BIOTECHNOLOGIES IN THE TREATMENT OF DEGENERATIVE …

[Pages:6]Article

DOI: 10.5504/bbeq.2011.0136

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BIOTECHNOLOGIES IN THE TREATMENT OF DEGENERATIVE DISC DISEASE OF THE CERVICAL SPINE

Dilyan Ferdinandov1, Iliya Tsekov2, Ventzeslav Bussarsky1, Zlatko Kalvatchev3 1Medical University ? Sofia, Department of Neurosurgery, Sofia, Bulgaria 2National Centre of Infectious and Parasitic Diseases, Department of Virology, Sofia, Bulgaria 3Military Medical Academy, Molecular Virology Laboratory, Sofia, Bulgaria Correspondence to: Dilyan Ferdinandov E-mail: d.ferdinandov@mu-sofia.bg

ABSTRACT

Degenerative disc disease of the cervical spine is a common medical condition in the population with underlying aging, genetic, mechanical and environmental factors. It is characterized with structural collapse, osteophyte formation and loss of the loading function of the intervertebral disc. As a consequence a compression of neural structures is observed with axial neck pain, cervical radiculopathy and myelopathy. Currently, the cervical interbody fusion and artificial disc implantation are the available operative treatment options when medications and physiotherapy are ineffective. New strategies focusing on biologic modalities would be appealing. We review the advanced biotechnologies in the treatment of degenerative disc disease of the spine. Many studies are focused on therapeutic molecules capable to influence the biochemical and structural processes of intervertebral disc degeneration. These factors are categorized as anticatabolics, mitogens, chondrogenic morphogens and intracellular regulators. Another step that further opposes the simple injection of tissues with therapeutic molecules is gene therapy seeking to transfer genetic material into target cells, which in turn become in situ factories for the long-term production of desired proteins. The use of tissue-engineered intervertebral disc constructs may prove helpful when treating degenerative cervical disc disease. An in vitro technology designed to produce cartilage or bone with desired biomechanical properties to repair damaged tissues is available nowadays. In the near future the golden standard might be the replacement of the degenerated disc with biological rather than polymer or metal prostheses. However, the cost-effectiveness of the reviewed approaches has not been definitively established. Regardless of its efficacy, the safety and economic issues may ultimately determine whether these therapies are viewed as an acceptable alternative to the recent treatment options.

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Keywords: degenerative disc disease, intervertebral disc, cervical spine, biotechnology

Introduction

Degenerative disc disease (DDD) of the cervical spine is a common medical condition in the population. The underlying mechanisms causing degeneration of the intervertebtal disc (IVD) are currently not completely understood, but risk factors include aging, genetic background, mechanical and environmental factors (15). This process begins with cellular loss, decreased ability to synthesize extracellular matrix, proteoglycan breakdown, and dehydration of the nucleus pulposus (NP). These changes lead to structural collapse, osteophyte formation and loss of the loading function of the disc at the advanced stage (6). Fissures of the annulus fibrosus (AF) appear which may lead to disc rupture and disc herniation. As a consequence a compression of neural structures is observed with axial neck pain, cervical radiculopathy and myelopathy.

Currently, the cervical interbody fusion and artificial disc implantation are the available operative treatment options when medications and physiotherapy are ineffective. However,

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this approach is limited to symptomatic relief. New strategies that focus on biologic modalities would be appealing.

We aim to review the advanced biotechnologies in the treatment of DDD of the cervical spine. Recent applications of biological products in support of surgery are also considered. This publication is a part of the PhD thesis of Dilyan Ferdinandov, MD.

Molecular and gene-based therapies

The hallmarks of disc degeneration include loss of proteoglycan, water, and type II collagen in the disc matrix. The qualitative changes in the disc structure such as loss of collagen crosslinking and proteoglycan organization are more difficult to assess. In general the change of the differentiated chondrocyte phenotype of the NP into a more fibrotic phenotype is important.

The focus of molecular therapy has been to prevent or reverse one or more aspects of the described biochemical and structural changes in the extracellular matrix of the disc. Although growth factors are usually used to describe this category of therapeutics, the more precise term is a therapeutic molecule. Effective therapeutic molecules may have activity unrelated to or beyond cell replication which growth factors

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imply. They can be categorized as anticatabolics, mitogens, chondrogenic morphogens and intracellular regulators. Molecules, which are capable to influence the process of IVD degeneration, are listed in Table 1.

