Sustained release of locally delivered celecoxib provides pain relief for osteoarthritis: a proof of concept in dog patients.

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 E2 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.

Nanoghosts: Mesenchymal Stem cells derived nanoparticles as a unique approach for cartilage regeneration.

Osteoarthritis (OA) is a chronic degenerative disease, which affects the joints and is characterized by inflammation, cartilage loss and bone changes. Nowadays, there are no treatments for OA, and current therapies are focused on relieving the symptoms. As a new therapy approach, micro and nanoparticles have been extensively explored and among all the studied particles, the use of cell-membrane-based particles is expanding. Another promising approach studied to treat OA, is the use of mesenchymal stem cells (MSCs) which play an important role modulating inflammation. We developed a novel kind of MSCs' cytoplasmic-membrane-based nanoparticles, termed nano-ghosts (NGs). Retaining MSCs' surface properties and lacking cells' internal machinery allow the NGs to have immunomodulatory capacity and to be immune-evasive while not susceptible to host-induced changes. In this study, we demonstrate NGs' ability to target cartilage tissues, in vitro and in vivo, while modulating the inflammatory process. In vivo studies demonstrated NGs ability to act as an immunomodulatory drug slowing down cartilage degeneration process. Our proof-of-concept experiments show that NGs system is a versatile nano-carrier system, capable of therapeutics loading, with targeting capabilities towards healthy and inflamed cartilage cells. Our results, along with previously published data, clearly reveal the NGs system as a promising nano-carrier platform and as a potential immunomodulatory drug for several inflammation-related diseases.

Efficacy of one-stage cartilage repair using allogeneic mesenchymal stromal cells and autologous chondron transplantation (IMPACT) compared to nonsurgical treatment for focal articular cartilage lesions of the knee: study protocol for a crossover randomized controlled trial.

BACKGROUND: Articular cartilage defects in the knee have poor intrinsic healing capacity and may lead to functional disability and osteoarthritis (OA). "Instant MSC Product accompanying Autologous Chondron Transplantation" (IMPACT) combines rapidly isolated recycled autologous chondrons with allogeneic MSCs in a one-stage surgery. IMPACT was successfully executed in a first-in-man investigator-driven phase I/II clinical trial in 35 patients. The purpose of this study is to compare the efficacy of IMPACT to nonsurgical treatment for the treatment of large (2-8 cm2) articular cartilage defects in the knee. METHODS: Sixty patients will be randomized to receive nonsurgical care or IMPACT. After 9 months of nonsurgical care, patients in the control group are allowed to receive IMPACT surgery. The Knee Injury and Osteoarthritis Outcome Score (KOOS), pain (numeric rating scale, NRS), and EuroQol five dimensions five levels (EQ5D-5 L) will be used to compare outcomes at baseline and 3, 6, 9, 12, and 18 months after inclusion. Cartilage formation will be assessed at baseline, and 6 and 18 months after inclusion using MRI. An independent rheumatologist will monitor the onset of a potential inflammatory response. (Severe) adverse events will be recorded. Lastly, the difference between IMPACT and nonsurgical care in terms of societal costs will be assessed by monitoring healthcare resource use and productivity losses during the study period. A health economic model will be developed to estimate the incremental cost-effectiveness ratio of IMPACT vs. nonsurgical treatment in terms of costs per quality adjusted life year over a 5-year time horizon. DISCUSSION: This study is designed to evaluate the efficacy of IMPACT compared to nonsurgical care. Additionally, safety of IMPACT will be assessed in 30 to 60 patients. Lastly, this study will evaluate the cost-effectiveness of IMPACT compared to nonsurgical care. TRIAL REGISTRATION: NL67161.000.18 [Registry ID: CCMO] 2018#003470#27 [EU-CTR; registered on 26 March 2019] NCT04236739 [ ClinicalTrials.gov ] [registered after start of inclusion; 22 January 2020].

Intradiscal delivery of celecoxib-loaded microspheres restores intervertebral disc integrity in a preclinical canine model.

