Delivery systems for the treatment of degenerated intervertebral discs.
Blanquer S B G,Grijpma D W,Poot A A
Advanced drug delivery reviews
The intervertebral disc (IVD) is the most avascular and acellular tissue in the body and therefore prone to degeneration. During IVD degeneration, the balance between anabolic and catabolic processes in the disc is deregulated, amongst others leading to alteration of extracellular matrix production, abnormal enzyme activities and production of pro-inflammatory substances like cytokines. The established treatment strategy for IVD degeneration consists of physiotherapy, pain medication by drug therapy and if necessary surgery. This approach, however, has shown limited success. Alternative strategies to increase and prolong the effects of bioactive agents and to reverse the process of IVD degeneration include the use of delivery systems for drugs, proteins, cells and genes. In view of the specific anatomy and physiology of the IVD and depending on the strategy of the therapy, different delivery systems have been developed which are reviewed in this article.
Advancing the cellular and molecular therapy for intervertebral disc disease.
Sakai Daisuke,Grad Sibylle
Advanced drug delivery reviews
The healthy intervertebral disc (IVD) fulfils the essential function of load absorption, while maintaining multi-axial flexibility of the spine. The interrelated tissues of the IVD, the annulus fibrosus, the nucleus pulposus, and the cartilaginous endplate, are characterised by their specific niche, implying avascularity, hypoxia, acidic environment, low nutrition, and low cellularity. Anabolic and catabolic factors balance a slow physiological turnover of extracellular matrix synthesis and breakdown. Deviations in mechanical load, nutrient supply, cellular activity, matrix composition and metabolism may initiate a cascade ultimately leading to tissue dehydration, fibrosis, nerve and vessel ingrowth, disc height loss and disc herniation. Spinal instability, inflammation and neural sensitisation are sources of back pain, a worldwide leading burden that is challenging to cure. In this review, advances in cell and molecular therapy, including mobilisation and activation of endogenous progenitor cells, progenitor cell homing, and targeted delivery of cells, genes, or bioactive factors are discussed.
Current trends in biologics delivery to restore intervertebral disc anabolism.
Fontana Gianluca,See Eugene,Pandit Abhay
Advanced drug delivery reviews
Low back pain is generally attributed to intervertebral disc (IVD) degeneration. This is a multifactorial disease induced by genetic and environmental factors and that progresses with aging. Disc degeneration is characterized by a limited ability of IVD cells to produce functional matrix while producing abnormal amounts of matrix-degrading enzymes. The prolonged imbalance between anabolism and catabolism in degenerative discs alters their composition and hydration. In turn, this results in increased angiogenesis and the loss of the disc's ability to maintain its aneural condition. Inflammation in the IVD, in particular the presence of pro-inflammatory cytokines, was found to favor innervation and also sensitization of the nociceptive pathways, thereby exacerbating degenerative symptoms. In this review, we discuss anti-inflammatory approaches to encounter disc catabolism, potential treatments to lower discogenic pain and pro-anabolic approaches in the form of protein delivery, gene therapy and cell delivery, to trigger regeneration in the IVD.
Mohawk promotes the maintenance and regeneration of the outer annulus fibrosus of intervertebral discs.
Nakamichi Ryo,Ito Yoshiaki,Inui Masafumi,Onizuka Naoko,Kayama Tomohiro,Kataoka Kensuke,Suzuki Hidetsugu,Mori Masaki,Inagawa Masayo,Ichinose Shizuko,Lotz Martin K,Sakai Daisuke,Masuda Koichi,Ozaki Toshifumi,Asahara Hiroshi
The main pathogenesis of intervertebral disc (IVD) herniation involves disruption of the annulus fibrosus (AF) caused by ageing or excessive mechanical stress and the resulting prolapse of the nucleus pulposus. Owing to the avascular nature of the IVD and lack of understanding the mechanisms that maintain the IVD, current therapies do not lead to tissue regeneration. Here we show that homeobox protein Mohawk (Mkx) is a key transcription factor that regulates AF development, maintenance and regeneration. Mkx is mainly expressed in the outer AF (OAF) of humans and mice. In Mkx(-/-) mice, the OAF displays a deficiency of multiple tendon/ligament-related genes, a smaller OAF collagen fibril diameter and a more rapid progression of IVD degeneration compared with the wild type. Mesenchymal stem cells overexpressing Mkx promote functional AF regeneration in a mouse AF defect model, with abundant collagen fibril formation. Our results indicate a therapeutic strategy for AF regeneration.
Development of a two-step protocol for culture expansion of human annulus fibrosus cells with TGF-β1 and FGF-2.
Chou Po-Hsin,Wang Shih-Tien,Ma Hsiao-Li,Liu Chien-Lin,Chang Ming-Chau,Lee Oscar Kuang-Sheng
Stem cell research & therapy
BACKGROUND:Different biologic approaches to treat disc regeneration, including growth factors (GFs) application, are currently under investigation. Human annulus fibrosus (hAF) repair or regeneration is one of the key elements for maintenance and restoration of nucleus pulposus function. However, so far there is no effective treatment for this purpose. The aim of the present study was to investigate the response of hAF cells to different combinations of GFs, and develop a protocol for efficient culture expansion. METHODS:hAF cells were harvested from degenerated disc tissues during surgical intervertebral disc removal, and hAF cells were expanded in a monolayer. The experiments were categorized based on different protocols with transforming growth factor (TGF-β1) and fibroblast growth factor (FGF-2) culture for 14 days: group 1 had no GFs (control group); group 2 received TGF-β1; group 3 received FGF-2; group 4 received both GFs; and group 5 (two-step) received both GFs for the first 10 days and TGF-β1 only for the next 4 days. Cell proliferation, collagen, and noncollagen extracellular matrix (ECM) production and genes expression were compared among these groups. RESULTS:At days 3, 7 and 10 of cultivation, groups 4 and 5 had significantly more cell numbers and faster cell proliferation rates than groups 1, 2, and 3. At 14 days of cultivation, significantly more cell numbers were observed in groups 3 and 4 than in group 5. The group 4 had the most cell numbers and the fastest proliferation rate at 14 days of cultivation. After normalization for cell numbers, group 5 (two-step) produced the most collagen and noncollagen ECM at 10 and 14 days of cultivation among the five groups. In group 5, ECM gene expression was significantly upregulated. High expression of matrix metalloproteinase-1 was upregulated with FGF-2 on the different days as compared to the other groups. Annulus fibrosus cell phenotypes were only marginally retained under the different protocols based on quantitative polymerase chain reaction results. CONCLUSION:Taken together, the two-step protocol was the most efficient among these different protocols with the most abundant ECM production after normalization for cell numbers for culture expansion of hAF cells. The protocol may be useful in further cell therapy and tissue engineering approaches for disc regeneration.
Diffusion characteristics of human annulus fibrosus-a study documenting the dependence of annulus fibrosus on end plate for diffusion.
Naresh-Babu J,Neelima G,Reshma Begum Sk,Siva-Leela Voleti
The spine journal : official journal of the North American Spine Society
BACKGROUND:Intervertebral disc being avascular depends on nutrition from either the end plate or the annulus fibrosus (AF). The role of the end plate on disc diffusion had been extensively studied. However, diffusion of human AF remains poorly understood because of the lack of reliable techniques to study AF in vivo and non-invasively. The present study for the first time evaluates the 24-hour diffusion characteristics of AF in radial, axial, and circumferential directions. PURPOSE:The study aimed to document the 24-hour diffusion characteristics of human AF. STUDY DESIGN:This is an in vivo human serial post-contrast magnetic resonance image study. METHODS:Twenty-five discs from five healthy volunteers (age <20 years) were studied. Diffusion over 24 hours following intravenous gadodiamide injection (0.3 mmol/kg) was studied at 10 minutes, and at 2, 4, 6, 12, and 24 hours. Axial images of the cranial, middle, and caudal zones of the discs were obtained. The vertebral body and end plate signal intensities were measured in sagittal sections. Thirty-nine regions of interest (24 in AF, 15 in nucleus pulposus) in each disc were analyzed. The peak enhancement percentage (EPmax) and the time to attain EPmax (Tmax) were calculated. Radial (outer vs. inner AF), axial (cranial vs. caudal vs. middle zone), and circumferential diffusions were analyzed. (The study received research grant from AOSpine India for US$6,000). RESULTS:Annulus fibrosus showed a biphasic pattern of diffusion with a characteristic "double peak." Early peak was seen at 10 minutes (coinciding with Tmax of the vertebral body) and delayed peak was seen at 6 hours (coinciding with Tmax of the nucleus pulposus), and characteristically noted after Tmax of the end plate (2 hours). The inner AF showed significant regional differences both at the early and delayed peaks, but the outer AF had no regional differences in the early peak. In axial direction, both outer and inner AF showed maximum enhancement percentage in the middle zone, followed by the caudal zone and least in the cranial zone. CONCLUSIONS:Annulus fibrosus characteristically showed a "double-peak" pattern of diffusion. Both the peaks had different characteristics, confirming two different sources of nutrition. The initial peak was contributed by periannular vascularity and the delayed one via the end plate from the vertebral body. The fact that even AF depends on the end plate for nutrition helps us better understand the complex nutritional pathways of intervertebral discs.
miR-221 attenuates the osteogenic differentiation of human annulus fibrosus cells.
