Tissue-engineered intervertebral disc and chondrogenesis using human nucleus pulposus regulated through TGF-beta1 in platelet-rich plasma

Human intervertebral disc (IVD) degeneration often initiated from the human nucleus pulposus (hNP) with aging leading to IVD destruction and extracellular matrix (ECM) depletion. Previously, we have successfully employed transforming growth factor-beta1 (TGF-beta1) to promote chondrogenesis of mesenchymal progenitor cells (MPCs) and immortalized human mesenchymal stem cells. In this study, we examine the role of TGF-beta1 in platelet-rich plasma (PRP) on disc regeneration, including proliferation, redifferentiation, and the reconstitution of tissue-engineered NP. hNP cells were isolated from volunteers with different ages and cultured in the presence of PRP. We found that the most effective concentration for hNP proliferation was 1 ng/ml TGF-beta1 in PRP, which was further applied in the following experiments. hNP cell proliferation in all age groups were increased time-dependently by PRP and cell morphologies showed aggregation. The mRNA of Sox9, type II collagen, and aggrecan were all significantly upregulated by PRP through RT-PCR. Glycosaminoglycan (GAG) accumulation reached the highest value at day 7 and continued to day 9 culture. PRP promoted NP regeneration via the Smad pathway was also determined and highly activated p-Smad2/3 at 30 min and continuously sustained to 120 min. Immunostaining of type II collagen indicates that PRP participates in chondrogenesis of tissue-engineered NP with collagen scaffolds. We concluded that growth factors in PRP can effectively react as a growth factor cocktail to induce hNP proliferation and differentiation, and also promote tissue-engineered NP formation. These findings are the first to demonstrate that PRP might be a therapeutic candidate for prevention of disc degeneration.     

The role of TGF‐β1/Smad2/3 pathway in platelet‐rich plasma in retarding intervertebral disc degeneration

Recent studies have suggested that platelet‐rich plasma (PRP) injections are an effective way to retard intervertebral disc degeneration, but the mechanism of action is unclear. Activated platelets release some growth factors, such as transforming growth factor‐β1 (TGF‐β1), which positively modulate the extracellular matrix of nucleus pulposus cells. The purpose of this study was to explore the mechanism underlying the PRP‐mediated inhibition of intervertebral disc degeneration. In an in vitro study, we found that the proliferation of nucleus pulposus cells was greatly enhanced with 2.5% PRP treatment. The TGF‐β1 concentration was much higher after PRP treatment. PRP administration effectively increased the collagen II, aggrecan and sox‐9 mRNA levels and decreased collagen X levels. However, Western blotting demonstrated that specifically inhibiting TGF‐β1 signalling could significantly prevent nucleus pulpous cellular expression of Smad2/3 and matrix protein. In a rabbit study, magnetic resonance imaging revealed significant recovery signal intensity in the intervertebral discs of the PRP injection group compared with the very low signal intensity in the control groups. Histologically, the PRP plus inhibitor injection group had significantly lower expression levels of Smad2/3 and collagen II than the PRP group. These results demonstrated that a high TGF‐β1 content in the platelets retarded disc degeneration in vitro and in vivo. Inhibiting the TGF‐β1/Smad2/3 pathway could prevent this recovery by inactivating Smad2/3 and down‐regulating the extracellular matrix. Therefore, the TGF‐β1/Smad2/3 pathway might play a critical role in the ability of PRP to retard intervertebral disc degeneration.     

Cartilage derived morphogenetic protein 2 – A potential therapy for intervertebral disc regeneration?

Low back pain is amongst the top ten risk factors that contribute to disability, ranking higher than diabetes and mental health disease globally as a contributor to years lost to disability (YLD), and escalating as Western societies age. Abundant evidence suggests that intervertebral disc (IVD) damage is central to the origin of pain in the spine. IVD degeneration involves the progressive deterioration of the highly organized disc tissue extracellular matrix, losing its elasticity and hence its' cushioning ability for the spine.Cartilage derived morphogenetic protein-2 (CDMP2) is a small peptide morphogen. Naturally occurring mutations segregate with skeletal defects in IVD development. CDMP2 signalling influences chondrogenic tissue determination, retards osteogenic tissue development and is crucial to early dorso–ventral axis defining events in zebrafish and Xenopus laevis.The potential of biological treatments to offer cutting edge early intervention, tissue regeneration and to preserve spinal motion segments shows great promise. The unique qualities of CDMP2 in IVD tissue formation, delineating discal matrix from vertebral bone, may prove adaptable in therapeutic applications to early discal degeneration.Here we explore the prevalence and origin of backache, the biology of CDMP2 and its potential application as an early intervention to arrest the disc degeneration sequelae.     

