A.I. Tsuchida

Matrix-Applied Characterized Autologous Cultured Chondrocytes Versus Microfracture: Two-Year Follow-up of a Prospective Randomized Trial

Background: Randomized controlled trials studying the efficacy and safety of matrix-applied characterized autologous cultured chondrocytes (MACI) versus microfracture (MFX) for treating cartilage defects are limited. Purpose: To compare the clinical efficacy and safety of MACI versus MFX in the treatment of patients with symptomatic cartilage defects of the knee. Study Design: Randomized controlled clinical trial; Level of evidence, 1. Methods: Patients enrolled in the SUMMIT (Demonstrate the Superiority of MACI implant to Microfracture Treatment) trial had ≥1 symptomatic focal cartilage defect (Outerbridge grade III or IV; ≥3 cm2) of the femoral condyles or trochlea, with a baseline Knee Injury and Osteoarthritis Outcome Score (KOOS) pain value <55. The co–primary efficacy endpoint was the change in the KOOS pain and function subscores from baseline to 2 years. Histological evaluation and magnetic resonance imaging (MRI) assessments of structural repair tissue, treatment failure, the remaining 3 KOOS subscales, and safety were also assessed. Results: Of the 144 patients treated, 137 (95%) completed the 2-year assessment. Patients had a mean age of 33.8 years and a mean lesion size of 4.8 cm2. The mean KOOS pain and function subscores from baseline to 2 years were significantly more improved with MACI than with MFX (pain: MACI, 37.0 to 82.5 vs MFX, 35.5 to 70.9; function: MACI, 14.9 to 60.9 vs MFX, 12.6 to 48.7; P = .001). A significant improvement in scores was also observed on the KOOS subscales of activities of daily living (MACI, 43.5 to 87.2 vs MFX, 42.6 to 75.8; P < .001), knee-related quality of life (MACI, 18.8 to 56.2 vs MFX, 17.2 to 47.3; P = .029), and other symptoms (MACI, 48.3 to 83.7 vs MFX, 44.4 to 72.2; P < .001) for patients treated with MACI compared with MFX. Repair tissue quality was good as assessed by histology/MRI, but no difference was shown between treatments. A low number of treatment failures (nonresponders: MACI, 12.5% vs MFX, 31.9%; P = .016) and no unexpected safety findings were reported. Conclusion: The treatment of symptomatic cartilage knee defects ≥3 cm2 in size using MACI was clinically and statistically significantly better than with MFX, with similar structural repair tissue and safety, in this heterogeneous patient population. Moreover, MACI offers a more efficacious alternative than MFX with a similar safety profile for the treatment of symptomatic articular cartilage defects of the knee.

Cytokine profiles in the joint depend on pathology, but are different between synovial fluid, cartilage tissue and cultured chondrocytes

IntroductionThis study aimed to evaluate whether profiles of several soluble mediators in synovial fluid and cartilage tissue are pathology-dependent and how their production is related to in vitro tissue formation by chondrocytes from diseased and healthy tissue.MethodsSamples were obtained from donors without joint pathology (n = 39), with focal defects (n = 65) and osteoarthritis (n = 61). A multiplex bead assay (Luminex) was performed measuring up to 21 cytokines: Interleukin (IL)-1α, IL-1β, IL-1RA, IL-4, IL-6, IL-6Rα, IL-7, IL-8, IL-10, IL-13, tumor necrosis factor (TNF)α, Interferon (IFN)γ, oncostatin M (OSM), leukemia inhibitory factor (LIF), adiponectin, leptin, monocyte chemotactic factor (MCP)1, RANTES, basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), vascular growth factor (VEGF).ResultsIn synovial fluid of patients with cartilage pathology, IL-6, IL-13, IFNγ and OSM levels were higher than in donors without joint pathology (P ≤0.001). IL-13, IFNγ and OSM were also different between donors with cartilage defects and OA (P <0.05). In cartilage tissue from debrided defects, VEGF was higher than in non-pathological or osteoarthritic joints (P ≤0.001). IL-1α, IL-6, TNFα and OSM concentrations (in ng/ml) were markedly higher in cartilage tissue than in synovial fluid (P <0.01). Culture of chondrocytes generally led to a massive induction of most cytokines (P <0.001). Although the release of inflammatory cytokines was also here dependent on the pathological condition (P <0.001) the actual profiles were different from tissue or synovial fluid and between non-expanded and expanded chondrocytes. Cartilage formation was lower by healthy unexpanded chondrocytes than by osteoarthritic or defect chondrocytes.ConclusionsSeveral pro-inflammatory, pro-angiogenic and pro-repair cytokines were elevated in joints with symptomatic cartilage defects and/or osteoarthritis, although different cytokines were elevated in synovial fluid compared to tissue or cells. Hence a clear molecular profile was evident dependent on disease status of the joint, which however changed in composition depending on the biological sample analysed. These alterations did not affect in vitro tissue formation with these chondrocytes, as this was at least as effective or even better compared to healthy chondrocytes.

