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Focus Paper

Cellular Therapy for Disc Degeneration

Anderson, David Greg MD; Albert, Todd J. MD; Fraser, John K. PhD; Risbud, Makarand PhD; Wuisman, Paul MD; Meisel, Hans-Jorg MD, PhD; Tannoury, Chadi MD; Shapiro, Irving PhD; Vaccaro, Alexander R. MD

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doi: 10.1097/01.brs.0000175174.50235.ba
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Disc degeneration is a universal part of human aging and in most cases results in minimal or self-limited symptoms. However, the small segment of the population that experiences disabling spinal pain thought to be in part attributable to disc degeneration represents a disproportionate medical, societal, and economic challenge. Current treatment options range from medicinal inflammation management, to invasive procedures including spine fusion and recently spinal arthroplasty. Unfortunately, all currently available approaches are limited to treating the symptoms of the degenerative process and not the underlying biologic alterations of the disc. Recently, there has been a growing interest in developing strategies to address the underlying biologic imbalances that lead to symptomatic disc degeneration.

The normal intervertebral disc is sandwiched between upper and lower osteochondral endplates and is composed of a tough, outer anulus fibrosus (AF) and gelatinous, proteoglycan-rich inner nucleus pulposus (NP), which attracts and retains water.1,2 The disc is a specialized biomechanical structure capable of converting axial spinal loads into tensile hoop stresses in the outer anulus while allowing motion of the vertebral segment.

With disc degeneration, the NP proteoglycans are progressively lost, leading to poor hydrodynamic transfer of axial stresses to the outer AF. Simultaneously, the integrity of the AF is degraded producing radial fissures, which extend outward toward the periphery of the disc. The vertebral endplates are also affected by the degenerative process and undergo an ossification process that may limit the nutritional supply to the disc. Although progressive disc degeneration results in dramatic changes to the biomechanical functioning of the disc, biochemical events probably play a role in producing disabling spinal pain. Biochemical changes possibly involved in discogenic pain production include the production and release of inflammatory mediators and cytokines from the disc, vascular ingrowth into anular fissures, and the stimulation of free nerve endings in the outermost region of the disc.

As the molecular basis of disc degeneration becomes increasingly understood, various biologic strategies to repair or regenerate the degenerative disc have been suggested.3,4 Because the disc has only a limited intrinsic capacity for regeneration, the therapeutic approaches are generally geared toward the stimulation of matrix production using growth factors, genes, or transplanting cells to repair the damaged disc matrix.5–9 This article will review the cell-based strategies for repair of the degenerated disc and discuss future trends in this rapidly developing area.

The Disc: A Challenging Environment for Cells

The normal NP has an acidic pH, low oxygen tension, and paucity of basic nutrients and thus is one of the most hostile biologic environments in the body. Mechanical loading of the disc subjects this environment to high pressures and complex shear stresses. The gel-like substance of the NP is normally devoid of blood vessels, making anaerobic metabolism a primary means of generating energy. Because the disc must rely on simple diffusion to exchange metabolites and waste products across the barrier of the vertebral endplates, the supply of nutrients is severely limited.10–12 Although disc cells can survive in an environment devoid of oxygen, they are highly dependent on the availability of glucose.13 The normal disc pH is acidic in the range of 6.9 to 7.2, as the result of lactate production; however, metabolic stress may cause the pH to fall as low as 6.1.14 Under highly acidic conditions, the production of matrix proteins by disc cells is suppressed.15

To survive in this harsh environment, disc cells are highly specialized. These cells function quite well in a mildly acidic environment with a lowered oxygen tension.15 Disc cells use specific signaling pathway activity, which facilitates survival in the specialized, low-oxygen microenvironment.16 Within the disc, the cell concentration is relatively sparse, making up only about 1% of the disc volume. This low concentration of cells may be adaptive to the environment, which has a limited supply of nutrients to support cell proliferation.

