Nucleus Replacement and Repair: Autologous Disc Chondrocyte Transplantation

14 Nucleus Replacement and Repair: Autologous Disc Chondrocyte Transplantation

Christian Hohaus, Timothy Ganey, and Hans Jörg Meisel

Abstract

Intervertebral disc (IVD) degeneration is in 40% of younger individuals and in more than 90% of individuals older than 50 years of age, a common disorder with a negative impact on life quality. Available treatment options, conservative as well as operative procedures, are limited and don’t treat the underlying biological reason, IVD degeneration. Total regeneration of the degenerated IVD is not currently considered a therapeutic assurance. The goals of regenerative medicine at this time are to prevent further progression of disc degeneration and its associated symptoms.

What has been shown to date, and therefore available in limited geographies and jurisdictions, is that expanded and transplanted autologous cells are safe and appear to arrest if not reverse degenerative disc disease (DDD) following treatment for sequestrectomy. In our clinical trial with more than 140 patients, the largest number of patients with degenerated IVD disease treated with cell transplantation under defined study conditions, all patients have benefited from transplantation in reduced back pain as one measure of an enhanced quality of life and were able to return to work after transplantation. Moreover, the reduction in reherniation rate was more than 50% better than the control group.

The use of autologous chondrocytes requires the ex vivo expansion of cells, adding burdens of cost, time, and regulation that add to the intricacy of the procedure beyond the medical interface. This limitation of the procedure could be avoided by the use of a one-step procedure, using stem cells obtained from autologous adipose tissue, with shows promising results in preclinical studies.

Cell transplantation appears to present an effective treatment option in DDD. The transplantation of autologous disc cells is currently the only biological treatment option with good clinical results under defined study conditions.

Keywords: autologous disc cell transplantation, cell therapy, clinical study, degenerative disc disease, intervertebral disc, low back pain

14.1 Introduction

Low back pain is extremely common, affecting nearly three quarters of the population sometime in their life. More than 80% of the population will suffer from lumbar back pain once in their lifetime.¹^,²^,³^,⁴ Although most people recover within 3 months, in some patients chronic back or leg pain leads to long-term physical disability, and the reduced quality of life that it imposes.

Disc anatomy would be expected to play a pivotal role and correlate with the underlying pain, yet abnormal spine and disc morphology including disc herniation has been described as a normal component of an asymptomatic population.⁵^,⁶

IVD degeneration is influenced by multiple factors such as age and genetic loading,⁷ biomechanical forces,⁸ and environmental factors such as immobilization, trauma, and application of nicotine.⁹ Age-related diseases such as diabetes, arterial hypertension, and complications attendant to vascular diseases have been defined as relevant factors as well.¹⁰

Accepting the vast prevalence of asymptomatic disc degeneration as noted, “normal” IVDs present an optimal balance between anabolic and catabolic processes that are regulated by anabolic growth factors, catabolic enzymes, and pro-inflammatory cytokines.¹¹ Among the key anabolic growth factors that have been noted are insulin-like growth factor (IGF)-1, transforming growth factor (TGF)-β, and bone morphogenetic proteins (BMPs).¹² Interleukin (IL)-1 and TGF-α clearly are involved in catabolic activities in the healthy IVD.¹³ Catabolic processes contribute to changing disc morphology and variations in matrix composition that initially alters biochemical composition, and ultimately result in the loss of proteoglycans and type II collagen. Following a restructuring of the matrix, an increase in cell death and decreasing nutrition further impose conditions and predisposition to mechanobiological consequence and precipitate a self-imposing insult that furthers the degenerative process.¹⁴^,¹⁵^,¹⁶^,⁷^,¹⁸