Low back pain, related to degeneration of the intervertebral disc (IVD), affects millions of people worldwide. Clinical studies using oral cyclooxygenase-2 (COX-2) inhibitors have shown beneficial effects, although side-effects were reported. Therefore, intradiscal delivery of nonsteroidal anti-inflammatory drugs can be an alternative treatment strategy to halt degeneration and address IVD-related pain. In the present study, the controlled release and biologic potency of celecoxib, a selective COX-2 inhibitor, from polyesteramide microspheres was investigated in vitro. In addition, safety and efficacy of injection of celecoxib-loaded microspheres were evaluated in vivo in a canine IVD degeneration model. In vitro, a sustained release of celecoxib was noted for over 28 days resulting in sustained inhibition of inflammation, as indicated by decreased prostaglandin E2 (PGE2) production, and anti-catabolic effects in nucleus pulposus (NP) cells from degenerated IVDs on qPCR. In vivo, there was no evidence of adverse effects on computed tomography and magnetic resonance imaging or macroscopic evaluation of IVDs. Local and sustained delivery of celecoxib prevented progression of IVD degeneration corroborated by MRI, histology, and measurement of NP proteoglycan content. Furthermore, it seemed to harness inflammation as indicated by decreased PGE2 tissue levels and decreased neuronal growth factor immunopositivity, providing indirect evidence that local delivery of a COX-2 inhibitor could also address pain related to IVD degeneration. In conclusion, intradiscal controlled release of celecoxib from polyesteramide microspheres prevented progression of IVD degeneration both in vitro and in vivo. Follow-up studies are warranted to determine the clinical efficacy of celecoxib-loaded PEAMs in chronic back pain.

Controlled release of celecoxib inhibits inflammation, bone cysts and osteophyte formation in a preclinical model of osteoarthritis.

Major hallmarks of osteoarthritis (OA) are cartilage degeneration, inflammation and osteophyte formation. COX-2 inhibitors counteract inflammation-related pain, but their prolonged oral use entails the risk for side effects. Local and prolonged administration in biocompatible and degradable drug delivery biomaterials could offer an efficient and safe treatment for the long-term management of OA symptoms. Therefore, we evaluated the disease-modifying effects and the optimal dose of polyesteramide microspheres delivering the COX-2 inhibitor celecoxib in a rat OA model. Four weeks after OA induction by anterior cruciate ligament transection and partial medial meniscectomy, 8-week-old female rats (n = 6/group) were injected intra-articular with celecoxib-loaded microspheres at three dosages (0.03, 0.23 or 0.39 mg). Unloaded microspheres served as control. During the 16-week follow-up, static weight bearing and plasma celecoxib concentrations were monitored. Post-mortem, micro-computed tomography and knee joint histology determined progression of synovitis, osteophyte formation, subchondral bone changes, and cartilage integrity. Systemic celecoxib levels were below the detection limit 6 days upon delivery. Systemic and local adverse effects were absent. Local delivery of celecoxib reduced the formation of osteophytes, subchondral sclerosis, bone cysts and calcified loose bodies, and reduced synovial inflammation, while cartilage histology was unaffected. Even though the effects on pain could not be evualated directly in the current model, our results suggest the application of celecoxib-loaded microspheres holds promise as novel, safe and effective treatment for inflammation and pain in OA.

Soluble and pelletable factors in porcine, canine and human notochordal cell-conditioned medium: implications for IVD regeneration.

