Quantitative analysis of collagen fiber in keratoconus

In order to identify the direct pathogenic factors involved in the stromal thinning of keratoconus, quantitative analysis of keratocytes, collagen fibers and collagen lamellae in keratoconus cornea was performed histologically by light and electron microscopy. Both normal and keratoconus corneas showed a similar cell density of keratocytes in the central stroma, therefore the total number of keratocytes in keratoconus cornea might be smaller than that of controls, because of the thinning of stroma in the keratoconus. The collagen lamellae in keratoconus corneas showed a significant decrease in number compared with controls. There was a direct relationship between the stromal thickness and number of collagen lamellae. On the other hand, there was no statistical significance between normal and keratoconus corneas in terms of the thickness of collagen lamellae. These results suggest that the thinning of the cornea in keratoconus might occur as the result of a defect of some collagen lamellae due to a disorganization during the process of collagen lamellae formation.     

Noninvasive corneal stromal collagen imaging using two-photon-generated second-harmonic signals

PURPOSE: To investigate the feasibility of using femtosecond-pulse lasers to produce second-harmonic generated (SHG) signals to noninvasively assess corneal stromal collagen organization. SETTING: The Eye Institute, University of California, Irvine, California, USA. METHODS: Mouse, rabbit, and human corneas were examined by two-photon confocal microscopy using a variable-wavelength femtosecond lasers to produce SHG signals. Two types were detected: forward scattered and backward scattered. Wavelength dependence of the SHG signal was confirmed by spectral separation using the 510 Meta (Zeiss). To verify the spatial relation between SHG signals and corneal cells, staining of cytoskeletons and nuclei was performed. RESULTS: Second-harmonic-generated signal intensity was strongest with an excitation wavelength of 800 nm for all 3 species. Second-harmonic-generated forward signals showed a distinct fibrillar pattern organized into bands suggesting lamellae, while backscattered SHG signals appeared more diffuse and indistinct. Reconstruction of SHG signals showed two patterns of lamellar organization: highly interwoven in the anterior stroma and orthogonally arranged in the posterior stroma. Unique to the human cornea was the presence of transverse, sutural lamellae that inserted into Bowman's layer, suggesting an anchoring function. CONCLUSIONS: Using two-photon confocal microscopy to generate SHG signals from the corneal collagen provides a powerful new approach to noninvasively study corneal structure. Human corneas had a unique organizational pattern with sutural lamellae to provide important biomechanical support that was not present in mouse or rabbit corneas.     

Transparency, swelling and scarring in the corneal stroma

PURPOSE: This paper briefly reviews current explanations for corneal transparency and uses a well-developed model to try to explain the increased light scattering either accompanying corneal swelling or following phototherapeutic keratectomy (PTK). METHODS: The direct summation of fields (DSF) method was used to compute light transmission as a function of wavelength. The method requires input of a number of structural parameters. Some of these were obtained from electron micrographs and others were calculated from X-ray diffraction data. RESULTS: By swelling sections of stroma cut from different depths in the tissue, we have shown that fluid entering the cornea causes more swelling in the posterior lamellae than in the anterior lamellae. Furthermore, posterior lamellae can reach a higher final hydration than anterior lamellae. Collagen-free regions ('lakes') exist in corneas swollen in vitro and in Fuch's dystrophy corneas, many of which may be caused by the death of cells. The DSF method shows that local fibril disordering, increased refractive index mismatch, and increased corneal thickness together can account for a 20% increase in light scattering in a Fuch's dystrophy cornea at H=5.8 compared to the normal cornea. Additional scattering is probably caused by 'lakes'. The DSF method applied to PTK rabbit stroma with high levels of haze suggests that the newly deposited collagen is not the cause of the increased light scattering. CONCLUSIONS: Fluid is not uniformly distributed within the corneal stroma when the cornea swells. Increased hydration of posterior lamellae may be because of known differences in the glycosaminoglycans between the anterior and posterior stroma. Lamellar interweave in the anterior stroma probably limits the extent to which the constituent lamellae can swell. The DSF method can be used to account for increased light scattering in oedematous corneas but cannot account for haze following PTK.     

