Dynamic loading leads to increased metabolic activity and spatial redistribution of viable cell density in nucleus pulposus tissue.

Background: Nucleus pulposus (NP) cell density is orchestrated by an interplay between nutrient supply and metabolite accumulation. Physiological loading is essential for tissue homeostasis. However, dynamic loading is also believed to increase metabolic activity and could thereby interfere with cell density regulation and regenerative strategies. The aim of this study was to determine whether dynamic loading could reduce the NP cell density by interacting with its energy metabolism. Methods: Bovine NP explants were cultured in a novel NP bioreactor with and without dynamic loading in milieus mimicking the pathophysiological or physiological NP environment. The extracellular content was evaluated biochemically and by Alcian Blue staining. Metabolic activity was determined by measuring glucose and lactate in tissue and medium supernatants. A lactate-dehydrogenase staining was performed to determine the viable cell density (VCD) in the peripheral and core regions of the NP. Results: The histological appearance and tissue composition of NP explants did not change in any of the groups. Glucose levels in the tissue reached critical values for cell survival (≤0.5 mM) in all groups. Lactate released into the medium was increased in the dynamically loaded compared to the unloaded groups. While the VCD was unchanged on Day 2 in all regions, it was significantly reduced in the dynamically loaded groups on Day 7 (p ≤ 0.01) in the NP core, which led to a gradient formation of VCD in the group with degenerated NP milieu and dynamic loading (p ≤ 0.05). Conclusion: It was demonstrated that dynamic loading in a nutrient deprived environment similar to that during IVD degeneration can increase cell metabolism to the extent that it was associated with changes in cell viability leading to a new equilibrium in the NP core. This should be considered for cell injections and therapies that lead to cell proliferation for treatment of IVD degeneration.

A bovine nucleus pulposus explant culture model.

Low back pain is a global health problem that is frequently caused by intervertebral disc degeneration (IVDD). Sulfated glycosaminoglycans (sGAGs) give the healthy nucleus pulposus (NP) a high fixed charge density (FCD), which creates an osmotic pressure that enables the disc to withstand high compressive forces. However, during IVDD sGAG reduction in the NP compromises biomechanical function. The aim of this study was to develop an ex vivo NP explant model with reduced sGAG content and subsequently investigate biomechanical restoration via injection of proteoglycan-containing notochordal cell-derived matrix (NCM). Bovine coccygeal NP explants were cultured in a bioreactor chamber and sGAG loss was induced by chondroitinase ABC (chABC) and cultured for up to 14 days. Afterwards, diurnal loading was studied, and explant restoration was investigated via injection of NCM. Explants were analyzed via histology, biochemistry, and biomechanical testing via stress relaxation tests and height measurements. ChABC injection induced dose-dependent sGAG reduction on Day 3, however, no dosing effects were detected after 7 and 14 days. Diurnal loading reduced sGAG loss after injection of chABC. NCM did not show an instant biomechanical (equilibrium pressure) or biochemical (FCD) restoration, as the injected fixed charges leached into the medium, however, NCM stimulated proliferation and increased Alcian blue staining intensity and matrix organization. NCM has biological repair potential and biomaterial/NCM combinations, which could better entrap NCM within the NP tissue, should be investigated in future studies. Concluding, chABC induced progressive, time-, dose- and loading-dependent sGAG reduction that led to a loss of biomechanical function. Keywords biomechanics | intervertebral disc | matrix degradation | low back pain | proteoglycans.

A comprehensive tool box for large animal studies of intervertebral disc degeneration.

Preclinical studies involving large animal models aim to recapitulate the clinical situation as much as possible and bridge the gap from benchtop to bedside. To date, studies investigating intervertebral disc (IVD) degeneration and regeneration in large animal models have utilized a wide spectrum of methodologies for outcome evaluation. This paper aims to consolidate available knowledge, expertise, and experience in large animal preclinical models of IVD degeneration to create a comprehensive tool box of anatomical and functional outcomes. Herein, we present a Large Animal IVD Scoring Algorithm based on three scales: macroscopic (gross morphology, imaging, and biomechanics), microscopic (histological, biochemical, and biomolecular analyses), and clinical (neurologic state, mobility, and pain). The proposed algorithm encompasses a stepwise evaluation on all three scales, including spinal pain assessment, and relevant structural and functional components of IVD health and disease. This comprehensive tool box was designed for four commonly used preclinical large animal models (dog, pig, goat, and sheep) in order to facilitate standardization and applicability. Furthermore, it is intended to facilitate comparison across studies while discerning relevant differences between species within the context of outcomes with the goal to enhance veterinary clinical relevance as well. Current major challenges in pre-clinical large animal models for IVD regeneration are highlighted and insights into future directions that may improve the understanding of the underlying pathologies are discussed. As such, the IVD research community can deepen its exploration of the molecular, cellular, structural, and biomechanical changes that occur with IVD degeneration and regeneration, paving the path for clinically relevant therapeutic strategies.

Optimization of hyaluronic acid-tyramine/silk-fibroin composite hydrogels for cartilage tissue engineering and delivery of anti-inflammatory and anabolic drugs.

