Antenatal differentiation of the human intervertebral disc

Summary To study the antenatal differentiation of the human intervertebral disc, the columns of forty eight embryos and fetuses were examined histologically. The primitive disc is composed of two structures: the notochord which shows a progressive expansion into the disc, and the fibrocartilaginous perinotochordal disc. No histological sign of interaction between notochordal and perinotochordal cells, which may explain the notochordal expansion into the discs, was seen. On the other hand, the notochordal intervention in the cartilaginous differentiation of the inner zone is probable.

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Différenciation anténatale du disque intervertébral humain

Résumé Cette étude de la différenciation anténatale du disque intervertébral humain repose sur l'examen de coupes histologiques de quarante huit colonnes vertébrales d'embryons et de foetus. Le disque primitif est composé de deux structures : la notochorde, qui présente une expansion progressive de son diamètre au sein du disque, et le disque périnotochordal, d'abord mésenchymateux puis fibrocartilagineux. Il n'a pas été mis en évidence de signe histologique témoignant d'une interaction entre les cellules notochordales et les cellules périnotochordales qui puisse expliquer l'expansion de la notochorde au sein des disques. Le rôle de la notochorde dans la différenciation cartilagineuse de la zone centrale est par contre probable.

     

Immunolocalization of major interstitial collagen types in human lumbar intervertebral discs of various ages

 We used complete transverse sections through 65 samples of human lumbar intervertebral discs for immunolocalization of the major interstitial collagen types I, II, III, V, VI and IX. The samples were selected from 47 patients ranging in age from 0 (fetuses) to 86 years. The results were compared with the histological findings in disc tissue degeneration and/or reparative alterations as indicated by tear and cleft formation, chondrocyte proliferation, mucous degeneration, granular matrix changes and fibrocartilage fibrillation. We observed a typical pattern for each antibody and each anatomical structure, with, however, remarkable inter- and intraindividual variability, which could be monitored only by use of the complete transverse sections. Accordingly, collagen I was seen in the normal annulus fibrosus and in the degeneratively altered nucleus pulposus, but not within the end-plate, regardless of degenerative changes. Collagens II and IX were found in the normal nucleus pulposus, the inner annulus fibrosus and the end-plate. The collagen II (and IX) staining seemed to be enhanced in areas of minor degenerative lesions, but reduced in advanced lesions and in the degenerated end-plate. Collagens III and VI were significantly increased in areas of minor to advanced degeneration in all anatomical settings, while collagen V showed only minor changes in its staining pattern. In general, histological signs of tissue degeneration coincided with significant quantitative, but also with certain qualitative, changes in the composition of the collagenous disc matrix. These observations indicate the association of degenerative and/or reparative alterations of the intervertebral disc and changes in the collagenous matrix, but document the variability in the extent of the abnormalities observed. Received: 5 December 1996 / Accepted: 30 May 1997     

Characterisation of cytoplasm-filled processes in cells of the intervertebral disc

We examined cells from the nucleus pulposus and annulus fibrosus of adult bovine intervertebral discs, using confocal laser scanning microscopy on living unfixed tissue. These cells were visualised using chloromethyl fluorescein diacetate, a membrane-impermeant fluorescent dye. The organisation of cells from the outer annulus was also determined using confocal microscopy after fixation and staining the actin-filaments with FITC-phalloidin. We found that cellular processes were a dominant feature of cells from all regions of the disc including the cells of the nucleus pulposus and inner annulus. These processes were also visible in histological sections of disc examined both at the light and electron microscope level, even though cells from the nucleus and inner annulus appeared chondrocyte-like, being rounded and enclosed in a capsule. The function of these processes is at present unknown. We suggest that they may serve to sense mechanical strain.     

