Anulus Fibrosus Cells Research Articles

Pro-Inflammatory Stimuli Influence Expression of Intercellular Adhesion Molecule 1 in Human Anulus Fibrosus Cells through FAK/ERK/GSK3 and PKCδ Signaling Pathways.

Objective: Intervertebral disc (IVD) degeneration and disc herniation are major causes of lower back pain, which involve the presence of inflammatory mediators and tissue invasion by immune cells. Intercellular adhesion molecule 1 (ICAM1, also termed CD54) is an adhesion molecule that mediates cell-cell interactions, particularly between immune cells and target tissue. The aim of this study was to examine the intracellular signaling pathways involved in inflammatory stimuli-induced ICAM1 expression in human anulus fibrosus (AF) cells. Methods: Quantitative reverse transcription-polymerase chain reaction (qPCR), western blotting, and flow cytometry were performed to dissect the roles of different signaling pathways in inflammatory stimuli-mediated ICAM1 expression. Results: Using qPCR and western blot analyses, a significant increase in ICAM1 expression was observed in AF cells after stimulation of lipopolysaccharide (LPS) plus interferon-gamma (IFNγ) in a time-dependent manner. Flow cytometry revealed ICAM1 upregulation on the surface of AF cells. Importantly, LPS plus IFNγ treatment also significantly promoted Chemokine ligand (CCL)2 expression, but not CCL3. The enhanced ICAM1 expression was abolished after incubation with antibody against CCL2. In AF cells, treatment with LPS plus IFNγ activated the FAK/ERK/GSK3 signaling pathways, promoted a time-dependent increase in PKCδ phosphorylation, and promoted PKCδ translocation to the nucleus. Treatment with the pharmacological PKCδ inhibitor; rottlerin, effectively blocked the enhanced productions of ICAM1 and CCL2. Conclusions: Inflammatory stimuli in AF cells are part of a specific pathophysiology in IVD degeneration and disc herniation that modulates CCL2/ICAM1 activation through the FAK/ERK/GSK3 and PKCδ signaling pathways in AF cells.

Lentivirus Is an Efficient and Stable Transduction Vector for Intervertebral Disc Cells

To evaluate transduction efficacy and sustainability of lentiviral vector for intervertebral disc cells both invitro and invivo. Human nuclear pulposus and anulus fibrosus cells isolated from disc tissue of 28 patients during surgical disc procedures were cultured and subsequently transduced using recombinant lentivirus carrying a gene for enhanced green fluorescent protein at multiplicities of infection of 0, 15, 30, 60, 90, and 150. Cell viability was determined using the trypan blue exclusion test. Transduction efficiency was measured by fluorescence-activated cell sorting analysis. Invivo experiments were done by injecting lentivirus into rat intervertebral discs. Disc tissue was harvested 7, 14, 21, and 28 days after transduction, and enhanced green fluorescent protein expression was examined using an inverted fluorescent microscope. Intervertebral disc cells transduced with different doses of lentivirus showed equally good viabilities compared with cells in the control group, as determined by cell morphology and growth curves after transduction. The transduction ratio for disc cells after transduction reached its optimum of 97% at 60 multiplicities of infection, independent of patient age, sex, surgical procedure, diagnosis, disc level, or degeneration grade. Invivo frozen sections revealed that enhanced green fluorescent protein expression peaked on the 7th day and remained detectable the 28th day after transduction. No significant systemic symptoms were observed during the invivo experiment. Lentivirus appears to be an efficient and stable transduction vector for intervertebral disc cells. It has potential as a gene therapy tool for treating human degenerative disc disease.

RANK/RANKL/OPG system in the intervertebral disc

BackgroundThe receptor activator of NF-κB ligand (RANKL), a member of the TNF ligand superfamily, is known to regulate bone metabolism. The expression of each component of the RANK/RANKL/osteoprotegerin (OPG) system in the intervertebral disc (IVD) has not been examined in detail. The purposes of this study were to examine the expression of the RANK/RANKL/OPG system and to evaluate the function of RANKL in the matrix metabolism of the rat IVD.MethodsSprague-Dawley, 12-week-old, male rats were used in this study. Anulus fibrosus (AF), nucleus pulposus (NP) and cartilaginous endplate (CEP) cells isolated from dissected thoracolumbar discs were monolayer-cultured. RANK/RANKL/OPG expression in rat IVDs was examined using real-time polymerase chain reaction (PCR) and immunohistochemical analysis (cultured cells and IVD tissues). To examine the effect of interleukin-1β (IL-1β) stimulation on the mRNA levels of RANK, RANKL and OPG, the cells were cultured with or without recombinant human IL-1β (rhIL-1β). To evaluate the effect of RANKL on the mRNA expression of catabolic factors (IL-1β, matrix metalloproteinase-3 (MMP-3) and MMP-13), the cells were cultured with RANKL in the presence or absence of rhIL-1β. The immunohistochemical expression of this system was also evaluated using human IVD tissues with different grades of degeneration.ResultsmRNA expression levels of RANK, RANKL, and OPG were clearly identified in AF, NP and CEP cells. Immunoreactivity to RANK, RANKL and OPG was distributed in the cell membranes and/or cytoplasm of the three types of cells. The mRNA level of RANKL was significantly upregulated by treatment with rhIL-1β of the three types of cells. Treatment with RANKL without rhIL-1β did not induce significant effects on the mRNA expression of catabolic factors by AF, NP and CEP cells. However, the expression was significantly upregulated by stimulation with RANKL in the presence of rhIL-1β. There was a general trend for more RANK/RANKL/OPG-positive cells in human IVD tissues in an advanced stage of degeneration compared to an early stage.ConclusionsOur study showed the possibility that the RANK/RANKL/OPG system may play a part in the process of intervertebral disc degeneration.

