Effects of cyclic dynamic tensile strain on previously compressed inner annulus fibrosus and nucleus pulposus cells of human intervertebral disc—an in vitro study

Our objective was to investigate whether dynamic tensile strain on previously compressed human intervertebral disc (IVD) cells can restore the biosynthetic effects of collagen and glycosaminoglycan. Inner annulus fibrosus (AF) and nucleus pulposus (NP) tissues of adolescent idiopathic scoliosis cases undergoing thoracoscopic discectomy and fusion were cultured on compressive plates. Compressive stress was applied using 0.4 MPa at 1 Hz, for 2 h twice a day for 7 days, to the inner AF and NP tissues, followed by equibiaxial cyclic tensile strain to deform the released cells onto the plate's flexible bottom. With 10% elongation at a rate of 1 Hz, for 2 h twice a day for 7 days, a significant increase in the level of collagen and glycosaminoglycan of the previously compressed inner AF, as well as the level of glycosaminoglycan of the previously compressed NP cells were found. The DNA content and number of endoplasmic reticulum under transmission electron micrograph of the previously compressed inner AF and NP cell were also significantly increased. The results suggested that equibiaxial cyclic tensile strain at a rate of 1 Hz with 10% tensile strain was capable of increasing collagen and glycosaminoglycan synthesis of previously compressed inner AF cells, and glycosaminoglycan synthesis of previously compressed NP cells. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:503–509, 2010     

Intervertebral disc and stem cells cocultured in biomimetic extracellular matrix stimulated by cyclic compression in perfusion bioreactor

Background contextIntervertebral disc (IVD) degeneration often causes back pain. Current treatments for disc degeneration, including both surgical and nonsurgical approaches, tend to compromise the disc movement and cannot fully restore functions of the IVD. Instead, cell-based IVD tissue engineering seems promising as an ultimate therapy for IVD degeneration.PurposeTo tissue-engineer an IVD ex vivo as a biological substitute to replace degenerative IVD.Study designAn extracellular matrix (ECM) structure-mimetic scaffold, cocultured human IVD cells and human mesenchymal stem cells (hMSCs), and mechanical stimulation were used to biofabricate a tissue-engineered IVD.MethodsAn optimal ratio of human annulus fibrosus (hAF) cells to hMSCs for AF generation within aligned nanofibers, and that of human nucleus pulposus (hNP) cells to hMSCs for NP generation within hydrogels were first determined after comparing different coculture ratios of hAF or hNP cells to hMSCs. Nanofibrous strips seeded with cocultured hAF cells/hMSCs were constructed into multilayer concentric rings, enclosing an inner core of hydrogel seeded with hNP cells/hMSCs. A piece of nonwoven nanofibrous mat seeded with hMSC-derived osteoblasts was assembled on the top of the cellular nanofiber/hydrogel assembly, as an interface layer between the cartilagenous end plate and vertebral body. The final assembled construct was then maintained in an osteochondral cocktail medium and stimulated with compressive loading to further enhance the hAF and hNP cells differentiation and increase the IVD ECM production.ResultsAmong all cocultured groups, hAF cells and hMSCs in the ratio of 2:1 cultured in nanofibers showed the closest mRNA expression levels of AF-related markers to positive control hAF cells, whereas hNP cells and hMSCs in the ratio of 1:2 cultured in hydrogels showed the closest expression levels of NP-related markers to positive control hNP cells. The effects of compressive loading on chondrogenesis of hAF or hNP cell and hMSC coculture were dependent on the scaffold structure; the expression of cartilage-related markers in AF nanofibers was downregulated, whereas that in NP hydrogel was upregulated. Interestingly, we found that hMSC-derived osteogenic cells in the interface layer were turned into chondrogenic lineage cells, with decreased expression of osteogenic markers and increased expression of chondrogenic markers.ConclusionsWe demonstrate a unique approach using a biomimetic scaffold, IVD and stem cell coculture, and mechanical stimulation to tissue-engineer a biological IVD substitute. The results show that our approach provides both favorable physical and chemical cues through cell-matrix and cell-cell interactions and mechanobiological induction to enhance IVD generation ex vivo. Our findings may lead to viable tissue engineering applications of generating a functional biological IVD for the treatment of disc degeneration.     

