Biological intervertebral disc replacement: an in vivo model and comparison of two surgical techniques to approach the rat caudal disc

 

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ABSTRACT

Study design: Prospective randomized animal study.

Objective: To determine a surgical technique for reproducible and functional intervertebral disc replacement in an orthotopic animal model.

Methods: The caudal 3/4 intervertebral disc (IVD) of the rat tail was approached by two surgical techniques: blunt dissection, stripping and retracting (Technique 1) or incising and repairing (Technique 2) the dorsal longitudinal tendons. The intervertebral disc was dissected and removed, and then either discarded or reinserted. Outcome measures were perioperative complications, spontaneous tail movement, 7T MRI (T1- and T2-sequences for measurement of disc space height (DSH) and disc hydration). Microcomputed tomographic imaging (micro CT) was additionally performed postmortem.

Results: No vascular injuries occurred and no systemic or local infections were observed over the course of 1 month. Tail movements were maintained. With tendon retraction (Technique 1) gross loss of DSH occurred with both discectomy and reinsertion. Tendon division (Technique 2) maintained DSH with IVD reinsertion but not without. The DSH was demonstrated on MRI measurement. A new scoring system to assess IVD appearances was described.

Conclusions: The rat tail model, with a tendon dividing surgical technique, can function as an orthotopic animal model for IVD research. Mechanical stimulation is maintained by preserved tail movements. 7T MRI is a feasible modality for longitudinal monitoring for the rat caudal disc.

* The experiments were performed at Weill Cornell Medical College, New York, NY, USA.

Roger Härtl and Harry Gebhard received a grant from AOSpine North America for this study.

STUDY RATIONALE AND CONTEXT Recent work introduced tissue-engineered intervertebral disc (IVD) constructs, which have shown promising characteristics in vitro 1, 2, 3, 4. An orthotopic model is the desirable next step to provide biological and mechanical stimulation. We describe a new surgical approach and animal study, to our knowledge not previously reported, to demonstrate feasibility of an in vivo model and to compare the influence of surgical disc space dissection on survival of a reimplanted disc 5. Using magnetic resonance imaging (MRI), sequential assessments are made and a new scoring system is described. CLINICAL QUESTIONS Does the surgical approach to the IVD in the rat tail, with discectomy or disc excision and reimplantation alter the postoperative disc height and appearance of the IVD on MRI? Is this a suitable animal model for preliminary IVD replacement testing using noninvasive means in longitudinal studies? METHODS Study design: Prospective randomized animal study. Local ethical and animal committee approval obtained. 24 nude (athymic) adult male rats, 250–300 g (Hsd: RH-Foxn1rnu) were included, the exclusion criterion was previous tail injury. No animals were excluded. Same surgeon for anesthesia (induction: 85 mg/kg Ketamine and 5 mg/kg Xylazine administered IP; maintenance: Isoflurane 2 %) and surgery. Intraoperative x-rays located the caudal 3/4 disc. The operative approach involved skin incisions and a midline muscle flap to be created identically in both groups (Fig 1). The paravertebral longitudinal tendons were then either: Retracted with blunt dissection and tissue stripping (Technique 1; Fig 2) Divided by sharp incision (Technique 2; Fig 3) Surgical sharp dissection was performed around the disc. Only then the annular fibers were sharply incised (cranial and caudal) and the disc was removed as a whole with blunt detachment of the nucleus (Fig 4). Next the disc was either discarded (discectomy) or reimplanted. Technique 2: Divided tendons underwent a tension-free surgical repair. In both techniques, a saline wash was performed, the muscle was reinserted, and the skin was sutured. Postoperative analgesia (Buprenorphine for 72 hours) was administered and the animals were under observation until active and feeding. Outcomes Clinical assessments: Daily wound observation (infection, delayed closure). Vascular assessment: Normal capillary blood flow at the rat tail tipp was assessed clinically by digital pressure and the ability of the tipp to reperfuse within 2 seconds. Function: At day 29 all animals were visually observed and tail movements counted by a single-skilled assessor during awake, day and night, at 30-minute periods. Internal control consisted of preoperative observations of six animals. MRI was performed day 1 and day 30. DSH was measured on T1 sequences (Fig 5) using an image analysis system (Osirix viewer®). IVD characteristics were measured on the T2 sequences, using a semiquantitative scoring system as shown in Table 2 (Fig 6). Magnetic resonance angiography (MRA) was conducted day 30 to assess for vascular injury (Fig 7). Two-blinded observers assessed MRI scans. The DSH was averaged, disagreement on characterization or vascular injury were decided by an independent third person, whose grading would be final. Micro CT: High-resolution images postmortem to assess injuries to vertebrae and end plates (Fig 8). Analysis: Distinct results were interpreted directly, while variations between groups were tested statistically. Clinical assessment: Wound complications (infection, delayed closure), incidence of capillary delay, and loss of vessel integrity were counted and compared with a t test. The tail-bending frequencies were analyzed with a Poisson regression model, and a multivariate analysis with stepwise variable selection between the groups. For disc height and disc grading, the adjacent level was the comparator. The strength of collapse measured by the relation of preoperative and postoperative surgical gap size was tested against a threshold of one, representing no change, by means of two-sided one-sample t tests. Comparisons of postoperative relations and corresponding values after 1 month were conducted by two-sided paired-samples t tests. Likewise it was tested for differences between relations observed with and without disc reinsertion, and between surgical approaches by two-sided two-sample t tests. In addition descriptive statistics are given by mean, standard deviation, and 95 % confidence intervals. All tests were performed in an explorative manner on a 5 % level of significance. Tabulation of scores and calculation of the Fleiss’ kappa coefficient for reliability testing in multirater settings. Number of injuries to the bony end plates was recorded.

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EDITORIAL STAFF PERSPECTIVE

The athymic rat tail model does appear to provide a potential animal model for future investigation into intervertebral disc replacement with appropriate technique in this prospective randomized animal study. This paper clearly demonstrates sharp dissection is superior to blunt detachment to maintaining intervertebral disc height, an interesting tangent as we learn more about soft-tissue dissection and maintenance of viable margins. There are, however, some significant limitations to this study.

One limitation lies in the inability of the rat tail model to provide significant axial loading upon the discs as would be the case in bipedal animals or in the actual spinal column of the investigated animals, as mentioned by the authors. In addition, the short-time frame of follow-up is of concern. Longer outcomes involving athymic rats may be necessary in order to understand if the replanted discs can withstand longer durations than just the 30 days. A 90-day window or even longer would seem to be more relevant to establish ingrowth and cartilage cell survival.

Another area of concern is the lack of significant data and limited discussion on the movements of the rats’ tails following discectomy or reinsertion. None of the data in Table [1] demonstrated significance (P < .05) from the control to the operated rats in regard to movement.

Detailed analysis of the results of the disc replacement or removal with clinical assessments, function, and imaging with MRI and micro CT is clearly one of the strengths of this paper. The comprehensive approach to imaging allows for very clear comparisons between the two surgical approaches of discs that were replanted or discarded.

There is no question of the importance of developing an appropriate animal model for the future quest for biological intervertebral disc replacements in an in vivo setting. A workable animal model will certainly facilitate future research opportunities and development of the disc replacements. Whether the athymic rat tail model will turn out to be the optimal model or not, this novel model certainly presents a laudable launching point for further investigations.