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 »  Introduction
 »  Material and methods
 »  Results
 »  Discussion
 »  Conclusion
 »  References

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Year : 2002  |  Volume : 50  |  Issue : 3  |  Page : 256-61

Immune response to dehydrated human dura mater : evaluation in a rabbit model.

Department of Neurosurgery, Ataturk University Medical School, Erzurum - 25240, Turkey.

Correspondence Address:
Department of Neurosurgery, Ataturk University Medical School, Erzurum - 25240, Turkey.
[email protected]

  »  Abstract

Ninety white hybrid rabbits, each weighing 2.5 to 3.5 kg, were used for this experimental model. Thirty rabbits were used for control, and sixty other rabbits were investigated for the response of host to the dural graft. In all animals, a dural defect, 1 x 1 cm in size, was created on the left parietal area following craniotomy. In the control group the excised free dural piece was then sutured again to the area from which it had been excised before. The dural defect was closed with dehydrated human dura mater (DHD) in the half of the rabbits in the group of study, and with autogenous fascia lata (AFL) in the other half. After operation, animals in each group were then subjected to one of five different groups comprising of 3,14,30,60 and 90 days follow-up periods. At the end of follow-up periods, histological, parameters such as cellular inflammatory response, development of fibrous tissue, capsulation, and calcification were examined in specimens obtained from the animals. There was no significant difference between AFL and DHD grafts. In conclusion, it seems that DHD is suitable as an ideal dural graft, because the immune response of host to DHD was almost similar to AFL.

How to cite this article:
Kadioglu H H, Takci E, Arik M, Gundogdu C, Aydin I H. Immune response to dehydrated human dura mater : evaluation in a rabbit model. Neurol India 2002;50:256

How to cite this URL:
Kadioglu H H, Takci E, Arik M, Gundogdu C, Aydin I H. Immune response to dehydrated human dura mater : evaluation in a rabbit model. Neurol India [serial online] 2002 [cited 2023 Feb 5];50:256. Available from: https://www.neurologyindia.com/text.asp?2002/50/3/256/1446

   »   Introduction Top

For a long time, different organic and inorganic materials have been used for repairing of dural defects. The varying porosity of the synthetic materials has allowed new tissue to grow from intact dura mater to the graft and better integrity of healing tissue.[1],[2],[3],[4],[5],[6],[7],[8],[9],[10],[11],[12],[13] Although all the graft materials used till now have useful properties, an ideal graft material is yet to be found. The recipients have tolerated the autogenous grafts very well, yet they required a second surgery, thus extending the duration of total operation and increase in postoperative morbidity due to the second surgery. Besides, some patients refused a second operation for cosmetic results.[5],[11],[12],[14] Both allografts and alloplasts, however, may cause a reaction between host and graft. There may be complications like resorption, distortion, migration, and rejection of grafts, infection, and necrosis secondary to use of different graft materials.[1] The dura mater grafts have been used approximately for a century of repairing dural defects.[12] A slow virus infection has been reported with use of dura mater grafts, due to limited cellular responses.[7],[15],[16],[17] We designed an experimental model to reveal the host response to dehydrated human dura mater (DHD) found as a result of the investigations carried out a long time ago.

