Rahul Gawri

Development of an organ culture system for long-term survival of the intact human intervertebral disc.

Study Design. Human intervertebral discs were used to develop an intact whole disc organ culture system with long-term cell viability. Objective. To develop and validate a long–term organ culture system for intact human intervertebral discs, in which the potential for biologic repair of disc degeneration can be studied. Summary of Background Data. Intervertebral disc degeneration is a common cause of back pain, which can be costly to the health care system and have a negative impact on the quality of life of the patient. Once injured the adult human intervertebral disc seems incapable of intrinsic repair, but the early stages of disc degeneration can potentially be retarded or even reversed by the administration of growth factors to promote new extracellular matrix synthesis. Methods. Intervertebral discs were prepared by three isolation techniques and placed in free swelling organ culture. Cell viability, disc swelling, glycosaminoglycan content, and extracellular matrix degradation were assessed under a variety of culture conditions. Results. Human intervertebral discs isolated with intact cartilage end plates retained cell viability and did not undergo matrix degradation when cultured for 4 weeks with both a high and low nutrient level. This contrasted with the excessive cell death that was observed if the cartilage end plates were removed before culture or if vertebral bone was retained. Conclusion. Retention of the cartilage end plates limits tissue swelling and permits efficient nutrient supply, thus allowing viable long-term organ culture. The availability of such a system will permit the repair potential of therapeutic candidates to be studied in human discs with naturally occurring degeneration. Furthermore, the system is simple and economical, as no apparatus is needed to limit the detrimental effects of excessive tissue swelling.

Chondroadherin fragmentation mediated by the protease HTRA1 distinguishes human intervertebral disc degeneration from normal aging

Background: Little is known about the molecular mechanisms involved in intervertebral disc degeneration (IVD). Results: CHAD fragmentation is found only in degenerate IVDs. HTRA1 is capable of generating the in vivo fragment. Conclusion: CHAD fragmentation can be a marker of degeneration, distinguishing between aging and degeneration. Significance: Inhibiting HTRA1 activity could be of value to slow down disc degeneration without influencing normal turnover. Chondroadherin, a member of the leucine-rich repeat family, has previously been demonstrated to be fragmented in some juveniles with idiopathic scoliosis. This observation led us to investigate adults with disc degeneration. Immunoblotting analysis demonstrated that non-degenerate discs from three different age groups show no chondroadherin fragmentation. Furthermore, the chondroadherin fragments in adult degenerate disc and the juvenile scoliotic disc were compared via immunoblot analysis and appeared to have a similar size. We then investigated whether or not chondroadherin fragmentation increases with the severity of disc degeneration. Three different samples with different severities were chosen from the same disc, and chondroadherin fragmentation was found to be more abundant with increasing severity of degeneration. This observation led us to the creation of a neoepitope antibody to the cleavage site observed. We then observed that the cleavage site in adult degenerate discs and juvenile scoliotic discs was identical as confirmed by the neoepitope antibody. Consequently, investigation of the protease capable of cleaving chondroadherin at this site was necessary. In vitro digests of disc tissue demonstrated that ADAMTS-4 and -5; cathepsins K, B, and L; and MMP-3, -7, -12, and -13 were incapable of cleavage of chondroadherin at this site and that HTRA1 was indeed the only protease capable. Furthermore, increased protein levels of the processed form of HTRA1 were demonstrated in degenerate disc tissues via immunoblotting. The results suggest that chondroadherin fragmentation can be used as a biomarker to distinguish the processes of disc degeneration from normal aging.

Physiological loading can restore the proteoglycan content in a model of early IVD degeneration.

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

Gene Expression Profiling Identifies Interferon Signalling Molecules and IGFBP3 in Human Degenerative Annulus Fibrosus

Low back pain is a major cause of disability especially for people between 20 and 50 years of age. As a costly healthcare problem, it imposes a serious socio-economic burden. Current surgical therapies fail to replace the normal disc in facilitating spinal movements and absorbing load. The focus of regenerative medicine is on identifying biomarkers and signalling pathways to improve our understanding about cascades of disc degeneration and allow for the design of specific therapies. We hypothesized that comparing microarray profiles from degenerative and non-degenerative discs will lead to the identification of dysregulated signalling and pathophysiological targets. Microarray data sets were generated from human annulus fibrosus cells and analysed using IPA ingenuity pathway analysis. Gene expression values were validated by qRT-PCR, and respective proteins were identified by immunohistochemistry. Microarray analysis revealed 238 differentially expressed genes in the degenerative annulus fibrosus. Seventeen of the dysregulated molecular markers showed log2-fold changes greater than ±1.5. Various dysregulated cellular functions, including cell proliferation and inflammatory response, were identified. The most significant canonical pathway induced in degenerative annulus fibrosus was found to be the interferon pathway. This study indicates interferon-alpha signalling pathway activation with IFIT3 and IGFBP3 up-regulation, which may affect cellular function in human degenerative disc.