Yeh Ching-Hua,Jin Li,Shen Francis,Balian Gary,Li Xudong Joshua
The spine journal : official journal of the North American Spine Society
BACKGROUND:In the moderate and end stages of intervertebral disc (IVD) degeneration, endochondral ossifications are found in the IVD. PURPOSE:The aim of this study was to investigate whether endochondral ossification in the late stages of disc degeneration is due to the differentiation of resident progenitor cell in the annulus fibrosus (AF) and the potential signaling pathways in vitro. STUDY DESIGN:This is an in vitro study of AF cell osteogenic differentiation and possible mechanisms METHODS:Normal annulus fibrosus (NAF) and degenerated annulus fibrosus (DAF) cells were isolated from tissue removed surgically from juvenile patients with idiopathic scoliosis and adult patients with degenerative scoliosis. Osteogenic differentiation was investigated using quantitative reverse transcription polymerase chain reaction (RT-PCR) and histology. The effects of miR-221 on osteogenesis were measured by overexpression of miR-221 with lentivirus. BMP2 and phospho-Smad proteins were detected by Western blotting. RESULTS:Both NAF and DAF cells underwent osteogenic differentiation, which was confirmed by detecting mineralization of the cell cultures and by an increase in the expression mRNAs for BMP2, runx2, alkaline phosphatase (ALP), and osteocalcin. DAF cells exhibited increased osteogenic differentiation potential over the NAF cells. By contrast to the elevated phospho-Smads, the basal level of miR-221 significantly decreased in DAF cells compared with that in NAF cells. Cultures of both cell types in osteogenic medium showed a decrease in miR-221 expression, and overexpression of miR-221 markedly decreased the level of BMP2, phospho-Smads, and the expression of osteogenic genes in DAF cells. The osteogenic potential of DAF cells diminished by the overexpression of miR-221. CONCLUSION:Compared with NAF cells, AF cells from degenerated discs have a greater tendency for osteogenic differentiation, which involves the BMP-Smad pathways and can be regulated by miR-221. These observations may be developed into a therapeutic to prevent the endochondral ossification.
Nano and micro biomechanical alterations of annulus fibrosus after in situ immobilization revealed by atomic force microscopy.
Liang Ting,Che Yan-Jun,Chen Xi,Li Hai-Tao,Yang Hui-Lin,Luo Zong-Ping
Journal of orthopaedic research : official publication of the Orthopaedic Research Society
Annulus fibrosus is critical to bear loads and resist fluid flow in the intervertebral disc. However, the detailed biomechanical mechanism of annulus fibrosus under abnormal loading is still ambiguous, especially at the micro and nano scales. This study aims to characterize the alterations of modulus at the nano scale of individual collagen fibrils in annulus fibrosus after in-situ immobilization, and the corresponding micro-biomechanics of annulus fibrosus. An immobilization model was used on the rat tail with an external fixation device. The elastic modulus of annulus fibrosus at both the nano- and micro-scale was examined using atomic force microscopy after fixation for 4 and 8 weeks, respectively. The fibrils in inner layer showed an alteration in elastic modulus from 91.38 ± 20.19 MPa in the intact annulus fibrosus to 110.64 ± 15.58 MPa (p < 0.001) at the nano scale after immobilization for 8 weeks, while the corresponding modulus at the micro scale also underwent a change from 0.33 ± 0.04 MPa to 0.47 ± 0.04 MPa (p < 0.001). The fibril disorder after immobilization was observed by hematoxylin/eosin staining. The gene expression of annulus fibrosus was also measured by real-time reverse transcription-polymerase chain reaction, which showed the upregulation of collagen II (p = 0.003) after immobilization. The results indicated that the immobilization not only influenced the individual fibril at the nanoscale, but also the micro-biomechanical property of annulus fibrosus which is critical to define the cell response to surrounding biomechanical environment. These alterations may also lead to the change in the mechanical property of the whole disc and the load-bearing function. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 9999:1-7, 2018.
Biologic Annulus Fibrosus Repair: A Review of Preclinical In Vivo Investigations.
Sloan Stephen R,Lintz Marianne,Hussain Ibrahim,Hartl Roger,Bonassar Lawrence J
Tissue engineering. Part B, Reviews
Lower back pain, the leading cause of workplace absences and disability, is often attributed to intervertebral disc degeneration, in which nucleus pulposus (NP) herniates through lesions in the annulus fibrosus (AF) and impinges on the spinal cord and surrounding nerves. Surgeons remove extruded NP via discectomy when indicated by local/radicular pain supported by radiographic evidence; however, current interventions do not alter the underlying disease or seal the AF. The reported rates of recurrent herniation or pain following discectomy cases range from 5% to 25%, which has pushed spine research in recent years toward annular repair and closure strategies. Synthetic implants designed to mechanically seal the AF have been subject to large animal and clinical trials, with limited success in preventing recurrent herniation. Like gold standard interventions, purely mechanical devices fail to promote tissue integration, long-term healing, or restore native biomechanical function to the spine. Biological repair strategies utilizing principles of tissue engineering have demonstrated success in overcoming the inadequacies of current interventions and mechanical implants, yet, none has reached clinical or proof-of-concept trials in humans. In this review, we will discuss annular repair strategies promoting biological healing that have been implemented in small and large animal models in vivo, and ways to enhance the efficacy of these treatments.
Quality Assessment of Surgical Disc Samples Discriminates Human Annulus Fibrosus and Nucleus Pulposus on Tissue and Molecular Level.
Schubert Ann-Kathrin,Smink Jeske J,Arp Mirko,Ringe Jochen,Hegewald Aldemar A,Sittinger Michael
International journal of molecular sciences
A discrimination of the highly specialised annulus fibrosus (AF) and nucleus pulposus (NP) cells in the mature human intervertebral disc (IVD) is thus far still not possible in a reliable way. The aim of this study was to identify molecular markers that distinguish AF and NP cells in human disc tissue using microarray analysis as a screening tool. AF and NP samples were obtained from 28 cervical discs. First, all samples underwent quality sorting using two novel scoring systems for small-sized disc tissue samples including macroscopic, haptic and histological evaluation. Subsequently, samples with clear disc characteristics of either AF or NP that were free from impurities of foreign tissue (IVD score) and with low signs of disc degeneration on cellular level (DD score) were selected for GeneChip analysis (HGU1332P). The 11 AF and 9 NP samples showed distinctly different genome-wide transcriptomes. The majority of differentially expressed genes (DEGs) could be specifically assigned to the AF, whereas no DEG was exclusively expressed in the NP. Nevertheless, we identified 11 novel marker genes that clearly distinguished AF and NP, as confirmed by quantitative gene expression analysis. The novel established scoring systems and molecular markers showed the identity of AF and NP in disc starting material and are thus of great importance in the quality assurance of cell-based therapeutics in regenerative treatment of disc degeneration.
Type XI collagen-perlecan-HS interactions stabilise the pericellular matrix of annulus fibrosus cells and chondrocytes providing matrix stabilisation and homeostasis.
Smith Susan M,Melrose James
Journal of molecular histology
The aim of this study was to ascertain whether, like many cell types in cartilaginous tissues if type XI collagen was a pericellular component of annulus fibrosus (AF) cells and chondrocytes. Fine fibrillar networks were visualised which were perlecan, HS (MAb 10E4) and type XI collagen positive. Heparitinase-III pre-digestion abolished the type XI collagen and 10E4 localisation in these fibrillar assemblies demonstrating a putative HS mediated interaction which localised the type XI collagen. Type XI collagen was confirmed to be present in the Heparitinase III treated AF monolayer media samples by immunoblotting. Heparitinase-III generated ΔHS stub epitopes throughout these fibrillar networks strongly visualised by MAb 3-G-10. Monolayers of murine hip articular chondrocytes from C57BL/6 and Hspg2 exon 3 null mice also displayed pericellular perlecan localisations, however type XI collagen was only evident in the Wild type mice. Perlecan was also immunolocalised in control and murine knee articular cartilage from the two mouse genotypes subjected to a medial meniscal destabilisation procedure which induces OA. This resulted in a severe depletion of perlecan levels particularly in the perlecan exon 3 null mice and was consistent with OA representing a disease of the pericellular matrix. A model was prepared to explain these observations between the NPP type XI collagen domain and HS chains of perlecan domain-I in the pericellular matrix of AF cells which likely contributed to cellular communication, tissue stabilization and the regulation of extracellular matrix homeostasis.
