Katherine Hudson

Recent advances in biological therapies for disc degeneration: tissue engineering of the annulus fibrosus, nucleus pulposus and whole intervertebral discs.

Advanced intervertebral disc (IVD) degeneration, a major cause of back pain in the United States, is treated using invasive surgical intervention which may cause further degeneration is the future. Because of the limitations of traditional solutions, tissue engineering therapies have become increasingly popular. IVDs have two distinct regions, the inner nucleus pulposus (NP) which is jelly-like and rich in glycosaminoglycans (GAGs) and the outer annulus fibrosus (AF) which is organized into highly collagenous lamellae. Tissue engineered scaffolds, as well as whole organ culture systems have been developed. These culture systems may help elucidate the initial causes of disc degeneration. To create an effective tissue engineered therapy, researchers have focused on designing materials that mimic the properties of these two regions to be used independently or in concert. The few in vivo studies show promise in retaining disc height and MRI T2 signal intensity, the gold standard in determining disc health.

Annular repair using high-density collagen gel: a rat-tail in vivo model.

Study Design. Animal in vivo study. Objective. To test the capability of high-density collagen gel to repair annular defects. Summary of Background Data. Annular defects are associated with spontaneous disc herniations and disc degeneration, which can lead to significant morbidity. Persistent annular defects after surgical discectomies can increase reherniation rates. Several synthetic and biological materials have been developed for annular repair. This is the first study to test an injectable biomaterial in vivo. Methods. We punctured caudal intervertebral discs in 42 athymic rats, using an 18-gauge needle to create an annular defect. High-density collagen (HDC), either alone or cross-linked with riboflavin (RF), was injected into the defect. There were 4 separate study groups: HDC, HDC cross-linked with either 0.25 mM RF or 0.50 mM RF, and a negative control that was punctured and not treated. The animals were followed for 5 weeks; radiographs were used to assess disc heights and magnetic resonance images were used to evaluate degenerative changes. We developed an algorithm on the basis of T2-relaxation time measurements to assess the size of the nucleus pulposus. Tails were collected for histological analysis to evaluate disc degeneration and measure the cross-sectional area of the nucleus pulposus. Results. After 5 weeks, the control and the uncross-linked HDC groups both showed signs of progressive degenerative changes with minimal or no residual nucleus pulposus tissue in the disc space. Cross-linking significantly improved the ability of HDC gels to repair annular defects. The 0.50 mM RF cross-linked group showed only a slight decrease in nuclear tissue when compared with healthy discs, with no signs of intervertebral disc (IVD) degeneration. The annulus fibrosus was partially repaired by a fibrous cap that bridged the defect. Host fibroblasts infiltrated and remodeled the injected collagen. Conclusion. HDC is capable of repairing annular defects induced by needle puncture. The stiffness of HDC can be modified by riboflavin cross-linking and seems to positively affect the repair mechanism. These results need to be replicated in a larger animal model. Level of Evidence: N/A

Tissue-engineered intervertebral discs: MRI results and histology in the rodent spine.

OBJECT Tissue-engineered intervertebral discs (TE-IVDs) represent a new experimental approach for the treatment of degenerative disc disease. Compared with mechanical implants, TE-IVDs may better mimic the properties of native discs. The authors conducted a study to evaluate the outcome of TE-IVDs implanted into the rat-tail spine using radiological parameters and histology. METHODS Tissue-engineered intervertebral discs consist of a distinct nucleus pulposus (NP) and anulus fibrosus (AF) that are engineered in vitro from sheep IVD chondrocytes. In 10 athymic rats a discectomy in the caudal spine was performed. The discs were replaced with TE-IVDs. Animals were kept alive for 8 months and were killed for histological evaluation. At 1, 5, and 8 months, MR images were obtained; T1-weighted sequences were used for disc height measurements, and T2-weighted sequences were used for morphological analysis. Quantitative T2 relaxation time analysis was used to assess the water content and T1ρ-relaxation time to assess the proteoglycan content of TE-IVDs. RESULTS Disc height of the transplanted segments remained constant between 68% and 74% of healthy discs. Examination of TE-IVDs on MR images revealed morphology similar to that of native discs. T2-relaxation time did not differ between implanted and healthy discs, indicating similar water content of the NP tissue. The size of the NP decreased in TE-IVDs. Proteoglycan content in the NP was lower than it was in control discs. Ossification of the implanted segment was not observed. Histological examination revealed an AF consisting of an organized parallel-aligned fiber structure. The NP matrix appeared amorphous and contained cells that resembled chondrocytes. CONCLUSIONS The TE-IVDs remained viable over 8 months in vivo and maintained a structure similar to that of native discs. Tissue-engineered intervertebral discs should be explored further as an option for the potential treatment of degenerative disc disease.