Human disc degeneration is associated with increased MMP 7 expression.

During intervertebral disc (IVD) degeneration, normal matrix synthesis decreases and degradation of disc matrix increases. A number of proteases that are increased during disc degeneration are thought to be involved in its pathogenesis. Matrix metalloproteinase 7 (MMP 7) (Matrilysin, PUMP-1) is known to cleave the major matrix molecules found within the IVD, i.e., the proteoglycan aggrecan and collagen type II. To date, however, it is not known how its expression changes with degeneration or its exact location. We investigated the localization of MMP 7 in human, histologically graded, nondegenerate, degenerated and prolapsed discs to ascertain whether MMP 7 is up-regulated during disc degeneration. Samples of human IVD tissue were fixed in neutral buffered formalin, embedded in paraffin, and sections stained with hematoxylin and eosin to score the degree of morphological degeneration. Immunohistochemistry was performed to localize MMP 7 in 41 human IVDs with varying degrees of degeneration. We found that the chondrocyte-like cells of the nucleus pulposus and inner annulus fibrosus were MMP 7 immunopositive; little immunopositivity was observed in the outer annulus. Nondegenerate discs showed few immunopositive cells. A significant increase in the proportion of MMP 7 immunopositive cells was seen in the nucleus pulposus of discs classified as showing intermediate levels of degeneration and a further increase was seen in discs with severe degeneration. Prolapsed discs showed more MMP 7 immunopositive cells compared to nondegenerated discs, but fewer than those seen in cases of severe degeneration.     

Human disc degeneration is associated with increased MMP 7 expression

During intervertebral disc (IVD) degeneration, normal matrix synthesis decreases and degradation of disc matrix increases. A number of proteases that are increased during disc degeneration are thought to be involved in its pathogenesis. Matrix metalloproteinase 7 (MMP 7) (Matrilysin, PUMP-1) is known to cleave the major matrix molecules found within the IVD, i.e., the proteoglycan aggrecan and collagen type II. To date, however, it is not known how its expression changes with degeneration or its exact location. We investigated the localization of MMP 7 in human, histologically graded, nondegenerate, degenerated and prolapsed discs to ascertain whether MMP 7 is up-regulated during disc degeneration. Samples of human IVD tissue were fixed in neutral buffered formalin, embedded in paraffin, and sections stained with hematoxylin and eosin to score the degree of morphological degeneration. Immunohistochemistry was performed to localize MMP 7 in 41 human IVDs with varying degrees of degeneration. We found that the chondrocyte-like cells of the nucleus pulposus and inner annulus fibrosus were MMP 7 immunopositive; little immunopositivity was observed in the outer annulus. Nondegenerate discs showed few immunopositive cells. A significant increase in the proportion of MMP 7 immunopositive cells was seen in the nucleus pulposus of discs classified as showing intermediate levels of degeneration and a further increase was seen in discs with severe degeneration. Prolapsed discs showed more MMP 7 immunopositive cells compared to nondegenerated discs, but fewer than those seen in cases of severe degeneration.     

Human disc degeneration is associated with increased MMP 7 expression

During intervertebral disc (IVD) degeneration, normal matrix synthesis decreases and degradation of disc matrix increases. A number of proteases that are increased during disc degeneration are thought to be involved in its pathogenesis. Matrix metalloproteinase 7 (MMP 7) (Matrilysin, PUMP-1) is known to cleave the major matrix molecules found within the IVD, i.e., the proteoglycan aggrecan and collagen type II. To date, however, it is not known how its expression changes with degeneration or its exact location. We investigated the localization of MMP 7 in human, histologically graded, nondegenerate, degenerated and prolapsed discs to ascertain whether MMP 7 is up-regulated during disc degeneration. Samples of human IVD tissue were fixed in neutral buffered formalin, embedded in paraffin, and sections stained with hematoxylin and eosin to score the degree of morphological degeneration. Immunohistochemistry was performed to localize MMP 7 in 41 human IVDs with varying degrees of degeneration. We found that the chondrocyte-like cells of the nucleus pulposus and inner annulus fibrosus were MMP 7 immunopositive; little immunopositivity was observed in the outer annulus. Nondegenerate discs showed few immunopositive cells. A significant increase in the proportion of MMP 7 immunopositive cells was seen in the nucleus pulposus of discs classified as showing intermediate levels of degeneration and a further increase was seen in discs with severe degeneration. Prolapsed discs showed more MMP 7 immunopositive cells compared to nondegenerated discs, but fewer than those seen in cases of severe degeneration.     