In vitro construction and biological properties of new tissue-engineered intervertebral discs

Objective To construct a novel tissue-engineered intervertebral disc complex and investigate the biological properties of the complex. Methods By the technique of tissue engineering, the anulus fibrosus scaffolds and nucleus pulposus cells were seeded in corresponding regions in the composite intervertebral discs. Then, the cell-scaffold hybrids were implanted in the subcutaneous space of the dorsum of athymic mice. At 4, 8, and 12 weeks after implantation, gross observation and histological morphology were performed and hydroxyproline, glycosaminoglycan, DNA content and biochemical properties were evaluated. Results Gross morphology and histology of the engineered discs resembled those of native intervertebral discs. Morphological studies revealed progressive tissue formation and junction integration between anulus fibrosus and nucleus pulposus cells. Scanning microscopy showed new extracellular matrix was rich in anulus fibrosus and nucleus pulposus cells. Content of hydroxyproline in anulus fibrosus and nucleus pulposus cells increased with time, i. e. before the implantation [(7.1±2.3)μg/mg, (5.9±2.5)μg/mg], at 4 weeks [(10.1±1.1)μg/mg, (6.6±2.0)μg/mg] and at 12 weeks [(12.6±2.1)μg/mg, (12.5±3.3)μg/mg] (P<0.05). Content of proteoglycan and DNA showed similar variation trends, i. e. in anulus fibrosus [(3.3±1.3)μg/mg, (10.9±2.7)μg/mg, (12.4±3.7)μg/mg; (52.2±5.8)ng/mg, (99.7±17.0)ng/mg, (149.5±12.0)ng/mg respectively], in nucleus pulposus cells [(13.4±1.7)μg/mg, (18.4±2.5)μg/mg, (22.7±3.6)μg/mg; (66.5±9.2)ng/mg, (148.6±9.8)ng/mg, (187.6±16.6)ng/mg respectively] (P<0.05). At 12 weeks, the maximum loads, compressive strength, yield strength and elastic modulus differed significantly from those before implantation (P<0.05). Biomechanical markers of the composite were substantially lowered with time compared to those of native tissues (P<0.05). Conclusion Tissue-engineered discs with similar morphological and biochemical properties to native discs provides a portability in tissue engineering. Key words: Intervertebral disc; Tissue engineering; Annular fibrosus

Micro-RNA Attenuates the Osteogenic Differentiation of Human Annulus Fibrosus Cells

