Abstract: The intervertebral disc (IVD) is the joint of the spine connecting vertebra to vertebra. It functions to transmit loading of the spine and give flexibility to the spine. It composes of three compartments: the innermost nucleus pulposus (NP) encompassing by the annulus fibrosus (AF), and two cartilaginous endplates connecting the NP and AF to the vertebral body on both sides. Discogenic pain possibly caused by degenerative intervertebral disc disease (DDD) and disc herniations has been identified as a major problem in our modern society. To study possible mechanisms of IVD degeneration, in vitro organ culture systems with live disc cells are highly appealing. The in vitro culture of intact bovine coccygeal IVDs has advanced to a relevant model system, which allows the study of mechano-biological aspects in a well-controlled physiological and mechanical environment. Bovine tail IVDs can be obtained relatively easy in higher numbers and are very similar to the human lumbar IVDs with respect to cell density, cell population and dimensions. However, previous bovine caudal IVD harvesting techniques retaining cartilaginous endplates and bony endplates failed after 1-2 days of culture since the nutrition pathways were obviously blocked by clotted blood. IVDs are the biggest avascular organs, thus, the nutrients to the cells in the NP are solely dependent on diffusion via the capillary buds from the adjacent vertebral body. Presence of bone debris and clotted blood on the endplate surfaces can hinder nutrient diffusion into the center of the disc and compromise cell viability. Our group established a relatively quick protocol to "crack"-out the IVDs from the tail with a low risk for contamination. We are able to permeabilize the freshly-cut bony endplate surfaces by using a surgical jet lavage system, which removes the blood clots and cutting debris and very efficiently reopens the nutrition diffusion pathway to the center of the IVD. The presence of growth plates on both sides of the vertebral bone has to be avoided and to be removed prior to culture. In this video, we outline the crucial steps during preparation and demonstrate the key to a successful organ culture maintaining high cell viability for 14 days under free swelling culture. The culture time could be extended when appropriate mechanical environment can be maintained by using mechanical loading bioreactor. The technique demonstrated here can be extended to other animal species such as porcine, ovine and leporine caudal and lumbar IVD isolation.
Abstract: INTRODUCTION: Notochordal cells and nucleus pulposus cells are co-existing in the intervertebral disc at various ratios among different mammalians. This fact rises the question about the interactions and the evolutionary relevance of this phenomenon. It has been described that these relatively large notochordal cells are mainly dominant in early lifetime of all vertebrates and then differences occur with ageing. Human, cattle, sheep, and goat lose the cells with age, whereas rodents and lagomorphs maintain these throughout their lifetime. MATERIALS AND METHODS: Here, we addressed the importance of cell ratio using alginate bead 3-D co-culture of bovine nucleus pulposus cells (bNPC) and porcine notochordal cells (pNCs) for 14Â days using culture inserts. RESULT: We found a significant stimulation of bNPC in the presence of pNC in terms of cell activity and glycosaminoglycan production, but not for proliferation (DNA content). Relative gene expression was significantly stimulated for collagen type 2 and aggrecan. CONCLUSION: The stimulating effect of NC was confirmed and the ideal ratio of NPC: NC was found to be ~50:50. This has direct implications for tissue-engineering approaches, which aim to repopulate discs with NP-like precursor cells.
Abstract: Loading is important to maintain the balance of matrix turnover in the intervertebral disc (IVD). Daily cyclic diurnal assists in the transport of large soluble factors across the IVD and its surrounding circulation and applies direct and indirect stimulus to disc cells. Acute mechanical injury and accumulated overloading, however, could induce disc degeneration. Recently, there is more information available on how cyclic loading, especially axial compression and hydrostatic pressure, affects IVD cell biology. This review summarises recent studies on the response of the IVD and stem cells to applied cyclic compression and hydrostatic pressure. These studies investigate the possible role of loading in the initiation and progression of disc degeneration as well as quantifying a physiological loading condition for the study of disc degeneration biological therapy. Subsequently, a possible physiological/beneficial loading range is proposed. This physiological/beneficial loading could provide insight into how to design loading regimes in specific system for the testing of various biological therapies such as cell therapy, chemical therapy or tissue engineering constructs to achieve a better final outcome. In addition, the parameter space of 'physiological' loading may also be an important factor for the differentiation of stem cells towards most ideally 'discogenic' cells for tissue engineering purpose.