During intervertebral disc (IVD) maturation, notochordal cells (NCs) are replaced by chondrocyte-like cells (CLCs) in the nucleus pulposus, suggesting that NCs play a role in maintaining tissue health. Affirmatively, NC-conditioned medium (NCCM) exerts regenerative effects on CLC proliferation and extracellular matrix (ECM) production. The aim of this study was to identify NC-secreted substances that stimulate IVD regeneration. By mass spectrometry of porcine, canine and human NCCM, 149, 170 and 217 proteins were identified, respectively, with 66 proteins in common. Mainly ECM-related proteins were identified, but also organelle-derived and membrane-bound vesicle proteins. To determine whether the effect of NCCM was mediated by soluble and/or pelletable factors, porcine and canine NCCM were separated into a soluble (NCCM-S; peptides and proteins) and pelletable (NCCM-P; protein aggregates and extracellular vesicles) fraction by ultracentrifugation, and tested on bovine and canine CLCs in vitro, respectively. In each model, NCCM-S exerted a more pronounced anabolic effect than NCCM-P. However, glycosaminoglycan (GAG) uptake from the medium into the carrier gel prevented more definite conclusions. While the effect of porcine NCCM-P on bovine CLCs was negligible, canine NCCM-P appeared to enhance GAG and collagen type II deposition by canine CLCs. In conclusion, porcine and canine NCCM exerted their anabolic effects mainly through soluble factors, but also the pelletable NCCM factors showed moderate regenerative potential. Although the regenerative potential of NCCM-P should not be overlooked, future studies should focus on unraveling the protein-based regenerative mechanism from NCCM produced from isolated NCs, e.g. by NCCM fractionation and pathway blocking studies.

Early evolving joint degeneration by cartilage trauma is primarily mechanically controlled.

BACKGROUND: Mechanical and inflammatory processes add to osteoarthritis (OA). To what extent both processes contribute during the onset of OA after a cartilage trauma is unknown. This study evaluates whether local cartilage damage leads to focally confined or more generalized cartilage damage with synovial inflammation in the early development of joint tissue degeneration. METHODS: In nine goats, cartilage damage was surgically induced on the weight bearing area of exclusively the medial femoral condyle of the right knee joint. The other tibio-femoral compartments, lateral femoral condyle and lateral medial tibial plateau, were left untouched. The contralateral left knee joint of each animal served as an intra-animal control. Twenty weeks post-surgery changes in cartilage matrix integrity in each of the four compartments, medial and lateral synovial tissue inflammation, and synovial fluid IL-1ß and TNFα were evaluated. RESULTS: In the experimental medial femoral plateau, significant macroscopic, histologic, and biochemical cartilage damage was observed versus the contralateral control compartments. Also the articulating cartilage of the experimental medial tibial plateau was significantly more damaged. Whereas, no differences were seen between the lateral compartments of experimental and contralateral control joints. Synovial tissue inflammation was mild and only macroscopically (not histologically) significantly increased in the experimental medial compartments. Synovial fluid IL-1ß level was not different between experimental and contralateral control joints, and TNFα was overall beneath the detection limit. CONCLUSIONS: Local cartilage damage is a trigger for development of OA, which in early onset seems primarily mechanically driven. Early treatment of traumatic cartilage damage should take this mechanical component into consideration.

The species-specific regenerative effects of notochordal cell-conditioned medium on chondrocyte-like cells derived from degenerated human intervertebral discs.

During intervertebral disc (IVD) maturation, the main cell type shifts from notochordal cells (NCs) to chondrocyte-like cells (CLCs). NCs secrete factors with regenerative potential, making them an interesting focus for regenerative treatments. During initial development, these strategies preferably employ non-human donors due to easy availability of their NC-rich nucleus pulposus (NP) tissue. To increase the success of translating these strategies for clinical application, this study aimed to delineate whether NC-secreted factors of different species have a regenerative effect on human CLCs. Human, canine and porcine NC-rich NP tissue and NC-conditioned medium (NCCM) were analysed biochemically and histologically. Human CLC micro-aggregates from degenerated IVDs were cultured in human, canine or porcine NCCM. Collagen, glycosaminoglycan (GAG) and DNA content was determined and histology was performed. Canine and porcine NPs were richer in NCs than human NPs. Human NPs contained the highest collagen content, whereas the DNA and GAG content of canine NPs was significantly higher than that of human or porcine NPs. NCCM from all species significantly increased the DNA and GAG content of the human CLC micro-aggregates. Porcine and canine NCCM were significantly more potent than human NCCM in inducing GAG deposition, whereas only human NCCM induced collagen type II production. Secreted factors from human, canine and porcine NC-rich NPs exerted regenerative effects on human CLCs, indicating a cross-species effect. Bioactive compound(s) are present in NCCM of different species that may reverse human IVD degeneration, supporting further research into strategies based on NC-technology employing canine or porcine models for their translation into humans.

