Gregory A. Helma b, D. Greg Andersonc
Departments of aNeurosurgery, bBiomedical Engineering and cOrthopedic Surgery, University of Virginia Health System, Charlottesville, Va., USA
A variety of evolving technologies are currently being evaluated in preclinical studies to promote tissue repair and/or regeneration in the spinal region. Cellular and genetic techniques to induce bone formation for interbody or posterolateral spinal arthrodesis are an attractive approach, since this technology could be employed through a minimally invasive approach with decreased morbidity and potentially higher fusion rates compared to traditional open procedures. Tissue engineering techniques are also being evaluated for disc repair and regeneration using various growth and differentiation factors, mesenchymal stem cells (MSCs) and genetic therapies. Percutaneous soft tissue stabilization techniques are also within the scope of current technologies, potentially allowing stabilization of the spine by the induction of ligamentous tissues, thus avoiding extensive spinal arthrodesis procedures. Clearly, tissue-engineering techniques will continue to evolve and certainly lead to more effective, and less invasive, procedures for the treatment of traumatic, neoplastic and degenerative spine problems.
The direct application of osteoinductive stem cells on a bioresorbable matrix, or scaffold, may be a useful strategy for engineering bone and soft tissues in the paraspinal region. Stem cells have several advantages for inducing osteogenesis compared with the application of bone growth factors alone. Pluripotent stem cells have the capacity to differentiate into the desired cell type and form appropriate matrix elements under the control of various growth and differentiation factors. By supplying stem cells to the site, the growth factor response is not dependent solely on the availability of local stem cell populations, which may be diminished due to senility, radiation, medical factors, or medications which might alter the intrinsic stem cell population. Therefore, direct implantation of pluripotent cells with or without growth factors has the potential to yield more rapid and uniform bony healing. Osteoprogenitor cells have been isolated from the bone marrow of rats, rabbits, dogs, and humans, as well as nonmarrow locations such as adipose tissue. These cells can be isolated and expanded in tissue culture, prior to implantation into a target location. Stem cells also have the potential to be genetically altered to express various growth factors or other therapeutic genes prior to implantation.
A variety of studies have demonstrated the utility of using stem cells to achieve bone formation. Bruder et al.  placed autologous canine MSCs obtained from bone marrow onto porous cylinders composed of hydroxyapatite and tricalcium phosphate. The implant was subsequently grafted into critical-sized femoral defects. Implants without MSCs produced atrophic nonunions in all treated animals. Implants containing the MSCs produced lamellar and woven bone within the carrier and resulted in a solid union at the site of the defect. Bruder et al.  also successfully achieved bone induction using human MSCs on a ceramic carrier in athymic nude rats. Using radiography, biome-chanical testing, and histologic analysis, the human MSCs were capable of healing critical-sized femoral defects. Quarto et al.  studied the use of autol-ogous bone marrow stromal cells delivered on a macroporous hydroxyapatite carrier for the healing of large bony defects (>4.0 cm) in a small human clinical series. The implant was placed within the defect, and the fracture was stabilized with external fixation. In each instance, the composite implant was able to achieve successful union of the bony defect, thus producing the first direct evidence that tissue engineering of bone can be successfully applied to humans in a clinical setting.
Bone Morphogenetic Protein Gene Therapy
Bone morphogenetic proteins (BMPs) are a group of secreted proteins that are members of the transforming growth factor-^ (TGF-P) superfamily based on their high degree of homology within the C-terminal seven-cysteine region [4-7]. These proteins were originally recognized for their ability to form ectopic bone by inducing primitive mesenchymal cell chemotaxis, proliferation, and differentiation into chondrocytes and osteoblasts [8-13]. The ectopic bone is formed primarily through endochondral mechanisms, recapitulating many of the events seen during embryonic development [7, 10, 11, 13-15]. However, when delivered in high concentrations, direct or intramembranous bone formation can also be induced at the treatment site. Other BMPs have been found experimentally to induce the formation of various other tissue types, including cartilage, tendon, and ligament [16-24]. Molecular cloning techniques have allowed the production of certain BMPs in large quantities, which has contributed to the rapid advancement of our collective understanding of BMP biology and potential for clinical applications .
BMPs are initially synthesized as monomeric precursors that, after proteolytic cleavage, undergo dimerization through disulfide bonding [15, 25]. These dimers, which can be either homologous or heterologous, are released extracellu-larly and act by binding to type I and II receptors [26-34]. Receptor binding and subsequent transphosphorylation leads to the phosphorylation and activation of various cytoplasmic Smad proteins [35-40]. These activated Smad proteins then translocate to the nucleus and initiate cellular responses, including chemotaxis, proliferation, and differentiation.
