Biological Repair of Intervertebral Disc (2003)

Back pain is one of the major causes of disability and, thus, has a major socioeconomic impact. The lifetime prevalence of low back pain is 75% to 80% of the population. Approximately 24% of people over 18 years of age report back pain. In the population over 64 years of age, 28.8% report back pain, and in this older age group, 58% of those who report pain of any kind complain of back pain. 1 In the middle age population (45–64 years), 30.1% report back pain, and 88% of the total number of people who report pain complain of back pain. Importantly, in the younger population (18–44 years) the prevalence of back pain is extremely high (19.9%) and is the only major source of pain. The significant restrictions on activity caused by back pain lead to major economic losses, such as an annual average of 297 million restricted days and 87 million disability days. Intervertebral disc (IVD) disorder is a major contributor to back pain; the direct relationship between that and pain generation needs to be studied. Approximately 5.7 million persons are diagnosed with IVD disorders each year, with the highest rate (4.6/100 persons) in the group 45 to 64 years old. Therefore, in addition to the direct costs for treatment of IVD disorders, the indirect costs, such as loss of productivity, cannot be ignored. 1 Of the IVD disorders, disc herniation is one of the major causes of low back pain. Current conventional treatment includes medication, steroid injection, physical therapy, and surgery. Recent progress in minimally invasive operations for disc herniation, such as endoscopic discectomy and percutaneous discectomy, has benefited patients, but these procedures still damage the disc structure. This, in turn, may cause further degeneration that eventually leads to instability of the motion segment that requires interbody fusion to stabilize those segments. The progression of disc degeneration cannot be prevented by these minimally invasive operative procedures. Considering the high prevalence of back pain in the young population, treatments that strategize to repair or regenerate the IVD biologically appear promising as a future treatment option. The IVD has a unique structure, composed of a tough outer ring (anulus fibrosus [AF]) and a gelatinous inner core (nucleus pulposus [NP]). The AF and NP, along with the endplates of the vertebrae above and below, provide the properties of flexibility and resiliency necessary to withstand mechanical loading. Morphologically, the AF consists of concentric lamellas rich in collagen fibers, whereas the NP is an inner gelatinous cushion rich in proteoglycans (PGs). 2 With increasing age, the NP is transformed from a turgid gel of PG into a more desiccated fibrocartilaginous structure that appears similar to the inner anulus. 3 Although these early biochemical and morphologic changes typically occur without a concomitant abnormality of the disc structure, recent progress in MRI techniques has resulted in the successful detection of radial tears of the AF in 21% to 35% of the IVDs in cadavers of people over the age of 40. 4 Those morphologic changes are thought to be associated with changes in biochemical properties, which are maintained by the proper balance between the synthesis of PGs and collagen by NP and AF cells and their degradation in the extracellular matrix (ECM). If we are able to understand the exact mechanism of the pathology of IVD disorders, the biologic repair or regeneration of the IVD can be, theoretically, achieved by the stimulation of synthesis of matrix molecules and/or the prevention of matrix degradation or cell death. In addition, the nutrition of the IVD is another important factor to be considered for biologic repair because changes in the nutrition of the IVD caused by sclerotic changes in the endplate or vascular changes might be occurring at advanced stages of IVD disorders. Recent advances in recombinant protein techniques, resulting in the relative mass production of growth factors, have enabled us to study the effect of growth factors on IVD cells and then to manipulate metabolic conditions of those cells to achieve repair or regeneration. In this article, we summarize recent progress in the use of growth factors and in tissue engineering of IVD tissues for biologic repair and regeneration of the IVD. Methods Strategy for Biologic Repair and Regeneration. Depending on the grade of degeneration of the IVD, different strategies for therapeutic approaches can be employed. The condition of the IVD structure and its matrix, as well as the viability and activity of IVD cells, are important factors to be considered. When nutrition through the endplate is impaired by sclerotic changes, the cells in the NP and the AF will be unable to properly maintain metabolic homeostasis. 5,6 If the stability of the motion segment is already compromised, biologic stimulation for repair cannot be achieved. Therefore, at each stage of IVD degeneration, a different treatment strategy should be considered. Treatment at the Early Stage. Metabolically impaired cells in an IVD exhibiting early degenerative changes or age-related changes might possibly repair their own matrix and the disc structure when exposed to growth factors. The growth factor protein injection, either as a single or repeated injection, will induce regeneration of an IVD exhibiting early stage degeneration. Another drug delivery system might be needed for prolonged exposure to growth factors. Our previous work demonstrated a stimulatory effect by rhOP-1 on matrix synthesis and accumulation (i.e., PG, collagen, and HA) by IVD cells in a dose-dependent manner. 