【摘要】目的：探讨聚羟乙基丙烯酸甲酯(PHEMA)海绵支架材料与角膜组织的生物相容性，评价改良聚羟乙基丙烯酸甲酯(PHEMA)—聚甲基丙烯酸甲酯（PMMA）一体化人工角膜植入兔与猴角膜的初步临床效果。方法：通过两个阶段化学聚合结合车床机械切削合成改良的一体化人工角膜。实验分为两个部分完成。第1部分（A组）：将PHEMA海绵边裙材料植入10只正常兔角膜板层间，术后2，4wk及2，3，4mo分别行组织病理、免疫组织化学及电镜检查，观察海绵边裙与角膜组织的生物学愈合情况。第2部分(B组)：8只兔眼和2只猴眼角膜囊袋内I期植入一体化人工角膜，术后临床观察材料与组织的愈合情况；术后3mo时行II期手术切除术眼角膜中央前板层角膜组织，暴露人工角膜中央镜柱，术后随访时间3～6mo，初步观察临床治疗效果。结果：1）A组接受角膜板层间边裙植入术的10只兔眼，随访期间未见并发症：组织病理学显示，PHEMA海绵边裙材料植入术后2wk始有成纤维细胞长入，术后2～3mo时多量成纤维细胞长入并伴有新生血管生长；免疫组织化学显示，海绵孔隙中长入的细胞和角膜基质细胞均对波形蛋白免疫反应呈阳性；电镜下可见，成纤维细胞在海绵材料间隙中生长，细胞生长状态良好，并分泌胶原和细胞外基质。2）B组接受一体化人工角膜移植术的，6只兔眼一体化人工角膜均在位，另2只兔眼I期术后有前板层角膜基质融解。接受一体化人工角膜移植术的2只猴眼，I期和II期术后均未见明显并发症。结论：PHEMA海绵能与角膜组织良好的生物学愈合；改良PHEMAPMMA一体化人工角膜植入术后能获得相对稳定的治疗效果。
【关键词】 聚羟乙基丙烯酸甲酯 聚甲基丙烯酸甲酯 人工角膜 兔 猴 生物相容性
INTRODUCTION
Corneal diseases are the second common cause of blindness all over the world, next to cataract. As a relatively immune privilege tissue, cornea can easily keep alive in allograft corneal transplantation. But routine keratoplasty is not successful in treating ocular chemical injury, heat injury, StevenJohnson syndrome, severe dry eye and other ocular surface diseases with neovascularization. Keratoprostheses (KPro) made of transparent materials can be used in keratoplasty to treat those diseases. But the biocompatibility between KPros and cornea tissue was found to be not good. At present there is no KPros which could be safely used in clinic without any complications.
Many materials have been used as skirts for KPro in the published studies，including carboform[1]，the mixture of polybutene and polypropylene，polyfluortetraethylene[2]，poly 2hydroxyethy methacrylate (PHEMA)[3, 4], scientists focused KPros on porous supports in recent years.
In China, majority of the corneal blindness patients cant be treated mainly during to shortage of cornea donor tissue .
Only few patients could be cured with traditional keratoplasty. So KPro surgery will be used widely in future for patients with severe ocular surface diseases resulting in blindness. At present most KPros were not safe enough, they always lead to serious complications due to poor histocompatibility. Comparing with other KPros, Chirila KPro from Australia has better histocompatibility to integrate with surrounding receipient cornea tissues. But the mechanical tensility of KPros sponge margin is lower, which results in difficulty for suturing and complications after surgery. Based on Chirila KPros, our research not only improved its disadvantages but also took advantage of its strong points. Utilizing PHEMA as chief material, we added some polymethyl methacrylate (PMMA) to strengthen the tensile intensity of KPro. In the meantime, we used this modified porous PHEMAPMMA KPro in rabbit and monkey corneas and observed the primary results.
MATERIALS AND METHODS
Manufacture of KPro The modified porous onepiece PHEMAPMMA coreandskirt KPro has been manufactured by the biomedical engineering Department of Jinan University (gifted). The KPro is made up of central optical core and peripheral porous sponge. The central core is polymerized by HEMA and MMA monomer(mass ratio is 10∶1); peripheral spongy skirt is polymerized by HEMA monomer in water. The two regions are joined at their interface by means of an interpenentrating polymer network, a strong chemical junction. There is a layer of PHEMAPMMA membrane which has been polymerized together with the central core in the bottom of spongy skirt. On this skirt membrane there are many laser holes of 200300μm size. The resulting device is cut to the required size, curvatured with a diamondtool lathing machine and stored in sterile balanced salt solution. After autoclaving, the finished KPro has a radius of 7.0mm, a thickness of 0.5mm, a whole diameter of 7.5mm with a central transparent diameter of 4.5mm (optic). The pores of peripheral spongy skirt are between 20 m and 60 m in size, and porosity is about 65% (Figure 1, 2).
Experimental Animals Eighteen New Zeland rabbits and two rhesus monkeys of either gender were used in this study. The experiment was divided into two groups. Group A: Corneal lamellar implantation of PHEMA sponges were done in ten rabbit eyes.
Group B: KPro implantation was done into the corneal lamellae in the other eight rabbit eyes and two monkeys eyes.
Surgerical Procedure
Implanting PHEMA porous sponges into the lamellar pockets: group A Under general anaesthesia, we dissected the partial anterior cornal(diameter is 9mm),implanted the PHEMA sponges between the cornea lamella, and sutured the anterior cornea with 100 nylon. Slit lamp pictures of these eyes were taken on 2, 4 weeks, 2, 3 and 4 months after operation. Two pieces of corneas were taken out at every period for histopathology under light microscopy and transmission electron microscopy (TEM); and immunohistochemistry was also done.