As opposed to simply injecting tissues with therapeutic molecules, gene therapy seeks to transfer genetic material into target cells, which in turn become in situ factories for the longterm production of desired proteins. For example, delivery of osteoinductive growth factors by genetically modified cells may serve for the purpose of enhancing spinal fusion. Gene therapy may provide a more potent osteoinductive signal than recombinant growth factors. These methods result in the sustained local release of osteogenic proteins at levels that are more physiologic than the administration of a single large dose, which may not be sufficient for achieving a spinal fusion.

There are three main components in gene therapy: DNA sequence of the protein of interest, target cells into which it will be inserted and a vector to transfer the gene into the cells. Nonviral vectors are easier to produce and are more stable than viruses. Moreover, infectious agents are not administered to the patient with this approach and nonviral constructs are also less antigenic, making them safer. Such vectors include liposomes and artificial matrices. Nevertheless, viral vectors are often favoured because of their superior transduction efficiencies. Retroviruses, adenoviruses and adeno-associated viruses have all been used to transmit DNA sequences to target cells (37).

The gene transfer may be accomplished by either in vivo or ex vivo techniques. It is easier to introduce the DNAcontaining vector directly into the patient but in vivo methods are limited by inefficient gene delivery, nonspecific targeting of cells and the possibility of a vigorous host inflammatory response. However, expression of BMP-2 by cells infected

with an adenoviral vector was shown to significantly stimulate the production of new bone in animals, suggesting that in vivo gene transfer may prove to be useful for enhancing spinal fusion (2).

Ex vivo techniques include harvesting of autologous target cells, cell culturing and transduction with genetic material before reimplantation of the cells into the patient. This approach allows high rates of transduction in selected certain target cells. Ex vivo gene delivery is considered to be safer, although it is more complex and expensive, because all of the genetic manipulation occurs out of the body and the genetically altered cells may be assessed before they are returned to the patient. This method contributes not only to deliver therapeutic molecules but also for selection of osteogenic or chondrogenic cells which express them.

Anticatabolics Matrix loss in IVD results from a disturbed balance between the processes of synthesis and degradation. The matrix metalloproteinases (MMPs) make up a particularly important group of catabolic enzymes which play a role in the normal turnover of molecules and are thought to be important in disc degeneration (30). An evidence to support this hypothesis is the elevated concentrations of MMPs in degenerated discs. MMP activity is normally inhibited within the matrix by tissue inhibitors of MMPs (TIMPs). In vitro experiments in human disc cell culture that used an adenoviral gene therapy approach revealed that TIMP-1 increased the accumulation of matrix proteoglycans (34).

Along with the balance of synthesis and degradation, the rate of disc matrix metabolism may also be important. Cytokines such as interleukin 1 (IL-1) and tumour necrosis

Therapeutic molecules

TABLE 1

Category Anticatabolics Mytogens

Chondrogenic morphogens

Intracellular regulators

Molecule

Tissue inhibitor of matrix metalloproteinase (TIMP-1 and TIMP-2)

Insulin-like growth factor-1 (IGF-1) Platelet-derived growth factor (PDGF) Epidermal growth factor (EGF) Fibroblast growth factor (FGF) Transforming growth factor b1(TGF-b1) Bone morphogenetic proteins (BMPs)

? BMP-2 ? BMP-6 ? BMP-7 (osteogenic protein 1, OP-1) ? BMP-12 ? BMP-13 (growth and differentiation factor 6, GDF-6, or cartilage-derived

morphogenetic protein 2,CDMP-2) ? GDF-5 (CDMP-1) Link N

Sma-Mad (Smad) proteins Sox 9 LIM mineralization protein 1 (LMP-1)

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factor a (TNF-a) may have critical roles in disc metabolism. Therefore, molecules such as IL-1 receptor antagonists and the available drug infliximab, which can block IL-1 and TNF-a, respectively, may be useful (21, 26). Further research into the field of anticatabolic molecules may yield important results.

Mitogens Mitogens are defined as molecules able to increase the rate of mitosis and they constitute the group of growth factors. Well known in vitro chondrogenic mitogens are insulin-like growth factor 1 (IGF-1), epidermal growth factor (EGF), fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF), (33). These molecules can increase the rates of mitosis and proteoglycan synthesis to various degrees depending on the region of the disc.

IGF-1 levels are found to decrease in an age-dependent manner in animals and human and it is speculated that matrix synthesis may be increased by restoring the IGF-1 in aging discs (25). In vivo experiments with growth factors in mouse disc degeneration model showed that IGF-1 had only mild and FGF had no effects on disc matrix protein synthesis (35). Another different potential mechanism of therapy is supposed to be the protection of cells from death induced by apoptosis. It is found that human disc cells placed in vitro in low-serum conditions underwent apoptosis which was prevented by a certain degree by addition of IGF-1 or PDGF (8). Unfortunately, IFG-1 also has catabolic effects decreasing the levels of TIMP-2 thus indicating its complex effect on disc matrix metabolism.

