Brandon Borde

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

Injectable, High Density Collagen Gels for Annulus Fibrosus Repair: An In Vitro Rat Tail Model

A herniated intervertebral disc often causes back pain when disc tissue is displaced through a damaged annulus fibrosus. Currently, the only methods available for annulus fibrosus repair involve mechanical closure of defect, which does little to address biological healing in the damaged tissue. Collagen hydrogels are injectable and have been used to repair annulus defects in vivo. In this study, high-density collagen hydrogels at 5, 10, and 15 mg/mL were used to repair defects made to intact rat caudal intervertebral discs in vitro. A group of gels at 15 mg/mL were also cross-linked with riboflavin at 0.03 mM, 0.07 mM, or 0.10 mM. These cross-linked, high-density collagen gels maintained their presence in the defect under loading and contributed positively to the mechanical response of damaged discs. Discs exhibited increases to 95% of undamaged effective equilibrium and instantaneous moduli as well as up to fourfold decreases in effective hydraulic permeability from the damaged discs. These data suggest that high-density collagen gels may be effective at restoring mechanical function of injured discs as well as potential vehicles for the delivery of biological agents such as cells or growth factors that may aid in the repair of the annulus fibrosus.

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.

Clinical doses of radiation reduce collagen matrix stiffness

Cells receive mechanical cues from their extracellular matrix (ECM), which direct migration, differentiation, apoptosis, and in some cases, the transition to a cancerous phenotype. As a result, there has been significant research to develop methods to tune the mechanical properties of the ECM and understand cell-ECM dynamics more deeply. Here, we show that ionizing radiation can reduce the stiffness of an ex vivo tumor and an in vitro collagen matrix. When non-irradiated cancer cells were seeded in the irradiated matrix, adhesion, spreading, and migration were reduced. These data have ramifications for both in vitro and in vivo systems. In vitro, these data suggest that irradiation may be a method that could be used to create matrices with tailored mechanical properties. In vivo, these suggest that therapeutic doses of radiation may alter tissue mechanics directly.

In vivo annular repair using high-density collagen gel seeded with annulus fibrosus cells

OBJECTIVE The aim is assessing the in vivo efficacy of annulus fibrosus (AF) cells seeded into collagen by enhancing the reparative process around annular defects and preventing further degeneration in a rat-tail model. SUMMARY OF BACKGROUND DATA Treating disc herniation with discectomy may relieve the related symptoms but does not address the underlying pathology. The persistent annular defect may lead to re-herniation and further degeneration. We recently demonstrated that riboflavin crosslinked high-density collagen gels (HDC) can facilitate annular repair in vivo. METHODS 42 rats, tail disc punctured with an 18-gauge needle, were divided into 3 groups: untreated (n = 6), injected with crosslinked HDC (n = 18), and injected with AF cell-laden crosslinked HDC (n = 18). Ovine AF cells were mixed with HDC gels prior to injection. X-rays and MRIs were conducted over 5 weeks, determining disc height index (DHI), nucleus pulposus (NP) size, and hydration. Histological assessments evaluated the viability of implanted cells and degree of annular repair. RESULTS Although average DHIs of both HDC gel groups were higher than those of the puncture control group at 5 weeks, the retention of disc height, NP size and hydration at 1 and 5 weeks was significant for the cellular group compared to the punctured, and at 5 weeks to the acellular group. Histological assessment indicated that AF cell-laden HDC gels have accelerated reparative sealing compared to acellular HDC gels. CONCLUSIONS AF cell-laden HDC gels have the ability of better repairing annular defects than acellular gels after needle puncture. STATEMENT OF SIGNIFICANCE This project addresses the compelling demand of a sufficient treatment strategy for degenerative disc disease (DDD) perpetuated by annulus fibrosus (AF) injury, a major cause of morbidity and burden to health care systems. Our study is designed to answer the question of whether injectable, photo-crosslinked, high density collagen gels can seal defects in the annulus fibrosus of rats and prevent disc degeneration. Furthermore, we investigated whether the healing of AF defects will be enhanced by the delivery of AF cells (fibrochondrocytes) to these defects. The use of cell-laden collagen gels in spine surgery holds promise for a wide array of applications, from current discectomy procedures to future nucleus pulposus reparative therapies, and our group is excited about this potential.

