Intact Annulus Fibrosus Research Articles

Comparative metagenomic analysis of human intervertebral disc nucleus pulposus and cartilaginous end plates.

Study designThe diversity of microflora inhabiting endplate (EP) and nucleus pulposus (NP) tissues of human intervertebral disc (IVD) was profiled through NGS-supported 16S rRNA amplicon sequencing. Sixteen EP and their corresponding NP were excised from the brain-dead voluntary organ donors with no clinical history of low back pain, and 12 herniated and 8 degenerated NP tissues isolated from the patients undergoing spinal surgery were subjected to study the alteration in the microbial diversity.Objective(s)To understand in normal IVD, whether the colonization of bacteria to the NP is through the EP in discs with intact annulus fibrosus. To identify significantly differing microbial population(s) between normal and diseased IVD (NP).Background of the studyThere is increasing evidence for subclinical infection by fastidious low, growing bacteria to be a cause of disc degeneration. Although the presence of bacteria in NP has been reported well in literature, the source of bacteria is not clearly proved as the disc is avascular in healthy condition. Documentation of similar bacterial populations in the EP and NP may add proof that bacterial inoculation of NP occurs via the EP.Materials and methodsSixteen EP and their corresponding NP excised from brain-dead voluntary organ donors with no history of back pain and 20 diseased discs collected from patients undergoing microdiscectomy/fusion surgery were used for profiling microbiome through 16S rRNA amplicon sequencing using primers specific for V1-V9 hypervariable regions. Changes in bacterial diversity and abundance were analysed to identify the key microbial populations in normal IVD NP and EP tissues and those significantly altered in diseased IVD (NP).ResultsNP and EP shared a similar spectrum of microbiome but with varying abundance. The five dominant phyla identified were Proteobacteria, Firmicutes, Actinobacteria, OD1, and Bacteroidetes. Proteobacteria was found to be the most abundant phyla in both NP (62%) and EP (53%) of the normal IVD. This was followed by Firmicutes (16%), Actinobacteriota (11%), OD1 (Parcubacteria) (7.6%), and Bacteroidetes (2%) in NP and Firmicutes (23.4%), OD1 (Parcubacteria) (17.6%), Actinobacteriota (2.8%), and Bacteroidetes (2.6%) in EP, respectively. Under diseased conditions, Proteobacteria (68%) was dominant when compared with other phyla. However, there was no significant difference in the abundance of Proteobacteria between the normal and diseased discs. Interestingly, the other dominant phyla such as Firmicutes (Normal-NP: 16.2%; Diseased-NP: 4.02%) and Actinobacteria (Normal-NP: 11%; Diseased-NP: 0.99%) showed a significant reduction in degenerated discs. To understand the key microbial populations that are significantly altered during disease, correlation analysis was performed among the three phyla, which revealed a negative correlation in the ratio of Actinobacteria + Firmicutes vs. Proteobacteria (p = 0.001) in DD.ConclusionResults of our study clearly demonstrated a similar bacterial diversity but with varying abundance between the EP and NP, suggesting the existence of the endplate–nucleus pulposus axis in the normal IVD microbiome. Further, our results have indicated that the changes in the abundance of Actinobacteria + Firmicutes vs. Proteobacteria during DDD need further investigation.

GAG content, fiber stiffness, and fiber angle affect swelling-based residual stress in the intact annulus fibrosus.

Biological tissues with a high glycosaminoglycan (GAG) content have an excellent ability to swell by absorbing water molecules from the surrounding environment. Our recent work showed that anisotropy in tissue swelling depends on the fiber-network architecture, including fiber angle, fiber stiffness, and lamellae structure. However, that work did not evaluate the effect of in situ boundary conditions, such as the kidney-bean shape of the annulus fibrosus (AF), on swelling behavior. The biochemical composition of intact AF is inhomogeneous with respect to GAG composition, collagen fiber angle, and fiber stiffness. Moreover, the GAG content in the inner AF decreases significantly with degeneration. In this study, we investigated the role of GAG content, fiber angle, and fiber stiffness in AF swelling and residual strain development using a finite element model based on a human lumbar disk. Our results showed that the annular ring structure had a great impact on swelling by developing region-dependent compressive stress/stretch in the inner layers and tensile stress/stretch in the outer AF. Swelling-based residual stretch was comparable to experimentally measured values, suggesting an important role of tissue swelling in maintaining residual stresses. Moreover, GAG loss in the inner AF, as observed with degeneration, decreased circumferential-direction stress by over 65%. Homogeneous distributions of fiber angle and stiffness overestimated or underestimated AF swelling behavior, such as swelling ratio, circumferential/axial stretch, and fiber stretch/reorientation. These findings demonstrate the need to include native fiber architecture in finite element models, to accurately predict tissue failure, as well as to cultivate engineered disks.

