The biological treatment of disc degeneration includes many different therapeutic approaches. Broadly speaking, therapeutic approaches include cellular, matrix, and molecular applications, and this chapter focuses on molecular therapies. Therapeutic molecules encompass the full range of therapeutic molecules rather than just the classical “growth factors”. This nomenclature is important because growth factors are named for their effect on cell mitosis, but the therapeutic effect of effective therapeutic molecules is not always achieved by affecting cell replication. Therapeutic molecules can be classified as antimetabolites, pro-apoptotic factors, chondroformers, and intracellular regulatory molecules. This chapter examines the primary literature and defines each of these classifications. Understanding the process of disc degeneration is helpful in understanding molecular therapy. The process of disc degeneration includes the loss of proteoglycans, water, and type II collagen in the medullary matrix. Some of the changes in the matrix that are uncertain, such as polymeric proteoglycans and others, are difficult to quantify. Changes in the fibrous rings include disruption of the annular laminae and defects in the collagen matrix. Generally, these matrix changes take many years and are mainly caused by an imbalance between synthesis and catabolism. The goal of molecular therapy is to stop the extracellular matrix changes in the disc by balancing synthesis and catabolism. The following are specific classifications. I. Antimetabolic effects Matrix loss is a balance between synthesis and catabolism, so the extracellular matrix of the disc can be increased by increasing synthesis or decreasing catabolism. One approach is to stop matrix loss by inhibiting catabolic enzyme activity. There are many catabolic enzymes in the extracellular matrix, of which matrix metalloproteinases (MMPs) are the most important class, which play an important role in the recycling of extracellular matrix molecules and disc degeneration. This hypothesis is supported by the significantly higher levels of MMPs in degenerated discs. In the extracellular matrix, inhibitors of MMPs are mainly tissue inhibitors of matrix metalloproteinases (TIMPs), and Wallach tested whether these anticatabolic molecules (TIMP-1) could increase the amount of proteoglycans in the matrix by an in vitro adenoviral gene transduction pathway. He found that expression of TIMP-1 in intervertebral disc cells did increase the amount of proteoglycans, while the rate of proteoglycan synthesis was also increased as measured. Another potent molecule is CPA-926, a precursor of hesperidin but with better pharmacokinetic properties.CPA-926 has anti-inflammatory and anti-tumor properties and stops cartilage degeneration in osteoarthritis.Okuma demonstrated that CPA-926 can stop or delay the onset of high disc degeneration by giving it orally to rabbits with disc degeneration and found histological evidence. The metabolic rate of the matrix is also important in the context of the balance between synthesis and catabolism. For example, disc metabolic rates are significantly higher in younger than in older adults, which can also cause changes in matrix composition, such as degenerated aggregated proteoglycans and newly synthesized aggregated proteoglycans (from cartilage). The total metabolic rate can also affect the nutritional requirements of the disc cells. Cytokines, such as IL-1 and TNF-a, both have important roles in disc metabolism. However, some molecules, such as IL-1Ra and infliximab, can block the effects of IL-1 and TNF-a, respectively, and are also useful therapeutically. Further studies on antimetabolic molecules may lead to more discoveries. Second, pro-apoptotic factors Pro-apoptotic factors are a class of molecules that increase the mitotic rate of cells, and they make up the true growth factors, so we can distinguish pro-apoptotic growth factors from chondrogenic molecules, which include insulin-like growth factor (IGF-1), epidermal growth factor (EGF), and fibroblast growth factor (FGF). Thompson demonstrated in vitro assays on adult dog intervertebral disc cells that pro-divisive growth factors increase the mitotic rate and proteoglycan synthesis rate of cells, and in general, EGF has higher potency. IGF-1 levels in rat intervertebral discs decreased with age, so the investigators concluded that matrix synthesis could be increased by increasing IGF-1 in older discs. the results of Walsh’s in vivo assays with growth factors in a degenerative model of caudal disc compression in rats were consistent with Thompson’s in vitro assays, both demonstrating a slight utility of IGF-1, while FGF had essentially no effect. Other growth factors may also prevent disc cell apoptosis by the same or different mechanisms. gruber demonstrated that disc cells are apoptotic in a low serum environment in vitro, but the addition of IGF-1 or PDGF reduced the rate of apoptosis. igf-1 