Histology after dural grafting with small intestinal submucosa

ELSEVIER

New Technique

HISTOLOGY AFTER DURAL GRAFTING WITH SMALL INTESTINAL SUBMUCOSA Mark A. Cobb, M.D., Stephen F. Badylak, M.D., Ph.D., D.V.M., W. Janas, B.S., and Frederick A. Boop, M.D. Departments of Neurosurgery (MAC, FAB), The University of Arkansas for Medical Sciencesand Arkansas Children’s Hospital, Little Rock, Arkansas; Hillenbrand Biomedical Engineering Center (SFB, WJ), Purdue University, West Lafayette, Indiana

Cobb MA, Badylak SF,Janas W, Boop FA. Histology after dural grafting with small intestinal submucosa. Surg Neurol 1996;46: 389-94. BACKGROUND

The search for the ideal dural substitute continues, inasmuch as available materials have significant limitations. Xenogeneic porcine small intestinal submucosa (SIS) has been successfully used as a soft tissue graft in several body organ systems, and it was logical to evaluate its use as a dural replacement. METHODS

Twenty rats underwent bihemispheric craniectomy with dural resection. SIS onlay grafting on one side was performed. Histologic assessment was obtained at 7 and 28 days after dural grafting and included descriptive evaluation and quantitative scoring of graft-site thickness, vascularity, and cellular density. The total scores for the respective groups were compared using the Student’s t test, significance being accepted for a p value < 0.05. RESULTS

Histologic evaluation showed graft infiltration by spindle shaped mononuclear cells, deposition of connective tissue, and neovascularity. This pattern is consistent with the previously described incorporation and remodeling of the SIS graft at other sites. A significant difference between the histologic scores of the SIS graft site and control site was found at 7 days (3.4 ? 0.8 versus 0.1 2 0.1) and at 28 days (4.6 t 1.1 versus 2.2 2 0.5). No evidence of adverse effect on the underlying cortex was observed. CONCLUSIONS

The results of this preliminary study utilizing porcine SIS as a dural substitute are promising and therefore justify further chronic studies. KEY WORDS

Dura substitute, xenogratl, small intestinal submucosa.

Address reprint requests to: Mark A. Cobb, M.D., Department of Neurosurgery, Slot 507, The University of Arkansas for Medical Sciences, 4301 West Markham, Little Rock, AR 72205. Received June 7, 1995; accepted February 26, 1996. 0 1996 by Elsevier Science Inc. 655 Avenue of the Americas, New York,

NY 10010

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he neurosurgeon is frequently confronted with the necessity of repairing dural defects due to trauma, tumor resection, and decompressive procedures. Over the last century, numerous materials have been investigated and discarded, as summarized in several recent studies [4,8,9,11,14,15]. Current options include autologous materials (e.g., pericranium, temporalis fascia, and tensor fascia lata), lyophilized cadaveric materials (e.g., dura mater and tensor fascia lata), and synthetic materials (e.g., Silastic sheets, Dacron sheets, Vicryl mesh); however, each of these materials is associated with significant limitations [4,14]. The success of small intestinal submucosa (SIS) as a graft for artery and vein [ 71, ligament and tendon [ 21, bone, and urinary bladder [ 61 raises the question of its utility as a dural substitute. This study was performed as an initial assessment of SIS as a dural substitute.

MATERIALSANDMETHODS EXPERlMENTAL DESIGN AND SURGICAL PROCEDURE This study was performed in accordance with the Guide for the Care and Use of Laboratory Animals [5] and was approved by the Purdue University Animal Care and Use Committee. Twenty medium size Sprague-Dawley laboratory rats were anesthetized (ketamine SO mg/kg and xylazine 10 mg/kg, intramuscularly) and placed in a stereotaxic head frame to stabilize the cranium. The scalp was shaved, prepped with chlorhexadine, and infiltrated with 1% lidocaine. Through a midline scalp incision and following incision of the fascia at the superior temporal line, the temporalis muscles were elevated laterally, exposing the parietal convexities. Bihemispheric parietal craniectomies, ap0090-3019/96/$15.00 PII S009&3019(96)00202-9

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Cobb et al

Histologic Assessment of SIS Dural Onlay Grafting vs Control

GROUP

THICKNFSS

VAsCULARITY

CELLULAR DENSITY

TOTAL SCORE

7-Day SIS graft (N = 8) 7-Day control (N = 8) 28-Day SE graft (N = 9) 28-Day control (N = 9)

1.1 + 0.2

0.9 2 0.2

1.4 5 0.2

3.4 5 0.8”