腰椎间盘的超微结构研究

本实验通过透射电镜对大鼠、家兔及人(胎儿及成人)腰椎间盘的观察,发现纤维环由纤维软骨组成。在640 A周期性的胶原原纤维之间,可见位于隐窝内的典型软骨细胞。这些细胞常随所在部位而异。靠近周边部位的细胞更接近成纤维细胞,细胞狭长,细胞膜的扇蛤样外形渐不明显,以至消失。靠内侧的细胞类似脊索细胞,细胞核固缩,细胞质内有浓集的糖原及脂滴。纤维环呈现细胞过渡的特点。纤维环内可见弹力纤维。髓核由包含细的原纤维、颗粒及特殊结构的疏松基质所组成。在各标本中,基质内均有脊索细胞出现。细胞可单独或成堆存在,细胞质内有众多的空泡,浓集的糖原颗粒及微丝。     

Integrin expression in cells of the intervertebral disc

In this study, we investigated the profile of integrin expression in human and porcine intervertebral disc tissue. Differences in extracellular matrix composition between anulus fibrosus (AF) and nucleus pulposus (NP) regions of the disc, as well as differences in cellular responses to environmental stimuli, suggest a role for integrins in presenting matrix signals that may mediate these responses. Human disc tissue and porcine AF and NP tissue were stained with antibodies to alpha integrin subunits 1-6, V and IIb, and beta integrin subunits 1-6 and graded for evidence of positive staining on a scale from 0 (no staining) to 3 (high incidence of staining). Human tissue expressed alpha and beta integrin subunits shown to be present in articular cartilage, including alpha(1), alpha(5) and alpha(V). Porcine AF tissue expressed similar integrin subunits to human disc, with both expressing alpha(1), alpha(5), beta(1), beta(3) and beta(5) subunits, whereas porcine NP tissue expressed higher levels of alpha(6), beta(1) and beta(4) than AF tissue. The expressed subunits are known to interact with proteins including collagens, fibronectin and laminin; however, additional studies will be required to characterize the interactions of the integrin subunits with specific matrix constituents, as well as their specific involvement in regulating environmental stimuli.     

体外培养人退变髓核与纤维环细胞增殖能力的研究

目的 探讨体外培养的人退变髓核与纤维环细胞的增殖能力,比较2种退变细胞体外培养中所表现的不同生物学行为,为椎间盘退变疾病的预防与治疗提供新的理论依据.方法 采集临床腰椎间盘突出症患者手术取材的椎间盘组织标本,病理学诊断评估其退变程度,酶消化法原代培养髓核与纤维环细胞,并鉴定.每例病例的髓核细胞与纤维环细胞分别体外培养至第5代,各代次细胞传代接种密度控制为1×105个.同条件培养48 h后,观察每一代细胞的形态学变化,同时应用流式细胞仪检测2种细胞的增殖能力.结果 体外培养的退变髓核与纤维环细胞生长状态良好.随着体外传代的进行,退变髓核与纤维环细胞S期细胞百分比和增殖指数(PI)均呈上升趋势,髓核细胞PI值于第3代时达到峰值,而纤维环细胞PI值于第5代时最高;且第2～4代的退变髓核细胞的增殖活性均高于退变纤维环的增殖活性(P＜0.05).结论 体外培养的人退变髓核与纤维环细胞有着不同的增殖特点,髓核与纤维环细胞对体内退变微环境有着不同的响应机制,并影响着整个椎间盘退变的进展.     

Three-dimensional morphology of the pericellular matrix of intervertebral disc cells in the rat

Intervertebral disc cells are surrounded by a pericellular matrix that is biochemically and morphologically distinct from other extracellular matrix regions. Although the function of the pericellular matrix is not fully understood, prior studies of pericellular matrix-chondrocyte regions in articular cartilage (termed 'chondrons') suggest that the size, shape, and mechanical properties of the pericellular matrix significantly influence the micromechanical environment of the contained cells. A first step in understanding the role of the pericellular matrix in the intervertebral disc is to quantify the three-dimensional morphology and zonal variations of these regions across the disc. In this study, three-dimensional reconstructions and morphometric measurements of pericellular matrix-cell regions were obtained in situ using fluorescence confocal microscopy of en bloc sections of nucleus pulposus and anulus fibrosus of the rat disc immunolabeled for type VI collagen. The morphology of the pericellular matrix and cells varied significantly across regions, with distinct pericellular matrix aspect ratios (largest/smallest diameter) showing shapes that were generally large and rounded in the nucleus pulposus (average of 1.9), and ellipsoidal and discoidal in the inner (2.4) and outer anulus fibrosus (2.8). The average pericellular matrix volume per cell was found to be significantly larger in the nucleus (6424 microm(3)) than that of inner (1903 microm(3)) and outer (1433 microm(3)) anulus. Pericellular matrix regions containing 1 or 2 cells were the dominant subgroup in the rat intervertebral disc at both 1 and 12 months of age. Multicellular pericellular matrix regions were present more often in the younger nucleus pulposus and outer anulus fibrosus. The orientation of the pericellular matrix regions further varied significantly across the disc, reflecting local collagen matrix architecture. These studies provide new information on the organization and shape of intervertebral disc cells and their surrounding pericellular matrix, which may provide new insights into the mechanisms that regulate cell-matrix interactions.     