Bovine and degenerated human annulus fibrosus: a microstructural and micromechanical comparison.
Vergari Claudio,Chan Daniel,Clarke Andrew,Mansfield Jessica C,Meakin Judith R,Winlove Peter C
Biomechanics and modeling in mechanobiology
The complex structure of the annulus fibrosus is strongly related to its mechanical properties. Recent work showed that it is possible to observe the relative movement of fibre bundles in loaded cow tail annulus; the aim of this work was to describe and quantify annulus fibrosus micromechanics in degenerated human disc, and compare it with cow tail annulus, an animal model often used in the literature. Second harmonic generation was used to image the collagen matrix in twenty strips of annulus fibrosus harvested from intervertebral disc of seven patients undergoing surgery. Samples were loaded to 6% tensile strain in 1% steps. Elastic modulus was calculated from loading curves, and micromechanical strains were calculated from the images using custom software. The same protocol was applied to twenty strips of annulus harvested from cow tail discs. Significant morphological differences were found between human and cow tail samples, the most striking being the lack of collagen fibre crimp in the former. Fibres were also observed bending and running from one lamella to the other, forming a strong flexible interface. Interdigitation of fibre bundles was also present at this interface. Quantitative results show complex patterns of inter-bundle and inter-lamellar behaviour, with inter-bundle sliding being the main strain mechanism. Elastic modulus was similar between species, and it was not affected by the degree of degeneration. This work gives an insight into the complex structure and mechanical function of the annulus fibrosus, which should be accounted for in disc numerical modelling.
Individual Collagen Fibril Thickening and Stiffening of Annulus Fibrosus in Degenerative Intervertebral Disc.
Liang Ting,Zhang Lin-Lin,Xia Wei,Yang Hui-Lin,Luo Zong-Ping
STUDY DESIGN:In vitro study using rat intervertebral discs (IVDs). OBJECTIVE:To explore the alteration of annulus fibrosus collagen fibrils after loading on IVD and to investigate the degeneration pathogenesis at the nanoscale. SUMMARY OF BACKGROUND DATA:Abnormal loading can lead to IVD degeneration, but the precise mechanism has been hitherto elusive, especially at the nanoscale. METHODS:A rat IVD loading model was used, which combined bending of the tail by 40° with compressive loading of 1.8, 4.5, and 7.2 N of the rat tail using an external fixation device. The structure and the elastic modulus of individual collagen fibrils within IVD Co8-Co9 was examined 2 weeks after loading at the nanoscale using atomic force microscopy. RESULTS:Significant fibril disorder and a decrease in cell number within the annulus fibrosus after loading was observed at the microscale as judged by hematoxylin/eosin staining, suggesting initiation of rupture of the structure and degradation of the IVD. The annulus fibrosus collagen fibrils underwent a change in diameter and elastic modulus from 170 ± 18 to 310 ± 24 nm (P < 0.001) and 0.86 ± 0.12 to 1.27 ± 0.30 GPa (P = 0.003), respectively when measured on the concave side after a loading of 7.2 N. Thus the loading process resulted in a thickening and stiffening of collagen fibrils with a difference between the inner and outer layers. CONCLUSION:The results of the present study indicated that abnormal loading was not only associated with disorder at the microscale, but also alteration of the collagen fibrils at the nanoscale, possibly leading to changes in the mechanical and physiological environment around the cells of the annulus fibrosus. LEVEL OF EVIDENCE:N/A.
In vivo annular repair using high-density collagen gel seeded with annulus fibrosus cells.
Moriguchi Yu,Borde Brandon,Berlin Connor,Wipplinger Christoph,Sloan Stephen R,Kirnaz Sertac,Pennicooke Brenton,Navarro-Ramirez Rodrigo,Khair Thamina,Grunert Peter,Kim Eliana,Bonassar Lawrence,Härtl Roger
OBJECTIVE:The aim is assessing the in vivo efficacy of annulus fibrosus (AF) cells seeded into collagen by enhancing the reparative process around annular defects and preventing further degeneration in a rat-tail model. SUMMARY OF BACKGROUND DATA:Treating disc herniation with discectomy may relieve the related symptoms but does not address the underlying pathology. The persistent annular defect may lead to re-herniation and further degeneration. We recently demonstrated that riboflavin crosslinked high-density collagen gels (HDC) can facilitate annular repair in vivo. METHODS:42 rats, tail disc punctured with an 18-gauge needle, were divided into 3 groups: untreated (n = 6), injected with crosslinked HDC (n = 18), and injected with AF cell-laden crosslinked HDC (n = 18). Ovine AF cells were mixed with HDC gels prior to injection. X-rays and MRIs were conducted over 5 weeks, determining disc height index (DHI), nucleus pulposus (NP) size, and hydration. Histological assessments evaluated the viability of implanted cells and degree of annular repair. RESULTS:Although average DHIs of both HDC gel groups were higher than those of the puncture control group at 5 weeks, the retention of disc height, NP size and hydration at 1 and 5 weeks was significant for the cellular group compared to the punctured, and at 5 weeks to the acellular group. Histological assessment indicated that AF cell-laden HDC gels have accelerated reparative sealing compared to acellular HDC gels. CONCLUSIONS:AF cell-laden HDC gels have the ability of better repairing annular defects than acellular gels after needle puncture. STATEMENT OF SIGNIFICANCE:This project addresses the compelling demand of a sufficient treatment strategy for degenerative disc disease (DDD) perpetuated by annulus fibrosus (AF) injury, a major cause of morbidity and burden to health care systems. Our study is designed to answer the question of whether injectable, photo-crosslinked, high density collagen gels can seal defects in the annulus fibrosus of rats and prevent disc degeneration. Furthermore, we investigated whether the healing of AF defects will be enhanced by the delivery of AF cells (fibrochondrocytes) to these defects. The use of cell-laden collagen gels in spine surgery holds promise for a wide array of applications, from current discectomy procedures to future nucleus pulposus reparative therapies, and our group is excited about this potential.
Qualitative and quantitative assessment of collagen and elastin in annulus fibrosus of the physiologic and scoliotic intervertebral discs.
Kobielarz Magdalena,Szotek Sylwia,Głowacki Maciej,Dawidowicz Joanna,Pezowicz Celina
Journal of the mechanical behavior of biomedical materials
The biophysical properties of the annulus fibrosus of the intervertebral disc are determined by collagen and elastin fibres. The progression of scoliosis is accompanied by a number of pathological changes concerning these structural proteins. This is a major cause of dysfunction of the intervertebral disc. The object of the study were annulus fibrosus samples excised from intervertebral discs of healthy subjects and patients treated surgically for scoliosis in the thoracolumbar or lumbar spine. The research material was subjected to structural analysis by light microscopy and quantitative analysis of the content of collagen types I, II, III and IV as well as elastin by immunoenzymatic test (ELISA). A statistical analysis was conducted to assess the impact of the sampling site (Mann-Whitney test, α=0.05) and scoliosis (Wilcoxon matched pairs test, α=0.05) on the obtained results. The microscopic studies conducted on scoliotic annulus fibrosus showed a significant architectural distortion of collagen and elastin fibres. Quantitative biochemical assays demonstrated region-dependent distribution of only collagen types I and II in the case of healthy intervertebral discs whereas in the case of scoliotic discs region-dependent distribution concerned all examined proteins of the extracellular matrix. Comparison of scoliotic and healthy annulus fibrosus revealed a significant decrease in the content of collagen type I and elastin as well as a slight increase in the proportion of collagen types III and IV. The content of collagen type II did not differ significantly between both groups. The observed anomalies are a manifestation of degenerative changes affecting annulus fibrosus of the intervertebral disc in patients suffering from scoliosis.
Shear-wave elastography can evaluate annulus fibrosus alteration in adolescent scoliosis.
Langlais Tristan,Vergari Claudio,Pietton Raphael,Dubousset Jean,Skalli Wafa,Vialle Raphael
OBJECTIVES:In vitro studies showed that annulus fibrosus lose its integrity in idiopathic scoliosis. Shear-wave ultrasound elastography can be used for non-invasive measurement of shear-wave speed (SWS) in vivo in the annulus fibrosus, a parameter related to its mechanical properties. The main aim was to assess SWS in lumbar annulus fibrosus of scoliotic adolescents and compare it to healthy subjects. METHODS:SWS was measured in 180 lumbar IVDs (L3L4, L4L5, L5S1) of 30 healthy adolescents (13 ± 1.9 years old) and 30 adolescent idiopathic scoliosis patients (13 ± 2 years old, Cobb angle: 28.8° ± 10.4°). SWS was compared between the scoliosis and healthy control groups. RESULTS:In healthy subjects, average SWS (all disc levels pooled) was 3.0 ± 0.3 m/s, whereas in scoliotic patients it was significantly higher at 3.5 ± 0.3 m/s (p = 0.0004; Mann-Whitney test). Differences were also significant at all disc levels. No difference was observed between males and females. No correlation was found with age, weight and height. CONCLUSION:Non-invasive shear-wave ultrasound is a novel method of assessment to quantitative alteration of annulus fibrosus. These preliminary results are promising for considering shear-wave elastography as a biomechanical marker for assessment of idiopathic scoliosis. KEY POINTS:• Adolescent idiopathic scoliosis may have an altered lumbar annulus fibrosus. • Shear-wave elastography can quantify lumbar annulus fibrosus mechanical properties. • Shear-wave speed was higher in scoliotic annulus than in healthy subjects. • Elastography showed potential as a biomechanical marker for characterizing disc alteration.