Assessment of intervertebral disc degeneration based on quantitative magnetic resonance imaging analysis: an in vivo study.

Study Design. Animal experimental study. Objective. To evaluate a novel quantitative imaging technique for assessing disc degeneration. Summary of Background Data. T2-relaxation time (T2-RT) measurements have been used to assess disc degeneration quanti-tatively. T2 values correlate with the water content of intervertebral disc tissue and thereby allow for the indirect measurement of nucleus pulposus (NP) hydration. Methods. We developed an algorithm to subtract out magnetic resonance imaging (MRI) voxels not representing NP tissue on the basis of T2-RT values. Filtered NP voxels were used to measure nuclear size by their amount and nuclear hydration by their mean T2-RT. This technique was applied to 24 rat-tail intervertebral discs (IVDs), which had been punctured with an 18-gauge needle according to different techniques to induce varying degrees of degeneration. NP voxel count and average T2-RT were used as parameters to assess the degeneration process at 1 and 3 months postpuncture. NP voxel counts were evaluated against radiograph disc height measurements and qualitative MRI studies on the basis of the Pfirrmann grading system. Tails were collected for histology to correlate NP voxel counts to histological disc degeneration grades and to NP cross-sectional area measurements. Results. NP voxel count measurements showed strong correlations to qualitative MRI analyses (R2 = 0.79, P < 0.0001), histological degeneration grades (R2 = 0.902, P < 0.0001), and histological NP cross-sectional area measurements (R2 = 0.887, P < 0.0001). In contrast to NP voxel counts, the mean T2-RT for each punctured group remained constant between months 1 and 3. The mean T2-RTs for the punctured groups did not show a statistically significant difference from those of healthy IVDs (63.55 ms ± 5.88 ms mo 1 and 62.61 ms ± 5.02 ms) at either time point. Conclusion. The NP voxel count proved to be a valid parameter to assess disc degeneration quantitatively in a needle puncture model. The mean NP T2-RT does not change significantly in needle-puncture–induced degenerated IVDs. IVDs can be segmented into different tissue components according to their innate T2-RT. Level of Evidence: N/A

Riboflavin crosslinked high-density collagen gel for the repair of annular defects in intervertebral discs: An in vivo study

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.

Hypoxic Expansion of Human Mesenchymal Stem Cells Enhances 3D Maturation of Tissue Engineered Intervertebral Discs

Culture of three-dimensional (3D) constructs in hypoxic conditions (1-5% O2) has been shown to increase production of extracellular matrix components in primary intervertebral disc (IVD) cells and drive chondrogenesis of human mesenchymal stem cells (hMSCs). Growing evidence suggests that two-dimensional (2D) expansion under hypoxic conditions may have an even greater influence on chondrogenesis in MSCs. This study aims to determine the effects of hypoxia during 2D expansion and subsequent 3D culture on the in vitro maturation of tissue-engineered IVDs (TE-IVDs) made with hMSCs, using a previously developed TE-IVD system. hMSCs were expanded in either hypoxic (5% O2) or normoxic (21% O2) conditions before construction of TE-IVDs. Discs were cultured in 3D in either hypoxic or normoxic conditions to create four experimental groups. Discs made from MSCs expanded in hypoxia were up to 141% stiffer than those made with normoxia-expanded MSCs. Similar patterns were seen in all mechanical properties. Increases in glycosaminoglycan content and collagen content in the nucleus pulposus (NP) were associated with 3D hypoxic culture. A boundary region between the manufactured fibrosus and NP regions developed by 2 weeks and mimicked the organization of the native disc. Hypoxic conditions in both 2D expansion and subsequent 3D culture improved the maturation of TE-IVDs made with hMSCs.