Single-Stage Cell-Based Cartilage Regeneration Using a Combination of Chondrons and Mesenchymal Stromal Cells Comparison With Microfracture

Background: Autologous chondrocyte implantation (ACI) is traditionally a 2-step procedure used to repair focal articular cartilage lesions. With use of a combination of chondrons (chondrocytes in their own territorial matrix) and mesenchymal stromal cells (MSCs), ACI could be innovated and performed in a single step, as sufficient cells would be available to fill the defect within a 1-step surgical procedure. Chondrons have been shown to have higher regenerative capacities than chondrocytes without such a pericellular matrix. Purpose: To evaluate cartilage formation by a combination of chondrons and MSCs in vitro and in both small and large animal models. Study Design: Controlled laboratory study. Methods: Chondrons and MSCs were cultured at different ratios in vitro containing 0%, 5%, 10%, 20%, 50%, or 100% chondrons (n = 3); embedded in injectable fibrin glue (Beriplast); and implanted subcutaneously in nude mice (n = 10; ratios of 0%, 5%, 10%, and 20% chondrons). Also, in a 1-step procedure, a combination of chondrons and MSCs was implanted in a freshly created focal articular cartilage lesion (10% chondrons) in goats (n = 8) and compared with microfracture. The effect of both treatments, after 6-month follow-up, was evaluated using biochemical glycosaminoglycan (GAG) and GAG/DNA analysis and scored using validated scoring systems for macroscopic and microscopic defect repairs. Results: The addition of MSCs to chondron cultures enhanced cartilage-specific matrix production as reflected by a higher GAG production (P < .03), both in absolute levels and normalized to DNA content, compared with chondrocyte and 100% chondron cultures. Similar results were observed after 4 weeks of subcutaneous implantation in nude mice. Treatment of freshly created cartilage defects in goats using a combination of chondrons and MSCs in Beriplast resulted in better microscopic, macroscopic, and biochemical cartilage regeneration (P ≤ .02) compared with microfracture treatment. Conclusion: The combination of chondrons and MSCs increased cartilage matrix formation, and this combination of cells was safely applied in a goat model for focal cartilage lesions, outperforming microfracture. Clinical Relevance: This study describes the bench-to-preclinical development of a new cell-based regenerative treatment for focal articular cartilage defects that outperforms microfracture in goats. In addition, it is a single-step procedure, thereby making the expensive cell expansion and reimplantation of dedifferentiated cells, as in ACI, redundant.

Interleukin-6 is elevated in synovial fluid of patients with focal cartilage defects and stimulates cartilage matrix production in an in vitro regeneration model

IntroductionThis study aimed to determine whether, as in osteoarthritis, increased levels of interleukin-6 (IL-6) are present in the synovial fluid of patients with symptomatic cartilage defects and whether this IL-6 affects cartilage regeneration as well as the cartilage in the degenerated knee.MethodsIL-6 concentrations were determined by ELISA in synovial fluid and in conditioned media of chondrocytes regenerating cartilage. Chondrocytes were obtained from donors with symptomatic cartilage defects, healthy and osteoarthritic donors. The effect of IL-6 on cartilage regeneration and on metabolism of the resident cartilage in the knee was studied by both inhibition of endogenous IL-6 and addition of IL-6, in a regeneration model and in osteoarthritic explants in the presence of synovial fluid, respectively. Readout parameters were DNA and glycosaminoglycan (GAG) content and release. Differences between controls and IL-6 blocked or supplemented samples were determined by univariate analysis of variance using a randomized block design.ResultsSynovial fluid of patients with symptomatic cartilage defects contained more IL-6 than synovial fluid of healthy donors (P = 0.001) and did not differ from osteoarthritic donors. IL-6 production of osteoarthritic chondrocytes during cartilage regeneration was higher than that of healthy and defect chondrocytes (P < 0.001). Adding IL-6 increased GAG production by healthy chondrocytes and decreased GAG release by osteoarthritic chondrocytes (P < 0.05). Inhibition of IL-6 present in osteoarthritic synovial fluid showed a trend towards decreased GAG content of the explants (P = 0.06).ConclusionsOur results support a modest anabolic role for IL-6 in cartilage matrix production. Targeting multiple cytokines, including IL-6, may be effective in improving cartilage repair in symptomatic cartilage defects and osteoarthritis.

