ОФТАЛЬМОЛОГИЧЕСКАЯ КОНФЕРЕНЦИЯ "Рефракция 2009. Пресбиопия"ИМПЛАНТАТ АМНИОТИЧЕСКОЙ МЕМБРАНЫ СИЛИКОВЫСУШЕННЫЙМатериалы по теме "Взаимосвязь гидродинамики и аккомодациии" готовятся к публикации на сайте. Обновление информации ожидается после 10 ноября 2008 года.
English  
О больницеУслуги и ценыАРТ-ВизиологияКонтакты

SILICA GEL DISSICATION OF AMNIOTIC MEMBRANE WITH RELATED EPITELIUM CELLS FOR OCULAR SURFACE RECONSTRUCTION

Cell and Tissue Banking 5:271-275, 2004.

SILICA GEL DISSICATION OF AMNIOTIC MEMBRANE WITH RELATED EPITELIUM CELLS FOR Ocular Surface Reconstruction

E.S. Miljudin, A.V. Zolotaryov, L.T.Volova*, U.M.Ahmerova

Ophthalmology hospital a.n. T.I.Eroshevsky

*Samara State medical university

Address for correspondence:

Miljudin E.S.

Novo-Sadovaja str., 158

Samara, 443068, Russia


Cornea reparative regeneration when in various pathological states needs creating certain conditions to intensify the potential of regional trunk cells mitotic activity. The search for materials to creat  these conditions led to  a rediscovery of amniotic membrane. The anatomic structure, peculiarities of  tissue and the functions  of  the amnion determine the qualities  that attract research -workers  and clinic  specialists and make  them use amniotic membrane when treating various pathological states.  Used for the making of the  transplantant the out-of-placenta zone of the amnion is carpeted v/with cubic epithelium.  Amnion epithelium produces glycoproteins,  interlinkings,  growth  factors (Champliaud M.F., et.al., 1996). These substances  intensify the prolypheration,  migration and differentiation of  trunk epithelium cells, prevent  epithelium apoptosis  and  suppress  the development of fibroais  (Keelan J.A., et.al.,1997 Shimazaki J, et.al.,1997; Na B.K, et.al.,1998).

Especially interesting for the problem under discussion are  the laminins found in epithelium cells. These glycoproteins are responsible for the cells of regenerating epithelium adhesion to  the out-of-cell matrix.

Consequently, the  layer of  epithelium  cells  of  the  amniotic membrane and the  active proteins found there  are of prime importance in creating conditions for intensifying the regional trunk cells mitotic  activity potential and  successful fixation of  the formed anew layer of  the  epithelium cells  on the cornea  surface.

However, the use of native amniotic membrane is restricted by the necessity of preliminary investigations, by the possibility of infectioning the recipient and the  short-term period of  epithelium preservation.

Amnion epithelium is well  preserved in nourishing surroundings at +4°С during 6-7  days with all the healing qualities being, preserved, too.

When preserved being frozen down to –80°С  the  amniotic membrane changes within 6 months: there are picknotic  changes in the nuclei, the destruction of  the nuclei,  the appearance of the polygonal cell form (Panda A. 1999, Mejia LF, et.al. 2001, Xu L, et.al., 2001). Further investigation with the cultivation of criopreserved amniotic membrane cells showed that the amnion epithelium cells used to lose their faculty for growth in vitro  (Taylor, R. J., et.al.,1998).

Conservation with the lyophilic drying used and further sterilization of  the amniotic membrane with gamma rays from  the very beginning presupposes the preservation only of the collagen structure (Момозе А., et.al.,2001).

However, as well as lyophilized amniotic membrane native and criopreserved membrane, when fixed on  the surface  of  the eye ball  have similar healing qualities (Tseng S.C., et.al.,1997; Момозе А., et.al.,2001).

A much simpler method  (in comparison with lyophilization) is the  one of drying over silica gel. This method of  conservation, as  well as  lyophilization, does not make  the  cells  of  the  amniotic membrane preserve their vital capacity; so, we may predict the preservation of  the amniotic membrane unique qualities  in having non-vital capacity epithelium.