Stromal cell-derived factor-1α-encapsulated albumin/heparin nanoparticles for induced stem cell migration and intervertebral disc regeneration in vivo.
Zhang Hua,Yu Shan,Zhao Xinlian,Mao Zhengwei,Gao Changyou
Intervertebral disc (IVD) degeneration may cause many diseases and pain. Stem cell migration toward the site of IVD degeneration is a key factor for IVD regeneration. In the current study, we prepared albumin/heparin nanoparticles (BHNPs) as injectable carriers of stromal cell-derived factor-1α (SDF-1α, also known as C-X-C motif chemokine 12), a powerful chemoattractant for the homing of bone marrow resident mesenchymal stem cells (MSCs), for protection of the molecule against degradation for a sustained release. The NPs have relatively uniform small size, with a diameter of about 110 nm. The NPs possess a high loading capacity of SDF-1α with a sustained release profile. The bioactivity of the obtained BHNPs/SDF was then studied in vitro and in vivo. The BHNPs/SDF can induce migration of MSCs in a dose-dependent manner in vitro. After injected into the damaged disc, BHNPs/SDF induce much better regeneration of annulus fibrosus and nucleus pulposus, compared to SDF-1α and BHNPs alone, evidenced with better histological grade scores and higher expression of SOX9, Aggrecan, and Collagen type II at the level of both mRNA and protein. This study provides a simple nanoplatform to load SDF-1α and protect it against degradation, with potential application in inductive tissue regeneration in vivo. STATEMENT OF SIGNIFICANCE:Stem cell migration toward the site of IVD degeneration is a key event to promote IVD regeneration. In the current study, we prepared albumin/heparin nanoparticles (BHNPs) as injectable carriers to protect SDF-1α against degradation and for the sustained release of the molecule. After injected into the damaged disc, BHNPs/SDF induced much better regeneration of IVD, compared to SDF-1α and BHNPs alone. This study provides a simple nanoplatform to load SDF-1α and protect it from degradation, with potential application in inductive tissue regeneration in vivo.
Riboflavin crosslinked high-density collagen gel for the repair of annular defects in intervertebral discs: An in vivo study.
Grunert Peter,Borde Brandon H,Towne Sara B,Moriguchi Yu,Hudson Katherine D,Bonassar Lawrence J,Härtl Roger
Open annular defects compromise the ability of the annulus fibrosus to contain nuclear tissue in the disc space, and therefore lead to disc herniation with subsequent degenerative changes to the entire intervertebral disc. This study reports the use of riboflavin crosslinked high-density collagen gel for the repair of annular defects in a needle-punctured rat-tail model. High-density collagen has increased stiffness and greater hydraulic permeability than conventional low-density gels; riboflavin crosslinking further increases these properties. This study found that treating annular defects with crosslinked high-density collagen inhibited the progression of disc degeneration over 18 weeks compared to untreated control discs. Histological sections of FITC-labeled collagen gel revealed an early tight attachment to host annular tissue. The gel was subsequently infiltrated by host fibroblasts which remodeled it into a fibrous cap that bridged the outer disrupted annular fibers and partially repaired the defect. This repair tissue enhanced retention of nucleus pulposus tissue, maintained physiological disc hydration, and preserved hydraulic permeability, according to MRI, histological, and mechanical assessments. Degenerative changes were partially reversed in treated discs, as indicated by an increase in nucleus pulposus size and hydration between weeks 5 and 18. The collagen gel appeared to work as an instant sealant and by enhancing the intrinsic healing capabilities of the host tissue.
MR Elastography-derived Stiffness: A Biomarker for Intervertebral Disc Degeneration.
Walter Benjamin A,Mageswaran Prasath,Mo Xiaokui,Boulter Daniel J,Mashaly Hazem,Nguyen Xuan V,Prevedello Luciano M,Thoman William,Raterman Brian D,Kalra Prateek,Mendel Ehud,Marras William S,Kolipaka Arunark
Purpose To determine the repeatability of magnetic resonance (MR) elastography-derived shear stiffness measurements of the intervertebral disc (IVD) taken throughout the day and their relationship with IVD degeneration and subject age. Materials and Methods In a cross-sectional study, in vivo lumbar MR elastography was performed once in the morning and once in the afternoon in 47 subjects without current low back pain (IVDs = 230; age range, 20-71 years) after obtaining written consent under approval of the institutional review board. The Pfirrmann degeneration grade and MR elastography-derived shear stiffness of the nucleus pulposus and annulus fibrosus regions of all lumbar IVDs were assessed by means of principal frequency analysis. One-way analysis of variance, paired t tests, concordance and Bland-Altman tests, and Pearson correlations were used to evaluate degeneration, diurnal changes, repeatability, and age effects, respectively. Results There were no significant differences between morning and afternoon shear stiffness across all levels and there was very good technical repeatability between the morning and afternoon imaging results for both nucleus pulposus (R = 0.92) and annulus fibrosus (R = 0.83) regions. There was a significant increase in both nucleus pulposus and annulus fibrosus MR elastography-derived shear stiffness with increasing Pfirrmann degeneration grade (nucleus pulposus grade 1, 12.5 kPa ± 1.3; grade 5, 16.5 kPa ± 2.1; annulus fibrosus grade 1, 90.4 kPa ± 9.3; grade 5, 120.1 kPa ± 15.4), and there were weak correlations between shear stiffness and age across all levels (R ≤ 0.32). Conclusion Our results demonstrate that MR elastography-derived shear stiffness measurements are highly repeatable, weakly correlate with age, and increase with advancing IVD degeneration. These results suggest that MR elastography-derived shear stiffness may provide an objective biomarker of the IVD degeneration process. RSNA, 2017 Online supplemental material is available for this article.
The ultra-structural organization of the elastic network in the intra- and inter-lamellar matrix of the intervertebral disc.
Tavakoli J,Elliott D M,Costi J J
The inter-lamellar matrix (ILM)-located between adjacent lamellae of the annulus fibrosus-consists of a complex structure of elastic fibers, while elastic fibers of the intra-lamellar region are aligned predominantly parallel to the collagen fibers. The organization of elastic fibers under low magnification, in both inter- and intra-lamellar regions, was studied by light microscopic analysis of histologically prepared samples; however, little is known about their ultrastructure. An ultrastructural visualization of elastic fibers in the inter-lamellar matrix is crucial for describing their contribution to structural integrity, as well as mechanical properties of the annulus fibrosus. The aims of this study were twofold: first, to present an ultrastructural analysis of the elastic fiber network in the ILM and intra-lamellar region, including cross section (CS) and in-plane (IP) lamellae, of the AF using Scanning Electron Microscopy (SEM) and second, to -compare the elastic fiber orientation between the ILM and intra-lamellar region. Four samples (lumbar sheep discs) from adjacent sections (30μm thickness) of anterior annulus were partially digested by a developed NaOH-sonication method for visualization of elastic fibers by SEM. Elastic fiber orientation and distribution were quantified relative to the tangential to circumferential reference axis. Visualization of the ILM under high magnification revealed a dense network of elastic fibers that has not been previously described. Within the ILM, elastic fibers form a complex network, consisting of different size and shape fibers, which differed to those located in the intra-lamellar region. For both regions, the majority of fibers were oriented near 0° with respect to tangential to circumferential (TCD) direction and two minor symmetrical orientations of approximately±45°. Statistically, the orientation of elastic fibers between the ILM and intra-lamellar region was not different (p=0.171). The present study used extracellular matrix partial digestion to address significant gaps in understanding of disc microstructure and will contribute to multidisciplinary ultrastructure-function studies. STATEMENT OF SIGNIFICANCE:Visualization of the intra-lamellar matrix under high magnification revealed a dense network of elastic fibers that has not been previously described. The present study used extracellular matrix partial digestion to address significant gaps in understanding of disc microstructure and will contribute to multidisciplinary ultrastructure-function studies.
A novel mouse model of intervertebral disc degeneration shows altered cell fate and matrix homeostasis.
