Morphological , comparative and qualitative tissue reaction analysis due to the implantation of alloplant TM to the eyelid and subcutaneous of rabbits

Objetivo: Analisar comparativamente através da microscopia óptica, a reação tecidual causada pelo Alloplant Ô na conjuntiva e dorso de 24 coelhos. Métodos: Todos os coelhos foram submetidos a implantação do AlloplantÔ no terço interno da pálpebra inferior, em substituição à conjuntiva tarsal de um dos olhos. Após trinta dias seis coelhos (grupo I) foram sacrificados e as pálpebras inferiores dos dois lados e um fragmento do dorso (controle) foram encaminhados para a microscopia óptica. Após sessenta dias foram sacrificados mais seis coelhos (grupo II) e suas pálpebras um fragmento do dorso (controle) foram encaminhados para estudo microscópico. Nesse dia também foram realizadas implantações cirúrgicas do aloimplanteÔ no dorso dos doze coelhos restantes, os quais foram sacrificados noventa (grupo III) e cento e vinte (grupo IV) dias após a primeira cirurgia para estudo microscópico das pálpebras e dorsos. Resultados: O “Alloplant Ô” (aloimplante), causou intensa reação inflamatória mista exsudativa e proliferativa com predominância de linfócitos, macrófagos e formação de granulomas de corpo estranho nas duas localizações, que evoluiu com a substituição do implante por tecido conjuntivo fibroso denso. Conclusão: na pálpebra a reação inflamatória foi mais intensa e mais prolongada do que no dorso e no dorso a formação de fibrose foi mais intensa, mostrando inadequação ao uso cirúrgico. Descritores:Implantes experimentais;Histologia; Pálpebras; Procedimentos cirúrgicos ambulatórios; Microscopia; Coelhos ABSTRACT

In 1976 Muldashev, Kamelieva and Vasilieva, proposed the use of anallograft, with very similar characteristics to the human tarsus, extracted from fascia, tendons, fat and plantar region of human cadavers, as another option for the reconstruction of the eyelid.According to these authors, the material"Alloplant ™ " (allomplant) was well incorporated into the host,with a quickly epithelization, stimulating the regeneration of the local tissue and could be used indifferent surgical procedures besides oculo plastics.(9)(10)(11)(12)(13)(14).
The aim of this study was to comparatively analyze the tissue reaction caused by implantation of "Alloplant ™ " (Figure 1A and 1B) in the eyelid and backs of rabbits, by light microscopy with qualitative and semi-quantitative analysis.

METHODS
The experimental study was performed in1997.Twenty four rabbits were divided into four groups (I, II, III and IV) of six rabbits each and two surgical modalities: surgery with implantation int he eyelid of all rabbits and surgery with implantationin the back of 12 rabbits.
Fora better understanding the study was divided into five times as follows (Table 1): Experimental time zero: 24 rabbit sunderwent surgery with implantation on the lower eyelidof one side, chosen random lyat time of surgery.
Experimental time one: (30th day after eyelid surgery), 6 rabbits in group I were sacrificed and lower eyelid sand fragments of the back (control) were sent to histopathological study.
Experimental time two: (60th day after eyelid surgery), 6 rabbitsin group II were sacrificed, lower eyelids and one fragment of the back (control), were sent to histopathological study.At this experimental time the surgery with implantation in the back of the rabbits in groups III and IV were performed.
Experimental time three: (90th day after eyelid surgery), 6 rabbits in group III were sacrificed, lower eyelids and framents of the back (30th day after dorsal surgery) were sent to histopathological study.
Experimental time four: (120th days after eyelid surgery), 6 rabbits of group IV were sacrificed, lower eyelids and fragments of the back (60th day after dorsal surgery)were sent to histopathological study.
These animals were kept inindividual appropriate cages and suitable environmental conditions (DPC Laboratory Medlab -São Paulo -Brazil) during the pre and postoperative times.The postoperative ocular health of each rabbit were followed up by clinical examination with drops of fluorescein and cobalt blue filter flash light, for corneal examination at the surgical site on the first, seventh, fifteenth and thirtieth postoperative day of the eyelid surgery.
The back was assessedin the first and seventh post operative when the 6 -0 mono nylon sutures were removed.