Effect of circumferential constraint on nucleus pulposus tissue in vitro

Background contextDegeneration of the intervertebral disc (IVD) involves structural changes in the annulus fibrosus (AF), which could alter the mechanical forces imposed on the nucleus pulposus (NP) tissue. This could contribute to degenerative changes that occur in the NP.PurposeThe purpose of the study was to determine whether circumferential constraint affects anabolic and catabolic gene expression, biochemical composition, and mechanical properties of NP tissue.Study designNucleus pulposus cells were isolated from bovine caudal IVD and allowed to form tissue for a period of two weeks. The effect of no, intermediate, or high circumferential constraint on biochemical composition (cellularity and proteoglycan and collagen synthesis), gene expression, and compressive mechanical properties was evaluated.ResultsIncreasing the rigidity of circumferential constraint surrounding in vitro formed NP tissue resulted in decreased gene expression of aggrecan and type II collagen and increased expression of MMP-1 and ADAMTS-5. This was associated with decreased accumulation of extracellular matrix and a deterioration of the compressive mechanical properties of the tissue.ConclusionsAs increased circumferential constraint can have a significant negative effect on the composition and quality of NP tissue and this raises the possibility that the AF may contribute to the degenerative or age-related alterations that occur in the NP. Further study in a functional spinal unit is required to validate this.     

Role of SHOX2 in the development of intervertebral disc degeneration

Intervertebral disc (IVD) degeneration is the most common cause of low back pain, which affect 80% of the population during their lives, with heavy economic burden. Many factors have been demonstrated to participate in IVD degeneration. In this study, we investigated the role of short stature homeobox 2 (SHOX2) in the development of IVD degeneration. First, we detected the expression of SHOX2 in different stages of human IVD degeneration; then explored the role of SHOX2 on nucleus pulposus (NP) cells proliferation and apoptosis, finally we evaluated the effect of SHOX2 on the production of extracellular matrix in NP cells. Results showed that the expression of SHOX2 is mainly in NP compared with AF tissues, its expression decreased with the severity of human IVD degeneration. TNF‐α treatment led to dose‐ and time‐dependent decrease in SHOX2 mRNA, protein expression and promoter activity in NP cells. The silencing of SHOX2 inhibited NP cells proliferation and induced NP cells apoptosis. Finally, SHOX2 silencing led to decreased aggrecan and collagen II expression, along with increased ECM degrading enzymes MMP3 and ADAMTS‐5 in NP cells. In summary, our results indicated that SHOX2 plays an important role in the process of IVD degeneration, and might be a protective factor for IVD degeneration. Further studies are required to confirm its exact role, and clarify the mechanism. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1047–1057, 2017.

     

A small-molecule inhibitor of the Wnt pathway,lorecivivint (SM04690), as a potential disease-modifying agent for the treatment of degenerative disc disease

BACKGROUND CONTEXTAbnormal Wnt signaling in intervertebral discs (IVDs) progresses degenerative disc disease (DDD) pathogenesis by impairing nucleus pulposus cell function, decreasing matrix deposition, and accelerating fibrosis.PURPOSEThis study was conducted to evaluate the effects of lorecivivint (LOR; SM04690), a small-molecule Wnt pathway inhibitor, on IVD cells and in an animal model of DDD.STUDY DESIGNWe used in vitro assays and a rat model of DDD to test the effects of LOR on nucleus pulposus cell senescence and viability, annulus fibrosus (AF) cell fibrosis, and cartilage regeneration and protection.METHODSWnt pathway gene expression was measured in human NP and AF cell cultures treated with LOR or DMSO (vehicle). Chondrocyte-like differentiation of rat and human NP cells, NP cell senescence and protection, and AF cell fibrosis were assessed using gene expression and immunocytochemistry. Disc and plasma pharmacokinetics were analyzed following intradiscal LOR injection in rats. In vivo effects of LOR and vehicle on AF integrity, AF/NP junction, NP cellularity and matrix, and disc height were compared using histopathology and radiography in a rat coccygeal IVD needle-puncture model of DDD.RESULTSIn NP and AF cell cultures, LOR-inhibited Wnt pathway gene expression compared with vehicle. In NP cells, LOR inhibited senescence, decreased catabolism, and induced differentiation into chondrocyte-like cells; in AF cells, LOR decreased catabolism and inhibited fibrosis. A single intradiscal LOR injection in rats resulted in therapeutic disc concentrations (~30 nM) for >180 days and minimal systemic exposure. DDD-model rats receiving LOR qualitatively demonstrated increased cartilage matrix and reduced AF lamellar disorganization and fragmentation with significantly (p<.05) improved histology scores and increased disc height compared with vehicle.CONCLUSIONSLOR showed beneficial effects on IVD cells in vitro and reduced disease progression in a rat model of DDD compared with vehicle, suggesting that LOR may have disease-modifying therapeutic potential.CLINICAL SIGNIFICANCEThe current therapeutic options for DDD are pain management and surgical intervention; there are no approved therapies that alter the progression of DDD. Our data support advancing LOR into clinical development as an injectable, small-molecule, potential disease-modifying treatment for DDD in humans.     

