Introduction

Post-meniscectomy syndrome is broadly characterised by intractable pain following the partial or total removal of a meniscus. There is a large treatment gap between the first knee pain after meniscectomy and the eligibility for a TKA. Hence, there is a strong unmet need for a solution that will relieve this post-meniscectomy pain. Goal of this first-in-man study was to evaluate the safety and performance of an anatomically shaped artificial medial meniscus prosthesis and the accompanying surgical technique.

Osteoarthritis is one of the major causes of immobility. Most commonly, osteoarthritis manifests at the knee joint. Prevalence of knee osteoarthritis (KNOA) increases with age. Another important risk factor for KNOA is obesity. Research has shown that obese subjects have almost four times the risk of developing KNOA, which may be explained by both an increased knee loading. In medial compartment KNOA, the knee adduction moment (KAM) during gait is considered a marker for disease severity. KAM is dependent of the magnitude of the ground reaction force and its moment arm relative to the knee joint centre. In addition, obesity has been reported to augment KAM during gait. However, after removal of the direct contributions of body weight, KAM parameters may be different due to obesity-related gait adaptations to limit knee loading. While KAM has been thoroughly investigated during gait, little is known about KAM during stair negotiation, during which knee loads are higher compared to gait. The aim of the current study is therefore to compare normalized KAM during the stance phase of stair negotiation between lean KNOA patients, obese KNOA patients, and healthy controls. This case control study included 20 lean controls, 14 lean KNOA patients, and 16 obese KNOA patients. All subjects ascended and descended a two-step staircase at a self-selected, comfortable speed. Radiographic imaging and MRI were used to evaluate knee cartilage and KNOA status. Motion analysis was performed with a three-dimensional motion capture system. Kinetic data were obtained by one force platform. The parameters of study included: stance phase duration, toe-out angle, KAM peaks and KAM impulse. During stair ascent obese KNOA patients showed a longer stance phase than healthy controls (P 0.050). Despite high between-subject variability, KAM impulse was found 45% higher in the obese KNOA group during stair descent, when compared to healthy controls (P =0.012). The absence of a significant effect of groups on the normalized KAM during stair negotiation may be explained by a lower ambulatory speed in the obese KNOA group, that effectively lowers GRFz. Decreasing ambulatory speed may be an effective strategy to lower KAM during stair negotiation.

Focal knee resurfacing implants (FKRIs) are typically intended to treat focal cartilage defects in middle-aged patients. All currently available FKRIs are (partly) composed of metal, which potentially leads to degeneration of the opposing articulating cartilage and hampers follow-up using magnetic resonance imaging (MRI). The purpose of this study was to investigate the in vivo osseointegration process of a novel non-degradable thermoplastic polycarbonate-urethane (TPU) osteochondral implant. Bi-layered implants measuring 6 mm in diameter, with a double-curvature to match the approximate curvature of the goat medial femoral condyle were fabricated. TPU implants were composed of an articulating Bionate® II 80A top layer, and a Bionate® 75D bottom layer (DSM Biomedical, Geleen, the Netherlands) which is intended to osseointegrate. A biphasic calcium phosphate coating formulation, optimized during a prior in vitro study, was applied to half of the TPU implants, while the other half was left uncoated. Bi-layered metal implants (articulating cobalt-chromium top layer and titanium bottom layer) were used as positive control implants. Eight implants per group were implanted bilaterally in the medial femoral condyle of the stifle joints in 12 Dutch milk goats. 18F-sodium fluoride (18F-NaF) positron emission tomography-computed tomography (PET-CT) scanning was performed at 3 and 12 weeks postoperatively, and the corrected maximum standard uptake values (cSUVmax) was calculated to assess the peri-implant bone metabolism. After sacrifice 12 weeks postoperatively, bone histomorphometric analysis was performed to assess the bone-to-implant contact area (BIC). Student's T-test was used in case of normal distribution and the Mann-Whitney-U-test was used in case of abnormal distribution for comparison of BIC and cSUVmax. The BIC value of 10.27 ± 4.50% (mean ± SD) for the BCP-coated TPU implants was significantly (P=0.03) higher than the 4.50 ± 2.61% for the uncoated TPU implants. The uncoated TPU implants scored significantly (P=0.04) lower than the BIC of 12.81 ± 7.55% for the metal implants, whereas there was no significant difference between BCP-coated TPU implants and the metal implants (P=0.68). There was a strong correlation between the cSUVmax values and the BIC values at 12 weeks (Pearson's R=0.74, P=0.001). The cSUVmax values significantly decreased between 3 and 12 weeks for the metal implants (p=0.04). BCP-coated TPU implants followed a similar trend but did not reach statistical significance (p=0.07). cSUVmax in the uncoated TPU implants did not show a significant difference between the time-points (p=0.31). Osseointegration of BCP-coated TPU implants did not significantly differ from metal implants. 18F-NaF PET-CT is a feasible modality to assess osseointegration patterns and showed a similar trend between the BCP-coated and metal implants. Hence, an implant fully composed of TPU may avoid the typical metal-related drawbacks of currently available FKRIs. Long-term follow-up studies are advocated to address the effects of the implant to the opposing cartilage, and are therefore warranted.