Introduction During the process of disc degeneration, there is bone/fibrocartilage formation in the intervertebral area. As degeneration of the IVD and consequent chronic low back pain progresses, nerves and blood vessels are found increasingly in the inner part of the annulus fibrosus (AF) region of the intervertebral disc. Our previous study demonstrated that multipotent cells can be isolated from the inner AF tissue and, when cultured in vitro, will undergo differentiation into different lineage such chondrocytes, osteoblasts, and endothelial cells. The underlying cellular mechanism, however, has not studied yet. The objective of this study is to investigate not only the behavior but also the mechanisms of human AF cells from normal and degenerated IVDs. Materials and Methods Normal and degenerated human AF cells were isolated respectively from discarded tissue of patients ( n = 3) who have undergone sclerosis or spinal fusion, respectively. Cells at passage 2 to 4 were cultured in osteogenic medium (OIM) in vitro, and cellular differentiation was investigated using real time RT-PCR and histology at different time points. To measure the level of micro-RNA 221, total RNA was isolated with Trizol, reverse transcribed with Qiagen micro-RNA and amplified with universal master mix. Degenerated AF cells overexpressing miR221 were infected with lentiviral particles carrying miR221 and selected with puromycin. Total and phospo-Smad were detected with Western blotting with specific antibodies. Data are presented as mean ± standard deviation. Statistical analyses were performed by one-way ANOVA assuming equal variance using Microsoft Excel 2010 software. A p-value of less than 0.05 was considered statistically significant. Results Both normal AF (NAF) and degenerated AF (DAF) cells demonstrated their ability to undergo osteogenic differentiation as confirmed by mineralization (Fig. 1) and increased mRNA expression of BMP2, Runx2, ALP, and OCN (Fig. 2). DAF cells expressed increased osteogenic differentiation potential over the NAF cells. In contrast to the elevated phosphor-Smad1 and Smad 1/5/8 in DAF cells, the basal level of miR221 significantly decreased in DAF compared with NAF cells (Figs. 3 and 4). Osteogenic differentiation reduced miRNA221 expression in NAF cells, but there was no significant further reduction in DAF cells. Overexpression of miR221 with lentiviral particles markedly decreased the level of p-Smads and bone marker genes in DAF cells (Fig. 5). Consistently, the osteogenic potential of AF cells was diminished by miR221 expression (Fig. 6). Conclusion We have demonstrated that human AF cells have the potential to differentiate into various cell lineages upon appropriate stimulation. Consistent with the observation in degenerated IVD, DAF cells showed a stronger potential for osteogenic differentiation than NAF cells. The osteogenic ability may be modulated by posttranscriptional regulation of Smads (1, 5, 8), which are crucial regulators for signal transduction by BMPs during the process of osteogenesis through morphogenesis and development and in tissue repair. Moreover, the elevation of p-Smads is attenuated by overexpressing miR221 in the DAF cells. miR221 has an important role in cell proliferation and migration, and may serve as a regulator of the mechanotransduction pathway. While loss of function of miR221 is still under investigation, our results demonstrate that the osteogenic potential of degenerated AF cells is regulated by BMP-Smad pathways that are potentially modified by the expression of miR221. Disclosure of Interest None declared References Feng G, Yang X, Shang H, et al. Multipotential differentiation of human anulus fibrosus cells: an in vitro study. J Bone Joint Surg Am 2010;92(3):675–685 Hong E, Reddi AH. MicroRNAs in chondrogenesis, articular cartilage, and osteoarthritis: implications for tissue engineering. Tissue Eng Part B Rev 2012;18(6):445–453

Characterization of Human Anulus Fibrosus– and Nucleus Pulposus–Derived Cells During Monolayer Expansion and in Hydrogel Cultures

In vitro-expanded intervertebral disc (IVD) cells could be a source for disc repair. However, IVD cell characterization still remains challenging and is demanded to detect phenotypical shifts. Therefore, the aim of the present study was to determine IVD cell expression profile during two- and three-dimensional culturing in direct comparison to in situ conditions. Human IVD tissue was analyzed immunohistologically and anulus fibrosus (AF) and nucleus pulposus (NP) cells were isolated and characterized for cytoskeletal architecture and expression of typical markers (type I, II, and III collagens, aggrecan, decorin, cartilage oligomeric protein, the chondrogenic transcription factor sox9, the tendon markers scleraxis and tenascin C) during 6 monolayer passages using real-time detection polymerase chain reaction and/or immunolabellings. Cells were introduced in alginate and collagen hydrogels and cell morphology and viablility was determined after 7 days. In addition to typical extracellular matrix components, IVD tissue and isolated cells revealed scleraxis expression. In early passages of cell expansion, genes of sox9, scleraxis, and the small proteoglycan decorin were expressed higher, but type I and III collagen genes were expressed lower in NP cells compared with AF cells. However, in passage 6, actin stress fibers increased and the expression levels of sox9 were nearly similar in NP and AF cells. The immunolabeling indicated that the fibroblast marker tenascin C could only be detected in vitro in both cell types but not in situ. Decorin protein expression decreased in both cell types in vitro in passage 6. IVD cells survived in both hydrogel cultures, and some cells elongated in collagen gels.

Changes in the Molecular Phenotype of Nucleus Pulposus Cells with Intervertebral Disc Aging

Intervertebral disc (IVD) disorder and age-related degeneration are believed to contribute to low back pain. Cell-based therapies represent a promising strategy to treat disc degeneration; however, the cellular and molecular characteristics of disc cells during IVD maturation and aging still remain poorly defined. This study investigated novel molecular markers and their age-related changes in the rat IVD. Affymetrix cDNA microarray analysis was conducted to identify a new set of genes characterizing immature nucleus pulposus (NP) cells. Among these markers, select neuronal-related proteins (Basp1, Ncdn and Nrp-1), transcriptional factor (Brachyury T), and cell surface receptors (CD24, CD90, CD155 and CD221) were confirmed by real-time PCR and immunohistochemical (IHC) staining for differential expression between IVD tissue regions and among various ages (1, 12 and 21 months). NP cells generally possessed higher levels of mRNA or protein expression for all aforementioned markers, with the exception of CD90 in anulus fibrosus (AF) cells. In addition, CD protein (CD24 and CD90) and Brachyury (T) expression in immature disc cells were also confirmed via flow cytometry. Similar to IHC staining, results revealed a higher percentage of immature NP cells expressing CD24 and Brachyury, while higher percentage of immature AF cells was stained positively for CD90. Altogether, this study identifies that tissue-specific gene expression and age-related differential expression of the above markers do exist in immature and aged disc cells. These age-related phenotype changes provide a new insight for a molecular profile that may be used to characterize NP cells for developing cell-based regenerative therapy for IVD regeneration.