Aims of the Research: to find out the decree of the epithelium AM preservation after preliminary processing and conservation by means of dehydration over silica gel with further sterilization; to study the effectiveness of clinical treatment of AM conserved in the surroundings with vital ability epithelium and AM dried over silica gel.

Materials and methods. There was carried out an investigation of 18 samples of native amnion treated with antibiotics and 18 total surface amnion samples conserved by drying over silica gel and then sterilized by gamma rays.  Clinic experiments were carried out on patients with severe chemical and thermal burns. In the years of 1999 - 2002 we watched 18 people (21 eyes) with severe cornea and eyeball conjunctiva burns.  All the patients were male with average age of 39. Thirteen patients  (15 eyes) had chemical affection; 5 patients  (6 eyes) had thermal affection.  The patients were divided into two groups. Four patients  (5 eyes) of the 1st group were treated with AM with vital capacity cells of the epithelium layer. Fourteen patients (16 eyes) of the 2nd group were treated with AM conserved by drying over silica gel with non-vital capacity epithelium layer. During the pre- and post-operation period all the patients had visometry, biomicroscopy, cytogram of  the cornea  epithelium. Either during the first 3 – 4 days or on the 10 - 14th day after the burn trauma all the patients underwent the standard procedure of necrectomy, the covering of the eye ball with amniotic membrane and the covering of the conjunctiva arches with the amniotic membrane dried over silica gel.

Preparation and preserve human amniotic membrane.

Human placentas were collected aseptically from selective caesarean sections in normal women in time, having seronegative for human immunodeficiency virus (HIV), hepatitis B and C viruses, and syphilis results. In the eye bank the amnion is separated from the remnants of chorion washed free of blood clots. After processing in the solution of antiseptics and antibiotics in addition the amniotic membrane is treated in glycerol. The scarps of the amniotic membrane are fixed on frames over silica gel for 18 – 24 hours. Then, under sterile conditions, the amniotic membrane is separated from the frame, paced and -sterilised. Under room conditions the preservation time of the amniotic membrane, thus conserved, is not less than 24 months in hermetic packing.

Results. Detailed study of the native amnion proved the existence in it of 5 layers; amniotic epithelium, balm membrane, compact layer, fibroblast layer and spongiosis layer (Van Herendael B.J., et.al., 1978).

The epithelium cells are, mainly, one -nucleus, but there can be met multi-nuclei as well.  In amniotic epithelium cells there can be wet mitosis  (approximately 1 per 100 cells). The epithelium cells are connected by bridges between which inter-cell gaps are located.

Under electronic-microscopic research it was found out that the diameter of inter-cell gaps is 7 - 10 mn.  These gaps are connected with the amnion cavity and inter-cell vacuoles, they are treated as inter-cell canals that take part in the processes of interchange (Ю. В.Гулькевич et.al., 1968).

The balm membrane is situated under amniotic epithelium and consists of a thin net of reticular fibers. In the under-epithelium layer there are blood vessels. To the balm membrane there adjusts itself the compact layer, which also  consists of  interwoven and closely connected reticular fibers. The fibroblast  layer consists of cells which are situated in the  thick net of collagen, reticular fibres and intercell  substance. Among the cells of this layer there are nitosis, gystiocids and Kastchenko – Gofbauer cells.

The spongiosis layer is connected with smooth chorion via conneetive-tissue fibres and inter-cell substance. The chorion consists  of reticular fibres betveen the loops of which there  is liquid.