Structural analysis of normal corneas and diseased corneas by applying second harmonic generation

We have established a second harmonic generation (SHG) microscopy system for imaging of the human cornea with a mode-locked femtosecond laser and a laser confocal microscope. This SHG microscopy system has allowed us to scan corneal tissue noninvasively ex vivo and to obtain three-dimensional images of corneal collagen lamellae. Such three-dimensional imaging of the normal anterior cornea revealed that collagen lamellae at the anterior stroma are inter-woven and adhere to Bowman membrane with these adherent lamellae being designated "sutural lamellae." Sutural lamellae adhere to Bowman membrane at an angle of approximately 19 degrees, whereas the angle of lamellae in the mid-stroma relative to Bowman membrane is smaller. We hypothesize that the structural unit consisting of both Bowman membrane and the sutural lamellae contributes to the rigidity and anterior curvature of the cornea. SHG imaging of keratoconic corneas revealed an either abnormal or a total lack of structure of the sutural lamellae, suggesting that this abnormality might be related to that of the corneal anterior curvature in such corneas. Furthermore, SHG imaging of corneas affected by stromal edema showed that the structure of the sutural lamellae was maintained, although abnormal collagen signals both above and below Bowman membrane were detected in corneas affected by clinical stromal edema for more than 12 months. SHG imaging of the structure of collagen lamellae in normal and diseased corneas thus has the potential to provide insight both into the mechanism for maintenance of corneal curvature as well as into the pathophysiology of corneal diseases.     

Second-harmonic imaging microscopy of normal human and keratoconus cornea

PURPOSE: The purpose of this study was to evaluate the ability of second-harmonic imaging to identify differences in corneal stromal collagen organization between normal human and keratoconus corneas. METHODS: Six normal corneas from eye bank donors and 13 corneas of patients with keratoconus, obtained after penetrating keratoplasty were examined. A femtosecond titanium-sapphire laser with 800-nm output was used to generate second-harmonic signals collected at 400 nm from central and paracentral corneal tissue blocks. Three-dimensional (3-D) data sets were collected and reconstructed to evaluate the location and orientation of stromal collagen lamellae. RESULTS: Imaging of second-harmonic signals combined with 3-D reconstruction of the normal cornea identified a high degree of lamellar interweaving, particularly in the anterior cornea. Of note was the detection of lamellae that inserted into Bowman's layer and were oriented transverse to the corneal surface, penetrating posteriorly approximately 120 mum. In keratoconus corneas, imaging second-harmonic signals identified less lamellar interweaving and a marked reduction or loss of lamellae inserting into Bowman's layer in 12 of 13 cases, particularly in regions associated with cone development without breaks in Bowman's layer or scarring. CONCLUSIONS: Compared with normal adult corneas, marked abnormalities were detected in the organization of the anterior corneal collagen lamellae of keratoconus corneas by second harmonic imaging. These structural abnormalities are consistent with the known changes in collagen organization and biomechanical strength of keratoconus.     

Ultrastructural analyses of enzyme-treated microfibrils in rabbit corneal stroma

Microfibrils have been identified within and between corneal collagen lamellae in a number of vertebrate species in a variety of developmental and pathological conditions, but they are relatively rare in normal adult animals. The present study was undertaken to analyze corneal microfibrils in adult rabbits using enzymatic digestion techniques. Transmission electron microscopy (TEM) showed clusters of 10-15 nm microfibrils arranged in quasi-parallel bundles within or between orthogonally arranged stromal collagen lamellae. When corneas were fixed with tannic acid/glutaraldehyde, the entire stroma showed increased electron density and microfibrillar bundles were heterogeneously stained. Peripheral fibrils were more electron-dense than those located more centrally. Following sequential detergent solubilization of unfixed corneas, all cellular elements were removed and collagen lamellae were distorted. Microfibrillar bundles remained intact, however, and resembled untreated controls. Subsequent treatment with pepsin, trypsin or elastase resulted in swollen corneal tissues in which collagen lamellae were no longer distinguishable but individual collagen fibrils maintained their morphological integrity. In these tissues microfibrillar bundles were rarely identifiable and were reduced to randomly oriented fragments or clusters of filamentous material. Testicular hyaluronidase or chondroitinase ABC did not affect the fibrils. These data indicate that rabbit corneal microfibrils are proteinaceous and that the tannic acid-staining component of the bundles is not glycosaminoglycan. The fibrils are indistinguishable from those identified as oxytalan in cornea and other ocular tissues. Moreover, their sensitivity to elastase and preferential staining with tannic acid/glutaraldehyde strongly suggest they may be related to the elastic system of fibrils.     