Disc cell clusters in pathological human intervertebral discs are associated with increased stress protein immunostaining

Intervertebral disc (IVD) cells within the annulus fibrosus (AF) and nucleus pulposus (NP) maintain distinct functional extracellular matrices and operate within a potentially noxious and stressful environment. How disc cells respond to stress and whether stress is responsible for triggering degeneration is unknown. Disc cell proliferation and cluster formation are most marked in degenerate IVDs, possibly indicating attempts at matrix repair. In other tissues, stress proteins increase rapidly after stress protecting cell function and, although implicated in degeneration of articular cartilage, have received little attention in degenerative IVD pathologies. We have compared the distribution of stress protein immunolocalization in pathological and control IVDs. Disc tissues were obtained at surgery from 43 patients with degenerative disc disease (DDD) and herniation, and 12 controls at postmortem. Tissues were immunostained with a polyclonal antibody for heat shock factor 1 (HSF-1) and monoclonal antibodies for the heat shock proteins, Hsp27 and Hsp72, using an indirect immunoperoxidase method. Positively stained cells were expressed as a percentage of the total. Cell cluster formation was also assessed. The proportion of cells in clusters was similar in the AF (both 2%) and NP (8 and 9%) of control and DDD samples, whereas in herniated tissues this was increased (AF 12%, NP 14%). Stress antigen staining tended to be more frequent in clustered rather than in single/doublet cells, and this was significant (P < 0.005) in both the AF and NP of herniated discs. Clustered cells, which are most common in herniated discs, may be mounting a protective response to abnormal environmental factors associated with disc degeneration. A better understanding of the stress response in IVD cells may allow its utilization in disc cell therapies.     

Effect of bupivacaine on intervertebral disc cell viability

Background contextBupivacaine is a local anesthetic commonly used to relieve or control pain in interventional spine procedures. Bupivacaine has been shown to be toxic to articular cartilage, which has similarities to intervertebral disc (IVD) cartilage, raising concern over a potentially negative effect of bupivacaine on the disc.PurposeTo determine bupivacaine's effect on cell viability of IVD cells in vitro and to elucidate whether this is through apoptosis or necrosis.Study designIn vitro controlled study of bupivacaine effect on cell viability in human and rabbit IVD cells.SubjectsRabbit annulus fibrosus (AF) tissue, nucleus pulposus (NP) cells, and knee articular chondrocytes were isolated from New Zealand white rabbits. Human AF and NP cells were isolated from stage 3 to 4 degenerative disc surgical specimens.Outcome measuresCell viability was assessed after exposure to bupivacaine via trypan blue staining or flow cytometry.MethodsAnnulus fibrosus and NP cells were grown in monolayer and alginate beads, respectively, to simulate their physiologic environment. The cells were then exposed to bupivacaine or saline control at 60 and 120 minutes and examined for cell viability.ResultsRabbit NP cell death demonstrated a time and dose dependence in response to bupivacaine. In addition, cell death was greater than that observed for articular chondrocytes. Rabbit AF tissue also demonstrated increased cell death in response to bupivacaine exposure. Human NP cells demonstrated time-dependent cell death, with greater necrosis than apoptosis. Annulus fibrosus cells grown in monolayers also resulted in similar effects, with greater necrosis rather than apoptosis.ConclusionsDespite its pain relieving properties, bupivacaine decreases cell viability in rabbit and human disc cells in a time-dependent manner. In addition, the changes observed are greater than that seen for articular chondrocytes. This increase in cell death appears to be related to an increase in necrosis rather than apoptosis. Whether bupivacaine exerts similar effects in vivo or how this relates to overall clinical outcome remains to be explored.     

Hypoxia suppresses serum deprivation‐induced degradation of the nucleus pulposus cell extracellular matrix through the JNK and NF‐κB pathways

Intervertebral disc (IVD) degeneration is associated with the imbalance between anabolism and catabolism of the nucleus pulposus (NP) extracellular matrix (ECM). Serum deprivation (SD) has been reported to exacerbate IVD degeneration; however, the effect of SD on ECM metabolism is not fully understood. Hypoxia plays important roles in maintaining the physiological functions of IVD cells; however, whether hypoxia has any effect on NP ECM production under conditions of SD is still unclear. In the current study, we established an in vitro SD model by exposing NP cells to serum‐free medium. SD decreased the expression of aggrecan and collagen II, as well as the production of sulfated glycosaminoglycan (sGAG) in a time‐dependent manner. However, hypoxia abolished SD‐mediated down‐regulation of aggrecan and collagen II expression via JNK1/2 activation. Moreover, hypoxia abolished SD‐induced MMP‐3 and MMP‐13 expression by inhibiting NF‐κB activation, p65 translocation, and MMP‐3 and MMP‐13 promoter activity. These results indicated that, hypoxia maintained ECM production under conditions of SD. This effect was elicited in part through JNK1/2‐mediated up‐regulation of matrix gene expression and down‐regulation of MMP expression, through the inhibition of NF‐κB. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2059–2066, 2017.