Tissue-engineered Total Disc Replacement: In vivo Outcomes of a Canine Cervical Disc Study

Introduction Despite being effective, the most commonly performed treatments for degenerative disc disease (DDD), fusion and prosthetic total disc replacement, still pose risks of pseudoarthrosis, implant dislodgement, and adjacent segment disease.1–3 Tissue engineered intervertebral discs (TE-IVD) are an alternative treatment option for DDD, and have been previously developed by our group as a biological TDR device.4 Presently, we evaluate the surgical conditions that promote implant stability and in-vivo efficacy of our TE-IVDs in a translational beagle cervical spine model. Material and Methods TE-IVD Construction: TE-IVD components were constructed in vitro using either annulus fibrosus (AF) or nucleus pulposus (NP) cultivated canine disc cells; the collagen gel based composite AF enclosed an alginate gel based composite NP, as previously described.4 Experimental and Surgical Protocol: 14 skeletally mature beagles underwent discectomy with whole IVD resection at a level between C3/4 and C6/7, and were divided into two groups: a solely discectomized control (n = 2) and a TE-IVD implanted group (n = 12). Discectomy and TE-IVD implantation were performed under segmental distraction. Implant stability was evaluated upon distraction release at the end of the procedure. Imaging: Postoperative imaging was performed with conventional X-rays and high-resolution 3-Tesla MRI under full anesthesia. Disc height indices were measured on X-rays using a pre-established method. 5 All MRIs were analyzed both qualitatively and quantitatively in accordance to T2-weighted images. Utilizing a novel algorithm developed by our group, we filtered out all MRI voxels unrepresentative of NP tissue using their T2-relaxation time (T2-RT), sequestering the extent of NP hydration based on the mean T2-RT within the NP voxel.6 Histological assessment: Animals were sacrificed either at 4 or 16 weeks. Histological staining was obtained using Safranin-O for proteoglycans. Statistics: A Chi-Squared test was performed to determine the correlation between implant stability and surgical level or posterior longitudinal ligament (PLL) resection. For the analyses of continuous outcomes in disc height index, NP size, and NP hydration, we employed linear regression models with a generalized estimating equation and robust standard errors to estimate differences in mean changes from baseline controls (discectomy) across displaced and stable implantation groups. Results TE-IVDs that demonstrated displacement of over 25% TE-IVD volume upon distraction release were defined as “displaced” implants and the remaining were termed as “stable” implants. There was a correlation between implant stability and surgical level but not between implant stability and PLL resection, with implants at C3/4 having the greatest stability (p < 0.05). Quantitative X-ray and MRI assessments showed that only the stable implants had significant retention of disc height and NP size as well as NP physiological hydration compared with discectomy controls. Both 4- and 16-week histology demonstrated that implanted TE-IVDs yielded AF-like and NP-like tissues in the treated segment. Integration into host tissue was confirmed over 16 weeks without any signs of immune reaction. Conclusion Despite significant biomechanical demands of the beagle cervical milieu, our in vivo TE-IVDs, when implanted successfully, maintained their position, structure and hydration in addition to disc height over 16 weeks. References Nesterenko SO, Riley LH III, Skolasky RL. Anterior cervical discectomy and fusion versus cervical disc arthroplasty: current state and trends in treatment for cervical disc pathology. Spine 2012;37(17):1470–1474 Sugawara T, Itoh Y, Hirano Y, Higashiyama N, Mizoi K. Long term outcome and adjacent disc degeneration after anterior cervical discectomy and fusion with titanium cylindrical cages. Acta Neurochir (Wien) 2009;151(4):303–309, discussion 309 Kelly MP, Mok JM, Frisch RF, Tay BK. Adjacent segment motion after anterior cervical discectomy and fusion versus Prodisc-c cervical total disk arthroplasty: analysis from a randomized, controlled trial. Spine 2011;36(15):1171–1179 Bowles RD, Gebhard HH, Härtl R, Bonassar LJ. Tissue-engineered intervertebral discs produce new matrix, maintain disc height, and restore biomechanical function to the rodent spine. Proc Natl Acad Sci U S A 2011;108(32):13106–13111 Kim JS, Kroin JS, Li X, et al. The rat intervertebral disk degeneration pain model: relationships between biological and structural alterations and pain. Arthritis Res Ther 2011;13(5):R165 Grunert P, Hudson KD, Macielak MR, et al. Assessment of intervertebral disc degeneration based on quantitative magnetic resonance imaging analysis: an in vivo study. Spine 2014;39(6):E369–E378