0.2 5 0.1

0.1 L 0.1

0.1 ? 0.1

0.4 ? 0.1

1.9 ? 0.3

1.9 ? 0.4

0.8 +- 0.3

4.6 ? 1.1”

1.1 2 0.3

0.8 2 0.2

0.3 + 0.2

2.2 5 0.5

“Value is significantly different from the value in the control group at a p value of cO.05. The SIS graft sites are compared with the control sites using thickness, vascularity, and cellular represent the mean value % S.E.M. CRITERIA

Thickness

Vascularity

Cellular

density

SCORE

o= l= 2= o= l= 2= o= l= 2=

proximately 4 mm X 8 mm, were made with an electric hand drill and burr. The dura, a thin, nearly transparent membrane in the rat, was resected at the craniectomy sites under loupe magnification. Care was taken not to injure the underlying cerebral cortex. SIS is an acellular material, consisting of collagen and extracellular matrix, which comprises the basement membrane of the tunica mucosa and the entirety of the tunica submucosa of the small intestine. The SIS graft material, harvested as previously described [3] and sterilized by exposure to 0.1% peracetic acid, was cut to the appropriate size and placed with the stratum compacturn surface toward the cerebral cortex as an onlay graft over one convexity. The contralateral hemisphere received no graft, thus serving as a control for host response to the operative procedure. In two animals, the bone fragment was replaced. The wound was irrigated with normal saline and closed with staples. A single postoperative dose of ampicillin (25 mg/kg, subcutaneously) was given. Immediate postoperative care included placement on a heating pad, covering with a towel, turning every 15 minutes until awake and moving, and monitoring heart rate and respiration. The animals were monitored daily for the occurrence of seizures or neurologic deficits, appetite and fluid intake, and weight. Ten of the rats were sacrificed by barbiturate overdose (150 mg/kg, intracardiac) 7 days after graft placement. The re-

density

as the scoring

DEWRIPTTON

~100 PM 100-200 /.LM >200 /.LM O-l vessel cross 2-4 vessel cross 25 vessel cross Total cell area: Total cell area: Total cell area:

sections/100 sections/100 sections/100 extracellular extracellular extracellular

criteria.

The values

listed

OF SCORE

PM’ PM* PM’ matrix matrix matrix

maining 10 rats were killed placement.

material material material

~0.5 OS-l.0 >l.O

28 days after graft

HISTOLOGIC PREPARATION AND EVALUATION Following sacrifice, the rats were perfused with formalin via carotid artery catheters. The cranium was then fixed in formalin and decalcified. Six micronthick sections were cut, stained with hematoxylin and eosin (H&E), and prepared for histologic examination. The tissues examined included the interfaces of the graft with the cortex, bone, and scalp. The control side was similarly prepared. Microscopic evaluation of the specimens was augmented with quantification of the cellular infiltrate, vascularity, and thickness of the defect site using an image analysis system (Optimus Image Analysis System; Bioscan, Inc; Edmonds, WA). Graft-versus-control values were compared at both the 7-day and 28day time points. The numerical scores given to the remodeling tissues were based upon criteria given in Table 1. The values were compared using the Student’s t test. The total score for the respective groups was used to test the null hypothesis that there was no difference between the morphologic changes seen in the SIS filled versus the non-SIS filled defect sites at either 7 or 28 days. A p value less than 0.05 was considered significant.

Surg Neurol 1996;46:389 -94

Small Intestinal Submucosa as a Dural Substitute

39 1

SIS tissue 7 days after implantation. Note the high degree of cellularity and vascularity. The cells are qmaterial.Remodeling a mixture of round mononuclear cells and spindle-shaped cells, deposited in a background of extracellular matrix (H&E, 100) X

RESULTS Three of the 20 rats died of anesthetic-related complications in the early postoperative period. The remaining 17 rats recovered uneventfully from the procedure without evidence of seizures, infection, or neurologic deficit. Eight rats were killed at day 7. Nine were killed at day 28. The histologic findings of this study showed distinct differences between the defects repaired with SIS versus those left to heal without any material placed at the defect site. At day 7, the main differences between the two groups were the cellular infiltrate, vascularity, and the thickness of the connective tissue deposited at the defect site. These morphologic changes were compared in a semiquantitative fashion as defined in Table 1. This method of comparison showed increased thickness, increased vascularity, and greater cellular infiltration of the SIS-treated defects versus the non%-treated control defects. The mononuclear cells, which were seen within and around the SIS material at day 7, often showed a spindle shape and were surrounded by eosinophilic staining extracellular matrix (ECM) material. The remodeling SIS material showed a large number of capillary sized blood vessels in contrast to those observed in the non-SIS control defects (Figure 1). By 28 days, the cellular infiltrate had moderated in the SISfilled defects and the amount of ECM had

