Decellularized porcine conjunctiva as an alternative substrate for tissue-engineered epithelialized conjunctiva

PurposeThe long-term success of visual rehabilitation in patients with severe conjunctival scarring is reliant on the reconstruction of the conjunctiva with a suitable substitute. The purpose of this study is the development and investigation of a re-epithelialized conjunctival substitute based on porcine decellularized conjunctiva (PDC).MethodsPDC was re-epithelialized either with pre-expanded human conjunctival epithelial cells (PDC + HCEC) or with a human conjunctival explant placed directly on PDC (PDC + HCEx). Histology and immunohistochemistry were performed to evaluate epithelial thickness, proliferation (Ki67), apoptosis (Caspase 3), goblet cells (MUC5AC), and progenitor cells (CK15, ΔNp63, ABCG2). The superior construct (PDC + HCEx) was transplanted into a conjunctival defect of a rabbit (n = 6). Lissamine green staining verified the epithelialization in vivo. Orbital tissue was exenterated on day 10 and processed for histological and immunohistochemical analysis to examine the engrafted PDC + HCEx. A human-specific antibody was used to detect the transplanted cells.ResultsFrom day-14 in vitro onward, a significantly thicker epithelium and greater number of cells expressing Ki67, CK15, ΔNp63, and ABCG2 were noted for PDC + HCEx versus PDC + HCEC. MUC5AC-positive cells were found only in PDC + HCEx. The PDC + HCEx-grafted rabbit conjunctivas were lissamine-negative during the evaluation period, indicating epithelial integrity. Engrafted PDC + HCEx showed preserved progenitor cell properties and an increased number of goblet cells comparable to those of native conjunctiva.ConclusionPlacing and culturing a human conjunctival explant directly on PDC (PDC + HCEx) enables the generation of a stable, stratified, goblet cell-rich construct that could provide a promising alternative conjunctival substitute for patients with extensive conjunctival stem and goblet cell loss.     

32例再生障碍性贫血的微循环及血液粘度

对３２例再障患者的甲襞、球结膜微循环及血粘度进行观察，结果表明：再障组均有不同程度的微循环异常改变（１００％），与正常对照组比较差异显著（Ｐ＜０．０１）；再障组全血粘度、全血还原粘度明显低于正常对照组（Ｐ＜０．０１），再障患者的Ｈｂ与全血粘度及血浆比粘度呈正相关。     

角膜缘干细胞与结膜移植治疗翼状胬肉

目的:观察角膜缘干细胞与结膜移植治疗翼状胬肉的疗效。方法:采用自体角膜缘干细胞与结膜移植术,对38例43眼初发和复发性翼状胬肉患者进行治疗,术后随访3～30月,平均10月。结果:38例43眼中34例38眼上皮愈合稳定,角膜恢复正常光滑、透明,胬肉无复发。4例5眼失访。结论:自体角膜缘上皮移植为病变区角膜和结膜提供新的干细胞来源,是治疗翼状胬肉的理想方法。眼科学报1999;15:89—90。     

自体角膜缘移植术治疗复发性翼状胬肉疗效观察

目的　观察自体角膜缘移植术治疗复发性翼状胬肉的疗效。方法　对 10例 (10眼 )复发性翼状胬肉患者施行患眼健侧带结膜的自体角膜缘移植术。结果　随访 6～ 12个月 ,10例患者角膜移植片均透明光滑 ,愈合良好 ,无一例复发。结论　自体角膜缘移植术治疗复发性翼状胬肉有效、安全、简便。     

老年人结膜囊细菌状况及药物敏感实验

目的:为了有效的预防术后眼内炎,对目前老年人结膜囊的细菌状况和对抗生素的敏感率进行评估。方法:北京市老年人(81.5±10.2)岁165眼,常规眼科检查除外眼前段,眼表面炎性疾患,行结膜囊细菌培养,对培养阳性者行细菌分离培养和药物(常用的16种抗生素)敏感实验;滴3g/L氧氟沙星滴眼液1wk,再培养。结果:首次培养阳性率为21.2%(35/165),第二次阳性率为7.3%(12/165)。首次培养中共41个菌株,11个菌种。葡萄球菌属最多,共27株(70.7%),其中表皮样葡萄球菌19株(46.3%),金黄色葡萄球菌7株(17.1%)。次之为棒状杆菌5株(12.3%)。万古霉素对G 菌全部敏感,亚胺培南对G-菌极敏感,头孢菌素和喹喏酮类药物耐药菌株增多,敏感率63.6%～78.8%。结论:术前滴抗生素眼液可显著减少结膜囊的细菌(P <0.01),但是不能达到完全无菌状态。目前头孢菌素和喹喏酮类药物耐药菌株增多,但仍可做为防治眼部感染和眼内炎的主要药物。     