Nano and micro biomechanical alterations of annulus fibrosus after in situ immobilization revealed by atomic force microscopy.

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.

Novel stepwise model of intervertebral disc degeneration with intact annulus fibrosus to test regeneration strategies.

Novel preclinical models that do not damage the annulus fibrosus (AF) of the intervertebral disc are required to study the efficacy of new regenerative strategies for the nucleus pulposus (NP). The aim of the study was to characterize a preclinical ovine model of intervertebral disc degeneration (IDD) induced by endplate (EP) damage and repair via the transpedicular approach, with or without partial nucleotomy, while keeping the AF intact. Twelve adult sheep were used. By the transpedicular approach, a 2 mm tunnel was drilled to the NP through the EP. A partial-nucleotomy was performed. The tunnel was sealed using a polyurethane scaffold. Lumbar discs were assigned to different groups: L1-2: nucleotomy; L2-3: EP tunnel; L3-4: nucleotomy + EP repair; L4-5: EP tunnel + repair; L5-6: control. X-Ray and MRI were performed at 0, 1, 3, and 6 months after surgery. Disc height and MRI indexes were calculated. Macro- and micro-morphology were analyzed. Pfirrmann and Thompson grades were assigned. The treated discs exhibited a progressive decrease in NP signal intensity and MRI index, displaying specific grades of degeneration based on the surgical treatment. According to Pfirrmann and Thompson grades different procedures were staged as: EP tunnel + repair: grade-II; EP tunnel: grade-III, nucleotomy + EP repair: grade-IV; nucleotomy: grade-V. A new stepwise model of IDD to study and test safety and efficacy of novel strategies for NP regeneration has been characterized. The different degrees of IDD have been observed similar to Pfirrmann and Thompson grading system. The intact AF allows for loading studies and eliminating the need for AF closure. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2460-2468, 2018.

Are Intervertebral Disc Tissue Cells Damaged When Attempting to Prevent Thrombus Formation Using Dabigatran, A New Oral Anticoagulant?

To investigate the effect of dabigatran, a new oral anticoagulant, on human primary cell cultures isolated from intact intervertebral disc tissue. Cell cultures were prepared from tissues obtained from six cases who had undergone surgery due to spinal trauma. Dabigatran, an active pharmacological agent, was applied to intact annulus fibrosus (AF)/nucleus pulposus (NP) primary cell cultures from the study group. After performing cell viability, toxicity, and proliferation tests on all cultures in the control and study groups, the surface morphologies of the samples were evaluated. Subsequently, chondroadherin (CHAD), cartilage oligomeric matrix protein (COMP), and matrix metalloproteinase (MMP)-13 and -19 expressions were measured via a real-time polymerase chain reaction (RT-PCR). Data were analyzed statistically. In the proliferation assays performed on the 20th day of the study, cells in the dabigatran-supplemented group were reported to have lost 46.37% more viability than those in the control group. Expressions of all genes examined except MMP-13 were evaluated in the control group by time, but in contrast to the control group results, COMP and MMP-19 gene expressions decreased in the dabigatran-treated group. No CHAD or MMP-13 expression was noted in these cultures. The potential for a systemically applied drug to accumulate in tissue and negatively affect surrounding tissues and microstructures must be emphasized.