The modified Hedgehog technique was previously used to reattach pure chondral shear-off fragments in the pediatric knee. In the modified Hedgehog technique, the calcified side of chondral fragments is multiple times incised and trimmed obliquely for an interlocking fit in the defect site. Fibrin glue with or without sutures is subsequently applied to fix the fragment to the defect. This preliminary report further elucidates the potential of the technique by evaluation of its application in young adults using patient reported outcome measures (PROMs) and high-field Magnetic Resonance Imaging (MRI) as outcome measures. Three patients with a femoral cartilage defect (2 medial, 1 lateral), and a concomitant pure chondral corpus liberum were operatively treated by the modified Hedgehog technique. Age at surgery ranged from 20.6–21.2 years, defect size ranged from 3.8–6.0 cm2. Patients were evaluated at three months and one year after surgery by PROMs and 7.0T MRI. PROMs included the Internation Knee Documentation (IKDC), Knee Injury and Osteoarthritis Outcome Score (KOOS) and Visual Analog Scale (VAS) questionnaires. 7.0T MRI (Magnetom, Siemens Healthcare, Erlangen, Germany) using a 28-channel proton knee coil (QED, Electrodynamics LLC, Cleveland, OH) included a proton density weighted turbo spin-echo sequence with fat suppression to assess morphological tissue structure andgagCEST imaging to measure the biochemical tissue composition in terms of glycosaminoglycans (GAG). Twelve months after surgery all patients reported no pain and showed full range of motion. While PROMs at three months showed large variability between patients, one year after surgery the scores were consistently improved. Over time, morphological MRI visualized improvements in integration of the cartilage fragment with the surrounding cartilage, which was supported by biochemical MRI showing increased GAG values at the defect edges. Statistics were not applied to the results because of the small sample size. The modified Hedgehog technique in young adults with an acute onset caused by a pure chondral corpus liberum can be considered promising. The improved PROM results over time were supported by 7.0T MRI that visualized improvements in tissue structure and biochemical composition. Inclusion of more patients in future studies would allow statistical analysis and more conclusive results. The etiology of loosening and time between onset of symptoms and surgery for successful graft integration may differ between pediatric and young adult patients and is subject for future studies.

Early detection of knee osteoarthritis (OA) is critical for possible preventive treatment, such as weight loss, physical activity and sports advice and restoring biomechanics, to postpone total knee arthroplasty (TKA). Specific biomarkers for prognosis and early diagnosis of OA are lacking. Therefore, in this study, we analyzed the lipid profiles of different tissue types within Hoffa's fat pad (HFP) of OA and cartilage defect (CD) patients, using matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging (MSI). The HFP has already been shown to play an important role in the inflammatory process in OA by prostaglandin release. Additionally, MALDI-MSI allows us to investigate on tissue lipid distribution at molecular level, which makes it a promising tool for the detection of disease specific biomarkers for OA development.