The ultrastructural changes in the corneal lens stroma of wound healing process following epikeratophakia in rabbits

We investigated the ultrastructural change of corneal lens+ stroma histologically following epikeratophakia in rabbits. Epikeratophakia was performed on rabbit corneas using cryolathed corneal lens+. At days 10, 16, 45, 63 and 90 after the operation, pachymetry was performed, and corneas were excised and analyzed histologically using an electron microscope. At days 16 to 63, collagen fibril density (CFD) in corneal lens+ stroma decreased 57 to 63% of that in the control cornea, but returned to 78% at day 90. The results of pachymetry revealed that postoperative increase of corneal thickness was greater than the expected value at days 10 and 16. However, after that the corneal thickness decreased gradually and became thinner than the expected value after 45 days postoperatively. At days 45 to 90, many activated keratocytes with dilated and well developed rough endoplasmic reticulum, suggestive of active collagen production, were observed in corneal lens+ stroma. Furthermore, at day 90, keratocytes with extended pseudopoidia that suggested phagocytic activity were also observed, especially in the subepithelial zone. These findings indicated that collagen fibrils in the corneal lens+ stroma were damaged under the influence of the cryolathing process and partially exfoliated postoperatively, however, collagen-producing activity increased gradually and the reconstruction of corneal lens+ stroma continued through day 90 after the operation.     

Ⅱ型胶原酶构建兔角膜离体扩张模型

目的探索使用Ⅱ型胶原酶建立兔角膜离体扩张模型的方法及其可行性。方法实验研究。20只离体兔角膜采用随机数字表法分为I组（阴性对照组）、II组（5 mg/ml Ⅱ型胶原酶组）、III组（10 mg/ml Ⅱ型胶原酶组）及IV组（15 mg/ml Ⅱ型胶原酶组）,每组5只角膜。刮除上皮后角膜置于人工前房上,各组采用不同浓度Ⅱ型胶原酶溶液浸泡60 min。分别记录处理前后各组角膜在不同人工前房内压力[模拟眼压（IOP）]下（15、30、45 mmHg）角膜平均曲率及角膜中央厚度,并对处理后各组角膜进行组织学检查。各组处理前后角膜平均曲率差值△Km及角膜中央厚度差值△CCT的变化采用单因素方差分析。结果不同IOP梯度下,4组角膜处理前后角膜平均曲率差值△Km差异有统计学意义（F=8.46、9.24、8.58,P＜0.01）;在各个IOP梯度下各组比较发现IV组与I组的△Km差异均有统计学意义（P＜0.01）,II组、III组与I组的△Km差异均无统计学意义（P＞0.05）。不同IOP梯度下,4组角膜处理前后角膜中央厚度差值△CCT差异均无统计学意义（F=0.22、0.66、1.60,P＞0.05）。与阴性对照组相比,胶原酶处理过的角膜基质胶原纤维排列疏松,基质水肿。结论15 mg/ml Ⅱ型胶原酶溶液浸泡角膜60min能使角膜平均曲率增加,可用于建立兔角膜离体扩张模型。     

Cultured human corneal endothelial cell transplantation

Researchers have demonstrated the feasibility of transplanting human cultured corneal endothelial cells (HCEC) in various animal models. This review provides an overview of recent advances in our understanding of cultured corneal endothelial cell transplantation. We propose HCEC transplantation with a collagen sheet as the substitute carrier of HCEC. We also propose a novel strategy for corneal endothelial cell deficiency with the injection of adult human corneal endothelial precursors (HCEP). Using white rabbits or nude rats as keratopathy models, cultured HCEC were seeded on a collagen sheet. Descemetorhexis was performed on rabbit eyes. The HCEC collagen sheet was brought into the anterior chamber and fixed to the posterior stroma (HCEC group). Rabbit corneas with collagen sheet transplantation after descemetorhexis(collagen group) and with only descemetorhexis(no transplantation group) were the controls, respectively. As for HCEP transplantation, HCEP, isolated from rabbit corneal endothelial cells by sphere-forming assay, were injected into the anterior chamber and a face-down position was maintained for 24 hours in the rabbits (HCEP group). Pump function parameters of the HCEC sheets were 76-95% of those of human donor corneas. Mean corneal thickness in the HCEC group was significantly less than in the collagen and no transplantation groups 1, 3, 7, 14, 21, and 28 days (p< 0.05) after surgery. Cells were spread over the rear corneal surface in the HCEC group. In HE staining, marked stromal edema was present in the collagen and in the no transplantation groups, but not in the HCEC group with collagen sheets bearing monolayer cells. In the HCEP group, injected spheres were spread over the rear surface of the cornea and corneal edema was markedly suppressed. Our findings indicate that transplantation of cultured HCEC from adult human donor cornea by means of a collagen sheet can maintain the function of corneal dehydration. This suggests the feasibility of transplantation using cultured HCEC with a collagen sheet for corneal endothelial cell dysfunction. Additionally, adult precursor injection therapy can be also an effective strategy for corneal endothelial cell deficiency in place of conventional full-thickness corneal transplantation.     

