Continuous hypoxic culturing maintains activation of Notch and allows long-term propagation of human embryonic stem cells without spontaneous differentiation.

OBJECTIVE: The maintenance of pluripotency of human embryonic stem cells (hESCs) requires a high efficiency of self-renewal. During in vitro propagation, however, hESCs have a propensity to differentiate spontaneously. In this study, we assessed the nature of hESC responses to hypoxic conditions. MATERIALS AND METHODS: Human embryonic stem cells were grown in normoxic and hypoxic conditions, and the cells expressing Oct4 and stage-specific embryonic antigen-1 were identified by indirect immunofluorescence. The transcriptional expression of Nanog, Notch1, and Oct4 was determined by a real-time reverse transcription-polymerase chain reaction, and the inhibition of Notch-mediated signalling was achieved with a gamma-secretase inhibitor. RESULTS: In contrast to culture at 21% oxygen, where the colonies displayed a marked degree of differentiation, we found that during exposure to 5% oxygen, the hESC colonies displayed a homogenous and flat morphology that was consistent with the presence of Oct4-positive phenotype, indicating no spontaneous differentiation. When cultured at 5% oxygen for either 4 weeks or up to 18 months, high levels of Nanog and Notch1 transcriptional expression were detected, albeit the expression was significantly lower during longer exposure. The suppression of differentiation was rapidly reversed on transfer of the hypoxic cultures to normoxic conditions. Looking into the molecular mechanisms of the maintenance of self-renewal at low oxygen tensions, we found that inhibition of Notch signalling fully abrogated the hypoxic induction of undifferentiated phenotype. CONCLUSION: Our data, thus, indicate that hypoxic exposure has the capacity to sustain long-term self-renewal of hESCs and that this effect is mediated through activation of Notch.

Evaluation of potential organ culture media for eye banking using human donor corneas.

AIM: To evaluate the ability of different commercially available cell culture solutions to preserve human donor corneas during 3 weeks of "closed system" organ culture at physiological temperature. This screening was performed in an attempt to establish a rational basis for the development of a serum-free organ culture medium for eye banking. METHODS: 72 normal human donor corneas were organ cultured for 21 days at 31 degrees C in eight different test media (nine corneas in each group). The basic culture solutions included: minimal essential medium (MEM), MEM with stabilised L-glutamine, M199, DIF-1000, SFM, F99, and F99 with ascorbic acid, insulin, bFGF, transferrin, selenium, and lipids (termed F99-Sr). All media were supplemented with 2% fetal calf serum (FCS), except for MEM, which was also studied at 8% FCS. The evaluation parameters included: (1) the endothelial cell loss as evaluated using trypan blue staining; (2) the ability of keratocytes and endothelial cells to incorporate tritiated uridine into RNA as evaluated using autoradiography and digital image analysis; (3) the leakage of immunogenic keratan sulphate as assessed using ELISA; and (4) changes in storage medium pH, glucose, and lactate content. RESULTS: SFM induced the lowest endothelial cell loss of 14% (SD 2%) and the highest RNA synthesis rates of all test solutions supplemented with 2% FCS. Corneas stored in SFM also showed the least leakage of keratan sulphate and the highest glucose consumption and lactate production. In five media (MEM with 2% FCS, MEM with stabilised L-glutamine, M199, F99, and F99-Sr), comparable and intermediate potentials for organ culture were observed with endothelial cell loss of 16-19%. By contrast, 29% (4%) of the endothelium was lost after storage in DIF-1000. Interestingly, the use of 8% FCS (in MEM) had a marked protective effect on the endothelium, which showed the highest RNA synthetic activity combined with a cell loss of only 11% (4%), compared with 19% (6%) at 2% FCS (p<0.05). CONCLUSION: Among the present test solutions, SFM appears to be the most prominent candidate for a new corneal organ culture medium and should be further tested and possibly refined to effectively substitute serum addition.

Treatment of post-keratoplasty astigmatism by topography supported customized laser ablation.