In Vivo Total Disc Replacement using Tissue-Engineered Intervertebral Discs in a Canine Model

Introduction Disc degeneration in the cervical spine is a prevalent clinical predicament often requiring surgery. Anterior cervical decompression and fusion (ACDF), the most commonly performed procedure, poses risks of pseudoarthrosis, and adjacent segment disease (ASD).1,2 An emerging alternative treatment option is prosthetic total disc replacement (TDR),3 which preserves segmental mobility. Our group previously developed a biological TDR device using composite AF/NP disc-like construct with viable cells and mechanical properties analogous to the native discs in a rat tail model.4 In this study, we evaluated the in vivo efficacy of this tissue-engineered intervertebral disc (TE–IVD) in a beagle cervical model assessing radiological and histological parameters. Material and Methods TE-IVD construction: Canine-sized TE–IVDs were constructed as previously described.4 Cervical IVDs from skeletally mature beagles were separated into AF and NP tissues; component cells were isolated and cultured in vitro. Cultured NP cells were seeded with alginate, injected into a predesigned mold, and encircled with two layers of an AF cell laden collagen gel. The combined construct was kept in media for 2 weeks as the surrounding annulus fibrous aligned and contracted until required TE–IVD diameter was attained. Experimental Protocol: Overall, eight skeletally mature beagles were divided into the following two groups: the control group (n = 2) underwent discectomy with fully resected IVDs, and the experimental group (n = 6) underwent TE–IVD implantation postdiscectomy. Adjacent proximal segments served as internal healthy controls. Postoperative X-ray and MRI were taken at 2 and 4 weeks; disc height indices5 and NP hydration using a pre-established algorithm6 were analyzed. Beagles were humanely killed at 4 weeks for histological assessment. Results TE–IVDs were successfully implanted postdiscectomy (Fig. 1A, B). At 2 weeks, MRIs of TE–IVDs revealed T2 high intensity with acute outer inflammation because of the surgical invasion, which faded by 4 weeks. At 4 weeks, TE–IVDs maintained position in the disc space with relatively increased T2 intensity, whereas discectomized segments manifested as black discs (Fig. 1C, D). These findings suggest that the implanted TE–IVDs engraft in the disc space despite significant biomechanical demands of the beagle cervical environment. In fact, disc height indices of the TE–IVDs and discectomized discs were 71 and 49%, respectively, of that of healthy control discs. Likewise, MRIs revealed that NP hydration of the implanted TE–IVDs was over 70% of that of healthy discs. Histological assessments further demonstrated chondrocyte-like cell viability in the TE–IVD, abundant proteoglycan content in the extracellular matrices, and substantial integration into host tissues without signs of immune reactions. Fig. 1 (A, B) TE–IVD. (C, D) Sagittal and axial T2-weighed MR images of control/experimental segments. Conclusion Despite the severe local milieu of the beagle cervical spine owing to mechanical loading, our in vivo TE–IVDs when appropriately implanted, maintained their position and structure at 4 weeks. The TE–IVDs displayed dynamic adaptation to the host environment, with extracellular matrix production and cell proliferation. They further maintained disc height as well as NP hydration at 4 weeks with up to 70% viability as the normal healthy discs. References Nesterenko SO, Riley LH III, Skolasky RL. Anterior cervical discectomy and fusion versus cervical disc arthroplasty: current state and trends in treatment for cervical disc pathology. Spine 2012;37(17):1470–1474 Sugawara T, Itoh Y, Hirano Y, Higashiyama N, Mizoi K. Long term outcome and adjacent disc degeneration after anterior cervical discectomy and fusion with titanium cylindrical cages. Acta Neurochir (Wien) 2009;151(4):303–309, discussion 309 Kelly MP, Mok JM, Frisch RF, Tay BK. Adjacent segment motion after anterior cervical discectomy and fusion versus Prodisc-c cervical total disk arthroplasty: analysis from a randomized, controlled trial. Spine 2011;36(15):1171–1179 Bowles RD, Gebhard HH, Härtl R, Bonassar LJ. Tissue-engineered intervertebral discs produce new matrix, maintain disc height, and restore biomechanical function to the rodent spine. Proc Natl Acad Sci U S A 2011;108(32):13106–13111 Kim JS, Kroin JS, Li X, et al. The rat intervertebral disk degeneration pain model: relationships between biological and structural alterations and pain. Arthritis Res Ther 2011;13(5):R165 Grunert P, Hudson KD, Macielak MR, et al. Assessment of intervertebral disc degeneration based on quantitative magnetic resonance imaging analysis: an in vivo study. Spine 2014;39(6):E369–E378