Towards biological anulus repair: TGF-β3, FGF-2 and human serum support matrix formation by human anulus fibrosus cells

Closure and biological repair of anulus fibrosus (AF) defects in intervertebral disc diseases is a therapeutic challenge. The aim of our study was to evaluate the anabolic properties of bioactive factors on cartilaginous matrix formation by AF cells. Human AF cells were harvested from degenerated lumbar AF tissue and expanded in monolayer culture. AF cell differentiation and matrix formation was initiated by forming pellet cultures and stimulation with hyaluronic acid (HA), human serum (HS), fibroblast growth factor-2 (FGF-2), transforming growth factor-β3 (TGF-β3) and TGF-β3/FGF-2 for up to 4 weeks. Matrix formation was assessed histologically by staining of proteoglycan, type I and type II collagens and by gene expression analysis of typical extracellular matrix molecules and of catabolic matrix metalloproteinases MMP-2 and MMP-13. AF cells, stimulated with HS, FGF-2 and most pronounced with TGF-β3 or TGF-β3/FGF-2 formed a cartilaginous matrix with significantly enhanced expression of matrix molecules and of MMP-13. Stimulation of AF cells with TGF-β3 was accompanied by induction of type X collagen, known to occur in hypertrophic cartilage cells having mineralizing potential. HA did not show any chondro-inductive characteristics. These findings suggest human serum, FGF-2 and TGF-β3 as possible candidates to support biological treatment strategies of AF defects.

Smad3 controls β‐1,3‐glucuronosyltransferase 1 expression in rat nucleus pulposus cells: Implications of dysregulated expression in disc disease

To study the regulation of expression of β-1,3-glucuronosyltransferase 1 (GlcAT-1), an important regulator of glycosaminoglycan (GAG) synthesis, by Smad3 in nucleus pulposus (NP) cells. GlcAT-1 expression was examined in rat NP and anulus fibrosus (AF) cells treated with transforming growth factor β (TGFβ). The effects of Smad signaling and Smad suppression on GlcAT-1 were examined in rat NP cells. GlcAT-1 expression was analyzed in the discs of Smad3-null mice and in degenerated human NP tissue. TGFβ increased the expression of GlcAT-1 in rat NP but not rat AF cells. Suppression of GlcAT-1 promoter activity was evident with dominant-negative ALK-5 (DN-ALK-5). Cotransfection with Smad3 strongly induced promoter activity independent of TGFβ. Bioinformatics analysis indicated the presence of several Smad binding sites in the promoter; deletion analysis showed that the region between -274 and -123 bp was required for Smad3 response. DN-Smad3, Smad 3 small interfering RNA, and Smad7 strongly suppressed basal as well as TGFβ-induced promoter activity. Induction of promoter activity by Smad3 was significantly blocked by DN-Smad3; Smad7 had a very small effect. Lentiviral transduction of NP cells with short hairpin RNA Smad3 resulted in a decrease in GlcAT-1 expression and accumulation of GAG. Compared to wild-type mice, significantly lower expression of GlcAT-1 was seen in the discs of Smad3-null mice. Analysis of degenerated human NP tissue specimens showed no positive correlation between GlcAT-1 and TGFβ expression. Moreover, isolated cells from degenerated human tissue showed a lack of induction of GlcAT-1 expression following TGFβ treatment, suggesting an altered response. Our findings demonstrate that in healthy NP cells, the TGFβ-Smad3 axis serves as a regulator of GlcAT-1 expression. However, an altered responsiveness to TGFβ during disc degeneration may compromise GAG synthesis.

Differential expression of galectin‐1 and its interactions with cells and laminins in the intervertebral disc

Galectin-1 (Gal-1), an endogenous β-galactoside-binding protein, binds to laminins, which are highly expressed in the nucleus pulposus (NP) of the intervertebral disc (IVD). The objective of this study is to evaluate the expression of Gal-1 protein in IVD tissues during aging and the effect of Gal-1 on IVD cell adhesion to laminins. Tissues from rat, porcine, and human (scoliosis or disc degeneration) IVDs were used to evaluate Gal-1 expression via immunostaining, RT-PCR, and Western blot analysis. Attachment of isolated IVD cells (porcine and human) on select laminin isoforms (LM-111 and LM-511) was compared with/without pre-incubation with exogenous Gal-1. A biotinylated Gal-1(B-Gal-1) was used to evaluate for binding to IVD cells and to select for IVD cells by magnetic activated cell sorting (MACS). NP cells expressed high levels of Gal-1 protein as compared to anulus fibrosus (AF) cells in immature tissues, while exogenous Gal-1 increased both NP and AF cell attachment to laminins and exhibited a similar binding to both cell types in vitro. With aging, Gal-1 levels in NP tissue appeared to decrease. In addition, incubation with B-Gal-1 was able to promote the retention of more than 50% of IVD cells via MACS. Our results provide new findings for the presence and functional role of Gal-1 within IVDs. Similar staining patterns for Gal-1 and LM-511 in IVD tissue suggest that Gal-1 may serve as an adhesion molecule to interact with both cells and laminins. This MACS protocol may be useful for selecting pure IVD cells from mixed cells of pathological tissue.