PURPOSE: To evaluate the clinical and optical efficiency of topography modulated customized corneal ablations for irregular corneal astigmatism. MATERIAL & METHODS: Sixteen eyes of 16 patients with iatrogenic corneal astigmatism (post keratoplasty) were consecutively included. Based on preoperative corneal topographic measurements height deviations from a spherical corneal shape were calculated and transferred to a flying-spot excimer laser. Photo-refractive keratectomy of the topographic irregularities was then performed. Clinical and optical efficiency was evaluated by best corrected visual acuity and by computation of corneal wavefront aberrations before and up to one year after treatment. Wavefront aberrations were decomposed by Zernike polynomial analysis. RESULTS: Before treatment the average best-corrected visual acuity was 0.23. Three and 12 months after PRK the average best-corrected visual acuity had increased to 0.37 (p<0.05) and 0.45 (p<0.05), respectively. Corneal wavefront aberrations (root-mean-square) were 3.35 before surgery and 1.88 (p<0.05) and 1.51 (p<0.05) at three and 12 months after treatment. Zernike polynomial decomposition of the wavefront aberrations revealed that regular corneal astigmatism was the most important aberration component before and after surgery. Regular astigmatism was significantly decreased by the procedure, whereas coma, spherical aberrations, and higher-order aberrations were not reduced significantly. CONCLUSION: Topography modulated photorefractive keratectomy of highly astigmatic corneal grafts can improve best corrected visual acuity and reduce corneal wavefront aberrations. Even in apparently irregular topographic astigmatism, regular astigmatic wavefront aberration may be the most important contributor to wavefront errors.

Human lens thioltransferase: cloning, purification, and function.

PURPOSE: To clone the human lens thioltransferase (TTase) gene and to purify, characterize and study the possible function of the recombinant human lens thioltransferase (RHLT). METHODS: The human lens TTase gene was cloned by using RT-PCR and verified by sequence and RNase protection assay. TTase overexpressed in Escherichia coli was isolated and purified to homogeneity by column chromatography and identified by Western blot analysis. The activity was assayed with a synthetic substrate hydroxyethyl disulfide. Its function in dethiolating and reactivating other key metabolic enzymes was studied by using pure glutathione S:-transferase (GST) and glutathione peroxidase (GPx) from commercial source and also with the cell extract of rabbit lens epithelial cells preexposed to H2O2. RESULTS: The cloned human lens TTase gene showed identical sequence to the TTase gene from other human tissues. The RNase protection assay displayed a single transcript from the total RNA of human lens epithelial cells. The purified RHLT had a molecular weight of 11.8 kDa and reacted positively with anti-pig liver TTase. It displayed similar structural, functional, and kinetic characteristics to those of TTases from other sources. It was shown that RHLT effectively regenerated the activities of GST and GPx, after each was inactivated by S-thiolation with cystine in vitro. Furthermore, RHLT was able to restore the activity of the oxidatively inactivated glyceraldehyde-3-phosphate dehydrogenase (G-3PD) in H2O2-exposed rabbit lens epithelial cells. CONCLUSIONS: The human lens TTase gene has been cloned for the first time. Its gene product showed the characteristics which support our speculation that TTase may play a major role in maintaining the homeostasis of lens protein thiols thus protecting against oxidative stress.

Evaluation of potential organ culture media for eye banking using a human corneal endothelial cell growth assay.