The study of  hystological preparations of AM dried over silica gel proved our predictions about the considerable changes in the epithelium layer. For one  thing, the  epithelium cells look thickened with homogeneous oxyfilled cytoplasm. The majority of the nuclei are of stick-like or oval form with rough blocks of chromatin. In some cells one can see the picknosis of the nuclei; in some cells there are near-cells vacuoles. The nuclei in such cells are changed - they are of irregular shape the surface is not smooth, with deformities end protuberances. In some areas the cells do not have nuclei. The balm membrane is well-contoured. The compact layer looks as if it were homogeneous oxyfilled band. In the connective tissue we see a great decrease of the number of cell elements. It is not seldom hat the shadows of  the nuclei are seen. The nuclei of the preserved fibroblasts are of stick-like form. The cells themselves are considerably thickened. The number of macrophagal elements is also decreased, but we managed to see, side by side with the  thickened  Kastchenko-Gofbauer cells, normal unchanged cells. In this zone one can see longitudinal fibres and areas of homogeneous oxyfilled colour. The spongiosis layer is seen as acre loose than the one described before. In it one can see loosely situated clusters of winding fibres which show weak oxyfillation. Between the clusters of fibres there are accidental fibroblasts. We cannot trace a strict borderline between these two layers - that  of fibroblasts and the spongiosis layer.

The cariopicknosis that we see in the preparation testifies to the fact that the amnion epithelium and the macrophagal row of cells lose their vital capacity after being dried over silica gel. So, side by side with rather  good preservation of structure, amnion cell  structure, dried over silica gel, we should note  the non-vital capacity of the cells  of its epithelium,  fibroblasts and the decrease of  the number of cells in all  the  layers. However, in connection with the preservation of the cell structure and the close links of cell membranes of the epithelium with the underneath layer there appear grounds to predict the preservation of certain qualities of AM dried over silica gel which are characteristic only of AM with vital capacity epithelium layer.

On the 3d day in both the groups the donor material preserved its close application to  the eye ball surface and became transparent. The patients noted the decrease of pain in the eye. During 8-10 days the inflammation process was stopped, the hypopyon resolved, there were infiltrates in the stroma of cornea. AM had been resolving for 15-21 days.  All the patients were seen to have quick epithelization of the eyeball covered with AM.  Patients with the 3d decree of burn and a partial affection of limbal area had epithelium formed by the 7-12 day of  the  post-operation period. Cytological investigation carried out on such patients proved  the  regeneration of  cornea epithelium.  Patients with  the 4th degree of burn had the surface of  the cornea covered with conjunctiva epithelium in all  the cases.  With all the patients AM coverage made it possible to avoid the lysis of the cornea and to partially preserve the conjunctiva arches.

When admitted for treatment, the majority of patients of the 1st group had the acuity of vision that allowed them  to  correctly identify light. One of the patients with the 4th decree of chemical burn had the acuity of vision that equaled –0,01.

In the 2d group the acuity of vision of patients with the 3d decree of burn equalled, on the average,  -0,05  (from correct light identification up to 0,3); patients with the 4th decree of burn had –0,03 acuity of vision  (from correct light identification up to 0,09).

When examined in three months time, all the patients demonstrated a well-formed wall-eye with rare newly formed vessels. There were no traces of relapse of cornea epithelium erosion. When cytological investigation was  carried out on patients with the affection of more  than 2/3  of  the limbal  zone diameter on  the  cornea surface  there  were found cells  characteristic of  the conjunctiva mucous membrane.

In three months time after the trauma acuity of vision of two patients in the 1st group improved from 1/p.l.c. up to 0,01.

During the same period of time acuity of vision of the patients with the 3d degree of  burn  increased in the majority of  eyes.

The range of acuity of vision varied from 0,8 to light identification. In one case acuity of vision dropped from 0,03 to 0,01. On  the eyes with the 4th degree of affection the improvement of acuity of vision was achieved only in one case, with a patient with a thermal burn, and it was from 0,09 up to 0,5. For all this, the centre of affection was situated a little bit eccentrically, in the lower outward area.  In all the remaining cases the acuity of vision became worse, dropped to light identification with correct light projection.  Low degree of acuity of vision is determined by temporary blepharoraphia and cornea opacity.

The usage of AM with vital capacity cells  of  the epithelium layer is, practically, no less effective  than the usage of AM dried over silica gel.  However, surgeons remark the complexity of cutting off and fixing up the native AM transplant on the eye ball surface.

Conclusion.

The drying out of the amniotic membrane over silica gel on frames without being fixed on nitrocellulose paper makes the process of the amniotic membrane conservation simpler and makes it possible to preserve its unique biological qualities.

