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INTRODUCTION: Low back pain (LBP) is a global health concern that affects as many as 75–80% of people during their lifetime. Although the causes of LBP are multifactorial, increasing evidence implicates intervertebral disc (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, the progenitor cell population was described in the nucleus pulposus (NP) of the IVD. These cells, positive for the Tie2 marker, have self-renewal capacity and in vitro multipotency potential. However, extremely low numbers of the NP progenitors limit the feasibility of cell therapy strategies. Here, we study the influence of the culture method and of the microenvironment on the human NP progenitors and their differentiation potential in vitro. METHODS:Cells were obtained from human NP tissue from trauma patients undergoing spinal surgery. Briefly, after mild overnight digestion, the NP tissue cells were cultured in 2D (monolayer) or 3D (alginate beads) conditions with medium supplemented in ascorbic acid. After 2 weeks, cells from 2D or 3D culture were expanded on fibronectin-coating flasks with medium supplemented in FGF-2 to mimic the native microenvironment of NP cells. Subsequently, expanded NP cells were then characterized by cytometry (CD105, CD90, CD73, CD45, CD34, and Tie2) and tri-lineage differentiation, which was analyzed by qPCR and histology. RESULTS: Cytometry analysis, after 2D- or 3D-expansion showed the presence of 0.1 % and 78.2 % of Tie2+ NP progenitors, respectively. Concerning the chondrogenic differentiation assay, the detection of glycosaminoglycans in the culture medium was drastically increased for 3D-expanded cells (11-fold) vs 2D-expanded cells. Moreover, the relative gene expression of collagen type 2 and aggrecan was also increased (600-fold and 2-fold, respectively). Regarding osteogenic differentiation assay, relative gene expression for osteopontin increased for 3D- (150-fold) vs 2D-expanded cells. However, no difference was observed between 2D and 3D expansion for the adipogenic differentiation assay. DISCUSSION & CONCLUSIONS: The present study shows that 3D expansion of NP cells better preserves the progenitor's cells population and increases the chondrogenic and osteogenic differentiation potential compared to 2D expansion. This project not only has a scientific impact by evaluating the role of native physiological niches on the functionality of NP progenitors but could also lead to an innovative clinical approach with cell therapy for IVD regeneration and repair. Acknowledgments: Financial support was received from iPSpine H2020 project #825925.

Osteoarthritis (OA) and chronic low back pain (CLBP) caused by intervertebral disc (IVD) degeneration are joint diseases that have become major causes for loss of quality of life worldwide. Despite the unmet need, effective treatments other than invasive, and often ineffective, surgery are lacking. Systemic administration of drugs entails suboptimal local drug exposure in the articular joint and IVD. This review provides an overview of the potency of biomaterial-based drug delivery systems as novel treatment modality, with a focus on the biological effects of drug release systems that have reached translation at the level of in vivo models and relevant ex vivo models. These studies have shown encouraging results of biomaterial-based local delivery of several types of drugs, mostly inhibitors of inflammatory cytokines or other degenerative factors. Prevention of inflammation and degeneration and pain relief was achieved, although mainly in small animal models, with interventions applied at an early disease stage. Less convincing data were obtained with the delivery of regenerative factors. Multidisciplinary efforts towards tackling the discord between in vitro and in vivo release, combined with adaptations in the regulatory landscape may be needed to enhance safe and expeditious introduction of more and more effective controlled release-based treatments with the OA and CLBP patients.