Link N is cleaved by human annulus fibrosus cells generating a fragment with retained biological activity

Presently, there are no established treatments to prevent, stop or even retard back pain arising from disc degeneration. Previous studies have shown that Link N can act as a growth factor and stimulate the synthesis of proteoglycans and collagens, in IVD. However, the sequences in Link N involved in modulating cellular activity are not well understood. To determine if disc cells can proteolytically process Link N, human disc cells were exposed to native Link N over a 48 h period and mass spectrometric analysis revealed that a peptide spanning residues 1–8 was generated in the presence of AF cells but not NP cells. Link N 1–8 significantly induced proteoglycan production in the presence of IL‐1β NP and AF cells, confirming that the biological effect is maintained in the first 8 amino acids of the peptide and indicating that the effect is sustained in an inflammatory environment. Thus Link‐N 1–8 could be a promising candidate for biologically induced disc repair, and the identification of such a stable specific peptide may facilitate the design of compounds to promote disc repair and provide alternatives to surgical intervention for early stage disc degeneration.

The Effects of Chlorhexidine Graft Decontamination on Tendon Graft Collagen and Cell Viability

Background: Chlorhexidine (CLX) has been reported as a popular and effective disinfectant of contaminated tendon grafts with no biomechanical sequelae; however, its biochemical effects on tendon collagen and fibroblasts remain unknown. Purpose: To determine whether CLX disinfection of contaminated tendon grafts has deleterious effects on tendon collagen or a toxic effect on fibroblast function. Study Design: Controlled laboratory study. Methods: Collagen fibrils prepared from purified bovine collagen type I were treated with various CLX concentrations (0.5%-4%) and incubation times (10-40 minutes), and the effects on fibril degradation and solubility were then examined using gel electrophoresis. Fresh bovine tendons were treated with sterile water or 2% CLX; then, fibroblast mobility and metabolic activity were evaluated using light microscopy and Alamar Blue assay, respectively. Results: No effect on collagen fibrils was observed when they were exposed to 0%, 0.5%, or 2% CLX at any exposure time. However, 4% CLX dissolved the fibrils even after short incubation times. Fibroblasts migrated out from the control tendon explants but not from explants treated with 2% CLX, and a 5-fold reduction in metabolic activity was observed throughout the tendon in explants exposed to 2% CLX, suggesting that CLX penetrated and killed cells throughout the tissue. Conclusion: Four-percent CLX caused collagen fibrils to dissolve in vitro, and tendon graft disinfection with 2% CLX was cytotoxic to the cells. Clinical Relevance: Because of its chemical effect on tendon collagen and cytotoxic effect on tendon fibroblasts, 4% CLX should not be used as a disinfectant. Two-percent CLX can be used to disinfect contaminated ACL grafts, but such treatment will drastically reduce the metabolic activity of the cells within the graft, making it similar to an acellular allograft tendon.

Chondroadherin Fragmentation as a Biochemical Marker for Early Stage Disk Degeneration