Abstract: Implantation of intervertebral disc (IVD) allograft or tissue engineered disc constructs in the spine has emerged as an alternative to artificial disc replacement for the treatment of severe degenerative disc disease (DDD). Establishment of a bank of cryopreserved IVD allografts enables size matching and facilitates logistics for effective clinical management. However, the biomechanical properties of cryopreserved IVDs have not been previously reported. This study aimed to assess if cryopreservation with different concentrations of cryopreservant agents (CPA) would affect the dynamic viscoelastic properties of the IVD. Whole porcine lumbar IVDs (n = 40) were harvested and processed using various concentrations of CPA, 0 % CPA, 10 % CPA and 20 % CPA. The discs were cryopreserved using a stepwise freezing protocol and stored in liquid nitrogen. After four weeks of storage, the cryopreserved IVDs were quickly thawed at 37 °C for dynamic viscoelastic testing. The apparent modulus, elastic modulus (G'), viscous modulus (G") and loss modulus (G"/G') were calculated and compared to a fresh control group. Cryopreserved IVD without cryopreservants was significantly stiffer than the control. In the dynamic viscoelastic testing, cryopreservation with the use of CPA was able to preserve both G' and G" of an IVD. No significant differences were found between fresh IVD and IVD cryopreserved with 10 % CPA or 20 % CPA. This study demonstrated that CPAs at an optimal concentration could preserve the mechanical properties of the IVD allograft and can provide further credence for the application of long-term storage of IVD allografts for disc transplantation or tissue engineered construct applications.
Abstract: Severe intervertebral disc (IVD) degeneration often requires disc excision and spinal fusion, which leads to loss of spinal segment mobility. Implantation of an allograft disc or tissue engineered disc construct emerges as an alternative to artificial disc replacement for preserving the motion of the degenerated level. Establishment of a bank of cadaveric or engineered cryopreserved discs enables size matching, and facilitates clinical management. However, there is a lack of understanding of the behaviour of disc cells during cryopreservation, as well as how to maximize their survival, such that disc graft properties can be preserved. Here, we report on the effect of alterations in cooling rates, cryoprotective agents (CPAs), and duration of pre-cryopreservation incubation in CPA on cellular activity in whole porcine lumbar discs. Our results indicated that cooling rates of -0.3 degrees C/min and -0.5 degrees C/min resulted in the least loss of metabolic activity in nucleus pulposus (NP) and annulus fibrosus (AF) respectively, while metabolic activity is best maintained by using a combination of 10% dimethylsulphoxide (DMSO) and 10% propylene-glycol (PG) as CPA. By the use of such parameters, metabolic activity of the NP and the AF cells could be maintained at 70% and 45%, respectively, of that of the fresh tissue. Mechanical testing and histological evaluation showed no significant differences in mechanical properties or alterations in disc structure compared to fresh discs. Despite the limitations of the animal model, our findings provide a framework for establishing an applicable cryopreservation protocol for human disc allografts or tissue-engineered disc constructs.
Abstract: A recent clinical study demonstrated that cryopreserved allogeneic intervertebral disc transplantation relieved pain and preserved motion, thus opening up a new treatment option for degenerative disc disease. However, these transplanted discs continued to degenerate, possibly due to a lack of viable cells. Bone marrow-derived stromal cell (BMSC) implantation has been shown to delay disc degeneration.
Abstract: The SRS-22 questionnaire is specifically designed for the assessment of quality of life in spinal deformity patients. This study is the first to use it to assess the quality of life of adolescent idiopathic scoliosis patients under brace treatment and compares the results with an observational group matched by age and curve magnitude. Forty-six patients were enrolled into each group. Overall, it was found that patients under observation had a significantly better quality of life than braced patients. Specifically, the domains for function/activity and self-image were most affected. This effect was most apparent in those with a curve magnitude of under 20 degrees . The scores did not improve significantly with the duration of brace wear, suggesting little adaptation. This study has implications for treatment, and more attention will need to be given to those with mild but progressive curves to help improve patients' understanding of their treatment and hence their compliance and satisfaction.
Abstract: Stainless steel and titanium alloys are the most common metallic orthopedic materials. Recently, nickel-titanium (NiTi) shape memory alloys have attracted much attention due to their shape memory effect and super-elasticity. However, this alloy consists of equal amounts of nickel and titanium, and nickel is a well known sensitizer to cause allergy or other deleterious effects in living tissues. Nickel ion leaching is correspondingly worse if the surface corrosion resistance deteriorates. We have therefore modified the NiTi surface by nitrogen plasma immersion ion implantation (PIII). The surface chemistry and corrosion resistance of the implanted samples were studied and compared with those of the untreated NiTi alloys, stainless steel, and Ti-6Al-4V alloy serving as controls. Immersion tests were carried out to investigate the extent of nickel leaching under simulated human body conditions and cytocompatibility tests were conducted using enhanced green fluorescent protein mice osteoblasts. The X-ray photoelectron spectroscopy results reveal that a thin titanium nitride (TiN) layer with higher hardness is formed on the surface after nitrogen PIII. The corrosion resistance of the implanted sample is also superior to that of the untreated NiTi and stainless steel and comparable to that of titanium alloy. The release of nickel ions is significantly reduced compared with the untreated NiTi. The sample with surface TiN exhibits the highest amount of cell proliferation whereas stainless steel fares the worst. Compared with coatings, the plasma-implanted structure does not delaminate as easily and nitrogen PIII is a viable way to improve the properties of NiTi orthopedic implants.