INTRODUCTION:Low back pain (LBP) is a major cause of disability in many countries, affecting more than half a billion people worldwide. A promising and future-oriented approach to treat LBP is cell therapy using stem or progenitor cells. Over the last decade, cells positive for Tie2 and mesenchymal stromal cell markers have been found within the nucleus pulposus (NP) of human intervertebral discs (IVD). However, little is known about the effect of expansion and cryo-preservation on here called “heterogenic” human NP cells (hNPCs) and their stemness in a context of cell therapy for regeneration of the IVD. Therefore, the aim of our study was to expand hNPCs whilst investigating their differentiation potential before and after cryo-preservation and to find and optimal approach to cryo-preserve them. METHODS:HNPCs from three human trauma patients (32, 55 and 69 years old) undergoing spinal surgery were isolated with a mild two-step digestion protocol. After subsequent expansion until complete confluency, hNPCs were separated and then differentiated into osteogenic, adipogenic or chondrogenic lineages for 21 days or were cryo-preserved for one week at -150°C with five cryo-preservation media (90% fetal bovine serum and 10% dimethyl sulfoxide (DMSO); 90% low glucose medium + 10% DMSO and three commercially available media) to compare their effect on the cell’s viability and differentiation potential. Cell viability was determined with trypan blue and by cytometry employing propidium iodide. The differentiation potential was assessed using histological analysis and qPCR. RESULTS:HNPCs cultured in osteogenic medium showed a significant (p

INTRODUCTION:Low back pain is the leading cause of disability worldwide (1). Nevertheless, the mechanism of the intervertebral disc (IVD) degeneration is still not clear. In this context, the nucleus pulposus (NP) and more precisely NP progenitor cells (NPPCs) present in the IVD, positive for angiopoietin-1 receptor (aka. Tie2) display multipotent and stem capacity (2,3). In this study, the first aim was to determine whether spheroid formation in suspension-culture will increase the amount/percentage of NPPCs during the expansion compared to traditional monolayer culture. The second aim of this study was to investigate if the percentage of NPPCs will be enriched even further by the resuspension of the spheroid-like cultured cells (=1st generation) and reformation of those spheroids one more time (= 2nd generation). METHODS:Human NP tissues from trauma patients (N=3) were obtained with written ethical consent and isolated by a two-step digestion protocol (3). The NP cells were resuspended and frozen at -150°C after reaching confluence of passage 0. At passage 1, NP cells were seeded in standard or ultra-low attachment tissue culture flasks with 2.5 ng/ml FGF-2 in low glucose - DMEM (supplemented with 10 % FBS). Flow cytometry was used to analyze and quantify the percentage of NPPCs using Tie2 antibody. We defined the spheroids formed after passage 1 NPCs as 1st generation spheroid. We obtained the 2nd generation spheroids by resuspending the 1st-generation-spheroid and reassembly. The NPCs from 1st and 2nd spheroid were quantified by CFU-assay. RESULTS:As a result, the percentage of NPPCs in monolayer culture condition was reaching 7 ± 2 % (Mean±SEM), however, in the 1st and 2nd generation spheroids culture condition, we were observing 20 ± 10 % and 28 ± 6% of Tie2+ cells, respectively. Concerning the CFU-assay, the NPCs from the 2nd generation spheroid formed 30 CFU-S per 1,000 cells, which were twice more CFU-S compared to the 1st generation spheroid. DISCUSSION & CONCLUSIONS:From these data we conclude than the spheroid-like formation of NPCs would be a more efficient method for expansion and enrichment of NPPCs than monolayer expansion in a context of future cell therapy. Acknowledgements:Financial support was received from iPSpine H2020 project under grant agreement #825925 and China Scholarship Council to X.Z.

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.

Cellular senescence, that is, the withdrawal from the cell cycle, combined with the acquirement of the senescence associated secretory phenotype has important roles during health and disease and is essential for tissue remodeling during embryonic development. Osteoclasts are multinucleated cells, responsible for bone resorption, and cell cycle arrest during osteoclastogenesis is well recognized. Therefore, the aim of this study was to investigate whether these cells should be considered senescent and to assess the influence of hypoxia on their potential senescence status. Osteoclastogenesis and bone resorption capacity of osteoclasts, cultured from CD14+ monocytes, were evaluated in two oxygen concentrations, normoxia (21% O2 ) and hypoxia (5% O2 ). Osteoclasts were profiled by using specific staining for proliferation and senescence markers, qPCR of a number of osteoclast and senescence-related genes and a bone resorption assay. Results show that during in vitro osteoclastogenesis, osteoclasts heterogeneously obtain a senescent phenotype. Furthermore, osteoclastogenesis was delayed at hypoxic compared to normoxic conditions, without negatively affecting the bone resorption capacity. It is concluded that osteoclasts can be considered senescent, although senescence is not uniformly present in the osteoclast population. Hypoxia negatively affects the expression of some senescence markers. Based on the direct relationship between senescence and osteoclastogenesis, it is tempting to hypothesize that contents of the so-called senescence associated secretory phenotype (SASP) not only play a functional role in matrix resorption, but also may regulate osteoclastogenesis.