兔眼准分子激光近视角膜切削术后角膜膨出的研究

目的：探讨兔眼在准分子激光近视角膜切削术（Excimer laser in-situ keratomileusis,LASIK)手术后在不同的眼压下角膜膨出的程度。方法：26兔52眼随机分为对照组，LASIK手术组，单盲法，术前，术后测量角膜厚度和角膜地形图后，再将两组随机交叉分为正常眼压组，低度高眼压组，高度高眼压组，并观察1个月，用角膜地形图对实验前后的角膜形态进行分析。并应用统计学方法将手术切削量和眼压两个因素对角膜扩张的影响做双因素方差分析。结果：LASIK手术的角膜切削量，术后高眼压以及两者的交互效应对角膜抵抗力都有显著影响。较大的切削量和术后持续高眼压后的兔眼角膜地形图经分析符合圆锥角膜模式，并出现前，后弹力膜断裂的形态学改变。结论：角膜较大的切削量和术后持续的高眼压是导致兔眼LASIK术后发生圆锥角膜的重要原因。     

Collagen fiber diameter in the rabbit cornea after collagen crosslinking by riboflavin/UVA

OBJECTIVE: Collagen crosslinking of the cornea has been developed recently as a quasiconservative treatment of keratoconus. Biomechanical in vitro measurements have demonstrated a significant increase in biomechanical stiffness of the crosslinked cornea. The aim of the present study was to evaluate the effect of this new procedure on the collagen fiber diameter of the rabbit cornea. METHODS: The corneas of the right eyes of 10 New Zealand White albino rabbits were crosslinked by application of the photosensitizer riboflavin and exposure to UVA light (370 nm, 3 mW/cm2) for 30 minutes. The left fellow control eyes were either left untreated (rabbits 1-4), deepithelialized (rabbits 5-7), or deepithelialized and treated with riboflavin/dextran solution (rabbits 8-10) to exclude an influence of epithelial debridement or hydration changes on the fiber diameter. On ultrathin sections of samples from the anterior and posterior cornea, the collagen fiber diameter was measured semiautomatically with the help of morphometric computer software. RESULTS: In the anterior stroma, the collagen fiber diameter in the treated corneas was significantly increased by 12.2% (3.96 nm), and in the posterior stroma by 4.6% (1.63 nm), compared with the control fellow eyes. In the crosslinked eyes, the collagen fiber diameter was also significantly increased by, on average, 9.3% (3.1 nm) in the anterior compared with the posterior stroma within the same eye. CONCLUSIONS: Collagen crosslinking using riboflavin and UVA leads to a significant increase in corneal collagen diameter. This alteration is the morphologic correlate of the crosslinking process leading to an increase in biomechanical stability. The crosslinking effect is strongest in the anterior half of the stroma because of the rapid decrease in UVA irradiance across the corneal stroma as a result of riboflavin-enhanced UVA absorption.     