Choi Hyowon,Tessier Steven,Silagi Elizabeth S,Kyada Rutvin,Yousefi Farzad,Pleshko Nancy,Shapiro Irving M,Risbud Makarand V
Matrix biology : journal of the International Society for Matrix Biology
Intervertebral disc degeneration and associated low back and neck pain is a ubiquitous health condition that affects millions of people world-wide, and causes high incidence of disability and enormous medical/societal costs. However, lack of appropriate small animal models with spontaneous disease onset has impeded our ability to understand the pathogenetic mechanisms that characterize and drive the degenerative process. We report, for the first time, early onset spontaneous disc degeneration in SM/J mice known for their poor regenerative capacities compared to "super-healer" LG/J mice. In SM/J mice, degenerative process was marked by decreased nucleus pulposus (NP) cellularity and changes in matrix composition at P7, 4, and 8 weeks with increased severity by 17 weeks. Distinctions between NP and annulus fibrosus (AF) or endplate cartilage were lost, and NP and AF of SM/J mice showed higher histological grades. There was increased NP cell death in SM/J mice with decreased phenotypic marker expression. Polarized microscopy and FTIR spectroscopy demonstrated replacement of glycosaminoglycan-rich NP matrix with collagenous fibrous tissue. The levels of ARGxx were increased in, indicating higher aggrecan turnover. Furthermore, an aberrant expression of collagen X and MMP13 was observed in the NP of SM/J mice, along with elevated expression of Col10a1, Ctgf, and Runx2, markers of chondrocyte hypertrophy. Likewise, expression of Enpp1 as well as Alpl was higher, suggesting NP cells of SM/J mice promote dystrophic mineralization. There was also a decrease in several pathways necessary for NP cell survival and function including Wnt and VEGF signaling. Importantly, SM/J discs were stiffer, had decreased height, and poor vertebral bone quality, suggesting compromised motion segment mechanical functionality. Taken together, our results clearly demonstrate that SM/J mouse strain recapitulates many salient features of human disc degeneration, and serves as a novel small animal model.
Injectable hyaluronic acid down-regulates interferon signaling molecules, IGFBP3 and IFIT3 in the bovine intervertebral disc.
Kazezian Zepur,Li Zhen,Alini Mauro,Grad Sibylle,Pandit Abhay
Low back pain which is a major cause of disability for people aged between 20 and 50years imposes a serious socio-economic burden. The current focus of regenerative medicine is on identifying molecular markers to facilitate the design of targeted therapeutics. Previously, we have demonstrated that expression of the anti-proliferative interferon-induced protein with tetratricopeptide repeats 3 (IFIT3) and pro-apoptotic insulin-like growth factor-binding protein-3 (IGFBP3), are up-regulated as downstream targets of the inflammatory cytokine interferon α (IFNα) signaling pathway in the human annulus fibrosus (AF). Here, we hypothesised that injection of hyaluronic acid (HA) would have an anti-inflammatory and matrix modulatory effect on injured and IFNαβ inflamed bovine intervertebral discs (IVD). Discs with an AF defect and challenged with IFNαβ were used in a bovine IVD organ culture model to test the effect of HA on the IFNαβ pathway, as well as the matrix proteins aggrecan and collagen I. qRT-PCR was used to assess the gene expression of IFNαβ signaling molecules. Additionally, immunostaining was used to measure protein expression. Our results show that HA treatment significantly down-regulates IFNAR1, IFNAR2, STAT1/2, JAK1, IFIT3 and IGFBP3 mRNA expression in the inflamed groups. Protein analysis confirmed the PCR results. In the extracellular matrix, aggrecan and collagen I were up-regulated while ADAMTS4 was down-regulated upon treatment of the injured and inflamed discs with HA. Hence, HA demonstrates both an anti-inflammatory role, resulting in the down-regulation of IFIT3 and IGFBP3 in the AF, and a matrix modulatory effect by up-regulating aggrecan and collagen I expression. STATEMENT OF SIGNIFICANCE:The pro-inflammatory environment of the degenerated IVD represents a challenge for regenerative therapies. The study demonstrates that hyaluronan acts as an anti-inflammatory molecule by down-regulating IFNAR1 and IFNAR2, the signaling molecules STAT1, STAT2, JAK1 and the downstream apoptotic targets IGFBP3 and IFIT3. We also demonstrated that hyaluronan modulates the disc matrix environment by increasing aggrecan and collagen I synthesis and down-regulating ADAMTS4 that degrades the matrix under inflammatory conditions. The significance of this work lies in the fact that hyaluronan acts as an anti-inflammatory molecule by shifting the disc environment towards a more anabolic state and by promoting native IVD matrix production.
Shape-memory porous alginate scaffolds for regeneration of the annulus fibrosus: effect of TGF-β3 supplementation and oxygen culture conditions.
Guillaume Olivier,Daly Andrew,Lennon Kerri,Gansau Jennifer,Buckley Shane F,Buckley Conor T
Disc herniation as a result of degenerative or traumatic injury is believed to be the primary instigator of low back pain. At present there is a lack of viable treatment options to repair damaged annulus fibrosus (AF) tissue. Developing alternative strategies to fill and repair ruptured AF tissue is a key challenge. In this work we developed a porous alginate scaffold with shape-memory properties which can be delivered using minimally invasive approaches and recover its original geometry once hydrated. Covalently cross-linked alginate hydrogels were created using carbodiimide chemistry, followed by a freeze-drying step to impart porosity and create porous scaffolds. Results showed that porous alginate scaffolds exhibited shape-memory recovery and mechanical behaviour that could be modulated depending on the cross-linker concentrations. The scaffold can be repeatedly compressed and expanded, which provides the potential to deliver the biomaterial directly to the damaged area of the AF tissue. In vitro experiments demonstrated that scaffolds were cytocompatible and supported cell seeding, penetration and proliferation under intervertebral-disc-like microenvironmental conditions (low glucose media and low oxygen concentration). Extracellular matrix (ECM) was secreted by AF cells with TGF-β3 stimulation and after 21days had filled the porous scaffold network. This biological matrix was rich in sulfated glycosaminoglycan and collagen type I, which are the main compounds of native AF tissue. Successful ECM deposition was also confirmed by the increase in the peak stress of the scaffold. However, the immaturity of the matrix network after only 21days of in vitro culture was not sufficient to attain native AF tissue mechanical properties. The ability to deliver porous scaffolds using minimal invasive approaches that can potentially promote the regeneration of AF defects provides an exciting new avenue for disc repair.
Modulation of the gene expression of annulus fibrosus-derived stem cells using poly(ether carbonate urethane)urea scaffolds of tunable elasticity.
Zhu Caihong,Li Jun,Liu Chen,Zhou Pinghui,Yang Huilin,Li Bin
UNLABELLED:Annulus fibrosus (AF) injuries commonly lead to substantial deterioration of the intervertebral disc (IVD). While tissue engineering has recently evolved into a promising approach for AF regeneration, it remains challenging due to the cellular, biochemical, and mechanical heterogeneity of AF tissue. In this study, we explored the use of AF-derived stem cells (AFSCs) to achieve diversified differentiation of cells for AF tissue engineering. Since the differentiation of stem cells relies significantly on the elasticity of the substrate, we synthesized a series of biodegradable poly(ether carbonate urethane)urea (PECUU) materials whose elasticity approximated that of native AF tissue. When AFSCs were cultured on electrospun PECUU fibrous scaffolds, the gene expression of collagen-I in the cells increased with the elasticity of scaffold material, whereas the expression of collagen-II and aggrecan genes showed an opposite trend. At the protein level, the content of collagen-I gradually increased with substrate elasticity, while collagen-II and GAG contents decreased. In addition, the cell traction forces (CTFs) of AFSCs gradually decreased with scaffold elasticity. Such substrate elasticity-dependent changes of AFSCs were similar to the gradual transition in the genetic, biochemical, and biomechanical characteristics of cells from inner to outer regions of native AF tissue. Together, findings from this study indicate that AFSCs, depending on the substrate elasticity, have strong tendencies to differentiate into various types of AF-like cells, thereby providing a solid foundation for the tissue engineering applications of AFSCs. STATEMENT OF SIGNIFICANCE:Repairing the annulus fibrosus (AF) of intervertebral disc (IVD) is critical for the treatment of disc degeneration disease, but remains challenging due to the significant heterogeneity of AF tissue. Previously, we have identified rabbit AF-derived stem cells (AFSCs), which are AF tissue-specific and hold promise for AF regeneration. In this study, we synthesized a series of poly(ether carbonate urethane)ureas of various elasticity (or stiffness) and explored the potential of induced differentiation of AFSCs using electrospun PECUU scaffolds. This work has, for the first time, found that AFSCs are able to present different gene expression patterns simply as a result of the elasticity of scaffold material. Therefore, our findings will help supplement current knowledge of AF tissue regeneration and may benefit a diversified readership from scientific, engineering, and clinical settings whose work involves the biology and tissue engineering of IVD.
Initial investigation of individual and combined annulus fibrosus and nucleus pulposus repair ex vivo.