Samples of HFP were obtained of patients undergoing surgical treatment for OA (n=3) (TKA) or CD (n=3) (cartilage repair). In all cases, tissue was obtained without patient harm. HFP samples were washed in phosphate buffered saline (PBS) and snap-frozen directly after surgical dissection to remove redundant blood contamination and to prevent as much tissue degradation as possible. Tissue sections were cut at 15 µm thickness in a cryostat (Leica Microsystems, Wetzlar) and deposited on indium tin oxide glass slides. Norharmane (Sigma-Aldrich) matrix was sublimed onto the tissue using the HTX Sublimator (HTX Technologies, Chapel Hill). µMALDI-MSI was performed using Synapt G2Si (Waters) at 50 µm resolution in positive ion mode. MS/MS fragmentation was performed for lipid identification. Data were processed with in-house Tricks for MATLAB and analyzed using principle component analysis (PCA) and verlan

OA and CD HFP specific lipid profiles were revealed by MALDI-MSI followed by PCA and DA. With these analyses we were able to distinguish different tissue types within HFP of different patient groups. Further discriminant analysis showed HFP intra-tissue heterogeneity with characteristic lipid profiles specific for connective and adipose tissues, but also for synovial tissue and blood vessels, revealing the high molecular complexity of this tissue. As expected, lipid signals were lower at the site of the connective tissue, compared to the adipose tissue. In particular, tri-acyl glycerol, di-acyl glycerol, sphingomyelin and phosphocholine species were differently abundant in the adipose tissue of HFP of OA compared to CD.

To our knowledge, this is the first study comparing lipid profiles in HFP of OA patients with CD patients using MALDI-MSI. Our results show different lipid profiles between OA and CD patients, as well as intra-tissue heterogeneity within HFP, rendering MALDI-MSI as a useful technology for OA biomarker discovery. Future research will focus on expanding the number of subjects and the improvement of lipid detection signals.

The incidence of osteoarthritis (OA) is increasing in our younger population. OA development early in life is often related to cartilage damage, caused by (sport) injury or trauma. Detection of early knee OA is therefore crucial to target early treatment. However, early markers for OA prognosis or diagnosis are lacking. Hoffa's fat pad (HFP) is an emerging source for knee biomarkers, as it is easily accessible and shows important interaction with the homeostasis of the knee. In this study, we used Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) as a first approach. MALDI-MSI allows the study of tissue-specific molecular distributions. Therefore, we used MALDI-MSI to analyze the lipid profiles in the HFP of three patients with OA and three patients undergoing cartilage regenerative treatment. We demonstrate that the lipid profile of patients with OA is different from patients with cartilage defects.

HFP of each patient were snap frozen directly after surgical resection and cryosectioned at 15 μm. Each slide was sublimed with Norharmane matrix and analyzed by MALDI-MSI in positive and negative ion modes at a lateral resolution of 50 μm on a RapifleX Tissue Typer. The difference between patient groups were analyzed using principle component analysis and linear discriminant analysis. Lipid identifications were obtained on an Orbitrap Elite™ Hybrid Ion Trap-Orbitrap Mass Spectrometer in data dependent acquisition mode and analyzed using Lipostar software.

Linear discriminant analysis showed a specific lipid profile for each group (variance 33.94%). Score projections revealed a differential lipid spatial distribution of OA patients compared to cartilage defect patients. Among the lipids that differed significantly, for instance, the m/z 760.59 [M+H]⁺ was associated to osteoarthritis and identified as glycerophospholipid (PC 34:1), a main component of biological membranes. Additionally, the samples were found to be intra-tissue heterogeneous, with molecular profiles found in adipose-, connective- and synovial tissue.

These results suggest that lipid profiles in HFP could be useful for early OA detection. However, intra-tissue heterogeneity in HFP should be recognized when using HFP as a biomarker source.

INTRODUCTION

Adequate osseointegration of knee resurfacing implants for the treatment of focal cartilage defects is an important prerequisite for good clinical outcomes. Inadequate initial fixation and sustained micromotion may lead to osteolysis and ultimately implant failure. PET/CT with the bone seeking tracer 18F-sodium fluoride (18F-NaF) allows for localisation and quantification of abnormalities in bone metabolism. 18F-NaF PET/CT has been shown to correlate with loosening of implants in the hip and spine. Here, we asses osseointegration of the knee resurfacing implants using micro-computed tomography (µCT) and correlate µCT parameters to 18F-NaF uptake on PET/CT scans taken 3 and 12 weeks after surgery. We hypothesize that 18F-NaF uptake at 12 weeks and its relative decrease between 3 and 12 weeks correlates with osseointegration at 12 weeks postoperatively. Polymer implants with Young”s moduli approximately equal to- and below the Young's modulus of bone, with- and without surface modification were used in this study next to a control metal implant.