Cultured human corneal endothelial cell transplantation with a collagen sheet in a rabbit model

PURPOSE: To evaluate the function of cultured human corneal endothelial cells (HCECs) in vivo and the feasibility of HCEC transplantation with a collagen sheet as the substitute carrier of HCECs. METHODS: Adult human donor cornea derived from cultured HCECs was labeled with the fluorescent tracker DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) and seeded on a collagen sheet. The pump function of the HCEC sheet was evaluated by measurement of the potential difference and short-circuit current. A 6-mm sclerocorneal incision and Descemetorhexis were performed on rabbit eyes. The HCECs on a collagen sheet was brought into the anterior chamber and fixed to the posterior stroma (HCEC group). Rabbit corneas with collagen sheet transplantation after Descemetorhexis (collagen group) and with only Descemetorhexis (no-transplantation group) were the control. Each group, observed for 28 days after surgery, underwent histologic and fluorescence microscopic examinations. RESULTS: Pump function parameters of the HCEC sheets were 76% to 95% of those of human donor corneas. Mean corneal thickness in the HCEC group was significantly less than in the collagen and no-transplantation groups 1, 3, 7, 14, 21, and 28 days (P < 0.05) after surgery. DiI-labeled cells were spread over the rear corneal surface in the HCEC group. Marked stromal edema was present in the collagen and no-transplantation groups with hematoxylin-eosin staining, but none in the HCEC group with collagen sheets bearing monolayer cells. CONCLUSIONS: The findings indicate that cultured HCECs transplanted from adult human donor cornea by means of a collagen sheet can retain their function of corneal dehydration in a rabbit model and suggest the feasibility of transplantation for CEC dysfunction using cultured HCECs with a collagen sheet.     

The role of dermatopontin in the stromal organization of the cornea

PURPOSE: Dermatopontin (DPT) is an abundant component of the stromal extracellular matrix; however, its function in the cornea is poorly understood. This study was conducted to determine whether DPT has a direct role in corneal matrix organization by investigating the ultrastructure of Dpt-null (Dpt(-/-)) mouse corneas. METHODS: Conventional light microscopy was used to compare the corneal thickness of Dpt(-/-) mice with that of the wild type. Collagen fibril distribution was studied using transmission electron microscopy and the datasets analyzed using image analysis software to determine fibrillar volume, fibril diameter, and spacing. RESULTS: Light microscopy demonstrated that Dpt(-/-) corneas in 2-month-old mice showed a 24% reduction in average stromal thickness compared with wild type (P < 0.001). The epithelium and Descemet's membrane appeared normal. Examination of Dpt(-/-) stroma by transmission electron microscopy indicated significant disruption of fibril spacing within the posterior lamellae, whereas the mid and anterior regions appeared largely unaffected compared with wild type. The collagen fibrils in Dpt(-/-) stroma showed a lower fibril volume fraction and a pronounced change in posterior fibrillar organization. There was no apparent difference in fibril diameter between Dpt(-/-) and wild-type mice. CONCLUSIONS: Collectively, these data suggest that DPT plays a key role in collagen fibril organization. The defects in collagen organization in Dpt(-/-) cornea appear to be most severe in the posterior stroma. It is possible that DPT interacts with corneal proteoglycans and that this interaction is involved in the maintenance of stromal architecture.     

Interaction between corneal invasion of polymorphonuclear leukocytes and corneal epithelium

The interaction between corneal invasion of polymorphonuclear leukocytes (PMNs) and corneal epithelium was investigated following different types of corneal injury. Histological examination of centrally denuded corneas demonstrated that it took 18-24 hours for PMNs to infiltrate into the center of the denuded stroma, approximately synchronous with the reepithelialization. On the other hand, in the centrally denuded corneas immediately covered by glue to prevent the reepithelialization, the time required for the appearance of PMNs at the central portion of the corneas was more than 96 hours. Chemotactic activities of PMNs in the conditioned media of normal cornea, completely denuded cornea and reepithelializing cornea were examined using a Boyden chamber. The highest chemotactic activity was defected in the conditioned medium of the cornea with reepithelialization. These data suggest the possibility that the corneal epithelium, especially the re-covering epithelium, stimulates infiltration of the stroma by PMNs.     

Cohesive strength of corneal lamellae

Strips of rabbit stroma were prepared, and the force required to tear them apart along their length was determined. This amounted to an average of 10 g mm-1 width of tissue and it is independent of the depth of the plane in which the splitting of the cornea takes place. It could not be determined, however, whether the cohesive strength of the tissue is due to occasional collagen fibrils binding it together, interweaving of the lamellae or enmeshing of the collagen fibrils by ground substances. Other corneas were split by blunt dissection in vivo and allowed to recover for various periods of time, when the reformed strength of the split was measured. It was found to be negligible for about 5 days and rose quickly to 0.25-0.5 of the value of the untouched cornea. No increase in the force of adhesion of the split stroma was observed if an extract of corneal epithelium or a suspension of platelets was injected into the wound.     