At least two morphologically distinct cells types populate the NP region of the disc in young animals. One cell type is small and round, similar to a chondrocyte. The second cell type is much larger, with a vacuolated appearance and prominent intracellular glycogen deposits.17 The larger cells are thought to be a remnant of the primitive notochord.18 At the ultrastructural level, the large NP cells contain multiple intracellular vacuoles and prominent Golgi apparatus, suggesting that these cells are actively involved in the synthesis of proteins. However, few mitochondria are seen, probably because of the relatively poor supply of oxygen and a reliance on anaerobic energy pathways (Figure 1).

Figure 1
Figure 1:
Electron micrograph of nucleus pulposus (NP) cell from adult rat disc. Note that cell is surrounded by dense matrix (M) and has many vesicles (V) filled with a ground substance. Cell shows extensive Golgi (GA) network but very few mitochondria suggesting its special metabolic status (original magnification ×10,000)

In humans, the population of notochordal cells diminishes rapidly during childhood, leading to a paucity of these cells in the adult disc. Until recently, no reliable distinguishing cell markers were available to definitely identify the NP or notochordal cell phenotype.11,19 Recently, however, certain cell markers, used in combination, have been suggested to define NP phenotype.20 Rajpurohit et al found that differential expression of the two HIF-1 (hypoxia inducible factor) isoforms, HIF-1α and HIF-1β, along with the expression of GLUT-1 and MMP-2 markers, can provide a phenotypic signature that permits NP cells to be distinguished from neighboring tissues.20

Whole Disc and Disc Tissue Transplantation

Frick et al attempted to address the degenerative process by transplanting whole discs along with the endplates to the spine of mature dogs.21 Although technically feasible, the biologic outcome of this study was disappointing in that proteoglycan synthesis and NP morphology were substantially degraded by the 4-month time point.

Katsuura and Hukuda implanted cryopreserved intact allogeneic discs to the intervertebral disc space of dogs following discectomy.22 As with the Frisk et al study,21 the authors observed modest preservation of disc architecture over the short-term, but progressive degeneration with longer follow-up. These findings may be due in part to the difficulty in maintaining adequate viably of the cryopreserved disc cells as suggested by the work of Nishimura and Mochida.23 These researchers compared cryopreserved NP to fresh tissue transplants in a rat nucleotomy model.23 Although both fresh and cryopreserved disc tissue decelerated degeneration compared with an artificial substitute, the fresh tissue transplants significantly outperformed the cryopreserved tissue in maintaining disc height.

Luk et al transplanted whole disc/endplate constructs to the spines of 14 Rhesus monkeys and demonstrated successful healing of the implants but noted early loss of disc height.24 However, with additional follow-up, there was partial reconstitution of disc height and only minimal disc degeneration noted by the 12-month time point.24

Transplantation of Cultured Disc Cells

Okuma et al documented slowing of the degenerative process in rabbit discs following the insertion of cocultured NP and AF cells.25 In another study, this group compared intact NP transplants with cultured NP cells in a degenerative, young rabbit model that had previously undergone NP aspiration.26 Two weeks after the induction of degeneration, intact NP tissue or cultured NP cells (up to 50,000 cells) from an allogeneic donor were injected into the central region of the rabbit discs. At the 14-week time point, the discs treated with intact NP tissue demonstrated less degeneration than discs treated with cultured NP cells or untreated controls. Unfortunately, data on the survival of cultured donor cells were not included, making it impossible to determine if the relatively poor performance of the transplanted cells was due to a lack of cell survival or was related to the efficacy of cell transplantation strategy. Cell viability has been shown to affect the outcome of other related tissue engineering strategies, making analysis of cell survival an important step in cell research design.27

Gruber et al analyzed the survival and function of cultured autologous disc cells in the sand rat (Psammomys obesus), an animal that undergoes spontaneous disc degeneration.28,29 In this study, an average of 10,000 cultured disc cells embedded in a 2-mm3 collagen matrix were implanted into a defect in the lumbar disc. The cells were labeled before implantation to allow postimplantation tracking. Labeled cells were seen in the discs of animals as late as 8 months postimplantation, but the efficacy of disc repair was not reported.