     

Influence of extracellular osmolarity and mechanical stimulation on gene expression of intervertebral disc cells.

Intervertebral discs (IVD) have a higher extracellular osmolarity than most other tissues; moreover their osmolarity changes by around 25% during each diurnal cycle. In this study, changes in aggrecan, collagen I and collagen II expression of IVD cells were examined after exposure to osmotic environment alterations or mechanical stimulation under different osmotic conditions. Human and bovine IVD cells seeded in three-dimensional (3D) collagen type I matrices were cultured under hypo-osmotic (300 mOsm), iso-osmotic (400 mOsm), or hyperosmotic (500 mOsm) conditions. Osmolarity-induced changes in gene expression of IVD cells were measured after 5 days. Load-induced changes in gene expression under the different osmotic conditions were measured after application of hydrostatic pressure (0.25 MPa, 0.1 Hz, 30 min) or cyclic strain (4%, 1 Hz, 24 h). The results showed that IVD cells respond strongly to changes in the osmotic environment by altering mRNA expression. Human cells cultured over 5 days increased expression of aggrecan and collagen II in both nucleus and annulus cells under increasing osmolarity. In contrast, collagen I expression was inhibited at high osmolarity in both cell types. Mechanically induced alterations in gene expression appear to have only modest effects on matrix protein expression, but the same stimulus partly resulted in an inhibition or stimulation of gene expression, depending on the osmotic conditions. This study showed that the osmotic environment does not only have an appreciable effect on gene expression but also affects responses to mechanical stimuli. This suggests that the osmotic conditions cannot be ignored when examining physiological and pathological behavior of IVD cells.     

Differential phenotypic behaviors of human degenerative nucleus pulposus cells under normoxic and hypoxic conditions: influence of oxygen concentration during isolation,expansion, and cultivation

Background contextIntervertebral discs (IVDs) are the largest avascular structures in the body; therefore, cells within these discs might be adapted to low-oxygen conditions. Although it has been demonstrated that a low oxygen concentration could promote synthesis of the extracellular matrix by IVD cells in the in vitro culture, isolation, expansion, and cultivation of IVD cells under classical tissue culture O₂ saturation could still be detrimental.PurposeTo investigate the phenotypic differences between human degenerative nucleus pulposus (NP) cells during isolation and expansion under normoxic (Nx: 21% O₂) or hypoxic (Hx: 3.5% O₂) conditions.Study designWe investigated in vitro isolation, expansion, and cultivation of human NP cells.MethodsHuman NP tissue samples were obtained from patients who underwent lumbar disc surgeries. Nucleus pulposus cells were then isolated, expanded, and cultivated under normoxic or hypoxic conditions. To determine whether the effects of normoxic expansion are reversible, another group of cells was isolated and expanded in normoxic conditions and then cultivated under hypoxic conditions (Nx→Hx group). Cellular proliferation, RNA expression of selected genes, and immunohistochemical staining were performed to evaluate the phenotypic behaviors of human NP cells under different conditions.ResultsExpressions of Type II collagen and aggrecan in the Nx→Hx group were significantly higher than those in the normoxic group but were significantly lower than those in the hypoxic group. The normoxic group showed higher expression of matrix metalloproteinase (MMP)-2 and MMP-13 than did the other groups. Expression levels of hypoxia-inducible factors (HIFs) were significantly higher in the normoxic groups; however, a greater degree of HIF-1α staining was found in the hypoxic group, whereas a greater degree of HIF-2α staining was found in the normoxic group.ConclusionsHuman degenerative NP cells isolated, expanded, and cultivated in hypoxic conditions could better preserve the cells' regenerative potential. Compromised properties that were observed during isolation and expansion under normoxic conditions could only be partially rescued by later hypoxic cultivation. The superior phenotypic behaviors of human NP cells under hypoxia may be related to higher HIF-1α production and lower HIF-2α production. Cells that are isolated, expanded, and cultivated under hypoxic conditions may show better regenerative results when transplanted; therefore, the isolation and expansion processes of human degenerative NP cells should be managed in a hypoxic environment.     