Restoring Segmental Biomechanics Through Nucleus Augmentation

In vitro biomechanical laboratory study. The purpose of this study is to evaluate a mechanical treatment to create a degenerative motion segment and the ability of nucleus augmentation to restore biomechanics. In cases with an intact annulus fibrosus, the replacement or augmentation of the nucleus pulposus alone may provide a less invasive option to restore normal biomechanics and disk height when compared with spinal fusion or total disk replacement. Laboratory testing allows these changes to be fully characterized. However, without preexisting pathology, nucleus augmentation therapies are difficult to evaluate in vitro. The present study evaluated pure moment bending and compressive biomechanics in 3 states (n=6): (1) intact, (2) after creep loading and nucleus disruption to induce degenerative biomechanical changes, and (3) after nucleus augmentation through an injectable polymer (DiscCell). Neutral zone and ROM were increased in all modes of bending after the degenerative treatment. The most sensitive mode of bending was lateral bending, with intact ROM (20.0±2.9 degrees) increased to 22.3±2.6 degrees after degenerative treatment and reduced to 18.4±1.6 degrees after injection of the polymer. All bending ROM and NZ changes induced by the degenerative treatment were reversed by nucleus augmentation. This material was shown to be effective at altering motion segment biomechanics and restoring disk height during time zero tests. This technique may provide a model to examine the time zero performance of a nucleus augmentation device/material.

Biomechanical Evaluation of the Transpedicular Nucleotomy with Intact Anulus Fibrosus

Introduction Mechanical loading represents a crucial part of intervertebral disc (IVD) homeostasis. However, traditional regenerative strategies require violation of the annulus fibrosus (AF) resulting in significant alteration of joint mechanics. The transpedicular nucleotomy represents a suitable method to create a cavity into the nucleus polposus (NP), as a model to study IVD regeneration with intact AF. The aim is to study how the transpedicular tunnel (TT) and the novel mechanical nucleotomy affects the biomechanics of the NP. Material and Methods 60 ovine lumbar FSUs (L1-L6) randomly assigned to 5 groups: Control; TT; TT + polymethylmethacrylate (PMMA), to repair the bone tunnel; Nucleotomy; Nucleotomy + PMMA. The robotic spine testing system consists of a robotic manipulator (Staubli RX90) and a six-axis load cell (JR3 Inc.). Flexion/extension, lateral bending and axial rotation were evaluated under adaptive displacement control. Axial compression was applied for 15 cycles of preconditioning followed by 1 hour of constant compression. Viscoelastic properties were determined. Results TT has minimal effects on rotational biomechanics. The nucleotomy increases ROM and NZ displacement width while decreasing NZ stiffness. TT + PMMA has small effects in terms of ROM. Nucleotomy + PMMA brings ROM back to the control, increases NZ stiffness and decreases NZ displacement width. The nucleotomy tends to increase the rate of early creep. TT reduces early and late damping. The use of PMMA increased late elastic damping (S2) and reduced viscous damping (η2) culminating in faster resolution of creep. Conclusion Biomechanical properties of NP are crucial for IVD repair. TT does not affect rotational stability. Mechanical changes that occur with nucleotomy are similar to those characteristics of early degeneration. These findings confirm that the NP is crucial for segmental stability in physiologic rotations.