七种动物正常角膜组织结构的比较性研究

目的 比较7种实验动物（BALB/c小鼠、豚鼠、新西兰大白兔、猫、狗、猪和牛）角膜的光镜和电镜下结构,探讨不同动物作为各种角膜实验模型的形态学依据。设计 实验研究。研究对象 BALB/c小鼠（8~12周）、豚鼠(350~450 g)、新西兰大白兔（2.5~3.5 kg）和成年猫、狗、猪、牛7种动物,各6只新鲜眼球。方法 制作各种动物眼球的角膜光镜、电镜切片,分别对HE染色切片摄制成的彩色照片和电镜照片进行比较分析。主要指标 角膜各层结构形态、厚度,有无前后弹力层,基底膜及半桥粒的特点。结果 角膜厚度的变化趋势：兔、狗、猪、牛角膜是中央薄、边缘厚,与人相似;中央厚、周边薄者有豚鼠和猫;小鼠角膜中央到周边厚度变化不明显。中央角膜上皮层厚度：豚鼠与人最相似。角膜上皮层平均厚度、中央区全层厚度、角膜中央区及旁中央区基质层厚度：兔与人最接近。角膜缘厚度：牛与人最接近。角膜基质层平均厚度：狗与人最相近。角膜后弹力层厚度：牛与人最相近。角膜内皮层厚度、后弹力层与内皮层厚度比例：猪与人最接近。角膜内皮细胞直径：豚鼠与人最接近。光镜下,7种动物角膜上皮细胞形态：复层扁平细胞为长扁形,翼细胞为多角形,基底细胞为单层柱状细胞。光镜下均无法判断是否有前弹力层,基质层内胶原纤维排列整齐,无定型结构的后弹力层可辨认,内皮细胞为单层扁平细胞。电镜下,BALB/c小鼠和狗的角膜未见明确前弹力层,兔、猫、猪和牛角膜有前弹力层。豚鼠角膜因照片质量不佳,无法判断是否有前弹力层。BALB/c小鼠的后弹力层内可见条梭状特殊结构,牛的后弹力层呈条栅状条纹。各动物角膜的半桥粒平均直径（单位：μm）分别为小鼠0.16±0.01,兔0.18±0.02,猫0.27±0.06,狗0.19±0.08,猪1.72±0.08,牛1.13±0.82。半桥粒平均面积（单位：μm2）分别为小鼠0.03,兔0.04,猫0.09,狗0.05,猪3.76,牛2.09。BALB/c小鼠角膜半桥粒的直径和面积与人角膜最为接近。结论 兔角膜拥有最多的与人角膜相近的测量指标,故7种动物中兔角膜在做角膜模型方面更有优势。其他动物则各有所长。 （眼科, 2015, 24： 341-347）     

Histology after lamellar keratoplasty and corneal excimer laser ablation.

PURPOSE: To correlate clinical and histological findings after lamellar keratoplasty, phototherapeutic keratectomy, and application of a donor lenticule on a human cornea. METHODS: A cornea was obtained during penetrating keratoplasty. The specimen was fixated, dehydrated and embedded in Epon resin. The tissue was cut in 0.5-microm-thick semi-thin sections, stained with toluidine blue, and studied with light microscopy. RESULTS: The central part of the photoablated cornea, which was covered by the donor lenticule, did not differ from a normal cornea. Peripherally, a hazy ring was found clinically. Histology showed an irregular epithelium. Where it was thickened, the epithelium was hyperplastic and showed an increased number of cell layers. In the hazy region, Bowman's layer was absent, indicating that the donor lenticule did not cover this part of the photokeratectomized cornea. The anterior-most part of the corneal stroma was vacuolized and contained amorphous extracellular material; swollen keratocytes were present in this region. Beneath this layer, collagen lamellae were wavy and interwoven and keratocytes were increased in number, appeared swollen, and some had assumed an atypical shape. Peripheral to the haze, the cornea was clear. Histologically, the epithelium was irregular and hyperplastic, Bowman's layer was absent, and stromal collagen lamellae were abnormally organized, but no vacuolization was found. CONCLUSIONS: The formation of haze after excimer laser photokeratectomy can be minimized if the ablated stroma is covered by a corneal lenticule.     

