Combined nucleus pulposus augmentation and annulus fibrosus repair prevents acute intervertebral disc degeneration after discectomy.

Tissue-engineered approaches for the treatment of early-stage intervertebral disc degeneration have shown promise in preclinical studies. However, none of these therapies has been approved for clinical use, in part because each therapy targets only one aspect of the intervertebral disc's composite structure. At present, there is no reliable method to prevent intervertebral disc degeneration after herniation and subsequent discectomy. Here, we demonstrate the prevention of degeneration and maintenance of mechanical function in the ovine lumbar spine after discectomy by combining strategies for nucleus pulposus augmentation using hyaluronic acid injection and repair of the annulus fibrosus using a photocrosslinked collagen patch. This combined approach healed annulus fibrosus defects, restored nucleus pulposus hydration, and maintained native torsional and compressive stiffness up to 6 weeks after injury. These data demonstrate the necessity of a combined strategy for arresting intervertebral disc degeneration and support further translation of combinatorial interventions to treat herniations in the human spine.

Imaging the local biochemical content of native and injured intervertebral disc using Fourier transform infrared microscopy.

Alterations to the biochemical composition of the intervertebral disc (IVD) are hallmarks of aging and degeneration. Methods to assess biochemical content, such as histology, immunohistochemistry, and spectrophotometric assays, are limited in their ability to quantitatively analyze the spatial distribution of biochemical components. Fourier transform infrared (FTIR) microscopy is a biochemical analysis method that can yield both quantitative and high-resolution data about the spatial distribution of biochemical components. This technique has been largely unexplored for use with the IVD, and existing methods use complex analytical techniques that make results difficult to interpret. The objective of the present study is to describe an FTIR microscopy method that has been optimized for imaging the collagen and proteoglycan content of the IVD. The method was performed on intact and discectomized IVDs from the sheep lumbar spine after 6 weeks in vivo in order to validate FTIR microscopy in healthy and degenerated IVDs. FTIR microscopy quantified collagen and proteoglycan content across the entire IVD and showed local changes in biochemical content after discectomy that were not observed with traditional histological methods. Changes in collagen and proteoglycans content were found to have strong correlations with Pfirrmann grades of degeneration. This study demonstrates how FTIR microscopy is a valuable research tool that can be used to quantitatively assess the local biochemical composition of IVDs in development, degeneration, and repair.

Proteoglycan removal by chondroitinase ABC improves injectable collagen gel adhesion to annulus fibrosus.

Intervertebral disc (IVD) herniations are currently treated with interventions that leave the IVD with persistent lesions prone to further herniations. Annulus fibrosus (AF) repair has become of interest as a method to seal defects in the IVD and prevent reherniation, but this requires strong adhesion of the implanted biomaterial to the native AF tissue. Our group has previously developed a high-density collagen (HDC) gel for AF repair and tested its efficacy in vivo, but its adhesion to the AF could be improved. Increased cell adhesion to cartilage has previously been reported through chondroitinase ABC (ChABC) digestion, which removes proteoglycans and increases access to cell binding motifs. Such approaches could also increase biomaterial adhesion to tissue, but the effects of ChABC digestion on AF have yet to be investigated. In this study, ovine AF tissue was digested with either 10 U/mL ChABC or saline for up to 10 min and the effect of this treatment on collagen adhesion between AF tissue samples was investigated by histology and mechanical testing in a lap-shear configuration. ChABC digestion removed proteoglycans within the AF in a time-dependent fashion and enhanced adhesion of the HDC gel to the AF. ChABC digestion increased the elastic toughness and total shear energy of the HDC gel-AF interface by 88% and 46% respectively. ChABC treatment enhanced the adhesion of the HDC gel to the AF without significantly decreasing native AF cell viability. Thus, ChABC digestion is a viable method to improve adhesion of biomaterials for AF repair. STATEMENT OF SIGNIFICANCE: Intervertebral disc herniations are currently treated with interventions that leave persistent lesions in the annulus fibrosus that are prone to further herniations. Annular repair is a promising method to seal lesions and prevent reherniation, but requires strong adhesion of the implanted biomaterial to native annulus fibrosus. Since large proteoglycans like aggrecan occupy regions of the extracellular matrix between collagen fibers in the annulus fibrosus, we hypothesized that removing proteoglycans via chondroitinase digestion would increase the adhesion of annular repair hydrogels. This investigation demonstrated that chondroitinase removed proteoglycans within annulus fibrosus tissue, enhanced the interaction of an injected collagen gel with the native tissue, and mechanically improved adhesion between the collagen gel and annulus fibrosus. This is the first study of its kind to evaluate the biochemical and mechanical effects of short-term chondroitinase digestion on annulus fibrosus tissue.