Abstract: Polyurethane (PU) components of cardiovascular devices are subjected to oxidation-initiated surface degradation, which leads to cracking and ultimately device failure. In the present study, we investigated a novel bromoalkylation chemical strategy to covalently attach the antioxidant, di-tert-butylphenol (DBP), and/or cholesterol (Chol) to the PU urethane nitrogen groups to hypothetically prevent oxidative degradation. These experiments compared PU, PU-DBP, PU-Chol, and PU-Chol-DBP. A series of comparative oxidative degradation studies involved exposing PU samples (modified and unmodified) to H2O2-CoCl2 for 15 days at 37 degrees C, to cause accelerated oxidative degradation. The extent and effects of degradation were assessed by attenuated total reflectance Fourier transformation infrared spectroscopy (FTIR), scanning electron microscopy (SEM), surface contact angle measurements, and mechanical testing. Both the Chol and DBP modification conferred significant resistance to oxidation related changes compared to unmodified PU per FTIR and SEM results. SEM demonstrated cavitation only in unmodified PU. However, contact angle analysis showed significant oxidation-induced changes only in the Chol-modified PU formulations. Most importantly, uniaxial stress-strain testing revealed that only PU-DBP demonstrated bulk elastomeric properties that were minimally affected by oxidation; PU, PU-Chol, PU-Chol-DBP showed marked deterioration of their stress-strain properties following oxidation. In conclusion, these results demonstrate that derivatizing PU with DBP confers significant resistance to oxidative degradation compared with unmodified PU.
Abstract: Intervertebral Disc (IVD) allograft transplantation is presented to be a practical treatment of severe Degenerative Disc Disease (DDD). Limited availability of fresh allograft tissues necessitate the establishment of tissue banking for long-term storage. However, the effects of the cryopreservants on the mechanical properties of the IVD remains unknown. We hypothesize that the appropriate use of cryopreservants can preserve the mechanical properties of the IVD. In this study porcine discs were cryopreserved in different cryopreservative agent (CPA) concentration following a stepwise freezing protocol. After four weeks of storage in liquid nitrogen, dynamic viscoelastic properties of the IVDs were tested using uniaxial compression at different frequencies. Compared to fresh IVDs, no significant differences were found in the elastic modulus (Eâ), viscous modulus (Eâ) and loss tangent (Eâ/Eâ) in the discs cryopreserved with 0-20% CPA. However, cryopreservation at 20% CPA mixture resulted in lower Eâ and Eâ. Our data suggest properly cryopreserved IVD is able to maintain both elastic and viscous characteristics and distribute loads as of a normal IVD.
Abstract: Degenerative disc disease (DDD) is one of the major causes of low back pain. Conventional surgical treatments for DDD can alleviate pain but usually results in fusion of the motion segment and consequently leads to adjacent level disc degeneration. Clinical result of transplanting cryopreserved allograft disc for DDD treatment has demonstrated satisfactory result in motion and stability preservation of the spinal segment but revealed degeneration of the transplanted disc. It is hypothesize that the decrease in cell activity after disc cryopreservation induces disc degeneration of the transplanted disc. This study aims to investigate means of improving cellularity of the cryopreserved intervertebral disc (IVD) either by injecting exogenous bone marrow stromal cells (BMSCs) or enhancing the metabolic activity of post-thaw disc endogenous cells through modification of the cryopreservation protocol.
A mechanical loading bioreactor has been used for ex-vivo disc cultivation. Evaluation of the cryopreserved bovine caudal IVD cell viability, the catabolic and anabolic gene expression profile, and histological changes during the culture has been performed. Bovine disc cryopreserved with the conventional cryopreservation protocol has resulted in cell viability between 30-40% in both NP and AF regions as assessed by live/dead staining and confocal microscopy. In addition, histogical and real-time RT-PCR assessment have suggested that the cryopreserved IVD has reduced matrix over a 7-day culture together with a trend of decrease in collagen type II production and increase in collagenase (MMP-13). Introduction of BMSCs to the cryopreserved IVD cannot demonstrate obvious beneficial effects in terms of gene expression profile.
Porcine lumbar IVD has been used for disc cryopreservation protocol optimization where the effects of various cryopreservation parameters, including cooling rates, constituents of the cryopreservation solution and incubation time, on the metabolic function of the post-thaw tissue have been tested by alamar blue assay. Different optimal cooling rates were found for NP and AF tissue, which were decreasing at the rate of 0.3 and 0.5 °C/min respectively. The difference in optimal cooling rate was probably due to the different in matrix composition and cell types of the two tissues. Increasing total cryoprotectants concentration by combining dimethyl sulphoxide and propylene glycol has improved the cryopreservation result in AF. The optimal disc cryopreservation protocol found in this study have a non-specific metabolic activity of 70.51 ± 13.03 % and 46.97 ± 16.17% of the fresh control in NP and AF respectively.
Considering the above two approaches, a modification of cryopreservation protocol appears to be more practical in preserving the functional integrity of the disc and to be adopted for use clinically. Moreover, BMSC injection may still be needed for replenishing the depleted cells in the disc transplant probably after nutrition re-establishment. Surely, investigations will be needed to understand the changes of the cryopreserved allograft disc after implantation as well as to investigate the timing of nutrition re-establishment to facilitate BMSCs injection. On the other hand, there are crucial needs to understand the fate of the injected BMSC and optimize the injection technique for maximizing function and survival of the injected BMSC.