Gene-based therapies attempt to deliver specific genes, known as the transgene, to target cells to change the existing physiologic state or disease process [41, 42]. Delivery of the genetic material to the target cell can be accomplished by both nonviral and viral vectors. Nonviral or synthetic delivery techniques include the use of molecular conjugates, liposomes, naked DNA, plasmids, electroporation, or the incorporation of the transgenes with viral proteins. To date, the majority of these techniques are limited by their relatively low cellular transduction rates. In contrast, viral vectors can be highly efficient in their delivery of genetic material. Some of the more common viruses used for gene therapy include the adenovirus, herpes virus, retrovirus, and adeno-associated viruses (AAV). These vectors are often genetically modified so that they are replication-deficient, through the removal of specific portions of the viral genome, which allow for the insertion of a therapeutic transgene. Although gene therapy as a concept continues to capture the imagination of basic scientists and clinical researchers, the advancement of the techniques toward human use has proven difficult [41-44]. Technical hurdles such as insufficient transgene expression, an inability to circumvent the host immune system, and a failure to achieve long-term transgene expression continue to present a challenge for many clinical applications. Fortunately, bone induction using BMPs may be an ideal application of gene therapy because long-term expression of the BMP is not required to achieve therapeutic bone formation. In fact, the elimination of BMP expression may be advantageous in that tissue overgrowth or toxicity is less likely to be encountered.
Adenoviral Vectors. Adenoviruses are double-stranded DNA viruses which bind to specific cell surface receptors, enter the cells by endocytosis, and subsequently release their DNA into the cytoplasm [45, 46]. The viral genome is divided into immediate early genes, early genes, and late genes according to the time in which the genes are expressed. The immediate early genes activate early gene transcription, while the early genes are involved both in subsequent viral replication and host immune evasion. The late genes code for the aden-oviruses' structural proteins . Most adenoviral vectors studied to date are derived from the adenovirus serotype-5, which are rendered replication-defective by deletion of the E1 region. The E3 region is often deleted as well to make room for larger transgenes. First generation adenoviral vectors can accommodate up to 8 kb of foreign DNA.
The advantages of adenoviral vectors include their ability to be produced in high titers, their extrachromosomal life cycle, which reduces the risk of insertional mutagenesis, and their ability to transfect numerous cell types [46, 47]. There are several potential disadvantages of the adenoviral vector for gene therapy. Because the virus does not integrate into the cellular genome, the length of gene expression is limited and therapeutic genes are not passed to the progeny of the transduced cells. Perhaps the most problematic issue with adenoviral vectors, however, is the robust humoral and cellular immune response that can occur at the treatment site resulting in reduced transgene expression. Intense research efforts are currently directed at blunting the immune response by alterations to the adenoviral vector such as deleting the viral DNA polymerase gene (Apol adenoviral vectors) or completely eliminating the viral genome (gutless adenoviral vectors).
AAV Vectors. AAV, which are defective single-stranded DNA parvoviruses, are also attractive vectors for BMP gene therapy studies . AAV vectors have the ability to integrate stably into the target cell's genome, transduce a variety of cell types, maintain high levels of gene expression, transfect both proliferating and quiescent cells, and be generated in high titers. Numerous studies have demonstrated that AAV can efficiently transduce muscle and other cells in vivo with little inflammatory response and no evidence of insertional mutagenesis. The production of AAV vectors was initially fraught with numerous technical difficulties; however, current techniques of vector production lead to a high yield of recombinant AAV, completely free of wild-type AAV
Retroviral Vectors. Retroviral vectors enter target cells through interactions between the viral envelope proteins and cell surface glycoproteins. Importantly, retroviruses contain single-stranded RNA. Once the viral genome is released into the cytoplasm, retroviral reverse transcriptase produces a double-stranded DNA copy, which subsequently integrates into the host genome during mitosis. One disadvantage to using retroviral vectors is that retroviruses can only integrate their genetic material into proliferating cells . Therefore, it may be difficult to achieve adequate cellular transduction at sites requiring bone induction by direct injection. Other major disadvantages of retroviral vectors are their low infectivity and instability of the virions. The utilization of retroviruses for BMP gene therapy will most likely be limited to ex vivo approaches, such as for the genetic modification of MSC populations.
