Genetic variants and altered expression of SERPINF1 confer disease susceptibility in patients with otosclerosis.

Otosclerosis (OTSC) is a focal and diffuse bone disorder of the human middle ear characterized by abnormal bone growth and deposition at the stapes' footplate. This hinders the transmission of acoustic waves to the inner ear leading to subsequent conductive hearing loss. The plausible convections for the disease are genetic and environmental factors with yet an unraveled root cause. Recently, exome sequencing of European individuals with OTSC revealed rare pathogenic variants in the Serpin Peptidase Inhibitor, Clade F (SERPINF1) gene. Here, we sought to investigate the causal variants of SERPINF1 in the Indian population. The gene and protein expression was also evaluated in otosclerotic stapes to ameliorate our understanding of the potential effect of this gene in OTSC. A total of 230 OTSC patients and 230 healthy controls were genotyped by single-strand conformational polymorphism and Sanger sequencing methods. By comparing the case controls, we identified five rare variants (c.72 C > T, c.151 G > A, c.242 C > G, c.823 A > T, and c.826 T > A) only in patients. Four variants c.390 T > C (p = 0.048), c.440-39 C > T (p = 0.007), c.643 + 9 G > A (p = 0.035), and c.643 + 82 T > C (p = 0.005) were found to be significantly associated with the disease. Down-regulation of SERPINF1 transcript level in otosclerotic stapes was quantified by qRT-PCR, ddPCR and further validated by in situ hybridization. Similarly, reduced protein expression was observed by immunohistochemistry and immunofluorescence in otosclerotic stapes that corroborate with immunoblotting of patients' plasma samples. Our findings identified that SERPINF1 variants are associated with the disease. Furthermore, reduced expression of SERPINF1 in otosclerotic stapes might contribute to OTSC pathophysiology.

Effect of Aerobic/Strength Training on RANKL Gene DNA Methylation Levels.

BACKGROUND: The osteoclastogenesis RANKL gene plays a key role in bone remodeling. The hypomethylation of its promoter region may cause osteoporosis. The present study aimed to elucidate the influence of physical activity on DNA methylation changes of RANKL promoter cytosine-phosphate-guanine (CpG)-rich region in active and sedentary adults and to assess the effect of aerobic and strength training on RANKL DNA methylation changes among Tunisian-North African adults. METHODS: A total of 104 participants including 52 adults (58% males and 42% females) and 52 adults (31% males and 69% females) were recruited for the observational and interventional part of the study, respectively. The intervention consisted of 12 weeks of aerobic training (30 min/session) followed by 10 minutes of strengthening exercises. All participants completed the International Physical Activity Questionnaire and provided blood samples for quantitative methylation-specific polymerase chain reaction (PCR) analysis. RESULTS: The study revealed a significant difference (P = 6 × 10-10) in the methylation level of the RANKL promoter region between active and sedentary adults, with a 6.68-fold increase observed in the active group. After the intervention, both the trained (P = 41 × 10-5) and untrained (P = .002) groups displayed high methylation levels in the RANKL promoter region. In addition, the trained group exhibited significant improvements in heart rate (P = 2.2 × 10-16), blood pressure (P = 39 × 10-3), maximal oxygen uptake (P = 1.5 × 10-7), and fat mass (P = 7 × 10-4). CONCLUSION: Exploring epigenetic modifications in the RANKL promoter region may contribute to a more comprehensive understanding of the complexity of osteoporosis. This suggests that aerobic/strength training could potentially improve the bone system, reducing its vulnerability to osteoporosis by increasing RANKL DNA methylation.

The risks of RELN polymorphisms and its expression in the development of otosclerosis.