Quality of scaffold fixation in a human cadaver knee model

OBJECTIVE Newly developed regenerative cartilage interventions based on the application of 3D-scaffolds require a further evaluation of the surgical techniques involved. The present study compared four different scaffold fixation techniques [fibrin glue (FG), transosseous (TS) fixation, biodegradable pin (BP) fixation and continuous cartilage sutures (CS)] to implant a custom-printed porous PEOT/PBT1000/70/30 scaffold in a human cadaver knee model. METHODS After implantation, the knees were subjected to a vertically oriented loaded continuous passive motion (CPM) protocol. The fixation techniques were evaluated after 60 and a subsequent 150 motion cycles, focusing on area coverage, outline attachment and scaffold integrity. After the total of 210 cycles, also an endpoint fixation test was performed. RESULTS The fixation techniques revealed marginal differences for area coverage and outline attachment after 60 and 150 cycles. The FG scored higher on scaffold integrity compared to TS (P<0.05) and CS (P=0.01). Endpoint fixation was highest for the CS, whereas FG showed a weak final fixation strength (P=0.01). CONCLUSIONS This study showed that optimal fixation cannot be combined always with high scaffold integrity. Special attention devoted to scaffold properties in relation to the fixation technique may result in an improvement of scaffold fixation, and thus clinical cartilage regenerative approaches involving these scaffolds.

dGEMRIC as a tool for measuring changes in cartilage quality following high tibial osteotomy: a feasibility study

OBJECTIVE The high tibial osteotomy (HTO) is an effective strategy for treatment of painful medial compartment knee osteoarthritis. Effects on cartilage quality are largely unknown. Delayed gadolinium-enhanced magnetic resonance imaging of cartilage (dGEMRIC) enables non-invasive assessment of cartilage glycosaminoglycan content. This study aimed to evaluate if dGEMRIC could detect relevant changes in cartilage glycosaminoglycan content following HTO. DESIGN Ten patients with medial compartment osteoarthritis underwent a dGEMRIC scan prior to HTO, and after bone healing and subsequent hardware removal. A dGEMRIC index (T1Gd) was used for changes in cartilage glycosaminoglycan content, a high T1Gd indicating a high glycosaminoglycan content and vice versa. Radiographic analysis included mechanical axis and tibial slope measurement. clinical scores [knee osteoarthritis outcome scale (KOOS), visual analogue score (VAS) for pain, Knee Society clinical rating system (KSCRS)] before, 3 and 6 months after HTO and after hardware removal were correlated to T1Gd changes. RESULTS Overall a trend towards a decreased T1Gd, despite HTO, was observed. Before and after HTO, lateral femoral condyle T1Gd was higher than medial femoral condyle (MFC) T1Gd and tibial cartilage T1Gd was higher than that of femoral cartilage (P < 0.001). The MFC had the lowest T1Gd before and after HTO. Clinical scores all improved significantly (P < 0.01), KOOS Symptoms and QOL were moderately related to changes in MFC T1Gd. CONCLUSIONS dGEMRIC effectively detected differences in cartilage quality within knee compartments before and after HTO, but no changes due to HTO were detected. Hardware removal post-HTO seems essential for adequate T(1)Gd interpretation. T(1)Gd was correlated to improved clinical scores on a subscore level only. Longer follow-up after HTO may reveal lasting changes. ClinicalTrials.gov registration ID: NCT01269944.

Pronounced biomaterial dependency in cartilage regeneration using nonexpanded compared with expanded chondrocytes

AIM We aimed to investigate freshly isolated compared with culture-expanded chondrocytes with respect to early regenerative response, cytokine production and cartilage formation in response to four commonly used biomaterials. MATERIALS & METHODS Chondrocytes were both directly and after expansion to passage 2, incorporated into four biomaterials: Polyactive™, Beriplast®, HyStem® and a type II collagen gel. Early cartilage matrix gene expression, cytokine production and glycosaminoglycan (GAG) and DNA content in response to these biomaterials were evaluated. RESULTS HyStem induced more GAG production, compared with all other biomaterials (p ≤ 0.001). Nonexpanded cells did not always produce more GAGs than expanded chondrocytes, as this was biomaterial-dependent. Cytokine production and early gene expression were not predictive for final regeneration. CONCLUSION For chondrocyte-based cartilage treatments, the biomaterial best supporting cartilage matrix production will depend on the chondrocyte differentiation state and cannot be predicted from early gene expression or cytokine profile.