Injury of articular cartilage leads to an imbalance in tissue homeostasis, and due to the poor self-healing capacity of cartilage the affected tissue often exhibits osteoarthritic changes. In recent years, injectable and highly tunable composite hydrogels for cartilage tissue engineering and drug delivery have been introduced as a desirable alternative to invasive treatments. In this study, we aimed to formulate injectable hydrogels for drug delivery and cartilage tissue engineering by combining different concentrations of hyaluronic acid-tyramine (HA-Tyr) with regenerated silk-fibroin (SF) solutions. Upon enzymatic crosslinking, the gelation and mechanical properties were characterized over time. To evaluate the effect of the hydrogel compositions and properties on extracellular matrix (ECM) deposition, bovine chondrocytes were embedded in enzymatically crosslinked HA-Tyr/SF composites (in further work abbreviated as HA/SF) or HA-Tyr hydrogels. We demonstrated that all hydrogel formulations were cytocompatible and could promote the expression of cartilage matrix proteins allowing chondrocytes to produce ECM, while the most prominent chondrogenic effects were observed in hydrogels with HA20/SF80 polymeric ratios. Unconfined mechanical testing showed that the compressive modulus for HA20/SF80 chondrocyte-laden constructs was increased almost 10-fold over 28 days of culture in chondrogenic medium which confirmed the superior production of ECM in this hydrogel compared to other hydrogels in this study. Furthermore, in hydrogels loaded with anabolic and anti-inflammatory drugs, HA20/SF80 hydrogel showed the longest and the most sustained release profile over time which is desirable for the long treatment duration typically necessary for osteoarthritic joints. In conclusion, HA20/SF80 hydrogel was successfully established as a suitable injectable biomaterial for cartilage tissue engineering and drug delivery applications.

Enhanced BMP-2-Mediated Bone Repair Using an Anisotropic Silk Fibroin Scaffold Coated with Bone-like Apatite.

The repair of large bone defects remains challenging and often requires graft material due to limited availability of autologous bone. In clinical settings, collagen sponges loaded with excessive amounts of bone morphogenetic protein 2 (rhBMP-2) are occasionally used for the treatment of bone non-unions, increasing the risk of adverse events. Therefore, strategies to reduce rhBMP-2 dosage are desirable. Silk scaffolds show great promise due to their favorable biocompatibility and their utility for various biofabrication methods. For this study, we generated silk scaffolds with axially aligned pores, which were subsequently treated with 10× simulated body fluid (SBF) to generate an apatitic calcium phosphate coating. Using a rat femoral critical sized defect model (CSD) we evaluated if the resulting scaffold allows the reduction of BMP-2 dosage to promote efficient bone repair by providing appropriate guidance cues. Highly porous, anisotropic silk scaffolds were produced, demonstrating good cytocompatibility in vitro and treatment with 10× SBF resulted in efficient surface coating. In vivo, the coated silk scaffolds loaded with a low dose of rhBMP-2 demonstrated significantly improved bone regeneration when compared to the unmineralized scaffold. Overall, our findings show that this simple and cost-efficient technique yields scaffolds that enhance rhBMP-2 mediated bone healing.

Characterization of biomaterials intended for use in the nucleus pulposus of degenerated intervertebral discs.

Biomaterials for regeneration of the intervertebral disc must meet complex requirements conforming to biological, mechanical and clinical demands. Currently no consensus on their characterization exists. It is crucial to identify parameters and their method of characterization for accurate assessment of their potential efficacy, keeping in mind the translation towards clinical application. This review systematically analyses the characterization techniques of biomaterial systems that have been used for nucleus pulposus (NP) restoration and regeneration. Substantial differences in the approach towards assessment became evident, hindering comparisons between different materials with respect to their suitability for NP restoration and regeneration. We have analysed the current approaches and identified parameters necessary for adequate biomaterial characterization, with the clinical goal of functional restoration and biological regeneration of the NP in mind. Further, we provide guidelines and goals for their measurement. STATEMENT OF SIGNIFICANCE: Biomaterials intended for restoration of regeneration of the nucleus pulposus within the intervertebral disc must meet biological, biomechanical and clinical demands. Many materials have been investigated, but a lack of consensus on which parameters to evaluate leads to difficulties in comparing materials as well as mostly partial characterization of the materials in question. A gap between current methodology and clinically relevant and meaningful characterization is prevalent. In this article, we identify necessary methods and their implementation for complete biomaterial characterization in the context of clinical applicability. This will allow for a more unified approach to NP-biomaterials research within the field as a whole and enable comparative analysis of novel materials yet to be developed.

Effect of fluid dynamics on decellularization efficacy and mechanical properties of blood vessels.

Decellularization of blood vessels is a promising approach to generate native biomaterials for replacement of diseased vessels. The decellularization process affects the mechanical properties of the vascular graft and thus can have a negative impact for in vivo functionality. The aim of this study was to determine how detergents under different fluid dynamics affects decellularization efficacy and mechanical properties of the vascular graft. We applied a protocol utilizing 1% TritonX, 1% Tributyl phosphate (TnBP) and DNase on porcine vena cava. The detergents were applied to the vessels under different conditions; static, agitation and perfusion with 3 different perfusion rates (25, 100 and 400 mL/min). The decellularized grafts were analyzed with histological, immunohistochemical and mechanical tests. We found that decellularization efficacy was equal in all groups, however the luminal ultrastructure of the static group showed remnant cell debris and the 400 mL/min perfusion group showed local damage and tearing of the luminal surface. The mechanical stiffness and maximum tensile strength were not influenced by the detergent application method. In conclusion, our results indicate that agitation or low-velocity perfusion with detergents are preferable methods for blood vessel decellularization.