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Purpose: Limited nutrient transport is hypothesized to be involved in intervertebral disc (IVD) degeneration. It is widely recognized that the dominant mode of transport of small molecules such as glucose is via diffusion, rather than convection. However, recent findings suggest a role for convection-induced by fast (motion-related) and slow (diurnal) dynamic loading in molecular transport of even such small solutes. The aim of this study was to investigate whether fluid exchange induced by simulated physiological loading (composed of both fast cyclic or slower diurnal loading) can influence the molecular transport of a small molecule through the cartilage endplate (CEP) into the nucleus pulposus (NP) of IVDs., Methods: The molecular transport of fluorescein through the CEP and into the NP was studied in a bovine CEP/NP explant model and loading was applied by an axial compression bioreactor. The loaded explants (convection and diffusion) were compared to unloaded explants (diffusion alone)., Results: In the initial 24 h, there were no differences between loaded and unloaded explants, indicating that convection did not enhance molecular transport of small solutes over diffusion alone. Notably, after 48 h which corresponds to two complete diurnal cycles of tissue compression, fluid exudation/imbibing and redistribution, the fluorescein concentration was significantly increased in the top and bottom layer of the explant, when compared to the unloaded explant., Conclusions: Slower diurnal cyclic compression of the IVD might enhance the transport of small molecules into the IVD although it could not be discerned whether this was due to diffusion/convection or a combination., Competing Interests: Declarations. Conflict of interest: The authors declare no conflicts of interest., (© 2024. The Author(s).)

Intradiscal drug delivery is a promising strategy for treating intervertebral disk degeneration (IVDD). Local degenerative processes and intrinsically low fluid exchange are likely to influence drug retention. Understanding their connection will enable the optimization of IVDD therapeutics. Release and retention of an inactive hydrophilic fluorine-19 labeled peptide ( 19 F-P) as model for regenerative peptides was studied in a whole IVD culture model by measuring the 19 F-NMR (nuclear magnetic resonance) signal in culture media and IVD tissue extracts. In another set-up, noninvasive near-infrared imaging was used to visualize IR-780, as hydrophobic small molecular drug model, retention upon injection into healthy and degenerative caudal IVDs in a rat model of disk degeneration. Furthermore, IR-780-loaded degradable polyester amide microspheres (PEAM) were injected into healthy and needle pricked degenerative IVDs, subcutaneously, and in knee joints with and without surgically-induced osteoarthritis (OA). Most 19 F-P was released from the IVD after 7 days. IR-780 signal intensity declined over a 14-week period after bolus injection, without a difference between healthy and degenerative disks. IR-780 signal declined faster in the skin and knee joints compared to the IVDs. IR-780 delivery by PEAMs enhanced disk retention beyond 16 weeks. Moreover, in degenerated IVDs the IR-780 signal was higher over time than in healthy IVDs while no difference between OA and healthy joints was noted. We conclude that the clearance of peptides and hydrophobic small molecules from the IVD is relatively fast. These results illustrate that development of controlled release formulations should take into account the target anatomical location and local (patho)biology.

Osteoarthritis (OA) is a common disease in dogs with severe impact on their welfare. The multimodal management of OA includes feeding therapeutic diets and nutraceuticals to slow down OA progression. Collagen hydrolysates (CH) are a nutritional supplement that may exert anabolic effects on osteoarthritic joint cartilage as well as disease-modifying effects. After oral intake, CH is absorbed, mainly as amino acids, di- and tripeptides that are transported amongst others to the joint. In addition to reducing cartilage degradation, CH metabolites may reduce synovial inflammation and subchondral bone sclerosis during OA. Preliminary evidence in dogs suffering from the consequences of OA support the clinical efficacy of CH with reported reductions in lameness. However, effects on biomarker level of cartilage metabolism and inflammation are inconclusive. Additionally, current studies show a lack of standardised dosing regimens and the use of not validated outcomes. Future work should therefore elucidate further on the bioavailability of CH in dogs in order to establish adequate dosing recommendations. Furthermore, high-quality placebo-controlled randomised controlled trials are essential to dstudies have evaluated the cetermine the clinical efficacy of CH to reduce lameness, prevent OA progression and thereby improve the level of evidence., (© 2024 The Author(s). Journal of Animal Physiology and Animal Nutrition published by Wiley‐VCH GmbH.)

The development of human induced pluripotent stem cell (iPSC)-based regenerative therapies is challenged by the lack of specific cell markers to isolate differentiated cell types and improve differentiation protocols. This issue is particularly critical for notochordal-like cells and chondrocytes, which are crucial in treating back pain and osteoarthritis, respectively. Both cell types produce abundant proteoglycan aggrecan (ACAN), crucial for the extracellular matrix. We generated two human iPSC lines containing an ACAN-2A-mScarlet reporter. The reporter cell lines were validated using CRISPR-mediated transactivation and functionally validated during notochord and cartilage differentiation. The ability to isolate differentiated cell populations producing ACAN enables their enrichment even in the absence of specific cell markers and allows for comprehensive studies and protocol refinement. ACAN's prevalence in various tissues (e.g., cardiac and cerebral) underscores the reporter's versatility as a valuable tool for tracking matrix protein production in diverse cell types, benefiting developmental biology, matrix pathophysiology, and regenerative medicine.

Lumbosacral intervertebral disc herniation (IVDH) is a common cause of lower back pain in dogs and humans. In humans, the vertebral endplate to annulus fibrosus (AF) attachment was implicated as an alternative failure site besides rupture through the dorsal AF (AFF). Endplate junction failure (EPJF) is characterized by IVDH, accompanied by endplate irregularities (type A), rim avulsions (type B), or larger bony avulsions on one (type C) or both endplates (type D), associated with an adjacent endplate defect. This retrospective study reports the CT prevalence of presumed EPJF in dogs and its associations with signalment and other lumbosacral CT abnormalities. CT scans, including the lumbosacral spine of dogs obtained at two institutions, were assessed, yielding 324 scans. Presumed EPJF was found in 69 dogs (21%) and AFF in 68 dogs (21%), commonly at the caudal endplate of the last lumbar vertebra (71%). The remaining 187 dogs did not show presumed EPJF or AFF. Presumed EPJF type A occurred in 49/69, type B in 19/69, and type C in 1/69 dogs. Univariable logistic regression showed that presumed EPJF was associated with significantly higher IVDH grades than AFF. In the multiple regression model, presumed EPJF and AFF remained associated with increasing age and spondylosis deformans. Presumed EPJF was associated with vertebral endplate sclerosis and AFF with zygapophyseal joint osteoarthritis. In conclusion, presumed EPJF was observed on CT in 21% of dogs with lumbosacral IVDH. Prospective studies correlating EPJF on CT with clinical, surgical, and histopathological findings are needed for a better understanding of the underlying pathology and clinical relevance., (© 2024 The Author(s). Veterinary Radiology & Ultrasound published by Wiley Periodicals LLC on behalf of American College of Veterinary Radiology.)