An immunohistochemical study of enthesis development in the medial collateral ligament of the rat knee joint

The changing distributions of collagens and glycosaminoglycans have been studied at the attachments of the medial collateral ligament during postnatal development. The ligament is of particular interest because it has a fibrocartilaginous attachment to the femoral epiphysis, but a fibrous one to the tibial metaphysis. Ligaments were examined in rats killed at birth and at 2, 4, 6, 8, 10, 20, 30, 45, 60, 90 and 120 days after birth. Cryosections were immunolabelled with monoclonal and polyclonal antibodies against types I and II collagen, chondroitin 4 and 6 sulfate, dermatan and keratan sulfate. Although the ligament is attached at both ends to bones that develop from cartilage, there was a striking difference in collagen labelling. Type II collagen was only found in spicules of calcified cartilage in bone beneath the tibial enthesis after ossification had commenced, but there was a continuous band of labelling at all stages of development at the femoral enthesis. Initially, the cartilage at the femoral attachment lacked type I collagen, but by 45 days labelling was continuous from ligament to bone. Continuity of labelling was seen much earlier at the tibial enthesis, as soon as bone had formed. There were also marked changes in glycosaminoglycan distribution. Keratan sulfate was present at both entheses up to 45 days, but only at the femoral enthesis thereafter. Both attachments labelled throughout life for dermatan sulfate, but chondroitin 4 and 6 sulfate were only found at the femoral end. The results suggest that enthesial cartilage at the femoral attachment was initially derived from the cartilaginous bone rudiment but was quickly eroded on its deep surface by endochondral ossification as bone formed at the attachment site. It was replaced by fibrocartilage developing in the ligament. This mechanism allows enthesis cartilage/fibrocartilage to contribute to the growth of a bone at a secondary centre of ossification in addition to dissipating stress at the ligament-bone junction.     

Photocrosslinkable laminin-functionalized polyethylene glycol hydrogel for intervertebral disc regeneration

Intervertebral disc (IVD) disorders and age-related degeneration are believed to contribute to lower back pain. There is significant interest in cell-based strategies for regenerating the nucleus pulposus (NP) region of the disc; however, few scaffolds have been evaluated for their ability to promote or maintain an immature NP cell phenotype. Previous studies have shown that NP cell–laminin interactions promote cell adhesion and biosynthesis, which suggests a laminin-functionalized biomaterial may be useful for promoting or maintaining the NP cell phenotype. Here, a photocrosslinkable poly(ethylene glycol)–laminin 111 (PEG-LM111) hydrogel was developed. The mechanical properties of PEG-LM111 hydrogel could be tuned within the range of dynamic shear moduli values previously reported for human NP. When primary immature porcine NP cells were seeded onto PEG-LM111 hydrogels of varying stiffnesses, LM111-presenting hydrogels were found to promote cell clustering and increased levels of sGAG production as compared to stiffer LM111-presenting and PEG-only gels. When cells were encapsulated in 3-D gels, hydrogel formulation was found to influence NP cell metabolism and expression of proposed NP phenotypic markers, with higher expression of N-cadherin and cytokeratin 8 observed for cells cultured in softer (<1 kPa) PEG-LM111 hydrogels. Overall, these findings suggest that soft, LM111-functionalized hydrogels may promote or maintain the expression of specific markers characteristic of an immature NP cell phenotype.     