Cytotoxicity of local anesthetics and nonionic contrast agents on bovine intervertebral disc cells cultured in a three-dimensional culture system

Background contextCarragee et al. reported an accelerated progression of lumbar intervertebral disc (IVD) degeneration after discography in a human trial. Local anesthetics and contrast agents have exhibited toxicity to cardiac, renal, and neuronal cells. We hypothesize that local anesthetics or contrast agents commonly injected into the disc space during discography may result in cytotoxicity in vitro. In this study, we compared the cytotoxicity of these agents, alone or in combination, using nucleus pulposus (NP) and annulus fibrosus (AF) cells in a three-dimensional (3D) culture system.PurposeThe purpose of this study was to examine the effects of local anesthetics and contrast agents on IVD cells to help guide their usage in future clinical practices.Study designOurs was an in vitro study to assess the cytotoxicity of local anesthetics and contrast agents commonly used in discography, using bovine NP and AF cells cultured in a 3D system.MethodsBovine NP and AF cells were isolated and encapsulated in alginate beads and cultured in media completed with serum and ascorbic acid. Beads were transferred to a 24-well plate and treated with local anesthetics, nonionic contrast agents, or with saline as a control for 2, 6, and 16 hours. Three different concentrations of local anesthetics, lidocaine and bupivacaine, were tested: 0.25%, 0.125%, and 0.0625%. Two different dilutions (1:2 or 1:4) of nonionic contras agents, iohexol and iopamidol, were tested. In a parallel study, beads were incubated with a combination of local anesthetics at equipotent concentrations and contrast agents for 6 hours. Cells were then examined with the LIVE/DEAD cell assay. Live cells (fluorescing green) and dead cells (fluorescing red) were visualized using fluorescent microscopy. The percentage of live cells after treatment was determined.ResultsMore cell death was observed when NP and AF cells were incubated with anesthetics than contrast agents at the concentrations tested. When tested at equipotent concentrations, 0.125% bupivacaine (N=8) resulted in significantly more cell death than 0.5% lidocaine (N=6) in NP cells (p<.05). In these studies, cell death caused by bupivacaine was both dose and time dependent. When tested at the same dilutions, iopamidol diluted 1:2 caused slightly more cell death than iohexol. When incubating the cells with a combination of contrast and anesthetic agent, the cytotoxic effects of the anesthetics and contrast agent were not synergistic. In this culture system, AF cells were more sensitive to some of the agents than NP cells.ConclusionsCell death was observed when AF and NP cells were incubated in a dose- and time-dependent manner with local anesthetics and contrast agents commonly used for discography. Relative toxicity of these compounds was noted in the order of bupivacaine, lidocaine, iopamidol, and iohexol. Future studies of the effects of these agents in organ culture or animal models are indicated to predict what happens in vivo.     

Comparison of toxicity effects of ropivacaine,bupivacaine, and lidocaine on rabbit intervertebral disc cells in vitro

Background contextIt has been shown that bupivacaine, the most commonly used local anesthetic to relieve or control pain in interventional spine procedures, is cytotoxic to intervertebral disc (IVD) cells in vitro. However, some other common local anesthetics, such as ropivacaine and lidocaine, are also frequently used in the treatment of spine-related pain, and the potential effects of these agents remain unclear.PurposeThe purpose of this study was to evaluate the effect of various local anesthetics on rabbit IVD cells in vitro and further compare the cytotoxicity of ropivacaine, bupivacaine, lidocaine, and saline solution control.Study designControlled laboratory study.SubjectsRabbit annulus fibrosus (AF) and nucleus pulposus (NP) cells were isolated from Japanese white rabbits.MethodsBoth AF and NP cells at the second generation maintained in monolayer were exposed to various concentrations of local anesthetics (eg, bupivacaine) or different durations of exposure and evaluated for cell viability by use of cell counting kit-8 (CCK-8). In addition, to compare the cytotoxicity of ropivacaine, bupivacaine, lidocaine, and saline solution control in commercial concentration, the viability was analyzed by flow cytometry after 60-minute exposure, and the morphologic changes were observed by the phase-contrast microscopy. Apoptosis and necrosis of IVD cells were confirmed by using fluorescence microscopy with double staining of Hoechst 33342 and propidium iodide.ResultsRabbit IVD cell death demonstrated a time and dose dependence in response to bupivacaine and lidocaine. However, ropivacaine only exerted a significant time-dependent effect on IVD cells. There was no significant difference in IVD viability after treatment with different doses of ropivacaine. In addition, the results showed that lidocaine was the most toxic of the three local anesthetics and that ropivacaine presented less cytotoxicity than lidocaine and bupivacaine. Fluorescence microscopy also confirmed that the short-term toxic effect of local anesthetics on both AF and NP cells was mainly caused by necrosis rather than apoptosis.ConclusionsResults show that bupivacaine and lidocaine decrease cell viability in rabbit IVD cells in a dose- and time-dependent manner. All local anesthetics should be avoided if at all possible. Ropivacaine may be a choice if necessary, but it is also toxic. The increase in cell death is more related with cell necrosis rather than cell apoptosis. If these results can be corroborated in tissue explant models or animal studies, caution regarding diagnosing, treating, and controlling spine-related pain with local anesthetics is prompted.     

