Elsevier

The Spine Journal

Volume 4, Issue 3, May–June 2004, Pages 281-286
The Spine Journal

Clinical Studies
Posterolateral lumbar fusions in athymic rats: characterization of a model

https://doi.org/10.1016/j.spinee.2003.10.001Get rights and content

Abstract

Background context

The athymic rat has been used to study the role of osteoinductive products in spinal fusions. This small animal model has been advocated to minimize potential inflammatory responses to allogeneic or xenogenic proteins. Despite past experience, this model has not yet been well characterized.

Purpose

To further define and validate a posterolateral lumbar fusion model in the athymic rat.

Study design/setting

Comparison of fusions after animal survival surgery.

Patient sample

Forty athymic and 20 normothymic rats.

Outcome measures

Manual palpation, radiography and histology at 3 and 6 weeks.

Methods

Single-level intertransverse fusions were performed at the L4–L5 level of 40 athymic rats. Twenty rats were implanted with autograft (athymic/autograft), and 20 had no graft placed (athymic/no graft). An additional 20 autograft fusions were performed on normothymic rats (normothymic/autograft). Half were sacrificed at 3 weeks; half were sacrificed at 6 weeks.

Results

At 3 weeks, 0% of the athymic/no graft rats fused, 20% of the athymic/autograft rats fused and 20% of the normothymic/autograft rats fused by manual palpation. At 6 weeks, 0% of the athymic/no graft rats fused, 30% of the athymic/autograft rats fused and 40% of the normothymic/autograft rats fused by manual palpation. Radiographs were of limited utility in determining fusion, and histology results were roughly concordant with those of manual palpation.

Conclusions

This work further characterizes the athymic rat posterolateral lumbar fusion model. The absence of a thymus does not appear to affect autograft fusion rates, and no spontaneous fusions were seen when no graft was placed.

Introduction

Spinal fusion is a common surgical procedure, and autograft is the gold standard grafting material. However, despite advances in surgical techniques, pseudarthrosis remains an issue [1]. Further, autograft may be limited in supply, and its harvest is associated with significant morbidity and increased operative time [2]. Attention has thus been directed toward refining alternative means of inducing bone formation.

Bone graft substitutes that are currently being studied include demineralized bone matrices (DBMs), individual recombinant bone morphogenetic proteins (BMPs) and autogenous blood product isolates. In addition to the active agent in these new products, the mode of delivery and type of carrier are important factors in determining efficacy.

With the many variables being studied, it has been necessary to use animal models. Noninstrumented posterolateral lumbar fusions are commonly studied, because they pose a challenging and clinically relevant model. Studies have been done in a number of species, including rats [3], [4], rabbits [5], [6] and dogs [7], [8]. Products are often tested in lower species and moved along to higher species only when promising results are found.

Some osteoinductive products, however, cannot be tested in certain models. For example, Aspenburg et al. [9] found xenogenic DBM to induce little or no bone formation when placed intramuscularly in normal rats. It was hypothesized that this was because of an immunologic response of the host to the nonconserved or differentially expressed donor proteins. Substantiating this hypothesis, increased bone induction was seen when athymic recipient rats were studied under otherwise identical circumstances. Although xenogenic DBMs are not used clinically, there is interest in studying them in animals as products are being developed. Other studies have shown human DBM to have a dose-dependent osteoinductive effect when placed intramuscularly or subcutaneously in athymic rats [10].

Additional work is ongoing to study the effectiveness of BMP containing adenoviral vectors (Ad) in inducing bone formation. When Ad-BMP-2 was injected intramuscularly into normothymic rats, significant immune response (without concomitant bone formation) was elicited by the first-generation adenoviral constructs [11], [12]. Conversely, bone formation without such immune response was seen when the same construct was studied in athymic rats.

Subsequently, the athymic rat has been used to study the potential role of osteoinductive products in spinal fusions by several authors. Athymic rat fusions have been studied with open implantation of xenogenic DBMs [4], [13], injection of adenoviral constructs [14], [15] and application of mammalian expression vectors [3].

Much evidence suggests that nonsteroidal anti-inflammatory agents can adversely affect fusion rates [16], [17]. Should a similar effect be expected if fusions are studied in the athymic rat where the immune system is compromised? Contrary to what might be expected, bone formation and metabolism does not appear to be affected by the absence of the thymus gland in rats [18]. Another study showed no significant differences in the healing of osteotomized tibias of athymic and normothymic rats [19].

Some small animal models have high fusion rates solely because of surgical exposure. This is not expected in the athymic rat model. Entire study groups have been described by Wang et al. [4] with no fusion (namely with Dynagraft [Citagenix, Inc., Lavai, QC, Canada], which was thought to be of insufficient osteoinductivity). By inference, fusion does not appear to be induced by surgical approach alone. This, however, has not yet been demonstrated as an independent variable.

Although used in previously reported studies, the model for open posterolateral lumbar fusion in the athymic rat has not, to our knowledge, been well characterized as performed for other fusion models [5]. The purpose of the current study is to clearly characterize a posterolateral lumbar fusion model in the athymic rat. Radiographic, manual palpation and histologic examinations are to be used to define the rates of autograft fusion, effect of an absent thymus and rates of spontaneous fusion in this model.

Section snippets

Overview

Sixty L4–L5 posterolateral lumbar fusions were performed (of note, rats have six lumbar vertebrae). Forty were mature female athymic nude rats (rnu/rnu), 8 to 9 weeks of age. Twenty received autograft (experimental study group, athymic/autograft). Twenty were opened and closed with no graft placed (negative control group, athymic/no graft). An additional 20 fusions were performed on 9- to 10-week-old immunologically intact female Sprague Dawley rats. These animals were similar to the athymic

Results

The athymic rats used in this experiment weighed between 170 and 200 g. The normothymic rats weighted between 190 and 200 g. The surgical procedures took approximately 20 to 30 minutes to perform.

Three rats were excluded from the study because of complications: two were lost perioperatively because of anesthesia, and one was lost approximately 1 week postoperatively with no clear cause. Of the three rats lost, all were normothymic. These three rats were replaced and are not included in the

Discussion

The athymic rat model has previously been used to study spinal fusion, but additional characterization was required before continuing study with this useful model. In what frame of reference should posterolateral fusion studies performed in these animals be interpreted?

The rat is a small and easily handled animal in which posterolateral fusion is easy to perform. Although much smaller than the human, the morphology of the rat lumbar vertebrae are roughly similar to that of the human (Fig. 3).

Acknowledgements

The authors thank Daniel Zelterman, PhD, for his statistical assistance and Chris Coady for her histologic assistance.

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Author JNG acknowledges a financial relationship (grant research support from Stryker Biotech, Hopkinton, MA), which may indirectly relate to the subject of this research. Additional support was received from the Yale Core Center for Musculoskeletal Disorders.

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