Biomaterials for intervertebral disc regeneration: past performance and possible future strategies.

Intervertebral disc (IVD) degeneration is associated with most cases of cervical and lumbar spine pathologies, amongst which chronic low back pain has become the number one cause of loss of quality-adjusted life years. In search of alternatives to the current less than optimal and usually highly invasive treatments, regenerative strategies are being devised, none of which has reached clinical practice as yet. Strategies include the use of stem cells, gene therapy, growth factors and biomaterial carriers. Biomaterial carriers are an important component in musculoskeletal regenerative medicine techniques. Several biomaterials, both from natural and synthetic origin, have been used for regeneration of the IVD in vitro and in vivo. Aspects such as ease of use, mechanical properties, regenerative capacity, and their applicability as carriers for regenerative and anti-degenerative factors determine their suitability for IVD regeneration. The current review provides an overview of the biomaterials used with respect to these properties, including their drawbacks. In addition, as biomaterial application until now appears to have been based on a mix of mere availability and intuition, a more rational design is proposed for future use of biomaterials for IVD regeneration. Ideally, high-throughput screening is used to identify optimally effective materials, or alternatively medium content comparative studies should be carried out to determine an appropriate reference material for future studies on novel materials.

Enhanced cell-induced articular cartilage regeneration by chondrons; the influence of joint damage and harvest site.

OBJECTIVE: Interactions between chondrocytes and their native pericellular matrix provide optimal circumstances for regeneration of cartilage. However, cartilage diseases such as osteoarthritis change the pericellular matrix, causing doubt to them as a cell source for autologous cell therapy. METHODS: Chondrons and chondrocytes were isolated from stifle joints of goats in which cartilage damage was surgically induced in the right knee. After 4 weeks of regeneration culture, DNA content and proteoglycan and collagen content and release were determined. RESULTS: The cartilage regenerated by chondrons isolated from the damaged joint contained less proteoglycans and collagen compared to chondrons from the same harvest site in the nonoperated knee (P < 0.01). Besides, chondrons still reflected whether they were isolated from a damaged joint, even if they where isolated from the opposing or adjacent condyle. Although chondrocytes did not reflect this diseased status of the joint, chondrons always outperformed chondrocytes, even when isolated from the damaged joints (P < 0.0001). Besides increased cartilage production, the chondrons showed less collagenase activity compared to the chondrocytes. CONCLUSION: Chondrons still outperform chondrocytes when they were isolated from a damaged joint and they might be a superior cell source for articular cartilage repair and cell-induced cartilage formation.

The fixation of a collagen type I/III membrane in the distal radioulnar joint of a human cadaver model.

Damage to the cartilage of the distal radioulnar joint frequently leads to pain and limitation of movement, therefore repair of this joint cartilage would be highly desirable. The purpose of this study was to investigate the fixation of scaffold in cartilage defects of this joint as part of matrix-assisted regenerative autologous cartilage techniques. Two techniques of fixation of collagen scaffolds, one involving fibrin glue alone and one with fibrin glue and sutures, were compared in artificially created cartilage defects of the distal radioulnar joint in a human cadaver. After being subjected to continuous passive rotation, the methods of fixation were evaluated for cover of the defect and pull out force. No statistically significant differences were found between the two techniques for either cover of the defect or integrity of the scaffold. However, a significantly increased mean pull out force was found for the combined procedure, 0.665 N (0.150 to 1.160) versus 0.242 N (0.060 to 0.730) for glue fixation (p = 0.001). This suggests that although successful fixation of a collagen type I/III scaffold in a distal radioulnar joint cartilage defect is feasible with both forms of fixation, fixation with glue and sutures is preferable.