Modulation of annulus fibrosus cell alignment and function on oriented nanofibrous polyurethane scaffolds under tension

Background contextAnnulus fibrosus (AF), a component of the intervertebral disc (IVD), is always under tension in vivo, a condition that must be taken into consideration when tissue engineering an IVD. Loss of the tensile forces has been implicated in the pathogenesis of disc degeneration characterized by mechanical and structural breakdown of the AF.PurposeIn this study, we hypothesize that tensile forces modulate cellular and molecular behavior of AF cells grown on nanofibrous scaffolds in vitro.Study design/settingBovine AF cells were seeded onto strained electrospun-aligned nanofibrous polycarbonate urethane (PU) scaffolds. Tension was either maintained throughout the culture duration (monotonic) or removed after 24 hours (relaxed).MethodsThe effect of tension on AF cells cultured on PU scaffolds was evaluated over 7 days by scanning electron microscopy, biochemical assays, immunofluorescence microscopy, and quantitative polymerase chain reaction.ResultsCells grown on the relaxed scaffold were significantly more proliferative, synthesized more collagen and had increased collagen type I and TGFβ-1 gene expression; however these cells were not as aligned as were the cells and matrix on monotonic strained scaffolds. The alignment of AF cells grown on monotonic scaffolds correlated with significantly greater scaffold elastic modulus on day 7. Additionally, the cellular response to the change in strain was delayed by 3 to 5 days after tension release, which correlated with the time at which changes in scaffold length were detected.ConclusionsThis study demonstrated that AF cells respond at the molecular and cellular level to the changes in matrix/scaffold tension. This suggests that it may be necessary to determine the optimal elastic modulus and applied tensile forces to tissue engineer an AF that mimics the native tissue. Furthermore, this study provides insight into how changes in tensile forces may lead to changes in the AF cell function.     

The effects of recombinant human bone morphogenetic protein-2, recombinant human bone morphogenetic protein-12, and adenoviral bone morphogenetic protein-12 on matrix synthesis in human annulus fibrosis and nucleus pulposus cells

BACKGROUND CONTEXT: Bone morphogenetic proteins (BMPs) are potential therapeutic factors for degenerative discs, and BMP-12 does not have the osteogenic potential of BMP-2, making it better suited for intradiscal injection. However, no reports have compared the actions of BMP-2 and -12 on human annulus fibrosus (AF) and nucleus pulposus (NP) cells nor evaluated adenoviral-mediated gene therapy in human AF cells. PURPOSE: To evaluate and compare the effects of recombinant human (rh) BMP-2, rhBMP-12, and adenoviral BMP-12 (Ad-BMP-12) on nucleus pulposus and annulus fibrosis cell matrix protein synthesis. STUDY DESIGN: In vitro study using rhBMP-2 and -12 and adenoviral BMP-12 with human intervertebral disc (IVD) cells. METHODS: Human NP and AF IVD cells were isolated, maintained in monolayer, and incubated with BMP-2 or -12 for 2 days. AF and NP cells were transduced with Ad-BMP-12, pellets formed, and incubated for 6 days. Growth factor-treated cells were labelled with either 35-S or 3H-proline to assay matrix protein synthesis. RESULTS: rhBMP-2 increased NP proteoglycan, collagen, and noncollagen protein synthesis to 355%, 388%, and 234% of control. RhBMP-12 increased the same NP matrix proteins' synthesis to 140%, 143%, and 160% of control. Effects on AF matrix protein synthesis were minimal. Ad-BMP-12 significantly increased matrix protein synthesis and DNA content of AF and NP cells in pellet culture. NP synthesis of all matrix proteins and AF synthesis of proteoglycans was increased when the data were normalized to pellet DNA. AF synthesis of noncollagen protein and collagen was not modulated by Ad-BMP-12 if the data are normalized to pellet DNA content. CONCLUSIONS: Both rhBMP-2 and -12 increase human NP cell matrix protein synthesis while having minimal effects on AF cells. However, Ad-BMP-12 did increase matrix protein synthesis in both NP and AF cells, making it a potential therapy for enhancing matrix production in the IVD. These responses plus the proliferative action of Ad-BMP-12 seen in the current studies, and the lack of an osteogenic action noted in other studies justifies future studies to determine if gene therapy with BMP-12 could provide protective and/or reparative actions in degenerating discs.     