The effectiveness of the regeneration of epithelium tissue of the eye ball surface with amniotic membrane dried over silica gel without  the vital  capacity cells  of  the  epithelium layer is analogous  to  the regeneration of epithelium cells with amniotic membrane with vital  capacity cells.

With eye burns AM coverage hinders the formation of rough conjunctiva cicatrix, provides a favorable out-of-cell matrix substrate for epithelium migration and leads to quicker regeneration of one"s own epithelium,  makes further visual rehabilitation simpler.

 

References.

1.     Addsa P.J, Huntb C., Hartleyc S.  Bacterial contamination of amniotic membrane //Br J Ophthalmol 2001;85:228-230.

2.     Mejia LF, Acosta C, Santamaria JP.Use of nonpreserved human amniotic membrane for the reconstruction of the ocular surface. //Cornea. 2001 Oct;20(7):773-4.

3.     Panda A. Amniotic membrane transplantation in ophthalmology (fresh v. preserved tissue) //Br J Ophthalmol. 1999 Dec;83(12):1410-1.

4.     van Herendael BJ, Oberti C, Brosens I. Microanatomy of the human amniotic membrane: a light microscopic, transmission and scanning microscopic study. Am J Obstet Gynecol 1978;131:872-880.

5.     Xu L, Zhou S, Chen J, Chen L, Zhang M. A study on the preservation of fresh amniotic membrane. //Yan Ke Xue Bao. 2001 Sep;17(3):158-62.

6.     Гулькевич Ю.В., Маккавеева М.Ю., Никифоров Б.И. Патология последа человека и ее влияние на плод. Минск Беларусь 1968; 232.

7.     Момозе А., Ксяо-Хонг К., Джунсуке А. Использование лиофилизированной амниотической оболочки человека для лечения поражений поверхности глазного яблока //Офтальмохирургия, 2001, №3. -С.3-9.


Abstract.

SILICA GEL DISSICATION OF AMNIOTIC MEMBRANE WITH RELATED EPITELIUM CELLS FOR Ocular Surface Reconstruction

E.S. Milyudin, A.V. Zolotaryov, L.T.Volova*, U.M.Ahmerova

Ophthalmology hospital a.n. T.I.Eroshevsky

*Samara State medical university

Cornea reparative regeneration when in various pathological states needs creating certain conditions to intensify the potential of regional trunk cells mitotic activity.

Aims of  the Research: to find out the decree of  the  epithelium AM preservation after preliminary processing and  conservation by means of dehydration over silica gel with further sterilization; to study the  effectiveness of clinical treatment of AM conserved in  the  surroundings with vital  ability epithelium and AM dried over silica gel.

Materials and methods. There was carried out an investigation of 18 samples of native amnion treated with antibiotics and 18 total surface amnion samples conserved by drying over silica gel and then sterilized by gamma rays.  Clinic experiments were carried out on patients with severe chemical and thermal burns – 18 people (21 eyes). After the burn trauma all the patients underwent the standard procedure of necrectomy, the covering of the eyeball with amniotic membrane dried over silica gel.

Conclusion.

The drying out of the amniotic membrane over silica gel on frames without being fixed on nitrocellulose paper makes the process of the amniotic membrane conservation simpler and makes it possible to preserve its unique biological qualities.

The effectiveness of the regeneration of epithelium tissue of the eye ball surface with amniotic membrane dried over silica gel without the vital capacity cells of the epithelium layer is analogous to the regeneration of epithelium cells with amniotic membrane with vital capacity cells.

With eye burns AM coverage hinders the formation of rough conjunctiva cicatrix, provides a favorable out-of-cell matrix substrate for epithelium migration and leads to quicker regeneration of one"s own epithelium, makes further visual rehabilitation simpler.

Key Words: Amniotic membrane, cornea, drying over silica gel, epithelium cells, eyeball burns

<-Назад

 



© Copyright 2005 «Областная клиническая больница им. Ерошевского»
© Веб-дизайн: GxG, © Разработка: InternetElite