Overexpression of hsa-miR-148a promotes cartilage production and inhibits cartilage degradation by osteoarthritic chondrocytes.

OBJECTIVE: Hsa-miR-148a expression is decreased in Osteoarthritis (OA) cartilage, but its functional role in cartilage has never been studied. Therefore, our aim was to investigate the effects of overexpressing hsa-miR-148a on cartilage metabolism of OA chondrocytes. DESIGN: OA chondrocytes were transfected with a miRNA precursor for hsa-miR-148a or a miRNA precursor negative control. After 3, 7, 14 and 21 days, real-time PCR was performed to examine gene expression levels of aggrecan (ACAN), type I, II, and X collagen (COL1A1, COL2A1, COl10A1), matrix metallopeptidase 13 (MMP13), a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) and the serpin peptidase inhibitor, clade H (heat shock protein 47), member 1 (SERPINH1). After 3 weeks, DNA content and proteoglycan and collagen content and release were determined. Type II collagen was analyzed at the protein level by Western blot. RESULTS: Overexpression of hsa-miR-148a had no effect on ACAN, COL1A1 and SERPINH1 gene expression, but increased COL2A1 and decreased COL10A1, MMP13 and ADAMTS5 gene expression. Luciferase reporter assay confirmed direct interaction of miR-148a and COL10A1, MMP13 and ADAMTS5. The matrix deposited by the miR-148a overexpressing cells contained more proteoglycans and collagen, in particular type II collagen. Proteoglycan and collagen release into the culture medium was inhibited, but total collagen production was increased. CONCLUSION: Overexpression of hsa-miR-148a inhibits hypertrophic differentiation and increases the production and deposition of type II collagen by OA chondrocytes, which is accompanied by an increased retention of proteoglycans. Hsa-miR-148a might be a potential disease-modifying compound in OA, as it promotes hyaline cartilage production.

A validated new histological classification for intervertebral disc degeneration.

UNLABELLED: Histology is an important outcome variable in basic science and pre-clinical studies regarding intervertebral disc degeneration (IVD). Nevertheless, an adequately validated histological classification for IVD degeneration is still lacking and the existing classifications are difficult to use for inexperienced observers. OBJECTIVE: Therefore the aim of this study was to develop and to validate a new histological classification for IVD degeneration. Moreover, the new classification was compared to the frequently used non-validated classification. METHODS: The new classification was applied to human IVD sections. The sections were scored twice by two independent inexperienced observers, twice by two experienced IVD researchers and once by a pathologist. For comparison, the sections were also scored according to the classification described by Boos et al. by two experienced IVD researchers. Macroscopic grading according Thompson et al., glycosaminoglycan (GAG) content and age were used for validation. RESULTS: The new classification had an excellent intra- and a good inter-observer reliability. Intraclass Correlation Coefficients (ICC) were 0.83 and 0.74, respectively. Intra- and inter-observer reliability were comparable for experienced and inexperienced observers. Statistically significant correlations were found between the new classification, macroscopic score, GAG content in the nucleus pulposus (NP) and age; Correlation coefficient (CC) 0.79, -0.62 and 0.68, respectively. The CCs of the Boos classification were all lower compared to the new classification. CONCLUSION: the new histological classification for IVD degeneration is a valid instrument for evaluating IVD degeneration in human IVD sections and is suitable for inexperienced and experienced researchers.

Inflammatory mediators in posttraumatic radiocarpal osteoarthritis.