Disc Regeneration Using MSC Transplanted via the Endplate Route

Introduction Stem cell based intervertebral disc (IVD) regeneration is quickly moving toward clinical applications.1 However, many aspects need to be investigated to routinely translate this therapy to clinical applications, in particular, the most efficient way to deliver cell to the IVD. Cells are commonly delivered to the IVD through the annulus fibrosus (AF) injection. However, recent studies have shown serious drawbacks of this approach2–4 suggesting that intradiscal injection through the AF route itself is not completely innocuous and may disable the treatments to therapeutic agents delivered. As an alternative we have described and tested a new surgical approach to the IVD via the endplate-pedicles (transpedicular approach). The Purpose of the study was to test MSCs/hydrogel transplantation for IVD regeneration in a grade IV preclinical model of IDD on large size animals via the transpeducular approach5 with cell dose escalation. Material and Methods Adult sheep ( n = 18) underwent bone marrow aspiration for autologous MSC isolation and expansion. MSC were suspended in autologous PRP and conjugated with Hyaluronic Acid and Batroxobin at the time of transplant (MSCs/hydrogel). Nucleotomy was performed via the transpedicular approach under fluoroscopy6 in four lumbar IVDs and that were injected with 1) hydrogel, 2) Low doses of MSC/hydrogel [5x106 cell/ml] 3) High doses of MSC/hydrogel [1x107 cell/ml], 4) no injection (CTRL). The endplate tunnel was sealed using a polyurethane scaffold.7 X-ray and MRI were performed at baseline and 1,3,6,12 months. Disc height and MRI indexes were calculated at each time point. Disc macro- and micro-morphology were analyzed at each time point. Results The MRI index showed a significant decrease in the untreated group, the disc injected with hydrogel and those injected with low MSC dose compared with healthy discs in all time points. The discs treated with high dose of MSC showed maintenance of the MRI index compared with the healthy disc. Morphologically, the grade of degeneration evaluated using the Thompson grade system8 was in agreement with the grades observed at the MRI. The High MSC dose treated discs demonstrated abundant cartilage formation at 3 months, and to a lesser extent at 6 and 12 months. For the carrier and low MSC dose treated groups, however, there was less proteoglycan matrix. Conclusion An effective dose of autologous MSC [1x107 cell/ml] delivered via the alternative transpedicular approach regenerates the NP in a preclinical model of grade IV IDD. These data highlight as the disc regeneration can be achieved maintaining the AF intact via the end-plate route. This preclinical study has high translational value as large animal model with the long fallow up were used, MSCs were expanded in GMP facility simulating the clinical scenario, and the hydrogel were composed of clinically available drags and materials. This study bring a significant contribution toward the translation of regenerative therapies for the biological restoration of degenerative changes in the IVD, which is crucial to improve present clinical treatment and life quality of several patients. Acknowledgment The support of the Italian Ministry of Instruction, University and Research Grant (PRIN-200938NT8Z), the Young Investigator Research Grant of the Italian Ministry of Health (GR-2010–2318448) and the BIOSPINA Award of the Italian Society of Spine Surgery (SICV&amp;GIS) are gratefully acknowledged. References Orozco L, Soler R, Morera C, Alberca M, Sánchez A, García-Sancho J. Intervertebral disc repair by autologous mesenchymal bone marrow cells: a pilot study. Transplantation 2011;92(7):822–828 Carragee EJ, Don AS, Hurwitz EL, Cuellar JM, Carrino JA, Herzog R. 2009 ISSLS Prize Winner: Does discography cause accelerated progression of degeneration changes in the lumbar disc: a ten-year matched cohort study. Spine 2009;34(21):2338–2345 Korecki CL, Costi JJ, Iatridis JC. Needle puncture injury affects intervertebral disc mechanics and biology in an organ culture model. Spine 2008;33(3):235–241 Vadalà G, Sowa G, Hubert M, Gilbertson LG, Denaro V, Kang JD. Mesenchymal stem cells injection in degenerated intervertebral disc: cell leakage may induce osteophyte formation. J Tissue Eng Regen Med 2012;6(5):348–355 Vadalà G, Russo F, Pattappa G, et al. The transpedicular approach as an alternative route for intervertebral disc regeneration. Spine 2013;38(6):E319–E324 Vadalà G, De Strobel F, Bernardini M, Denaro L, D'Avella D, Denaro V. The transpedicular approach for the study of intervertebral disc regeneration strategies: in vivo characterization. Eur Spine J 2013;22(Suppl 6):S972–S978 Vadala G, et al. A nucleotomy model with intact annulus fibrosus to test intervertebral disc regeneration strategies. Tissue Eng Part C Methods 2015;21(11):1117–1124 Thompson JP, Pearce RH, Schechter MT, Adams ME, Tsang IK, Bishop PB. Preliminary evaluation of a scheme for grading the gross morphology of the human intervertebral disc. Spine 1990;15(5):411–415

A Nucleotomy Model with Intact Annulus Fibrosus to Test Intervertebral Disc Regeneration Strategies.

