectional Studies ; Enzyme-Linked Immunosorbent Assay ; Humans ; Immunoassay/methods ; Male ; Osteocalcin/blood ; Spondylitis, Ankylosing/*metabolism

Infrared radiation is increasingly and uncritically used for cosmetic and wellness purposes, despite the poorly understood biologic effects of such treatments on humans. In the present study, we investigated the effects of infrared radiation on collagen and elastin production in dermal fibroblasts, as well as the clinical and histopathologic effects of infrared radiation on photo-aged facial skin lesions. In order to determine the effects of infrared radiation on collagen and elastin production, dermal fibroblasts were exposed to infrared radiation for varying lengths of time and collagen and elastin contents were subsequently determined. Additionally, 20 patients with mild to moderate facial wrinkles and hyperpigmented lesions received daily treatments of far infrared radiation (900 to 1000micronm) for six-months. During the treatment, patients and a medical observer conducted independent photographic and clinical evaluations every 4 weeks, and skin biopsies were obtained for histological analysis at baseline and one month post-treatment. We found that the content of collagen and elastin produced by the fibroblasts increased after infrared radiation, and that this increase was proportional to the duration of irradiation exposure. Following 6 months of treatment, all patients reported good (51-75%) improvements in skin texture and roughness. Additionally, patients noted fair (25-50%) improvement in color tone of the skin; however, improvements in hyperpigmented lesions were not observed. Objective medical evaluation of the patients indicated that roughness and laxity were fairly improved, but there was no significant improvement in hyperpigmented lesions. Histological examination failed to reveal any differences as well. These results suggest that infrared radiation may have beneficial effects on skin texture and wrinkles by increasing collagen and elastin contents from the stimulated fibroblasts. Therefore, skin treatment with infrared radiation may be an effective and safe non-ablative remodeling method, and may also be useful in the treatment of photo-aged skin.
Time Factors ; Skin Pigmentation/*radiation effects ; *Skin Aging ; Skin/*radiation effects ; Middle Aged ; *Light ; *Infrared Rays ; Humans ; Fibroblasts/metabolism ; Female ; Elastin/metabolism ; Collagen/metabolism ; Biopsy ; Adult