INTRODUCTION

The generation of cartilage from progenitor cells for the purpose of cartilage repair is often hampered by unwanted hypertrophic differentiation of the generated tissue due to endochondral ossification. Continuing on our earlier studies, our goal is to further improve the engineering of hyaline cartilage for the treatment of a cartilage defect in our in vivo model for subperiosteal generation of cartilage, by tuning the differentiation status of the generated cartilage and prevent hypertrophic differentiation. As a healthy cartilage matrix contains high amounts of aggrecan we hypothesise that aggrecan supplementation of the bio-gel used in the generation of the subperiosteal cartilage, mimics the composition of the extracellular matrix environment of cartilage with potential beneficial properties for the engineered cartilage.

Introduction

Cartilage homeoprotein 1 (CART-1) is a homeoprotein which has been suggested to play a role in chondrocyte differentiation and in skeletal development. It is expressed mainly in prechondrocytic mesenchymal condensations. Patients with mutations in the CART-1 gene display several craniofacial abnormalities, suggesting that CART-1 has a functional role in craniofacial skeletal development. However, its target genes and position in the established chondrogenic pathways is poorly documented. Given the fact that CART-1 is expressed predominantly in the chondrocyte lineage and its role in skeletal development, we hypothesized that CART-1 regulates expression of several pivotal genes involved in chondrogenic differentiation.

INTRODUCTION

The generation of cartilage from progenitor cells for the purpose of cartilage repair is often hampered by unwanted ossification of the generated tissue due to endochondral ossification. Our in vitro data show that celecoxib is able to suppress the hypertrophic differentiation phase of endochondral ossification in differentiating human bone marrow stem cells via inhibition of prostaglandin signalling. Continuing on our earlier studies our goal is to further improve the engineering of hyaline cartilage for the treatment of cartilage defects, by determining if celecoxib released from poly(D,L-lactic acid)microspheres is able to prevent unwanted ossification in an in vivo model for the subperiosteal cartilage generation.

Introduction

In vitro expansion of human articular chondrocytes (HACs) is required for cell-based strategies to treat cartilage defects. We have earlier shown that culturing HACs at increased osmolarity (i.e., 380 mOsm), as compared to plasma osmolarity (i.e., 280 mOsm), increases collagen type II (COL2A1) expression in vitro. Our earlier results showed that knockdown of TGF-β2, a prototypic member of the TGF-β superfamily and an accepted key regulator of chondrocyte differentiation, resulted in increased COL2A1 production. BMPs are members of the TGF-β superfamily which are known to be involved in the regulation of COL2A1 expression. In this study, we aimed to elucidate the role of BMP signaling, in the upregulation of COL2 production upon TGF-β2 knockdown (KD) under hyperosmotic culture conditions.

Background

Treatment of cartilage defects requires in vitro expansion of human articular chondrocytes (HACs) for autologous chondrocyte implantation (ACI). During standard expansion culture (i.e. plasma osmolarity, 280 mOsm) chondrocytes inevitably lose their specific phenotype (i.e. collagen type II (COL2) expression). This de-differentiation makes them inappropriate for ACI. Physiological osmolarity (i.e. 380 mOsm) improves COL2 expression in vitro, but the underlying reason is unknown. However, an accepted key regulator of chondrocyte differentiation, transforming growth factor beta (TGFβ), is known to stimulate COL2 production. In this study we aimed to elucidate if TGFβ signaling could potentially be driving the COL2 expression under physiological culture conditions.

As cartilage has poor intrinsic repair capacity, in vitroexpansion of human articular chondrocytes (HACs) is required for cell-based therapies to treat cartilage pathologies. During standard expansion culture (i.e. plasma osmolarity, 280 mOsm) chondrocytes inevitably lose their specific phenotype and de-differentiate, which makes them inappropriate for autologous chondrocyte implantation. It has been shown that physiological osmolarity (i.e. 380 mOsm) increases collagen type II (COL2) expression in vitro, but the underlying molecular mechanism is unknown. Transforming growth factor beta (TGFβ) super family members are accepted key regulators of chondrocyte differentiation and known to stimulate COL2 production. In this study we aimed to elucidate the role of TGFβ superfamily member signalling as a molecular mechanism potentially driving the COL2 expression under physiological (380 mOsm) culture conditions.