Platelet-Rich Plasma Effects on Degenerative Disk Disease: Analysis of Histology and Imaging in an Animal Model

Introduction The current conservative and surgical modalities of treating degenerative disk disease are aimed at relief of symptoms. At this time, there is no cure for the degenerative process. Platelet-rich plasma (PRP) is a bioactive component of autologous whole blood. The indications for the use of PRP in the treatment of musculoskeletal diseases continue to expand. The mechanism of action for PRP is due to the release of healing factors from platelets. While clinical evidence strongly supports the use of PRP for certain conditions, the potential benefit of PRP in treating or preventing progression of degenerative disk disease has not been critically evaluated. There is a preliminary evidence that certain growth factors in PRP, such as PDGF and IGF-1, may be beneficial in slowing the progression of disease. The objective of this study is to analyze the early and late phase effects of PRP injection into the intervertebral disk using an animal model. Materials and Methods After obtaining IRB and Institutional Animal Care and Use Committee (IACUC) approval, the L4-L5 intervertebral disk of adult Sprague Dawley rats were injured with a 21-gauge needle in 24 rats. After injury, animals received either an acute injection of PRP collected from matched donor animals, a delayed (2 week) injection of PRP after the degenerative process had commenced, or no further intervention (sham PRP). Magnetic resonance imaging was performed for each group at 0, 2, and 4 weeks. Three rats were euthanized at 2 weeks post-PRP injection, as well as three shams and a control rat. We collected traumatized and adjacent discs in test subjects, along with discs from control subjects. Each disk was sectioned axially and sagittally followed by histopathological and immunohistochemical analysis. Results The disks treated with PRP, both in the acute and delayed administration, had less degeneration than the sham group. The sham group had clear degenerative changes with near total loss of organizational structure, large amounts of empty space, fibrous (scar) tissue, and large numbers of inflammatory cells (Fig. 2). The PRP-treated group had annulus fibers that were damaged with some empty spaces and inflammatory cells. However, there was a maintenance of the ring structure and the nucleus pulposus, while having irregular borders and overall deceased size, appeared to have a healthy central portion (Fig. 3). Overall, the PRP treated group retained more normal morphologic features of the control disks (Fig. 1), contained fewer inflammatory cells than those not receiving PRP, and did not appear as damaged on MRI. The disk height did not seem to be greatly different between all three groups at the 2 weeks interval. Conclusion The needle puncture technique was validated as an effective method for creating a degenerative disk model. The administration of PRP has a protective effect on damaged disks and decreases the amount of inflammation at the 2-week interval and the 6-week interval. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest R. Mcguire Grant/Research support from: spinal usa, stryker, Consultant for: synthes Chen WH, Liu HY, Lo WC, et al. Intervertebral disk regeneration in an ex vivo culture system using mesenchymal stem cells and platelet-rich plasma. Biomaterials 2009;29:5523–33. Epub 2009 Jul 30 Sawamura K, Ikeda T, Nagae M, et al. Characterization of in vivo effects of platelet-rich plasma and biodegradable gelatin hydrogel microspheres on degenerated intervertebral discs. Tissue Engineering Part A 2009;12:3719–3727 Nagae M, Ikeda T, Mikami Y, et al. Intervertebral disk regeneration using platelet-rich plasma and biodegradable gelatin hydrogel microspheres. Tissue Engineering 2007;1:147–158 Akeda K, An HS, Pichika R, et al. Platelet-rich plasma (PRP) stimulates the extracellular matrix metabolism of porcine nucleus pulposus and anulus fibrosus cells cultured in alginate beads. Spine 2006;31(9):959–966 Chen WH, Lo WC, Lee JJ, et al. Tissue-engineered intervertebral disk and chondrogenesis using human nucleus pulposus regulated through TGF-beta1 in platelet-rich plasma. Journal of Cellular Physiology 2006;209(3):744–754 Elliot DM, Yerramalli CS, Beckstein JC, Boxberger JI, Johannessen W, Vresilovic EJ. The effect of relative needle diameter in puncture and sham injection animal models of degeneration. Spine 2008;33(6):588–596

Platelet-Rich Plasma Effects on Degenerative Disk Disease: Analysis of Histology and Imaging in an Animal Model