Therapeutic options for treating degenerative disc disease (DDD) resulting from this knowledge have informed strategies including the substitution of growth factors, gene therapy tissue engineering, and cell transplantation. Some of these efforts have met with suggestive success to the extent that proteoglycan synthesis has been shown to increase in a canine model after direct injection of recombinant TGF-β in combination with epidermal growth factor.¹⁹ Similarly, the direct stimulation of cells with BMP-7 in rabbit IVDs resulted in an increase in proteoglycan synthesis as well as restoration of disc height.²⁰^,²¹^,²² The intradiscal application of BMP-7 for regeneration of IVD degeneration in a natural canine model, however, did not promote disc regeneration but instead resulted in the formation of extradiscal bone.²³ A pilot clinical study combining matrix components and growth factors that was directly injected into degenerated discs stimulated IVD regeneration that was durable over the course of a 12-month follow-up.²⁴ Despite the success achieved, the concern for therapeutic support for such techniques seemed severely limited by the availability of viable cells and the limitation in application only in early stages of disc degeneration.²⁵ Transplantation of mesenchymal stem cells (MSCs) has also gained increasing attention,²⁶^,²⁷^,²⁸^,²⁹ although critics of that strategy have weighed in on limitations of cell viability as concerning.²⁵

Given that disc herniation is thought to be an extension of progressive disc degeneration that attends the normal aging process, seeking an effective therapy that staves disc degeneration has been considered a logical attempt to reduce back pain. Disc herniation is the most common reason for radicular symptoms in the lumbar spine leading to an operative treatment. Herniated material from the degenerated disc has been considered a potential donor for nucleus pulposus (NP) cells and chondrocytes because of their extended viability.³⁰ Culturing and expanding these cells is not simple but is possible, and following successful transplantation cells produce both type-II collagen as well as appropriate proteoglycans.³¹ Transplantation of these expanded cultured cells would create an option to enlarge the viable cell content in the degenerated disc and to increase the necessary extracellular matrix (ECM).

14.2 Preclinical Study Using Autologous Disc Cell Transplantation for Nucleus Regeneration in the Dog

This study³¹ was performed to test the hypothesis that restoration of IVD morphology could be achieved by transplantation of cultured autologous chondrocytes into the NP. The study addressed the question of whether the introduction of cultured autologous disc–derived cells would repair a damaged disc and inhibit degenerative changes. The dogs were divided into two basic groups; 4 animals receiving autologous cells containing bromodeoxyuridine (BrdU) as a nuclear marker, the other 14 receiving autologous cells without a nuclear marker. Animals were radiographed to establish a baseline for preexisting spine pathology.

Lumbar IVDs at three levels (L1–2, L2–3, and L3–4) were identified as study levels for the procedure and disc tissue was collected. The sampled disc cells were expanded in culture through several passages, producing approximately 6 million cells with the goal of establishing a population of disc cells capable of producing matrix and sustaining an expanded volume within the damaged disc. In this study, the L1–2 IVD had tissue removed but did not receive chondrocyte transplantation, the L2–3 disc was approached but not violated and served as a surgical control, and the L3–4 level had disc material removed and received chondrocyte transplantation 12 weeks later via left-side minimally invasive puncture of the IVD under fluoroscopic imaging.

An important criterion for evaluating the success of cell transplantation in the disc repair procedure was identifying that matrix regeneration was attributable to transplanted, cultured, expanded disc cells rather than a result of inherent disc capacity for self-repair. BrdU, an analog nucleotide of thymidine, was incorporated into the nucleus during deoxyribonucleic acid (DNA) synthesis and could later be identified by immunohistochemical techniques. As such, it was possible to analyze morphology in situ after repair, and delineate cells that were transplanted from those already present in the host tissue.

The animals were humanely euthanized 3, 6, 9, and 12 months following the cell transplantation. Immediately after the dogs were killed, their lumbar spines were removed and the tissue analyzed (▶ Fig. 14.1). Magnetic resonance imaging (MRI) and X-ray analysis with coronal slices of the spinal column were performed to interpret disc height. Tissue analyses included light microscopy and immunohistochemistry for assessing BrdU content (▶ Fig. 14.2) and collagen expression.

Fig. 14.1 Gross pathology at 12-month follow-up after autologous chondrocyte transplantation in the canine model. Level L3–4 was transplanted, level L1–2 received no treatment and displayed more scar tissue, and level L2–3 was the control level with a normal intervertebral disc.