BACKGROUND: To evaluate the ability of different commercially available cell culture media to induce proliferation and morphological changes in primary cultures of human corneal endothelial cells (HCEC). This screening model was used in an attempt to establish a rational basis for the development of well-defined, serum-free preservation media for long-term organ culture of human donor corneas. METHODS: A total of 11 different culture media enriched with 0%, 2%, 5%, and 10% fetal calf serum (FCS) were compared. The test media were divided into three groups: Group 1: Media based on minimal essential medium (MEM), currently used for long-term corneal organ culture in European eye banks; Group 2: F99-based media, enriched for growth of corneal endothelial cells at serum-reduced conditions; and Group 3: Media designed for growth of special cell types or for short-term corneal organ culture. The growth-promoting capacity of each test medium was quantified using an HCEC proliferation assay, whereas changes in cell morphology were evaluated by phase-contrast microscopy. RESULTS: The morphological characteristics of HCEC were best maintained in the group of F99-based media, which also induced the highest level of cell proliferation under serum-reduced conditions. Specifically, the medium F99-Sr (F99 enriched with ascorbic acid, insulin, bFGF, transferrin, selenium, and lipids) induced a two- to three-fold higher HCEC density at both 0% and 2% FCS when compared to all other test media, and it also maintained the most endothelial cell-like morphology. Also, at higher serum concentrations (5% and 10% FCS), the cell growth was most prominent in F99-Sr, as well as in the medium SFM that originally was designed for serum-free growth of vascular endothelial cells. CONCLUSION: This study suggests that the media F99-Sr and SFM should be further tested and refined as potential new storage solutions for long-term corneal organ culture at physiological temperatures.

Grafting of the posterior cornea. Description of a new technique with 12-month clinical results.

PURPOSE: To describe the technique of grafting only the posterior cornea and to report 12-month clinical results. METHOD: A two-layer technique with an anterior recipient flap created by a microkeratome and a posterior penetrating donor graft allows for a watertight wound closure and at the same time a peroperative correction of astigmatism. Four eyes (3 patients) were followed for 12 months. RESULTS: The surgical technique could be completed in all cases without complications. The postoperative course was uneventful. The intrastromal absorbable sutures disappeared spontaneously and completely. Graft thickness showed the expected 6-month minimum while recipient flap thickness remained constant. After 1 year endothelial cell densities were 1200-2300 cells/mm2. Confocal microscopy showed activated keratocytes in the flap and quiescent keratocytes in the donor tissue by one year. The anterior chamber depth was normal in all cases. The optical quality of the cornea was studied by automatic keratometry and keratoscopy (TMS). The obtained optical properties were not optimal. CONCLUSIONS: The developed novel technique gives a better wound closure and a complication free postoperative course. It may allow for better control of postoperative astigmatism. In order to disseminate the use of the technique, eyebanks should supply posterior corneas to the surgeon.

Refractive results of radial keratotomy: a ten-year retrospective study.

PURPOSE: To investigate the long-term effects and stability of refraction after radial keratotomy procedure. METHODS: Radial keratotomy was performed on 123 persons to reduce myopia (range: -1 to -13 diopters) in 1986 to 1989. A mean of 11.5 years later (range 10 to 13), 61 of these patients with 102 eyes underwent a standardised refractive examination where subjective spherical equivalent refraction was measured and compared to the preoperative and the one month postoperative refractive measurement collected from the patients records. RESULTS: There was a reduction in spherical equivalent from an average of -5.46 diopters (SD 2.38) preoperatively to -2.32 diopters (SD 1.96) 11.5 years postoperatively. The mean change in direction of myopia between 1 month and 11.5 years postoperatively was 0.17 diopters (SD 1.18). This change was not statistically significant. From 1 month to 11.5 years, 10 of the eyes had developed more than 1 diopter hyperopia, and 20% more than 1 diopter myopia. When asked directly, all patients were satisfied with the result of their operation in general; 2 patients still complained of glare. CONCLUSION: No significant changes in refraction were found between 1 month and 11.5 years after radial keratotomy. Previously reported long-term studies on this field have found a trend toward progressive hyperopia. No evidence of such change can be supported by this study.

[Surgery for nearsightedness]. / Operation for naersynethed.

Myopia can today be reduced or eliminated by refractive surgery. Excimer laser surgery of the cornea by surface sculpturing (photorefractive keratectomy) or intrastromal tissue removal (LASIK) are the most widely used techniques, although implantation of intra corneal ring segments for low myopia also appears promising. Treatment of high myopia (> 10 diopters) is still difficult although epikeratoplasty or phakic IOL implantation are present possibilities. The perfect surgery for myopia remains to be developed, but the existing techniques will without doubt be further optimised. In 10 years time, supra normal visual acuity may even be obtained when surgical, optical, and biological variables can be described and controlled in each individual undergoing refractive surgery.