Collection of tissue
The lower eyelids and backs were taken from all rabbits of the study when they were sacrificed, and the non-operated eyelids and backs were considered controls.

Histological examination of the eyelids
All specimens, includinga fragment of Alloplant were fixed Table 1 Twenty four rabbits were divided into four groups (I, II, III and IV) of six rabbits each and two surgical modalities: surgery with implantation in the eyelid of all rabbits and surgery with implantation in the back of 12 rabbits.

Surgical techniques
Each rabbit was placed separate lyin a suitable location for thecontainmentand anesthetized with intravenous injection of sodium pentobarbitalat a dose of 33mg/kg and one drop of proparacaine 0.5% instilled in the lower conjunctival fornix for lower eyelids surgery,wherea fragment of approximately 5x7mm of the middle third tarsal conjunctiva was ressected (Figure 1C) and in its place it was sutured a similar fragment of the alloplant with interrupted 6 -0 Vycrilsutures.
For surgeryin the back it was used 2% lidocaine with 1:200,000 epinephrine injected into the subcutaneous before the   (15).Received coded identification numbers, so that the pathologisthad no informationon the time elapsed between the graft and biopsy (double blind).The research was conducted at DPC MEDLAB PRODUTOS DIAGNÓSTICOS, at São Paulo, Brazil.
The scores were given for the presence of inflammation: 0 = no inflammatory reaction; 1 = mild inflammation; 2 = moderate inflammatory reaction; 3 = severe inflammatory reaction.

Statistical analysis
For statistical analysisWilcox on tests were applied; Analysis of Variance points by Kruskal -Wallis and the Fisher exact test was set at 0.05 or 5% (a<0.05) the level of rejectionof the null hypothesis, indicating with an asterisk a significant values outcomes.

RESULTS
Regarding the ocular health of the rabbits it was observed in the early day salight to moderate punctate keratitis on the lower third of the operated side cornea which decreased significantly on the seventh day disappearing completely altogetherin the fifteenth postoperative.

Histologic controls
Eyelid Controls: the cross-section showed up: skin, orbicularis, tarsal plate permeated by Meibo miangland scovered by cylindrical conjunctival epithelium, very similar to human eyelid (Figures 2A and B).
Backs Controls (group I and II): no abnormalities (Figures 2 C and D)

Histology of the Backs
On the 30th postoperative day (group III), moderate chronic mixed inflammatory process, with presence of foreign body granulomas, mostly lymphocytes and macrophages and severe fibrosis (Figure 3A and B).

Histologic of the eyelids
On the 30th postoperative day (group I), anintense mixed, proliferative and exudative inflammatory reaction, with a predominance of lymphocytes and macrophages, the presence of foreign body granulomas, dissociation of collagen fibers of the implant within macrophages in all the pieces.The conjunctiva was complete reepitheliased with squamous epithelium with stratified non-keratinized "metaplasia" (Figure 4 A and B).
On the 60th postoperative day (group II), a persistence of intense mixed inflammatory process and the beginning of fibrosis formation (Figure 4 C and D).
On the 90th postoperative day (group III) chronic mixed inflammatory process starts to decrease and increases the fibrosis formation (Figure 4 E and F).
On the 120th postoperatively day (group IV): persistence ofchronic mixed inflammatory process and moderate degrees of fibrosis formation (Figure 4 Gand H).