IntroductionThe current conservative and surgical modalities of treating degenerative disk disease are aimed at relief of symptoms. At this time, there is no cure for the degenerative process. Platelet-rich plasma (PRP) is a bioactive component of autologous whole blood. The indications for the use of PRP in the treatment of musculoskeletal diseases continue to expand. The mechanism of action for PRP is due to the release of healing factors from platelets. While clinical evidence strongly supports the use of PRP for certain conditions, the potential benefit of PRP in treating or preventing progression of degenerative disk disease has not been critically evaluated. There is a preliminary evidence that certain growth factors in PRP, such as PDGF and IGF-1, may be beneficial in slowing the progression of disease. The objective of this study is to analyze the early and late phase effects of PRP injection into the intervertebral disk using an animal model.Materials and MethodsAfter obtaining IRB and Institutional Animal Care and Use Committee (IACUC) approval, the L4-L5 intervertebral disk of adult Sprague Dawley rats were injured with a 21-gauge needle in 24 rats. After injury, animals received either an acute injection of PRP collected from matched donor animals, a delayed (2 week) injection of PRP after the degenerative process had commenced, or no further intervention (sham PRP). Magnetic resonance imaging was performed for each group at 0, 2, and 4 weeks. Three rats were euthanized at 2 weeks post-PRP injection, as well as three shams and a control rat. We collected traumatized and adjacent discs in test subjects, along with discs from control subjects. Each disk was sectioned axially and sagittally followed by histopathological and immunohistochemical analysis.ResultsThe disks treated with PRP, both in the acute and delayed administration, had less degeneration than the sham group. The sham group had clear degenerative changes with near total loss of organizational structure, large amounts of empty space, fibrous (scar) tissue, and large numbers of inflammatory cells (Fig. 2). The PRP-treated group had annulus fibers that were damaged with some empty spaces and inflammatory cells. However, there was a maintenance of the ring structure and the nucleus pulposus, while having irregular borders and overall deceased size, appeared to have a healthy central portion (Fig. 3). Overall, the PRP treated group retained more normal morphologic features of the control disks (Fig. 1), contained fewer inflammatory cells than those not receiving PRP, and did not appear as damaged on MRI. The disk height did not seem to be greatly different between all three groups at the 2 weeks interval.ConclusionThe needle puncture technique was validated as an effective method for creating a degenerative disk model. The administration of PRP has a protective effect on damaged disks and decreases the amount of inflammation at the 2-week interval and the 6-week interval.I confirm having declared any potential conflict of interest for all authors listed on this abstractYesDisclosure of Interest R. Mcguire Grant/Research support from: spinal usa, stryker, Consultant for: synthesChen WH, Liu HY, Lo WC, et al. Intervertebral disk regeneration in an ex vivo culture system using mesenchymal stem cells and platelet-rich plasma. Biomaterials 2009;29:5523–33. Epub 2009 Jul 30Sawamura K, Ikeda T, Nagae M, et al. Characterization of in vivo effects of platelet-rich plasma and biodegradable gelatin hydrogel microspheres on degenerated intervertebral discs. Tissue Engineering Part A 2009;12:3719–3727Nagae M, Ikeda T, Mikami Y, et al. Intervertebral disk regeneration using platelet-rich plasma and biodegradable gelatin hydrogel microspheres. Tissue Engineering 2007;1:147–158Akeda K, An HS, Pichika R, et al. Platelet-rich plasma (PRP) stimulates the extracellular matrix metabolism of porcine nucleus pulposus and anulus fibrosus cells cultured in alginate beads. Spine 2006;31(9):959–966Chen WH, Lo WC, Lee JJ, et al. Tissue-engineered intervertebral disk and chondrogenesis using human nucleus pulposus regulated through TGF-beta1 in platelet-rich plasma. Journal of Cellular Physiology 2006;209(3):744–754Elliot DM, Yerramalli CS, Beckstein JC, Boxberger JI, Johannessen W, Vresilovic EJ. The effect of relative needle diameter in puncture and sham injection animal models of degeneration. Spine 2008;33(6):588–596

A 3D environment for anulus fibrosus regeneration

Biological repair strategies for the treatment of degenerated intervertebral discs are of growing interest. In addition to the development of nucleus pulposus implants to restore disc height and relieve pain, there is growing demand for an appropriate method for reconstructing the anulus fibrosus (AF). The aim of this pilot study was to evaluate the applicability of a resorbable 3D polymer of pure polyglycolic acid (PGA) combined with hyaluronan for the use in cell-free and cell-based regeneration and repair of the AF. Adult human AF cells were expanded in vitro using human serum and rearranged three dimensionally in hyaluronan-PGA scaffolds that were stabilized with fibrin for in vitro analyses. The capacity of dedifferentiated AF cells to redifferentiate was evaluated after 2 weeks of culture, using propidium iodide/fluorescein diacetate staining, gene expression analysis of typical marker genes, and histological staining of proteoglycans. The propidium iodide/fluorescein diacetate staining demonstrated that vital human AF cells were evenly distributed within the construct. The induction of typical AF marker genes such as collagen Types I-III indicated the initiation of AF redifferentiation by 3D assembly in hyaluronan-PGA. Histological analysis of the constructs showed initial formation of an AF-like matrix comprising proteoglycans. The results suggest that the 3D arrangement of human AF cells in resorbable hyaluronan-PGA scaffolds cultured in the presence of human serum is an excellent system for AF cell redifferentiation.