The influence of corneal stromal matrix proteins on the migration of human corneal fibroblasts.

Motivated by the alterations seen in the corneal matrix composition after photorefractive keratectomy and the migration of corneal keratocytes seen following this procedure, the locomotor response of corneal stromal fibroblasts to various extracellular matrix proteins was determined. In addition, the involvement of integrin mediated attachment to the matrix proteins was investigated. Quantitative invasion assays were performed using collagen gels, supplemented with either fibronectin, tenascin, collagen type V, collagen type VI, chondroitin sulfate or keratan sulfate. The ultrastructure of the gels was visualized by scanning electron microscopy and related to the migration results. The extent of alpha(1)beta(1), alpha(2)beta(1), alpha(3)beta(1)and alpha(5)beta(1)integrin mediated attachment to the matrix proteins was evaluated using blocking antibodies. Fibronectin increased corneal fibroblast migration significantly, and served as an excellent substrate for cellular attachment, mediated by the alpha(5)beta(1)integrin. Addition of tenascin to the fibronectin-containing gels disrupted these effects, while attachment to this matrix also involved the integrins alpha(2)beta(1)and alpha(3)beta(1). Chondroitin sulfate and collagen types V and VI primarily altered the structure of the collagen matrix, resulting in an inhibition of migration by the collagens and an increase by chondroitin sulfate. They all served as poor substrates for attachment. Thus, the migratory activity of corneal fibroblasts in vitro is influenced by the composition of the surrounding extracellular matrix, either by integrin mediated cell-matrix interactions or through matrix-matrix interactions. This study provides evidence that the provisional matrix deposited in a corneal stromal wound may facilitate the entry of migrating corneal fibroblasts.

A clinical comparison of the Xpert non-contact tonometer with the Goldmann applanation tonometer after penetrating keratoplasty.

PURPOSE: To assess the agreement between the Xpert non-contact tonometer (NCT) and the Goldmann applanation tonometer in patients who have undergone penetrating keratoplasty. METHODS: The study material consisted of 42 consecutive patients (43 eyes) who had undergone penetrating keratoplasty within the previous 13 months. RESULTS: The slope of the linear relationship between the two measurement methods did not differ significantly from 1.0. The mean difference between the methods of 0.96 mmHg was not statistically significant. The range of intraindividual differences between the methods was from -9.8 to 22.8 mmHg. The standard deviation of differences was 6.62 mmHg. The 95% limits of agreement were -12.00 to 13.94 mmHg. There was no significant correlation between the central corneal thickness, astigmatism or transplant size and the difference between the methods CONCLUSION: The Xpert NCT shows considerable variation from the Goldmann values The degree of agreement with the true IOP value remains to be shown.

Prolonged persistence on the ocular surface of fortified gentamicin ointment as compared to fortified gentamicin eye drops.

PURPOSE: A comparative study on the elimination of gentamicin from the ocular surface and the concentration of gentamicin in the anterior chamber following application of either an ointment or eye drops containing equal concentrations (1.5%) of gentamicin. METHODS: A disc-diffusion test was used to determine the concentration of gentamicin in fornix inferior of 10 persons. The anterior chamber concentration of gentamicin was determined in 5 cataract patients by the TDX analyzer, Abbot Laboratories, II., USA. RESULTS: Ten minutes following application, the concentration of gentamicin was significantly higher in the eyes receiving ointment (310.6 mg/L) compared to drops (45 mg/L) (p<0.01). Furthermore, gentamicin could be detected 40 minutes after application in the eyes receiving ointment compared to 10 minutes in the eyes receiving drops. The anterior chamber concentration of gentamicin after application of either drops or ointment was lower than 0.6 mg/L and thus below detection limit. CONCLUSIONS: The persistence of gentamicin ointment was significantly longer on the ocular surface as compared to gentamicin eye drops. Gentamycin ointment may thus provide a means to reduce the high application frequency presently in use with eye drops to treat bacterial keratitis and thereby reduce patient inconvenience, especially during nighttime.