HACs from OA patients (p1) were cultured in cytokine-free medium of 280 or 380 mOsm, under standard 2D in vitroconditions, with or without lentiviral TGFβ2 knockdown (RNAi). Expression of TGFβ isoforms, BMPs and chondrocyte marker genes was evaluated by QPCR. TGFβ2 protein secretion was evaluated using ELISA and bioactivity was determined using an established reporter cell line. Involvement of BMP signaling was investigated by culturing OA HACs (p1) in the presence or absence of dorsomorphin (10 µM).

Physiological osmolarity increased TGFβ2 and TGFβ3 mRNA expression, TGFβ2 protein secretion as well as general TGFβ activity by 380 mOsm. Upon TGFβ2 isoform-specific knockdown COL2 mRNA expression was induced. TGFβ2 RNAi induced expression of several BMPs (e.g. BMP2,-4,-6) and this induction was enhanced in culture conditions with physiological osmolarity. Dorsomorphin inhibited physiological osmolarity induced COL2 mRNA expression.

TGFβ2 knockdown under 380 mOsm increases COL2 expression in human osteoarthritic chondrocytes in vitromost likely through a regulatory feedback loop via BMP signaling, which is involved in osmolarity-induced COL2 expression. Future studies will further elucidate the BMP-mediated regulatory feedback loop after TGF β2 knockdown and its influence on COL2 expression.

Heterotopic ossi?cation is the abnormal formation of bone in soft tissues and is a frequent complication of hip replacement surgery. Heterotopic ossi?cations are described to develop via endochondral ossification and standard treatment is administration of indomethacin. It is currently unknown how indomethacin influences heterotopic ossi?cation on a molecular level, therefore we aimed to determine whether indomethacin might influence heterotopic ossi?cation via impairing the chondrogenic phase of endochondral ossification.

ATDC5, human bone marrow stem cells (hBMSCs) and rabbit periosteal agarose cultures were employed as progenitor cell models; SW1353, human articular chondrocytes and differentiated ATDC5 cells were used as matured chondrocyte cell models. All cells were cultured in the presence of (increasing) concentrations of indomethacin. The action of indomethacin was confirmed by decreased PGE2 levels in all experiments, and was determined by specific PGE2 ELISA. Gene- and protein expression analyses were employed to determine chondrogenic outcome.

Progenitor cell models differentiating in the chondrogenic lineage (ATDC5, primary human bone marrow stem cells and ex vivo periosteal agarose cultures) were treated with increasing concentrations of indomethacin and a dose-dependent decrease in gene- and protein expression of chondrogenic and hypertrophic markers as well as decreased glycosaminoglycan content was observed. Even when hypertrophic differentiation was provoked the addition of indomethacin resulted in decreased hypertrophic marker expression. Interestingly, when mature chondrocytes were treated with indomethacin, a clear increase in collagen type 2 expression was observed. Similarly, when ATDC5 cells and bone marrow stem cells were pre-differentiated to obtain a chondrocyte phenotype and indomethacin was added from this time point onwards, low concentrations of indomethacin also resulted in increased chondrogenic differentiation.

Indomethacin induces differential effects on in vitro endochondral ossification, depending on the chondrocyte's differentiation stage, with complete inhibition of chondrogenic differentiation as the most pronounced action. This observation may provide a rationale behind the elusive mode of action of indomethacin in the treatment of heterotopic ossifications.

Summary

The quantification of T1Rho relaxation times is not related with internal loading. Improvements in modeling and imaging techniques might lead to better understanding of the pathomechanics of the knee.

Summary Statement

This paper is the first to compare the results of unicompartmental to total knee arthroplasty revision surgery between cases with explained pain and cases with unexplained pain. Revision surgery for unexplained pain usually results in a less favourable outcome.

Summary

Indomethacin has differential effects on chondrogencic outcome depending on differentiation stage