Sloan Stephen R,Galesso Devis,Secchieri Cynthia,Berlin Connor,Hartl Roger,Bonassar Lawrence J
Novel tissue engineered and biomaterial approaches to treat intervertebral disc (IVD) degeneration focus on single aspects of the progressive disease and hence are insufficient repair strategies. In this study, annulus fibrosus (AF) and nucleus pulposus (NP) biomaterial repair strategies were used individually and combined to treat IVD degeneration modeled in ex vivo rat-tail motion segments by annulotomy and nucleotomy. An injectable riboflavin cross-linked high-density collagen gel patched defects in the AF, while NP repair consisted of injections of a modified hyaluronic acid (HA) hydrogel. Qualitative imaging showed the annulotomy and nucleotomy successfully herniated NP material, while the HA NP injections restored intact NP morphology and the collagen AF patches sealed AF defects. Assessed by quantitative T2 magnetic resonance imaging, combined repair treatments yielded disc hydration not significantly different than intact hydration, while AF and NP repairs alone only restored ∼1/3 of intact hydration. Mechanical testing showed NP injections alone recovered on average ∼35% and ∼40% of the effective instantaneous and equilibrium moduli. The combined treatment comprising biomaterial AF and NP repair was effective at increasing NP hydration from NP repair alone, however HA injections alone are sufficient to improve mechanical properties. STATEMENT OF SIGNIFICANCE:Intervertebral disc degeneration affects an estimated 90% of individuals throughout their life, and is a candidate pathology for tissue engineered repair. The current standard of clinical care reduces spinal articulation and leads to further degeneration along the spine, hence great interest in a regenerative medicine therapy. Literature studies focused on biomaterial repair strategies for treating degenerated discs have partially restored native disc function, however no studies have reported the use of combined therapies to address multiple aspects of disc degeneration. This initial investigation screened injectable biomaterial repair strategies ex vivo, and through complementary outcome measures showed a combined therapy restores disc function better than individual approaches. This study is the first of its kind to address multiple aspects of disc degeneration, using clinically-oriented biomaterials in a well-established animal model.
Angle-ply biomaterial scaffold for annulus fibrosus repair replicates native tissue mechanical properties, restores spinal kinematics, and supports cell viability.
Borem Ryan,Madeline Allison,Walters Joshua,Mayo Henry,Gill Sanjitpal,Mercuri Jeremy
Annulus fibrosus (AF) damage commonly occurs due to intervertebral disc (IVD) degeneration/herniation. The dynamic mechanical role of the AF is essential for proper IVD function and thus it is imperative that biomaterials developed to repair the AF withstand the mechanical rigors of the native tissue. Furthermore, these biomaterials must resist accelerated degradation within the proteolytic environment of degenerate IVDs while supporting integration with host tissue. We have previously reported a novel approach for developing collagen-based, multi-laminate AF repair patches (AFRPs) that mimic the angle-ply architecture and basic tensile properties of the human AF. Herein, we further evaluate AFRPs for their: tensile fatigue and impact burst strength, IVD attachment strength, and contribution to functional spinal unit (FSU) kinematics following IVD repair. Additionally, AFRP resistance to collagenase degradation and cytocompatibility were assessed following chemical crosslinking. In summary, AFRPs demonstrated enhanced durability at high applied stress amplitudes compared to human AF and withstood radially-directed biaxial stresses commonly borne by the native tissue prior to failure/detachment from IVDs. Moreover, FSUs repaired with AFRPs and nucleus pulposus (NP) surrogates had their axial kinematic parameters restored to intact levels. Finally, carbodiimide crosslinked AFRPs resisted accelerated collagenase digestion without detrimentally effecting AFRP tensile properties or cytocompatibility. Taken together, AFRPs demonstrate the mechanical robustness and enzymatic stability required for implantation into the damaged/degenerate IVD while supporting AF cell infiltration and viability. STATEMENT OF SIGNIFICANCE:The quality of life for millions of individuals globally is detrimentally impacted by IVD degeneration and herniation. These pathologies often result in the structural demise of IVD tissue, particularly the annulus fibrosus (AF). Biomaterials developed for AF repair have yet to demonstrate the mechanical strength and durability required for utilization in the spine. Herein, we demonstrate the development of an angle-ply AF repair patch (AFRP) that can resist the application of physiologically relevant stresses without failure and which contributes to the restoration of functional spinal unit axial kinematics following repair. Furthermore, we show that this biomaterial can resist accelerated degradation in a simulated degenerate environment and supports AF cell viability.
Intervertebral disc swelling maintains strain homeostasis throughout the annulus fibrosus: A finite element analysis of healthy and degenerated discs.
Yang Bo,O'Connell Grace D
Tissues in the intervertebral disc have a large capacity to absorb water, partially due to the high glycosaminoglycan (GAG) content, which decreases linearly from the nucleus pulposus (NP) in the center to the outer annulus. Our recent work showed that fiber network and GAG distribution contributes to development of residual stresses and strains that were compressive in the inner annulus to tensile in the outer annulus. GAG loss in the inner annulus, as observed with early to moderate degeneration, reduced swelling capacity and circumferential-direction stress by over 50%. However, our previous model was not capable of evaluating interactions between the NP and annulus fibrosus (AF) during swelling. In this study, we evaluated the effect of degeneration (GAG content or swelling capacity) on residual stress development throughout the disc. Simulations of moderate to severe degeneration showed a 40% decrease in NP swelling capacity, with a 25% decrease in AF and cartilaginous endplate swelling. Together, these changes in tissue swelling resulted in a decrease in NP pressure (healthy = 0.21 MPa; severe degeneration = 0.03 MPa) that was comparable to observations in human discs. There was a 60% decrease in circumferential-direction residual deformations with early degeneration. Radial-direction stretch switched from compressive to tensile with degeneration, which may increase the risk for tears or delamination. Degeneration had a significant impact on residual stress/stretch and fiber stretch in the posterior AF, which is important for understanding herniation risk. In conclusion, degenerative changes in disc geometry and intradiscal deformations was recreated by only altering NP and AF GAG composition. Since most computational models simulate degeneration by altering material stiffness, this work highlights the importance of directly simulating biochemical composition and distribution to study disc biomechanics with degeneration. STATEMENT OF SIGNIFICANCE: Tissues in the intervertebral disc have a large swelling capacity, due to its high glycosaminoglycan content. Our recent work demonstrated the importance of fiber network and glycosaminoglycan distribution residual stresses and strains development. In this study, we evaluated the effect of swelling on intradiscal deformations between the nucleus pulposus and annulus fibrosus. We also investigated the effect of degenerative glycosaminoglycan loss on swelling-based intradiscal deformations of the intact disc and its subcomponents. Decreases in nucleus glycosaminoglycan content resulted in morphological changes observed with degenerated discs and may help to explain mechanisms behind the increases in annular tears and mechanical dysfunction with degeneration.
miR-640 aggravates intervertebral disc degeneration via NF-κB and WNT signalling pathway.
Dong Wengang,Liu Jun,Lv Yang,Wang Fei,Liu Tao,Sun Siguo,Liao Bo,Shu Zhen,Qian Jixian
OBJECTIVES:Low back pain becomes a common orthopaedic disease today. It is mainly induced by the degeneration of the intervertebral disc. In this study, we tried to reveal the pathogenesis of the degeneration and the relative therapeutic strategy, which are still elusive. MATERIALS AND METHODS:We collected 15 degenerative intervertebral tissues and five healthy donors. Nucleus pulposus and annulus fibrosus cells were subcultured. miR-640 expression was determined by qPCR. Computer analysis and luciferase reporter assay were used to confirm miR-640 target genes. Immunohistochemical and immunocytochemical staining was used to trace the proinflammatory cytokines and key transductor of signalling pathways. We also used β-galactosidase staining, flow cytometry, and cell viability assay to monitor the degenerative index. RESULTS:miR-640 overexpressed in patients derived degenerative nucleus pulposus tissues and cells. The inflammatory environment promoted miR-640 expression via NF-κB signalling pathway. In addition, miR-640 targeted to LRP1 and enhances NF-κB signal activity, which built a positive feedback loop. miR-640 inhibited the expression of β-catenin and EP300, therefore, restrained WNT signal and induced the degeneration in nucleus pulposus cells. miR-640 inhibitor treatment exhibited the effects of anti-inflammation, reverse WNT signalling pathway exhaustion, and remission of degenerative characteristics in vitro. CONCLUSIONS:miR-640 plays an important role in the degeneration of intervertebral disc and the relative inflammatory microenvironment. It is a promising potential therapeutic target for the low back pain biotherapy.
AMOT130 linking F-actin to YAP is involved in intervertebral disc degeneration.