Physiological Loading Can Restore the Proteoglycan Content in a Model of Early IVD Degeneration

A hallmark of early IVD degeneration is a decrease in proteoglycan content. Progression will eventually lead to matrix degradation, a decrease in weight bearing capacity and loss of disc height. In the final stages of IVD degradation, fissures appear in the annular ring allowing extrusion of the NP. It is crucial to understand the interplay between mechanobiology, disc composition and metabolism to be able to provide exercise recommendations to patients with early signs of disc degeneration. This study evaluates the effect of physiological loading compared to no loading on matrix homeostasis in bovine discs with induced degeneration. Bovine discs with trypsin-induced degeneration were cultured for 14 days in a bioreactor under dynamic loading with maintained metabolic activity. Chondroadherin abundance and structure was used to confirm that a functional matrix was preserved in the chosen loading environment. No change was observed in chondroadherin integrity and a non-significant increase in abundance was detected in trypsin-treated loaded discs compared to unloaded discs. The proteoglycan concentration in loaded trypsin-treated discs was significantly higher than in unloaded disc and the newly synthesised proteoglycans were of the same size range as those found in control samples. The proteoglycan showed an even distribution throughout the NP region, similar to that of control discs. Significantly more newly synthesised type II collagen was detected in trypsin-treated loaded discs compared to unloaded discs, demonstrating that physiological load not only stimulates aggrecan production, but also that of type II collagen. Taken together, this study shows that dynamic physiological load has the ability to repair the extracellular matrix depletion typical of early disc degeneration.     

Association of the Myosin-binding Subunit of Myosin Phosphatase and Moesin: Dual Regulation of Moesin Phosphorylation by Rho-associated Kinase and Myosin Phosphatase

To examine the role of bone morphogenetic protein (BMP) signaling in chondrocytes during endochondral ossification, the dominant negative (DN) forms of BMP receptors were introduced into immature and mature chondrocytes isolated from lower and upper portions of chick embryo sternum, respectively. We found that control sternal chondrocyte populations expressed type IA, IB, and II BMP receptors as well as BMP-4 and -7. Expression of a DN-type II BMP receptor (termed DN-BMPR-II) in immature lower sternal (LS) chondrocytes led to a loss of differentiated functions; compared with control cells, the DN-BMPR- II–expressing LS chondrocytes proliferated more rapidly, acquired a fibroblastic morphology, showed little expression of type II collagen and aggrecan genes, and upregulated type I collagen gene expression. Expression of DN-BMPR-II in mature hypertrophic upper sternal (US) chondrocytes caused similar effects. In addition, the DN-BMPR-II–expressing US cells exhibited little alkaline phosphatase activity and type X collagen gene expression, while the control US cells produced both alkaline phosphatase and type X collagen. Both DN-BMPR-II–expressing US and LS chondrocytes failed to respond to treatment with BMP-2 . When we examined the effects of DN forms of types IA and IB BMP receptors, we found that DN-BMPR-IA had little effect, while DN-BMPR-IB had similar but weaker effects compared with those of DN-BMPR-II. We conclude that BMP signaling, particularly that mediated by the type II BMP receptor, is required for maintenance of the differentiated phenotype, control of cell proliferation, and expression of hypertrophic phenotype.     