   »   Material and methods Top

Ninety, white hybrid rabbits, each weighing between 2.5 to 3.5 kg, were used for the study. Thirty rabbits were used as the control group and the remaining sixty rabbits were used as the study group. The study group was first divided into two main groups, according to grafts to be used, and each of these main groups was also divided into five subgroups of 3,14,30,60 and 90 days followup and analysis.[18] The surgery was performed on animals after six hours fasting and anesthesia with intramuscular injections of 30 mg/kg of ketamine HCl (Ketalar, 50 mg/kg, 10 ml flacon, Eczacibasi Ilaç San, Istanbul, Türkiye). The forehead and parietal regions were shaved. In half of the animals of the study group, the lateral region of the left thigh was also shaved and all of these shaved regions were prepared with polivinylprolidoniode (Batticon, Adeka Ilaç San., Istanbul, Türkiye). In animals of the control group, scalp was incised by using coronal incision. After the pericranial tissues were elevated, left parietal craniectomy of two square centimeters was performed. A one cm square piece of dura mater was excised by using microscope to avoid cortical injury. The excised free dural piece was then resutured again into its position with 6/0 back silk suture. The tissues were closed with 4/0 black silk suture.
Two dura mater grafts were prepared for use in the study groups. In the first group DHD (Tutoplast Dura, Biodinamics International, GmbH Wetter Kruez 19.a.D-91058 Erlangen-Tennenlohe, Germany) was used. Human cadaver dura mater after excision gets dehydrated when exposed to acetone, H2O2, and NaOH at 37oC. This dehydrated dura mater is irrigated by sterile water and irradiated by 2.5 Mrad ?-ray. A piece of DHD of 1 cm[2] in size, prepared before implantation and incubated for rehydratation in the solution of 0.09% of NaCl for 15 to 20 minutes, was sutured to the edges of dural defect with 6/0 black silk suture in a watertight way. In the animals of the second group, before a dural defect was performed, a graft was prepared from left fascia lata (AFL) and incubated in a solution of 0.09% of NaCl until surgery as performed in the first group. After the surgery, procaine penicillin was administered intramuscularly to all of the animals for three days. All of the animals were observed for seven days and complications of wound such as erythema, hematoma, rejection of graft, and failure in the sealing of wound were recorded. At the end of follow-up periods, animals were scarified with intravenous administration of high dose phenobarbital. To remove the cerebral cortex and graft together as a whole, 2 ml formaldehyde (10%) was injected into the surgical area. Two hours later, the graft and entire surrounding area of 5 mm was retrieved en bloc. The retrieved piece of graft and cerebral tissue was fixed in 2% formaldehyde solution. After preparation, the specimen was stained with hematoxylen-eosin and Masson tricrome. The stained specimens, as described by Nordstrom et al,[14] were examined for cellular inflammatory response, capsulation, connective tissue infiltration and calcification. The evaluation of cellular inflammatory response, connective tissue infiltration and foreign body giant cell were made according to a grading system of 0 to 3 (grade 0 : nil, grade1 : minimal, Grade : 2 moderate, and grade 3 : severe). The capsulation or calcification was evaluated according to its presence or absence.

   »   Results Top

Macroscopic results: In all of the three groups, wounds sealed completely. There was no hematoma, erythema, and leakage of CSF, suppuration, or graft rejection. In the control group, there was a minimal dural thickening at the edges of incision. In animals with fascia lata or DHD, the graft could not be distinguished from host's dura mater, but had a slightly more dense and clearer color than dura mater. The thickening was found most evidently on 3 and 14 days. This thickness, decreased in 30 days. The thickening was maximum on 14 and 30 days in animals where AFL was implanted, and it decreased gradually. The thickening of graft in animals where DHD was used was similar to when fascia lata was used. There was minimal to no difference as for as the thickness of graft was concerned between the two groups [Table I]. In the control group, no animals developed cortical adhesion. In 70% of the animals with AFL implantion, the cortical adhesion was very marked at day 60. However, at day 90, these cortical adhesion got reduced by 50%. At day 30, in the group using DHD, no adhesion was seen. At day 60, in the half of animals with DHD, a moderate cortical adhesion was observed. However, at day 90, the adhesion was milder as compared to 60 days.

