Chemonucleolysis combined with dynamic loading for inducing degeneration in bovine caudal intervertebral discs.

Chemonucleolysis has become an established method of producing whole organ culture models of intervertebral disc (IVD) degeneration. However, the field needs more side-by-side comparisons of the degenerative effects of the major enzymes used in chemonucleolysis towards gaining a greater understanding of how these organ culture models mimic the wide spectrum of characteristics observed in human degeneration. In the current work we induced chemonucleolysis in bovine coccygeal IVDs with 100 µL of papain (65 U/mL), chondroitinase ABC (chABC, 5 U/mL), or collagenase II (col'ase, 0.5 U/mL). Each enzyme was applied in a concentration projected to produce moderate levels of degeneration. After 7 days of culture with daily dynamic physiological loading (0.02-0.2 MPa, 0.2 Hz, 2 h), the cellular, biochemical and histological properties of the IVDs were evaluated in comparison to a PBS-injected control. Papain and collagenase, but not chABC, produced macroscopic voids in the tissues. Compared to day 0 intact IVDs, papain induced the greatest magnitude glycosaminoglycan (GAG) loss compared to chABC and col'ase. Papain also induced the greatest height loss (3%), compared to 0.7%, 1.2% and 0.4% for chABC, col'ase, and PBS, respectively. Cell viability in the region adjacent to papain and PBS-injection remained at nearly 100% over the 7-day culture period, whereas it was reduced to 60%-70% by chABC and col'ase. Generally, enzyme treatment tended to downregulate gene expression for major ECM markers, type I collagen (COL1), type II collagen (COL2), and aggrecan (ACAN) in the tissue adjacent to injection. However, chABC treatment induced an increase in COL2 gene expression, which was significant compared to the papain treated group. In general, papain and col'ase treatment tended to recapitulate aspects of advanced IVD degeneration, whereas chABC treatment captured aspects of early-stage degeneration. Chemonucleolysis of whole bovine IVDs is a useful tool providing researchers with a robust spectrum of degenerative changes and can be utilized for examination of therapeutic interventions.

A Novel Fiber-Reinforced Poroviscoelastic Bovine Intervertebral Disc Finite Element Model for Organ Culture Experiment Simulations.

Intervertebral disc (IVD) degeneration and methods for repair and regeneration have commonly been studied in organ cultures with animal IVDs under compressive loading. With the recent establishment of a novel multi-axial organ culture system, accurate predictions of the global and local mechanical response of the IVD are needed for control system development and to aid in experiment planning. This study aimed to establish a finite element model of bovine IVD capable of predicting IVD behavior at physiological and detrimental load levels. A finite element model was created based on the dimensions and shape of a typical bovine IVD used in the organ culture. The nucleus pulposus (NP) was modeled as a neo-Hookean poroelastic material and the annulus fibrosus (AF) as a fiber-reinforced poroviscoelastic material. The AF consisted of 10 lamella layers and the material properties were distributed in the radial direction. The model outcome was compared to a bovine IVD in a compressive stress-relaxation experiment. A parametric study was conducted to investigate the effect of different material parameters on the overall IVD response. The model was able to capture the equilibrium response and the relaxation response at physiological and higher strain levels. Permeability and elastic stiffness of the AF fiber network affected the overall response most prominently. The established model can be used to evaluate the response of the bovine IVD at strain levels typical for organ culture experiments, to define relevant boundaries for such studies, and to aid in the development and use of new multi-axial organ culture systems.

Physiological and degenerative loading of bovine intervertebral disc in a bioreactor: A finite element study of complex motions.

Intervertebral disc (IVD) degeneration and regenerative therapies are commonly studied in organ-culture experiments with uniaxial compressive loading. Recently, in our laboratory, we established a bioreactor system capable of applying loads in six degrees-of-freedom (DOF) to bovine IVDs, which replicates more closely the complex multi-axial loading of the IVD in vivo. However, the magnitudes of loading that are physiological (able to maintain cell viability) or mechanically degenerative are unknown for load cases combining several DOFs. This study aimed to establish physiological and degenerative levels of maximum principal strains and stresses in the bovine IVD tissue and to investigate how they are achieved under complex load cases related to common daily activities. The physiological and degenerative levels of maximum principal strains and stresses were determined via finite element (FE) analysis of bovine IVD subjected to experimentally established physiological and degenerative compressive loading protocols. Then, complex load cases, such as a combination of compression + flexion + torsion, were applied on the FE-model with increasing magnitudes of loading to discover when physiological and degenerative tissue strains and stresses were reached. When applying 0.1 MPa of compression and ±2-3° of flexion and ±1-2° of torsion the investigated mechanical parameters remained at physiological levels, but with ±6-8° of flexion in combination with ±2-4° of torsion, the stresses in the outer annulus fibrosus (OAF) exceeded degenerative levels. In the case of compression + flexion + torsion, the mechanical degeneration likely initiates at the OAF when loading magnitudes are high enough. The physiological and degenerative magnitudes can be used as guidelines for bioreactor experiments with bovine IVDs.

Toward the Next Generation of Spine Bioreactors: Validation of an Ex Vivo Intervertebral Disc Organ Model and Customized Specimen Holder for Multiaxial Loading.