SARS-CoV S蛋白功能性受体ACE2在人、兔角膜、结膜中的表达

目的 SARS冠状病毒(SARS-CoV)的S蛋白介导了病毒与宿主细胞的结合。最近已鉴定出SARS-CoV S蛋白的功能性受体是血管紧张素转化酶2(angiotensin-converting enzyme2，ACE2)。本研究检测了ACE2在人、兔眼结膜、角膜中的表达，由此来初步推断SARS-CoV由眼部入侵的可能。方法 分别采取：(1)成人眼球破裂伤摘除的眼角膜和结膜组织；(2)4～6月中期引产胎儿的眼角膜、结膜、心、肺组织；(3)兔眼角膜、结膜、心、肺组织；(4)培养的人结膜成纤维细胞和兔角膜上皮细胞。分别采用RT-PCR和免疫组化方法检测各组织、细胞中ACE2的表达。取尸体解剖的成人心、肺组织作为对照、结果 RT-PCR法在上述各种组织和细胞中均检测到ACE2的扩增条带，其中胎儿、成人和兔的角膜、结膜扩增条带较心、肺稍弱，兔各组织的扩增条带亮度与对应的人体组织的扩增条带亮度相近。ACE2免疫组化反应产物主要位于胞浆和胞膜，呈棕黄色或棕褐色．角膜和结膜的上皮细胞及角膜内皮细胞呈明显的阳性表达，角膜和结膜的成纤维细胞呈弱阳性表达，培养的人结膜成纤维细胞和兔角膜上皮细胞亦可见阳性表达。兔组织中的ACE2表达强度及组织定位与人相近。结论 本研究从mRNA和蛋白质水平证实了眼组织存在SARS冠状病毒S蛋白的功能性受体ACE2，由此初步推断SARS-CoV存在由眼部入侵的可能，为临床进一步研究SARS的防护和致病机制提供了线索。同时，为新西兰大白兔可能成为研究SARS-CoV入侵途径的动物模型提供了初步的实验依据。     

Heterogeneity in the ultrastructure of the mucous (goblet) cells of the rabbit palpebral conjunctiva

Aim: The purpose of this study was to assess objectively the ultrastructure of the secretory granules in rabbit conjunctival mucin‐producing ‘goblet’ cells. Method: The upper eyelids from five young adult dioestrous female rabbits were dissected out, stretched onto a cardboard support and prepared for transmission electron microscopy by repeated application of an isotonic two per cent glutaraldehyde fixative at room temperature. Post‐fixation treatment included osmium tetroxide and staining with uranyl acetate and lead citrate. Low magnification micrographs were taken of the goblet cells of the conjunctiva, printed at a magnification of approximately 6,000 and the number, size and features of the secretory granules assessed. Results: Across the entire palpebral conjunctiva of ail five rabbits, the majority of mucous cells displayed a goblet shape and the secretory granules were uniformly pale in staining. The average width of the goblet cells was 10.8 ± 1.1 μm and the diameter of the secretory granules was 0.82 ± 0.16 μm. However, in localised regions across the palpebral conjunctiva of two of the rabbits, some goblet cells were different in that the secretory granules had either a denser‐staining core, in which some of the granules were densely staining (while others were pale) or most of the granules were densely staining. These mucous cells had an average diameter of 10.3 ± 1.7 μm and the granule diameters averaged 0.88 ± 0.01 μm. For these abnormal goblet cells, inflammatory cells were found in their immediate vicinity. Occasionally, goblet cells were seen to be in the process of degranulation with associated apparent cell necrosis and the mucin granule diameter was close to 1 μm. Conclusions: The ultrastructure of the mucin‐containing secretory granules of the conjunctival mucous cells is not necessarily homogeneous in character and further attention needs to be given to the effects of localised inflammation in the tissue and to possible hormonal influences.     