Otosclerosis (OTSC) is the primary form of conductive hearing loss characterized by abnormal bone remodelling within the otic capsule of the human middle ear. A genetic association of the RELN SNP rs3914132 with OTSC has been identified in European population. Previously, we showed a trend towards association of this polymorphism with OTSC and identified a rare variant rs74503667 in a familial case. Here, we genotyped these variants in an Indian cohort composed of 254 OTSC cases and 262 controls. We detected a significant association of rs3914132 with OTSC (OR = 0.569, 95%CI = 0.386-0.838, p = 0.0041). To confirm this finding, we completed a meta-analysis which revealed a significant association of the rs3914132 polymorphism with OTSC (Z = 6.707, p<0.0001) across different ethnic populations. Linkage analysis found the evidence of linkage at RELN locus (LOD score 2.1059) in the OTSC family which has shown the transmission of rare variant rs74503667 in the affected individuals. To understand the role of RELN and its receptors in the development of OTSC, we went further to perform a functional analysis of RELN/reelin. Here we detected a reduced RELN (p = 0.0068) and VLDLR (p = 0.0348) mRNA levels in the otosclerotic stapes tissues. Furthermore, a reduced reelin protein expression by immunohistochemistry was confirmed in the otosclerotic tissues. Electrophoretic mobility shift assays for rs3914132 and rs74503667 variants revealed an altered binding of transcription factors in the mutated sequences which indicates the regulatory role of these variations in the RELN gene regulation. Subsequently, we showed by scanning electron microscopy a change in stapes bone morphology of otosclerotic patients. In conclusion, this study evidenced that the rare variation rs74503667 and the common polymorphism rs3914132 in the RELN gene and its reduced expressions that were associated with OTSC.

Genetic Association of rs1021188 and DNA Methylation Signatures of TNFSF11 in the Risk of Conductive Hearing Loss.

Otosclerosis (OTSC) is a complex bone disorder of the otic capsule, which causes conductive hearing impairment in human adults. The dysregulation of the signaling axis mediated by the receptor activator of nuclear factor-kappa-B (RANK), RANK ligand (RANKL), and osteoprotegerin has been widely attributed to the context of metabolic bone disorders. While genetic associations and epigenetic alterations in the TNFSF11 gene (RANKL) have been well-linked to metabolic bone diseases of the skeleton, particularly osteoporosis, they have never been addressed in OTSC. This study aimed to assess whether the genetic association of rs1021188 polymorphism in the upstream of TNFSF11 and the DNA methylation changes in its promoter CpG-region reveal the susceptibility of OTSC. Peripheral blood DNA samples were collected from unrelated Tunisian-North African subjects for genotyping (109 cases and 120 controls) and for DNA methylation analysis (40 cases and 40 controls). The gender-stratified analysis showed that the TNFSF11 rs1021188 C/T was associated with OTSC in men (p = 0.023), but not in women (p = 0.458). Individuals with CC genotype were more susceptible to OTSC, suggesting an increased risk to disease development. Using publicly available data, the rs1021188 was within a cluster grouping the subpopulations with African ethnicity. Moreover, 26 loci in the TNFSF11 gene were in linkage disequilibrium with rs1021188, revealing relative similarities between different populations. Significant differences in both DNA methylation and unmethylation status were detected with 4.53- and 4.83-fold decreases in the global DNA methylation levels in female and male OTSC groups, respectively. These changes could contribute to an increased risk of OTSC development. Bioinformatic analyses indicated that each of the rs1021188 variations and the DNA methylation changes in the promoter CpG-sites within TNFSF11 may play an important role in its transcription regulation. To our knowledge, this is the first study that investigates an independent effect of the rs1021188 polymorphism and DNA hypomethylation of TNFSF11 promoter in OTSC. Genetic and epigenetic changes in the regulatory regions of TNFSF11 could offer new molecular insights into the understanding of the complexity of OTSC.

Inhibition of the extracellular signal-regulated kinase pathway reduces the inflammatory component in nucleus pulposus cells.