New cells/hydrogel-based treatments for intervertebral disc (IVD) regeneration need to be tested on animal models before clinical translation. Ovine IVD represents a good model but does not allow the injection of a significant volume into intact IVD. The aim of this study was to compare different methods to create a cavity into ovine nucleus pulposus (NP) by enzymatic digestion (E), mechanical nucleotomy (N), or a combining technique (E+N), as a model to study IVD regeneration strategies with intact annulus fibrosus (AF) in functional spinal units (FSUs) in vitro. The transpedicular approach via the endplate route (2 mm tunnel) was performed on ovine FSU (IVD and superior and inferior endplate) to access the NP. FSUs were treated by N (Arthroscopic shaver), E (Trypsin/Collagenase), or E+N. Treatments were evaluated macro- and microscopically. The degradation of proteoglycan (PG) around the cavity was assessed by gel electrophoresis. Cell viability was evaluated using the lactate dehydrogenase (LDH) assay. Cavity volume was quantified through computerized tomography after injection of agarose gel/contrast agent. A cavity with intact AF was successfully created with all three methods. The N group showed high reproducibility, low PG degradation, and no endplate thinning. Histological analysis demonstrated NP matrix degradation in enzyme-treated groups, while the PG content was homogenous using mechanical discectomy. Cell viability was affected only in the E group. The cavity volume normalized to the total IVD volume was 5.2% ± 1.6% in E, 5.0% ± 1.4% in E+N, and 4.2% ± 0.1% in N. Mechanical nucleotomy leads to a more reproducible and less destructive cavity in the NP. Enzymatic methods perform better in terms of cavity volume; however, the cells and PG of the surrounding tissue may be affected. The mechanical nucleotomy enables the creation of a cavity into the IVD while keeping the AF intact, allowing the injection of reproducible volumes of hydrogel and tissue engineering construct for preclinical tests.

Papain-induced in vitro disc degeneration model for the study of injectable nucleus pulposus therapy

Background contextProteolytic enzyme digestion of the intervertebral disc (IVD) offers a method to simulate a condition of disc degeneration for the study of cell-scaffold constructs in the degenerated disc. PurposeTo characterize an in vitro disc degeneration model (DDM) of different severities of glycosaminoglycans (GAG) and water loss by using papain, and to determine the initial response of the human mesenchymal stem cells (MSCs) introduced into this DDM. Study designDisc degeneration model of a bovine disc explant with an end plate was induced by the injection of papain at various concentrations. Labeled MSCs were later introduced in this model. MethodsPhosphate-buffered saline (PBS control) or papain in various concentrations (3, 15, 30, 60, and 150 U/mL) were injected into the bovine caudal IVD explants. Ten days after the injection, GAG content of the discs was evaluated by dimethylmethylene blue assay and cell viability was determined by live/dead staining together with confocal microscopy. Overall matrix composition was evaluated by histology, and water content was visualized by magnetic resonance imaging. Compressive and torsional stiffness of the DDM were also recorded. In the second part, MSCs were labeled with a fluorescence cell membrane tracker and injected into the nucleus of the DDM or a PBS control. Mesenchymal stem cell viability and distribution were evaluated by confocal microscopy. ResultsA large drop of GAG and water content of the bovine disc were obtained by injecting >30 U/mL papain. Magnetic resonance imaging showed Grade II, III, and IV disc degeneration by injecting 30, 60, and 150 U/mL papain. A cavity in the center of the disc could facilitate later injection of the nucleus pulposus tissue engineering construct while retaining an intact annulus fibrosus. The remaining disc cell viability was not affected. Mesenchymal stem cells injected into the protease-treated DDM disc showed significantly higher cell viability than when injected into the PBS-injected control disc. ConclusionsBy varying the concentration of papain for injection, an increasing amount of GAG and water loss could be induced to simulate the different severities of disc degeneration. MSC suspension introduced into the disc has a very low short-term survival. However, it should be clear that this bovine IVD DDM does not reflect a clinical situation but offers exciting possibilities to test novel tissue engineering protocols.