Microscopic studies
Inflammatory Response in Grafts : The inflammatory response was minimal in 67% of the animals in the control group on day 3. In the remaining subjects it was moderate. In both the groups, polymorphonuclear leukocytes (PNL) were the dominant inflammatory cell type. The cellular inflammatory reaction observed in all the animals was composed mainly of mononuclear elements at days 14. It decreased at days 30, and there was no animal with such a reaction at days 60 to 90. In the group where AFL was implanted the inflammatory response was minimal, and was composed mainly of PNL. At day 14, the dominant element of inflammatory response was also PNL, but inflammatory reaction was much less prominent. After 30 days, inflammatory reaction decreased gradually. At day 3, the cellular reaction in those with DHD implantation was maximal in 50%, moderate in 17%, and minimal in 17% of animals and it was composed mainly of mononuclear cells. The prominence of mononuclear elements was observed on 14th day, and at 30 and 60th days, this prominence continued to exist on day 30 and 60. However, this decreased after 60 days and was minimal at 90 days [Table II].
Fibroblastic Proliferation in Grafts : In the evaluation of specimens obtained from the animals of the control group, a minimal fibroblastic proliferation (FP) was seen in 33% of the animals at days 3. FP was minimal in 50% and moderate in the other half, in brains seen at 14 days. It was minimal in all the animals at 30 and 60 days. At 90 days, FP was minimal in 17% subjects only. The FP was seen in 33% of the animals with AFL implant. This proliferation was maximal in half of the animals but was moderate in the other half. The proliferation was moderate at 30 days and minimal at 60 days. At 90 days, the observed FP was moderate in 67% and minimal in 33% of the animals. The FP in the animals with DHD implantation was at a lower level in 3 day group. This proliferation was moderate in half of the animals at 14 days, but maximal in the other half. The FP at day 60 was minimal, moderate, and maximal in equal number of animals [Table III].
Foreign Body Reaction in Grafts : There was no foreign body giant cell, an evidence of foreign body reaction, in the control group at day 3. At days 14 and 30, it was ocassionally seen in areas adjacent to sutures in 67% of the control group. However, it was of various degrees in 83% of the animals at days 60 to 90. None of the animals with AFL implant had foreign body giant cell at day 3. At day 14, 50% of the animals had foreign body giant cells (mild reaction). However, 67% of the animals at days 30 and 60 and 83% of the animals at day 90 had foreign body giant cell in variable number. In the group which received DHD grafts at days 3 to 14, there were no foreign body giant cells at day 3 and 14. However, there was foreign body giant cell in 50% of them at 14 days, in 67% at 60 days and in all of them at 90 days.
Capsulation and Calcification in Grafts : No animal in the control group had capsulation and calcification. No animals implanted with AFL developed capsulation at 3,14 or 30 days. However, in 17% of animals at 60 and 90 days, encapsulation was present along with vascular proliferation. Although mild calcification was observed in half of animals at day 3, 14, and 30, it was to a moderate degree in the other half. At day 90, the calcification was marked in 33% of animals; in the remaining, it was of moderate in degree. There was no encapsulation in the animals at 3, 14 ,30, and 60 days in those with implanted DHD. At 90 days, however, encapsulation, was very marked. Capsulation was determined in 67% of animals in this group.
No calcification was determined at 'day 3' in animals implanted with DHD. Although moderate calcification was observed in half of the animals of '14 days' group. It was mild degree in the other half. All the animals of day 30 group developed moderate calcification. Howewer, at 60 days, calcification was moderate in 34% of animals and was mild in the remaining animals. All the animals at 90 days developed moderate to severe calcification. In the control group, no animal developed cortical adhesion. 70% of animals implanted with AFL had very marked cortical adhesions at day 60. However, at day 90, the cortical adhesions reduced to 50%. At day 30, in the group with DHD, no adhesion was seen. At day 60, in the half of animals with DHD, moderate cortical adhesions were seen. However, at day 90, these adhesion were less than those at 60 days.