Comparison of endothelial cell density estimated by contact and non-contact specular microscopy.

PURPOSE: To compare a contact and a non-contact specular microscope in the determination of endothelial cell density. SUBJECTS AND METHODS: One hundred and twenty-one eyes from 70 patients who had undergone various degrees of photorefractive keratectomy for myopia were included. The endothelium was imaged by contact (Konan Clinical Specular Microscope) and non-contact (Topcon SP-1000) specular microscopy and the endothelial cell density estimated. RESULTS: The average endothelial cell density achieved by the contact specular microscope was 3011+/-298 cells/mm2 (mean+/-SD, n=121) and by the non-contact specular microscope 3015+/-265 cells/mm2 (n= 121). The difference in endothelial cell density between the contact and the non-contact specular microscope (contact minus non-contact) was -4+/-175 cells/mm2 (t=0.26, 2p>0.05 in a paired t-test). The sampling error on the estimated endothelial cell density was 76 cells/mm2 for the contact specular microscope and 74 cells/mm2 for the non-contact specular microscope. CONCLUSION: The average endothelial cell density and the precision of the measuring technique were similar for the contact and the non-contact specular microscope. Furthermore, the endothelial cell densities estimated by the two instruments at various values of anterior central corneal refractive power and central corneal thickness were similar. The two instruments can be used interchangeably.

Stability of graft refractive power after penetrating keratoplasty.

PURPOSE: Patients needing penetrating keratoplasty (PK) and cataract extraction with intraocular lens (IOL) implantation may be handled with a single triple procedure or a two-stage procedure with initial keratoplasty and cataract surgery in a later session. The latter approach is considered more safe by some surgeons and allows adjustment of the IOL power to the power of the actual corneal graft. The purpose of this study was to estimate the optimal timing of cataract surgery with IOL implantation by studying the refractive stability of 8 mm penetrating keratoplasty grafts. METHODS: Penetrating keratoplasty (8.0 mm graft and recipient bed) was performed in 28 eyes of 28 patients. Corneal topography (TMS-1) was studied at 1, 2, 3, 6, and 12 months after surgery and after suture removal (30 months). The central spherical equivalent graft power was computed from the topographical data (rings 2 through 4). RESULTS: On average, the spherical equivalent graft power was stable from one month after surgery up to suture removal (range: 41.9 to 42.7 diopters). After suture removal the graft steepened slightly (0.7 diopters). Corneal refractive power of single grafts fluctuated considerably over time. The standard deviation on time-dependent changes in graft power was from 3 months efter PK smaller than the standard deviation on the graft powers at 12 months. CONCLUSION: The average central spherical equivalent power of an 8.0 mm donor graft in an 8.0 mm recipient bed was stable from one month after penetrating keratoplasty until suture removal. A two-stage procedure with cataract surgery performed 3 months after PK can, compared to the triple procedure, reduce postoperative ametropia at 12 months if graft topography is taken into consideration at the time of cataract surgery. We recommend that cataract surgery with IOL implantation takes place from 3 months after penetrating keratoplasty.

Magnification changes in specular microscopy after corneal refractive surgery.

PURPOSE: To describe the effect of corneal refractive surgery on the magnification of a contact and a non-contact specular microscope. METHOD: The magnification of a contact specular microscope (Konan Clinical Specular Microscope) and a non-contact specular microscope (Topcon SP-1000) was experimentally and theoretically studied as a function of anterior corneal refractive power and central corneal thickness. RESULTS: The magnification of the contact and non-contact specular microscope was found to decrease slightly with decreasing central corneal thickness. In addition, the magnification of the non-contact specular microscope decreased slightly with decreasing anterior corneal refractive power. CONCLUSION: As the preoperative and postoperative measuring conditions are different in patients undergoing corneal refractive surgery a correction for magnification changes is necessary when small changes in endothelial cell density are looked for.