Zhang Cong,Wang Feng,Xie Zhiyang,Chen Lu,Sinkemani Arjun,Yu Haomin,Wu Xiaotao
OBJECTIVES:Dysregulation of YAP by the Hippo signalling is associated with intervertebral disc degeneration (IDD). However, the relationship between the F-actin and Hippo pathway in IDD, and their effects on YAP remain poorly understood. METHODS:The characteristics of Hippo pathway and F-actin the in the NP (nucleus pulposus) and annulus fibrosus of immature, mature, ageing and disc degeneration model rats were observed by immunofluorescence, western blot and qPCR. Nucleus pulposus cells (NPCs) were transfected with lentivirus Sh-LATS A, Sh-LATS B and harvested for SA-β-gal staining, qPCR, western blotting and immunofluorescence staining to investigate the mechanism of Hippo pathway and F-actin interact in NPCs. RESULTS:We observed moderate decreases in F-actin and YAP expression with age in healthy intervertebral discs (IVDs). F-actin stress fibres distributed throughout the cytoplasm disappeared following treatment with latrunculin B (Lat B), resulting in a punctate distribution. Depletion of large tumour suppressor homologues 1/2 (LATS1/2) did not decrease the rate of cellular senescence, and YAP remained in the cytoplasm following Lat B treatment. Furthermore, angiomotin 130 (AMOT130) was associated with F-actin through a conserved actin-binding domain to retain YAP in the cytoplasm. CONCLUSIONS:This study showed that a mechanism by which Hippo pathway and F-actin synergize to modulate YAP activation and localization in the context of IDD and help to control NPCs proliferation.
Decoding the intervertebral disc: Unravelling the complexities of cell phenotypes and pathways associated with degeneration and mechanotransduction.
Kerr Geoffrey J,Veras Matthew A,Kim Min Kyu M,Séguin Cheryle A
Seminars in cell & developmental biology
Back pain is the most common cause of pain and disability worldwide. While its etiology remains unknown, it is typically associated with intervertebral disc (IVD) degeneration. Despite the prevalence of back pain, relatively little is known about the specific cellular pathways and mechanisms that contribute to the development, function and degeneration of the IVD. Consequently, current treatments for back pain are largely limited to symptomatic interventions. However, major progress is being made in multiple research directions to unravel the biology and pathology of the IVD, raising hope that effective disease-modifying interventions will soon be developed. In this review, we will discuss our current knowledge and gaps in knowledge on the developmental origin of the IVD, the phenotype of the distinct cell types found within the IVD tissues, molecular targets in IVD degeneration identified using bioinformatics strategies, and mechanotransduction pathways that influence IVD cell fate and function.
Neonatal mouse intervertebral discs heal with restored function following herniation injury.
Torre Olivia M,Das Rohit,Berenblum Ramy E,Huang Alice H,Iatridis James C
FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Adult intervertebral discs (IVDs) have poor endogenous healing capacity, because of their challenging microenvironment and complex mechanical demands, which can result in painful IVD herniation. There are no regenerative strategies available to improve IVD healing and restore its function. Neonatal mice are excellent models of mammalian regeneration, but there are no studies of the regenerative capacity of neonatal IVDs. In this study, we developed a neonatal model of improved IVD healing to inform repair strategies after herniation. In vivo puncture injuries were performed to simulate herniation with complete annulus fibrosus (AF) tears in caudal IVDs of neonatal (postnatal d 5) and adult (4-6 mo) Scleraxis green fluorescent protein ( ScxGFP) mice. Acute and long-term healing responses were assessed with histologic, radiologic, and biomechanical measurements. Neonates underwent accelerated IVD healing compared to adults with functional restoration and enhanced structural repair after herniation. A population of ScxGFP cells identified in the neonatal repair site may be associated with this improved healing and warrants future investigation. In summary, function of neonatal IVDs was restored after herniation injury, whereas that of adult discs was not. This improved healing response is likely driven by multiple mechanisms that may include differences in mechanical loading and available repair cells during growth.-Torre, O. M., Das, R., Berenblum, R. E., Huang, A. H., Iatridis, J. C. Neonatal mouse intervertebral discs heal with restored function following herniation injury.
Strategies for Annulus Fibrosus Regeneration: From Biological Therapies to Tissue Engineering.
Chu Genglei,Shi Chen,Wang Huan,Zhang Weidong,Yang Huilin,Li Bin
Frontiers in bioengineering and biotechnology
Intervertebral disc (IVD) is an avascular tissue which contributes to the weight bearing, motion, and flexibility of spine. However, IVD is susceptible to damage and even failure due to injury, pathology, and aging. Annulus fibrosus (AF), the structural and functional integrity of which is critically essential to confine nucleus pulpous (NP) and maintain physiological intradiscal pressure under mechanical loading, plays a critical role in the biomechanical properties of IVD. AF degeneration commonly results in substantial deterioration of IVD. During this process, the biomechanical properties of AF and the balance between anabolism and catabolism in IVD are progressively disrupted, leading to chronic back pain, and even disability of individuals. Therefore, repairing and regenerating AF are effective treatments to degeneration-associated pains. However, they remain highly challenging due to the complexity of natural AF tissue in the aspects of cell phenotype, biochemical composition, microstructure, and mechanical properties. Tissue engineering (TE), by combining biological science and materials engineering, shed lights on AF regeneration. In this article, we review recent advances in the pro-anabolic approaches in the form of cell delivery, bioactive factors delivery, gene therapy, and TE strategies for achieving AF regeneration.
Rapamycin prevents the intervertebral disc degeneration via inhibiting differentiation and senescence of annulus fibrosus cells.
Gao Changhong,Ning Bin,Sang Chenglin,Zhang Ying
The effects of bleomycin and rapamycin on cellular senescence and differentiation of rabbit annulus fibrosus stem cells (AFSCs) were investigated using a cell culture model. The results showed that bleomycin induced cellular senescence in AFSCs as evidenced by senescence-associated secretory phenotype. The morphology of AFSCs was changed from cobblestone-like cells to pancake-like cells. The senescence-associated β-galactosidase activity, the protein expression of P16 and P21, and inflammatory-related marker gene levels IL-1β, IL-6, and TNF-α were increased in bleomycin-treated AFSCs in a dose-dependent manner. Rapamycin treatment decreased the gene expression of MMP-3, MMP-13, IL-1β, IL-6, TNF-α, and protein levels of P16 and P21 in bleomycin-treated AFSCs. Furthermore, neither bleomycin nor rapamycin changed the ribosomal S6 protein level in AFSCs. However, the phosphorylation of the ribosomal S6 protein was increased in bleomycin-treated AFSCs and decreased in rapamycin-treated AFSCs. AFSCs differentiated into adipocytes, osteocytes, and chondrocytes when they were cultured with respective differentiation media. Rapamycin inhibited multi-differentiation potential of AFSCs in a concentration-dependent manner. Our findings demonstrated that mammalian target of rapamycin (mTOR) signaling affects cellular senescence, catabolic and inflammatory responses, and multi-differentiation potential, suggesting that potential treatment value of rapamycin for disc degenerative diseases, especially lower back pain.
Bioengineering a multicomponent spinal motion segment construct--a 3D model for complex tissue engineering.
Chik Tsz Kit,Chooi Wai Hon,Li Yuk Yin,Ho Fu Chak,Cheng Hiu Wa,Choy Tsz Hang,Sze Kam Yim,Luk Keith Kei Dip,Cheung Kenneth Man Chi,Chan Barbara Pui
Advanced healthcare materials
Intervertebral disc degeneration is an important clinical problem but existing treatments have significant drawbacks. The ability to bioengineer the entire spinal motion segment (SMS) offers hope for better motion preservation strategies but is extremely challenging. Here, fabrication of a multicomponent SMS construct with complex hierarchical organization from mesenchymal stem cells and collagen-based biomaterials, using a module-based integrative approach, is reported. The construct consists of two osteochondral subunits, a nucleus pulposus (NP-)-like core and a multi-lamellae annulus fibrosus (AF-)-like component. Chondrogenic medium is crucial for stabilizing the osteochondral subunits, which are shown to allow passive nutrient diffusion, while cyclic compression is necessary for better fiber matrix organization. Cells adhere, survive, and interact with the NP-like core. Cyclic torsional loading stimulates cell alignment in the AF-like lamellae and the number of lamellae affects the mechanical properties of the construct. This work represents an important milestone in SMS tissue engineering and provides a 3D model for studying tissue maturation and functional remodeling.
Prediction of Lumbar Disk Herniation and Clinical Outcome Using Quantitative Magnetic Resonance Imaging: A 5-Year Follow-Up Study.