DISCUSSION
Despite numerous published surgical options, depending on the extentand location of the eyelid defect, using an implant would eliminate themorbidity of the donor site and the lack of adequate amounts of tissue for reconstruction (2).
Among several implant possibilities,"Alloplant™" was chosen, and according to the authors (10) was a material taken from tendons, fascias and plantar region of human cadavers, with similar consistency to the human tarsus and easy handling, allowing its use as a natural tissue regeneration stimulator in any part of the human body (12).
As the literature about "Alloplant™" was scarce and not clarifying, it was decided to put an experimental study that could demonstrate the histological changes at the site of implantation.
The choiceof the rabbit as experimental animals was dueto the fact that the rabbit eyelid exhibits major histological similarities with human eyelid, and they are docile and easy handling animals.
The implant was placed in the middle third of the lower eyelid in direct contact with the anterior ocular surface (Figure 1C) without conjunctival covering, which allowed the assessment of repairof the conjunctiva at the siteand also the traumatic changes caused to the cornea.As occurred in the experimental study of Jordan et al.( 6) who use dirradiated human tarsal implantin the lower eyelids of monkey sin direct contact with the cornea, light superficial punctate keratitis was observed in the first day safter surgery, neither of which cases progressed to persistent epithelial defect and corneal ulceration.Histologically, the conjunctiva of the site was completely healed 30 day post operatively.
Tissue was treated with Hematoxylin-Eosin (HE) and also Masson's Tricromium (TM), this last one revealed to be the best coloration to better disclosure of collagen fibers.(15) The histological inflammatory reaction observed was much more intense and persistent in the eyelid than in the back, possibly because in the back the implant was protected from theexternal environment, facilitating phagocytosis and hence speed of fibrosis formation, but in both locations the implant caused the same type of reaction: mixed exudative and proliferative inflammatory, with predominance of lymphocytes, macrophages and formation of foreign body granuloma (Figure 3 C).These findings are very similar to those reported by Muldashev et al. (9), who observed that in the first weeks after surgery there was a major inflammatory reaction with proliferation of fibroblasts "Alloplant™" was being completely reabsorbed and replaced by host tissue growth, but only in the later postoperative (14).
The number of lids was higher than the backs because the back allowed us to assess the antigenicity of the implant, that is, if the rabbits would havepreviously been sensitized during the eyelid surgery, as demonstrated in the experimental study with preserved duramater by Dias et al. (16).
Russian researchers, with the aid of histochemical electron microscopy, found that inflammatory immune response decreases considerably with the extraction of glycosaminoglycans collagen fibers molecules from the all omplant which inhibits the migration of lymphocytes, reducing the infiltration of macrophages and fibroblasts by increasing the ability of natural regeneration of normal tissue of adjacent areas.(12,13).The understanding of this process was acquired after this study.
The microscopicaspect observedin the corresponding area on the back implantation was very similar to the eyelid.If there had been prior sensitization at the time of the first implant (17), one would expecta cellular responsetriggered by the second implant, which would remove the possibility of "Alloplant ™" be have asimmunologically inactive substance (6,7).
Removal of glycosaminoglycans from collagen fibers of different tissues (fascia, tendons and connective tissue) contributes to this type of implant present low immunological properties (12,13).Thusit could be considered as an alternative choice in the eyelids reconstructions involving the restoration of posterior lamella because it would avoid the use of ear cartilage nasocondral, preserved sclera and the hard palate.Also avoid the occlusion, even if temporaly one eye and also a second surgical intervention (2).Some authors employed similar material that could be a good choice in the case in eyelid reconstruction which is the "AlloDerm (Life CellCorp., Woodlands, Texas, USA) alloderm TM.Consists of acellular human dermis, and can be used both in eyelid reconstruction of the anterior and the posterior lamella (18,19).

CONCLUSION
The Alloplant™ when implanted in the eyelids and backs of rabbits caused an intense mixed exudative and proliferative inflammatory reaction with a predominance of lymphocytes, macrophages and formation of foreign body granuloma.The microscopic findings observed in the correspond in garea on the eyelid implant showed histological pattern of more intense inflammation than the back and in both cases the implant was replaced by dense fibrous connective tissue.
The implant site was repithelized with conjunctival epithelium of the non-keratinized stratified squamous type(squamous metaplasia), revealing that the implant was unsuitable for use on the eyelids.

Figure 1 :
Figure 1: The aim of this study was to comparatively analyze the tissue reaction caused by implantation of "Alloplant ™ " in the eyelid and backs of rabbits, by light microscopy with qualitative and semiquantitative analysis.