PURPOSE: To identify the mediator profile in healthy, pre-osteoarthritis (OA) and end-stage OA radiocarpal joints. We hypothesized that there would be an increase in soluble mediators in posttraumatic wrist OA. METHODS: We obtained radiocarpal synovial fluid samples from 3 groups of patients: healthy control (n = 12) samples were collected during wrist ganglion resection; pre-osteoarthritic (n = 16) samples, during a 3-ligament tenodesis procedure for complete scapholunate dissociation; and end-stage OA (n = 20) samples in patients with proven radiological OA changes. Using a multiplex enzyme-linked immunosorbent assay, we measured 12 mediators: interleukin (IL)-1ß, tumor necrosis factor-α, oncostatin-M, interferon-γ, IL-4, IL-6, IL-7, IL-8, IL-10, IL-13, IL-1RA, and osteoprotegerin. Statistical analysis was performed using analysis of variance and Bonferroni-corrected post hoc tests. RESULTS: Mediators IL-6, IL-10, and interferon-γ were increased in OA wrists compared to healthy and pre-OA samples. Tumor necrosis factor-α, oncostatin-M, osteoprotegerin, IL-8, and IL-1RA were detected but not at increased levels in OA wrists. We found no differences between healthy and pre-OA joints in all 12 mediators. Mediators IL-4, IL-7, IL-13, and IL-1ß were not detected in either healthy, pre-OA or end-stage OA samples. CONCLUSIONS: We identified no differences between healthy and pre-OA samples, suggesting no alteration in inflammatory status at the time of the 3-ligament tenodesis procedure. Consequently, mechanical disturbance seems to be the driving force toward OA and OA-associated inflammation in this stage of scapholunate dissociation. Increased levels of interferon-γ, IL-6, and IL-10 confirm inflammatory changes in the mechanically disturbed posttraumatic radiocarpal joint.

An explorative study comparing levels of soluble mediators in control and osteoarthritic synovial fluid.

OBJECTIVE: Soluble mediators in synovial fluid (SF) are acknowledged as key players in the pathophysiology of osteoarthritis (OA). However, a wide-spectrum screening of such mediators in SF is currently lacking. In this study, the levels of 47 mediators in the SF of control donors and osteoarthritic (OA) patients were compared. MATERIALS & METHODS: SF was collected from control donors (n = 16) and end-stage knee OA patients (n = 18) and analysed for 47 cytokines, chemokines and growth factors using several multiplex enzyme-linked immunosorbent assays (ELISAs). A Mann-Whitney U test was used to determine differences between OA and control controls. A principal component analysis (PCA) was performed to cluster the 47 mediators. RESULTS: The majority of the mediators could be detected in both control and OA SF. Interleukin (IL)-6, interferon inducible protein (IP)-10, macrophage derived chemokine (MDC), platelet derived growth factor (PDGF)-AA and regulated on activation normal T cell expressed and secreted (RANTES) levels were found to be higher in OA compared to control SF (P < 0.001). Leptin, IL-13, macrophage inflammatory protein (MIP)-1ß, soluble CD40 (sCD40L) levels were higher and eotaxin and granulocyte colony-stimulating factor (G-CSF) levels were lower in OA SF than in control SF, albeit borderline significant (P < 0.05). The PCA enabled identification of six clusters of mediators, which explained 76% of the variance. CONCLUSIONS: The current study provides the first extensive profile of cytokines, chemokines and growth factors present in control and OA SF. Increased levels of mediators such as MDC and IL-6 imply involvement of inflammatory processes and might be associated with the influx of inflammatory cells in OA synovial tissue. Moreover, the performed cluster analysis indicated multiple clusters, which could indicate different pathophysiological pathways in the joint.

One-stage focal cartilage defect treatment with bone marrow mononuclear cells and chondrocytes leads to better macroscopic cartilage regeneration compared to microfracture in goats.

