The Application of Mesenchymal Stromal Cells and Their Homing Capabilities to Regenerate the Intervertebral Disc.

Chronic low back pain (LBP) remains a challenging condition to treat, and especially to cure. If conservative treatment approaches fail, the current "gold standard" for intervertebral disc degeneration (IDD)-provoked back pain is spinal fusion. However, due to its invasive and destructive nature, the focus of orthopedic research related to the intervertebral disc (IVD) has shifted more towards cell-based therapeutic approaches. They aim to reduce or even reverse the degenerative cascade by mimicking the human body's physiological healing system. The implementation of progenitor and/or stem cells and, in particular, the delivery of mesenchymal stromal cells (MSCs) has revealed significant potential to cure the degenerated/injured IVD. Over the past decade, many research groups have invested efforts to find ways to utilize these cells as efficiently and sustainably as possible. This narrative literature review presents a summary of achievements made with the application of MSCs for the regeneration of the IVD in recent years, including their preclinical and clinical applications. Moreover, this review presents state-of-the-art strategies on how the homing capabilities of MSCs can be utilized to repair damaged or degenerated IVDs, as well as their current limitations and future perspectives.

In Situ Cell Signalling of the Hippo-YAP/TAZ Pathway in Reaction to Complex Dynamic Loading in an Intervertebral Disc Organ Culture.

Recently, a dysregulation of the Hippo-YAP/TAZ pathway has been correlated with intervertebral disc (IVD) degeneration (IDD), as it plays a key role in cell survival, tissue regeneration, and mechanical stress. We aimed to investigate the influence of different mechanical loading regimes, i.e., under compression and torsion, on the induction and progression of IDD and its association with the Hippo-YAP/TAZ pathway. Therefore, bovine IVDs were assigned to one of four different static or complex dynamic loading regimes: (i) static, (ii) "low-stress", (iii) "intermediate-stress", and (iv) "high-stress" regime using a bioreactor. After one week of loading, a significant loss of relative IVD height was observed in the intermediate- and high-stress regimes. Furthermore, the high-stress regime showed a significantly lower cell viability and a significant decrease in glycosaminoglycan content in the tissue. Finally, the mechanosensitive gene CILP was significantly downregulated overall, and the Hippo-pathway gene MST1 was significantly upregulated in the high-stress regime. This study demonstrates that excessive torsion combined with compression leads to key features of IDD. However, the results indicated no clear correlation between the degree of IDD and a subsequent inactivation of the Hippo-YAP/TAZ pathway as a means of regenerating the IVD.

Spheroid-Like Cultures for Expanding Angiopoietin Receptor-1 (aka. Tie2) Positive Cells from the Human Intervertebral Disc.

Lower back pain is a leading cause of disability worldwide. The recovery of nucleus pulposus (NP) progenitor cells (NPPCs) from the intervertebral disc (IVD) holds high promise for future cell therapy. NPPCs are positive for the angiopoietin-1 receptor (Tie2) and possess stemness capacity. However, the limited Tie2+ NPC yield has been a challenge for their use in cell-based therapy for regenerative medicine. In this study, we attempted to expand NPPCs from the whole NP cell population by spheroid-formation assay. Flow cytometry was used to quantify the percentage of NPPCs with Tie2-antibody in human primary NP cells (NPCs). Cell proliferation was assessed using the population doublings level (PDL) measurement. Synthesis and presence of extracellular matrix (ECM) from NPC spheroids were confirmed by quantitative Polymerase Chain Reaction (qPCR), immunostaining, and microscopy. Compared with monolayer, the spheroid-formation assay enriched the percentage of Tie2+ in NPCs' population from ~10% to ~36%. Moreover, the spheroid-formation assay also inhibited the proliferation of the Tie2- NPCs with nearly no PDL. After one additional passage (P) using the spheroid-formation assay, NPC spheroids presented a Tie2+ percentage even further by ~10% in the NPC population. Our study concludes that the use of a spheroid culture system could be successfully applied to the culture and expansion of tissue-specific progenitors.

Conditioned Medium of Intervertebral Disc Cells Inhibits Osteo-Genesis on Autologous Bone-Marrow-Derived Mesenchymal Stromal Cells and Osteoblasts.