Intervertebral disc (IVD) degeneration is a spinal disorder that triggers an inflammatory response and subsequent development of spinal pseudoarthrosis. The aim of the present study is to elucidate the role of the extracellular signal-regulated kinase (ERK) pathway in inflammation-induced IVD cells. Inflammatory human nucleus pulposus (NP) cells (NPCs) were induced using tumor necrosis factor-α and the ERK pathway was blocked using a selective molecule-based inhibitor U0126. Gene expression of catabolic and anabolic markers, proinflammatory, and NPCs markers was investigated. The enzymatic activity of matrix metalloproteinases (MMP)2/MMP9 was determined by gelatin zymography and nitrite production was assessed by Griess reaction. The NPC metabolic activity and viability were assessed using resazurin sodium-salt and live/dead assays, and subsequently, the specificity of U0126 on ERK1/2 signaling was determined. The catabolic enzyme MMP3 (p = 0.0001) and proinflammatory cytokine interleukin 6 (p = 0.036) were downregulated by U0126 in NPCs under inflammatory conditions. A significant increase of the cytokeratin 19 (p = 0.0031) was observed, suggesting a partial and possible recovery of the NP phenotype. U0126 does not seem to have an effect on prostaglandin production, aggrecanases, or other anabolic genes. We confirmed that U0126 selectively blocks the ERK phosphorylation and only affects the cell metabolic activity without the reduction of viable cells. Inhibition of ERK signaling downregulates important metalloproteinases and proinflammatory cytokines, and upregulates some NP markers, suggesting its potential to treat IVD degeneration.

A Novel Bioreactor System Capable of Simulating the In Vivo Conditions of Synovial Joints.

Any significant in vitro evaluation of cartilage tissue engineering and cartilage repair strategies has to be performed under the harsh conditions encountered in vivo within synovial joints. To this end, we have developed a novel automated physiological robot reactor system (PRRS) that is capable of recapitulating complex physiological motions and load patterns within an environment similar to that found in the human knee. The PRRS consists of a mechanical stimulation unit (MSU) and an automatic sample changer (ASC) within an environment control box in which the humidity, temperature, and gas composition are tightly regulated. The MSU has three linear (orthogonal) axes and one rotational degree of freedom (around the z-axis). The ASC provides space for up to 24 samples, which can be allocated to individual stimulation patterns. Cell-seeded scaffolds and ex vivo tissue culture systems were established to demonstrate the applicability of the PRRS to the investigation of the effect of load and environmental conditions on engineering and maintenance of articular cartilage in vitro. The bioreactor is a flexible system that has the potential to be applied for culturing connective tissues other than cartilage, such as bone and intervertebral disc tissue, even though the mechanical and environmental parameters are very different.

Osteoprotegerin gene polymorphisms and otosclerosis: an additional genetic association study, multilocus interaction and meta-analysis.

BACKGROUND: Otosclerosis (OTSC) is among the most common causes of a late-onset hearing loss in adults and is characterized by an abnormal bone growth in the otic capsule. Alteration in the osteoprotegerin (OPG) expression has been suggested in the implication of OTSC pathogenesis. METHODS: A case-control association study of rs2228568, rs7844539, rs3102734 and rs2073618 single nucleotide polymorphisms (SNPs) in the OPG gene was performed in a Tunisian-North African population composed of 183 unrelated OTSC patients and 177 healthy subjects. In addition, a multilocus association and a meta-analysis of existing studies were conducted. RESULTS: Rs3102734 (p = 0.013) and rs2073618 (p = 0.007) were significantly associated with OTSC, which were predominantly detected in females after multiple corrections. Among the OPG studied SNPs, the haplotypes A-A-C-G (p = 0.0001) and A-A-C-C (p = 0.0004) were significantly associated with OTSC in females. Multilocus association revealed that the SNPs: rs2073618 in OPG, rs1800472 in TGFß1, rs39335, rs39350 and rs39374 in RELN, and rs494252 in chromosome 11 showed significant OTSC-associated alleles in Tunisian individuals. In addition, meta-analysis of the rs2073618 SNP in Tunisian, Indian and Italian populations revealed evidence of an association with OTSC (OR of 0.826, 95% CI [0.691-0.987], p = 0.035). CONCLUSIONS: Our findings suggest that rs3102734 and rs2073618 variants are associated with OTSC in North African ethnic Tunisian population. Meta-analysis of the rs2073618 in three different ethnic population groups indicated an association with OTSC.

Evaluation of the Genetic Association and mRNA Expression of the COL1A1, BMP2, and BMP4 Genes in the Development of Otosclerosis.