7 This result was confirmed in an in vivo experiment using an injection of OP-1 into a rabbit IVD. In the normal rabbit, the intradiscal administration of OP-1 in vivo resulted in increases in the disc height and in the PG content of the NP when compared with intradiscal saline injections. Treatment at the Intermediate Stage. In the case of a degenerated disc, prolonged exposure to growth factors may indeed be needed. To achieve this prolonged exposure, cells or tissues that carry specific genes for a growth factor can be introduced to serve as a source for that growth factor. In our feasibility study, we showed the effectiveness of a novel gene gun-mediated gene transfer method to rabbit IVD cells in vitro using a reporter gene. 8 This nonviral transfection method is suitable for use with a nonlife-threatening disease, such as disc degeneration. Our in vitro study showed that transgene expression was sustained for 3 weeks without significant alterations in the metabolic activity of the cells. We have also shown that the transgene expression of the OP-1 gene resulted in the stimulation of matrix synthesis in bovine IVD cells. 9 These findings suggest that this approach has great promise as a form of gene therapy when the goal is to use growth factors to promote regeneration of disc tissues. Severe Stage Treatment. Even if the gene therapy approach is applied, the biologic results will depend on the metabolic status of cells in the disc. The cells in the severely degenerated disc may have an impaired metabolic function and fail to respond to any biologic stimulation. In this case, the supplementation of healthy cells or gene-manipulated cells into the IVD after microdiscectomy or even percutaneous procedures such as percutaneous discectomy can be considered. However, this procedure may be problematic because the “supplemented” cells may move out through the anulus defect generated by the procedure itself. Thus, in order to replenish the matrix in the IVD after those surgical procedures, tissue-engineering techniques may be utilized to produce, in vitro, a functional tissue for transplantation into the defect. There is only a little information in the literature on tissue-engineered IVDs. Recently, we have developed a novel two-step culture method, the ARC method, for the production of cartilaginous tissue in vitro, including IVD tissue, which does not require exogenous matrices. 10,11 This technique is utilized to form a cohesive tissue for transplantation. We hypothesize that the gene therapy approach can be used in combination with the tissue-engineering approach to achieve the best therapeutic result. Results Effect of Growth Factors on IVD Cells In Vitro The use of growth factors, such as transforming growth factor-β (TGF–Tβ), in IVD tissue was first pioneered by Thompson et al, 12 who studied the effects of growth factors on the metabolism of the ECM by dog IVD cells. The rate of PG synthesis by IVD cells in vitro increases several-fold on the addition of TGF–Tβ and epidermal growth factor. 12 Insulin-like growth factor-1 is also reported to stimulate proliferation and matrix synthesis in vitro. 13 Gruber et al 14 also confirmed the positive effect of TGF–Tβ in human IVD cells in a three-dimensional culture, such as agarose or alginate culture. Recently, similar results were shown in pellet culture. 15 Osteogenic protein-1 is a member of the transforming growth factor-β (TGF–Tβ) superfamily that exerts potent effects on chondrocyte and osteocyte differentiation and metabolism. 16 Recombinant human OP-1 (rhOP-1) has been shown to enhance the synthesis of PG and type II collagen by bovine chondrocytes. 17 In another study, the effects of rhOP-1 on mature human articular chondrocytes cultured in alginate beads were also examined. 18 Osteogenic protein-1 stimulated the assembly of the cell-associated matrix (CM) by normal human articular chondrocytes as well as the up-regulation of hyaluronan synthase, CD44, and aggrecan. 19,20 Having observed that rhOP-1 is a potent stimulator of PG and collagen synthesis, we have conducted a series of studies to prove the usefulness of OP-1 to serve as a model of growth factor treatment. Effect of OP-1 on Matrix Metabolism of IVD Cells In Vitro Recombinant human OP-1 was effective in promoting matrix synthesis and matrix formation by rabbit NP and AF cells cultured in alginate beads. 7 The effects of culturing the cells in the presence of fetal bovine serum (10%) and various concentrations of rhOP-1 were assessed by measuring changes in cell proliferation, PG and collagen synthesis and mRNA expression, and in the matrix contents of PG and collagen, as indicators of matrix accumulation. At high concentrations, rhOP-1 had a moderate mitogenic effect on both NP and AF cells. rhOP-1 also stimulated the synthesis of PG and collagen in a dose-dependent manner, with corresponding increases in the expression of mRNA for aggrecan core protein and collagen type II. The stimulation of PG synthesis was more apparent than that of collagen synthesis. In addition, continuous treatment with rhOP-1 led to an increase in the total PG and collagen contents in both NP and AF cultures. The results provided evidence of the ability of OP-1 to stimulate the metabolism of both NP and AF cells. Our studies also showed that OP-1 was effective in promoting matrix re-establishment after depletion of the matrix by IL-1, which is a major catabolic regulator. 