Keratoprostheses implantation: group B The KPro was implanted through a twostage surgery procedure. The interval between stage I and stage II was 3 months.
Stage I: Under general anaesthesia, 9mm trephine was used to imprint an annular mark on the cornea with the papillary as central point. A partial cut was made in the cornea involving peripheral superior 270° according the mark. The depth of cutting was about half of corneal thickness. We dissected the anterior corneal flap from posterior cornea, the dissection continued as a pocket of 9mm diameter within the inferior cornea. The flap was retracted inferiorly, allowing a central trephination of 3mm to be made in the posterior lamella of the cornea. The KPro was then positioned within the pocket so that the center of its optic lay over the posterior opening. The skirt of the KPro was sutured to posterior lamellar cornea with 100 nylon. The anterior lamellar cornea was replaced and sutured with posterior lamellar cornea in situ with 100 nylon. At that time, KPro was held between anterior and posterior lamellar cornea, injecting physiological saline solution into anterior chamber from side incision of cornea. Sub conjunctival injections of dexamethasone 2.5mg and gentamicin 20mg were given. Tarsorrhaphy was done and kept for 48 hours. Topical Tobradex eyedrop was given four times daily for the first postoperative month, and Tobramycin 3g/L ointment was given every night until the second stage of surgery.
Figure 1 The PHEMA sponges（略）
Figure 2 Modified PHEMAPMMA Keratoprosthese KPro has a radius of 7.0mm, a thickness of 0.5mm, a whole diameter of 7.5mm with a central transparent diameter of 4.5mm（略）
Figure 3 Three months after sponge into intra lamellar cornea, new vessel invasion into sponge pore（略）
Stage II: The anterior layers over the KPro optic were trephined with a 3mm dermatologic punch to expose the optic as a fullthickness corneal replacement.Then tarsorrhaphy was done and kept for 48 hours. Postoperatively, TobraDex eyedrops 4 times per day and 5g/L tetracycline ointment every night to animals for 46 weeks.
Histopathology examination Cornea tissue was fixed in 40g/L paraformaldehyde, and then embedded in paraffin. After the sections were made, haematoxylineeosine staining was done and observed under light microscope.
Immunohistochemistry examination Streptavidin biotinperoxidase complex (SABC) technique was carried out. First antibody was mouse antirabbit monoclonal antibody (dilution was 1∶50), second antibody was goat antimouse IgG. All the reagents were come from Wuhan Boster Company.
Transmission electron microscope examination Cornea tissue was fixed in electron microscopy stationary liquid, dehydrated, soaked, blocked, sectioned, stained, and observation under TEM. KPro and neighboring tissue (including cells) were seen with low power lens. Structure of cell organs, extracellular matrix, collagen and angiogenesis were observed with high power lens.
Ultrasonic biomicroscope and Bmode ultrasound examination Three months after the second stage of operation, the eyes of twomonkeys were subjected to Ultrasonic biomicroscope(UBM) and Bmode ultrasound (Bscan) to see the situation of anterior chamber angle, vitreous body and retina.
RESULTS
Group A
Slit lamp examination We didnt detect the complication after sponge intralamellar implantation in 10 rabbit eyes. In early stage after surgery, there was no obviously response in either anterior chamber or pupil. Ten to fourteen days later, there was angiogenesis in upper part of limbus. Till three weeks later, there was much angiogenesis; some of it came near to the edge of PHEMA sponge. Six to eight weeks later, plenty of angiogenesis appeared in corneal stroma around the sponges. Three to four months later, PHEMA sponges remained stable in lamellar pocket and there were no new blood vessels (Figure 3).
Histopathology results Two to four weeks after implantation, cells were seen growing into the margin of PHEMA sponge; most of those cells were fibroblasts, others were inflammatory cells. We also found some inflammatory cells, mostly lymphocytes, in nearby corneal tissue. Two months later, fibroblasts and new blood vessels dispersed in the PHEMA sponge; collagen fibers in nearby cornea arranged orderly with few inflammatory cells infiltrated. Three to four months later, lots of fibroblasts and angiogenesis occurred in sponge without inflammatory cell infiltration (Figure 4A, 4B). Corneal tissue near the sponge showed regularly arranged collagen fibers and few eosinophilic granulocytes.
Immunohistochemistry results We got sections of corneal tissue at different periods. In each time, stroma cells of cornea were positive to Vimitin and full of filemot cytoplasm. The fibroblasts in sponge tissue held between lamellar corneas were positive to Vimitin immunoreactions. Their cytoplasm was filemot and nucleolus was blue (Figure 5).
TEM results Fibroblasts grew into the holes of sponge two to four weeks after surgery. Till two months after operation, most fibroblasts grew up between the pores in the sponge.
Figure 4 A: Haematoxyline eosine stain, fibroblast and new vessel seen into the pore of the sponge 3 months after sponge into intra lamellar cornea（200×）; B: Haematoxyline eosine stain, no obvious inflammatory cells intiltrated into the pore of sponge 3 months after sponge into intra lamellar cornea (200×)（略）
Figure 5 Immunohistochemistry, fibroblast in the sponge is positive for Vimitin (400×)（略）
Plenty of chondriosomes and rough endoplasmic reticulums were observed. Three to four months later, more cells growing into the pores of sponge. We could see cytoplasmic processes extend along the interspace and some of them superpose each other under low power microscope. We also observed that cell organs, chondriosomes and endoplasmic reticulums were active under high power microscope (Figure 6). There were angiogenesis and vessel endothelial cells in some fields of view.
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