The acetabular rim extension (ACE-X) implant is a custom-made three-dimensionally printed titanium device designed for the treatment of canine hip dysplasia. In this study, 34 dogs (61 hips) underwent ACE-X implantation, and assessments were conducted using computed tomography, force plate analysis, Ortolani's test, and the Helsinki chronic pain index (HCPI) questionnaires at five intervals: the pre-operative day, the surgery day, and the 1.5-month, 3-month, and 12-month follow-ups. Statistically significant increases in femoral head coverage with a negative Ortolani subluxation test were observed immediately after surgery and persisted throughout the study. Osteoarthritis (OA) scores remained stable, but osteophyte size significantly increased between the surgery day and the 12-month follow-up, especially in hips with a baseline OA score of 2 compared to those with a score of 1. The force plate data showed no significant changes during the study. The HCPI demonstrated a significant decrease in pain score from pre-operative value to six-week follow-up and gradually decreased over time. Major complications were identified in six hips (9.8%) of four dogs. In conclusion, the ACE-X implant effectively increased femoral head coverage, eliminated subluxation, and provided long-term pain relief with minimal complications, benefiting over 90% of the study population. The study supports the ACE-X implant as a valuable alternative treatment for canine hip dysplasia.

Hip dysplasia causes major disability in dogs. Treatment options are limited to palliative treatment (e.g., pain relief, physical exercise, lifestyle changes, and weight control) or invasive surgeries such as pelvic osteotomies and total hip arthroplasty. Hence, a strong unmet need exists for an effective and dog-friendly solution that enhances the quality of life of man's best friend. We fill this treatment gap by offering a minimally traumatic and extraarticular, dog-specific, 3-dimensional-printed, hip implant (3DHIP) that restores hip joint stability. The surgical treatment using a 3DHIP implant is less invasive than osteotomies and can be performed bilaterally in one surgical session. The 3DHIP implant extends the dorsal acetabular rim of the dysplastic hip joint thereby increasing coverage of the femoral head and inhibiting joint subluxation with fast recovery. Sufficient access to the dorsal acetabular rim and ventral border of the iliac body together with optimal fitting and fixation of the implant are key steps for a successful 3DHIP implantation and imply the need for a specific approach. The present article aims to showcase this innovative surgical technique with tips and tricks as a surgical manual for implantation of the 3DHIP implant in dogs affected by hip dysplasia.

Background: Intervertebral disc degeneration is frequent in dogs and can be associated with symptoms and functional impairments. The degree of disc degeneration can be assessed on T2-weighted MRI scans using the Pfirrmann classification scheme, which was developed for the human spine. However, it could also be used to quantify the effectiveness of disc regeneration therapies. We developed and tested a deep learning tool able to automatically score the degree of disc degeneration in dog spines, starting from an existing model designed to process images of human patients., Methods: MRI midsagittal scans of 5991 lumbar discs of dog patients were collected and manually evaluated with the Pfirrmann scheme and a modified scheme with transitional grades. A deep learning model was trained to classify the disc images based on the two schemes and tested by comparing its performance with the model processing human images., Results: The determination of the Pfirrmann grade showed sensitivities higher than 83% for all degeneration grades, except for grade 5, which is rare in dog spines, and high specificities. In comparison, the correspondent human model had slightly higher sensitivities, on average 90% versus 85% for the canine model. The modified scheme with the fractional grades did not show significant advantages with respect to the original Pfirrmann grades., Conclusions: The novel tool was able to accurately and reliably score the severity of disc degeneration in dogs, although with a performance inferior than that of the human model. The tool has potential in the clinical management of disc degeneration in canine patients as well as in longitudinal studies evaluating regenerative therapies in dogs used as animal models of human disorders., Competing Interests: The authors declare no conflicts of interest., (© 2024 The Authors. JOR Spine published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

Understanding the emergence of human notochordal cells (NC) is essential for the development of regenerative approaches. We present a comprehensive investigation into the specification and generation of bona fide NC using a straightforward pluripotent stem cell (PSC)-based system benchmarked with human fetal notochord. By integrating in vitro and in vivo transcriptomic data at single-cell resolution, we establish an extended molecular signature and overcome the limitations associated with studying human notochordal lineage at early developmental stages. We show that TGF-β inhibition enhances the yield and homogeneity of notochordal lineage commitment in vitro . Furthermore, this study characterizes regulators of cell-fate decision and matrisome enriched in the notochordal niche. Importantly, we identify specific cell-surface markers opening avenues for differentiation refinement, NC purification, and functional studies. Altogether, this study provides a human notochord transcriptomic reference that will serve as a resource for notochord identification in human systems, diseased-tissues modeling, and facilitating future biomedical research., Competing Interests: The authors declare no competing interests., (© 2024 The Authors.)

Low back pain is the leading cause of disability worldwide, but current therapeutic interventions are palliative or surgical in nature. Loss of notochordal cells (NCs) and degradation of the healthy matrix in the nucleus pulposus (NP), the central tissue of intervertebral discs (IVDs), has been associated with onset of degenerative disc changes. Recently, we established a protocol for decellularization of notochordal cell derived matrix (NCM) and found that it can provide regenerative cues to nucleus pulposus cells of the IVD. Here, we combined the biologically regenerative properties of decellularized NCM with the mechanical tunability of a poly(ethylene glycol) hydrogel to additionally address biomechanics in the degenerate IVD. We further introduced a hydrolysable PEG-diurethane crosslinker for slow degradation of the gels in vivo. The resulting hydrogels were tunable over a broad range of stiffness's (0.2 to 4.5 kPa), matching that of NC-rich and -poor NP tissues, respectively. Gels formed within 30 min, giving ample time for handling, and remained shear-thinning post-polymerization. Gels also slowly released dNCM over 28 days as measured by GAG effusion. Viability of encapsulated bone marrow stromal cells after extrusion through a needle remained high. Although encapsulated NCs stayed viable over two weeks, their metabolic activity decreased, and their phenotype was lost in physiological medium conditions in vitro. Overall, the obtained gels hold promise for application in degenerated IVDs but require further tuning for combined use with NCs., (© 2023 The Authors. Journal of Biomedical Materials Research Part A published by Wiley Periodicals LLC.)

Background: The regulation of inflammatory mediators in the degenerating intervertebral disc (IVD) and corresponding ligamentum flavum (LF) is a topic of emerging interest. The study aimed to investigate the expression of a broad array of inflammatory mediators in the degenerated LF and IVD using a dog model of spontaneous degenerative disc disease (DDD) to determine potential treatment targets., Methods: LF and IVD tissues were collected from 22 normal dogs (Pfirrmann grades I and II) and 18 dogs affected by DDD (Pfirrmann grades III and IV). A qPCR gene array was used to investigate the expression of 80 inflammatory genes for LF and IVD tissues, whereafter targets of interest were investigated in additional tissue samples using qPCR, western blot (WB), and immunohistochemistry., Results: Tumor necrosis factor superfamily (TNFSF) signaling was identified as a regulated pathway in DDD, based on the significant regulation ( n -fold ± SD) of various TNFSF members in the degenerated IVD, including nerve growth factor ( NGF ; -8 ± 10), CD40LG (464 ± 442), CD70 (341 ± 336), TNFSF Ligand 10 (9 ± 8), and RANKL/TNFSF Ligand 11 (85 ± 74). In contrast, TNFSF genes were not significantly affected in the degenerated LF compared to the control LF. Protein expression of NGF (WB) was significantly upregulated in both the degenerated LF (4.4 ± 0.5) and IVD (11.3 ± 5.6) compared to the control group. RANKL immunopositivity was significantly upregulated in advanced stages of degeneration (Thompson grades IV and V) in the nucleus pulposus and annulus fibrosus of the IVD, but not in the LF., Conclusions: DDD involves a significant upregulation of various TNFSF members, with tissue-specific expression profiles in LF and IVD tissues. The differential involvement of TNFSF members within multiple spinal tissues from the same individual provides new insights into the inflammatory processes involved in DDD and may provide a basis to formulate hypotheses for the determination of potential treatment targets., Competing Interests: The authors declare no conflicts of interest., (© 2023 The Authors. JOR Spine published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