OBJECTIVE: The combination of chondrocytes and mononuclear fraction (MNF) cells might solve the expansion induced dedifferentiation problem of reimplanted cells in autologous chondrocytes implantation as sufficient cells would be available for direct, one-stage, implantation. Earlier in vitro work already showed a positive stimulation of cartilage specific matrix production when chondrocytes and MNF cells were combined. Therefore, this study aimed to evaluate cartilage regeneration using a one-stage procedure combining MNF cells and primary chondrocytes for the treatment of focal cartilage lesions in goats compared to microfracture treatment. DESIGN: Freshly created focal cartilage defects were treated with either a combination of chondrocytes and MNF cells embedded in fibrin glue or microfracture treatment. After 6 months follow-up local regeneration as well as the general joint cartilage health were evaluated using validated scores and biochemical assays. RESULTS: Macroscopic (P = 0.015) scores for the cartilage surface at the treated defect were, after 6 months, significantly higher for the chondrocyteMNF treatment compared to microfracture-treated defects, but microscopic scores were not (P = 0.067). The articulating cartilage showed more (P = 0.005) degeneration following microfracture treatment compared to chondrocyteMNF treatment. Biochemical glycosaminoglycans (GAG) evaluation did not reveal differences between the treatments. Both treatments had resulted in a slight to moderate cartilage degeneration at other locations in the joint. CONCLUSION: In conclusion, treatment of focal articular cartilage lesions in goats using a combination of MNF cells from bone marrow and unexpanded chondrocytes leads to better macroscopic regeneration compared to microfracture, however needs further fine-tuning to decrease the negative influence on other joint compartments.

Delayed gadolinium enhanced MRI of cartilage (dGEMRIC) can be effectively applied for longitudinal cohort evaluation of articular cartilage regeneration.

OBJECTIVE: Delayed gadolinium enhanced magnetic resonance imaging (MRI) of cartilage (dGEMRIC) facilitates non-invasive evaluation of the glycosaminoglycan content in articular cartilage. The primary aim of this study was to show that the dGEMRIC technique is able to monitor cartilage repair following regenerative cartilage treatment. DESIGN: Thirty-one patients with a focal cartilage lesion underwent a dGEMRIC scan prior to cartilage repair surgery and at 3 and 12 months follow-up. At similar time points clinical improvement was monitored using the Knee injury and Osteoarthritis Outcome Score (KOOS) and Lysholm questionnaires. Per MRI scan several regions-of-interest (ROIs) were defined for different locations in the joint. The dGEMRIC index (T1gd) was calculated for each ROI. Repeated-measures analysis of variance (RMANOVA) analysis was used to evaluate improvement in clinical scores and MRI T1gd over time. Also regression analysis was performed to show the influence of local repair on cartilage quality at distant locations in the knee. RESULTS: Clinical scores and the dGEMRIC T1gd per ROI showed a statistically significant improvement (P < 0.01), from baseline, at 12 months follow-up. Also, improvement from baseline in T1gd of the ROI defining the treated cartilage defect showed a direct relationship (P < 0.007) to the improvement of the T1gd of ROI at other locations in the joint. CONCLUSIONS: The dGEMRIC MRI protocol is a useful method to evaluate cartilage repair. In addition, local cartilage repair influenced the cartilage quality at other location in the joint. These findings validate the use of dGEMRIC for non-invasive evaluation of the effects of cartilage regeneration.

Intervertebral disc disease in dogs - part 1: a new histological grading scheme for classification of intervertebral disc degeneration in dogs.

Intervertebral disc (IVD) degeneration is common in dogs and can lead to serious disorders. Current treatments can relieve clinical signs of disease, but do not restore IVD function. The development of regenerative strategies for IVD dysfunction requires detailed knowledge of the pathogenesis of IVD degeneration and its underlying mechanisms. Histological examination of IVDs at different stages of degeneration might provide this knowledge, but as there is currently no histological grading scheme for canine IVD degeneration, the aim of this study, which is the first of a two-part series, was to design and validate an appropriate scheme. Three independent observers evaluated 35 IVDs at different stages of degeneration using the scheme. Glycosaminoglycan contents of the nucleus pulposus and macroscopic grading according to Thompson, which are considered 'gold standards' for IVD degeneration, were used to validate the scheme. Reproducibility was assessed by analysing the inter-observer reliability of all individual variables of the grading scheme, using a weighted κ analysis. Significant correlations were found between Thompson grading and total histological score (r=0.94; P<0.01) and between glycosaminoglycan content and total histological score (r=-0.72; P<0.01). Most individual histological variables showed 'moderate' to 'almost perfect' inter-observer reliability. The high correlation with the gold standards in combination with the high reproducibility indicates that the proposed histological grading scheme is reliable and objective for classification of IVD degeneration in both chondrodystrophic and non-chondrodystrophic dog breeds.