Ganey et al transplanted autologous cells to the disc of skeletally mature dogs following a posterolateral aspiration injury (∼100 mg of AF and ∼100 mg of NP were removed).30 Twelve weeks later, approximately 6 million autologous cells cultured from tissue aspiration were injected into the NP region of the injured discs. The animals were followed for 12 months and disc height was measured with high resolution radiography. Significant improvements in disc height were noted at the 12-month time point in the cell-treated discs compared with control discs, although prior time points failed to show a significant difference. This delay may reflect the slow biosynthetic rate of NP cells in the disc. Histologic evaluation at the 6-, 9-, and 12-month time points indicated less scarring and more proteoglycan content in the cell-treated discs compared with controls. The labeled cells survived in vivo at least 6 months. Despite the improvements in the cell-treated discs, none of the treated specimens regained normal architecture by the 12-month time point.

Using a similar approach, Ganey and Meisel conducted a human pilot safety study and have initiated a pivotal study using autologous NP cells derived from therapeutic discectomy that were cultured under Good Manufacturing Practices and delivered 12 weeks following discectomy.31 In this nonrandomized and unblinded study, a preliminary data analysis has demonstrated some examples of MR imaging improvements consistent with increased proteoglycan matrix within the NP.31 Although encouraging, these findings require validation from prospective, randomized clinical studies before this approach can be widely recommended in humans.

Implantation of Disc Cell/Scaffold Constructs

Many different types of scaffolds have been used for the growth of disc cells. The purpose of a cellular scaffold is to provide an optimal microenvironment for cellular migration and proliferation that allows the cells to maintain the appropriate phenotype. It is well known that NP cells will alter their phenotype depending on the culture technique used; therefore, selection of an appropriate scaffold and culture technique is crucial.

Alini et al successfully proliferated bovine coccygeal disc cells using a collagen/hyaluronate and chitosan-based scaffold.32,33 Séguin et al grew bovine caudal NP cells using a sintered calcium polyphosphate scaffold and reported successful production of proteoglycans and collagen but noted that the engineered tissue fell short of reaching levels of proteoglycan production found in the native disc.34

Sato et al used a honeycomb-shaped scaffold of modified Type II collagen to grow AF cells and later reported placing these tissue constructs into laser-mediated defects in rabbit discs.35,36 Using a lipophilic dye to track the cells, survival of the implants was documented out to the 12-week time point along with robust proteoglycan production and disc height maintenance.

Mizuno et al used a composite scaffold composed of an outer region of polyglycolic acid polymer and an inner region of alginate/calcium sulfate to grow AF and NP cells, respectively.37 After a 24-hour culture period, the constructs were implanted to the subcutaneous space of athymic mice. Cell survival and matrix protein production were documented over the 12-week follow-up period; however, the amount of proteoglycans produced fell well short of the levels expected in the native intervertebral disc.

Use of Non–Disc-Derived Cells for Disc Tissue Engineering

Because autologous disc cells cannot be obtained without causing morbidity to the disc and because disc cells from a degenerative disc may be functionally impaired, alternative cell types have been considered for disc tissue engineering. Candidate cell populations must be capable of producing large amounts of proteoglycans and Type II collagen (chondrogenic) when placed under suitable conditions. Two good cellular candidates for disc tissue engineering include chondrocytes and mesenchymal stem cells (MSCs), which may be rendered chondrogenic depending on the microenvironment and/or culture conditions used. MSCs may be obtained from several sources, but bone marrow38,39 and adipose tissue40–43 (adipose-derived stem cells) appear to be the most promising clinically. Although MSCs from different sources have distinct cell culture requirements42,43 and differ in the expression of certain cell surface markers,44,45 they share the ability to generate chondrogenic cells.40,46 Some data suggest that adipose tissue may be a richer source of progenitor and stem cells than marrow.44,45,47

Recently, Risbud et al have demonstrated that bone marrow-derived MSCs can be differentiated into NP-like cells using microenvironmental conditions similar to those found in the intact disc.48 Using a combination of low oxygen tension and high osmolarity, the MSCs took on the morphologic appearance of NP cells and formed clusters similar to that seen in the central region of the disc. In addition, the expression profile of the engineered MSCs was noted to be similar to NP cells from the disc (Figure 2). In theory, these engineered cells might be used to repopulate the NP region of the disc after degeneration has ensued.