In regenerative medicine, extracellular vesicles (EVs) are considered as a promising cell-free approach. EVs are lipid bilayer-enclosed vesicles secreted by cells and are key players in intercellular communication. EV-based therapeutic approaches have unique advantages over the use of cell-based therapies, such as a high biological, but low immunogenic and tumorigenic potential. To analyze the purity and biochemical composition of EV preparations, the International Society for Extracellular Vesicles (ISEV) has prepared guidelines recommending the analysis of multiple (EV) markers, as well as proteins coisolated/recovered with EVs. Traditional methods for EV characterization, such as Western blotting, require a relatively high EV sample/protein input for the analysis of one protein. We here evaluate a combined Western and bead-based multiplex platform, called DigiWest, for its ability to detect simultaneously multiple EV markers in an EV-containing sample with inherent low protein input. DigiWest analysis was performed on EVs from various sources and species, including mesenchymal stromal cells, notochordal cells, and milk, from human, pig, and dog. The study established a panel of nine antibodies that can be used as cross-species for the detection of general EV markers and coisolates in accordance with the ISEV guidelines. This optimized panel facilitates the parallel evaluation of EV-containing samples, allowing for a comprehensive characterization and assessment of their purity. The total protein input for marker analysis with DigiWest was 1 μg for all nine antibodies, compared with ∼10 μg protein input required for traditional Western blotting for one antibody. These findings demonstrate the potential of the DigiWest technique for characterizing various types of EVs in the regenerative medicine field.

Background: Chronic low back pain, a leading contributor to disease burden worldwide, is often caused by intervertebral disc (IVD) degeneration. Modic changes (MCs) are MRI signal intensity changes due to lesions in vertebral bone marrow adjacent to degenerated IVDs. Only a few studies described the histopathological changes associated with MC to date. MC type 1 is suggested to be associated with bone marrow infiltration of fibrovascular tissue, type 2 with fatty infiltration, and type 3 with bone sclerosis in humans., Methods: This study investigated whether the dog can be a valuable animal model to research MCs, by examining the prevalence, imaging, and histological characteristics of lumbar MCs in dogs (340 dogs, 2496 spinal segments)., Results: Logistic regression analysis indicated that the presence of lumbosacral MCs was associated with age and disc herniation (annulus fibrosis protrusion and/or nucleus pulposus extrusion). According to MRI analysis, MCs were mostly detected at the lumbosacral junction in dogs. Most signal intensity changes represented MC type 3, while previous spinal surgery seemed to predispose for the development of MC type 1 and 2. Histological analysis (16 dogs, 39 spinal segments) indicated that IVDs with MCs showed more histopathological abnormalities in the endplate and vertebral bone marrow than IVDs without MCs. Mostly chondroid proliferation in the bone marrow was encountered, while the histologic anomalies described in humans associated with MCs, such as fibrovascular or fatty infiltration, were scarcely detected., Conclusions: Dogs spontaneously develop MCs, but may exhibit other pathological processes or more chronic bone marrow pathologies than humans with MCs. Therefore, more research is needed to determine the translatability of the MCs encountered in dog low-back-pain patients., Competing Interests: The authors declare no conflicts of interest., (© 2023 The Authors. JOR Spine published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

Background: Lineage-tracing experiments have established that the central region of the mature intervertebral disc, the nucleus pulposus (NP), develops from the embryonic structure called "the notochord". However, changes in the cells derived from the notochord which form the NP (i.e., notochordal cells [NCs]), in terms of their phenotype and functional identity from early developmental stages to skeletal maturation are less understood. These key issues require further investigation to better comprehend the role of NCs in homeostasis and degeneration as well as their potential for regeneration. Progress in utilizing NCs is currently hampered due to poor consistency and lack of consensus methodology for in vitro NC extraction, manipulation, and characterization., Methods: Here, an international group has come together to provide key recommendations and methodologies for NC isolation within key species, numeration, in vitro manipulation and culture, and characterization., Results: Recommeded protocols are provided for isolation and culture of NCs. Experimental testing provided recommended methodology for numeration of NCs. The issues of cryopreservation are demonstrated, and a pannel of immunohistochemical markers are provided to inform NC characterization., Conclusions: Together we hope this article provides a road map for in vitro studies of NCs to support advances in research into NC physiology and their potential in regenerative therapies., Competing Interests: The authors have no relevant conflicts of interest to declare in relation to this article., (© 2023 The Authors. JOR Spine published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

Degeneration of the intervertebral disc is an age-related condition. It also accompanies the disappearance of the notochordal cells, which are remnants of the developmental stages of the nucleus pulposus (NP). Molecular changes such as extracellular matrix catabolism, cellular phenotype, and glycosaminoglycan loss in the NP have been extensively studied. However, as one of the most significant co- and posttranslational modifications, glycosylation has been overlooked in cells in degeneration. Here, we aim to characterize the N -glycome of young and mature NP and identify patterns related to aging. Accordingly, we isolated N -glycans from notochordal cell-rich NP from porcine discs, characterized them using a combined approach of exoglycosidase digestions and analysis with hydrophilic interaction ultra-performance liquid chromatography and mass spectrometry. We have assigned over 300 individual N -glycans for each age group. Moreover, we observed a notable abundance of antennary structures, galactosylation, fucosylation, and sialylation in both age groups. In addition, as indicated from our results, increasing outer arm fucosylation and decreasing α(2,3)-linked sialylation with aging suggest that these traits are age-dependent. Lastly, we have focused on an extensive characterization of the N -glycome of the notochordal cell-rich NP in aging without inferred degeneration, describing glycosylation changes specific for aging only. Our findings in combination with those of other studies, suggest that the degeneration of the NP does not involve identical processes as aging., Competing Interests: The authors of this manuscript declare no conflict of interest., (©2023 The Authors FASEB BioAdvances published by The Federation of American Societies for Experimental Biology.)