Mesenchymal Stem Cell-Seeded High-Density Collagen Gel for Annular Repair: 6-Week Results From In Vivo Sheep Models.

BACKGROUND: Our group has previously demonstrated in vivo annulus fibrosus repair in animal models using an acellular, riboflavin crosslinked, high-density collagen (HDC) gel. OBJECTIVE: To assess if seeding allogenic mesenchymal stem cells (MSCs) into this gel yields improved histological and radiographic benefits in an in vivo sheep model of annular injury. METHODS: Fifteen lumbar intervertebral discs (IVDs) were randomized into 4 groups: intact, injury only, injury + acellular gel treatment, or injury + MSC-seeded gel treatment. Sheep were sacrificed at 6 wk. Disc height index (DHI), Pfirrmann grade, nucleus pulposus area, and T2 relaxation time (T2-RT) were calculated for each IVD and standardized to healthy controls from the same sheep. Quantitative histological assessment was also performed using the Han scoring system. RESULTS: All treated IVDs retained gel plugs on gross assessment and there were no adverse perioperative complications. The MSC-seeded gel treatment group demonstrated statistically significant improvement over other experimental groups in DHI (P = .002), Pfirrmann grade (P < .001), and T2-RT (P = .015). There was a trend for greater Han scores in the MSC-seeded gel-treated discs compared with injury only and acellular gel-treated IVDs (P = .246). CONCLUSION: MSC-seeded HDC gel can be delivered into injured IVDs and maintained safely in live sheep to 6 wk. Compared with no treatment and acellular HDC gel, our data show that MSC-seeded HDC gel improves outcomes in DHI, Pfirrmann grade, and T2-RT. Histological analysis shows improved annulus fibrosus and nucleus pulposus reconstitution and organization over other experimental groups as well.

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.

Biologic Annulus Fibrosus Repair: A Review of Preclinical In Vivo Investigations.

Lower back pain, the leading cause of workplace absences and disability, is often attributed to intervertebral disc degeneration, in which nucleus pulposus (NP) herniates through lesions in the annulus fibrosus (AF) and impinges on the spinal cord and surrounding nerves. Surgeons remove extruded NP via discectomy when indicated by local/radicular pain supported by radiographic evidence; however, current interventions do not alter the underlying disease or seal the AF. The reported rates of recurrent herniation or pain following discectomy cases range from 5% to 25%, which has pushed spine research in recent years toward annular repair and closure strategies. Synthetic implants designed to mechanically seal the AF have been subject to large animal and clinical trials, with limited success in preventing recurrent herniation. Like gold standard interventions, purely mechanical devices fail to promote tissue integration, long-term healing, or restore native biomechanical function to the spine. Biological repair strategies utilizing principles of tissue engineering have demonstrated success in overcoming the inadequacies of current interventions and mechanical implants, yet, none has reached clinical or proof-of-concept trials in humans. In this review, we will discuss annular repair strategies promoting biological healing that have been implemented in small and large animal models in vivo, and ways to enhance the efficacy of these treatments.

Annulus Fibrosus Repair Using High-Density Collagen Gel: An In Vivo Ovine Model.

STUDY DESIGN: Ovine in vivo study. OBJECTIVE: To 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 DATA: Biological-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. METHODS: Forty 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. RESULTS: The 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. CONCLUSION: Injectable 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 EVIDENCE: N /A.

Depth-Dependent Out-of-Plane Young's Modulus of the Human Cornea.