Diversity of intervertebral disc cells: phenotype and function

The intervertebral disc (IVD) is a moderately moving joint that is located between the bony vertebrae and provides flexibility and load transmission throughout the spinal column. The disc is composed of different but interrelated tissues, including the central highly hydrated nucleus pulposus (NP), the surrounding elastic and fibrous annulus fibrosus (AF), and the cartilaginous endplate (CEP), which provides the connection to the vertebral bodies. Each of these tissues has a different function and consists of a specific matrix structure that is maintained by a cell population with distinct phenotype. Although the healthy IVD is able to balance the slow matrix turnover of synthesis and degradation, this balance is often disturbed, leading to degenerative disorders. Successful therapeutic management of IVD degeneration requires a profound understanding of the cellular and molecular characteristics of the functional IVD. Hence, the phenotype of IVD cells has been of significant interest from multiple perspectives, including development, growth, remodelling, degeneration and repair. One major challenge that complicates our understanding of the disc cells is that both the cellular phenotype and the extracellular matrix strongly depend on disc maturity and health and as a consequence are continuously evolving. This review delineates the diversity of the cell types found in the intervertebral disc, with emphasis on human, but with reference to other species. The cells of the NP appear rounded and express a proteoglycan-rich matrix, whereas the more elongated AF cells are embedded in a collagen fibre matrix and the CEPs represent a layer of cartilage. Even though all disc cells have often been referred to as ‘intervertebral disc chondrocytes’, distinct phenotypical differences in comparison with articular chondrocytes exist and have been reported recently. The availability of more specific markers has also improved our understanding of progenitor cell differentiation towards an IVD cell phenotype. Ultimately, new cell- and tissue-engineering approaches to regenerative therapies will only be successful if the specific characteristics of the individual tissues and their context in the function of the whole organ, are taken into consideration.     

Monitoring of the effect of intervertebral disc nucleus pulposus ablation by MRI

In order to investigate intervertebral disc (IVD) degeneration and repair, a quantitative non‐invasive tool is needed. Various MRI methods including qCPMG, which yields dipolar echo relaxation time (T_DE), magnetization transfer contrast (MTC), and ¹H and ²H double quantum filtered (DQF) MRI were used in the present work to monitor changes in rat IVD after ablation of the nucleus pulposus (NP), serving as a model of severe IVD degeneration. In the intact IVD, a clear distinction between the annulus fibrosus (AF) and the NP is obtained on T₂ and T_DE weighted images as well as on MTC maps, reflecting the high concentration of ordered collagen fibers in the AF. After ablation of the NP, the distinction between the compartments is lost. T₂ and T_DE relaxation times are short throughout the disc and MTC is high. ¹H and ²H DQF signal, which in intact discs is obtained only for the AF, is now observable throughout the tissue. These results indicate that after ablation, there is an ingression of collagen fibers from the AF into the area that was previously occupied by the NP, as was confirmed by histology. Copyright © 2010 John Wiley & Sons, Ltd.     

The potential role of melatonin in retarding intervertebral disc ageing and degeneration: A systematic review

Intervertebral disc degeneration (IDD) is a common degenerative disease of the musculoskeletal system that develops with age. It is regarded as the main cause of chronic low back pain in the elderly. IDD has various causes, including ageing, mechanical overloading, and nutritional deficiency. Melatonin is a pleiotropic indole hormone secreted by the pineal gland and plays an important role in resisting various degenerative diseases. The serum levels of melatonin decline with age and are reported to be negatively correlated with the symptomatic and histopathological scores of IDD. In vivo studies have shown that exogenous administration of melatonin could maintain the structural integrity of the intervertebral disc and inhibit the development of IDD. Mechanistically, by interacting with its membrane or intracellular receptors, melatonin can promote autophagic flux, scavenge free radicals, inhibit the release of pro-inflammatory factors, and block apoptotic pathways, thereby enhancing anti-stress abilities and matrix anabolism in different types of disc cells. Therefore, melatonin supplementation may be a promising therapeutic strategy for IDD. This review aimed to summarize the latest findings regarding the therapeutic potential of melatonin in IDD.     