Introduction Intervertebral disk (IVD) degeneration has been strongly associated with and named a major cause of back pain. At present, little is known about the molecular mechanisms involved in the degeneration of IVD and how these may differ from normal turnover of the tissue. As a result of this, a biomarker for disk degeneration has not yet been identified and we propose chondroadherin (CHAD) fragmentation as a potential option. CHAD, a protein of the leucine rich repeat (LRR) family, is one of the proteins predominantly expressed in the extracellular matrix of cartilaginous tissue, including that of the IVD. This restricted distribution is unusual among LRR proteins, which commonly show a wide tissue distribution. CHAD is primarily found close to the cells, where it can interact with collagen fibrils of the ECM and molecules at the cell surface, providing a mechanism for regulating cell metabolism and ECM structure. These interactions may also aid in promoting matrix homeostasis, and variation in CHAD abundance or structure might therefore lead to pathological changes in the tissue over time. The aims of this study were to determine whether CHAD fragmentation occurs and is unique to disk degeneration, and to characterize the cleavage site within CHAD at which fragmentation occurs. Materials and Methods Healthy and degenerate lumbar IVDs were obtained through organ donations via Transplant Quebec. IVDs from patients with degenerative disk disease and from patients with scoliosis were obtained at the time of surgery. Punches of 4 mm were taken and disk tissue then extracted using 15 volumes of extraction buffer (4 M GuCl, 10 mM EDTA, COMPLETE, 50 mM NaAc, pH 5.8) on a wet weight per volume basis. Extracted proteins were ethanol precipitated, and CHAD fragmentation was studied using SDS-PAGE and western blotting in combination with specific antibodies. To characterize the CHAD cleavage site, a degenerate disk extract was subjected to CsCl density gradient centrifugation to remove proteoglycans. CHAD was purified from the protein fraction by chromatography through carboxymethyl 52. The CHAD-containing samples were then fractionated on an SDS-PAGE gel and stained with Coomassie Blue. Gel portions containing the CHAD fragment were excised, then lyophilized, reduced and alkylated, and subjected to trypsin digestion. Peptides were then identified by liquid chromatography mass spectrometry. Antineoepitope antibodies specifically recognizing the fragmented CHAD were generated by immunizing rabbits with synthetic peptides conjugated to KLH. The peptides represented the terminal amino acid sequences at the site of CHAD fragmentation. Results Proteolytic fragmentation of CHAD was observed in IVDs from patients with DDD and in some individuals with adolescent idiopathic scoliosis (AIS). Its presence appeared to be related to the degree of degeneration in both cases (Fig. 1). This phenomenon was found to be specific to disk degeneration, as CHAD fragmentation was not observed in healthy adolescent and adult disks from organ donors. Within the degenerate disk, fragmentation was evident in tissue with signs of degeneration but not in tissue that had no signs of degeneration. This same trend was also seen when comparing normal and degenerate disk tissue from different levels of the spine from the same donor. Upon analysis with the antineoepitope antibody, it was apparent that CHAD fragmentation occurred at the same site in degenerate disks from Transplant Quebec donors, surgical samples from adults with DDD, and surgical samples from adolescents with AIS. Normal tissue samples showed no antineoepitope antibody binding, confirming that CHAD fragmentation at this site was not present in the healthy disk. Conclusion CHAD fragmentation is associated with disk degeneration present both in the adult with DDD and the adolescent with scoliosis, and fragmentation is created by cleavage at the same site within both disorders. Fragmentation is not, however, present in normal disk tissue. Thus, CHAD fragmentation may distinguish catabolic processes leading to disk degeneration from those associated with normal turnover of the tissue, and as such serve as a marker of disk degeneration. It is necessary to recognize the biochemical processes that specifically contribute to disk degeneration, if degeneration is to be prevented or retarded, and if novel treatments initiating disk regeneration are to be developed. The mechanism leading to fragmentation and the proteinase involved are currently under investigation. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared

Link-N Peptide: A Stepping Stone towards Biological Repair of Intervertebral Disk Degeneration