Response of cyclic tensile strain on the human degenerative anulus fibrosus cells

Objective To investigate the effects of different cyclic tensile strain on the proliferation of human anulus fibrosus cells from degenerated discs.Methods Anulus fibrosus(AF) cells were isolated from a degenerated human IVD,expanded in monolayer,and cyclically strained for 3 hours,applying 0,5％,10％,15％ and 20％ strains at a frequency of 0.25 Hz with the use of the DioDynamic test instrument.The flow cytometry method was used to examine the Af cells proliferation at 24 hours following application of the cyclic tensile strains.Results The proliferative index (PI) increased with the magnitude value of cyclic tensile strain except 20％ group.The most significant increase of proliferation index were found in 15％ group.Conclusion There might be some corelationships between magnitude of cyclic tensile strain and the proliferation of the degenerative AF cells. Key words: Intervertebral disc degeneration ; Cyclic tensile strain ; Anulus fibrosus cells ; Proliferative ;

Identification of cell surface-specific markers to target human nucleus pulposus cells: Expression of carbonic anhydrase XII varies with age and degeneration

Back pain is a major cause of disability, affecting millions of people worldwide. One cause of axial back pain is degeneration of the nucleus pulposus (NP) of the intervertebral disc. This study was undertaken to investigate associations of NP cells with cell surface-specific proteins that differ from proteins in closely related cell types, i.e., intervertebral disc anulus fibrosus (AF) cells and articular cartilage (AC) chondrocytes, in order to identify potential surface molecules for directed delivery of therapeutic agents. We conducted a complementary DNA microarray analysis of 16 human samples from 6 donors, followed by gene list reduction using a systematic approach. Genes that were more highly expressed in NP than AC cells, contained transmembrane domains, and appeared attractive for targeting were assessed by quantitative reverse transcription-polymerase chain reaction (RT-PCR). As a viable candidate, carbonic anhydrase XII (CAXII) was analyzed at the protein level by immunohistochemistry and functional study. Microarray results demonstrated a clear divide between the AC and AF and between the AC and NP samples. However, the transcriptomic profile of AF and NP samples displayed a greater intersubject similarity. Of the 552 genes with up-regulated expression in NP cells, 90 contained transmembrane domains, and 28 were quantified by RT-PCR. Most intense CAXII labeling was observed in the NP of discs from young subjects and in degenerative tissue. CAXII may be considered for detection or targeting of degenerating disc cells. Furthermore, CAXII may be involved in pH regulation of NP cells. Its potential for directed delivery of regenerative factors and its functional role in NP cell homeostasis warrant further investigation.

Age-Related Variation in Cell Density of Human Lumbar Intervertebral Disc

changes in cell density of endplate (EP), nucleus pulposus (NP), and anulus fibrosus (AF) during ageing were systematically investigated in defined regions of interest in complete human motion segments. to elucidate cell density and total cell number in distinct anatomic regions of the intervertebral disc; to test effects of gender, level and age on cell density; and to correlate changes in cell density with histologic signs of disc degeneration. the available information on the cell density within intervertebral discs and its age-related changes is sparse. This knowledge, however, is a crucial prerequisite for cell-based tissue engineering approaches of the intervertebral disc. in 49 complete cross-sections from lumbar motion segments (newborn to 86 years) from 22 specimens, cell density was determined by the Abercrombie method in EP, NP, and AF, and total cell number was counted per region of interest. cell density in EP, NP, and AF decreased significantly from 0 to 16 years with the main changes occuring from 0 to 3 years for NP and AF. No significant variations were observed thereafter. We found a significant correlation of cell density and histologic degeneration score between 0 and 1, but not for scores >1. Gender and disc level did not influence cell density. This study provides data concerning the total number of cells in the various regions of the intervertebral disc for different age groups. This knowledge will be beneficial for cell-based treatment approaches, which may evolve in the future.

The response of human anulus fibrosus cells to cyclic tensile strain is frequency‐dependent and altered with disc degeneration

