Pieter J. Emans

Redifferentiation of dedifferentiated human articular chondrocytes: comparison of 2D and 3D cultures.

OBJECTIVE Three-dimensional (3D) cultures are widely used to redifferentiate chondrocytes. However, the rationale behind the choice for 3D above two-dimensional (2D) cultures is poorly systematically investigated and mainly based on mRNA expression and glycosaminoglycan (GAG) content. The objective was to determine the differential redifferentiation characteristics of human articular chondrocytes (HACs) in monolayer, alginate beads and pellet culture by investigating mRNA expression, protein expression, GAG content and cell proliferation. DESIGN Dedifferentiated HACs from six individuals were redifferentiated in identical medium conditions for 7 days in monolayer, alginate beads or pellet culture. Read-out parameters were expression of chondrogenic and hypertrophic mRNAs and proteins, GAG content and cell proliferation. RESULTS 3D cultures specifically expressed chondrogenic mRNAs [collagen type II (COL2A1), SRY (sex determining region Y)-box 9 (SOX9), aggrecan (ACAN)), whereas 2D cultures did not. Hypertrophic mRNAs (collagen type X (COL10A1), runt-related transcription factor 2 (RUNX2), matrix metalloproteinase 13 (MMP13), vascular endothelial growth factor A (VEGFA), osteopontin (OPN), alkaline phosphatase (ALP)) were highly increased in 2D cultures and lower in 3D cultures. Collagen type I (COL1A1) mRNA expression was highest in 3D cultures. Protein expression supports most of the mRNA data, although an important discrepancy was found between mRNA and protein expression of COL2A1 and SOX9 in monolayer culture, stressing on the importance of protein expression analysis. GAG content was highest in 3D cultures, whereas chondrocyte proliferation was almost specific for 2D cultures. CONCLUSIONS For redifferentiation of dedifferentiated HACs, 3D cultures exhibit the most potent chondrogenic potential, whereas a hypertrophic phenotype is best achieved in 2D cultures. This is the first human study that systematically evaluates the differences between proliferation, GAG content, protein expression and mRNA expression of commonly used 2D and 3D chondrocyte culture techniques.

Degradation of mesh coatings and intraperitoneal adhesion formation in an experimental model

In laparoscopic ventral hernia repair a mesh is placed in direct contact with the viscera, often leading to substantial adhesions. In this experimental study the ability of different coated and uncoated meshes to attenuate adhesion formation was examined.

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.

Autologous engineering of cartilage

Treatment of full-thickness damage to hyaline cartilage is hampered by the limited availability of autologous healthy cartilage and the lengthy, cost-prohibitive cell isolation and expansion steps associated with autologous cartilage implantation (ACI). Here we report a strategy for de novo engineering of ectopic autologous cartilage (EAC) within the subperiosteal space (in vivo bioreactor), through the mere introduction of a biocompatible gel that might promote hypoxia-mediated chondrogenesis, thereby effectively overcoming the aforementioned limitations. The EAC is obtained within 3 wk post injection of the gel, and can be press-fit into an osteochondral defect where it undergoes remodeling with good lateral and subchondral integration. The implanted EAC showed no calcification even after 9 mo and attained an average O’Driscoll score of 11 (versus 4 for controls). An “on demand” autologous source of autologous cartilage with remodeling capacity is expected to significantly impact the clinical options in repair of trauma to articular cartilage.

NSAIDs and fracture healing

Purpose of reviewPublished data raise concerns about the use of nonselective NSAIDs and selective cyclo-oxygenase (COX)-2 inhibitors as anti-inflammatory or analgesic drugs in patients after a recent fracture or who are undergoing (uncemented) arthroplasty or osteotomy. However, clinical reports on the effect of COX-2 inhibition on fracture healing in humans have been variable and inconclusive. This review gives an overview of the published data and an advice when to avoid NSAIDs. Recent findingsProstaglandins play an important role as mediators of inflammation and COX are required for their production. Inflammation is an essential step in the fracture healing process in which prostaglandin production by COX-2 is involved. Data from animal studies suggest that NSAIDs, which inhibit COX-2, can impair fracture healing due to the inhibition of the endochondral ossification pathway. Animal data suggest that the effects of COX-2 inhibitors are dependent on the timing, duration, and dose, and that these effects are reversible. SummaryThese animal data, together with the view of limited scientifically robust clinical evidence in humans, indicate that physicians consider only short-term administration of COX-2 inhibitors or other drugs in the pain management of patients who are in the phase of fracture or other bone defect healing. COX-2-inhibitors should be considered a potential risk factor for fracture healing, and therefore to be avoided in patients at risk for delayed fracture healing.