   »   Discussion Top

Dural defects produced as a result of any cause are always closed using autologous, homologous, or heterologous graft materials. Use of inert and nonabsorbable substitutes can cause many complications e.g. persistent foreign body reaction, diffuse inflammatory response, unfamiliar neovascularization (that can produce late hemorrhage or hematoma) and compression of neural structures due to endurance of graft.[3],[9] Fascia lata, muscle, or pericranium is used as autologous graft for dural defects repair. Autologous grafts have an advantage of having no immunoreactivity and immunogenitciy. But, they have also some disadvantages such as herniation of muscle from the site of removal of fascia.[10],[19] In addition, autologous graft tissues may not be of sufficient size for large defects.[9] For these reasons, an ideal dural graft must be immunologically neutral, having a high capability of bending and strain resistances, prevent CSF leakage, conducive to minimal scar tissue formation, and usable in large size. Allografts may have a risk of infection by slow viral diseases as well as immune reactions.[7],[15],[19] Therefore, these should be treated for diminishing their antigenicity.[15] Host immune response is less with lyophilized human dura mater (LHD) because tissue antigens and proteins have been degraded, and altered due to denaturation during process of freezedrying while preparing LHD.[3],[11] Dehydrated human dura mater (DHD) has lesser antigenity than LHD because the DHD grafts used in the study presented here were not processed by freeze-drying, but were dehydrated by immersion at 37oC in acetone, hydrogen peroxide, and sodium hydroxide. The tissue is thoroughly rinsed with sterile water to remove all residues of the chemical agents. Terminal ?-irradiation eliminates the use of ethylene oxide, which may have a residue effect.[3] After dural homotransplantations, graft rejection has not been reported in literature.[3],[4] However, it might be observed that the immune-type menengeal reactions do exist with perivascular lymphocytic infiltration, foreign body giant cells, eosinophilia, and pleocytosis in CSF. [3],[16]
We found that host inflammatory response to autogenous fascia lata (AFL) grafts was more intense than DHD grafts. This response was characteristic in polymorphonuclear leukocyte dominance in the first days. It transformed to mononuclear dominance in the course of time. Foreign body giant cells were seen in the areas close to suturing in both the groups. Meddings et al[18] reported that foreign body reaction was not observed in collagen grafts, while it was maintained in porcine biomembranes and LHD grafts 3 and 6 months after repair of experimental dural defects. Macfarlane any Symon[11] repaired the dural defects created in baboons with LHD. Histological examination of the grafts 12 months after placement showed foreign-body giant cells with mild perivascular infiltration. Abbott et al[1] observed nonspecific minimal inflammatory response and foreignbody reactions in 13 patients with dural defects, which were repaired with LHD, and concluded that these excellent results were due to dura mater having a relatively inert structure. The lack of antigenicity of LHD has been attributed to the alteration or elimination of histocompatibility antigens.
Although we found that the rate of capsule formation was higher in DHD grafts than AFL grafts 90 days after placement; the difference between two groups was statistically insignificant. It has been well known that capsule formation occur extensively with use of non-viable materials.[2],[8] On the contrary, it was reported that capsule formation was also observed in rabbits treated with DHD grafts.[14] The dystrophic calcifications, because of accumulation of calcium in injured tissues, can be observed even if serum calcium concentrations were normal.[6] We obtained high level of calcifications in all DHD grafts followed-up for 90 days, as reported by Macfarlane and Symon,[11] and Meddings.[18] This process might occur in more specimens in DHD grafts than in AFL. Therefore, one may think that injury or damage to tissue is a result of host immune reaction.
Cruetzfeld-Jakob disease (CJD) has been reported in a few cases that had received LHD implants.[7],[15],[20] However some alterations secondary to production methods of the cadaveric dura used in the case reported include chemical treatment of grafts in addition to very meticulously selection of donors. There is no report of the contraction of CJD in cases using cadaveric dura mater once the methology has been perfect.[20] Parizek et al[12] pointed out that the cadaveric dural grafts should be selected after a very careful serological investigation, and neurological and psychiatric examinations. On the other hand, WHO also recommended against the use of cadaveric dura mater, because slow virus infections such as bovine spongioform encephalopathies were widespread in those days.[17]
In conclusion, the DHD has come close to fulfilling the criteria for the ideal dural substitute as the host immune response in cases where used DHD grafts was used, were similar to case received AFL implants. However, in considerations of new concepts on the contraction of the slow virus infections, we think that autogenous dural grafts should be particularly used in dural repair; DHD and other new developed substitutes can be used in such cases having large dural defects, or those who refuse getting autogenous graft for cosmetical reasons. It becomes necessary for the future investigations in dural substitutes to aim at getting ideal dural graft materials.