Annular Repair Using High-density Collagen Gel with Riboflavin Crosslinkage: Preliminary Data from an in vivo Ovine Model

Introduction Discectomy of herniated intervertebral discs (IVDs) successfully alleviates neurological symptoms but fails to repair the iatrogenic annulotomy. This persistent annular defect post-discectomy is associated with increased risk of reherniation, progressive IVD degeneration, and chronic low back pain. Previously we demonstrated the ability of high-density collagen (HDC) gels to facilitate annular repair in a rodent model.1,3 This study aims to use HDC gel in an ovine model which has previously been used as a surrogate for the human spine.2 The goal of this study is create an effective ovine model for a herniated nucleus pulposus through a generated annular defect and to show that our HDC helps induce annular repair reduces the degenerative changes. Methods Sheep have had intervertebral discs violated to create an annular defect and induce a herniated nucleus pulposus. These sheep were laid in a lateral position with their right-side up. A longitudinal incision was made from their most caudal rib to their iliac crest 1-cm ventral to their transverse processes. Using a lateral approach, the soft tissue and muscle were dissected of their transverse processes to expose their vertebral bodies. The annular fibrosis (AF) defect was created with a 3.2 mm drill inserted to a 9–11 mm depth and then an 18-gauge needle was inserted to a maximum depth of 9–10 mm to injure the nucleus pulposus (NP) and induce a herniation through the annular defect. The violated IVDs were then randomized to either treatment with riboflavin-crosslinked HDC gel or no treatment. Thus far 4 IVDs were randomized to the control group and 4 IVDs to the treatment group. An in vivo MRI and post-mortem MRI were performed 6 weeks after the surgery to assess the degree of herniation of the nucleus pulposus. A post-mortem X-Ray was also obtained 6 weeks after the surgery to assess for any changes in disc height, which is a surrogate for degenerative disc changes. Results Of the 8 intervertebral disc which we induced an annular defect, we were able to induce nucleus pulposus herniation through the annulotomy in all levels as in the example in Fig. 1. Our preliminary data so far shows no difference in the disc height index of the intervertebral discs that received HDC versus those that received no treatment at 6 weeks after the surgery. The average disc height index of the treated intervertebral discs was 0.78 ± 0.005, 0.77 ± 0.004 for the untreated, puncture-only intervertebral discs, and 0.82 ± 0.006 for the healthy discs. Conclusion Lumbar discectomy to treat disc herniation is one of the most commonly performed spinal procedures,1 with an estimated 300,000 cases per year in the United States.2 A ~5% to 15% of discectomy cases result in a reherniation of their nucleus pulposus3 through the annulotomy and is associated with compromised patients outcomes and increased health care costs.4 Our preliminary data show that we are able to effectively use an ovine model to simulate a herniated nucleus pulposus via a lateral approach to the lumbar vertebral. Experiments are ongoing to analyze the disc levels using T2 mapping on MRI to assess changes in the NP hydration and overall area as our group has done previously in the rat-tail model.5 If HDC is found to be efficacious in inducing annular repair in a large mammal model, the next step would be to progress to clinical trials in humans. Fig. 1 L4/5 Disc level on sheep A showing annulus fibrosis (blue arrow), nucleus pulposus (star), and the annular defect with herniating nucleus pulposus (red arrow). References Laus M, Bertoni F, Bacchini P, Alfonso C, Giunti A. Recurrent lumbar disc herniation: what recurs? (A morphological study of recurrent disc herniation). Chir Organi Mov 1993;78(3):147–154 Carragee EJ, Han MY, Suen PW, Kim D. Clinical outcomes after lumbar discectomy for sciatica: the effects of fragment type and anular competence. J Bone Joint Surg Am 2003;85-A(1):102–108 Swartz KR, Trost GR. Recurrent lumbar disc herniation. Neurosurg Focus 2003;15(3):E10 Ambrossi GLG, McGirt MJ, Sciubba DM, et al. Recurrent lumbar disc herniation after single-level lumbar discectomy: incidence and health care cost analysis. Neurosurgery 2009;65(3):574–578, discussion 578 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