Annular elastolytic giant cell granuloma of conjunctiva: A case report

Annular elastolytic giant cell granuloma is a condition characterized histologically by damaged elastic fibers associated with preponderance of giant cells along with absence of necrobiosis, lipid, mucin, and pallisading granuloma. It usually occurs on sun-damaged skin and hence the previous name actinic granuloma. A similar process occurs on the conjunctiva. Over the past three decades only four cases of conjunctival actinic granuloma have been documented. All the previous patients were females with lesions in nasal or temporal bulbar conjunctiva varying 2-3 mm in size. We report a male patient aged 70 years presenting with a 14 mm × 7 mm fleshy mass on right lower bulbar conjunctiva. Clinical differential diagnoses were lymphoma, squamous cell carcinoma in situ and amyloidosis. Surgical excision followed by histopathology confirmed it to be a case of actinic granuloma. This is the first case of isolated conjunctival actinic granuloma of such a large size reported from India.     

Conjunctival Melanoma in Asian Indians: A Study of 42 Patients

Purpose: To describe the clinical features, treatment, and outcomes of conjunctival melanoma in Asian Indians.

Methods: Retrospective study of 42 patients.

Results: The mean age at presentation of conjunctival melanoma was 43 years (median, 45 years; range, 9–78 years). There were 20 (48%) males and 22 (52%) females. Nineteen patients (45%) had a known history of a preexisting pigmented conjunctival lesion. Bulbar conjunctiva (n = 28; 67%) was the most common tumor epicenter, and medial ocular surface quadrant (n = 15; 36%) was more commonly involved. The mean tumor basal diameter was 12 mm (median, 10 mm; range, 4–30 mm), and the mean tumor thickness was 4 mm (median, 2 mm; range, 1–30 mm). Majority of the patients had a pigmented tumor (n = 33; 79%). The tumors arose de novo (n = 17, 41%) or were associated with conjunctival nevus (n = 9; 21%) or primary acquired melanosis (n = 16, 38%). Wide excisional biopsy, adjunctive cryotherapy, and amniotic membrane grafting were performed in 27 (71%) patients, 11 (29%) underwent orbital exenteration, and 4 were lost to follow-up prior to definitive treatment. Over a mean follow-up period of 24 months (median, 9 months; range, <1 to 136 months), four (11%) patients had tumor recurrence, seven (18%) had locoregional lymph node metastasis, and four (11%) developed systemic metastasis and died due to metastatic disease.

Conclusion: Conjunctival melanoma predominantly occurs in middle-aged Asian Indians and is associated with a high rate of systemic metastasis and death.     

Development of decellularized conjunctiva as a substrate for the ex vivo expansion of conjunctival epithelium

This study was performed to develop a method to decellularize human conjunctiva and to characterize the tissue in terms of its deoxyribose nucleic acid (DNA) content, tensile strength, collagen denaturation, basement membrane, extracellular matrix components and its potential to support conjunctival epithelial growth. Human conjunctival tissues were subjected to a decellularization process involving hypotonic detergent and nuclease buffers. Variations in sodium dodecyl sulfate concentration (0.05–0.5%, w/v) were tested to determine the appropriate concentration of detergent buffer. DNA quantification, collagen denaturation, cytotoxicity and tensile strength were investigated. Human conjunctival cell growth by explant culture on the decellularized tissue substrate was assessed after 28 days in culture. Samples were fixed and paraffin embedded for immunohistochemistry including conjunctival epithelial cell markers and extracellular matrix proteins. Conjunctival tissue from 20 eyes of 10 donors (age range 65–92 years) was used. Decellularization of human conjunctiva was achieved to 99% or greater DNA removal (p < 0.001) with absence of nuclear staining. This was reproducible at the lowest concentration of sodium dodecyl sulfate (0.05% w/v). No collagen denaturation (p = 0.74) and no difference in tensile strength parameters was demonstrated following decellularization. No significant difference was noted in the immunolocalization of collagen IV, laminin and fibronectin, or in the appearance of periodic acid–Schiff‐stained basement membranes following decellularization. The decellularized tissue did not exhibit any cytotoxicity and explant culture resulted in the growth of stratified conjunctival epithelium. Allogeneic decellularized human conjunctiva can be successfully decellularized using the described protocol. It represents a novel substrate to support the expansion of conjunctival epithelium for ocular surface cellular replacement therapies. Copyright © 2017 John Wiley & Sons, Ltd.