The socioeconomic burden of chronic back pain related to intervertebral disc (IVD) disease is high and current treatments are only symptomatic. Minimally invasive strategies that promote biological IVD repair should address this unmet need. Notochordal cells (NCs) are replaced by chondrocyte-like cells (CLCs) during IVD maturation and degeneration. The regenerative potential of NC-secreted substances on CLCs and mesenchymal stromal cells (MSCs) has already been demonstrated. However, identification of these substances remains elusive. Innovatively, this study exploits the regenerative NC potential by using healthy porcine NC-derived matrix (NCM) and employs the dog as a clinically relevant translational model. NCM increased the glycosaminoglycan and DNA content of human and canine CLC aggregates and facilitated chondrogenic differentiation of canine MSCs in vitro. Based on these results, NCM, MSCs and NCM+MSCs were injected in mildly (spontaneously) and moderately (induced) degenerated canine IVDs in vivo and, after six months of treatment, were analyzed. NCM injected in moderately (induced) degenerated canine IVDs exerted beneficial effects at the macroscopic and MRI level, induced collagen type II-rich extracellular matrix production, improved the disc height, and ameliorated local inflammation. MSCs exerted no (additive) effects. In conclusion, NCM induced in vivo regenerative effects on degenerated canine IVDs. NCM may, comparable to demineralized bone matrix in bone regeneration, serve as 'instructive matrix', by locally releasing growth factors and facilitating tissue repair. Therefore, intradiscal NCM injection could be a promising regenerative treatment for IVD disease, circumventing the cumbersome identification of bioactive NC-secreted substances.

The optimal release profile of locally delivered bone morphogenetic protein-2 (BMP-2) for safe and effective clinical application is unknown. In this work, the effect of differential BMP-2 release on bone formation was investigated using a novel biomaterial oligo[(polyethylene glycol) fumarate] bis[2-(methacryloyloxy) ethyl] phosphate hydrogel (OPF-BP) containing poly(lactic-co-glycolic acid) microspheres. Three composite implants with the same biomaterial chemistry and structure but different BMP-loading methods were created: BMP-2 encapsulated in microspheres (OPF-BP-Msp), BMP-2 encapsulated in microspheres and adsorbed on the phosphorylated hydrogel (OPF-BP-Cmb), and BMP-2 adsorbed on the phosphorylated hydrogel (OPF-BP-Ads). These composites were compared with the clinically used BMP-2 carrier, Infuse® absorbable collagen sponge (ACS). Differential release profiles of bioactive BMP-2 were achieved by these composites. In a rat subcutaneous implantation model, OPF-BP-Ads and ACS generated a large BMP-2 burst release (>75%), whereas a more sustained release was seen for OPF-BP-Msp and OPF-BP-Cmb (~25% and 50% burst, respectively). OPF-BP-Ads generated significantly more bone than did all other composites, and the bone formation was 12-fold higher than that of the clinically used ACS. Overall, this study clearly shows that BMP-2 burst release generates more subcutaneous bone than do sustained release in OPF-BP-microsphere composites. Furthermore, composites should not only function as a delivery vehicle but also provide a proper framework to achieve appropriate bone formation.