Energy metabolism of intervertebral disc under mechanical loading

Intervertebral disc (IVD) degeneration is closely associated with low back pain (LBP), which is a major health concern in the U.S. Cellular biosynthesis of extracellular matrix (ECM), which is important for maintaining tissue integrity and preventing tissue degeneration, is an energy demanding process. Due to impaired nutrient support in avascular IVD, adenosine triphosphate (ATP) supply could be a limiting factor for maintaining normal ECM synthesis. Therefore, the objective of this study was to investigate the energy metabolism in the annulus fibrosus (AF) and nucleus pulposus (NP) of porcine IVD under static and dynamic compressions. Under compression, pH decreased and the contents of lactate and ATP increased significantly in both AF and NP regions, suggesting that compression can promote ATP production via glycolysis and reduce pH by increasing lactate accumulation. A high level of extracellular ATP content was detected in the NP region and regulated by compressive loading. Since ATP can serve not only as an intra‐cellular energy currency, but also as a regulator of a variety of cellular activities extracellularly through the purinergic signaling pathway, our findings suggest that compression‐mediated ATP metabolism could be a novel mechanobiological pathway for regulating IVD metabolism. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:1733–1738, 2013     

Effect of extracellular matrix protein on the rate of proteoglycan synthesis in rabbit intervertebral disc cells

OBJECTIVE: The extracellular matrix (ECM) is very important for fundamental cellular processes. However, the effects of ECM proteins on intervertebral disc (IVD) cell proliferation and metabolism have not been clarified. To verify the effects of ECM proteins on DNA and proteoglycan (PG) synthesis of IVD cells, PG synthesis rate was measured in IVD cells cultured in monolayer with or without ECM protein. METHODS: Nucleus pulposus (NP) cells and anulus fibrosus (AF) cells isolated from adolescent rabbits were cultured in monolayer with or without ECM protein and at different concentrations of ECM protein for 4-6 days. [S]Sulfate incorporation into PG in the cell-associated matrix (CM) formed around cells and the further-removed matrix (FRM) in labeling medium was measured and standardized to DNA content. CONCLUSIONS: NP cells in type I or type II collagen-coated plates significantly increased the rate of PG synthesis in both the CM and the FRM compared with those in uncoated plates and in fibronectin-coated plates; however, AF cells with ECM proteins did not increase the rate significantly. The rate of PG synthesis of nucleus cells was contra-dose dependent on both type I and type II collagen.     

Mechanical loading affects the energy metabolism of intervertebral disc cells

Research has shown that mechanical loading affects matrix biosynthesis of intervertebral disc (IVD) cells; however, the pathway(s) to this effect is currently unknown. Cellular matrix biosynthesis is an energy demanding process. The objective of this study was to investigate the effects of static and dynamic compressive loading on energy metabolism of IVD cells. Porcine annulus fibrosus (AF) and nucleus pulposus (NP) cells seeded in 2% agarose were used in this experiment. Experimental groups included 15% static compression and 0.1 and 1 Hz dynamic compression at 15% strain magnitude for 4 h. ATP, lactate, glucose, and nitric oxide (NO) contents in culture media, and ATP content in cell–agarose construct were measured using biochemical assays. While the total ATP content of AF cells was promoted by static and dynamic loading, only 1 Hz dynamic loading increased total ATP content of NP cells. Increases in lactate production and glucose consumption of AF cells suggest that ATP production via glycolysis is promoted by dynamic compression. ATP release and NO production of AF and NP cells were significantly increased by dynamic loading. Thus, this study clearly illustrates that static and dynamic compressive loading affect IVD cell energy production while cellular responses to mechanical loading were both cell type and compression type dependent. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29:1634–1641, 2011     