Background: Otosclerosis (OTSC) is a genetically heterogeneous disorder, characterized by abnormal bone growth in the middle ear, affecting the stapes bone. Previous studies have shown that single nucleotide polymorphisms (SNPs) of the COL1A1, BMP2, and BMP4 genes are linked to susceptibility of OTSC, musculoskeletal degenerative diseases, and bone remodeling. Aims: To evaluate the genetic association and expression levels of COL1A1, BMP2, and BMP4 genes with OTSC in the Indian population. Methods: A total of 320 otosclerotic and 320 control samples were screened for four SNPs (rs1107946, rs11327935, rs2269336, and rs1800012) of the COL1A1 gene; rs3178250 of the BMP2 gene; and rs17563 of the BMP4 gene using single-strand conformation polymorphism analysis, and restriction fragment length polymorphism analyses. Genotypic, haplotypic, and linkage disequilibrium analyses were performed to assess the potential associations of these SNPs with OTSC. COL1A1, BMP2, and BMP4 mRNA expression levels were analyzed by semiquantitative RT-PCR and real-time PCR. Results: Genotypes of two SNPs, rs1800012 and rs17563, were found to be associated with OTSC (the rs1800012 GT genotype, p = 0.0022, OR = 0.481; and the rs17563 TC genotype, p = 0.0225, OR = 1.471). Haplotypic analyses revealed that the COL1A1 haplotype G-T-C-T (p = 0.021) was significantly increased among controls. Functional studies revealed an unexpected decrease in mRNA expression of COL1A1 but an increased expression of the BMP2 and BMP4 genes in otosclerotic stapes tissues. Conclusions: Our findings suggest that OTSC is a heterogeneous disorder, but that the GT genotype of the rs1800012 locus is protective and that the TC genotype at the rs17563 locus is a risk factor. In addition, our studies indicate that changes in the expression of the COL1A1, BMP2, and BMP4 genes may contribute to the genetic susceptibility of OTSC by regulating their mRNA levels.

Application of Cytokines of the Bone Morphogenetic Protein (BMP) Family in Spinal Fusion - Effects on the Bone, Intervertebral Disc and Mesenchymal Stromal Cells.

Low back pain is a prevalent socio-economic burden and is often associated with damaged or degenerated intervertebral discs (IVDs). When conservative therapy fails, removal of the IVD (discectomy), followed by intersomatic spinal fusion, is currently the standard practice in clinics. The remaining space is filled with an intersomatic device (cage) and with bone substitutes to achieve disc height compensation and bone fusion. As a complication, in up to 30% of cases, spinal non-fusions result in a painful pseudoarthrosis. Bone morphogenetic proteins (BMPs) have been clinically applied with varied outcomes. Several members of the BMP family, such as BMP2, BMP4, BMP6, BMP7, and BMP9, are known to induce osteogenesis. Questions remain on why hyper-physiological doses of BMPs do not show beneficial effects in certain patients. In this respect, BMP antagonists secreted by mesenchymal cells, which might interfere with or block the action of BMPs, have drawn research attention as possible targets for the enhancement of spinal fusion or the prevention of non-unions. Examples of these antagonists are noggin, gremlin1 and 2, chordin, follistatin, BMP3, and twisted gastrulation. In this review, we discuss current evidence of the osteogenic effects of several members of the BMP family on osteoblasts, IVD cells, and mesenchymal stromal cells. We consider in vitro and in vivo studies performed in human, mouse, rat, and rabbit related to BMP and BMP antagonists in the last two decades. We give insights into the effects that BMP have on the ossification of the spine. Furthermore, the benefits, pitfalls, and possible safety concerns using these cytokines for the improvement of spinal fusion are discussed.

Fluorescence-Activated Cell Sorting Is More Potent to Fish Intervertebral Disk Progenitor Cells Than Magnetic and Beads-Based Methods.