has a pro-synthetic effect, but also a pro-catabolic effect. In tissue culture assays, IGF-1 decreased the level of TIMP-2, so it is said to have a dual effect on intervertebral disc matrix metabolism III. Chondrocytoplasmin Chondrocytoplasmin is a class of cytokines that have pro-apoptotic ability but are primarily characterized by an increase in chondrocyte-specific phenotypes. These specific chondrocyte properties are determined by type II collagen, the Sox9 gene, aggregated proteoglycans, and sulfated aminoglucan. Most of the studies on chondrocyte-forming elements have been on transforming growth factors (TGF-b), osteogenic proteins (BMPs), and growth differentiation factors (GDFs). Chondrocytoplasins have attracted attention because of their ability to reverse the fibrous phenotype of normal intervertebral disc cells to a chondrocyte phenotype. These molecules act in an autocrine, paracrine, and endocrine manner, and they are able to exert great effects by diffusing and acting on different cells. The activity of the prototypical chondrocyte formers is determined by the extracellular surface receptors and active intracellular messaging systems of the cells involved, whereas intervertebral disc cells are able to express TGF, BMP molecules and receptors, and the level of expression changes with the aging of the disc. TGF-b1 was the first disc-forming molecule to be identified, and Thompson reported that TGF-b1 is not only a mitogen but also a pro-anabolic molecule that increases cellular proteoglycan synthesis, and its ability to increase proteoglycan synthesis is superior to that of growth factors such as EGF, IGF-1, PDGF, and FGF. Later, Nishida demonstrated that an adenoviral vector carrying the TGF-b1 gene could be injected directly into the intervertebral discs of immune rabbits and could increase proteoglycan synthesis by expressing TGF-b1. In vitro assays with human degenerated disc cells also demonstrated that TGF-b1 increased the rate of proteoglycan and collagen synthesis, suggesting that degenerated disc cells remain sensitive to TGF-b1. In vitro assays on rat caudal disc cells demonstrated a proliferative effect of TGF-b1 on inner fibro-ring cells, but it did not significantly alter the height of the disc. Although TGF-b1 has this effect, its role in human living intervertebral discs remains unproven. Hutton reported that recombinant human BMP-2 increased proteoglycan synthesis in rat disc cells and significantly increased the chondrocyte phenotype of disc cells, as well as increased gene expression of aggregated proteoglycans and type II collagen, but no significant changes in type I collagen.Kim reported that BMP-2 partially reversed the effects of Kim reported that BMP-2 partially reversed the inhibitory effect of nicotine on intervertebral disc cell proteoglycan synthesis, as we all know that BMP-2 promotes the terminal differentiation of osteoblasts during bone formation, but it remains to be confirmed whether BMP-2 also promotes the differentiation of intervertebral disc cells. In vitro tests on human intervertebral disc cells demonstrated that BMP-2 increased chondrocyte gene expression but had no effect on osteoblast genes. The pro-anabolic effect of BMP-2 was further demonstrated by the selective application of gene therapy to intervertebral disc cells in vitro during spinal surgery. To date, there are still no studies on the effects of BMP-2 on human living degenerated discs, which is a hot topic of research. BMP-7 (also known as osteogenic protein OP-1) is also an intervertebral disc cytoformer, and Masuda reported a dose-dependent relationship between the growth of intervertebral disc cells of rabbit annulus fibrosus and nucleus pulposus origin and BMP-7 in vitro. Takegami demonstrated that growth of rat disc cells in an alginate environment with the addition of the inflammatory factor IL-1 resulted in a loss of proteoglycan and collagen in the alginate, whereas the proteoglycan and collagen in the medium after the addition of 200 mg/ml BMP-7 was even higher than in the absence of IL-1. Zhang reported increased proliferation of all three fractions of bovine intervertebral disc cells (from the outer zone of the annulus fibrosus, inner zone, and nucleus pulposus, respectively) cultured in vitro in response to BMP-7. However, only cells from the outer zone of the annulus fibrosus and the nucleus pulposus showed an increased rate of proteoglycan synthesis. Trials of BMP-7 application in rabbit disc degeneration models have been initiated, and these trials have demonstrated that direct intradiscal injection of BMP-7 increases disc height and proteoglycan levels in rabbits. Data from these trials also suggest that intradiscal injections of BMP-7 are also effective in degenerating discs in rats. BMP-13 is also known as growth differentiation factor (GDF-6) or cartilage-derived morphogenetic protein-2 (CDMP-2). Although BMP-13 belongs to the BMP family, its amino acid sequence