椎间融合器治疗腰椎间盘突出症的效果及其临床意义

目的 分析腰椎间盘突出症髓核摘除术后脊柱生物力学改变，并评价置入椎间融合器的效果。方法 1998年3月2001年4月对22例腰椎间盘突出症患者采用后路双侧扩大半椎板间盘摘除椎间融合器置入术，22例均行自体骨移植置入椎间合器合，并对置入结果进行评价。结果 随访9-34个月，平均14.5个月，通过放射学检查，证实了术后椎体间骨性融合形成，恢复或保持术后椎间盘的高度，椎间孔水平的通道，结论 椎间融合器的应用可以解决腰椎间盘突出症椎间盘摘除术后椎间隙变窄，椎体间相对移位，椎间孔水平的通道狭窄等问题。     

In situ oxygen utilization in the rat intervertebral disc

Nucleus pulposus cells of the intervertebral disc have no endogenous vasculature and have thus been hypothesized to be hypoxic. This hypothesis was tested using 2-nitroimidazole, EF5, a drug that at low oxygen concentrations forms covalent adducts with cellular proteins. After administrating EF5 to rats, sections of the intervertebral disc were analysed for EF5 adducts. Drug adducts were quantified in tissue sections using a fluorescent monoclonal antibody. Although the level of EF5 fluorescence in all intervertebral disc tissues was low, the transition zone at the periphery of the nucleus pulposus exhibited the highest level of EF5 binding. To substantiate this result, tissue nitroreductase levels and drug pharmacology were evaluated. Nitroreductase levels were measured in whole discs under severe hypoxia. We noted that there was robust EF5 binding to cells in the annulus fibrosus and transition zone with modest binding to cells of the nucleus pulposus and endplate. High-performance liquid chromatography analysis indicated limitations in EF5 access to the nucleus pulposus, most probably related to the lack of vasculature and slow drug distribution through the gel-like interior of the disc. However, despite diffusion problems, the drug dose was determined to be sufficient to report the oxygen status of the nucleus pulposus cells. Based on these findings, we conclude that despite poor vascularization, the disc cells accommodate to the local environment by displaying a limited need for oxygen. Accordingly, the cells of the intervertebral disc are not severely hypoxic.     

去细胞化基质材料修复椎间盘的研究与进展

文题释义：去细胞化：是指利用物理、化学和酶学方法裂解细胞，去除细胞内容物，保留细胞外基质、组织天然构架。通常联合运用以上多种方法去除同种异体或异种细胞成分，理论上可利用由此获得的细胞外基质制造出具备合适生物力学和生物相容性的最低免疫原性支架。 椎间盘：是由位于中央的髓核(蛋白聚糖和Ⅱ型胶原组成的集中高度水化的纤维胶状核)及环绕其周围分层排列的纤维环(主要由Ⅰ型胶原组成的多层纤维软骨环)和上下软骨终板构成的复杂结构，是无血运器官，自身代谢缓慢，损伤后的自我修复、愈合能力极其有限，一般的物理修复与手术治疗无法从根本上修复椎间盘，效果欠佳，容易复发，椎间盘去细胞化组织工程为之提供了新思路。 背景：随着组织工程学的发展，椎间盘组织的修复与再生成为可能，将组织去细胞后的细胞外基质作为修复材料是进行椎间盘组织工程再生的重要手段。 目的：总结近年来用于椎间盘再生去细胞化基质材料的制备工艺、质量控制、应用效果，并进行展望。 方法：检索PubMed、Web of Science和CNKI数据库等收录的有关去细胞化方法及去细胞基修复椎间盘的文章，英文检索词为“Intervertebral disc，Decellularization，Extracellular matrix，Scaffold material，Tissue engineering”，中文检索词为“椎间盘，去细胞化，细胞外基质，支架材料，组织工程”。根据纳入与排除标准对所有文章进行初筛后，保留相关性较高的文章进行综述。 结果与结论：目前的椎间盘去细胞化组织工程旨在最大程度地保留生理相关的生物活性物质，提高力学性能和生物相容性，降低免疫原性。去细胞化基质材料能够模拟椎间盘内细胞外基质的天然微环境，作为细胞载体能对种子细胞起到良好的诱导分化用，其修复椎间盘已经取得了一定进展，但材料的孔隙率、免疫排斥反应、植入体内方式及效果评估等问题还有待进一步的深入研究。 ORCID: 0000-0003-3225-3611(金筱妤) 中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程     