Corneal transplantation with donor tissue kept in organ culture for 7 weeks.

PURPOSE: To study the fate of corneal grafts after extended organ culture (7 weeks). METHODS: Six patients with symmetrical eye diseases were grafted bilaterally, in one eye with a cornea prepared by routine organ culture (mean 16 days), in the other eye with a donor cornea kept for 7 weeks (mean 49 days) in organ culture. The outcome was evaluated by biomicroscopy, graft thickness, endothelial cell density and visual performance after an observation time of at least 1 year. RESULTS: Penetrating 7-8 mm grafting was uncomplicated in all cases. The endothelial densities were in both groups in the range 1000-2000 cells/mm2, and visual acuity 0.2-0.9 in cases with no other ocular pathology. Postoperative graft thickness and deswelling did not differ between 2- and 7-week cultured corneas. At final examination the thicknesses were 0.50 mm and 0.49 mm for 2- and 7-weeks cultured corneas. CONCLUSION: Seven-week cultured corneas give clinical results comparable to those obtained using shorter culture periods. An extended culture period may be used to improve other qualities of the graft (compatibility, cell number, cell metabolism) and microbiological control.

Endothelial cell loss after photorefractive keratectomy for myopia.

PURPOSE: To study the long-term effect of 193 nm excimer laser photorefractive keratectomy (PRK) on the human corneal endothelial cell density. SUBJECTS AND METHODS: One hundred and twenty-four eyes from 71 patients underwent photorefractive keratectomy for myopia or myopic-astigmatism. Endothelial cell density was examined a short time before the operation and on an average of 50 months after the operation using a contact specular microscope. A subgroup of 32 eyes from 20 patients treated only once was examined preoperatively and 7 and 52 months postoperatively. The endothelial cell densities were corrected for the changing magnification of the contact specular microscope with changing central corneal thickness and for the expected physiological cell loss with time. RESULTS: The average endothelial cell density was preoperatively 3098+/-283 cells/mm2 (mean+/-SD) and postoperatively 3048+/-294 cells/mm2 corresponding to a change of -50+/-157 cells/mm2. This was statistically significantly different from zero in a paired t-test (n=124, t=3.58, 2p<0.001). The average changes in endothelial cell density for the subgroup were -34+/-159 cells/mm2 for the postoperative time interval 0-7 months and -20+/-188 cells/mm2 for the postoperative time interval 7-52 months. These results were not statistically significantly different from zero in a paired t-test (n=32, t=1.21 and t=0.60, 2p>0.05). A statistically significant negative correlation between preoperative cell density and the change in cell density was found (n=124, r=-0.21, 2p<0.05). CONCLUSION: This study suggests a potentially harmful effect of PRK on the human corneal endothelium. It appears that most cells are lost during ablation or within the first period of time after PRK.

A morphological and functional study of Congenital Hereditary Endothelial Dystrophy.

PURPOSE: To contribute to the basic understanding of Congenital Hereditary Endothelial Dystrophy (CHED) by clinical, functional, and histopathological examinations of three cases. METHODS: Prior to grafting, corneas were evaluated by slit lamp examination and by assessment of endothelial permeability to fluorescein. Following penetrating keratoplasty, corneal buttons were evaluated by light- and electron microscopy and by assessment of stromal swelling pressure. RESULTS: Patients with CHED had a markedly increased corneal thickness (0.93-0.98 mm) with epithelial oedema and a stromal swelling pressure close to zero; suggesting that the stroma was maximally swollen in vivo. Corneal endothelium showed an increased permeability to fluorescein; suggesting a functional barrier defect. Histopathological evaluation revealed: 1) a normal endothelial cell density; 2) an abnormal endothelial morphology with irregular and multi-nucleated cells containing abnormal cell organelles; and 3) a profound thickening of Descemet's membrane, 16-18 microm, with multiple focal areas of abnormal fibrillar deposits in the posterior half. CONCLUSIONS: This study suggests that the primary defect in patients with CHED is a degenerated and dysfunctional corneal endothelium, characterized by an increased permeability and an abnormal and accelerated Descemet's membrane secretion. The underlying pathophysiological mechanism(s) may be related to an abnormal endothelial barrier function, leading to secondary swelling of the stroma and epithelium. Further studies are needed to identify the specific functional defect(s) and the embryological origin of the abnormal corneal endothelium in CHED.