21 Alginate beads containing rabbit NP and AF cells were first treated by interleukin-1α to deplete matrices and were then cultured further with or without OP-1 in the presence of 10% fetal bovine serum. The initial treatment with interleukin-1α resulted in a significant decrease in PG and collagen contents in the matrix formed by both NP and AF cells. However, subsequent treatment with OP-1 led, in both cases, to the rapid recovery of the matrix content of PGs and collagens, whose levels returned to those seen in cells not previously exposed to IL-1α. By the end of the culture period (day 21), those values reached levels higher than those found in beads containing cells never exposed to IL-1α. The study on the rate of synthesis essentially showed the same results. Chondroitinase ABC (C-ABC) is commonly used to degrade the glycosaminoglycan chains, primarily chondroitin sulfate, found in PGs, to unsaturated disaccharides. C-ABC has been suggested as an alternative agent for chemonucleolysis. 22–24 Although the regeneration of the cartilaginous matrix after treatment with C-ABC occurs earlier and to a greater extent than it does with chymopapain, disc height and PG content do not reflect full biomechanical recovery. 25 We have tested the hypothesis that repair after chemonucleolysis by C-ABC can be markedly stimulated by exposing the cells to a low dose of OP-1 in vitro26 (Figure 1). Alginate beads containing rabbit NP and AF cells were first treated with C-ABC, and after several washes, the cultures were maintained in media containing 10% FBS and OP-1. As we have seen in the IL-1 treatment experiment described above, OP-1 was very effective in promoting in vitro the repair of the C-ABC–treated ECM around NP and AF cells. These results suggest that rhOP-1 could prove most useful as a therapeutic agent in promoting repair of the matrix of degenerated human IVD or in promoting matrix repair in conjunction with chemonucleolysis. Figure 1: Effect of OP-1 on the recovery of PG content after C-ABC–induced PG depletion. The alginate beads encapsulating either anulus fibrosus (AF) or nucleus pulposus (NP) cells were cultured for 14 days to re-establish the matrix around the cells. The control treatment consisted of beads cultured for an additional 12 days in complete medium (W/O C-ABC, W/O OP-1). The beads in the other two treatment groups were first cultured for 2 hours in the presence of the enzyme chondroitinase-ABC [C-ABC (0.1 U/mL)] to deplete PGs from the matrix, followed by 3 × 30 minutes washes before culturing the beads for the next 12 days in complete medium either in the presence or absence of osteogenic protein-1 (OP-1) at 200 ng/mL (C-ABC with C-ABC W/O At various and 12 the PG content in the beads was using the method after further the effect of OP-1 on matrix metabolism more we have a method that cultures a disc with endplates and the effects of an intradiscal OP-1 The IVD with endplate was and cultured in media containing 10% or OP-1 was into the NP in vitro and cultured for an additional 14 days. The NP and AF were then The injection of OP-1 resulted in a higher of PGs in the NP when compared with the injection of The PG content of the AF of the OP-1 group was also higher than that in the saline group on 7 and However, after 14 days of culture the PG content in NP and AF of the OP-1 group was not different from the control that OP-1 to the of metabolism. Those in vitro results suggested that the use of OP-1 might to the repair of matrix degeneration in IVD or of changes chemonucleolysis. Effect of OP-1 on Matrix Metabolism of IVD Cells In To on our in vitro the effects on the metabolism of the IVD by in vivo OP-1 into a rabbit IVD were studied. Osteogenic protein-1 or saline was into each mature rabbit IVD, and changes in disc height disc height and biochemical in the NP were at to 8 weeks after to 8 weeks after injection, the of was higher in the OP-1 group than in the saline group (Figure the PG content in the NP of the OP-1 group was higher than that of the saline group at the The effects of OP-1 in vivo were also confirmed in the IVD after chemonucleolysis with earlier and complete recovery of disc height was observed after the injection of the of OP-1 in the rabbit IVD Figure Intervertebral disc height after OP-1 With the rabbit were exposed through a approach as previously 4 and osteogenic protein-1 (OP-1) at 2 in of saline or saline only was into the of the nucleus pulposus (NP) of IVDs At and 8 weeks after the injection, of the were with the By 2 weeks after the injection, the of in the OP-1 group was higher than that in the saline group This significant in was maintained at 4 and 8 weeks in vivo and in vitro results suggest the of use of growth factors, such as to promote the regeneration of IVD tissue, at earlier stages of degeneration. the degenerated IVD does not healthy cells, those studies of impaired cells or cells can to growth factors or or not those cells a prolonged exposure to growth factors. Current studies are to the effect of OP-1 injection in a disc the therapy an alternative to a specific gene for a growth factor into genes and induce the production of those growth factors by the at the of regeneration. studies using an have reported that IVD cells could be by gene To achieve gene transfection for IVD we have a nonviral a gene transfer using as gun-mediated gene gene transfection using the gene system is thought to be than gene gene transfer using a gene is a nonviral alternative for transfection that showed great promise in the early This was first developed by and led to the production of of major