Potential biomarkers of the mature intervertebral disc identified at the single cell level

Intervertebral disc (IVD) degeneration and trauma is a major socio‐economic burden and the focus of cell‐based regenerative medicine approaches. Despite numerous ongoing clinical trials attempting to replace ailing IVD cells with mesenchymal stem cells, a solid understanding of the identity and nature of cells in a healthy mature IVD is still in need of refinement. Although anatomically simple, the IVD is comprised of heterogeneous cell populations. Therefore, methods involving cell pooling for RNA profiling could be misleading. Here, by using RNA in situ hybridization and z proportion test, we have identified potential novel biomarkers through single cell assessment. We quantified the proportion of RNA transcribing cells for 50 genetic loci in the outer annulus fibrosus (AF) and nucleus pulposus (NP) in coccygeal bovine discs isolated from tails of four skeletally mature animals. Our data reconfirm existing data and suggest 10 novel markers such as Lam1 and Thy1 in the outer AF and Gli1, Gli3, Noto, Scx, Ptprc, Sox2, Zscan10 and LOC101904175 in the NP, including pluripotency markers, that indicate stemness potential of IVD cells. These markers could be added to existing biomarker panels for cell type characterization. Furthermore, our data once more demonstrate heterogeneity in cells of the AF and NP, indicating the need for single cell assessment by methods such as RNA in situ hybridization. Our work refines the molecular identity of outer AF and NP cells, which can benefit future regenerative medicine and tissue engineering strategies in humans.     

An injectable hydrogel incorporating mesenchymal precursor cells and pentosan polysulphate for intervertebral disc regeneration

Intervertebral disc (IVD) degeneration is one of the leading causes of lower back pain and a major health problem worldwide. Current surgical treatments include excision or immobilisation, with neither approach resulting in the repair of the degenerative disc. As such, a tissue engineering-based approach in which stem cells, coupled with an advanced delivery system, could overcome this deficiency and lead to a therapy that encourages functional fibrocartilage generation in the IVD. In this study, we have developed an injectable hydrogel system based on enzymatically-crosslinked polyethylene glycol and hyaluronic acid. We examined the effects of adding pentosan polysulphate (PPS), a synthetic glycosaminoglycan-like factor that has previously been shown (in vitro and in vivo) to this gel system in order to induce chondrogenesis in mesnchymal precursor cells (MPCs) when added as a soluble factor, even in the absence of additional growth factors such as TGF-β. We show that both the gelation rate and mechanical strength of the resulting hydrogels can be tuned in order to optimise the conditions required to produce gels with the desired combination of properties for an IVD scaffold. Human immunoselected STRO-1+ MPCs were then incorporated into the hydrogels. They were shown to retain good viability after both the initial formation of the gel and for longer-term culture periods in vitro. Furthermore, MPC/hydrogel composites formed cartilage-like tissue which was significantly enhanced by the incorporation of PPS into the hydrogels, particularly with respect to the deposition of type-II-collagen. Finally, using a wild-type rat subcutaneous implantation model, we examined the extent of any immune reaction and confirmed that this matrix is well tolerated by the host. Together these data provide evidence that such a system has significant potential as both a delivery vehicle for MPCs and as a matrix for fibrocartilage tissue engineering applications.     

Elastic fibre organization in the intervertebral discs of the bovine tail

Elastic fibres have been revealed by both elastin immunostaining and conventional histological orcein-staining in the intervertebral discs of the bovine tail. These fibres are distributed in all regions of the disc but their organization varies from region to region. In the centre of the nucleus, long (> 150 microm) elastic fibres are orientated radially. In the transitional region between nucleus and annulus, the orientation of the elastic fibres changes, producing a criss-cross pattern. In the annulus itself, elastic fibres appear densely distributed in the region between the lamellae and also in 'bridges' across the lamellae, particularly in the adult. Elastic fibres are apparent within the lamellae, orientated parallel to the collagen fibres of each lamella, particularly in the young (12-day-old) discs. In the region between the disc and the cartilaginous endplate, elastic fibres appear to anchor into the plate and terminate there. The results of this study suggest that elastic fibres contribute to the mechanical functioning of the intervertebral disc. The varying organization of the elastic fibres in the different regions of the disc is likely to relate to the different regional loading patterns.     