In Vivo Repair Of Rat Annulus Fibrosus Defects Using High Density Collagen Gels

Introduction Although a discectomy successfully relieves the neurological symptoms of a herniated intervertebral disc (IVD), it does not treat the underlying degenerative process; the annular defect remains unrepaired. Persistent annular defect is associated with an increased risk of recurrent herniation and progressive degenerative changes to the IVD. It may also be the primary cause of chronic low back pain following discectomy. To date, there is no established method for repairing annular defects in vivo. The presented study aims to determine whether injectable high-density collagen (HDC) gels can reduce further disc herniation and inhibit degenerative changes in a needle-punctured rat-tail model, and whether riboflavin (RF) cross-linking of injected collagen influences the repair process. Materials and Methods A total of 31 athymic rats were punctured with an 18-gauge needle in C3/4 of the caudal spine. They were divided into four groups: group 1 punctured and injected with HDC cross-linked with 0.5 mM (n = 6) or 0.75 mM (n = 7) RF; group 2 punctured and injected with noncross-linked collagen (n = 6); group 3 punctured and untreated (n = 8); group 4 punctured and injected with FITC-labeled cross-linked collagen (n = 4). Degenerative changes to punctured discs, NP size, and NP hydration were analyzed using X-ray, MRI), and histology. Functionality of repaired AF tissue was measured by mechanical tests, by comparing the hydraulic permeability of treated discs to that of healthy discs. Results After 5 weeks, untreated discs showed signs of terminal degenerative changes on MRI and histological sections (Fig. 1). No NP tissue remained in the disc space. In contrast, discs treated with RF cross-linked collagen gels retained 63% of NP and 80% of disc height at 5 weeks, and showed minimal degenerative changes on histological section. Interestingly, a series of in vivo imaging showed that after 5 weeks, discs treated with cross-linked HDC demonstrated consistent improvement in both volume and hydration over time, while untreated discs and discs treated with noncross-linked HDC, lost volume, and water content. Injected collagen was seen to form a zipper-like adhesion to the host AF and connective tissue after 1 week and by 5 weeks a fibrous cap that persisted until 18 weeks. NP hydration and IVD functionality of treated discs were found to be similar to those of adjacent healthy discs at 18 weeks. Fig. 1 An 18-week outcome examples from all punctured groups. Adjacent segments served as healthy controls. Specimen from noncross-linked and punctured, untreated groups showed degenerative changes on X-ray and MRI; these changes were attenuated in the RF cross-linked group. NP tissue from the RF cross-linked group appeared hyperintense on MRI and retained its ovular shape. MRI, magnetic resonance imaging; RF, riboflavin; NP, nucleus pulposus. Conclusion Injection of high-density collagen gel cross-linked with RF can repair annular defects, prevent the degenerative cascade, and maintain the functionality of IVDs in a rat-tail spine. Further studies will be performed to confirm the ability of HDC gels to repair annular defects in a large animal model.