Chondrogenic morphogens Chondrogenic morphogens are cytokines with a mitogenic capability, an ability to promote the differentiation of chondrocyte-specific phenotype and to reverse the fibrotic phenotype of the disc cells. By definition, these molecules are secreted and act via receptor molecules on the extracellular surface of the responding cells and an intracellular messenger system.

Transforming growth factor b1 (TGF-b1) is a mitogen and a highly anabolic molecule that significantly increases the synthesis of proteoglycans per cell (33). The effect of TGF-1 is superior to that of other growth factors such as EGF, IGF-1, PDGF, and FGF. When an adenoviral vector is used it is possible to achieve in vivo transduction with DNA for TGF-b1 in NP cells. Animal studies more than 10 years ago showed dramatic increases in TGF-b1 expression, as well as a double increase in proteoglycan synthesis when compared to nontransduced cells (23).

In vitro experiments with cells from degenerated human IVD indicated that TGF-1 can increase the rates of proteoglycan and collagen synthesis, suggesting that cells from degenerated discs are capable to respond to TGF-1 (32). Furthermore, cells of human discs transduced with adenoviral TGF-b1 vector construct were shown to demonstrate significantly greater synthesis of both proteoglycans and collagen, up to three times more that of nontransduced cells (19). The decreased proteoglycan synthesis is known to be a

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hallmark of disc degeneration and TGF-1 has the potential to delay the progression or even prevent the disease.

Bone morphogenic protein 2 (BMP-2) is another prototypic chondrogenic morphogen. The commercially available recombinant human BMP-2 (rh-BMP-2) increases the proteoglycan production and initiates the chondrocytic phenotype of the disc cells (38). Furthermore, the transduction with adenoviral IGF-1 and BMP-2 vectors resulted in similar upregulation of proteoglycan and collagen production and an additive effect was observed when they were cotransduced in human cells (18). BMP-2 molecule is also found to partially reverse the inhibitory effect that nicotine plays on the synthesis of proteoglycans. It is hypothesized that it has the potential to prevent the degeneration of IVD associated with this risk factor (11). Although this is a highly active area of research there are still limited reports with in vivo models on the efficacy of BMP-2 in treating disc degeneration.

Because BMP-2 is well known to promote the terminal differentiation of osteoblasts during bone formation, there is a concern that it can lead to disc cell differentiation along with osteoblastic lineage. However, in vitro experiments with human disc cells showed that BMP-2 enhances the expression of chondrocytic genes but not of osteogenic ones (10).

Nowadays, rhBMP-2 is widely used with questionable outcome in augmenting spinal fusion in patients, especially in the cervical spine. Perri et al. (28) reported a case of anterior cervical discectomy and fusion using rhBMP-2 that resulted in a life threatening adverse event of postoperative diffuse and intense soft-tissue swelling. Other serious side effects including acute renal insufficiency, supraventricular tachycardia, and confusion are reported in a single patient (20). Cases of retropharyngeal swelling are also reported by other authors as well as local inflammatory reactions, transient dysphagia, heterotopic ossification, prevertebral haematoma, and formation of seroma (4, 14). Adverse events are supposed to be dose-dependent and are more likely in the region of the cervical than the lumbar spine due to anatomical considerations.

BMP-6 is also characterized with potent osteoinductive properties. Adenoviral BMP-6 vector construct showed induction of local bone tissue deposition and fusion after multiple level percutaneous injections in the spine of rabbits (12). The same study also demonstrates that BMP-6 gene therapy can induce bone formation in immunocompetent animals, even when administered with an antigenic vector. It could be speculated that an anatomically precise fusion can be accomplished by percutaneous administration of the proposed gene therapy.

Another potent disc cell morphogen is BMP-7, also known as osteogenic protein 1 (OP-1). It stimulates both AF and NP cell growth in vitro (16). These cells increase proteoglycan synthesis and expression of the aggrecan and type II collagen chondrocytic genes. In vivo experiments in rabbit models of disc degeneration showed that direct intradiscal injection of BMP-7 increases disc height and proteoglycan content (3, 16).