Intervertebral Disc Repair by Autologous Mesenchymal Bone Marrow Cells: A Pilot Study

Degenerative disc disease may cause severe low-back pain, a large public health problem with significant economic and life quality impact. Chronic cases often require surgery, which may lead to biomechanical problems and accelerated degeneration of the adjacent segments. Cell-based therapies may circumvent these problems and have exhibited encouraging results in vitro and in animal studies. We designed a pilot study to assess feasibility and safety and to obtain early indications on efficacy of treatment with mesenchymal stem cells (MSC) in humans. Ten patients with chronic back pain diagnosed with lumbar disc degeneration with intact annulus fibrosus were treated with autologous expanded bone marrow MSC injected into the nucleus pulposus area. Clinical evolution was followed for 1 year and included evaluation of back pain, disability, and quality of life. Magnetic resonance imaging measurements of disc height and fluid content were also performed. Feasibility and safety were confirmed and strong indications of clinical efficacy identified. Patients exhibited rapid improvement of pain and disability (85% of maximum in 3 months) that approached 71% of optimal efficacy. This outcome compares favorably with the results of other procedures such as spinal fusion or total disc replacement. Although disc height was not recovered, water content was significantly elevated at 12 months. MSC therapy may be a valid alternative treatment for chronic back pain caused by degenerative disc disease. Advantages over current gold standards include simpler and more conservative intervention without surgery, preservation of normal biomechanics, and same or better pain relief.

In vitro measurement of pressure in intervertebral discs and annulus fibrosus with and without annular tears during discography

Background context Discogram studies have shown that pain reproduction correlates with the extent of annular disruption. However, it has not been assessed if pressure changes in the annulus fibrosus vary incrementally with intradiscal pressure. Purpose To determine the relationship between intradiscal pressure and outer annular pressure during discography in intervertebral discs with and without annular tears. Study design Intradiscal and periannular pressures were measured simultaneously in vitro during intradiscal injection in porcine cadaver spines. Methods Twenty fresh porcine cadaver lumbar spines with intervening discs were tested. Intradiscal and periannular pressures were measured simultaneously using two pressure sensors during intradiscal contrast injection. The tip of a 25-gauge needle connected with a pressure manometer was placed in the center of the nucleus pulposus. A second pressure transducer was located at the outer third of the annulus fibrosus. Needle and transducer locations were confirmed by fluoroscopy. To compare the intact and torn annulus fibrosus, annular disruptions were created with a 20-gauge needle and confirmed by fluoroscopy. Results During intradiscal injections of discs with an intact annulus, annulus fibrosus pressure remained low and a sharp increase in intradiscal pressure was observed. Significantly higher pressures were noted in the outer annulus in discs with annular tears (p<.01). Mean pressures at the central nucleus pulposus, the outer third of intact annuli and torn annuli were 93.4±40.9 psi, 14.8±1.9 psi and 85.7±24.8 psi, respectively. Conclusions Volumetric injection of intervertebral discs with a torn annulus fibrosis during discography may increase intra-annular pressure similar to the increase in pressure that may occur during spinal loading activities. This effect may not occur in discs with an intact annulus fibrosus.

Heat-induced changes in porcine annulus fibrosus biomechanics

The intervertebral disc is implicated as the source of low-back pain in a substantial number of patients. Because thermal therapy has been thought to have a therapeutic effect on collagenous tissues, this technique has recently been incorporated into several minimally invasive back pain treatments. However, patient selection criteria and precise definition of optimum dose are hindered by uncertainty of treatment mechanisms. The purpose of this study was to quantify acute changes in annulus fibrosus biomechanics after a range of thermal exposures, and to correlate these results with tissue denaturation. Intact annulus fibrosus (attached to adjacent vertebrae) from porcine lumbar spines was tested ex vivo. Biomechanical behavior, microstructure, peak of denaturation endotherm, and enthalpy of denaturation (mDSC) were determined before and after hydrothermal heat treatment at 37°C, 50°C, 60°C, 65°C, 70°C, 75°C, 80°C, and 85°C. Shrinkage of excised annular tissue (removed from adjacent vertebrae) was also measured after treatment at 85°C. Significant differences in intact annulus biomechanics were observed after treatment, but the effects were much smaller in magnitude than those observed in excised annulus and those reported previously for other tissues. Consistent with this, intact tissue was only minimally denatured by treatment at 85°C for 15 min, whereas excised tissue was completely denatured by this protocol. Our data suggest that in situ constraint imposed by the joint structure significantly retards annular thermal denaturation. These findings should aid the interpretation of clinical outcomes and provide a basis for the future design of optimum dosing regimens.