Hydrogel-based 3D cell cultures are an emerging strategy for the regeneration of cartilage. In an attempt to regenerate dysfunctional intervertebral discs, nucleus pulposus (NP) cells can be cultured in hydrogels of various kinds and physical properties. Stiffness sensing through focal adhesions is believed to direct chondrogenesis, but the mechanisms by which this works are largely unknown. In this study we compared focal adhesion formation and glycosaminoglycan (GAG) deposition by NP cells in a range of hydrogels. Using a focal adhesion kinase (FAK) inhibitor, we demonstrated that focal adhesion signaling is involved in the response of NP cells in hydrogels that contain integrin binding sites (i.e. methacrylated gelatin (gelMA) and type II collagen), but not in hydrogels deplete from integrin binding sites such as alginate and agarose, or CD44-binding hydrogels based on hyaluronic acid. As a result of FAK inhibition we observed enhanced proteoglycan production in gelMA, but decreased production in type II collagen hydrogels, which could be explained by alteration in cell fate as supported by the increase in the adipogenic marker peroxisome proliferator-activated receptor gamma (PPARy). Furthermore, GAG deposition was inversely proportional to polymer concentration in integrin-binding gelMA, while no direct relationship was found for the non-integrin binding gels alginate and agarose. This corroborates our finding that focal adhesion formation plays an important role in NP cell response to its surrounding matrix. Statement of Significance Biomaterials are increasingly being investigated for regenerative medicine applications, including regeneration of the nucleus pulposus. Cells interact with their environment and are influenced by extracellular matrix or polymer properties. Insight in these interactions can improve regeneration and helps to understand degeneration processes. The role of focal adhesion formation in the regenerative response of nucleus pulposus cells is largely unknown. Therefore, the relation between materials, stiffness and focal adhesion formation is studied here.

Parathyroid hormone‐related protein (PTHrP) and hedgehog signaling play an important role in chondrocyte development, (hypertrophic) differentiation, and/or calcification, but their role in intervertebral disc (IVD) degeneration is unknown. Better understanding their involvement may provide therapeutic clues for low back pain due to IVD degeneration. Therefore, this study aimed to explore the role of PTHrP and hedgehog proteins in postnatal canine and human IVDs during the aging/degenerative process. The expression of PTHrP, hedgehog proteins and related receptors was studied during the natural loss of the notochordal cell (NC) phenotype during IVD maturation using tissue samples and de‐differentiation in vitro and degeneration by real‐time quantitative polymerase chain reaction (RT‐qPCR) and immunohistochemistry. Correlations between their expression and calcification levels (Alizarin Red S staining) were determined. In addition, the effect of PTHrP and hedgehog proteins on canine and human chondrocyte‐like cells (CLCs) was determined in vitro focusing on the propensity to induce calcification. The expression of PTHrP, its receptor (PTHR1) and hedgehog receptors decreased during loss of the NC phenotype. N‐terminal (active) hedgehog (Indian hedgehog/Sonic hedgehog) protein expression did not change during maturation or degeneration, whereas expression of PTHrP, PTHR1 and hedgehog receptors increased during IVD degeneration. Hedgehog and PTHR1 immunopositivity were increased in nucleus pulposus tissue with abundant vs no/low calcification. In vitro, hedgehog proteins facilitated calcification in CLCs, whereas PTHrP did not affect calcification levels. In conclusion, hedgehog and PTHrP expression is present in healthy and degenerated IVDs. Hedgehog proteins had the propensity to induce calcification in CLCs from degenerated IVDs, indicating that in the future, inhibiting hedgehog signaling could be an approach to inhibit calcification during IVD degeneration., Hedgehog and PTHrP expression decreases duringmaturation to increase back again in advanced stages of IVD degeneration; herein hedgehogs facilitate calcification