A novel biomimetic artificial intervertebral disc (bioAID) replacement implant has been developed containing a swelling hydrogel representing the nucleus pulposus, a tensile strong fiber jacket as annulus fibrosus and titanium endplates with pins to primarily secure the device between the vertebral bodies. In this study, the design safety of this novel implant was evaluated based on several biomechanical parameters, namely compressive strength, shear-compressive strength, risk of subsidence and device expulsion as well as identifying the diurnal creep-recovery characteristics of the device. The bioAID remained intact up to 1 kN under static axial compression and only 0.4 mm of translation was observed under a compressive shear load of 20 N. No subsidence was observed after 0.5 million cycles of sinusoidal compressive loading between 50 and 225 N. After applying 400 N in antero-posterior direction under 100 N axial compressive preload, approximately 2 mm displacement was found, being within the range of displacements reported for other commercially available cervical disc replacement devices. The diurnal creep recovery behavior of the bioAID closely resembled what has been reported for natural intervertebral discs in literature. Overall, these results indicate that the current design can withstand (shear-compression loads and is able to remain fixed in a mechanical design resembling the vertebral bodies. Moreover, it is one of the first implants that can closely mimic the poroelastic and viscoelastic behavior of natural disc under a diurnal loading pattern., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Hip dysplasia (HD) is a common orthopedic problem in young dogs. To decrease the laxity of the hip joint related to HD, the surgical treatments are recommended to increase femoral head coverage. ACEtabular rim eXtension (ACE-X) using a personalized 3-dimensional printed titanium shelf implant is a new surgical treatment to increase femoral head coverage and decrease laxity of the dysplastic hip joint, however, the efficacy is less know. Client-owned dogs older than 6 months with clinical signs of coxofemoral joint subluxation and radiographic evidence of HD with no or mild osteoarthritis (OA) were included. The Norberg angle (NA), linear percentage of femoral head overlap (LFO), and percentage of femoral head coverage (PC) were investigated radiographically and with computed tomography (CT) before and after surgery. OA was graded (scores 0-3) according to the maximum osteophyte size measured on CT. In addition, joint laxity (Ortolani) test results, gait analysis, and the Helsinki chronic pain index (HCPI) questionnaire were obtained at preoperative, immediately postoperative and at 1.5- and 3-month evaluations. Acetabular rim extension was performed in 61 hips of 34 dogs; NA, LFO, and PC were significantly higher immediately postoperatively and at the 1.5- and 3-month follow-up examinations compared with preoperative values ( p < 0.05). Osteophyte size gradually increased over time ( p < 0.05). The OA score significantly increased between preoperatively and directly postoperatively, and between preoperatively and at 3-month follow-up ( p < 0.05). The laxity test normalized in 59 out of 61 hips after surgery, and the HCPI questionnaire showed that the pain score decreased significantly at 1.5 and 3 months, postoperatively. The force plate showed no significant improvement during the 3 months follow-up. Although pain reduction by the implant was unclear in short-term results, a personalized shelf implant significantly increased femoral head coverage and eliminated subluxation of the dysplastic hip joint. Further studies are required to study the long-term efficacy of gait, chronic pain, and progression of osteoarthritis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Kwananocha, Magré, Willemsen, Weinans, Sakkers, How, Verseijden, Tryfonidou, van der Wal and Meij.)

Purpose: Various sustained-release formulations incorporate high bupivacaine concentrations but data on local toxicity is lacking. This study explores local toxic effects of highly concentrated (5%) bupivacaine compared to clinically used concentrations in vivo following skeletal surgery, to assess the safety of sustained-release formulations with high bupivacaine concentrations., Methods: Sixteen rats underwent surgery, in which screws with catheters affixed were implanted in the spine or femur in a factorial experimental design, allowing single-shot or continuous 72 h local administration of 0.5%, 2.5% or 5.0% bupivacaine hydrochloride. During the 30-day follow-up, animal weight was recorded and blood samples were obtained. Implantation sites underwent histopathological scoring for muscle damage, inflammation, necrosis, periosteal reaction/thickening and osteoblast activity. Effects of bupivacaine concentration, administration mode and implantation site on local toxicity scores were analyzed., Results: Chi-squared tests for score frequencies revealed a concentration-dependent decrease in osteoblast count. Moreover, spinal screw implantation led to significantly more muscle fibrosis but less bone damage than femoral screw implantation, reflecting the more invasive muscle dissection and shorter drilling times related to the spinal procedure. No differences between bupivacaine administration modes regarding histological scoring or body weight changes were observed. Weight increased, while CK levels and leukocyte counts decreased significantly during follow-up, reflecting postoperative recovery. No significant differences in weight, leukocyte count and CK were found between interventional groups., Conclusion: This pilot study found limited concentration-dependent local tissue effects of bupivacaine solutions concentrated up to 5.0% following musculoskeletal surgery in the rat study population., (© 2023. The Author(s).)

Objective: Drug delivery platforms that allow for gradual drug release after intra-articular administration have become of much interest as a treatment strategy for osteoarthritis (OA). The aim of this study was to investigate the safety and efficacy of an intra-articular sustained release formulation containing celecoxib (CXB), a cyclooxygenase-2 (COX-2) selective inhibitor., Methods: Amino acid-based polyesteramide microspheres (PEAMs), a biodegradable and non-toxic platform, were loaded with CXB and employed in two in vivo models of arthritis: an acute inflammatory arthritis model in rats (n = 12), and a randomized controlled study in chronic OA dog patients (n = 30). In parallel, the bioactivity of sustained release of CXB was evaluated in monolayer cultures of primary dog chondrocytes under inflammatory conditions., Results: Sustained release of CXB did not alleviate acute arthritis signs in the rat arthritis model, based on pain measurements and synovitis severity. However, in OA dog patients, sustained release of CXB improved limb function as objective parameter of pain and quality of life based on gait analysis and owner questionnaires. It also decreased pain medication dependency over a 2-month period and caused no adverse effects. Prostaglandin E 2 levels, a marker for inflammation, were lower in the synovial fluid of CXB-treated dog OA patients and in CXB-treated cultured dog chondrocytes., Conclusion: These results show that local sustained release of CXB is less suitable to treat acute inflammation in arthritic joints, while safe and effective in treating pain in chronic OA in dogs., (Copyright © 2022 Utrecht University. Published by Elsevier Ltd.. All rights reserved.)