Purpose/Aim: Despite their importance in accurate mechanical modeling of the cornea, the depth-dependent material properties of the cornea have only been partially elucidated. In this work, we characterized the depth-dependent out-of-plane Young's modulus of the central and peripheral human cornea with high spatial resolution. MATERIALS AND METHODS: Central and peripheral corneal buttons from human donors were subjected to unconfined axial compression followed by stress relaxation for 30 min. Sequences of fluorescent micrographs of full-thickness corneal buttons were acquired throughout the experiment to enable tracking of fluorescently labeled stromal keratocyte nuclei and measurements of depth-dependent infinitesimal strains. The nominal (gross) out-of-plane Young's modulus and drained Poisson's ratio for each whole specimen was computed from the equilibrium stress and overall tissue deformation. The depth-dependent (local) out-of-plane Young's modulus was computed from the equilibrium stress and local tissue strain based on an anisotropic model (transverse isotropy). RESULTS: The out-of-plane Young's modulus of the cornea exhibited a strong dependence on in-plane location (peripheral versus central cornea), but not depth. The depth-dependent out-of-plane Young's modulus of central and peripheral specimens ranged between 72.4-102.4 kPa and 38.3-58.9 kPa. The nominal out-of-plane Young's modulus was 87 ± 41.51 kPa and 39.9 ± 15.28 kPa in the central and peripheral cornea, while the drained Poisson's ratio was 0.05 ± 0.02 and 0.07 ± 0.04. CONCLUSIONS: The out-of-plane Young's modulus of the cornea is mostly independent of depth, but not in-plane location (i.e. central vs. peripheral). These results may help inform more accurate finite element computer models of the cornea.

Initial investigation of individual and combined annulus fibrosus and nucleus pulposus repair ex vivo.

Novel tissue engineered and biomaterial approaches to treat intervertebral disc (IVD) degeneration focus on single aspects of the progressive disease and hence are insufficient repair strategies. In this study, annulus fibrosus (AF) and nucleus pulposus (NP) biomaterial repair strategies were used individually and combined to treat IVD degeneration modeled in ex vivo rat-tail motion segments by annulotomy and nucleotomy. An injectable riboflavin cross-linked high-density collagen gel patched defects in the AF, while NP repair consisted of injections of a modified hyaluronic acid (HA) hydrogel. Qualitative imaging showed the annulotomy and nucleotomy successfully herniated NP material, while the HA NP injections restored intact NP morphology and the collagen AF patches sealed AF defects. Assessed by quantitative T2 magnetic resonance imaging, combined repair treatments yielded disc hydration not significantly different than intact hydration, while AF and NP repairs alone only restored ∼1/3 of intact hydration. Mechanical testing showed NP injections alone recovered on average ∼35% and ∼40% of the effective instantaneous and equilibrium moduli. The combined treatment comprising biomaterial AF and NP repair was effective at increasing NP hydration from NP repair alone, however HA injections alone are sufficient to improve mechanical properties. STATEMENT OF SIGNIFICANCE: Intervertebral disc degeneration affects an estimated 90% of individuals throughout their life, and is a candidate pathology for tissue engineered repair. The current standard of clinical care reduces spinal articulation and leads to further degeneration along the spine, hence great interest in a regenerative medicine therapy. Literature studies focused on biomaterial repair strategies for treating degenerated discs have partially restored native disc function, however no studies have reported the use of combined therapies to address multiple aspects of disc degeneration. This initial investigation screened injectable biomaterial repair strategies ex vivo, and through complementary outcome measures showed a combined therapy restores disc function better than individual approaches. This study is the first of its kind to address multiple aspects of disc degeneration, using clinically-oriented biomaterials in a well-established animal model.

The location- and depth-dependent mechanical response of the human cornea under shear loading.

PURPOSE: To characterize the depth-dependent shear modulus of the central and peripheral human cornea along the superior-inferior and nasal-temporal directions with a high spatial resolution. METHODS: Cylindrical explants from the central and peripheral corneas of 10 human donors were subjected to a 5% shear strain along the superior-inferior and nasal-temporal directions using a microscope-mounted mechanical testing device. Depth-dependent shear strain and shear modulus were computed through force measurements and displacement tracking. RESULTS: The shear modulus G of the human cornea varied continuously with depth, with a maximum occurring roughly 25% of the way from the anterior surface to the posterior surface. G also varied with direction in the superior region and (at some depths) was significantly higher for superior-inferior shear loading. In the anterior half of the cornea, the shear modulus along the nasal-temporal direction (GNT) did not vary with location; however, the superior region had significantly higher GNT in posterior cornea. In contrast, the shear modulus along the superior-inferior direction (GSI) was independent of location at all depths. CONCLUSIONS: This study demonstrates that the peak shear modulus of the human cornea occurs at a substantial distance within the corneal stroma. Depth-dependent differences between central and peripheral cornea possibly reflect the location-dependent mechanical environment of the cornea. Moreover, the cornea is not a transverse isotropic material, and must be characterized by more than a single shear modulus due to its dependence on loading direction. The material properties measured in this study are critical for developing accurate mechanical models to predict the vision-threatening morphological changes that can occur in the cornea.