Activation of NF-κB/p65 Facilitates Early Chondrogenic Differentiation during Endochondral Ossification

Background NF-κB/p65 has been reported to be involved in regulation of chondrogenic differentiation. However, its function in relation to key chondrogenic factor Sox9 and onset of chondrogenesis during endochondral ossification is poorly understood. We hypothesized that the early onset of chondrogenic differentiation is initiated by transient NF-κB/p65 signaling. Methodology/Principal Findings The role of NF-κB/p65 in early chondrogenesis was investigated in different in vitro, ex vivo and in vivo endochondral models: ATDC5 cells, hBMSCs, chicken periosteal explants and growth plates of 6 weeks old mice. NF-κB/p65 activation was manipulated using pharmacological inhibitors, RNAi and activating agents. Gene expression and protein expression analysis, and (immuno)histochemical stainings were employed to determine the role of NF-κB/p65 in the chondrogenic phase of endochondral development. Our data show that chondrogenic differentiation is facilitated by early transient activation of NF-κB/p65. NF-κB/p65-mediated signaling determines early expression of Sox9 and facilitates the subsequent chondrogenic differentiation programming by signaling through key chondrogenic pathways. Conclusions/Significance The presented data demonstrate that NF-κB/p65 signaling, as well as its intensity and timing, represents one of the transcriptional regulatory mechanisms of the chondrogenic developmental program of chondroprogenitor cells during endochondral ossification. Importantly, these results provide novel possibilities to improve the success of cartilage and bone regenerative techniques.

Decreased bone tissue mineralization can partly explain subchondral sclerosis observed in osteoarthritis

For many years, pharmaceutical therapies for osteoarthritis (OA) were focused on cartilage. However, it has been theorized that bone changes such as increased bone volume fraction and decreased bone matrix mineralization may play an important role in the initiation and pathogenesis of OA as well. The mechanisms behind the bone changes are subject of debate, and a better understanding may help in the development of bone-targeting OA therapies. In the literature, the increase in bone volume fraction has been hypothesized to result from mechanoregulated bone adaptation in response to decreased mineralization. Furthermore, both changes in bone volume fraction and mineralization have been reported to be highest close to the cartilage, and bone volume fraction has been reported to be correlated with cartilage degeneration. These data indicate that cartilage degeneration, bone volume fraction, and bone matrix mineralization may be related in OA. In the current study, we aimed to investigate the relationships between cartilage degeneration, bone matrix mineralization and bone volume fraction at a local level. With microCT, we determined bone matrix mineralization and bone volume fraction as a function of distance from the cartilage in osteochondral plugs from human OA tibia plateaus with varying degrees of cartilage degeneration. In addition, we evaluated whether mechanoregulated bone adaptation in response to decreased bone matrix mineralization may be responsible for the increase in bone volume fraction observed in OA. For this purpose, we used the experimentally obtained mineralization data as input for bone adaptation simulations. We simulated the effect of mechanoregulated bone adaptation in response to different degrees of mineralization, and compared the simulation results to the experimental data. We found that local changes in subchondral bone mineralization and bone volume fraction only occurred underneath severely degenerated cartilage, indicating that bone mineralization and volume fraction are related to cartilage degeneration at a local level. In addition, both the experimental data and the simulations indicated that a depth-dependent increase in bone volume fraction could be caused by decreased bone matrix mineralization. However, a quantitative comparison showed that decreased mineralization can only explain part of the subchondral sclerosis observed in OA.

Osmolarity determines the in vitro chondrogenic differentiation capacity of progenitor cells via nuclear factor of activated T-cells 5