Paired arcuate keratotomy for congenital and post-keratoplasty astigmatism.

PURPOSE: To study the effect of arcuate keratotomy on corneal astigmatism in previously grafted eyes compared to eyes with naturally occurring astigmatism. SUBJECTS AND METHODS: Twenty-three eyes with naturally occurring astigmatism and 21 eyes with post-keratoplasty astigmatism were treated by arcuate keratotomies in the steepest corneal meridian. Visual acuity, spherically equivalent refraction, and refractive cylinder were measured before surgery and 1 to 10 years after the operation. RESULTS: The preoperative refractive cylinder was reduced from 5.0 dioptres (median) to 1.25 dioptres in eyes with natural astigmatism and from 7.0 dioptres to 3.25 dioptres in post-keratoplasty eyes. Spherical equivalent refraction changed from -0.6 dioptres to -1.5 dioptres in eyes with natural astigmatism and from -3.5 dioptres to -4.5 dioptres in previously grafted eyes. The induced change in astigmatism, as calculated by Fourier analysis, correlated strongly with the existing preoperative astigmatism. The effect of the procedure did not correlate with the type of astigmatism (congenital vs. post-keratoplasty), time after surgery, or with patient age or sex. CONCLUSION: Arcuate keratotomy is a simple procedure to reduce naturally occurring astigmatism as well as induced astigmatism after keratoplasty. Parallel to the astigmatic change, negligible changes in the spherical equivalent are induced.

Chemotaxis of human keratocytes is increased by platelet-derived growth factor-BB, epidermal growth factor, transforming growth factor-alpha, acidic fibroblast growth factor, insulin-like growth factor-I, and transforming growth factor-beta.

PURPOSE: Following corneal wounding, early migration of keratocytes into the wound area is of pivotal importance in the healing process, but the nature of this migration is not well understood. The influence of peptide growth factors on the chemotactic and chemokinetic migration of human corneal keratocytes was investigated, using the following growth factors: platelet derived growth factor-BB (PDGF-BB), epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), insulin-like growth factor-I (IGF-I), and transforming growth factor-beta-1 (TGF-beta 1). METHODS: The chemotactic stimulation was investigated in the Boyden blind-well chemotaxis chamber, and the chemokinetic effect of the growth factors determined by a modified checker-board analysis. RESULTS: PDGF-BB, EGF and TGF-beta 1 stimulated chemotaxis towards a peak value, with a subsequent decline at higher concentrations. PDGF-BB and EGF peaked at 1 ng/ml with a 2.0 and a 2.5-fold increase respectively in the number of keratocytes migrating, whereas TGF-beta 1 reached a maximum response at 0.1 ng/ml, with a 1.7-fold increase. Chemotaxis reached an early plateau and remained constant at concentrations between 1 ng/ml and 100 ng/ml when stimulating with TGF-alpha (2.7-fold), bFGF (2.0-fold), aFGF (2.7-fold), and IGF-I (4.5-fold). Checkerboard analysis revealed that all growth factors were chemotactic agents for human keratocytes, except bFGF, which principally stimulated chemokinesis. CONCLUSION: These in vitro results demonstrate that PDGF-BB, EGF, TGF-alpha, aFGF, IGF-I, and TGF-beta 1 increase keratocyte chemotaxis, and they may play an important role in the early recruitment of keratocytes to the corneal wound site in vivo.