Background Context: Cervical disc replacement (CDR) aims to restore motion of the treated level to reduce the risk of adjacent segment disease (ASD) compared with spinal fusion. However, first-generation articulating devices are unable to mimic the complex deformation kinematics of a natural disc. Thus, a biomimetic artificial intervertebral CDR (bioAID), containing a hydroxyethylmethacrylate (HEMA)-sodium methacrylate (NaMA) hydrogel core representing the nucleus pulposus, an ultra-high-molecular-weight-polyethylene fiber jacket as annulus fibrosus, and titanium endplates with pins for primary mechanical fixation, was developed., Purpose: To assess the initial biomechanical effect of the bioAID on the kinematic behavior of the canine spine, an ex vivo biomechanical study in 6-degrees-of-freedom was performed., Study Design: A canine cadaveric biomechanical study., Methods: Six cadaveric canine specimens (C3-C6) were tested in flexion-extension (FE), lateral bending (LB) axial rotation (AR) using a spine tester in three conditions: intact, after C4-C5 disc replacement with bioAID, and after C4-C5 interbody fusion. A hybrid protocol was used where first the intact spines were subjected to a pure moment of ±1 Nm, whereafter the treated spines were subjected to the full range of motion (ROM) of the intact condition. 3D segmental motions at all levels were measured while recording the reaction torsion. Biomechanical parameters studied included ROM, neutral zone (NZ), and intradiscal pressure (IDP) at the adjacent cranial level (C3-C4)., Results: The bioAID retained the sigmoid shape of the moment-rotation curves with a NZ similar to the intact condition in LB and FE. Additionally, the normalized ROMs at the bioAID-treated level were statistically equivalent to intact during FE and AR while slightly decreased in LB. At the two adjacent levels, ROMs showed similar values for the intact compared to the bioAID for FE and AR and an increase in LB. In contrast, levels adjacent to the fused segment showed an increased motion in FE and LB as compensation for the loss of motion at the treated level. The IDP at the adjacent C3-C4 level after implantation of bioAID was close to intact values. After fusion, increased IDP was found compared with intact but did not reach statistical significance., Conclusion: This study indicates that the bioAID can mimic the kinematic behavior of the replaced intervertebral disc and preserves that for the adjacent levels better than fusion. As a result, CDR using the novel bioAID is a promising alternative treatment for replacing severely degenerated intervertebral discs., Competing Interests: Keita Ito is an Editorial Board member of JOR Spine and a co‐author of this article. To minimize bias, they were excluded from all editorial decision‐making related to the acceptance of this article for publication. [Correction added on 22 June 2023, after first online publication: Conflict of Interest statement was revised], (© 2023 The Authors. JOR Spine published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

Background: In vitro studies using nucleus pulposus (NP) cells are commonly used to investigate disc cell biology and pathogenesis, or to aid in the development of new therapies. However, lab-to-lab variability jeopardizes the much-needed progress in the field. Here, an international group of spine scientists collaborated to standardize extraction and expansion techniques for NP cells to reduce variability, improve comparability between labs and improve utilization of funding and resources., Methods: The most commonly applied methods for NP cell extraction, expansion, and re-differentiation were identified using a questionnaire to research groups worldwide. NP cell extraction methods from rat, rabbit, pig, dog, cow, and human NP tissue were experimentally assessed. Expansion and re-differentiation media and techniques were also investigated., Results: Recommended protocols are provided for extraction, expansion, and re-differentiation of NP cells from common species utilized for NP cell culture., Conclusions: This international, multilab and multispecies study identified cell extraction methods for greater cell yield and fewer gene expression changes by applying species-specific pronase usage, 60-100 U/ml collagenase for shorter durations. Recommendations for NP cell expansion, passage number, and many factors driving successful cell culture in different species are also addressed to support harmonization, rigor, and cross-lab comparisons on NP cells worldwide., Competing Interests: The authors have no relevant conflicts of interest to declare in relation to this article., (© 2023 The Authors. JOR Spine published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

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., Competing Interests: The authors declare no conflicts of interest., (© 2022 The Authors. JOR Spine published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

Background: Cartilage regenerative mechanisms initiated by knee joint distraction (KJD) remain elusive. Animal experiments that are representative for the human osteoarthritic situation and investigate the effects of KJD at consecutive time points could be helpful in this respect but are lacking. This study investigated the effects of KJD on the osteoarthritic joint of dogs on two consecutive timepoints., Methods: Osteoarthritis was bilaterally induced for 10 weeks in 12 dogs using the groove model. Subsequently, KJD was applied to the right hindlimb for 8 weeks. The cartilage, subchondral bone and synovial membrane were investigated directly after KJD treatment, and after 10 weeks of follow-up after KJD treatment. Macroscopic and microscopic joint tissue alterations were investigated using the OARSI grading system. Additionally, proteoglycan content and synthesis of the cartilage were assessed biochemically. RT-qPCR analysis was used to explore involved signaling pathways., Results: Directly after KJD proteoglycan and collagen type II content were reduced accompanied by decreased proteoglycan synthesis. After 10 weeks of follow-up, proteoglycan and collagen type II content were partly restored and proteoglycan synthesis increased. RT-qPCR analysis of the cartilage suggests involvement of the TGF-β and Notch signalling pathways. Additionally, increased subchondral bone remodelling was found at 10 weeks of follow-up., Conclusion: While the catabolic environment in the cartilage is still present directly after KJD, at 10 weeks of follow-up a switch towards a more anabolic joint environment was observed. Further investigation of this timepoint and the pathways involved might elucidate the regenerative mechanisms behind KJD., The Translational Potential of This Article: Further elucidation of the regenerative mechanisms behind KJD could improve the existing KJD treatment. Furthermore, these findings could provide input for the discovery or improvement of other joint regenerative treatment strategies., Competing Interests: FPJGL is consultant as UMCU employee for Synerkine Pharma BV and co-founder of ArthroSave BV without further relations. All authors declare that there is no conflict of interest., (© 2022 The Authors.)

Background: Intervertebral disc (IVD) degeneration is suggested as a major cause of chronic low back pain (LBP). Intradiscal delivery of growth factors has been proposed as a promising strategy for IVD repair and regeneration. Previously, BMP-4 was shown to be more potent in promoting extracellular matrix (ECM) production than other BMPs and TGF-β in human nucleus pulposus (NP) cells, suggesting its applicability for disc regeneration., Methods: The effects of BMP-4 on ECM deposition and cell proliferation were assessed in sheep NP and annulus fibrosus (AF) cells in a pellet culture model. Further, a nuclectomy induced sheep lumbar IVD degeneration model was used to evaluate the safety and effects of intradiscal BMP-4 injection on IVD regeneration. Outcomes were assessed by magnetic resonance imaging, micro-computed tomography, histological and biochemical measurements., Results: In vitro, BMP-4 significantly increased the production of proteoglycan and deposition of collagen type II and proliferation of NP and AF cells. Collagen type I deposition was not affected in NP cells, while in AF cells it was high at low BMP-4 concentrations, and decreased with increasing concentration of BMP-4. Intradiscal injection of BMP-4 induced extradiscal new bone formation and Schmorl's node-like changes in vivo. No regeneration in the NP nor AF was observed., Conclusion: Our study demonstrated that although BMP-4 showed promising regenerative effects in vitro, similar effects were not observed in a large IVD degeneration animal model., The Translational Potential of This Article: The contradictory results of using BMP-4 on IVD regeneration between in vitro and in vivo demonstrate that direct BMP-4 injection for disc degeneration-associated human chronic low back pain should not be undertaken. In addition, our results may also shed light on the mechanisms behind pathological endplate changes in human patients as a possible target for therapy., Competing Interests: The authors have no conflicts of interest relevant to this article., (© 2022 The Authors.)

Porcine notochordal cell-derived matrix (NCM) has anti-inflammatory and regenerative effects on degenerated intervertebral discs. For its clinical use, safety must be assured. The porcine DNA is concerning because of (1) the transmission of endogenous retroviruses and (2) the inflammatory potential of cell-free DNA. Here, we present a simple, detergent-free protocol: tissue lyophilization lyses cells, and matrix integrity is preserved by limiting swelling during decellularization. DNA is digested quickly by a high nuclease concentration, followed by a short washout. Ninety-four percent of DNA was removed, and there was no loss of glycosaminoglycans or collagen. Forty-three percent of the total proteins remained in the decellularized NCM (dNCM). dNCM stimulated as much GAG production as NCM in nucleus pulposus cells but lost some anti-inflammatory effects. Reconstituted pulverized dNCM yielded a soft, shear-thinning biomaterial with a swelling ratio of 350% that also acted as an injectable cell carrier (cell viability &gt;70%). dNCM can therefore be used as the basis for future biomaterials aimed at disc regeneration on a biological level and may restore joint mechanics by creating swelling pressure within the intervertebral disc.