Low back pain (LBP) is associated with the degeneration of human intervertebral discs (IVDs). Despite progress in the treatment of LBP through spinal fusion, some cases still end in non-fusion after the removal of the affected IVD tissue. In this study, we investigated the hypothesis that the remaining IVD cells secrete BMP inhibitors that are sufficient to inhibit osteogenesis in autologous osteoblasts (OBs) and bone marrow mesenchymal stem cells (MSCs). A conditioned medium (CM) from primary human IVD cells in 3D alginate culture was co-cultured with seven donor-matched OB and MSCs. After ten days, osteogenesis was quantified at the transcript level using qPCR to measure the expression of bone-related genes and BMP antagonists, and at the protein level by alkaline phosphatase (ALP) activity. Additionally, cells were evaluated histologically using alizarin red (ALZR) staining on Day 21. For judging ALP activity and osteogenesis, the Noggin expression in samples was investigated to uncover the potential causes. The results after culture with the CM showed significantly decreased ALP activity and the inhibition of the calcium deposit formation in alizarin red staining. Interestingly, no significant changes were found among most bone-related genes and BMP antagonists in OBs and MSCs. Noteworthy, Noggin was relatively expressed higher in human IVD cells than in autologous OBs or MSCs (relative to autologous OB, the average fold change was in 6.9, 10.0, and 6.3 in AFC, CEPC, and NPC, respectively; and relative to autologous MSC, the average fold change was 2.3, 3.4, and 3.2, in AFC, CEPC, and NPC, respectively). The upregulation of Noggin in residual human IVDs could potentially inhibit the osteogenesis of autologous OB and MSC, thus inhibiting the postoperative spinal fusion after discectomy surgery.

The bone morphogenetic protein 2 analogue L51P enhances spinal fusion in combination with BMP2 in an in vivo rat tail model.

Bone morphogenic protein 2 (BMP2) is known to induce osteogenesis and is applied clinically to enhance spinal fusion despite adverse effects. BMP2 needs to be used in high doses to be effective due to the presence of BMP2 inhibitors. L51P is a BMP2 analogue that acts by inhibition of BMP2 inhibitors. Here, we hypothesized that mixtures of BMP2 and L51P could achieve better spinal fusion outcomes regarding ossification. To test whether mixtures of both cytokines are sufficient to improve ossification, 45 elderly Wistar rats (of which 21 were males) were assigned to seven experimental groups, all which received spinal fusion surgery, including discectomy at the caudal 4-5 level using an external fixator and a porous ß-tricalcium phosphate (ßTCP) carrier. These ßTCP carriers were coated with varying concentrations of BMP2 and L51P. X-rays were taken immediately after surgery and again six and twelve weeks post-operatively. Histological sections and µCT were analyzed after twelve weeks. Spinal fusion was assessed using X-ray, µCT and histology according to the Bridwell scale by voxel-based quantification and a semi-quantitative histological score, respectively. The results were congruent across modalities and revealed high ossification for high-dose BMP2 (10 µg), while PBS induced no ossification. Low-dose BMP2 (1 µg) or 10 µg L51P alone did not induce relevant bone formation. However, all combinations of low-dose BMP2 with L51P (1 µg + 1/5/10 µg) were able to induce similar ossificationas high-dose BMP2. These results are of high clinical relevance, as they indicate L51P is sufficient to increase the efficacy of BMP2 and thus lower the required dose for spinal fusion. STATEMENT OF SIGNIFICANCE: Spinal fusion surgery is frequently applied to treat spinal pathologies. Bone Morphogenic Protein-2 (BMP2) has been approved by the U .S. Food and Drug Administration (FDA-) and by the "Conformité Européenne" (CE)-label. However, its application is expensive and high concentrations cause side-effects. This research targets the improvement of the efficacy of BMP2 in spinal fusion surgery.

Repairing Annulus Fibrosus Fissures Using Methacrylated Gellan Gum Combined with Novel Silk.

Intervertebral disc (IVD) herniation often causes severe pain and is frequently associated with the degeneration of the IVD. As the IVD degenerates, more fissures with increasing size appear within the outer region of the IVD, the annulus fibrosus (AF), favoring the initiation and progression of IVD herniation. For this reason, we propose an AF repair approach based on methacrylated gellan gum (GG-MA) and silk fibroin. Therefore, coccygeal bovine IVDs were injured using a biopsy puncher (⌀ 2 mm) and then repaired with 2% GG-MA as a filler material and sealed with an embroidered silk yarn fabric. Then, the IVDs were cultured for 14 days either without any load, static loading, or complex dynamic loading. After 14 days of culture, no significant differences were found between the damaged and repaired IVDs, except for a significant decrease in the IVDs' relative height under dynamic loading. Based on our findings combined with the current literature that focuses on ex vivo AF repair approaches, we conclude that it is likely that the repair approach did not fail but rather insufficient harm was done to the IVD.

Textile Design of an Intervertebral Disc Replacement Device from Silk Yarn.