Fig. 14.2 Staining of paraffin sections of the regenerated intervertebral disc 6 months following cell transplantation. Bromodeoxyuridine (BrdU)-containing chondrocytes were detected and stained by immunohistochemical procedures using 3,3′-diaminobenzidine (DAB) as the chromogen. Sections were counterstained by eosin. BrdU-positive cells are colored black. (a) Nucleus regenerate overview (25 ×). (b) BrdU-stained transplanted cells (200 ×), (c,d) single BrdU-stained transplanted chondrocytes, pericellular de novo synthesis of nucleus matrix (1,000 ×).

The results of this animal study were extremely promising for further clinical applications: Autologous disc cells were expanded in culture and returned to the disc by a minimally invasive procedure after 12 weeks. Under defined conditions, it was possible to assure phenotype and assess metabolic capacity of the cells prior to transplantation. These transplanted disc cells remained viable after transplantation as shown by BrdU incorporation and maintained a capacity for proliferation after transplantation as depicted by histology. They produced an ECM that contained components similar to normal IVD tissue. Both type I and type II collagens were demonstrated in the regenerated IVD matrix by immunohistochemistry following chondrocyte transplantation. And there was a statistically significant correlation between transplanting cells and retention of disc height that was demonstrated at longer intervals following transplantation.³¹

14.3 Clinical Application of Autologous Disc Chondrocyte Transplantation in Degenerative Disc Disease

After these positive and promising results demonstrating both safety and efficacy, a randomized controlled study (the Euro Disc Randomized Trial) was designed to embrace a representative patient group, examining not only the traumatic, less degenerative disc, but also to include patients with persistent symptoms that had not responded to conservative treatment where an indication for surgical treatment was given. Between 2002 and 2006 a total number of 148 patients were included in this clinical trial from seven German spine centers.³²^,³³

14.3.1 Patient Selection

Eligibility to participate in the study was limited to patients having exclusively one level requiring surgical intervention. Criteria for surgical therapy were progressive sciatic pain with futile conservative treatment or neurological deficits caused by root nerve compression. Magnetic resonance imaging (MRI) of the lumbar spine was mandatory. The trial was limited to patients between 18 and 60 years of age, with a body mass index (BMI) below 28.

Exclusion criteria to participating in the study included sclerotic changes, edema, Modic changes of type 2 or 3 in preoperative MRI, and spondylolisthesis. Patients with generalized diseases or progressive neurological deficits were excluded, as well as pregnancy.

14.3.2 Operative Treatment

Each patient participating in the clinical trial underwent surgical treatment for prolapsed disc. This was done as a minimally invasive open sequestrectomy using a tubular retractor system for minimal affection of the lumbar muscles. The use of an operative microscope after opening the yellow ligament was mandatory. This procedure was performed by an experienced neurosurgeon with the patient under general anesthesia. Randomization was done after closure of the fascia thoracolumbalis, directly from the operating room to prevent surgical bias in the evaluation of patients. The harvested cells from the sequestered disc material were cultured by co.Don AG (Teltow, Germany) under Good Manufacturing Practice (GMP) conditions. Patients were not blinded to their treatment.

14.3.3 Transplantation

Cell implantation was performed 3 months after the primary operative procedure to assure that the anulus had healed and would contain the cells. Prior to intervention with the cells, an MRI was used to detect possible early recurrent sequestration or progressive degeneration. In the prepared solution for the transplantation were more than 5 million living disc cells.

Under fluoroscopic control the affected disc was punctured with a minimal caliber cannula opposite the side of the previous disc herniation procedure (▶ Fig. 14.3). Using a pressure-volume test³⁴ prior to the delivery of any chondrocytes, surgeons were able to place the cells with confidence that they would be retained at the site of delivery.

Fig. 14.3 Intraoperative picture of the fluoroscopically guided minimally invasive puncture of the intervertebral disc. (a) Fluoroscopic view after puncture of the disc. (b) Pressure-volume test. (c) Transplantation.

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