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

ABSTRACT: Animal in vivo study.To test the capability of high-density collagen gel to repair annular defects.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.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.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.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.N/A.

SUBMITTER: Grunert P

PROVIDER: S-EPMC4523893 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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

Grunert Peter P Borde Brandon H BH Hudson Katherine D KD Macielak Michael R MR Bonassar Lawrence J LJ Härtl Roger R

Spine 20140201 3

<h4>Study design</h4>Animal in vivo study.<h4>Objective</h4>To test the capability of high-density collagen gel to repair annular defects.<h4>Summary of background data</h4>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 ...[more]

PMID: 24253790

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Project description:Study designOvine in vivo study.ObjectiveTo perform lateral approach lumbar surgery in an ovine model to administer an injectable riboflavin cross-linked high-density collagen (HDC) gel and to assess its ability to mitigate intervertebral disc (IVD) degeneration after induced annulus fibrosus (AF) injury.Summary of background dataBiological-based injectable gels have shown efficacy in restoring biomechanical, radiographic, and histological parameters in IVD-injured animal models. Riboflavin cross-linked HDC gel has previously demonstrated retention of nucleus pulposus (NP) tissue, reduced loss of disc height, and prevention of terminal cellular degenerative changes in rat-tail spines. However, this biological therapy has never been tested in large animal models.MethodsForty lumbar IVDs were accessed from eight sheep via lateral approach surgery. IVDs were randomly assigned to healthy control, injury and HDC treatment, or negative control with injury and no treatment. IVD injury was carried out using a drill-bit through the AF followed by needle puncture of the NP. Sheep were followed for 16 weeks and underwent qualitative/quantitative magnetic resonance imaging, x-ray, and histological analyses of collagen and proteoglycan content.ResultsThe lateral approach to the ovine lumbar spine to deliver HDC gel proved to be safe and reproducible. IVDs treated with the HDC gel revealed less degenerative changes at the microscopic level based on AF and NP histology. However, mean Pfirrmann grade, T2 relaxation time, NP voxel size, and disc height index were not significantly different between the two injury groups.ConclusionInjectable HDC gel can be administered safely via lateral approach surgery in an ovine AF injury model. IVDs treated with HDC gel demonstrated less degeneration at the microscopic level though radiographic changes were slight when comparing treated to untreated IVDs. Future studies will need to elucidate the role of injury technique and time frame for follow-up in correlating histological and radiographical outcomes.Level of evidenceN /A.

