Stem Cell–Based Intervertebral Disc Restoration

51 Stem Cell–Based Intervertebral Disc Restoration

Mengqiao Alan Xi and Mick J. Perez-Cruet

Abstract

Biologic restoration of the degenerative intervertebral disc is the goal for restoring the natural integrity of the intervertebral disc. This chapter discusses the latest developments in science and technology to achieve this goal. A review of the current literature as well as the latest science on stem cell–based intervertebral disc restoration and possible future medical technology is conducted. It is hoped that the future may offer patients a biologic treatment that restores the normal anatomical function of the intervertebral disc for those suffering from debilitating spinal conditions.

Keywords: biologic intervertebral disc regeneration, stem cells, annular traction, disc degeneration, chronic back pain

51.1 Introduction

Strides in basic science research continue to shape the way health care is delivered. Stem cell technology has gained significant ground in the past few decades with the promise to restore the anatomical and physiological properties of the spine. Studies on intervertebral disc (IVD) restoration, in particular, have witnessed great success in both in vitro and in vivo settings, with some of the pilot projects beginning to explore the first human-subject stem cell implants. This chapter reviews pertinent spine studies that demonstrate the potential of stem cell–based IVD regeneration.

51.2 Fundamentals of Stem Cell Biology

Stem cells are known for their ability to proliferate indefinitely and to differentiate down different lineages. For this reason, many researchers have proposed various models to investigate the possibility of using stem cells to replace or regenerate damaged human tissue including components of the IVD. The fate of a stem cell is a result of the chemical and environmental milieu it is exposed to during development. Although much unknown remains, many of these lineage-determining factors have been elucidated so that a variety of targeted cell types can be procured by manipulating the growth environment.¹

The IVD consists of two distinct structures. The outer annulus fibrosus (AF) contains abundant collagen fibers. The nucleus pulposus (NP) contains glycosaminoglycans that act like a sponge to maintain intradiscal fluid pressure. Age-related deterioration of IVD occurs slowly but eventually results in dysfunction and apoptosis of NP cells, which are associated with reduced extracellular matrix (ECM) maintenance and loss of structural integrity.² This pathologic process is referred to as degenerative disc disease (DDD). Humans are born with notochordal cells, which are gradually replaced by NP cells during development ( Fig. 51.1). Adult NP cells resemble chondrocytes morphologically and functionally. For this reason, the primary aim of IVD restoration is to regenerate chondrocyte-like cells from stem cells to replenish the senescent or depleted NP cell pool in DDD.³ Hunter et al⁴ showed that notochordal cell retention in various mammalian species is lost with age, after which degeneration of the IVD can occur ( Table 51.1).

Fig. 51.1 As humans age, the notochordal cell population (a) disappears and is replaced by sparse chondrocyte-like cell populations (b). (c,d) Apoptosis with aging and degeneration. (c) Disc at 13 years of age and (d) disc at 26 years of age.

A few viable sources of stem cell have been investigated for this purpose, among which mesenchymal stem cells (MSCs) and embryonic stem cells (ESCs) are the most frequently studied ( Table 51.2). MSCs are multipotent adult cells derived from bone marrow (called bone marrow stromal cells or BMSCs), adipose tissue (called adipose-derived stem cells or ADSCs), peripheral blood, and the umbilical cord.¹ BMSCs and ADSCs are most commonly used for disc restoration. They share many common characteristics in gene expression, developmental pattern, and cell surface profile.⁵ It remains controversial which of the two is more chondrogenic. ADSCs are more abundant and accessible than BMSCs, but at the same time require a larger sample to yield the same quantity of cells. In contrast with MSCs, ESCs are primitive, pluripotent cells derived from the blastocyst during early development. Fig. 51.2 illustrates the various fates of an ESC when subjected to patterned biochemical environments. Our group is designing methods to develop chondroprogenitor stem cell lines that can restore the functional capability of the IVD. Our preliminary results show the ability of stem cells to differentiate into a chondrocyte cell line.

The immune-modulatory function of MSCs affords another avenue to combat disc degeneration. It is known that DDD is characterized by a cytokine-mediated response that involves classical proinflammatory factors such as interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α).⁶^,⁷ MSCs are capable of mitigating inflammation by virtue of three mechanisms.⁸ First, they secrete IL-1 receptor antagonists that directly inhibit the IL-1 signaling cascade. Second, they serve as a relay mediator of a negative feedback loop where MSCs produce antiphagocyte molecules in response to inflammation. These molecules cause macrophages to either deactivate or produce the immunosuppressive interleukin-10 (IL-10). Third, MSCs reduce the generation of reactive oxygen species (ROS), which are critical for inflammation and apoptosis.