Biomaterials that act as both protein delivery vehicle and scaffold, can improve the safety and efficacy of bone morphogenetic protein-2 (BMP-2) for clinical applications. However, the optimal scaffold characteristics are not known. The osteoinductive and osteoconductive capacity of a fixed electrically charged surface is thus far unexplored. Therefore, in this study we aim to investigate the effect of different electrical states on BMP-2 induced bone formation in oligo[(polyethylene glycol) fumarate] (OPF) hydrogels. Neutral, negatively or positively charged scaffolds were fabricated using unmodified OPF (neutral charge), sodium methacrylate (SMA) crosslinked OPF (negative charge), or [2-(methacryloyloxy) ethyl] trimethylammonium chloride (MAE) crosslinked OPF (positive charge), respectively. To allow investigation of surface charge for different BMP-2 release rates, three BMP-2 release profiles were generated by protein encapsulation into poly(lactic-co-glycolic acid) (PLGA) microspheres and/or adsorption on the OPF composite. Release of radiolabeled 125I-BMP-2 was analyzed in vitro and in vivo and bone formation was assessed after 9 weeks of subcutaneous implantation in rats. Negatively charged OPF generated significantly more bone formation compared to neutral and positively charged OPF. This effect was seen for all three loading methods and subsequent BMP-2 release profiles. Along with charge modifications, a more sustained release of BMP-2 improved bone formation in OPF composites. Overall, this study clearly shows that negative charge enhances bone formation compared to neutral and positive charge in OPF composites.

Notochordal cells (NCs) reside in the core of the healthy disc and produce soluble factors that can stimulate nucleus pulposus cells (NPCs). These NC-derived factors may be applied in intervertebral disc regeneration for treatment of low-back pain. However, identification of the active soluble factors is challenging. Therefore a novel approach to directly use porcine NC-rich NP matrix (NCM) is introduced. We explored porcine NCM's anabolic effects on bovine NPCs harvested from caudal discs of adolescent and adult (2-2.5 vs. 4-6 year old) cows. NC-conditioned medium (NCCM) and NCM were produced from porcine NC-rich NP tissue. Bovine NPCs were cultured in alginate beads for 4 weeks in base medium (BM), NCCM, and NCM to investigate NCM's regenerative potential. Porcine NCM increased glycosaminoglycan (GAG) content of both adolescent and adult bovine NPCs. This was through increased proliferation of adolescent bovine NPCs, whereas in adult bovine NPCs, it was mostly through increased GAG production per NPC. Furthermore, adolescent bovine NPCs were cultured in BM and porcine NCM treated with interleukin (IL)-1β to investigate NCM's potential in an inflammatory environment. Addition of IL-1β enhanced IL1β and CXCL8 (IL8) gene expression, while NCM diminished IL1β gene expression. IL-1β reduced GAG and DNA content, but the addition of NCM relative to BM improved GAG and DNA content. Altogether, porcine NCM exerts bovine NPC-age dependent effects, and NCM's anabolic effect on adult NPCs is stronger compared with NCCM. Furthermore, porcine NCM induced an anabolic response of bovine NPCs in an inflammatory environment and may have anti-inflammatory properties. Therefore, NCM has potential in a regenerative therapy for disc degeneration, and warrants additional in vivo studies.

The intervertebral disc (IVD) is a highly hydrated tissue, the rich proteoglycan matrix imbibes water, enabling the disc to withstand compressive loads. During ageing and degeneration increased matrix degradation leads to dehydration and loss of function. Aquaporins (AQP) are a family of transmembrane channel proteins that selectively allow the passage of water in and out of cells and are responsible for maintaining water homeostasis in many tissues. Here, the expression of all 13 AQPs at gene and protein level was investigated in human and canine non‐degenerate and degenerate IVDs to develop an understanding of the role of AQPs during degeneration. Furthermore, in order to explore the transition of notochordal cells (NCs) towards nucleus pulposus (NP) cells, AQP expression was investigated in canine IVDs enriched in NCs to understand the role of AQPs in IVD maturation. AQP0, 1, 2, 3, 4, 5, 6, 7 and 9 were expressed at gene and protein level in both non‐degenerate and degenerate human NP tissue. AQP2 and 7 immunopositivity increased with degeneration in human NP tissue, whereas AQP4 expression decreased with degeneration in a similar way to AQP 1 and 5 shown previously. All AQP proteins that were identified in human NP tissue were also expressed in canine NP tissue. AQP2, 5, 6 and 9 were found to localise to vacuole‐like membranes and cell membranes in NC cells. In conclusion, AQPs were abundantly expressed in human and canine IVDs. The expression of many AQP isotypes potentially alludes to multi‐faceted functions related to adaption of NP cells to the conditions they encounter within their microenvironment in health and degeneration. The presence of AQPs within the IVD may suggest an adaptive role for these water channels during the development and maintenance of the healthy, mature IVD.