Low back pain related to intervertebral disk (IVD) degeneration has a major socioeconomic impact on our aging society. Therefore, stem cell therapy to activate self-repair of the IVD remains an exciting treatment strategy. In this respect, tissue-specific progenitors may play a crucial role in IVD regeneration, as these cells are perfectly adapted to this niche. Such a rare progenitor cell population residing in the nucleus pulposus (NP) (NP progenitor cells [NPPCs]) was found positive for the angiopoietin-1 receptor (Tie2+), and was demonstrated to possess self-renewal capacity and in vitro multipotency. Here, we compared three sorting protocols; that is, fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), and a mesh-based label-free cell sorting system (pluriSelect), with respect to cell yield, potential to form colonies (colony-forming units), and in vitro functional differentiation assays for tripotency. The aim of this study was to demonstrate the efficiency of three widespread cell sorting methods for picking rare cells (<5%) and how these isolated cells then behave in downstream functional differentiation in adipogenesis, osteogenesis, and chondrogenesis. The cell yields among the isolation methods differed widely, with FACS presenting the highest yield (5.0% ± 4.0%), followed by MACS (1.6% ± 2.9%) and pluriSelect (1.1% ± 1.0%). The number of colonies formed was not significantly different between Tie2+ and Tie2- NPPCs. Only FACS was able to separate into two functionally different populations that showed trilineage multipotency, while MACS and pluriSelect failed to maintain a clear separation between Tie2+ and Tie2- populations in differentiation assays. To conclude, the isolation of NPPCs was possible with all three sorting methods, while FACS was the preferred technique for separation of functional Tie2+ cells. Impact Statement Tissue-specific progenitor cells such as nucleus pulposus progenitor cells of the IVD could become an ultimate cell source for tissue engineering strategies as these cells are presumably best adapted to the tissue's microenvironment. Fluorescence-activated cell sorting seemed to outcompete magnetic-activated cell sorting and pluriSelect concerning selecting a rare cell population from IVD tissue as could be demonstrated by improved cell yield and functional differentiation assays.

Correction to: Angiopoietin-1 receptor Tie2 distinguishes multipotent differentiation capability in bovine coccygeal nucleus pulposus cells.

Following publication of the original article in Stem Cell Research & Therapy [1], we would like to alert the reader that the immune-histological sections shown in Figure 2 bottom line are mistakenly the images from an experiment using a different Tie2+ antibody than originally reported in the manuscript (i.e. R&D, anti-human Tie2 labeled APC, cat.No:FAB3131A, clone:83715, mouse IgG) for the florescence associated cell sorting (FACS). This antibody has been previously tested in the group of Prof. Dr. Daisuke Sakai and was performed by Ms Tomoko Nakai, Tokai University. This antibody, however, was not found to be specific for bovine Tie2+ cells. The immune-histology procedure was correctly described using the PG antibody from Millipore. However, the pictures presented in Figure 2 in the last raw in the article of Tekari et al. [1] are not from the same experiment using the Tie2 antibody from Bioss, inc. clone bs-1300R, Bioss Antibodies, Woburn, MA, USA, as the publication reported.

Genipin-Enhanced Fibrin Hydrogel and Novel Silk for Intervertebral Disc Repair in a Loaded Bovine Organ Culture Model.

(1) Background: Intervertebral disc (IVD) repair represents a major challenge. Using functionalised biomaterials such as silk combined with enforced hydrogels might be a promising approach for disc repair. We aimed to test an IVD repair approach by combining a genipin-enhanced fibrin hydrogel with an engineered silk scaffold under complex load, after inducing an injury in a bovine whole organ IVD culture; (2) Methods: Bovine coccygeal IVDs were isolated from ~1-year-old animals within four hours post-mortem. Then, an injury in the annulus fibrosus was induced by a 2 mm biopsy punch. The repair approach consisted of genipin-enhanced fibrin hydrogel that was used to fill up the cavity. To seal the injury, a Good Manufacturing Practise (GMP)-compliant engineered silk fleece-membrane composite was applied and secured by the cross-linked hydrogel. Then, IVDs were exposed to one of three loading conditions: no load, static load and complex load in a two-degree-of-freedom bioreactor for 14 days. Followed by assessing DNA and matrix content, qPCR and histology, the injured discs were compared to an uninjured control IVD that underwent the same loading profiles. In addition, the genipin-enhanced fibrin hydrogel was further investigated with respect to cytotoxicity on human stem cells, annulus fibrosus, and nucleus pulposus cells; (3) Results: The repair was successful as no herniation could be detected for any of the three loading conditions. Disc height was not recovered by the repair DNA and matrix contents were comparable to a healthy, untreated control disc. Genipin resulted being cytotoxic in the in vitro test but did not show adverse effects when used for the organ culture model; (4) Conclusions: The current study indicated that the combination of the two biomaterials, i.e., genipin-enhanced fibrin hydrogel and an engineered silk scaffold, was a promising approach for IVD repair. Furthermore, genipin-enhanced fibrin hydrogel was not suitable for cell cultures; however, it was highly applicable as a filler material.