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The member of the same family of molecules, BMP-12, also has the potential to increase the synthesis of structural proteins of IVD. An adenoviral BMP-12 vector transfected human disc cells in vitro are found to produce twice more matrix proteins and to increase cell proliferation significantly comparing to controls (1). Interestingly, the effect of BMP-12 gene was more pronounced in the NP cells regarding proteoglycan and type II collagen synthesis. On the other hand, more pronounced synthesis of type I and VI collagen is found in the AF cells. The authors concluded that the observed increase in cell numbers might also help regenerate the degenerated disc.

BMP-13, known also as growth and differentiation factor 6 (GDF-6) or cartilage-derived morphogenetic protein 2 (CDMP-2), has only 50% homology to BMP-2 in its amino acid sequence (13). Experiments with a cartilage cell line indicated that BMP-13 also increases the proteoglycan synthesis rate, however, it was much less potent than BMP-2. Although additive effect of BMP-2 and BMP-13 is found no synergism was observed in proteoglycan production or chondrocytic gene expression (13). Research in this direction is still in an early stage.

Growth and differentiation factor 5 (GDF-5), known also as cartilage-derived morphogenetic protein 1 (CDMP-1), is a member of the TGF-b superfamily. GDF-5 is a molecule found to increase the disc height in mouse disc degeneration model (35). Furthermore, some increase in cellular proliferation in the middle and inner anulus and transitional zone was observed. However, the authors observed an inflammatory response with repeated injection of the protein. It was also shown that GDF-5 delivered by an adenovirus vector promoted the growth of human disc cells cultured in vitro (36). GDF-5 is now commercially available for spinal fusion application in humans.

Link N is an amino-terminal fragment of a link protein and has stimulatory activity on disc cells (22). The molecule modestly increases the proteoglycan production but not the cell number. Interestingly, the same group found that type II collagen production is significantly increased in cells derived from the NP and moderately in cells derived from the AF. The mechanism of the specific up-regulation is not clear but it is also observed with other therapeutic molecules from the group of chondrogenic morphogens.

Intracellular regulators

Intracellular regulators are molecules that are not secreted and do not work through transmembrane receptors. They typically control one or more aspects of cellular differentiation. For instance, Sma-Mad (Smad) proteins are intracellular molecules that mediate BMP-receptor signalling (24). Smad-1 and Smad5 proteins are predicted to induce similar effects on disc cells as BMP-2, such as increasing proteoglycan and type II collagen synthesis, but the intracellular regulators are still an uninvestigated area (9).

Sox9 protein is a chondrocyte marker and a positive regulator of type II collagen synthesis. Transduction of an adenovirus

Sox9 vector construct is found to increase NP cell production of type II collagen in vitro and to prevent degenerative changes in the disc in an animal model (27). Sox9-treated discs had a more chondrocyte-like phenotype with significantly increased cell number as compared with controls but the effect on the composition of disc matrix was unknown. As a gradual decline in the number of nucleus cells is thought to play a major role in degeneration, this potential to positively influence disc cell number and phenotype is an important step toward the goal of using gene therapy in the treatment of disc disease.

LIM mineralization protein-1 (LMP-1) is an intracellular molecule that has positive effect on bone formation and osteoblast differentiation. This intracellular regulatory protein has been shown to influence the BMP family, upregulating their expression and enhancing their anabolic activity. Both in vitro and in vivo after transduction with an adenoviral vector construct containing LMP-1 up-regulation of BMP2 and BMP-7 production is observed as well as synthesis of aggrecan and proteoglycans in disc cells (39). Even more, it is hypothesized that by this approach heterodimers of the BMP-2 and BMP-7 are produced which are 20 times more effective than homodimers of the same molecules. This molecule might be useful not only to treat degenerated IVD but also to promote fusion in spinal surgery.

Cell and tissue transplantation

An in vitro technology to produce a cartilaginous tissue to repair damaged tissues is now available. These methods have been combined with molecular factors to tissue-engineer IVD and to achieve the maximum stimulation of cell activity (16, 17). Many studies showed that the biomechanical strength and the biochemical properties of the tissue-engineered AF and NP constructs reflected the characteristics of the original tissues.

An improvement of this technology is the development of three-dimensional (3D) in vitro models for culturing of IVD cells (29). Both AF and NP cells are able to regain the cell morphology, the expression of specific genes and to increase the deposition of matrix proteins. The physiologic relevance and importance of culturing cells in a 3D configuration is pointed out by many authors. It is important that using this approach a reconstitution of collagen fibrous meshwork with cell density comparable with that of the native tissue is possible (29, 40). Furthermore, 3D microspheres can be produced into injectable size (200-300 microns in diameter) and used as a carrier for mesenchymal stem or NP cells. More physiologically relevant matrix microenvironment of the microsphere may facilitate the graft integration within the host tissue and therefore may promote the regenerative effects.