Objective To report the long‐term outcome of nine dogs treated for caudal cervical spondylomyelopathy (CCSM) with surgical spinal fusion. Study design Short case series. Animals Nine large‐breed dogs. Methods Medical records of dogs treated for disc‐associated CCSM (2013‐2016) were reviewed. The surgery objective was spinal distraction by implantation of a SynCage and fixation with two Unilock plates. Follow‐up included the Helsinki pain score questionnaire, neurological grading, radiography, computed tomography (CT), and micro‐CT (μCT) with subsequent histopathology (two dogs). Results Clinical follow‐up was obtained between 9 and 51 months (27.4 ± 13.4 months). The Helsinki pain score and neurological Griffith score improved (P

No standardized treatment guidelines are reported in veterinary medicine for dogs with large pituitary corticotroph neoplasms causing neurological signs, and such dogs usually have a short overall survival. When these dogs undergo pituitary surgery and the tumor regrows there are few reports of subsequent treatments. A 7 yr old male Maltese diagnosed with pituitary-dependent hypercortisolism developed seizures in conjunction with a large pituitary corticotroph adenoma and underwent transsphenoidal hypophysectomy. After 3 yr of clinical remission, hypercortisolism recurred, and trilostane therapy was initiated. One year later, the dog developed new neurological signs and computed tomography revealed regrowth of a large pituitary mass that was then treated with radiation therapy. The dog lived disease-free for 3 more yr. At postmortem examination, a more aggressive pituitary neoplasm than the one examined at the time of surgery was found, which is suggestive of malignant transformation into a carcinoma despite the absence of convincing metastasis.

Episodes of inflammation and pain are predominant features of arthritic joint diseases. Drug delivery systems (DDS) could reduce inflammation and pain long-term without chances of infection upon multiple injections. To allow for long-term evaluation of DDS, we modified a previously published acute arthritis model by extending follow-up periods between flare-ups. Unilateral synovial inflammation of the knee was induced by intra-articular injection of streptococcal cell wall peptidoglycan polysaccharide (PGPS), and flare-ups were induced by intravenous PGPS injections every 4 weeks for a total duration of 84 days. In PGPS-reactivated animals, joint swelling, pain behavior, post mortem synovitis, and osteophyte formation were notable features. Hepatitis, splenitis and inflammation of non-primed joints were observed as systemic side effects. To test the applicability of the modified arthritis model for long-term testing of DDS, the duration of anti-inflammatory and analgesic effects of a corticosteroid released from two different polymer-based platforms was evaluated. The current modified arthritis model has good applicability for testing of DDS for a prolonged period of time. Furthermore, the novel autoregulatory polyesteramide (PEA) microsphere platform releasing triamcinolone acetonide (TAA) was benchmarked against poly lactic-co-glycolic acid (PLGA) and reduced joint swelling and pain behavior more potently compared to TAA-loaded PLGA microspheres.

The main challenge in BMP-2 based application lies in finding strategies that prolong its effective period, as it has a short biological half-life. Several BMP-2 release profiles have shown to enhance bone formation at various application sites. However, it remains to be determined which BMP-2 release profile best augments bone formation and whether this effect is location-dependent. Therefore, the aim of this study was to investigate the effect of BMP-2 release from oligo[(polyethylene glycol) fumarate] bis(2-(methacryloyloxy)ethyl) phosphate (OPF-BP) composites on the osteoinductive efficacy at ectopic versus orthotopic application. By varying the BMP-2 loading method, three different OPF-BP composites were created with varied release profiles. The composites were compared to unloaded OPF-BP as negative control, and to the clinically used Infuse® absorbable collagen sponge (ACS) as positive control. Bone formation was assessed by micro-computed tomography after 9 weeks of subcutaneous implantation and 3, 6, and 9 weeks of orthotopic implantation in rats (n=48). Whereas a BMP-2 burst release of >49% generated significantly more bone compared to sustained release (burst release