Micro-CT quantification of subchondral endplate changes in intervertebral disc degeneration.

BACKGROUND: The intervertebral disc (IVD) is dependent on nutrient provision through a cartilage layer with underlying subchondral bone, analogous to joint cartilage. In the joint, subchondral bone remodeling has been associated with osteoarthritis (OA) progression due to compromised nutrient and gas diffusion and reduced structural support of the overlaying cartilage. However, subchondral bone changes in IVD degeneration have never been quantified before. OBJECTIVE: The aim of this study is to determine the subchondral bone changes at different stages of IVD degeneration by micro-CT. METHODS: Twenty-seven IVDs including the adjacent vertebral endplates were obtained at autopsy. Midsagittal slices, graded according the Thompson score, were scanned. Per scan 12 standardized cylindrical volumes of interest (VOI) were selected. Six VOIs contained the bony endplate and trabeculae (endplate VOIs) and six accompanying VOIs only contained trabecular bone (vertebral VOIs). Bone volume as percentage of the total volume (BV/TV) of the VOI, trabecular thickness (TrTh) and connectivity density (CD) were determined. RESULTS: An increase in BV/TV and TrTh was found in endplate VOIs of IVDs with higher Thompson score whereas these values remained stable or decreased in the vertebral VOIs. CONCLUSION: The increase in bone volume combined with the increase in TrTh in endplate VOIs strongly suggest that the subchondral endplate condenses to a more dense structure in degenerated IVDs. This may negatively influence the diffusion and nutrition of the IVD. The endplate differences between intact and mild degenerative IVDs (grade II) indicate an early association of subchondral endplate changes with IVD degeneration.

Hypertrophic differentiation and calcification during intervertebral disc degeneration.

BACKGROUND: In degenerative intervertebral discs (IVDs) collagen type X expression and calcifications have been demonstrated, resembling advanced osteoarthritis (OA), which is associated with hypertrophic differentiation, characterized by the production of collagen type X, Runt-related transcription factor 2 (Runx2), osteoprotegerin (OPG), alkaline phosphatase (ALP) and calcifications. OBJECTIVE: The aim of this study was to determine if hypertrophic differentiation occurs during IVD degeneration. METHODS: IVDs from all Thompson degeneration grades were prepared for histology, extraction of nucleus pulposus (NP) and annulus fibrosis (AF) tissue (N=50) and micro-CT (N=27). The presence of collagen type X, OPG and Runx2 was determined by immunohistochemistry, with OPG levels also determined by Enzyme-linked immunosorbent assay (ELISA). The presence of calcification was determined by micro-CT, von Kossa and Alizarin Red staining. RESULTS: Immunohistochemical staining for collagen type X, OPG, Runx2 appeared more intense in the NP of degenerative compared to healthy IVD samples. OPG levels correlated significantly with degeneration grade (NP: P<0.000; AF: P=0.002) and the number of microscopic calcifications (NP: P=0.002; AF: P=0.008). The extent of calcifications on micro-CT also correlated with degeneration grade (NP: P<0.001, AF: P=0.001) as did von Kossa staining (NP: P=0.015, AF: P=0.016). ALP staining was only incidentally seen in the transition zone of grades IV and V degenerated IVDs. CONCLUSION: This study for the first time demonstrates that hypertrophic differentiation occurs during IVD degeneration, as shown by an increase in OPG levels, the presence of ALP activity, increased immunopositivity of Runx2 and collagen type X.