has also been for use on cells for of different reporter and therapeutic genes have been using this we have reported the effectiveness of gene gun-mediated transfection of rabbit AF and NP cells in vitro using with the gene. 8 We also studied transfection of the gene for the growth factor OP-1 by this technique the metabolism of bovine IVD disc cells in vitro. fibrosus and NP cells were with a human OP-1 expression and PG synthesis was Cells with the OP-1 gene PGs at a rate in both AF of and NP of groups 9 (Figure gene of demonstrated a of for the NP cells and for the AF cells. The gene system is a method of gene transfer when properly tested and has the to an effective of gene therapy using growth factors for tissue, even the is than that of it can be that cells as well as cells are both and those that were not can still serve as cells in the Figure Effect of OP-1 gene transfer on PG synthesis by bovine articular and NP cells. To study gene transfection can the metabolism of bovine disc cells (anulus fibrosus and nucleus pulposus and bovine articular cells, these cells were in vitro, with the human osteogenic protein-1 using a nonviral gene gun-mediated 3 after the rate of synthesis was higher in cell and NP, in the OP-1 cells compared with the control cells. Cells from the NP were more than either AF cells or cells to the transfection of the OP-1 of the IVD To little thought has been to the of biologic repair by tissue-engineered IVD tissues. This tissue-engineering approach to disc because it the for of disc function that can possibly or and degeneration of at other Considering the of biomechanical in IVD tissues and the of culturing IVD cells with the proper only a that to IVD disc tissue in vitro have been whereas the same approach in articular repair has in the the of the treatment of is a initial because method of of the NP is to be successful in the of a AF repair is a approach to the treatment of the of disc will In an advanced with an without herniation, repair of the AF using AF tissue, followed by an treatment such as an injection of growth may be disc disease, in the presence of herniation of the NP, repair of the AF and NP with AF and NP tissue may be treatment be a to the over that are in the In the if a disc, as a structure can be which will be very to regeneration of the IVD might be However, at the of biologic repair for very advanced stage disc degeneration is and therapeutic approaches such as interbody fusion or a be the of Recently, we have developed a novel two-step culture the method, that the in vitro formation of tissue by mature bovine chondrocytes without the of a is that this method does not require the use of exogenous matrices. The first consists of culturing chondrocytes conditions that maintain their and their as assessed by type II collagen and aggrecan This was by culturing the chondrocytes in alginate beads the cells had a cell-associated matrix The consists of culturing the cells with their after recovery from the on a tissue culture with a The ARC method has several over other approaches of engineering tissues. The mechanical properties, and the of tissues are by the and of their The expression of the cells is necessary for the production of ECM with the and The is by the physical and of cells the The ARC method of culture, by the proper and proper expression and matrix By two the ARC method and the use of OP-1 7 in culture, we have demonstrated the feasibility of these approaches to form in vitro a cohesive IVD tissue for transplantation in Intervertebral disc cells from the of to bovine were using the ARC method, in the presence of FBS and OP-1 to form in vitro. 2 tissues from NP and AF cells had a structure and were to from the (Figure the between these two tissues the of biochemical of the source tissues. the collagen content was higher and the of PG to collagen was in the AF than in the NP tissue, with the that AF cells form a more tissue than do NP cells in vitro. The mechanical properties of the AF tissue were than that of the NP tissue, also with the observed in mechanical properties of the tissues using AF and NP cells. Our have also shown that the mechanical of the tissues with prolonged culture Figure method and tissue-engineered IVD (IVD) cells were from the of to The ARC method was used to form tissue in vitro. IVD cells (anulus fibrosus and nucleus pulposus were cultured in alginate gel for days. The cells and their associated matrix were and a tissue culture in the presence of fetal bovine serum and osteogenic protein-1 at 200 2 the tissues from AF and NP cells showed the in biochemical and mechanical the of tissue formation with growth factors might be for formation of a because the activity to form the matrix is compared with articular chondrocytes. The combination of two therapeutic gene therapy and tissue may provide to patients, those who have degeneration of the IVD. We have described our approach to the biologic repair and regeneration of the IVD and results. Although the in IVD degeneration might not be as as the conditions which we those the results of each experiment a promise for the biologic treatment of IVD is that in the the use of growth factors, cells, or a combination of these will be therapeutic in IVD degeneration. Another is that the mechanism of pain generation in disc disorder and its relationship to disc degeneration is not well The combination of the treatments for pain and biologic for the of C-ABC and will treatment. is needed to further the effects of these therapeutic in different and eventually in The for in the of the