  »   References Top

1.Abbott WM, Dupree EL : Clinical results of lyophilized human cadaver dura transplantation. J Neurosurg 1971; 34 : 770-773.   Back to cited text no. 1    
2.Adegbite AB, Paine KWE, Rozdilsky B : The role of neomembranes in formation of hematoma around silastic dura substitute (case report). J Neurosurg 1983; 58 : 295-297.   Back to cited text no. 2    
3.Alleyne CH, Barrow DL : Immune response in hosts with cadaveric dural grafts. Report of two cases. J Neurosurg 1994; 81 : 610-613.   Back to cited text no. 3    
4.Cantore G, Guidetti B, Delfini R : Neurosurgical use of human dura mater sterilized by gamma rays and stored in alcohol: Long-term results. J Neurosurg 1987; 66 : 93-95.   Back to cited text no. 4    
5.Crawford H : Dura replacement. An experimental study of derma autografts and preserved dura homografts. Plastic and Reconstructive Surgery 1957; 19 : 299-320.   Back to cited text no. 5    
6.Dodge HW, Grindlay JH, Craig WM et al : Use of polyvinyl sponge in neurosurgery. J Neurosurg 1954; 11 : 258-261.   Back to cited text no. 6    
7.Esmonde T, Lueck CJ, Symon L et al : Creutzfeldt-Jakob disease and lyophilized dura mater grafts: report of two cases. J Neurol Neurosurg Psychiatry 1993; 56 : 999-1000.   Back to cited text no. 7    
8.Gudmundsson G, Sogaard I : Complications to the use of vicryl-collagen dural substitute. Acta Neurochir 1995; 132 : 145-147.   Back to cited text no. 8    
9.Laquerriere A, Yun J, Tioillier J et al : Experimental evaluation of bilayered human collagen as a dural substitute. J Neurosurg 1993; 78 : 487-491.   Back to cited text no. 9    
10.Laun A, Tonn JC, Jerusalem C : Comparative study of lyophilized human dura mater and lyophilized bovine pericardium as a dural substitutes in neurosurgery. Acta Neurochir 1990; 107 : 16-21.   Back to cited text no. 10    
11.Macfarlane MR, Symon L : Lyophilized dura mater: experimental implantation and extended clinical neurosurgical use. J Neurol Neurosurg Psychiatry 1979; 42 : 854-858.   Back to cited text no. 11    
12.Parizek J, Mericka P, Husek Z et al : Detailed evaluation of 2959 allogenic and xenogeneic dense connective tissue grafts (fascia lata, pericardium, and dura mater) used in the course of 20 years for duraplasty in neurosurgery. Acta Neurochir 1997; 139 : 827-838.   Back to cited text no. 12    
13.Sharkey PC, Usher FC, Robertson RCL et al : Lyophilized human dura mater as a dural substitute. J Neurosurg 1958; 15 : 192-198.   Back to cited text no. 13    
14.Nordstrom MR, Wang TD, Neel III HB : Dura mater for softtissue augmentation: evaluation in a rabbit model. Arch Otolaryngol Head Neck Surgery 1993; 119 : 208-214.   Back to cited text no. 14    
15.Thadani V, Penar PL, Partington J et al : Creutzfeldt-Jakob disease probably acquired from a cadaveric dura mater graft. J Neurosurg 1988; 69 : 766-769, 1988   Back to cited text no. 15    
16.Vanaclocha V, Saiz-Sapena N : Duraplasty with freezedried cadaveric dura versus occipital pericranium for Chiari type I malformation: comparative study. Acta Neurochir 1997; 139 : 112-119.   Back to cited text no. 16    
17.WHO : Spongioform encephalopathies : New recommendations on medical products. WHO Press 1997; 27.   Back to cited text no. 17    
18.Meddings N, Scott R, Bullock R et al : Collagen vicryl- A new dural prosthesis. Acta Neurochir 1992; 117 : 53-58.   Back to cited text no. 18    
19.San-Galli F, Darrouzet V, Rivel J et al : Experimental evaluation of a collagen-coated vicryl mesh as a dural substitute. Neurosurgery 1992; 30 : 396-401.   Back to cited text no. 19    
20.Kniepkamp HE : Safety of lyodura. J Oral Maxillofac Surg 1994; 52 : 896-897.   Back to cited text no. 20    


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