Herpes Viral Vectors. Herpes viruses are double-stranded DNA viruses which can cause significant human pathology, including cold sores and encephalitis. Gene therapy studies utilizing herpes viral vectors typically utilize genetically modified herpes simplex type 1. In the normal life cycle of these viruses, the virion fuses with the cell membrane and is transported to the nucleus where, after several phases of gene transcription, the cell lyses and releases progeny viral particles. Herpes viruses also have the ability to enter a latency phase, during which time the viral genome is not actively transcribed. This latency phase can be lost during cell division . Herpes viral vectors have the advantage of being able to accommodate up to 40 kb of foreign DNA and can be utilized to insert foreign DNA into a variety of cell populations, including myocytes, with limited toxicity. Herpes viral vectors may, therefore, be a reasonable vector for BMP gene therapy .
Direct Plasmid Injection. Wolff  was the first to show that the direct intramuscular injection of plasmid DNA could lead to low level, short-term gene expression. Other researchers have now demonstrated the successful transduction of the myocardium, brain, thyroid, and various tumors using this technique [52-55]. Inadequate cellular transduction rates can be significantly increased by pretreatment of the injected area with hypertonic saline or bupi-vacaine . Systemic gene expression can also be obtained by the direct intravenous injection of naked DNA in adults, although rapid degradation of the DNA prior to reaching the target cell remains problematic in the absence of a delivery system, such as cationic liposomes . Currently, no published studies have demonstrated successful bone formation using direct injections of osteogenic plasmid into either orthotopic or heterotopic sites.
Electroporation. The diffusion of extracellular DNA into a cell in vitro and in vivo can be significantly increased by permeabilizing the cell's membrane using short, high intense electric pulses. The technique essentially opens small pores in the cell's membrane, through which molecules can diffuse down concentration gradients. When the pores spontaneously close, the DNA is sealed within the cell's cytoplasm, where it can be transported to the nucleus . This technique can increase the transduction rate over a 1,000-fold compared to direct plasmid injection, and has been utilized to transduce liver, melanoma, skin, and muscle cells [59, 60]. The ideal parameters for cellular transduction appear to vary between tissues. In addition, only short-term gene expression has been achieved. The utilization of this approach for the delivery of osteogenic genes has yet to be defined.
Gene Gun. Another interesting technology which is currently under investigation for transducing cells with foreign DNA is the gene gun. The technique involves the coating of gold particles with plasmid DNA, which are subsequently bombarded into the tissue of interest . Under optimal conditions, the gene gun can be utilized to transduce 10-20% of the cells at the treatment site. Although gene expression of up to 60 days has been achieved, the depth of tissue penetration is limited to <0.5 mm . In addition, low levels of gene expression are generally achieved, which is problematic for BMP gene therapy, where relatively high levels of local BMP expression are required for tissue induction.
Liposomes. Liposomes are commonly used to deliver DNA to cells in vitro . Cationic derivatives of diacylglycerol and cholesterol, lipid derivatives of polyamines, and quaternary ammonium detergents are typically used to form the cationic lipid-DNA complexes. The cationic-lipid compounds serve to decrease the negative changes of the DNA plasmids and facilitate entrance of the plasmids through the cell membrane. Liposome preparations also contain neutral or 'helper' lipids, including cholesterol or dioleoylphos-phatidylethanolamine, to improve DNA release from the endosome into the cytoplasm . Liposomes can transduce a wide variety of cells and tissues, including vascular endothelium, lung, brain, and skin [65, 66]. The attachment of cell-specific antibodies to the liposomal membrane may improve tissue specificity of this transduction technique. Intense research efforts are currently directed at improving liposome production and delivery, which may render these vectors ideal for BMP gene delivery in the near future.
Polymer-DNA Complexes. High-molecular-weight cationic polymers, such as poly-L-lysine, poly-L-ornithine, polyethylenimine, and chitosan, can improve DNA delivery to cells via nonspecific absorptive uptake [67, 68]. Various synthetic polymers can also be utilized to improve cellular transduction rates and can be designed to be biodegradable, thermosensitive, and biocompatible . Polymers can also be constructed with targeting ligands, such as antibodies, transferrin, and asialoglycoprotein to improve tissue specificity. Additional modifications to these molecules can also be made to improve cellular uptake and cytoplasmic trafficking of the therapeutic gene.