Introduction Back pain is a fairly common problem which affects a large portion of the population across all ages and has an impact on quality of life. Intervertebral disk (IVD) degeneration is the single most common implicated cause of back pain. Presently, there is no medical treatment or therapeutic agent to address this problem and surgery is the only offered option. Link-N peptide represents the 16 amino acid sequence from the N-terminus of the link protein that stabilizes the proteoglycan aggregates present in cartilage and disk. Link-N peptide is released from the link protein as a result of proteolysis, and has been suggested to play a role in matrix homeostasis by promoting new matrix synthesis. We evaluated its regenerative potential in intact human IVD. Materials and Methods Lumbar IVDs were obtained through organ donations via Transplant Quebec. Disks from seven individuals, five disks per spine, were harvested. Cells were isolated from nucleus pulposus (NP) and inner annulus fibrosus regions of the disks. Single cells were beaded in 1.2% alginate and cultured in DMEM containing 1 g/L glucose and 10% FBS. Alginate beads were exposed to 10–10000 ng/mL Link-N peptide for 48 hours. Intact disks were prepared for organ culture by parallel cuts through the adjacent vertebral bodies close to the end plates, and the remaining bone and the calcified part of the cartilage endplates were removed using a high-speed bone burr. Disks were maintained and cultured with no external load applied in DMEM containing 1 g/L glucose and supplemented with 1% FBS. Link-N was conjugated with 5-TAMRA dye then injected into the center of the disk. The distribution of Link-N in the medium and within the disk was studied to determine whether Link-N is retained in the disk. Disks from adjacent levels were matched for the degree of degeneration and were injected in their NP region with 50µCi 35SO4 along with 0.1 mg or 1 mg of Link-N in 100 µL of medium per disk and harvested after 48 hours. Sustained effect of Link-N was evaluated by injecting the disk with Link-N and injecting 35SO4 1 week later. Proteoglycan synthesis was evaluated by measuring 35SO4 incorporation. Results When human lumbar disk cells from NP and iAF regions beaded in alginate were exposed to Link-N peptide for 48 hours, proteoglycan synthesis was observed to increase in a dose-dependent manner with the maximal response at 1000 ng/mL Link-N. Fluorescently labelled Link-N peptide was injected into the disks to determine if Link-N is retained in the disks matrix or freely diffuses throughout the tissue and equilibrates with surrounding medium. Samples were taken continually from the surrounding medium and from the disk tissue at the termination of the experiment. Fluorescent-Link-N was detectable in the medium at 24 hours and reached equilibrium after 48 hours. The fluorescent peptide was found in the NP and NP/iAF junction but not in the remaining AF. Thus loss of Link-N appears to occur by diffusion through the endplates (Fig. 1). Cell viability was maintained in the NP, at >96%, after injection of 1 mg of Link-N/disk. Disks injected with Link-N showed increased proteoglycan synthesis in the NP and iAF compared to adjacent level control disks matched for grade of degeneration. To evaluate the duration of the effect, disks were injected with 35SO4 1 week after the injection of Link-N. Proteoglycan synthesis remained elevated in Link-N injected disks compared to adjacent level control disks suggesting a sustained effect. Conclusion Link-N peptide has previously been shown to promote matrix protein synthesis by bovine disk cells in monolayer and pellet cultures. In this work, we show that Link-N can promote proteoglycan synthesis not only in human disk cells cultured in 3D constructs, but also in intact adult human disks where the cells are in their native environment. Recently, an increase in disk height measured by MRI was shown in an in vivo rabbit model, where degenerated disks were injected with Link-N. If a similar restoration of disk function could be achieved in the human disks, then Link-N could be a promising candidate for biologically induced disk repair, and could provide an alternative to surgical intervention for early stage disk degeneration. Link-N has a significant cost advantage over growth factors, such as BMP7, TGFβ and, GDF5. Based on prior in vivo studies in the rabbit, Link-N is over 100 times less expensive than recombinant growth factors that have a similar repair response. Thus, Link-N peptide injection could be both effective and cost-efficient therapy for retarding the ongoing degenerative process in early stage disk disease and help relieve back pain. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared

Evaluation of Tissue Repair Using MRI Imaging of Human Intervertebral Discs Cultured in a Bioreactor

Introduction Low back pain is a major problem world-wide, affecting the quality of life for millions of people. Low back pain also has a tremendous impact on direct and indirect global healthcare costs. Intervertebral disc (IVD) degeneration has been strongly associated with low back pain. Long-term organ culture of human IVDs is essential to study IVD degeneration and repair. Using an ex vivo approach, the relationship between mechanobiology, disc matrix composition and metabolism can be better understood in the context of degenerative disease. We have developed a bioreactor where intact human discs can be cultured in a controlled dynamically loaded environment. Here, we aimed to determine the most suitable loading parameters for human discs culture by assessing IVD tissue integrity and cell viability under low, medium and high magnitude cyclic load. Furthermore, we investigated the suitability of this model toward cell supplementation strategies for tissue repair and developed a novel, single disc MRI imaging sequence aimed at direct visualization of tissue repair. Materials and Methods Human IVDs were isolated from lumbar spine segments as previously described. Spines were obtained with consent through the Transplant Quebec Organ Donation Program from individuals who had undergone sustained brain death. Discs were cultured under 3 different loading schemes to mimic a sedentary lifestyle: low 0.1–0.3, medium 0.1–0.6 and high 0.1–1.2 MPa loads. Cell viability and matrix stability was assessed following 10 days of loading. Feasibility of cell/hydrogel implantation was determined over 14 days of medium dynamic loading. To determine whether isolated discs could be imaged by MRI, extracted individual discs were visualized for T1 and T2 signals using a novel sequence using a small animal Bruker 7.5 Tesla MRI. Results Cell viability was maintained at greater than 80% throughout the discs at low and medium loads. Viability dropped to ~60–70% throughout the discs under high loads. Proteoglycan content remained stable in all loading protocols (~50 μg sGAG/mg tissue), as did CHAD and newly synthesized collagen II protein. To test for feasibility of cell therapies in the bioreactors, NP cells combined with a hydrogel were injected into discs and cultured under medium load. 14 days after dynamic culture, the injected cells were mainly localized to the NP region with greater than 90% viability. The small animal MRI was able to obtain well-defined images of isolated discs, with details of tissue integrity and proteoglycan content. Conclusion Our ex vivo model of dynamic human IVD culture can be used as a platform on which to study mechanisms of degeneration as well as for novel avenues aimed at biological repair using bioactive substances or cell based therapies. Cells and bioactive substances can be administered within hydrogels thereby enhancing the reparative properties. Furthermore, it is feasible to assess repair potential of the therapies by comparing MRI scans pre- and post-therapy.