Background: Synovial membrane-derived mesenchymal progenitor cells (SM-MPCs) are a promising candidate for the cell-based treatment of osteoarthritis (OA) considering their in vitro and in vivo capacity for cartilage repair. However, the OA environment may adversely impact their regenerative capacity. There are no studies for canine (c)SM-MPCs that compare normal to OA SM-MPCs, even though dogs are considered a relevant animal model for OA. Therefore, this study compared cSM-MPCs from normal and OA synovial membrane tissue to elucidate the effect of the OA environment on MPC numbers, indicated by CD marker profile and colony-forming unit (CFU) capacity, and the impact of the OA niche on tri-lineage differentiation., Methods: Normal and OA synovial membrane were collected from the knee joints of healthy dogs and dogs with rupture of the cruciate ligaments. The synovium was assessed by histopathological OARSI scoring and by RT-qPCR for inflammation/synovitis-related markers. The presence of cSM-MPCs in the native tissue was further characterized with flow cytometry, RT-qPCR, and immunohistochemistry, using the MPC markers; CD90, CD73, CD44, CD271, and CD34. Furthermore, cells isolated upon enzymatic digestion were characterized by CFU capacity, and a population doublings assay. cSM-MPCs were selected based on plastic adherence, expanded to passage 2, and evaluated for the expression of MPC-related surface markers and tri-lineage differentiation capacity., Results: Synovial tissue collected from the OA joints had a significantly higher OARSI score compared to normal joints, and significantly upregulated inflammation/synovitis markers S100A8/9, IL6, IL8, and CCL2. Both normal and OA synovial membrane contained cells displaying MPC properties, including a fibroblast-like morphology, CFU capacity, and maintained MPC marker expression over time during expansion. However, OA cSM-MPCs were unable to differentiate towards the chondrogenic lineage and had low adipogenic capacity in contrast to normal cSM-MPCs, whereas they possessed a higher osteogenic capacity. Furthermore, the OA synovial membrane contained significantly lower percentages of CD90+, CD44+, CD34+, and CD271+ cells., Conclusions: The OA environment had adverse effects on the regenerative potential of cSM-MPCs, corroborated by decreased CFU, population doubling, and chondrogenic capacity compared to normal cSM-MPCs. OA cSM-MPCs may be a less optimal candidate for the cell-based treatment of OA than normal cSM-MPCs., (© 2022. The Author(s).)

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., (© 2021 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

Background: Repopulating the degenerated intervertebral disc (IVD) with tissue-specific nucleus pulposus cells (NPCs) has already been shown to promote regeneration in various species. Yet the applicability of NPCs as cell-based therapy has been hampered by the low cell numbers that can be extracted from donor IVDs and their potentially limited regenerative capacity due to their degenerated phenotype. To optimize the expansion conditions, we investigated the effects of increasing culture medium osmolarity during expansion on the phenotype of dog NPCs and their ability to produce a healthy extracellular matrix (ECM) in a 3D culture model., Methods: Dog NPCs were expanded in expansion medium with a standard osmolarity of 300 mOsm/L or adjusted to 400 or 500 mOsm/L in both normoxic and hypoxic conditions. Following expansion, NPCs were cultured in a 3D culture model in chondrogenic culture medium with a standard osmolarity. Read-out parameters included cell proliferaton rate, morphology, phenotype and healthy ECM production., Results: Increasing the expansion medium osmolarity from 300 to 500 mOsm/L resulted in NPCs with a more rounded morphology and a lower cell proliferation rate accompanied by the expression of several healthy NPC and progenitor markers at gene ( KRT18, ACAN, COL2, CD73, CD90 ) and protein (ACAN, PAX1, CD24, TEK, CD73) level. The NPCs expanded at 500 mOsm/L were able to retain most of their phenotypic markers and produce healthy ECM during 3D culture independent of the oxygen level used during expansion., Conclusions: Altogether, our findings show that increasing medium osmolarity during expansion results in an NPC population with improved phenotype, which could enhance the potential of cell-based therapies for IVD regeneration., Competing Interests: The authors declare no conflicts of interest., (© 2022 The Authors. JOR Spine published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

The mouse outer annulus fibrosus (AF) was previously shown to contain CD146 + AF cells, while in vitro culture and exposure to transforming growth factor-beta (TGF-β) further increased the expression of CD146. However, neither the specific function of CD146 nor the underlying mechanism of TGF-β upregulation of CD146 + AF cells have been elucidated yet. In the current study, CD146 expression and its role in cultured human AF cells was investigated studying the cells' capacity for matrix contraction and gene expression of functional AF markers. In addition, TGF-β pathways were blocked by several pathway inhibitors and short hairpin RNAs (shRNAs) targeting SMAD and non-SMAD pathways to investigate their involvement in TGF-β-induced CD146 upregulation. Results showed that knockdown of CD146 led to reduction in AF cell-mediated collagen gel contraction, downregulation of versican and smooth muscle protein 22α (SM22α), and upregulation of scleraxis. TGF-β-induced CD146 upregulation was significantly blocked by inhibition of TGF-β receptor ALK5, and partially inhibited by shRNA against SMAD2 and SMAD4 and by an Protein Kinase B (AKT) inhibitor. Interestingly, the inhibition of extracellular signal-regulated kinases (ERK) pathway induced CD146 upregulation. In conclusion, CD146 was shown to be crucial to maintain the cell contractility of human AF cells in vitro. Furthermore, TGF-β upregulated CD146 via ALK5 signaling cascade, partially through SMAD2, SMAD4, and AKT pathway, whereas, ERK was shown to be a potential negative modulator. Our findings suggest that CD146 can potentially be used as a functional marker in AF repair strategies., (© 2021 Orthopaedic Research Society. Published by Wiley Periodicals LLC.)

Background: The perspectives of researchers and patients regarding roles and responsibilities in stem cell research are rarely studied, but these could offer insights about responsible research conduct. Method: We have conducted a qualitative study consisting of focus groups with both early- (n = 7) and late-career stem cell researchers (n = 11) that are primarily based in Europe, and with Dutch patients with chronic lower back pain (n = 9). These focus groups have been analyzed thematically. Results: Four themes were identified: 1) roles and responsibilities in the laboratory, 2) responsibilities of and toward patients and the public, 3) the role of regulation and 4) structural hurdles for responsibility. Discussion: The results suggest that responsible research conduct could be improved by addressing grant application procedures, publication pressure and by providing support of dissemination activities for researchers. Conclusion: Responsibility in stem cell research could be enhanced by embracing open science initiatives and targeted training.