To date no disease-modifying drugs for osteoarthritis (OA) are available, with treatment limited to the use of pain killers and prosthetic replacement. The ADAMTS (A Disintegrin and Metallo Proteinase with Thrombospondin Motifs) enzyme family is thought to be instrumental in the loss of proteoglycans during cartilage degeneration in OA, and their inhibition was shown to reverse osteoarthritic cartilage degeneration. Locked Nucleic Acid (LNA)-modified antisense oligonucleotides (gapmers) released from biomaterial scaffolds for specific and prolonged ADAMTS inhibition in co-delivered and resident chondrocytes, is an attractive therapeutic strategy. Here, a gapmer sequence identified from a gapmer screen showed 90% ADAMTS5 silencing in a monolayer culture of human OA chondrocytes. Incorporation of the gapmer in a fibrin-hyaluronic acid hydrogel exhibited a sustained release profile up to 14 days. Gapmers loaded in hydrogels were able to transfect both co-embedded chondrocytes and chondrocytes in a neighboring gapmer-free hydrogel, as demonstrated by flow cytometry and confocal microscopy. Efficient knockdown of ADAMTS5 was shown up to 14 days in both cell populations, i.e. the gapmer-loaded and gapmer-free hydrogel. This work demonstrates the use applicability of a hydrogel as a platform for combined local delivery of chondrocytes and an ADAMTS-targeting gapmer for catabolic gene modulation in OA.

Introduction: Nucleus Pulposus Progenitor Cells (NPPCs), positive for the angiopoietin-1 receptor (Tie2), were demonstrated in human, mouse, canine and bovine NP tissue. Tie2+ NPPCs possess a multi-lineage differentiation potential, and regeneration potential is attributed to them. However, the isolation of Tie2+ NPPCs can be cumbersome. Hence, three isolation methods were compared. Methods: Bovine NP cells were isolated from 10-14-month-old animals. Cell sorting was performed with an antibody against Tie2 (bs-1300R, Bioss) using FACS, magnetic-activated cell sorting (MACS) and pluriSelect, a size-based sorting method. Outcomes were evaluated by cell yield of Tie2+ cells, the ability of sorted cells to form colonies and tri-lineage differentiation assays. Results: FACS resulted in the highest Tie2+ cell yield (5.0 ± 4.0%) followed by MACS (1.6 ± 2.9%) and pluriSelect (1.1 ± 1.4%). Colony forming ability did not differ between Tie2+ and Tie2- cells for any isolation method. However, Tie2+ cells obtained by MACS resulted in more colonies than pluriSelect (p < 0.05). Osteogenic and adipogenic differentiation of Tie2+ and Tie2- cells did not result in a clear distinction for MACS and pluriSelect; Tie2+ FACS-sorted cells demonstrated superior osteogenic and adipogenic differentiation over Tie2- cells. Also for chondrogenesis, the Tie2+ FACS-sorted NPPCs tended to produce more proteoglycan vs Tie2- NPPCs, whereas for MACS and pluriSelect no difference was found. Conclusion: Isolation of Tie2+ NPPC is possible with all three methods tested. However, FACS resulted in the highest cell yield and a clearer separation after differentiation making it the method of choice for Tie2+ NPPC isolation.

Low back pain related to intervertebral disc (IVD) degeneration has a major socioeconomical impact on our ageing society. Therefore, stem cell therapy to activate self-repair of the IVD remains an exciting treatment strategy. In this respect, tissue-specific progenitors may play a crucial role for IVD regeneration, as these cells are perfectly adapted to this niche. Such a rare progenitor cell population residing in the nucleus pulposus (nucleus pulposus progenitor cells or NPPCs) was found positive for the angiopoietin-1 receptor (Tie2+) and was demonstrated to possess self-renewal capacity and in vitro multipotency. Here, we compared three sorting protocols, i.e., fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS) and a mesh-based label-free cell sorting system (pluriSelect), with respect to cell yield, potential to form colonies (colony forming units = CFUs) and in vitro functional differentiation assays for tripotency. The aim of this study was to demonstrate efficiency of three wide-spread cell sorting methods for picking rare cells (