Low back pain is often due to degeneration of the intervertebral discs (IVD). It is one of the most common age- and work-related problems in today's society. Current treatments are not able to efficiently restore the full function of the IVD. Therefore, the aim of the present work was to reconstruct the two parts of the intervertebral disc-the annulus fibrosus (AF) and the nucleus pulposus (NP)-in such a way that the natural structural features were mimicked by a textile design. Silk was selected as the biomaterial for realization of a textile IVD because of its cytocompatibility, biodegradability, high strength, stiffness, and toughness, both in tension and compression. Therefore, an embroidered structure made of silk yarn was developed that reproduces the alternating fiber structure of +30° and -30° fiber orientation found in the AF and mimics its lamellar structure. The developed embroidered ribbons showed a tensile strength that corresponded to that of the natural AF. Fiber additive manufacturing with 1 mm silk staple fibers was used to replicate the fiber network of the NP and generate an open porous textile 3D structure that may serve as a reinforcement structure for the gel-like NP.

Biomedical applications of silk and its role for intervertebral disc repair.

Intervertebral disc (IVD) degeneration (IDD) is the main contributor to chronic low back pain. To date, the present therapies mainly focus on treating the symptoms caused by IDD rather than addressing the problem itself. For this reason, researchers have searched for a suitable biomaterial to repair and/or regenerate the IVD. A promising candidate to fill this gap is silk, which has already been used as a biomaterial for many years. Therefore, this review aims first to elaborate on the different origins from which silk is harvested, the individual composition, and the characteristics of each silk type. Another goal is to enlighten why silk is so suitable as a biomaterial, discuss its functionalization, and how it could be used for tissue engineering purposes. The second part of this review aims to provide an overview of preclinical studies using silk-based biomaterials to repair the inner region of the IVD, the nucleus pulposus (NP), and the IVD's outer area, the annulus fibrosus (AF). Since the NP and the AF differ fundamentally in their structure, different therapeutic approaches are required. Consequently, silk-containing hydrogels have been used mainly to repair the NP, and silk-based scaffolds have been used for the AF. Although most preclinical studies have shown promising results in IVD-related repair and regeneration, their clinical transition is yet to come.

The effects of 3D culture on the expansion and maintenance of nucleus pulposus progenitor cell multipotency.

INTRODUCTION: Low back pain (LBP) is a global health concern. Increasing evidence implicates intervertebral disk (IVD) degeneration as a major contributor. In this respect, tissue-specific progenitors may play a crucial role in tissue regeneration, as these cells are perfectly adapted to their niche. Recently, a novel progenitor cell population was described in the nucleus pulposus (NP) that is positive for Tie2 marker. These cells have self-renewal capacity and in vitro multipotency potential. However, extremely low numbers of the NP progenitors limit the feasibility of cell therapy strategies. OBJECTIVE: Here, we studied the influence of the culture method and of the microenvironment on the proliferation rate and the differentiation potential of human NP progenitors in vitro. METHOD: Cells were obtained from human NP tissue from trauma patients. Briefly, the NP tissue cells were cultured in two-dimensional (2D) (monolayer) or three-dimensional (3D) (alginate beads) conditions. After 1 week, cells from 2D or 3D culture were expanded on fibronectin-coated flasks. Subsequently, expanded NP cells were then characterized by cytometry and tri-lineage differentiation, which was analyzed by qPCR and histology. Moreover, experiments using Tie2+ and Tie2- NP cells were also performed. RESULTS: The present study aims to demonstrate that 3D expansion of NP cells better preserves the Tie2+ cell populations and increases the chondrogenic and osteogenic differentiation potential compared to 2D expansion. Moreover, the cell sorting experiments reveal that only Tie2+ cells were able to maintain the pluripotent gene expression if cultured in 3D within alginate beads. Therefore, our results highly suggest that the maintenance of the cell's multipotency is mainly, but not exclusively, due to the higher presence of Tie2+ cells due to 3D culture. CONCLUSION: This project not only might have a scientific impact by evaluating the influence of a two-step expansion protocol on the functionality of NP progenitors, but it could also lead to an innovative clinical approach.

Effect of different cryopreservation media on human nucleus pulposus cells' viability and trilineage potential.