INTRODUCTION Previous studies have shown that human articular chondrocytes in vitro are osmolarity-responsive and increase matrix synthesis under cartilage-specific physiological osmolarity. The effects of increased osmolarity on chondrogenesis of progenitor cells in vitro are largely unknown. We therefore aimed to elucidate whether hyperosmolarity facilitates their chondrogenic differentiation and whether Nfat5 is involved. MATERIALS AND METHODS ATDC5 cells and human bone marrow stem cells (hBMSCs) were differentiated in the chondrogenic lineage in control and increased osmolarity conditions. Chondrogenic outcome was measured by gene- and protein expression analysis. RNAi was used to determine the role of Nfat5 in chondrogenic differentiation under normal and increased osmolarity. RESULTS Increasing the osmolarity of differentiation medium with 100mOsm resulted in significantly increased chondrogenic marker expression (Col2a1, Col10a1, Acan, Sox9, Runx2 and GAGs) during chondrogenic differentiation of the two chondroprogenitors, ATDC5 and hBMSCs. Nfat5 knockdown under both control and increased osmolarity affected chondrogenic differentiation and suppressed the osmolarity-induced chondrogenic induction. Knockdown of Nfat5 in early differentiation significantly decreased early Sox9 expression, whereas knockdown of Sox9 in early differentiation did not affect early Nfat5 expression. CONCLUSIONS Increasing the osmolarity of chondrogenic culture media by 100mOsm significantly increased chondrogenic gene expression during the course of chondrogenic differentiation of progenitor cells. Nfat5 may be involved in regulating chondrogenic differentiation of these cells under both normal and increased osmolarities and might regulate chondrogenic differentiation through influencing early Sox9 expression.

Tuning the differentiation of periosteum-derived cartilage using biochemical and mechanical stimulations

OBJECTIVE In this study, we aim at tuning the differentiation of periosteum in an organ culture model towards cartilage, rich in collagen type II, using combinations of biochemical and mechanical stimuli. We hypothesize that addition of TGF-β will stimulate chondrogenesis, whereas sliding indentation will enhance collagen synthesis. DESIGN Periosteum was dissected from the tibiotarsus of 15-day-old chick embryos. Explants were embedded in between two agarose layers, and cultured without stimulation (control), with biochemical stimulation (10 ng/ml TGF-β1), with mechanical stimulation (sliding indentation), or both biochemical and mechanical stimulations. Sliding indentation was introduced as a method to induce tensile tissue strain. Analysis included quantification of DNA, collagen and GAG content, conventional histology, and immunohistochemistry for collagen type I and II at 1 or 2 weeks of culture. RESULTS Embedding the periosteal explants in between agarose layers induced cartilage formation, confirmed by synthesis of sGAG and collagen type II. Addition of TGF-β1 to the culture medium did not further enhance this chondrogenic response. Applying sliding indentation only to the periosteum in between agarose layers enhanced the production of collagen type I, leading to the formation of fibrous tissue without any evidence of cartilage formation. However, when stimulated by both TGF-β1 and sliding indentation, collagen production was still enhanced, but now collagen type II, while sGAG was found to be similar to TGF-β1 or unloaded samples. CONCLUSIONS The type of tissue produced by periosteal explants can be tuned by combining mechanical stimulation and soluble factors. TGF-β1 stimulated a chondrocyte phenotype and sliding indentation stimulated collagen synthesis. Such a combination may be valuable for improvement of the quality of tissue-engineered cartilage.

Identifying Phenotypes of Knee Osteoarthritis by Separate Quantitative Radiographic Features May Improve Patient Selection for More Targeted Treatment

Objective. Expression of osteoarthritis (OA) varies significantly between individuals, and over time, suggesting the existence of different phenotypes, possibly with specific etiology and targets for treatment. Our objective was to identify phenotypes of progression of radiographic knee OA using separate quantitative features. Methods. Separate radiographic features of OA were measured by Knee Images Digital Analysis (KIDA) in individuals with early knee OA (the CHECK cohort: Cohort Hip & Cohort Knee), at baseline and at 2-year and 5-year followup. Hierarchical clustering was performed to identify phenotypes of radiographic knee OA progression. The phenotypes identified were compared for changes in joint space width (JSW), varus angle, osteophyte area, eminence height, bone density, for Kellgren-Lawrence (K-L) grade, and for clinical characteristics. Logistic regression analysis evaluated whether baseline radiographic features and demographic/clinical characteristics were associated with each of the specific phenotypes. Results. The 5 clusters identified were interpreted as “Severe” or “No,” “Early” or “Late” progression of the radiographic features, or specific involvement of “Bone density.” Medial JSW, varus angle, osteophyte area, eminence height, and bone density at baseline were associated with the Severe and Bone density phenotypes. Lesser eminence height and bone density were associated with Early and Late progression. Larger varus angle and smaller osteophyte area were associated with No progression. Conclusion. Five phenotypes of radiographic progression of early knee OA were identified using separate quantitative features, which were associated with baseline radiographic features. Such phenotypes might require specific treatment and represent relevant subgroups for clinical trials.