软骨寡聚基质蛋白过表达对BMP-2诱导骨髓间充质干细胞分化的影响

目的：研究软骨寡聚基质蛋白（cartilage oligomeric matrix protein,COMP）过表达对BMP-2诱导骨髓间充质干细胞成骨及成软骨分化的影响。方法：BMP-2诱导骨髓间充质干细胞分化,通过脂质体转染含人COMP基因的质粒使骨髓间充质干细胞过表达COMP,采用实时定量PCR和Western blotting分析COMP基因过表达、成骨相关基因Ⅰ型胶原、RUNX2、骨钙蛋白以及成软骨相关基因Ⅱ型胶原、SOX9、蛋白聚糖、X型胶原的表达变化;通过茜素红染色观察成骨终末阶段矿化结节的生成情况,阿利新蓝染色观察细胞基质蛋白多糖的合成情况。结果：质粒转染后骨髓间充质干细胞COMP基因蛋白和mRNA表达水平显著提高（P<0.05）。COMP基因过表达后,成骨标记基因RUNX2、Ⅰ型胶原（Col1a1）mRNA水平均显著低于对照组（P<0.05）,RUNX2、骨钙蛋白（Osteocalcin）蛋白表达水平明显低于对照组（P<0.05）,而成软骨标记基因SOX9、蛋白聚糖（Aggrecan）mRNA水平均显著高于对照组（P<0.05）,SOX9、Ⅱ型胶原（Col2a1）蛋白表达均明显多于对照组（P<0.05）。细胞成骨茜素红染色弱于对照组,而阿利新蓝染色强于对照组。过表达组细胞X型胶原（Col10a1）基因表达显著低于对照组（P<0.05）,结论：骨髓间充质干细胞COMP基因过表达可抑制BMP-2诱导其成骨分化,促进骨髓间充质干细胞成软骨分化,并抑制软骨细胞的成熟肥大,为软骨组织工程研究提供新的方向。     

Short aggrecan gene repetitive alleles associated with lumbar degenerative disc disease in Turkish patients

We investigated a possible association between aggrecan gene polymorphism and lumbar degenerative disc disease in Turkish patients. One hundred 20-30-year-old patients with or without low back pain were selected for the study. Lumbar magnetic resonance imaging was performed on all patients. The patient group had low back pain clinically and degenerative disc disease radiographically. The control group included patients with and without low back pain: all were negative radiographically for degenerative disc disease. Genomic DNA was extracted from all participants. A PCR assay were used to evaluate variable number of tandem repeat polymorphism of aggrecan gene alleles to determine if there was any correlation with degenerative disc disease. Significant associations were found between short repeated alleles of the aggrecan gene and severe disc degeneration. A significant association was also found between short repeated alleles of the aggrecan gene and multilevel disc herniation as well as extrusion and sequestration types of disc herniation. In Turkish population, short repeated alleles of the aggrecan gene are associated with increased disc degeneration and disc herniation.     

Cell shape and gene expression in human intervertebral disc cells: in vitro tissue engineering studies

The objective of the present study was to examine the relation between gene expression and the shape of human intervertebral disc cells cultured in vitro in three-dimensional (3D) scaffolds. Disc cells from 19 subjects were seeded into either a collagen sponge or collagen gel and cultured for 10 days. In situ hybridization was performed on serial sections of paraffin embedded specimens and assessed for expression of selected genes important for extracellular matrix formation: Types I and II collagen, aggrecan and chondroitin-6 sulfotransferase. Rounded cells grown in collagen gel showed expression of Types I and II collagen, aggrecan and chondroitin-6 sulfotransferase; expression of these genes was absent in spindle shaped cells. Cells in the collagen sponge that lay on the sponge margin were frequently spindle shaped; these cells expressed type I collagen, but not type II collagen, aggrecan or chondroitin-6 sulfotransferase. Results presented here provide novel data concerning disc cell gene expression with collagen 3D constructs. This information is useful for future tissue engineering studies that have the challenging goal of selectively modulating gene expression.     

Cell shape and gene expression in human intervertebral disc cells: in vitro tissue engineering studies

The objective of the present study was to examine the relation between gene expression and the shape of human intervertebral disc cells cultured in vitro in three-dimensional (3D) scaffolds. Disc cells from 19 subjects were seeded into either a collagen sponge or collagen gel and cultured for 10 days. In situ hybridization was performed on serial sections of paraffin embedded specimens and assessed for expression of selected genes important for extracellular matrix formation: Types I and II collagen, aggrecan and chondroitin-6 sulfotransferase. Rounded cells grown in collagen gel showed expression of Types I and II collagen, aggrecan and chondroitin-6 sulfotransferase; expression of these genes was absent in spindle shaped cells. Cells in the collagen sponge that lay on the sponge margin were frequently spindle shaped; these cells expressed type I collagen, but not type II collagen, aggrecan or chondroitin-6 sulfotransferase. Results presented here provide novel data concerning disc cell gene expression with collagen 3D constructs. This information is useful for future tissue engineering studies that have the challenging goal of selectively modulating gene expression.     