Introduction: Low back pain (LBP) is a significant cause of disability in many countries, affecting more than half a billion people worldwide. In the past, progenitor cells have been found within the nucleus pulposus (NP) of the human intervertebral disc (IVD). However, in the context of cell therapy, little is known about the effect of cryopreservation and expansion on here called "heterogenic" human NP cells (hNPCs), and whether commercially available cryopreservation media are more efficient than "commonly used" media in terms of cell viability. Materials: In this study, hNPCs from four trauma patients (age 40.5 ± 14.3 years) and two patients with degenerated IVDs (age 24 and 46 years), undergoing spinal surgery, were collected. To isolate hNPCs, the tissue was digested with a mild two-step protocol. After subsequent expansion, hNPCs at passages 2-5 were separated and either cryo-preserved for 1 week at -150°C or differentiated into osteogenic, adipogenic, or chondrogenic lineages for 21 days. Cryopreservation was performed with five different media to compare their effect on the cell's viability and differentiation potential. Cell viability was determined with flow cytometry using propidium iodide and the trilineage differentiation potential was assessed by quantitative polymerase chain reaction and histological analysis. Results: After 1 week of cryopreservation, the hNPC's cell viability was comparable for all conditions, that is, independent of the cryopreservation medium used (82.3 ± 0.8% of cell viability). Furthermore, hNPCs from trauma patients showed some evidence for adipogenic and chondrogenic differentiation and at lower levels, this and evidence of osteogenic differentiation could be confirmed with hNPCs from degenerated discs. Moreover, cryopreservation did not affect the cell's differentiation potential in the majority of the cases tested. Conclusion: "Commonly used" cryopreservation media seem to perform just as well as commercially available media in terms of cell viability and the overall maintenance of the hNPCs trilineage differentiation potential.

Establishment of a Novel Method for Spinal Discectomy Surgery in Elderly Rats in an In Vivo Spinal Fusion Model.

The rat model is a common model for intervertebral disc (IVD) and spinal research. However, complications remain challenging. Standard Operating Procedures (SOPs) are validated methods to minimize complications and improve safety and quality of studies. However, a SOP for rat spinal fusion surgery has been missing until now. Therefore, the aim of the study was to develop a SOP for spinal tail disc surgery in elderly Wistar rats (419.04 ± 54.84 g). An initial preoperative, intraoperative, and postoperative surgical setup, including specific anaesthesia and pain management protocols, was developed. Anaesthesia was induced by subcutaneous injection of a pre-mixture of fentanyl, midazolam, and medetomidin with the addition of 0.5% isoflurane in oxygen and caudal epidural analgesia. The surgery itself consisted of the fixation of a customized external ring fixator with ⌀ 0.8 mm Kirschner wires at the proximal rat tail and a discectomy and replacement with bone morphogenetic protein coated beta-tricalcium-phosphate carrier. The postoperative setup included heating, analgesia with buprenorphine, and meloxicam, as well as special supplementary food. Anaesthesia, surgery, and pain management were sufficient. In the presented optimized SOP, no animals developed any complications. A SOP for spinal surgery in elderly rats in an in vivo spinal fusion model was developed successfully. This novel protocol can improve transparency, reproducibility, and external validity in experimental rat spinal surgery experiments.

Non-viral Gene Delivery Methods for Bone and Joints.

Viral carrier transport efficiency of gene delivery is high, depending on the type of vector. However, viral delivery poses significant safety concerns such as inefficient/unpredictable reprogramming outcomes, genomic integration, as well as unwarranted immune responses and toxicity. Thus, non-viral gene delivery methods are more feasible for translation as these allow safer delivery of genes and can modulate gene expression transiently both in vivo, ex vivo, and in vitro. Based on current studies, the efficiency of these technologies appears to be more limited, but they are appealing for clinical translation. This review presents a summary of recent advancements in orthopedics, where primarily bone and joints from the musculoskeletal apparatus were targeted. In connective tissues, which are known to have a poor healing capacity, and have a relatively low cell-density, i.e., articular cartilage, bone, and the intervertebral disk (IVD) several approaches have recently been undertaken. We provide a brief overview of the existing technologies, using nano-spheres/engineered vesicles, lipofection, and in vivo electroporation. Here, delivery for microRNA (miRNA), and silencing RNA (siRNA) and DNA plasmids will be discussed. Recent studies will be summarized that aimed to improve regeneration of these tissues, involving the delivery of bone morphogenic proteins (BMPs), such as BMP2 for improvement of bone healing. For articular cartilage/osteochondral junction, non-viral methods concentrate on targeted delivery to chondrocytes or MSCs for tissue engineering-based approaches. For the IVD, growth factors such as GDF5 or GDF6 or developmental transcription factors such as Brachyury or FOXF1 seem to be of high clinical interest. However, the most efficient method of gene transfer is still elusive, as several preclinical studies have reported many different non-viral methods and clinical translation of these techniques still needs to be validated. Here we discuss the non-viral methods applied for bone and joint and propose methods that can be promising in clinical use.