Project description:ObjectiveTo evaluate how well different magnitudes of compression-induced degenerative changes using a bent rat tail model simulated human lumbar lordosis. It has been shown that compression plays an important role in intervertebral disc degeneration (IDD).MethodsSprague-Dawley rats (n = 25) were instrumented with a special compressive apparatus that was used to bend the intervertebral disc between the 8th and the 10th caudal vertebral bodies using two Kirschner wires inserted percutaneously into the middle of two tail vertebrae. Then, rats were divided into five different static compression loads (control, sham, 1.8 N, 4.5 N, and 7.2 N). The degeneration of the discs was evaluated by magnetic resonance imaging (MRI), histology, gene expression of anabolism and catabolism after 2 weeks. We used the signal characteristics of the disc in T2-weighted MRI to reflect the changes caused by degeneration as this is the most relevant and clinically recognized way to assess IDD. Pfirrmann classification was used to classify disc images. The tail discs from C8-9 and C9-10 with their two adjacent half vertebrae were carefully cut out and decalcified. Then the sections were paraffin-embedded and cut into 5-μm sections by histotome. Finally, they were stained with Safranin O-Fast Green and hematoxylin, and hematoxylin and eosin, respectively. Images were taken using a microscope and staining and compression-induced changes were assessed by a Masuda's grading scale. The relative expression levels of mRNA encoding rat anabolic genes and catabolic genes were evaluated by real-time reverse transcription (RT)-polymerase chain reaction (PCR). The mRNA expression fold change of the target gene was calculated using the 2-ΔΔCt method in the loaded and unloaded disc.ResultsAs the loading magnitude increased, static compression produced a significantly progressive decrease in nucleus intensity on T2-weighted MRI, a decrease of aggrecan and Type II collagen, an increase in Matrix metallopeptidase-3 (MMP-3) and MMP-13 expressions, and a histomorphological degeneration. The sham group had a score of 1.4 ± 0.3, the 1.8 N group had a score of 2.4 ± 0.3, the 4.5 N group had a score of 3.2 ± 0.3, and the 7.2 N group had a score of 4.4 ± 0.3, which was based on the Pfirrmann classification score, in which the control group had a score of 1. These results demonstrated that the sham group was not significantly different from the control group. Histological analysis showed that in the loaded disc, the size of the nucleus was reduced and that the annular layer was disorganized. Based on the Masuda grading scale, scores were as follows: for the control group, 3.8 ± 0.35; sham, 4.2 ± 0.35; 1.8 N, 5.4 ± 0.35; 4.5 N, 7.6 ± 0.35; and 7.2 N, 10 ± 0.35. The gene expression was divided into the following: anabolic genes (aggrecan, collagen type1-α1, and collagen type2-α1) and catabolic genes (MMP-3 and MMP-13). Aggrecan and collagen type 2 were, respectively, downregulated from 0.42 ± 0.04 to 0.21 ± 0.04 and from 0.93 ± 0.06 to 0.17 ± 0.06 as the magnitude of compression increased, whereas collagen type 1 was significantly upregulated, from 2.49 ± 0.19 to 4.40 ± 0.19, when compared with the control group (from 1.8 to 7.2 N, P < 0.05). Catabolic genes MMP-3 and MMP-13 were significantly upregulated in all experimental groups (P < 0.05, MMP-3: from 1.46 ± 0.18 to 3.44 ± 0.18; MMP-13: from 1.19 ± 0.12 to 2.82 ± 0.13); however, MMP-13 exhibited no significant changes but tended to be upregulated when compared with the 1.8 N group with the 4.5 N group.ConclusionsDifferent stresses led to different processes of degenerative changes, the concave disc degenerating more severely as stress gradually increased.

Project description:AIM:To explore the methods of hepatocytes culture in a collagen gel mixture or between double layers of collagen sandwich configuration and to examine the functional and cytomorphological characteristics of cultured hepatocytes. METHODS:A two-step collagenase perfusion technique was used to isolate the hepatocytes from Wistar rats or newborn Chinese experimental piglets. The isolated hepatocytes were cultured in a collagen gel mixture or between double layers of collagen sandwich configuration respectively. The former was that rat hepatocytes were mixed with type I rat tail collagen solution till gelled, and the medium was added onto the gel. The latter was that swine hepatocytes were seeded on a plate precoated with collagen gel for 24 h, then another layer of collagen gel was overlaid, resulting in a sandwich configuration. The cytomorphological characteristics, albumin secretion, and LDH-release of the hepatocytes cultured in these two models were examined. RESULTS:Freshly isolated rat hepatocytes were successfully mixed and fixed in collagen gel, and cultured in the gel condition. During the culture period, the urea synthesized and secreted by rat hepatocytes was detected throughout the period. Likewise, newborn experimental piglet hepatocytes were successfully fixed between the double layers of collagen gel, forming a sandwich configuration. Within a week of culture, the albumin secreted by swine hepatocytes was detected by SDS/PAGE analysis. The typical cytomorphological characteristics of the hepatocytes cultured by the above two culture models were found under a phase-contrast microscope. There was little LDH-release during the culture period. CONCLUSION:Both collagen gel mixture and double layers of collagen sandwich configuration can provide cultural conditions much closer to in vivo environment, and are helpful for maintaining specific hepatic functions and cytomorphological characteristics. A collagen gel mixture culture may be more eligible for the study of bioartificial livers.

Dataset Information

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

Publications

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

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