increased. The eosinophilic staining connective tissue in and around the SIS showed orientation in the direction that would extend from one edge of the cut calvaria to the opposite edge (Figure 2). There was also moderate organization of the connective tissue seen in the non-SIS defects; however, the amount of material present was much less than the SIS defect sites. The SIS material itself was not discernible by day 28. The ECM appeared homogeneous in these H&Estained sections. The cellular infiltrate was much less at day 28 than at day 7, and virtually all of the cells present were consistent with spindle-shaped mesenchymal cells (Figure 3). Occasional adhesions were noted between the ECM within the defect site and the underlying cerebral cortex in both the SIS and non-SIS sides. None of the specimens showed changes consistent with encephalitis, degeneration, or necrosis.

DISCUSSION The search for an ideal dural substitute has been arduous. The list of materials that have been investigated and discarded or modified has been summarized by several authors [ 4,8,14]. Presently available options have significant limitations. The autologous materials are frequently inadequate in quantity and are obtained with the associated mor-

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Cobb et al

Partially organized SIS material, 28 days after implantation, at the point of attachment to the cut edge of the calvarium. Note the moderation of the cellular infiltrate and the orientation of the connective tissue. (H&E, X 12.5)

bidity of additional incisions. The handling characteristics of synthetic sheets are poor compared to biologic materials. In addition, concern has been raised regarding long-term risks of hemorrhage from tissne reaction adjacent to the graft [l]. Cadaveric dura is expensive, occasionally limited in

supply, and has only fair handling characteristics. Of greater concern is its documented role as a vector in the transmission of the slow viruses such as Jakob-Creutzfeldt disease [10,13,16]. Extensive laboratory investigations have been performed on the novel biomaterial, designated SIS,

Remodeling SIStissue 28 days after implantation. Note the homogeneous appearance of the extracellular matrix El material, the preponderance of spindle-shaped mesenchymal cells, and the benign-appearing brain/graft interface. (H&E, x 100)

Small Intestinal Submucosa as a Dural Substitute

harvested from the small intestinal submucosa of various mammalian species [2]. A series of reports describes its successful use as a graft in artery and vein patching [ 71, ligament and tendon repair [2], bone repair [2], body wall hernia repair [2], and in urinary bladder wall replacement [ 61. In addition to the absence of an adverse immunologic response, a distinctive remodeling of the SIS graft material and incorporation into the host tissue has been demonstrated [ 21. That porcine SIS might prove a suitable dural substitute warrants investigation, inasmuch as it is a biomaterial for which considerable xenogeneic grafting experience now exists. The rat host provides an appropriate model to screen for unanticipated catastrophic responses of mammalian cortex to porcine SIS dural xenografting. Theoretically, the cerebral cortex should not respond adversely to SIS. In addition, other type I porcine and bovine collagen preparations have produced neither acute immune reactions nor epileptogenic scars when applied to rabbit, dog, and human cortex [4,8,11,15]. This study demonstrates that this is likewise true for SIS in a 28day rat model of onlay grafting following dural resection. Furthermore, the previously reported remodeling response to SIS, characterized by the presence of spindle cells, aggressive neovascularization, and an eosinophilic staining connective tissue matrix [2,12], has similarly been demonstrated in this rat model of dural replacement. Although the final form of the remodeled dural graft has yet to be elucidated (such was not the aim of this study), the histologic findings demonstrate a time course suggesting an evolution consistent with the behavior of SIS at other graft sites [2]. The ultimate objective is to provide neurosurgeons with a safe, effective, reliable dural substitute for human use. This animal study is a necessary step toward that goal. Further research on the use of SIS as a dural substitute is justified.

CONCLUSION This study evaluates the histologic response of the rat to cerebral onlay grafting with porcine small intestinal submucosa (SIS) following dural resection. The previously described incorporation and remodeling of the graft takes place in the absence of any adverse effects on the underlying cerebral cortex. The results of this preliminary study utilizing SIS as a dural substitute are promising; however, further long-term studies are warranted, prior to its use in humans.

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COMMENTARY

The use of nonvascularized tissues is commonplace in the practice of surgery. These tissues include bone marrow, cornea, skin, dura mater, fascia, and