BACKGROUND CONTEXT Local corticosteroids have been used to relieve symptoms of chronic low back pain, although treatment effects have been shown to wear off relatively fast. Prolonging corticosteroid presence by controlled release from biomaterials may allow for longer pain relief while circumventing adverse effects such as high bolus dosages. PURPOSE The purpose of this study was to evaluate the safety and efficacy of intradiscal controlled release of triamcinolone acetonide (TAA) by poly(esteramide) microspheres in a canine degenerated intervertebral disc (IVD) model. STUDY DESIGN In a preclinical experimental large animal model, the effect of prolonged glucocorticoid exposure on disc degeneration was evaluated. METHODS Degeneration was accelerated by nucleotomy of lumbar IVDs of Beagle dogs. After 4 weeks, microspheres loaded with 8.4 µg TAA, and 0.84mg TAA were administered to the degenerated IVDs by intradiscal injection (n=6 per group). Empty microspheres (n=6) and all adjacent non-nucleotomized noninjected IVDs were included as controls (n=24). Immediately prior to TAA administration and after 12 weeks, magnetic resonance imaging was performed. Degenerative changes were evaluated by disc height index, Pfirrmann grading, T1ρ and T2 mapping values, postmortem CT scans, macroscopic and microscopic grading, and biochemical/immunohistochemical analysis of inflammation and extracellular matrix content. In addition, nerve growth factor (NGF) protein expression, a biomarker for pain, was scored in nucleus pulposus (NP) tissues. The study was funded by a research grant from Health Holland (1.3million euros = 1.5million US dollars). RESULTS Macroscopic evaluation and CT images postmortem were consistent with mild disc degeneration. Other abnormalities were not observed. Nucleotomy-induced degeneration and inflammation was mild, reflected by moderate Pfirrmann grades and PGE2 levels. Regardless of TAA dosage, local sustained delivery did not affect disc height index nor Pfirrmann grading, T1ρ and T2 mapping values, PGE2 tissue levels, collagen, GAG, and DNA content. However, the low dosage of TAA microspheres significantly reduced NGF immunopositivity in degenerated NP tissue. CONCLUSIONS This is the first in vivo application in a preclinical large animal model of a controlled release formulation of corticosteroids in mild IVD degeneration. Sustained release of TAA locally in the IVD appeared safe and reduced NGF expression, suggesting its potential applicability for pain relief, although beneficial effects were absent on tissue degeneration. CLINICAL SIGNIFICANCE The present platform seems to be promising in extending the local controlled delivery of TAA with the potency to provide long-standing analgesia in the subset of LBP patients suffering from discogenic pain.

Introduction: Nucleus pulposus progenitor cells (NPPC), Tie2+ cells (positive for angiopoietin receptor), which possess multi-lineage differential potential is a potential cell population for cell therapy. However, the number of Tie2+ cells in NP is extremely limited. Referring to the recent research of Tie2+ hematopoietic stem cells we attempted to increase the Tie2+ cell sub-population in nucleus pulposus cells (NPC) by PPARδ agonist treatment and increasing mitophagy. Methods: Cells were isolated from fresh human IVD tissue from spinal surgery with written consent. The passage 1 human NP cells were cultured in low glucose Dulbecco’s Modified Eagle’s Medium media containing PPARδ agonist (GW501516, Sigma), i.e., 25 µM, or vehicle control (N = 2 donors). After 10 days NP, the Tie2 marker expression was then detected by flow cytometry cells and relative gene expression was determined by real-time qPCR, i.e. at ACAN, col1, col2, and PTEN-induced kinase 1 (PINK1). Results: PPARδ-agonist-treated NP population had ~3 times more Tie2+ cells and PINK1 gene expression tended to be higher than in the vehicle control group. Conclusion: PPARδ agonist possibly increases the Tie2+ cell population in NPC by increasing mitophagy similar to hematopoietic stem cells.