Osteoarthritis (OA) and chronic low back pain due to degenerative (intervertebral) disc disease (DDD) are two of the major causes of disabilities worldwide, affecting hundreds of millions of people and leading to a high socioeconomic burden. Although OA occurs in synovial joints and DDD occurs in cartilaginous joints, the similarities are striking, with both joints showing commonalities in the nature of the tissues and in the degenerative processes during disease. Consequently, repair strategies for articular cartilage (AC) and nucleus pulposus (NP), the core of the intervertebral disc, in the context of OA and DDD share common aspects. One of such tissue engineering approaches is the use of injectable hydrogels for AC and NP repair. In this review, the state-of-the-art and recent developments in injectable hydrogels for repairing, restoring, and regenerating AC tissue suffering from OA and NP tissue in DDD are summarized focusing on cell-free approaches. The various biomaterial strategies exploited for repair of both tissues are compared, and the synergies that could be gained by translating experiences from one tissue to the other are identified. Impact statement Joints affected by osteoarthritis (OA) and degenerative (intervertebral) disc disease (DDD) share similarities in tissue composition and in the degenerative disease processes. This has led to the development of similar tissue engineering approaches to repair the articular cartilage (AC) and the nucleus pulposus (NP), in the context of OA and DDD, such as injectable hydrogels. In this review, recent developments in injectable hydrogels for repair of AC and NP tissues are summarized, biomaterial strategies are compared, and synergies are identified focusing on cell-free approaches. The summarized developments are expected to inspire more cross talk between both research fields.

Chronic low back pain is the number one cause of years lived with disability. In about 40% of patients, chronic lower back pain is related to intervertebral disc (IVD) degeneration. The standard-of-care focuses on symptomatic relief, while surgery is the last resort. Emerging therapeutic strategies target the underlying cause of IVD degeneration and increasingly focus on the relatively overlooked notochordal cells (NCs). NCs are derived from the notochord and once the notochord regresses they remain in the core of the developing IVD, the nucleus pulposus. The large vacuolated NCs rapidly decline after birth and are replaced by the smaller nucleus pulposus cells with maturation, ageing, and degeneration. Here, we provide an update on the journey of NCs and discuss the cell markers and tools that can be used to study their fate and regenerative capacity. We review the therapeutic potential of NCs for the treatment of IVD-related lower back pain and outline important future directions in this area. Promising studies indicate that NCs and their secretome exerts regenerative effects, via increased proliferation, extracellular matrix production, and anti-inflammatory effects. Reports on NC-like cells derived from embryonic- or induced pluripotent-stem cells claim to have successfully generated NC-like cells but did not compare them with native NCs for phenotypic markers or in terms of their regenerative capacity. Altogether, this is an emerging and active field of research with exciting possibilities. NC-based studies demonstrate that cues from developmental biology can pave the path for future clinical therapies focused on regenerating the diseased IVD., Competing Interests: KI is a paid consultant and shareholder at NC Biomatrix BV. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Bach, Poramba-Liyanage, Riemers, Guicheux, Camus, Iatridis, Chan, Ito, Le Maitre and Tryfonidou.)

Gross morphology of healthy and degenerated intervertebral discs (IVDs) is largely similar in horses as in dogs and humans. For further comparison, the biochemical composition and the histological and biochemical changes with age and degeneration were analyzed in 41 warmblood horses. From 33 horses, 139 discs and 2 fetal vertebral columns were evaluated and scored histologically. From 13 horses, 73 IVDs were assessed for hydration, DNA, glycosaminoglycans, total collagen, hydroxyl-lysyl-pyridinoline, hydroxylysine, and advanced glycation end-product (AGE) content. From 7 horses, 20 discs were assessed for aggrecan, fibronectin, and collagen type 1 and 2 content. Histologically, tearing of the nucleus pulposus (NP) and cervical annulus fibrosus (AF), and total histological score (tearing and vascular proliferation of the AF, and chondroid metaplasia, chondrocyte-like cell proliferation, presence of notochordal cells, matrix staining, and tearing of the NP) correlated with gross degeneration. Notochordal cells were not seen in IVDs of horses. Age and gross degeneration were positively correlated with AGEs and a fibrotic phenotype, explaining gross degenerative changes. In contrast to dogs and humans, there was no consistent difference in glycosaminoglycan content and hydration between AF and NP, nor decrease of these variables with age or degeneration. Hydroxylysine decrease and collagen 1 and AGEs increase were most prominent in the NP, suggesting degeneration started in the AP. In caudal cervical NPs, AGE deposition was significantly increased in grossly normal IVDs and total collagen significantly increased with age, suggesting increased biomechanical stress and likelihood for spinal disease in this part of the vertebral column.

The concept of a novel patient-specific 3D-printed shelf implant should be evaluated in a relevant large animal model with hip dysplasia. Therefore, three dogs with radiographic bilateral hip dysplasia and a positive subluxation test underwent unilateral acetabular augmentation with a 3D-printed dog-specific titanium implant. The contralateral side served as control. The implants were designed on CT-based pelvic bone segmentations and extended the dysplastic acetabular rim to increase the weight bearing surface without impairing the range of motion. Outcome was assessed by clinical observation, manual subluxation testing, radiography, CT, and gait analysis from 6 weeks preoperatively until termination at 26 weeks postoperatively. Thereafter, all hip joints underwent histopathological examination. The implantation and recovery from surgery was uneventful. Clinical subluxation tests at the intervention side became negative. Imaging showed medialization of the femoral head at the intervention side and the mean (range) CE-angle increased from 94° (84°-99°) preoperative to 119° (117°-120°) postoperative. Gait analysis parameters returned to pre-operative levels after an average follow-up of 6 weeks. Histology showed a thickened synovial capsule between the implant and the femoral head without any evidence of additional damage to the articular cartilage compared to the control side. The surgical implantation of the 3D shelf was safe and feasible. The patient-specific 3D-printed shelf implants restored the femoral head coverage and stability of dysplastic hips without complications. The presented approach holds promise to treat residual hip dysplasia justifying future veterinary clinical trials to establish clinical effectiveness in a larger cohort to prepare for translation to human clinic., (© 2022. The Author(s).)

Objective: Folate receptor beta (FR-β) has been used as a clinical marker and target in multiple inflammatory diseases, including osteoarthritis (OA) and rheumatoid arthritis (RA). However, the conditions under which FR-β + macrophages arise remain unclear and could be affected by corticosteroids. Therefore, we studied FR-β expression in vitro in macrophage subtypes and determined their response to triamcinolone acetonide (TA), a clinically often-used corticosteroid., Design: Human monocyte-derived macrophages were differentiated to the known M0, M1, or M2 macrophage phenotypes. The phenotype and FR-β expression and plasticity of the macrophage subtypes were determined using flow cytometry, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and enzyme-linked immunosorbent assay (ELISA)., Results: FR-β expression was low in granulocyte-macrophage colony-stimulating factor (GM-CSF)-generated (M1-like) macrophages and high in macrophage colony-stimulating factor (M-CSF)-generated (M0 and M2-like) macrophages. FR-β expression remained high once the M0 or M2 macrophages were stimulated with pro-inflammatory stimuli (interferon-γ plus lipopolysaccharide) to induce M1-like macrophages. On the contrary, anti-inflammatory TA treatment skewed GM-CSF macrophage differentiation toward an M2 and FR-β + phenotype., Conclusions: As corticosteroids skewed monocytes toward an FR-β-expressing, anti-inflammatory phenotype, even in an M1 priming GM-CSF environment, FR-β has potential as a biomarker to monitor success of treatment with corticosteroids. Without corticosteroid treatment, M-CSF alone induces high FR-β expression which remains high under pro-inflammatory conditions. This explains why pro-inflammatory FR-β + macrophages (exposed to M-CSF) are observed in arthritis patients and correlate with disease severity.