The promotion of osteogenesis through the direct injection of adenoviral vectors in vivo has been successfully achieved in both immunosuppressed and immune-competent animals [70, 71]. All immune-competent animals tested with BMP adenoviral vectors, to date, have demonstrated evidence of an immune response at the treatment site. The immune response appears to be both humoral and cellular, and is directed against both the injected adenoviral particles and cells transduced by the adenoviral vector. It remains unclear whether there is a major immune response directed against the foreign BMP (i.e. human BMP gene in animal models) expressed using gene therapy techniques. A variety of different BMP adenoviral vectors have been shown to induce bone formation in athymic nude rodents, including Ad-BMP-2, Ad-BMP-4, Ad-BMP-6, Ad-BMP-7 and Ad-BMP-9. In immune-competent rodents, Ad-BMP-6 and Ad-BMP-9 are able to overcome the host immune response and induce significant bone formation. Figure 1 demonstrates the mechanisms involved in osteogenesis using direct BMP gene therapy with an adenoviral vector.
In the paraspinal region, direct injection of adenoviral vectors, including Ad-BMP-2 and Ad-BMP-9, have been shown to induce spinal fusion in athymic nude rodents, without producing central or lateral stenosis caused by exuberant bone formation [71, 72]. The fusion mass completely integrates with the adjacent laminae and spinal processes, without evidence of pseudoarthrosis, suggesting that decortication of cortical bone is not required for bony fusion when BMPs are applied in the paraspinal region. Stereotactically injected BMP vectors into the paraspinal musculature is a compelling approach, since multiple, percutaneous injections could be performed to produce a fusion mass with a predetermined three-dimensional shape and in locations specific for each pathological process. Postprocedural pain would be minimal since the technique would not require muscular dissection. Patients requiring neural decompression could be taken to surgery following successful bony fusion, where extensive laminectomies, facetectomies, and foraminotomies could be performed without leading to spinal instability. Figure 2 demonstrates a posterolateral spinal fusion in the lumbar region of a rabbit using percutaneous, direct gene therapy of a BMP-6 adenoviral vector, demonstrating significant bone formation with excellent union with the host transverse processes.
Another approach that is currently being investigated by several research groups is ex vivo gene therapy, a technique in which osteogenic genes are inserted into cells in tissue culture, and the genetically altered cells are subsequently implanted into regions requiring bone formation in experimental animals . The cellular implants express and secrete bone morphogens, which in turn induce the osteogenic response. Lieberman et al. [74, 75] demonstrated that a murine bone marrow stromal cell line, which had been transduced with BMP-2 cDNA using an adenoviral vector, could induce both heterotopic and orthotopic bone formation in severe combined immune-deficient (SCID) mice.
Fig. 1. Diagram showing mechanisms of bone formation using direct, percutaneous injection of an adenoviral vector containing the BMP-9 gene. Expression of BMP-9 by the transduced cells leads to migration, proliferation, and differentiation of MSCs, ultimately leading to successful osteogenesis.
Fig. 2. Three-dimensional CT reconstruction of rabbit lumbar spine treated with bilateral, percutaneous injections of a BMP-6 adenoviral vector using a posterolateral approach, demonstrating significant bone induction and a successful transverse process spinal fusion. The data demonstrates that successful osteogenesis can be achieved in immune-competent rabbits using current gene therapy technologies.
In a similar study, Riew et al.  transduced marrow-derived MSCs with the BMP-2 gene and autologously reimplanted the genetically modified cells in the paraspinal region in rabbits. One of the five treated rabbits demonstrated as successful intertransverse process spinal fusion, which was assessed radiographi-cally and histologically. Although only 20% of the treated animals showed significant bone formation, this study clearly demonstrated the potential of ex vivo gene therapy techniques. This group subsequently demonstrated that the rate of bone formation could be increased to 100% in the treated region by implanting the cells 7 days following viral transfection .
Musgrave  transduced mesenchymal cell populations obtained from human muscle with the BMP-2 gene using an adenoviral vector. The transduced cells were implanted into SCID mice and demonstrated successful ectopic bone formation. Lee et al.  inserted the BMP-2 gene into muscle-derived mesenchymal cells in mice and implanted the cells into mouse critical-sized cranial defects. The transduced cells significantly increased the healing rate of defects compared to control cells. In addition, fluorescent in situ hybridization was utilized to demonstrate incorporation of the transduced cells into the induced bone. In another compelling study, Turgeman  isolated human MSCs from the bone marrow of normal patients, as well as patients suffering from osteoporosis. The cells were transduced with the BMP-2 gene and subsequently grafted into ectopic and orthotopic locations, leading to successful osteogenesis. Utilizing a retroviral BMP-2 vector, Laurencin et al.  demonstrated successful heterotopic bone induction by BMP-2-transduced cells delivered on a PLAGA-HA scaffold in a SCID mouse model. The expression of the human bone morphogenetic protein-7 gene by periosteal-derived rabbit mesenchymal cells has also been shown to induce bone and articular cartilage repair in a rabbit knee osteochondral defect model . In an elegant study, Moutsatsos et al.  genetically engineered a murine MSC line to secrete BMP-2 in a regulated fashion. The BMP-2 gene was under control of a doxycycline-responsive promoter, such that the presence of doxycycline in vitro and in vivo would downregulate BMP-2 expression. In vivo implantation of this cell line led to both orthotopic and ectopic bone formation in a regulated fashion, suggesting that long-term regulation of bone induction may be possible.