is only 50% homologous to BMP-2. Chondrocyte cell line assays have demonstrated that BMP-13 does increase proteoglycan synthesis and chondrocyte phenotype, but it is much less potent than BMP-2. Although BMP-13 does not synergize with BMP-2 in proteoglycan synthesis and chondrocyte gene expression, the effects of the two together are cumulative. BMP-13 is not a recombinant protein that can be produced in large quantities, thus studies that require large amounts of BMP-13 are difficult to complete. GDF-5, also known as CDMP-1, is concentrated in the anterior cartilaginous mesenchyme during embryogenesis and is found throughout the cartilaginous core of developing long bones.Walsh compared the effects of GDF-5, TGF-b1, IGF-1, and FGF on a rat model of caudal disc degeneration, and GDF-5 was the only molecule that increased disc height compared to the saline control. In addition, the proliferation of cells in the medial zone of the fibrous annulus and the migratory zone was seen on tissue sections. However, repeated injections were prone to an inflammatory response, which the authors attribute to injection injury rather than GDF-5, as the same inflammatory response was seen even in the saline control group.Wang used gene therapy to demonstrate that adenoviral transduction of GDF-5 promotes the growth of cultured disc cells in vitro.GDF-5 is being developed commercially for spinal fusion, however, there is still a need for a large number of recombinant proteins for clinical trial studies. Link N is an amino-terminal fragment of the linker protein, which Mwale demonstrated to have stimulatory activity on intervertebral disc cells. pellet culture assays demonstrated that Link N increased proteoglycan synthesis but not cell number, with a 113% increase in type II collagen in nucleus pulposus-derived cells and a 25% increase in annulus fibrosus-derived cells. The mechanism by which Link N induces specific upregulation of an important marker of chondrocytes (type II collagen) but does not increase cell numbers remains unclear. However, these findings already suggest that Link N is also a pro-chondrogenic formulator. Intracellular regulatory molecules are a special class of molecules that do not act through transmembrane receptors. It is neither a cytokine nor a growth factor, but they have the same role as those secreted molecules discussed earlier. These molecules generally control one or more directions of cell differentiation. For example, SMADs are a class of intracellular molecules that regulate BMP-R. Although there is still nothing written about the role of SMADs on intervertebral disc cells, it is hypothesized that it can also have the same effect on intervertebral disc cells as BMP-2 in increasing proteoglycan and type II collagen synthesis. Paul demonstrated that adenoviral transduction of the Sox9 gene increased Sox9 expression and type II collagen synthesis in intervertebral disc cells in vitro. When injected into the body, adenovirus carrying Sox9 prevented degenerative changes in a rabbit disc fibrous annulus model. The degenerative disc model was used with a 27-gauge puncture needle, which caused minimal damage to the disc thus producing minimal degeneration. The most significant finding of this in vivo trial was that Sox-9-treated discs had a more chondrocyte-like phenotype compared to controls; however, this report does not list quantitative data on its effect on the disc matrix or the composition of the in vivo trial. Mineralizing protein (LMP-1) is a class of intracellular molecules identified for its role in promoting bone formation and osteoblast differentiation. yoon found that in intervertebral disc cells, LMP-1 upregulated the production of BMP-2, BMP-7, and proteoglycans not only in monolayer cultures but also in prolonged (3-week) alginate culture assays. Yoon demonstrated that the mechanism of action of LMP-1 is BMP-dependent. Subsequent in vivo experiments in rabbit intervertebral discs demonstrated that small doses of adenovirus transducing LMP-1 increased mRNA levels of BMP-2, BMP-7, and proteoglycans in intervertebral disc tissue. Because LMP-1 stimulates the formation of heterodimers of BMP-2 and BMP-7, its effect is 20-fold greater than that of homodimers. It is hypothesized that the reduction of the risk of adenoviral gene therapy can be achieved by reducing the dose of adenovirus under the effect of induced super BMP. V. Summary The molecules used for disc degeneration are no longer just classical growth factors, but at least four classes of molecules with a restorative effect on the disc. There are antimetabolites, pro-apoptotic factors, chondrocyte-derived formagens, and intracellular regulatory molecules. Although there are only some in vitro test data and few results from in vivo disc degeneration model tests in animals, the current screening tests are coming to an end and, in the near future, studies in animal disc degeneration models will start before those in humans.