Correlating Material Properties with Tissue Composition in Enzymatically Digested Bovine Annulus Fibrosus and Nucleus Pulposus Tissue

Aging and degeneration of the intervertebral disk are accompanied by decreases in water and proteoglycan contents, and structural alterations. The aim of this study was to determine the impact of compositional changes on the material properties of intervertebral disk tissues. Confined compression stress-relaxation experiments were applied to bovine caudal annulus fibrosus and nucleus pulposus tissue specimens that were separated into three experimental groups: in situ, free-swelling control (PBS), and digestion (chondroitinase-ABC). Measurements of glycosaminoglycan (GAG) and water content, as well as nonlinear finite deformation biphasic theory and multiple linear regression analyses were performed. The compressive modulus H _A0 and permeability k ₀ of in situ specimens were 0.37±0.06 MPa and 0.49±0.08×10⁻¹⁵ m⁴ N⁻¹ s⁻¹ for nucleus, and 0.74±0.13 MPa and 0.42±0.05×10⁻¹⁵ m⁴ N⁻¹ s⁻¹ for annulus, respectively. There was a larger effect of swelling and digestion on the material properties and biochemical composition of nucleus pulposus than for annulus fibrosus. Alterations in proteoglycan and water content affected the compressive modulus and permeability, although the permeability was somewhat more strongly affected by water content than by proteoglycan content. Correlation coefficients r≤0.75 for the multiple regression indicated water and GAG content can moderately predict material properties, however other compositional and structural factors must be considered.     

Effects of bound versus soluble pentosan polysulphate in PEG/HA-based hydrogels tailored for intervertebral disc regeneration

Previous reports in the literature investigating chondrogenesis in mesenchymal progenitor cell (MPC) cultures have confirmed the chondro-inductive potential of pentosan polysulphate (PPS), a highly sulphated semi-synthetic polysaccharide, when added as a soluble component to culture media under standard aggregate-assay conditions or to poly(ethylene glycol)/hyaluronic acid (PEG/HA)-based hydrogels, even in the absence of inductive factors (e.g. TGFβ). In this present study, we aimed to assess whether a ‘bound’ PPS would have greater activity and availability over a soluble PPS, as a media additive or when incorporated into PEG/HA-based hydrogels. We achieved this by covalently pre-binding the PPS to the HA component of the gel (forming a new molecule, HA-PPS). We firstly investigated the activity of HA-PPS compared to free PPS, when added as a soluble factor to culture media. Cell proliferation, as determined by CCK8 and EdU assay, was decreased in the presence of either bound or free PPS whilst chondrogenic differentiation, as determined by DMMB assay and histology, was enhanced. In all cases, the effect of the bound PPS (HA-PPS) was more potent than that of the unbound form. These results alone suggest wider applications for this new molecule, either as a culture supplement or as a coating for scaffolds targeted at chondrogenic differentiation or maturation. We then investigated the incorporation of HA-PPS into a PEG/HA-based hydrogel system, by simply substituting some of the HA for HA-PPS. Rheological testing confirmed that incorporation of either HA-PPS or PPS did not significantly affect gelation kinetics, final hydrogel modulus or degradation rate but had a small, but significant, effect on swelling. When encapsulated in the hydrogels, MPCs retained good viability and rapidly adopted a rounded morphology. Histological analysis of both GAG and collagen deposition after 21 days showed that the incorporation of the bound-PPS into the hydrogel resulted in increased matrix formation when compared to the addition of soluble PPS to the hydrogel, or the hydrogel alone. We believe that this new generation injectable, degradable hydrogel, incorporating now a covalently bound-PPS, when combined with MPCs, has the potential to assist cartilage regeneration in a multitude of therapeutic targets, including for intervertebral disc (IVD) degeneration.