Hip dysplasia (HD) is common in both humans and dogs. This interconnection is because humans and dogs descended from a common ancestor and therefore have a similar anatomy at micro- and macroscopic levels. Furthermore, dogs are the animals of choice for testing new treatments for human hip dysplasia and orthopedic surgery in general. However, little literature exists comparing HD between the two species. Therefore, the aim of this review is to describe the anatomy, etiology, pathogenesis, diagnostics, and treatment of HD in humans and dogs. HD as an orthopedic condition has many common characteristics in terms of etiology and pathogenesis and most of the differences can be explained by the evolutionary differences between dogs and humans. Likewise, the treatment of HD shows many commonalities between humans and dogs. Conservative treatment and surgical interventions such as femoral osteotomy, pelvic osteotomy and total hip arthroplasty are very similar between humans and dogs. Therefore, future integration of knowledge and experiences for HD between dogs and humans could be beneficial for both species., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Willemsen, Möring, Harlianto, Tryfonidou, van der Wal, Weinans, Meij and Sakkers.)

Objective: Joint distraction triggers intrinsic cartilage repair in animal models of osteoarthritis (OA), corroborating observations in human OA patients treated with joint distraction. The present study explores the still largely elusive mechanism initiating this repair process., Design: Unilateral OA was induced in the knee joint of 8 dogs using the groove model; the contralateral joint served as a control. After 10 weeks, 4 animals received joint distraction, the other 4 serving as OA controls. Halfway the distraction period (after 4 weeks of a standard 8-week distraction treatment), all animals were euthanized, and joint tissues were collected. A targeted quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis was performed of commonly involved processes including matrix catabolism/anabolism, inflammation, and known signaling pathways in OA. In addition, cartilage changes were determined on tissue sections using the canine OARSI (Osteoarthritis Research Society International) histopathology score and collagen type II (COL2A1) immunostaining., Results: Midway distraction, the distracted OA joint showed an upregulation of proteolytic genes, for example, ADAMTS5 , MMP9 , MMP13 , compared to OA alone and the healthy joints, which correlated with an increased OARSI score. Additionally, genes of the transforming growth factor (TGF)-β and Notch pathway, and markers associated with progenitor cells were increased., Conclusions: Joint distraction initiates both catabolic and anabolic transcriptional responses. The enhanced turnover, and thereby renewal of the matrix, could be the key to the cartilage repair observed in the months after joint distraction.

Lower back pain (LBP) occurs in 80% of adults in their lifetime; resulting in LBP being one of the biggest causes of disability worldwide. Chronic LBP has been linked to the degeneration of the intervertebral disc (IVD). The current treatments for chronic back pain only provide alleviation of symptoms through pain relief, tissue removal, or spinal fusion; none of which target regenerating the degenerate IVD. As nucleus pulposus (NP) degeneration is thought to represent a key initiation site of IVD degeneration, cell therapy that specifically targets the restoration of the NP has been reviewed here. A literature search to quantitatively assess all cell types used in NP regeneration was undertaken. With key cell sources: NP cells; annulus fibrosus cells; notochordal cells; chondrocytes; bone marrow mesenchymal stromal cells; adipose-derived stromal cells; and induced pluripotent stem cells extensively analyzed for their regenerative potential of the NP. This review highlights: accessibility; expansion capability in vitro; cell survival in an IVD environment; regenerative potential; and safety for these key potential cell sources. In conclusion, while several potential cell sources have been proposed, iPSC may provide the most promising regenerative potential., Competing Interests: The authors declare no conflicts of interest., (© 2021 The Authors. JOR Spine published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

Background: Cushing's disease (CD) is defined as hypercortisolemia caused by adrenocorticotropic hormone (ACTH)-secreting pituitary adenomas (corticotroph PA) that afflicts humans and dogs. In order to map common aberrant genomic features of CD between humans and dogs, we performed genomic sequencing and immunostaining on corticotroph PA., Methods: For inclusion, humans and dog were diagnosed with CD. Whole exome sequencing (WES) was conducted on 6 human corticotroph PA. Transcriptome RNA-Seq was performed on 6 human and 7 dog corticotroph PA. Immunohistochemistry (IHC) was complete on 31 human corticotroph PA. Corticotroph PA were compared with normal tissue and between species analysis were also performed., Results: Eight genes (MAMLD1, MNX1, RASEF, TBX19, BIRC5, TK1, GLDC, FAM131B) were significantly (P < 0.05) overexpressed across human and canine corticotroph PA. IHC revealed MAMLD1 to be positively (3+) expressed in the nucleus of ACTH-secreting tumor cells of human corticotroph PA (22/31, 70.9%), but absent in healthy human pituitary glands., Conclusions: In this small exploratory cohort, we provide the first preliminary insights into profiling the genomic characterizations of human and dog corticotroph PA with respect to MAMLD1 overexpression, a finding of potential direct impact to CD microadenoma diagnosis. Our study also offers a rationale for potential use of the canine model in development of precision therapeutics., (© 2021. The Author(s).)

There is an increasing interest in controlled release systems for local therapy in the treatment of human and equine joint diseases, aiming for optimal intra-articular concentrations with no systemic side effects. In this study, the intra-articular tolerability and suitability for local and sustained release of tacrolimus (FK506) from monospheres composed of [PDLA-PEG 1000 ]- b -PLLA multiblock copolymers were investigated. Unloaded and tacrolimus-loaded (18.4 mg tacrolimus/joint) monospheres were injected into the joints of six healthy horses, with saline and hyaluronic acid (HA) in the contralateral joints as controls. Blood and synovial fluid were analysed for the tacrolimus concentration and biomarkers for inflammation and cartilage metabolism. After an initial burst release, sustained intra-articular tacrolimus concentrations (>20 ng/mL) were observed during the 42 days follow-up. Whole-blood tacrolimus levels were below the detectable level (<0.5 ng/mL). A transient inflammatory reaction was observed for all substances, evidenced by increases of the synovial fluid white blood cell count and total protein. Prostaglandin and glycosaminoglycan release were increased in joints injected with unloaded monospheres, which was mitigated by tacrolimus. Both tacrolimus-loaded monospheres and HA transiently increased the concentration of collagen II cleavage products (C2C). A histologic evaluation of the joints at the endpoint showed no pathological changes in any of the conditions. Together, these results indicate the good biocompatibility of intra-articular applied tacrolimus-loaded monospheres combined with prolonged local drug release while minimising the risk of systemic side effects. Further evaluation in a clinical setting is needed to determine if tacrolimus-loaded monospheres can be beneficial in the treatment of inflammatory joint diseases in humans and animals.