Figure 2
Figure 2:
Differentiation of mesenchymal stem cells (MSC) into NP-like cells in vitro. MSC were cultured in alginate beads in hypoxia (Hx) or normoxia (Nx) for 7 days and assessed for their morphologic and phenotypic characters. I, Celltracker Green staining of MSC. A, Untreated cells after 24 hours in alginate. B, Cells treated with TGF-β1 in normoxia after 7 days in culture. C, Cells treated with TGF-β1 in hypoxia after 7 days in culture. Note that after 7 days in hypoxia, cells forms large aggregates resembling NP cell clusters (arrow). II, Western blot analysis of hypoxia responsive gene products expressed by MSC. MSC express HIF-1α and MMP-2 in hypoxia. Note that all these genes are expressed by NP cells in vivo and therefore indicate differentiation of MSC toward an NP-like phenotype. III, RT-PCR analysis of genes expressed by MSC cultured in alginate beads in hypoxia and normoxia. After 1 week in culture, in hypoxia, there is a significant increase in the level of expression of aggrecan, Type II and Type XI collagen, and Sox-9.

Sakai et al cultured autologous, bone marrow-derived MSCs on a honeycomb-shaped scaffold of modified Type II collagen.49 The constructs were implanted into rabbit discs 2 weeks following aspiration of the NP and then followed for an 8-week period. Using the lac Z reporter gene, cell viability was documented for at least 4 weeks, and disc height maintenance and proteoglycan production were observed.

Crevensten et al used MSCs grown in a hyaluronate gel to populate the coccygeal discs of Sprague-Dawley rats.50 Using cell labeling, viable cells were detected over the 28-day study period. The cell numbers initially decreased and then increased, suggesting proliferation of the cells within the disc. Although normal disc height was not maintained, this study was limited by the small size of the rat discs and the relatively small volume of injected cells.

Leo et al labeled fat and bone marrow-derived MSCs with the luciferase marker gene and encapsulated the cells in alginate beads before placing the cell containing beads into a small defect created in the anterior region of the intervertebral disc.51 Using a specialized, noninvasive detection system, the fate of the implanted cells was able to be followed in the live animals over a 14-day period (Figure 3). Although cell survival and retention in the disc region were documented, imaging also detected cells in the lungs and liver suggesting leakage or migration of some of the labeled cells. Given the large defect in the disc used in this study, it is not particularly surprising that some of the implanted cells were not retained within the disc.

Figure 3
Figure 3:
In vivo bioluminescent imaging of a rat on postoperative day 7 following implantation of fat stem cells transduced ex vivo with Ad-luc virus carrying the luciferase marker gene.

In addition to the direct production of matrix proteins, MSCs have been shown to stimulate proteoglycan production by native disc cells. In one experiment, Yamamoto et al cocultured MSCs in contact with autologous NP cells, using a 0.4-μm mesh to separate the two cell types.52 The cocultured NP cells up-regulated the production of both DNA and proteoglycan synthesis, suggesting that soluble factors released by the MSCs stimulated the metabolic output from the NP cells.

Enhancement of Cell Function with Growth Factors and/or Genes

Multiple growth factors including transforming growth factor-beta (TGF-β), epidermal growth factor (EGF), bone morphogenetic protein-2 (BMP-2), and bone morphogenetic protein-7 (BMP-7, also referred to as OP-1)53–58 have also been shown to stimulate matrix production by disc cells. For instance, Kim et al found a 200% increase in proteoglycan synthesis and 450% increase in aggrecan mRNA in human disc cells exposed to high doses (up to 2,000 ng/mL) of recombinant human BMP-2.58 These data suggest that using growth factors or growth factor genes may improve the success of a cellular approach to disc tissue engineering.

Paul et al performed adenovirus-mediated transduction of rabbit NP cells with the Sox 9 gene and injected the gene vector directly into the intervertebral disc of skeletally mature New Zealand white rabbits immediately following a stab injury.59 The transduced cells exhibited increased production of collagen II, while the injected discs exhibited less scarring and improved retention of matrix at the 5-week time point.