A new generation of bioreactors with integrated six degrees of freedom (6 DOF) aims to mimic more accurately the natural intervertebral disc (IVD) load. We developed and validated in a biological and mechanical study a specimen holder and corresponding ex vivo IVD organ model according to the bioreactor requirements for multiaxial loading and a long-term IVD culture. IVD height changes and cell viability were compared between the 6 DOF model and the standard 1 DOF model throughout the 3 weeks of cyclic compressive loading in the uniaxial bioreactor. Furthermore, the 6 DOF model and holder were loaded for 9 days in the multiaxial bioreactor under development using the same conditions, and the IVDs were evaluated for cell viability. The interface of the IVD model and specimen holder, enhanced with fixation screws onto the bone, was tested in compression, torsion, lateral bending, and tension. Additionally, critical motions such as tension and bending were assessed for a combination of side screws and top screws or side screws and adhesive. The 6 DOF model loaded in the uniaxial bioreactor maintained similar cell viability in the IVD regions as the 1 DOF model. The viability was high after 2 weeks throughout the whole IVD and reduced by more than 30% in the inner annulus fibrous after 3 weeks. Similarly, the IVDs remained highly viabile when cultured in the multiaxial bioreactor. In both models, IVD height changes after loading were in the range of typical physiological conditions. When differently directed motions were applied, the holder-IVD interface remained stable under hyper-physiological loading levels using a side screw approach in compression and torsion and the combination of side and top screws in tension and bending. We thus conclude that the developed holding system is mechanically reliable and biologically compatible for application in a new generation of multiaxial bioreactors.

Hyaluronic acid-based interpenetrating network hydrogel as a cell carrier for nucleus pulposus repair.

Hyaluronic acid (HA) is a key component of the intervertebral disc (IVD) that is widely investigated as an IVD biomaterial. One persisting challenge is introducing materials capable of supporting cell encapsulation and function, yet with sufficient mechanical stability. In this study, a hybrid interpenetrating polymer network (IPN) was produced as a non-covalent hydrogel, based on a covalently cross-linked HA (HA-BDDE) and HA-poly(N-isopropylacrylamide) (HA-pNIPAM). The hybrid IPN was investigated for its physicochemical properties, with histology and gene expression analysis to determine matrix deposition in vitro and in an ex vivo model. The IPN hydrogel displayed cohesiveness for at least one week and rheological properties resembling native nucleus pulposus (NP) tissue. When implanted in an ex vivo IVD organ culture model, the IPN supported cell viability, phenotype expression of encapsulated NP cells and IVD matrix production over four weeks under physiological loading. Overall, our results indicate the therapeutic potential of this HA-based IPN hydrogel for IVD regeneration.

Morphological and Molecular Evaluation of the Tissue Repair following Nasal Septum Biopsy in a Sheep Model.

OBJECTIVE: Nasal septal pathologies requiring surgical intervention are common in the population. Additionally, nasal chondrocytes are becoming an important cell source in cartilage tissue engineering strategies for the repair of articular cartilage lesions. These procedures damage the nasal septal cartilage whose healing potential is limited due to its avascular, aneural, and alymphatic nature. Despite the high incidence of various surgical interventions that affect septum cartilage, limited nasal cartilage repair characterizations have been performed to date. METHODS: To evaluate the healing of the nasal septum cartilage perforation, a septal biopsy was performed in 14 sheep. Two and 6 months later, the tissue formed on the place of perforation was explanted and compared with the native tissue. Tissue morphology, protein and gene expression of explanted tissue was determined using histological, immunohistochemical and real-time quantitative polymerase chain reaction analysis. RESULTS: Tissue formed on the defect site, 2 and 6 months after the biopsy was characterized as mostly connective tissue with the presence of fibroblastic cells. This newly formed tissue contained no glycosaminoglycans and collagen type II but was positively stained for collagen type I. Cartilage-specific genes COL2, AGG, and COMP were significantly decreased in 2- and 6-month samples compared with the native nasal cartilage. Levels of COL1, COL4, and CRABP1 genes specific for perichondrium and connective tissue were higher in both test group samples in comparison with native cartilage. CONCLUSIONS: Newly formed tissue was not cartilage but rather fibrous tissue suggesting the role of perichondrium and mucosa in tissue repair after nasal septum injury.

Nasal Chondrocyte-Based Engineered Grafts for the Repair of Articular Cartilage "Kissing" Lesions: A Pilot Large-Animal Study.

BACKGROUND: Bipolar or "kissing" cartilage lesions formed on 2 opposite articular surfaces of the knee joint are commonly listed as exclusion criteria for advanced cartilage therapies. PURPOSE: To test, in a pilot large-animal study, whether autologous nasal chondrocyte (NC)-based tissue engineering, recently introduced for the treatment of focal cartilage injuries, could provide a solution for challenging kissing lesions. STUDY DESIGN: Controlled laboratory study. METHODS: Osteochondral kissing lesions were freshly introduced into the knee joints of 26 sheep and covered with NC-based grafts with a low or high hyaline-like extracellular matrix; a control group was treated with a cell-free scaffold collagen membrane (SCA). The cartilage repair site was assessed at 6 weeks and 6 months after implantation by histology, immunohistochemistry, and magnetic resonance imaging evaluation. RESULTS: NC-based grafts, independently of their composition, induced partial hyaline cartilage repair with stable integrity in surrounding healthy tissue at 6 months after treatment. The SCA repaired cartilage to a similar degree to that of NC-based grafts. CONCLUSION: Kissing lesion repair, as evidenced in this sheep study, demonstrated the feasibility of the treatment of complex cartilage injuries with advanced biological methods. However, the potential advantages of an NC-based approach over a cell-free approach warrant further investigations in a more relevant preclinical model. CLINICAL RELEVANCE: NC-based grafts currently undergoing phase II clinical trials have a high potential to replace existing cartilage therapies that show significant limitations in the quality and reproducibility of the repair method. We have brought this innovative concept to the next level by addressing a new clinical indication.