Raudner Marcus,Schreiner Markus M,Juras Vladimir,Weber Michael,Stelzeneder David,Kronnerwetter Claudia,Windhager Reinhard,Trattnig Siegfried
OBJECTIVES:The aim of this study was to assess the predictive value of T2 mapping at baseline with regard to the development of disk herniation and clinical outcome at a 5-year follow-up in patients with low back pain. MATERIALS AND METHODS:Twenty-five symptomatic patients (13 male; mean age, 44.0 years; range, 24-64 years at baseline) were examined at 3 T magnetic resonance imaging, with a 5-year follow-up. Region of interest analysis was performed on 125 lumbar intervertebral disks on 2 central sagittal T2 maps. Absolute T2 relaxation times and a T2 value ratio of the posterior annulus fibrosus as a percentage of the nucleus pulposus (NPAF) were evaluated for each disk. All disks were graded morphologically using the Pfirrmann score. Roland-Morris Disability Questionnaires (RMDQ) and a visual analogue scale (VAS) were assessed for each patient at follow-up as a clinical end point and compared with diagnosed lumbar disk herniation. Statistical analysis was conducted by a biomedical statistician. RESULTS:Using the baseline NPAF ratio, follow-up development of herniation was predicted with an area under the curve (AUC) of 0.893 in a receiver operating characteristic curve. The same was done using the baseline nucleus pulposus T2, resulting in an AUC of 0.901. Baseline and follow-up NPAF, as well as baseline and follow-up nucleus pulposus T2, differed significantly (P < 0.001) between disks with no herniation, disks with herniation at baseline, and disks with new herniation at follow-up. Difference was still significant (all P < 0.001), when only testing for difference in degenerated discs with Pfirrmann score III to V. Calculating sensitivity and specificity for herniation prediction only in discs with Pfirmann III to V using a receiver operating characteristic, AUC was 0.844 with baseline herniations excluded.The lowest baseline nucleus pulposus T2 per patient correlated significantly with follow-up RMDQ (r = -0.517; P = 0.008) and VAS (r = -0.494; P = 0.012). The highest baseline NPAF correlated significantly with RMDQ (r = 0.462; P = 0.020), but not VAS (r = 0.279; P = 0.177). CONCLUSIONS:Quantitative T2 mapping may serve as a clinically feasible, noninvasive imaging biomarker that can indicate disks at risk for herniation and correlates with clinical outcome and subjective patient burden in a representative cohort of patients with low back pain.
Prolactin inhibits the progression of intervertebral disc degeneration through inactivation of the NF-κB pathway in rats.
Wu Xiexing,Liu Yu,Guo Xiaobin,Zhou Wei,Wang Liangliang,Shi Jiawei,Tao Yunxia,Zhu Mo,Geng Dechun,Yang Huilin,Mao Haiqing
Cell death & disease
Intervertebral disc degeneration (IVDD) is one of the key predisposing factors for low back pain. Although the exact mechanism remains unclear, inflammatory response and nucleus pulposus (NP) apoptosis are known to play important roles in this process. Prolactin protects against inflammation-associated chondrocyte apoptosis in arthritis. Based on prior studies, we hypothesized that prolactin might have therapeutic effects on IVDD by inhibiting the apoptosis of degenerative human disc NP cells. An experimental model of IVDD was established in 3-month-old Sprague-Dawley rats by submitting them to percutaneous disc puncture with a 20-gauge needle on levels 7-8 and 8-9 of the coccygeal vertebrae. Then the rats were injected with 20 or 200 ng prolactin on a weekly basis. Radiologic and histologic analyses were performed on days 4, 7, 14, and 28. The expression of prolactin and its receptor was analyzed in human tissue obtained from symptomatic patients undergoing microencoscopy discectomy, or from scoliosis patients undergoing deformity correction surgery. The results showed that intradiscal injection of prolactin maintained disc height and the mean signal intensity of the punctured disc. Histological analysis indicated that prolactin treatment significantly retained the complete structure of the NP and annulus fibrosus compared with the vehicle group. In addition, more collagen II, but fewer collagen I-containing tissues were detected in the prolactin treatment groups compared to the vehicle group. Moreover, low levels of tumor necrosis factor-α, interleukin-1β, cleaved-caspase 3, and TUNEL staining were observed in the prolactin treatment groups. We also demonstrated that prolactin impaired puncture-induced inflammation and cell apoptosis by downregulating activation of the NF-κB pathway. The degenerated NP tissues from patients had decreased expression of prolactin and its receptor, whereas expression was increased in the NP tissues removed from scoliosis patients. These results suggest that prolactin may be a novel therapeutic target for the treatment of IVDD.
Transgenic mice overexpressing human TNF-α experience early onset spontaneous intervertebral disc herniation in the absence of overt degeneration.
Gorth Deborah J,Shapiro Irving M,Risbud Makarand V
Cell death & disease
There is a well-established link between cytokine expression and the progression of intervertebral disc degeneration. Among these cytokines, interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) are the most commonly studied. To investigate whether systemic hTNF-α overexpression affects intervertebral disc health, we studied the spine phenotype of Tg197 mice, a widely used hTNF-α transgenic line. These mice were studied at 12-16 weeks of age using comprehensive histochemical and immunohistological analysis of the spinal motion segment. Micro-CT analysis was performed to quantify vertebral trabecular bone architecture. The Tg197 mice evidenced spontaneous annular tears and herniation with increased vascularity in subchondral bone and significant immune cell infiltration. The full-thickness annular tear without nucleus pulposus (NP) extrusion resulted in neutrophil, macrophage, and mast cell infiltration into the disc, whereas the disc with full-thickness tear and pronounced NP herniation showed additional presence of CD4+ and CD8+ T cells. While the observed defects involved failure of the annular, endplate, and vertebral junction, there were no obvious alterations in the collagen or aggrecan content in the NP and annulus fibrosus or the maturity of collagen fibers in Tg197 mice. Despite elevated systemic inflammation and pronounced loss of trabecular bone in the vertebrae, intact Tg197 discs were healthy and showed an increase in NP cell number. The NP cells in intact discs preserved expression of phenotypic markers: CAIII, Glut1, and Krt19. In conclusion, elevated systemic TNF-α increases the susceptibility of mice to spontaneous disc herniation and possibly radiculopathy, without adversely affecting intact intervertebral disc health.
Ageing affects chondroitin sulfates and their synthetic enzymes in the intervertebral disc.
Collin Estelle C,Carroll Oliver,Kilcoyne Michelle,Peroglio Marianna,See Eugene,Hendig Doris,Alini Mauro,Grad Sibylle,Pandit Abhay
Signal transduction and targeted therapy
The depletion of chondroitin sulfates (CSs) within the intervertebral disc (IVD) during degenerative disc disease (DDD) results in a decrease in tissue hydration, a loss of fluid movement, cell apoptosis, a loss of nerve growth inhibition and ultimately, the loss of disc function. To date, little is known with regards to the structure and content of chondroitin sulfates (CSs) during IVD ageing. The behavior of glycosaminoglycans (GAGs), specifically CSs, as well as xylosyltransferase I (XT-I) and glucuronyltransferase I (GT-I), two key enzymes involved in CS synthesis as a primer of glycosaminoglycan (GAG) chain elongation and GAG synthesis in the nucleus pulposus (NP), respectively, were evaluated in a bovine ageing IVD model. Here, we showed significant changes in the composition of GAGs during the disc ageing process (6-month-old, 2-year-old and 8-year-old IVDs representing the immature to mature skeleton). The CS quantity and composition of annulus fibrosus (AF) and NP were determined. The expression of both XT-I and GT-I was detected using immunohistochemistry. A significant decrease in GAGs was observed during the ageing process. CSs are affected at both the structural and quantitative levels with important changes in sulfation observed upon maturity, which correlated with a decrease in the expression of both XT-I and GT-I. A progressive switch of the sulfation profile was noted in both NP and AF tissues from 6 months to 8 years. These changes give an appreciation of the potential impact of CSs on the disc biology and the development of therapeutic approaches for disc regeneration and repair.
Fibrin in intervertebral disc tissue engineering.
Colombini Alessandra,Ceriani Cristina,Banfi Giuseppe,Brayda-Bruno Marco,Moretti Matteo
Tissue engineering. Part B, Reviews
Fibrin is clinically employed as a versatile, safe, and clinically applicable sealant and cell carrier. It has been able to support disc cell survival, favor extracellular matrix production, and enhance the efficiency of cell transfer in the intervertebral disc (IVD). The aim of this review was to evaluate how fibrin has been used in vitro, in vivo, and in clinical trials for IVD tissue engineering. Within the in vitro studies, disc cells were cultured in fibrin alone or combined with other materials and a difference in the behavior of nucleus pulposus (NP) and annulus fibrosus (AF) cells was sometimes reported, but in general, the formation of fibrocartilaginous matrix was observed. Moreover, data concerning the fibrin long-term stability and its anti-inflammatory properties were found. Disc cells of human origin were never employed in combination with fibrin in vivo or in clinical trials. In vivo, disc degeneration models used to test the fibrin properties essentially involved NP injuries. The addition of cells, in particular if terminally differentiated, to the injected fibrin seemed to promote a more physiological matrix in comparison with fibrin alone. Important aspects should be further investigated in future studies such as the use of fibrin to treat AF lesions as well as the mechanical properties of the fibrin-based biomaterials and of the neoformed tissue. Finally, in vivo studies and clinical trials with in situ injection of fibrin and human disc cells should be performed.