High-Density Collagen Gel Can Repair Annular Defects and Restore Biomechanical Function to Needle-Punctured Intervertebral Discs

Introduction Unrepaired annular defects potentially increase the reherniation rate after lumbar discectomies and have been shown to accelerate degenerative changes after discographies. This is the first study to test an injectable biological substance to repair annular defects in vivo. We used high-density collagen gel in a needle-punctured rat-tail model. Needle puncturing leads to extrusion of NP tissue with subsequent degenerative changes (4). Restoring annular integrity will help retain the NP material and therefore inhibit these changes. Materials and Methods We punctured the S3/S4 intervertebral disc (IVD) of 30 athymic rats using an 18-gauge needle. Subsequently high-density collagen (HDC) gel was injected to seal the defect. Riboflavin (RF) was added to increase the stiffness of the collagen gel by inducing chemical cross-link formation. Animals were subdivided into four groups. The first group was injected with uncross-linked HDC gel (n = 6), the second with cross-linked collagen using 0.5 mM (n = 8) and the third 0.75 mM (n = 8) concentration of RF. The fourth group (n = 8) served as control and was left untreated after needle puncture. The animals were followed up at weeks 1, 2, 5, 12, and 18 with X-ray measurements to assess the disc heights and MR imaging to evaluate degenerative changes according to a modified Pffirmann grading system. We developed an algorithm based on T2-relaxation time measurements to assess the size of the nucleus by the number of NP voxels that compose it (Fig. 1). Animals were sacrificed at 1, 2, 5 (n = 10), and 18 weeks (n = 20). Histological analysis of the collected tails was performed to study the fate of the collagen using Safranin O and fluorescent stains: FITC for collagen and DAPI for host cells. Disc degeneration was assessed histologically according to the established Han grading system and NP size according to cross-sectional area measurements. Half of the tails from the 18-week time point underwent mechanical testing. We performed stress relaxation tests to measure the stiffness of the discs and their ability to pressurize. Damping quality was measured using frequency sweep tests. Explanted segments were exposed to sinusoidal strains at variable frequencies ranging from 0.01 to 0.3 Hz at amplitudes of ± 10% strain. Results Disc Degeneration Over 18 weeks RF cross-linked groups retained significantly more NP tissue in the disc space than the uncross-linked and the control groups according to NP voxel count and histological NP cross-section measurements (p < 0.05) (Fig. 1). There was no significant difference in NP size between 0.5 and 0.75 mM groups (p > 0.05). Both groups retained approximately 70% of NP size when compared with healthy discs and maintained a disc height of over 80% compared with the prepuncture state. Both cross-linked groups showed no significant histological degenerative changes (Fig. 1). The NP showed regular cellularity and matrix morphology. The AF maintained its organization and lamellar structure. Both groups showed similar damping qualities according to frequency sweep tests as well as a similar stiffness and ability to pressurize when compared with healthy adjacent discs. After 18 weeks, the uncross-linked collagen and the control groups showed no residual NP tissue in the disc space, and terminal degenerative changes on histological sections and according to radiological assessments (Fig. 1). MRIs showed black discs, X-rays showed disc height drop of approximately 50% (Fig. 1). Histological sections showed extruded NP tissue in the paravertebral space. The NP in the disc space was replaced by connective tissue. The IVDs of both groups were stiffer then healthy discs and showed reduced damping qualities. Fate of the Collagen At 1 week, the cross-linked collagen had formed a macroscopically visible patch sealing the defect. FITC-stained cross-linked collagen showed a tight, zipper-like adhesion to the host AF and surrounding scar tissue. At 2 weeks, the collagen gel was infiltrated with host fibroblasts which remodeled the gel into organized fibrous tissue. At 5 weeks, cross-linked collagen formed a fibrous cap which bridged the fibers of the outer anulus and thereby repaired it. This fibrous cap consisted of fibroblasts embedded in a dense fibrous matrix. This fibrous cap was still visible after 18 weeks. In the uncross-linked and control groups, there was no repair tissue visible at the outer part of the AF at any time point. The disrupted annular fibers infiltrated the surrounding scar tissue. Conclusion Annular defects induced by needle puncture do not heal over 18 weeks in the described model and lead to extrusion of nuclear tissue, with subsequent degenerative changes affecting the entire IVD. HDC is capable of repairing annular defects in a rat-tail model, thereby inhibiting these changes and improving the mechanical properties of injured discs. The repair mechanism appears to be induced by host fibroblasts which infiltrate and remodel the injected collagen gel. Annular tissue repair restored the mechanical properties of the injured discs. Although the presented results are promising, experiments in larger animals with a spinal axial load similar to humans will be necessary to evaluate true clinical potential. Disclosure of Interest None declared