51.2.1 In Vitro Studies

The harsh IVD microenvironment poses a challenge for cell survival. These unyielding conditions are further accentuated in DDD.² Due to its avascular structure that provides limited blood supply, the NP is bathed in a hypoxic and low-glucose niche that promotes anaerobic respiration and low pH formation.⁹ Although NP cells are surprisingly well adapted to living in such conditions, MSCs demonstrate variable response to different chemical factors. For instance, IVD-like low glucose and hypoxia positively regulate cell viability and ECM deposition in BMSC and ADSC, while high osmolarity and low pH are negative regulators.¹⁰^,¹¹^,¹² Therefore, it is believed that predifferentiation of MSC into NP-like cells prior to implantation may be necessary to increase in vivo viability of cell grafts. Despite the difficulty in culturing chondrocyte-like NP cells, they have been successfully differentiated from MSCs with two mainstream methods, growth factors and cell–cell interaction.¹³

Table 51.2 Pros and cons for use of stem cells

Embryonic stem cells	Induced pluripotent stem cells	Adult stem cells
Isolated from embryo at blastocyst stage	Generated by ectopic expressions of pluripotent genes in primary cells	Isolated from various niches such as bone marrow
Unlimited self-renewal and differentiation potential into all somatic cell types	Unlimited self-renewal but limited differentiation potential	Limited self-renewal but limited differentiation potential
Potential to form teratoma	Little or no therapeutic potential due to viral vectors and changes in DNA	Alternative source for tissue engineering and cell therapy

The most commonly used exogenous growth factors in vitro include transforming growth factor-beta (TGF-β), insulinlike growth factor-1 (IGF-1), fibroblast growth factor-2 (FGF-2), platelet-derived growth factor-BB (PDGF-BB), and bone morphogenetic protein-2 (BMP-2).¹⁴^,¹⁵ Other factors such as cis-retinoic acid and ascorbic acid are also used. In addition, growth and differentiation factor-5 (GDF-5) may be more specific for promoting differentiation toward NP cell–like lines.¹⁶ Notochordal cell-conditioned medium has been shown to induce MSC differentiation toward a phenotype that is characteristic of earlier NP development stages, including elevated expression of type III collagen (compared with type II collagen in mature NP cells) and sulfated glycosaminoglycans.¹⁷ This finding is likely mediated by secreted soluble growth factors yet to be characterized.

Differentiation of MSC can also be achieved via cell–cell interactions. When co-cultured with degenerate NP cells, both BMSCs and ADSCs differentiate to acquire NP cell–like traits.¹⁸^,¹⁹ Interestingly, this interaction is reciprocal in that MSCs also enhance self-repair in dysfunctional NP cells via transfer of membrane components, resulting in increased ECM production.²⁰ Via direct cell-to-cell contact, autologous BMSCs can also activate degenerate NP cells from human patients to induce DNA replication, proteoglycan synthesis, and cell proliferation.²¹

51.2.2 In Vivo Animal Studies

A number of animal species have been used in preclinical studies on the effect of stem cell implantation to regenerate IVD tissue. These include rabbits, rats, dogs, sheep, goats, pigs, and mice. Several models of DDD have been proposed using either physical trauma (e.g., needle puncture²²) or biochemical lesion (e.g., injection of bromodeoxyuridine²³ or protease²⁴). In most of these studies, parametric analysis of disc degeneration was based on four factors: disc height index, MRI T2 signal intensity (which reflects water content), production of NP-related products such as type II collagen, and histological disc degeneration grades.

Fig. 51.2 The differential potential of stem cells and their therapeutic applications including intervertebral disc regeneration.

Most animal research on stem cell–based disc regeneration focused on BMSCs and only some on ADSCs. The relatively immunoprivileged IVD provides a suitable platform for xenographic implants. In rats, human BMSCs can survive and differentiate into collagen-producing, chondrocyte-like cells.²⁵ Similarly, human ADSC implant following iatrogenic injury to rat IVDs is associated with less pronounced disc degeneration and elevated ECM production compared with injured discs without implantation.²⁶ These findings are reproduced in rabbits and other mammalian species, as well as with use of ESCs.²⁷^,²⁸^,²⁹ The degeneration-protective effect is accentuated when the cells are injected via a three-dimensional carrier compared with simple suspensory media.³⁰ On the other hand, stem cell grafts have been used as a vehicle to introduce transfected genes into the IVD.³¹ This raises the possibility of weaving targeted gene therapy into stem cell technology to produce implants with a greater regenerative potential. Recent animal experiments have invoked a more conscientious, minimally invasive approach in eliciting artificial injury in order to recapitulate the slow, progressive disc degeneration in humans.³²

In a representative study conducted by our group,²⁸ a novel in vivo percutaneous animal model of disc degeneration was developed by performing needle punctures on healthy, intact discs of New Zealand white rabbits ( Fig. 51.3). This model reproduced the degenerative disc process that occurs in the human lumbar spine ( Fig. 51.4). Interestingly, degenerated discs in humans are seen in the setting of annular tears as illustrated with lumbar discography, making one speculate whether annular tears are responsible for initiating the cascade of disc degeneration ( Fig. 51.5). MRIs were obtained preoperatively and at 2, 4, and 8 weeks postoperatively. Before implantation, ESCs were cultured with cis-retinoic acid, TGF-β, ascorbic acid, and insulin growth factor to induce differentiation into a chondrocyte lineage. After MRI was used to confirm the level of disc degeneration, the discs were injected with murine ESCs that were labeled with a mutant green fluorescent protein (GFP). Alcian blue staining and other histologic analyses confirmed that viable chondrogenic progenitor cells existed before implantation.