Successful fishing for nucleus pulposus progenitor cells of the intervertebral disc across species.

BACKGROUND: Recently, Tie2/TEK receptor tyrosine kinase (Tie2 or syn. angiopoietin-1 receptor) positive nucleus pulposus progenitor cells were detected in human, cattle, and mouse. These cells show remarkable multilineage differentiation capacity and direct correlation with intervertebral disc (IVD) degeneration and are therefore an interesting target for regenerative strategies. Nevertheless, there remains controversy over the presence and function of these Tie2+ nucleus pulposus cells (NPCs), in part due to the difficulty of identification and isolation. PURPOSE: Here, we present a comprehensive protocol for sorting of Tie2+ NPCs from human, canine, bovine, and murine IVD tissue. We describe enhanced conditions for expansion and an optimized fluorescence-activated cell sorting-based methodology to sort and analyze Tie2+ NPCs. METHODS: We present flow cytometry protocols to isolate the Tie2+ cell population for the aforementioned species. Moreover, we describe crucial pitfalls to prevent loss of Tie2+ NPCs from the IVD cell population during the isolation process. A cross-species phylogenetic analysis of Tie2 across species is presented. RESULTS: Our protocols are efficient towards labeling and isolation of Tie2+ NPCs. The total flow cytometry procedure requires approximately 9 hours, cell isolation 4 to 16 hours, cell expansion can take up to multiple weeks, dependent on the application, age, disease state, and species. Phylogenetic analysis of the TEK gene revealed a strong homology among species. CONCLUSIONS: Current identification of Tie2+ cells could be confirmed in bovine, canine, mouse, and human specimens. The presented flow cytometry protocol can successfully sort these multipotent cells. The biological function of isolated cells based on Tie2+ expression needs to be confirmed by functional assays such as in vitro differentiation. in vitro culture conditions to maintain and their possible proliferation of the Tie2+ fraction is the subject of future research.

Efficient Nonviral Transfection of Primary Intervertebral Disc Cells by Electroporation for Tissue Engineering Application.

Low back pain (LBP) is an increasing global health problem associated with intervertebral disc (IVD) trauma and degeneration. Current treatment options include surgical interventions with partial unsatisfactory outcomes reported such as failure to relieve LBP, nonunions, nerve injuries, or adjacent segment disease. Cell-based therapy and tissue engineered IVD constructs supplemented with transfected disc cells that incorporate factors enhancing matrix synthesis represent an appealing approach to regenerate the IVD. Gene delivery approaches using transient nonviral gene therapy by electroporation are of a high clinical translational value since the incorporated DNA is lost after few cell generations, leaving the host's genome unmodified. Human primary cells isolated from clinically relevant samples were generally found very hard to transfect compared to cell lines. In this study, we present a range of parameters (voltage pulse, number, and duration) from the Neon® Transfection System for efficient transfection of human and bovine IVD cells. To demonstrate efficiency, these primary cells were exemplarily transfected with the commercially available plasmid pCMV6-AC-GFP tagged with copepod turbo green fluorescent protein. Flow cytometry was subsequently applied to quantify transfection efficiency. Our results showed that two pulses of 1400 V for 20 ms revealed good and reproducible results for both human and bovine IVD cells with efficiencies ≥47%. The presented parameters allow for successful human and bovine IVD cell transfection and provide an opportunity for subsequent regenerative medicine application.

Angiopoietin-1 receptor Tie2 distinguishes multipotent differentiation capability in bovine coccygeal nucleus pulposus cells.