Nucleus pulposus degeneration alters properties of resident progenitor cells

Background contextThe intervertebral disc (IVD) possesses a minimal capability for self-repair and regeneration. Changes in the differentiation of resident progenitor cells can represent diminished tissue regeneration and a loss of homeostasis. We previously showed that progenitor cells reside in the nucleus pulposus (NP). The effect of the degenerative process on these cells remains unclear.PurposeWe sought to explore the effect of IVD degeneration on the abundance of resident progenitor cells in the NP, their differentiation potential, and their ability to give rise to NP-like cells. We hypothesize that disc degeneration affects those properties.Study designNucleus pulposus cells derived from healthy and degenerated discs were methodically compared for proliferation, differentiation potential, and ability to generate NP-like cells.MethodsIntervertebral disc degeneration was induced in 10 skeletally, mature mini pigs using annular injury approach. Degeneration was induced in three target discs, whereas intact adjacent discs served as controls. The disc degeneration was monitored using magnetic resonance imaging for 6 to 8 weeks. After there was a clear evidence of degeneration, we isolated and compared cells from degenerated discs (D-NP cells [NP-derived cells from porcine degenerated discs]) with cells isolated from healthy discs (H-NP cells) obtained from the same animal.ResultsThe comparison showed that D-NP cells had a significantly higher colony-forming unit rate and a higher proliferation rate in vitro. Our data also indicate that although both cell types are able to differentiate into mesenchymal lineages, H-NP cells exhibit significantly greater differentiation toward the chondrogenic lineage and NP-like cells than D-NP cells, displaying greater production of glycosaminoglycans and higher gene expression of aggrecan and collagen IIa.ConclusionsBased on these findings, we conclude that IVD degeneration has a meaningful effect on the cells in the NP. D-NP cells clearly go through the regenerative process; however, this process is not powerful enough to facilitate full regeneration of the disc and reverse the degenerative course. These findings facilitate deeper understanding of the IVD degeneration process and trigger further studies that will contribute to development of novel therapies for IVD degeneration.     

Exogenous and autocrine growth factors stimulate human intervertebral disc cell proliferation via the ERK and Akt pathways

Intervertebral disc (IVD) degeneration is accompanied by growth factor-overexpression and increased cell proliferation, probably representing a tissue repair process. Accordingly, we studied the effect of exogenous and autocrine growth factors on the proliferation of human IVD cells. We observed that Platelet-Derived Growth Factor (PDGF), basic Fibroblast Growth Factor (bFGF), and Insulin-like Growth Factor-I (IGF-I) stimulate DNA synthesis of human IVD cells, through the activation of the MEK/ERK and the PI-3K/Akt signal transduction pathways. Furthermore, medium conditioned (CM) by IVD cells induced DNA synthesis in the same cells, indicating the secretion of autocrine growth factors. The MEK/ERK and PI-3K/Akt pathways were also induced by CM, while their inhibition reversed in large part the DNA synthesis induction by CM. These responses to the exogenous and autocrine growth factors were qualitatively similar in both nucleus pulposus (NP) and annulus fibrosus (AF) cell cultures. Immunohistochemical studies in human biopsies showed significant activation of both signaling pathways, which was most prominent in the clusters of proliferating cells. These in vitro and in vivo data indicate that the proliferation of human IVD cells is regulated by exogenous and autocrine growth factors mainly via the MEK/ERK and PI-3K/Akt pathways; this may contribute to the design of future interventional approaches.     

Quantitative T2* (T2 star) relaxation times predict site specific proteoglycan content and residual mechanics of the intervertebral disc throughout degeneration

Degeneration alters the biochemical composition of the disc, affecting the mechanical integrity leading to spinal instability. Quantitative T2* MRI probes water mobility within the macromolecular network, a potentially more sensitive assessment of disc health. We determined the relationship between T2* relaxation time and proteoglycan content, collagen content, and compressive mechanics throughout the degenerative spectrum. Eighteen human cadaveric lumbar (L4–L5) discs were imaged using T2* MRI. The T2* relaxation time at five locations (nucleous pulposus or NP, anterior annulus fibrosis or AF, posterior AF, inner AF, and outer AF) was correlated with sulfated‐glycosaminoglycan (s‐GAG) content, hydroxyproline content, and residual stress and strain at each location. T2* relaxation times were significantly correlated with s‐GAG contents in all test locations and were particularly strong in the NP (r = 0.944; p < 0.001) and inner AF (r = 0.782; p < 0.001). T2* relaxation times were also significantly correlated with both residual stresses and excised strains in the NP (r = 0.857; p < 0.001: r = 0.816; p < 0.001), inner AF (r = 0.535; p = 0.022: r = 0.516; p = 0.028), and outer AF (r = 0.668; p = 0.002: r = 0.458; p = 0.041). These strong correlations highlight T2* MRI's ability to predict the biochemical and mechanical health of the disc. T2* MRI assessment of disc health is a clinically viable tool showing promise as a biomarker for distinguishing degenerative changes. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1083–1089, 2014.