Our lab has recently demonstrated that human MSCs transduced with the BMP-9 gene can induce robust bone formation in athymic nude animals. In an ectopic model, the implanted cells survive long-term, as assessed by an antihuman mitochondrial stain, and contribute to the cellular composition of the ectopic bone. Local host stem cells at the injection site are also stimulated by the secreted BMP, and differentiate into chondrocytes and osteoblasts at the treatment site. The genetically modified cells were also capable of forming significant bone formation in the paraspinal region following percutaneous injection. The fusion mass integrated completely with the adjacent host spine, similar to the direct BMP adenoviral vector treatment sites, without evidence of posttreatment neural compression (unpubl. data).
In another set of interesting studies, Boden et al. [84, 85] have reported a novel ex vivo gene therapy technique which utilizes the insertion of the osteogenic LMP-1 gene into allogenic bone marrow cells. These investigators demonstrated significant bone formation by the transduced cells in the paraspinal region of rodents, in spite of relatively low transduction rates. LMP-1 gene therapy is unique in that it is thought to induce the secretion of a variety of osteogenic growth factors, which in turn stimulate bone formation.
These ex vivo techniques have the advantage of not only expressing osteogenic morphogens, but also supplying the treated region with bone precursor cells, which may be of limited supply at the treatment site. For example, it is unclear whether pluripotent stem cells are uniformly present throughout the body in the adult human, which could make direct BMP or BMP gene therapy treatments ineffective. Also, the number of MSCs may decrease with age, which might decrease the physiologic activity of BMPs. The introduction of stem cells, which are genetically modified to secrete bone morphogens, is, therefore, a compelling approach. The harvest and expansion of autologous stem cells for widespread human use may be hampered by high costs, cellular contamination, and other technical difficulties. Therefore, other approaches such as the genetic modification of traditional bone grafts (which contain cellular precursors such as stem cells and osteoblasts) at the time of surgery may be a more reasonable near-term approach.
Recent studies have clearly demonstrated the potential utility of MSCs and BMP gene therapy for the promotion of bone formation for spinal applications. However, the field of molecular spine surgery is certainly in its infancy. Many of the techniques are under continuing development and may require refinement prior to clinical application. The establishment of an allogenic or xeno-geneic source of stem cells, which could be modified to attenuate potential host immune responses, would have significant advantages compared to autologous cells for clinical application. Various groups are currently studying the use of genetically modified porcine cells, which are genetically altered to decrease their expression of foreign antigens. In addition, ex vivo gene therapy approaches are currently being developed to induce local immunosuppression around the transplanted cells. The efficacy of BMP gene therapy might also be improved
BMP I ce homodimer^ BMP I/II
+ heterodimers BMP II -,
BMP I ce homodimer^ BMP I/II
+ heterodimers BMP II -,
Fig. 3. Diagram demonstrating the local production of potent BMP heterodimers using combinational gene therapy techniques with two BMP adenoviral vectors. Two homodimeric and one heterodimeric species are produced, which may be more effective at promoting bone formation than treatment with a single BMP vector.
Osteoblast by achieving the expression of a cocktail of different growth factors at the treatment site. Several growth factors, including BMP-6 and TGF-^3, have been shown to have synergistic effects on stem cells in vitro. In addition, the generation of potent BMP heterodimers could be produced using a combination of vectors, which may have improved osteogenic effects compared to their respective BMP homodimers alone (fig. 3). Finally, the optimal gene delivery system for osteogenic genes has certainly not been firmly established for human use. Although viral vectors have clearly been the most effective approach in animal studies, their use in humans may be limited by their antigenicity. Therefore, the development of novel nonviral approaches which are able to achieve short-term, high-level transgene expression would be ideal for human use. It is anticipated that many of these issues will be addressed in the near future and that cutting edge tissue engineering technologies can be successfully applied to treat human spine pathology.
This work was supported by an NIH grant (1 RO1 AR/AI46488-01A2) and Medtronic-Sofamor Danek; we thank Mona Banton for her editorial support.
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