BACKGROUND: The intervertebral disc (IVD) has limited self-healing potential and disc repair strategies require an appropriate cell source such as progenitor cells that could regenerate the damaged cells and tissues. The objective of this study was to identify nucleus pulposus-derived progenitor cells (NPPC) and examine their potential in regenerative medicine in vitro. METHODS: Nucleus pulposus cells (NPC) were obtained from 1-year-old bovine coccygeal discs by enzymatic digestion and were sorted for the angiopoietin-1 receptor Tie2. The obtained Tie2- and Tie2+ fractions of cells were differentiated into osteogenic, adipogenic, and chondrogenic lineages in vitro. Colony-forming units were prepared from both cell populations and the colonies formed were analyzed and quantified after 8 days of culture. In order to improve the preservation of the Tie2+ phenotype of NPPC in monolayer cultures, we tested a selection of growth factors known to have stimulating effects, cocultured NPPC with IVD tissue, and exposed them to hypoxic conditions (2 % O2). RESULTS: After 3 weeks of differentiation culture, only the NPC that were positive for Tie2 were able to differentiate into osteocytes, adipocytes, and chondrocytes as characterized by calcium deposition (p < 0.0001), fat droplet formation (p < 0.0001), and glycosaminoglycan content (p = 0.0095 vs. Tie2- NPC), respectively. Sorted Tie2- and Tie2+ subpopulations of cells both formed colonies; however, the colonies formed from Tie2+ cells were spheroid in shape, whereas those from Tie2- cells were spread and fibroblastic. In addition, Tie2+ cells formed more colonies in 3D culture (p = 0.011) than Tie2- cells. During expansion, a fast decline in the fraction of Tie2+ cells was observed (p < 0.0001), which was partially reversed by low oxygen concentration (p = 0.0068) and supplementation of the culture with fibroblast growth factor 2 (FGF2) (p < 0.0001). CONCLUSIONS: Our results showed that the bovine nucleus pulposus contains NPPC that are Tie2+. These cells fulfilled formally progenitor criteria that were maintained in subsequent monolayer culture for up to 7 days by addition of FGF2 or hypoxic conditions. We propose that the nucleus pulposus represents a niche of precursor cells for regeneration of the IVD.

Bovine Osteochondral Tissues: A Questionable Model to Evaluate Mechanical Loading In Vitro.

Articular cartilage exists within synovial joints to adsorb and distribute mechanical loads to the subchondral bone. Mechanical loading is one aspect of a wide range of microenvironmental stressors that contribute to the maintenance of articular cartilage. The aim of the current study was to characterize bovine osteochondral tissues and to assess their suitability to serve as a model for investigating the effects of mechanical loading on cartilage tissue in vitro using a custom-made reactor system. Osteochondral tissues were harvested from bovine knee joints and cultured up to 24 days in loaded and unloaded conditions. Notably, we found a considerable zone-specific heterogeneity between cartilage explants harvested from the same joint as evidenced by histology and gene expression levels. Results using the reactor system revealed that differences observed after mechanical loading varied within the range of the heterogeneity observed amongst the different cartilage explants. Thus, it may be difficult to obtain reliable and reproducible data in mechanical loading experiments from these tissues in vitro, especially in cases where small variations between the experimental groups are expected. This will likely lead to the reporting of false positives or negatives in studies investigating the effect of mechanical load on the function of cartilage tissue.

Transforming growth factor beta signaling is essential for the autonomous formation of cartilage-like tissue by expanded chondrocytes.

Cartilage is a tissue with limited self-healing potential. Hence, cartilage defects require surgical attention to prevent or postpone the development of osteoarthritis. For cell-based cartilage repair strategies, in particular autologous chondrocyte implantation, articular chondrocytes are isolated from cartilage and expanded in vitro to increase the number of cells required for therapy. During expansion, the cells lose the competence to autonomously form a cartilage-like tissue, that is in the absence of exogenously added chondrogenic growth factors, such as TGF-ßs. We hypothesized that signaling elicited by autocrine and/or paracrine TGF-ß is essential for the formation of cartilage-like tissue and that alterations within the TGF-ß signaling pathway during expansion interfere with this process. Primary bovine articular chondrocytes were harvested and expanded in monolayer culture up to passage six and the formation of cartilage tissue was investigated in high density pellet cultures grown for three weeks. Chondrocytes expanded for up to three passages maintained the potential for autonomous cartilage-like tissue formation. After three passages, however, exogenous TGF-ß1 was required to induce the formation of cartilage-like tissue. When TGF-ß signaling was blocked by inhibiting the TGF-ß receptor 1 kinase, the autonomous formation of cartilage-like tissue was abrogated. At the initiation of pellet culture, chondrocytes from passage three and later showed levels of transcripts coding for TGF-ß receptors 1 and 2 and TGF-ß2 to be three-, five- and five-fold decreased, respectively, as compared to primary chondrocytes. In conclusion, the autonomous formation of cartilage-like tissue by expanded chondrocytes is dependent on signaling induced by autocrine and/or paracrine TGF-ß. We propose that a decrease in the expression of the chondrogenic growth factor TGF-ß2 and of the TGF-ß receptors in expanded chondrocytes accounts for a decrease in the activity of the TGF-ß signaling pathway and hence for the loss of the potential for autonomous cartilage-like tissue formation.