Mechanical loads are important for homeostasis of the intervertebral disc (IVD) cell matrix, with physiologic and nonphysiologic loads leading to matrix anabolism and catabolism, respectively. Previous investigations into the effects of load on disc cells have predominantly used animal models, with the limited number of human studies focusing primarily on nucleus pulposus cells. The aim of this study was to examine the effect of cyclic tensile strain (CTS) on human anulus fibrosus (AF) cells to ascertain whether the response was frequency-dependent and to compare AF cells derived from nondegenerated and degenerated tissue samples. AF cells were isolated from nondegenerated and degenerated human IVDs, expanded in monolayer, and cyclically strained for 20 minutes, applying 10% strain at a frequency of 1.0 Hz or 0.33 Hz with the use of a Flexcell strain device. Total RNA was extracted from the cells at baseline (control) and at 1, 3, and 24 hours following application of CTS. Real-time quantitative polymerase chain reaction was used to analyze gene expression of matrix proteins (aggrecan, type I collagen, and type II collagen) and enzymes (matrix metalloproteinases [MMPs] 3, 9, 13, and ADAMTS-4). The expression of catabolic genes (MMP-3 and ADAMTS-4) in AF cells derived from nondegenerated tissue decreased in response to 1.0 Hz of CTS, whereas changing the frequency to 0.33 Hz resulted in a shift toward matrix catabolism. Application of 1.0 Hz of CTS reduced anabolic gene expression (aggrecan and type I collagen) in AF cells derived from degenerated tissue, with 0.33 Hz of CTS resulting in increased catabolic gene expression. The response of human AF cells to CTS is frequency-dependent and is altered by degeneration.

Multipotential Differentiation of Human Anulus Fibrosus Cells

The existence of fibrocartilage, bone-like tissues, nerves, and blood vessels in the anulus fibrosus during intervertebral disc degeneration has been well documented. Migration of differentiated cells from outside the intervertebral disc has been hypothesized as a possible mechanism for the formation of these tissues. We hypothesized that the normal anulus fibrosus tissue contains multipotent progenitor cells, which are able to differentiate into cartilage and/or fibrocartilage cells, osteoblasts, neurons, and blood vessel cells. We isolated anulus fibrosus cells from the nondegenerative intervertebral discs of adolescent (thirteen to sixteen-year-old) patients with idiopathic scoliosis and cultured the cells in vitro in induction media containing different stimuli. Immunophenotypic analysis of cell surface markers was performed by flow cytometry. Expression of markers of adipogenesis, osteogenesis, chondrogenesis, neurogenesis, and differentiation into endothelial lineages was determined with use of immunostaining, cytohistological staining, and reverse transcription-polymerase chain reaction. Anulus fibrosus cells expressed several of the cell surface antigens that are sometimes associated with mesenchymal stem cells, including CD29, CD49e, CD51, CD73, CD90, CD105, CD166, CD184, and Stro-1, and two neuronal stem cell markers, nestin and neuron-specific enolase. Furthermore, varying the stimulants added to the induction media determined whether anulus fibrosus cells differentiated into adipocytes, osteoblasts, chondrocytes, neurons, or endothelial cells. Anulus fibrosus cells isolated from nondegenerative intervertebral discs can differentiate into adipocytes, osteoblasts, chondrocytes, neurons, and endothelial cells in vitro.

56. Primary Bovine Intervertebral Disc Cells Transduced with Adenovirus Overexpressing Twelve BMPs and SOX9 Maintain Their Chondrocyte-Like Phenotype

BACKGROUND CONTEXT: To identify individual gene therapy agents effective in IVD repair, a culture system that allows rapid assessment of transfection efficiency and, subsequently, gene expression and extracellular matrix production is essential. The traditional three-dimensional culture system is excellent for maintaining IVD cell phenotype and matrix production, but it is difficult to assess transduction efficiency without dissolving the culture sphere. In this study, we have used primary nucleus pulposus (NP) and anulus fibrosus (AF) cells cultured in monolayer.

Pericellular Matrix Mechanics in the Anulus Fibrosus Predicted by a Three-Dimensional Finite Element Model and In Situ Morphology

Anulus fibrosus (AF) cells have been demonstrated to exhibit dramatic differences in morphology and biologic responses to different types of mechanical stimuli. AF cells may reside as single cell, paired or multiple cells in a contiguous pericellular matrix (PCM), whose structure and properties are expected to have a significant influence on the mechanical stimuli that these cells may experience during physiologic loading of the spine, as well as in tissue degeneration and regeneration. In this study, a computational model was developed to predict the micromechanical stimuli, such as stress and strain, fluid pressure and flow, of cells and their surrounding PCM in the AF tissue using three-dimensional (3D) finite element models based on in situ morphology. 3D solid geometries of cell-PCM regions were registered from serial confocal images obtained from mature rat AF tissues by custom codes. Distinct cell-matrix units were modeled with a custom 3D biphasic finite element code (COMSOL Multiphysics), and simulated to experience uni-axial tensile strain along the local collagen fiber direction. AF cells were predicted to experience higher volumetric strain with a strain amplification ratio (relative to that in the extracellular matrix) of ~ 3.1 - 3.8 at equilibrium, as compared to the PCM domains (1.3 - 1.9). The strain concentrations were generally found at the cell/PCM interface and stress concentration at the PCM/ECM interface. Increased numbers of cells within a contiguous PCM was associated with an apparent increase of strain levels and decreased rate of fluid pressurization in the cell, with magnitudes dependent on the cell size, shape and relative position inside the PCM. These studies provide spatio-temporal information on micromechanics of AF cells in understanding the mechanotransduction in the intervertebral disc.