Discussion

Clearly, the biology of the intervertebral disc is complex and represents a significant challenge to those seeking to achieve successful tissue repair in this relatively hostile environment. At the current time, the science of disc cell transplantation is in its infancy. Issues that need to be worked out include defining the optimal cell type for disc repair, determining whether cellular scaffolds are necessary or beneficial, determining the optimal mechanobiologic environment for cell proliferation and proteoglycan production, and understanding which growth factors or genes may be used to enhance the biologic performance of the implanted constructs.

Many questions remain. For instance, it is not yet known whether MSCs are truly capable of forming mature disc cells or whether these engineered cells only mimic NP cells. Also, long-term survival of MSCs transplanted to the disc has not yet been shown. Because both autologous and allograft MSCs are readily available, the characterization of MSCs as a potential source of cells to repair the disc is an important issue. As better (more specific) cellular markers of the NP phenotype become known and longer-term studies using MSC cell transplants to the disc are performed, it should be possible to make this determination.

The lack of an ideal animal model of human disc degeneration also represents a challenge to the field of cellular disc repair. Virtually all available animal models have significant differences from the human degenerative condition. Although much useful information has been obtained through animal research, most of the existing studies have been performed on relatively young rodents or rabbits with normal or recently injured discs. Compared to humans, these models may be more readily able to achieve tissue repair. Studies using larger animals and involving discs with more advanced stages of degeneration are necessary as researchers move toward the goal of achieving meaningful disc tissue repair in humans.

Another limitation of the current approach to regeneration of the degenerated disc is the relatively poor link between disc degeneration and spinal symptoms. In humans, the real clinical problem is spinal pain and not disc degeneration per se. The assumption has been that many patients have pain from disc degeneration and, therefore, disc repair is a reasonable goal to alleviate the clinical symptoms of spinal pain. However, this assumption remains unproven. Because it is not possible to reliably measure pain in animal models, researchers have been forced to use surrogate methods to evaluate the success of disc repair experiments by quantifying the histologic or biochemical status of the disc.

Another issue that must be defined before tissue repair can be widely adopted in the clinical arena is the optimal stage at which to treat a patient with painful disc degeneration. It is theoretically possible that intervention will work best if applied at a very early stage of degeneration, but often the presence of symptoms is minimal in early disc degeneration. Alternatively, different disc repair strategies may have to be developed for different stages of disc degeneration. Clearly, substantial work remains to be done in the area of disc tissue engineering.

Fortunately, the field of tissue engineering is rapidly evolving, and successes thought impossible only a few years ago have already been achieved. Therefore, continued research into the area of disc repair is clearly warranted. There is reason for optimism as we continue to search for a successful, biologically sound method of addressing the problems of human disc degeneration.

Conclusion

Given our current state of knowledge, it appears that several cell types have the ability to survive, proliferate, and participate in matrix production within the disc for at least short periods of time. Some studies suggest that cell implantation to the disc may help to maintain disc height and improve biomechanical properties of the disc following early induced degeneration. Currently, however, the optimal cell therapy and the use of adjuvants such as cell scaffolds, genes, or growth factors remain to be defined. Further research is necessary to define the optimal combination of cells, scaffolds, and growth factors that will provide meaningful, long-term improvement in the biologic integrity of the intervertebral disc.

Key Points

  • Disc degeneration is a ubiquitous process that involves failure of the intrinsic disc cells to maintain normal matrix architecture within the disc.
  • Implantation of cells to the disc is a promising approach that may be useful in repairing the degenerative disc.
  • Various cell types, including disc cells, chondrocytes, and stem cells, have been investigated for disc repair.
  • Cellular scaffolds, which provide a microenvironment for the implanted disc cells may be useful in the repair strategy.
  • Growth factors and genes may also be used to modulate the repair process within the disc.

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Keywords:

disc degeneration; cell; repair; scaffold

© 2005 Lippincott Williams & Wilkins, Inc.