脱细胞猪角膜基质的生物相容性与组织工程化兔角膜上皮移植的研究

目的 探讨脱细胞猪角膜基质的生物相容性,评价组织工程化角膜上皮组织作供体的可行性,观察支架材料的细胞化情况和种子细胞的存活情况.方法 实验研究.采用完全随机化设计的方法,用Dispase-Triton-X-100处理猪角膜基质,脱去角膜细胞;以角膜基质囊袋内植入的方法,观察异种角膜基质植入后的生物相容性,A组:脱细胞猪角膜基质,B组:新鲜猪角膜基质,C组:空白对照组.以组织工程化雄性角膜上皮组织为供体,同种雌性为受体,作板层角膜移植,观察角膜的混浊、水肿、新生血管等情况;组织病理学和免疫组化方法检测支架材料的细胞化情况,Y染色体性别决定基因(SRY)-聚合酶链反应(PCR)方法追踪种子细胞的存活情况.结果 猪角膜基质植入兔角膜囊袋后,角膜逐渐恢复透明,排斥反应指数＜6,组织病理学观察角膜结构完整,胶原纤维平行排列,少许细胞长入脱细胞猪角膜基质边缘,各组免疫组化检测未见CIM+、CD8+T淋巴细胞浸润.组织工程化角膜上皮作异体板层角膜移植后,3～4 d上皮光滑,10～20 d变为透明;15 d时角膜上皮、基质、内皮完整,上皮细胞约4或5层结构,少许基质细胞长入支架,1个月时可见角膜上皮细胞约7或8层细胞,基质纤维排列规则,多量细胞长入脱细胞角膜基质.上皮细胞表达CK3,支架内新生细胞表达波形蛋白.SRY-PCR结果显示种子细胞可以在受体内长期存活.结论 脱细胞猪角膜基质生物相容性良好,组织工程化角膜上皮可作为板层角膜移植的供体,脱细胞猪角膜基质细胞化良好,种子细胞可以在受体内长期存活.     

组织工程角膜基质的体外构建及移植的实验研究

目的探讨利用组织工程技术体外构建角膜基质进行板层角膜移植的可行性和有效性。方法将猪角膜基质去细胞处理后制备成组织工程角膜基质载体;取幼兔角膜基质细胞体外培养,将其种植在载体上,体外构建成组织工程角膜基质,用PKH26荧光标记兔角膜基质细胞示踪角膜基质的构建;将16只兔的角膜基质内植入壳聚糖膜使之形成无菌性角膜溃疡,随机从16只兔中选8只,进行组织工程角膜基质移植;另外8只作为对照组,进行新鲜的同种异体兔板层角膜移植。术后对角膜进行裂隙灯、光学显微镜、透射电镜观察。结果体外构建的组织工程角膜的基质细胞具有活性,其结构与正常角膜基质相近。移植治疗无菌性角膜溃疡术后,1~2周有新生血管侵入组织工程角膜基质植片边缘,植片为灰白色半透明状;3~4周随着新生血管减退,组织工程角膜基质植片局部开始透明变薄;术后8~10周角膜溃疡完全修复,角膜恢复透明性,角膜神经可再生;观察最长达10月,角膜仍保持透明,无免疫排斥发生,与对照组疗效相同。结论体外构建的组织工程角膜基质无免疫原性、具有良好的生物相容性,可作为临床治疗角膜溃疡的移植材料。     

组织工程角膜片移植治疗无菌性角膜溃疡的实验研究

目的探讨组织工程角膜片治疗角膜溃疡的可行性及疗效。方法将16只新西兰大白兔制作成角膜溃疡模型,随机分为2组,每组8只,分别作单纯羊膜移植和以羊膜为载体的组织工程角膜片移植。术后进行大体观察、裂隙灯观察、组织学观察、扫描电镜观察。结果羊膜为载体组织工程角膜片移植治疗角膜溃疡,平均7.5周溃疡完全愈合,角膜恢复透明性和屈光性,较单纯羊膜移植组(平均9周)角膜溃疡修复快,效果好结论羊膜为载体构建的组织工程角膜片可以有效治疗角膜溃疡。