Osteogenic differentiation of bone marrow stromal cells is hindered by the presence of intervertebral disc cells.

BACKGROUND: Clinical observations indicate that the presence of nucleus pulposus (NP) tissue during spinal fusion hinders the rate of disc ossification. While the underlying mechanism remains unknown, this observation could be due to incomplete removal of NP cells (NPCs) that secrete factors preventing disc calcification, such as bone morphogenetic protein (BMP) antagonists including noggin and members of the DAN (differential screening selected gene aberrative in neuroblastoma) family. METHODS: Monolayer human bone marrow-derived mesenchymal stem cells (MSCs) were cocultured withNPCs and annulus fibrosus cells (AFCs) embedded in alginate for 21 days. At the end of coculture, MSCs were stained for mineral deposition by alizarin red, and relative expression of bone-related genes [Runt-related transcription factor 2, (RUNX2), Osteopontin (OPN), and Alkaline phosphatase (ALP)] and ALP activity were analyzed. Relative expression of three BMP antagonists, chordin (CHRD), gremlin (GREM1), and noggin (NOG), was determined in primary human NPCs and AFCs. These cells were also stained for Gremlin and Noggin by immunocytochemistry. RESULTS: Alizarin red staining showed that MSC osteogenesis in monolayer cultures was inhibited by coculture with NPCs or AFCs. ALP activity and RT-PCR analyses confirmed these results and demonstrated inhibition of osteogenesis of MSC in the presence of disc cells. NOG was significantly up-regulated in MSCs after coculture. Relative gene expression of intervertebral disc (IVD) cells showed higher expression of GREM1 in NPCs than in AFCs. CONCLUSIONS: We show that primary IVD cells inhibit osteogenesis of MSCs. BMP inhibitors NOG, GREM1 and CHRD were expressed in IVD cells. GREM1 appears to be differentially expressed in NPCs and AFCs. Our results have implications for the design and development of treatments for non-union in spinal fusion.

Chondrocytes expressing intracellular collagen type II enter the cell cycle and co-express collagen type I in monolayer culture.

For autologous chondrocyte transplantation, articular chondrocytes are harvested from cartilage tissue and expanded in vitro in monolayer culture. We aimed to characterize with a cellular resolution the synthesis of collagen type II (COL2) and collagen type I (COL1) during expansion in order to further understand why these cells lose the potential to form cartilage tissue when re-introduced into a microenvironment that supports chondrogenesis. During expansion for six passages, levels of transcripts encoding COL2 decreased to <0.1%, whereas transcript levels encoding COL1 increased 370-fold as compared to primary chondrocytes. Flow cytometry for intracellular proteins revealed that chondrocytes acquired a COL2/COL1-double positive phenotype during expansion, and the COL2 positive cells were able to enter the cell cycle. While the fraction of COL2 positive cells decreased from 70% to <2% in primary chondrocytes to passage six cells, the fraction of COL1 positive cells increased from <1% to >95%. In parallel to the decrease of the fraction of COL2 positive cells, the cells' potential to form cartilage-like tissue in pellet cultures steadily decreased. Intracellular staining for COL2 enables for characterization of chondrocyte lineage cells in more detail with a cellular resolution, and it may allow predicting the effectiveness of expanded chondrocytes to form cartilage-like tissue.