Chondrogenic potential of articular chondrocytes depends on their original location in the knee

OBJECTIVE This study aimed to investigate the regenerative capacity of chondrocytes derived from debrided defect cartilage and healthy cartilage from different regions in the joint to determine the best cell source for regenerative cartilage therapies. METHODS Articular cartilage was obtained from Outerbridge grade III and IV cartilage lesions and from macroscopically healthy weight-bearing and nonweight-bearing (NWB) locations in the knee. Chondrocytes isolated from all locations were either pelleted directly (P0 pellets) or after expansion (P2 pellets) and analyzed for glycosaminoglycan (GAG), DNA, and cartilage-specific gene expression. Harvested cartilage samples and cultured pellets were also analyzed by Safranin O histology and immunohistochemistry for collagen I, II, and X. Immunohistochemical stainings were quantified using a computerized pixel-intensity staining segmentation method. RESULTS After 4 weeks of culture, the P0 pellets derived from grade III or healthy weight-bearing chondrocytes contained more (p<0.015) GAG and GAG normalized per DNA compared to those from grade IV and NWB locations. After expansion, these differences were lost. Cartilage-specific gene expression was higher (p<0.04) in P0 pellets from grade III chondrocytes compared to grade IV chondrocytes. Semiquantitative immunohistochemistry showed a more intense (p<0.033) collagen I and X staining for grade IV debrided cartilage compared to grade III and weight-bearing cartilage. Also, collagen type X staining intensity was higher (p<0.033) in NWB cartilage compared to grade III and weight-bearing regions. CONCLUSION Chondrocytes derived from debrided cartilage perform better than cells from the NWB biopsy site, however, this difference is lost upon expansion. Based thereon, the debrided defect cartilage could be a viable donor site for regenerative cartilage surgery.

SUMMIT Prospective, Randomized, Controlled Trial: Response Rates To Matrix-induced Autologous Chondrocyte Implant (MACI) Versus Microfracture (MFX) By Lesion Characteristics

Objectives: To compare the response rates of patients treated with the MACI implant with those treated with MFX for the repair of symptomatic articular cartilage defects of the knee, based on lesion characteristics, in the phase 3 SUMMIT (Superiority of Matrix-induced autologous chondrocyte implant versus Microfracture for Treatment of symptomatic articular cartilage defects) trial (sponsored by Sanofi/Genzyme Biosurgery). Methods:: Patients with ≥1 symptomatic focal articular cartilage defect of the femoral condyles (MFC/LFC) and/or trochlea and baseline Knee Injury and Osteoarthritis Outcome Score (KOOS) pain score ≤55 participated in this prospective, randomized, controlled trial (NCT00719576). The co-primary endpoint was improvement in KOOS pain and function (sports/recreational activities) at 2-year follow up. A patient was considered a responder if they had at least a 10-point improvement over baseline in KOOS pain and function scores. The Cochran-Mantel-Haenszel χ2 (α=0.05) was used to analyze differences in response rates between groups by lesion size (>4 cm2, >5 cm2), lesion location (MFC/LFC/trochlea), and osteochondritis dissecans (OCD) etiology (yes/no). Results:: Patients (N=144) had a mean age of 33.8 years and 51% were male. The mean lesion size was 4.8 cm2, and most lesions were on the MFC (74%). The KOOS pain and function (sports/recreational activities) co-primary endpoint improvement was clinically and statistically significantly better in patients treated with the MACI implant versus MFX (P=0.001) at 2-year follow up. Significantly better improvements were also observed for KOOS activities of daily living (P<0.001), quality of life (P=0.029) and symptoms (P<0.001) with the MACI implant compared with MFX. Response rates for the overall and subgroup analyses are shown in the Table. The incidence of adverse events was similar between the treatment groups and no unexpected safety findings were reported. Based on the small patient numbers in the subgroups, further research on response predictors is warranted. Conclusion:: Clinically and statistically significantly better results in KOOS pain and function co-primary scores were observed with the MACI implant versus MFX for treating cartilage defects of the knee 2 years following surgery. More patients responded with the MACI implant compared with MFX when their lesions were >4 cm2, on the MFC, and not of OCD origin.

222 INHIBITION OF PROSTAGLANDIN E2 BY CELECOXIB DECREASES GLYCOSAMINOGLYCAN RELEASE, HOWEVER DOES NOT STIMULATE REPAIR OF OSTEOARTHRITIC CARTILAGE TISSUE

Results: BAP level was higher in OA synovial fluid than in simple synovitis, and d-ROM level was slightness lower in OA than control synovitis, but this differences were not significant. BAP and d-ROM levels were not related with OA grade. After intra-articular injection of HA, BAP level was significantly increased and d-ROM level was reduced. On the other hand, BAP and d-ROM level were significantly decreased after injection of corticosteroid. Conclusions: Oxidative stress and anti-oxidative potential in synovial fluid were not significant difference between OA and simple synovitis. Our results showed that hyaluronan increase the anti-oxidant potential and decreased oxidative stress, but corticosteroid decreased both oxidative stress and the anti-oxidative stress potential.