Modeling the role of IGF-1 on extracellular matrix biosynthesis and cellularity in intervertebral disc

The insulin-like growth factor-1 (IGF-1) is a well-known anabolic agent for intervertebral disc (IVD), promoting both proteoglycan (PG) biosynthesis and cell proliferation. Accordingly, it is believed that IGF-1 may play a central role in IVD homeostasis. Furthermore, the exogenous administration of IGF-1 has been proposed as a possible therapeutic strategy for disc degeneration. The objectives of this study were to develop a new computational framework for describing the mechanisms regulating IGF-mediated homeostasis in IVD, and to apply this numerical tool for investigating the effectiveness of exogenous administration of IGF-1 for curing disc degeneration. A diffusive–reactive model was developed for describing competitive binding of IGF-1 to its binding proteins and cell surface receptors, with the latter reaction initiating the intracellular signaling mechanism leading to PG production and cell proliferation. Because PG production increases cell metabolic rate, and cell proliferation increases nutritional demand, nutrients transport and metabolism were also included into the model, and co-regulated, together with IGF-1, IVD cellularity. The sustainability and the effectiveness of IGF-mediated anabolism were investigated for conditions of pathologically insufficient nutrient supply, and for the case of exogenous administration of IGF-1 to degenerated IVD. Results showed that pathological nutrients deprivation, by decreasing cellularity, caused a reduction of PG biosynthesis. Also, exogenous administration of IGF-1 was only beneficial in well-nourished regions of IVD, and exacerbated cell mortality in malnourished regions. These findings remark the central role of nutrition in IVD health, and suggest that adequate nutritional supply is paramount for achieving a successful IGF-based therapy for disc degeneration.     

Accelerated intervertebral disc degeneration in scoliosis versus physiological ageing develops against a background of enhanced anabolic gene expression

Molecular consequences of long-term deformation and altered mechanical loading of intervertebral disc (IVD) tissue in scoliosis have yet to be elucidated. We hypothesized that histological disc degeneration is faster in scoliosis than in normal ageing and that this is reflected by an altered gene expression profile. A semiquantitative histodegeneration score (HDS) revealed significantly enhanced degeneration in scoliosis (HDS 5.3) versus age-matched control IVDs (HDS 2.25; p = 0.001). Gene expression analysis by cDNA array and RT-PCR demonstrated higher mRNA levels for extracellular-matrix molecules like aggrecan, biglycan, decorin, lumican, chondromodulin, and COL2A1 in scoliotic discs versus normal discs of identical degeneration score. No differences were evident for catabolic molecules like MMP3, MMP13, MMP17, and TIMP1. In sum, morphologic disc degeneration was accelerated by about 2 decades in scoliosis versus physiological ageing and developed against a background of stronger anabolic matrix metabolism at younger age or in response to the altered mechanical environment of the tissue.     

Functions of transforming growth factor-beta family type I receptors and Smad proteins in the hypertrophic maturation and osteoblastic differentiation of chondrocytes

We investigated the effects of bone morphogenetic protein (BMP)-2, a member of the transforming growth factor-beta superfamily, on the regulation of the chondrocyte phenotype, and we identified signaling molecules involved in this regulation. BMP-2 triggers three concomitant responses in mouse primary chondrocytes and chondrocytic MC615 cells. First, BMP-2 stimulates expression or synthesis of type II collagen. Second, BMP-2 induces expression of molecular markers characteristic of pre- and hypertrophic chondrocytes, such as Indian hedgehog, parathyroid hormone/parathyroid hormone-related peptide receptor, type X collagen, and alkaline phosphatase. Third, BMP-2 induces osteocalcin expression, a specific trait of osteoblasts. Constitutively active forms of transforming growth factor-beta family type I receptors and Smad proteins were overexpressed to address their role in this process. Activin receptor-like kinase (ALK)-1, ALK-2, ALK-3, and ALK-6 were able to reproduce the hypertrophic maturation of chondrocytes induced by BMP-2. In addition, ALK-2 mimicked further the osteoblastic differentiation of chondrocytes induced by BMP-2. In the presence of BMP-2, Smad1, Smad5, and Smad8 potentiated the hypertrophic maturation of chondrocytes, but failed to induce osteocalcin expression. Smad6 and Smad7 impaired chondrocytic expression and osteoblastic differentiation induced by BMP-2. Thus, our results indicate that Smad-mediated pathways are essential for the regulation of the different steps of chondrocyte and osteoblast differentiation and suggest that additional Smad-independent pathways might be activated by ALK-2.