The Ability of Physiological Load to Restore Disc Function

Introduction The nucleus pulposus (NP) of a healthy intervertebral disc (IVD) is rich in collagen type II fibers that are arranged in a random fashion to entrap the highly anionic proteoglycan aggrecan which confers the swelling properties important for resistance to compression. A hallmark of IVD degeneration is the decrease in proteoglycan content. Matrix degradation and proteoglycan loss from the NP result in a decrease in weight bearing capacity and loss of disc height. In the final stages of IVD degradation fissures appear in the annular ring allowing extrusion of the NP. It is crucial to understand the interplay between mechanobiology, disc composition, and metabolism to understand the underlying cause of disc degeneration and to be able to study ways to regenerate the degenerate disc. To address such questions, a bioreactor has been developed that facilitates organ culture of intact discs in a controlled dynamically loaded environment.1 The bioreactor can be used in combination with an isolation and degeneration method which maintains the integrity of the intervertebral discs by preserving the noncalcified part of the cartilage endplate.2,3 To allow repair strategies to be studied, a degeneration method utilizing trypsin injection to deplete proteoglycans (PGs) from the matrix has been established.2 Materials and Methods Intact bovine and human discs were prepared as previously described.2,3 Degeneration was induced by trypsin in the bovine discs.2 Human and bovine discs were loaded in the bioreactor system over 14 days. Cell viability or metabolic activity was assessed using a Live/Dead and/or Alamar Blue metabolic assay. The proteoglycan content was assessed by DMMB assay and Safranin O staining of histological sections. CHAD and collagen type II were examined by western blotting. Stress profilometry was performed at 0.6 MPa static load.4 Results After PG depletion, the discs were loaded in the bioreactor under physiological cyclic dynamic load. Cell viability and metabolic activity at the end of the culture period was assessed. Bovine discs treated with trypsin maintained high cell viability when cultured for 14 days both loaded and unloaded. PG content was measured using the DMMB assay and by Safranin O staining of tissue sections. Unloaded discs lost approximately 60% of their proteoglycan content, whereas discs loaded under physiological dynamic load completely replenished the proteoglycan content. Collagen type II and CHAD protein levels were also increased under physiological dynamic loading. In a separate set of discs, stress profilometry was performed at 0.6 MPa static load and stress profiles were generated. No significant change in the load profiles were found for a low trypsin dose, however, the discs treated with the experimental dose of trypsin showed an 11% reduction of the internal pressure. The system has also been used to culture and load intact human discs under three different load magnitudes (high 0-3-1.2 MPa, medium 0.1-0.6, and low 0.1-0.3 MPa). The load curves and cell viability were followed over a 2-week period. Discs cultured under low load maintained a viability of > 90%, discs loaded under medium load showed a viability of > 86%, whereas the cell viability decreased to below 55% in discs loaded under high loads. Conclusion This study shows that physiological load has the ability to stimulate PG synthesis and to fully restore PG content after 14 days of axial dynamic loading at a physiological level. It also allows the response to load to be evaluated. The bioreactor can also be used to evaluate changes in mechanical properties of the disc following biologically induced changes, or to induce biomechanical stimulus of the disc to generate a biological change. As such, it is equally useful for studying the role of load in inducing disc degeneration or the role of biological stimuli in restoring disc function. It provides an experimental platform useful to evaluate if biologic repair is feasible over a range of loading conditions, or is impaired outside this range. Such knowledge is important for patient advice on lifestyle following a biological repair procedure. Disclosure of Interest None declared References Haglund L, Moir J, Beckman L, et al. Development of a bioreactor for axially loaded intervertebral disc organ culture. Tissue Eng Part C Methods 2011;17(10):1011–1019 Jim B, Steffen T, Moir J, Roughley P, Haglund L. Development of an intact intervertebral disc organ culture system in which degeneration can be induced as a prelude to studying repair potential. Eur Spine J 2011;20(8):1244–1254 Gawri R, Mwale F, Ouellet J, et al. Development of an organ culture system for long-term survival of the intact human intervertebral disc. Spine 2011;36(22):1835–1842 McNally DS, Adams MA. Internal intervertebral disc mechanics as revealed by stress profilometry. Spine 1992;17(1):66–73