The transplantation of NP and mesenchymal stem cells is proven to be effective in a rabbit model of IVD degeneration (5). Disc regeneration was confirmed regarding the elevation of the disc height, signal intensities on MR imaging, and histological assessment. It is found that both mesenchymal stem cells and NP cells are effective and their application is comparable.

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Another interesting approach is proposed by the group of Goldschlager (7). They test the hypothesis in an ovine model that adult allogeneic mesenchymal progenitor cells differentiated with a chondrogenic agent could synthesize a cartilaginous matrix when implanted into a biodegradable carrier. Over time, this implant might serve as a dynamic interbody spacer following anterior cervical discectomy. Their study demonstrated the feasibility and safety of the technique to produce cartilaginous tissue to replace the cervical IVD. This biological approach may offer a means preserving spinal motion and gives an alternative to fusion and to artificial prostheses.

A great step in this field is done with a trial with allograft disc transplants from cadavers in a small group of five patients in an attempt to provide a biological alterative to artificial cervical disc prosthesis (31). Cervical discs, together with endplates and uncovertebral joints, were removed from donors and stored frozen in liquid nitrogen before the procedure. Despite signs of mild disc degeneration, the motion and stability of the spinal unit was preserved after transplantation for the 5-years follow-up of the patients. Although these interventions did not represent actual biotechnology, it was shown that such transplants can survive and preserve function for a long time.

Discussion

The molecules used to treat IVD degeneration have expanded beyond the classic growth factors. Now, at least four different classes may be effective in disc repair. Although all of these molecules were studied in vitro and some of them are used in humans to promote spinal fusion, few have been tested to treat DDD in animal models and none in humans. The search continues both for targets capable of altering the imbalance of synthesis and catabolism within degenerating discs and for approaches of clinical application of this knowledge.

Gene therapy has been validated by many preclinical studies as an effective technique for treating degenerated IVD or enhancing bone formation but significant concerns remain regarding its safety in humans, especially the potential risks related to the use of viruses. Viral vectors may elicit a substantial host inflammatory response and their long-term systemic effects have not been well characterized. Despite that virus vectors are unable to replicate it is still a possibility that these viruses may trigger an uncontrollable infection or even malignant transformation of cells.

In recent years research has been focused on the possibility of regulation of transgene expression after gene transfer. Ideally, once cells become capable of producing increased levels of desired proteins, the amount of synthesized product could be exogenously controlled on the basis of need. Inducible systems by linking ligand-activated promoter regions to a particular gene of interest within a single vector construct are under development. The two basic strategies, termed "on" and "off", are intimately related to the larger issue of safety in gene therapy. Although many practical issues were resolved before

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any wide-scale effort to transfer this technology to the clinic, gene therapy must be shown to be not only effective, but also safe.

For repair of a larger defect, the use of tissue-engineered IVD constructs may prove helpful in treating cervical disc disease. In the near future the golden standard might be the replacement of the degenerated disc with biological rather than polymer or metal prosthesis. Because a reduced cell number is one of the characteristics of IVD degeneration, the transplantation of healthy functional cells and tissues might be required. Although great success in this field is achieved there are still significant practical problems that need to be resolved. The transplants will not survive if proper nutrition is not available. The degenerative processes are not restricted to the disc and in advanced stage of the disease the supply of nutrients is disturbed by sclerosis of the endplate. Another factor to be considered is the segmental instability and the biomechanical stress leading to production of undesirable factors. The transplanted transfected cells with growth factors and tissue constructs might not induce disc regeneration. An interesting idea is to combine 3D cell cultured microspheres or tissue-engineered grafts with a biodegradable implants to support the last.

Conclusions

The cost-effectiveness of the reviewed approaches has not been definitively established. Regardless of its efficacy, the safety and economic issues may ultimately determine whether these therapies are viewed as an acceptable alternative to the recent treatment options including spinal fusion and disc arthroplasty.

Although many issues remain unresolved and many questions unanswered, exciting progress certainly has been made. Up to date gene and cell based therapies as well as tissue engineering have shown the potential to become a powerful tool in the treatment of DDD in humans. Furthermore, not only the replacement or restoration of the IVD but also its prevention from degeneration is a future goal.

Acknowledgements

This collection has been compiled with the financial support of the "Human Resources Development" Operational Programme, co-financed by the European Union through the European Social Fund. The whole responsibility for the collection contents lies with the Beneficiary and under no circumstances should this collection be regarded as representing the official position of the European Union and the Contract Body.

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