In the Treatment of Lateral Epicondylitis by Percutaneous Perforation, Injectables Have No Added Value.

BACKGROUND: No single injection therapy has been proven to be superior in the treatment of lateral epicondylitis. In most studies, the injection technique is not standardized, which makes it challenging to compare outcomes. QUESTIONS/PURPOSES: (1) Does injection with autologous blood, dextrose, or needle perforation only at the extensor carpi radialis brevis tendon origin produce better VAS pain scores during provocation testing at 5 months of follow-up? (2) Which percutaneous technique resulted in better secondary outcome measures: VAS during rest and activity, VAS during maximum grip, Oxford elbow score (OES), QuickDASH, Patient-related Tennis Elbow Evaluation (PRTEE), or EuroQol-5D (EQ-5D)? METHODS: In this multicenter, randomized controlled trial performed from November 2015 to January 2020, 166 patients with lateral epicondylitis were included and assigned to one of the three treatment groups: autologous blood, dextrose, or perforation only. Complete follow-up data were available for the primary outcome measures at the 5-month follow-up interval for 77% (127 of 166) of patients. Injections of the extensor carpi radialis brevis tendon were conducted in an accurate and standardized way. The three groups did not differ in terms of key variables such as age, gender, duration of symptoms, smoking habits, pain medication, and physiotherapy use. Data were collected at baseline and 8 weeks, 5 months, and 1 year after treatment and compared among the groups. The primary endpoint was the VAS pain score with provocation at 5 months. Our secondary study outcomes were VAS pain scores during rest, after activity, and after maximum grip strength; functional recovery; and quality of life. Therefore, we report the VAS pain score (0 to 100, with higher scores representing more-severe pain, minimum clinically important difference [MCID] 10), OES (0 to 48, with higher scores representing more satisfactory joint function, MCID 10), QuickDASH (0 to 100, with higher scores representing more severe disability, MCID 5.3), PRTEE (0 to 100, with higher scores representing more pain or more disability, MCID 20), EQ-5D/QALY (EQ-5D sumscore 0 to 1, with the maximum score of 1 representing the best health state, MCID 0.04), and EQ-5D VAS (0 to 100, with higher scores representing the best health status, MCID 8). For analysis, one-way analysis of variance and a linear mixed-model analysis were used. The analyses were performed according to the intention-to-treat principle. Four patients from the perforation group opted to crossover to autologous blood after 5 months. RESULTS: No injection therapy proved to be superior to any other in terms of VAS pain scores during the provocation test at 5 months of follow-up (VAS for perforation: 25 ± 31; autologous blood: 26 ± 27; dextrose: 29 ± 32; p = 0.35). For the secondary outcomes, only a clinically important difference was found for the QuickDASH score. Both the perforation-only group (-8 [98% CI -4 to -12]) and autologous blood (-7 points [98% CI -3 to -11]) had improved QuickDASH scores over time compared with the dextrose group (MCID 5.3; p < 0.01). For the other outcomes, no clinically important differences were found. CONCLUSION: There is no benefit to injectable autologous blood and dextrose over perforation alone to treat lateral epicondylitis, and they are therefore not indicated for this condition. LEVEL OF EVIDENCE: Level I, therapeutic study.

Patients undergoing revisions for total knee replacement malposition are younger and more often female: An analysis of data from the Dutch Arthroplasty register.

Background: The proportion of patients who are dissatisfied with the outcome following total knee replacement (TKR) is high. Malalignment is considered a probable cause for persistent pain, but whether this reason for revision could be the result of specific patient characteristics remains unclear. Therefore, we aim to assess whether specific patient characteristics are associated with revision for symptomatic TKR malalignment. Methods: Data was obtained from the Dutch Arthroplasty Register (LROI), which contains data from all Dutch hospitals. All patients who underwent TKR revision surgery between 2008 and 2019 were included in this study. The (primary) reason for revision as well as patient characteristics (age, gender, ASA classification, and pre-operative patient-reported outcome measures) were abstracted. The Chi-square test for categorical variables, and ANOVA or Kruskal-Wallis for continuous variables were used to determine differences in patient characteristics between the subgroups based on reason for revision. Results: A total of 11,044 TKR revisions were registered in The Netherlands between 2008 and 2019. Malalignment was registered as the primary reason for revision in 13% of the patients. Subgroup analyses showed that patients who underwent TKR revisions for malalignment where younger (63.8 year, SD ± 9.3) and more often female (70%) compared to other major reasons for TKR revisions. Conclusion: Patients who had a TKR revision for malalignment were younger and more often female. This suggests that patient characteristics may play a role when reasons to perform revision surgery are considered. Surgeons should invest in the expectation management in (young) patients and inform patients of these potential risk factors as part of shared decision-making.

The synovial microenvironment suppresses chondrocyte hypertrophy and promotes articular chondrocyte differentiation.

During the development of the appendicular skeleton, the cartilaginous templates undergo hypertrophic differentiation and remodels into bone, except for the cartilage most adjacent to joint cavities where hypertrophic differentiation and endochondral bone formation are prevented, and chondrocytes instead form articular cartilage. The mechanisms that prevent hypertrophic differentiation and endochondral bone formation of the articular cartilage have not been elucidated. To explore the role of the synovial microenvironment in chondrocyte differentiation, osteochondral allografts consisting of articular cartilage, epiphyseal bone, and growth plate cartilage from distal femoral epiphyses of inbred Lewis rats expressing enhanced green fluorescent protein from a ubiquitous promoter were transplanted either in inverted or original (control) orientation to matching sites in wildtype littermates, thereby allowing for tracing of transplanted cells and their progenies. We found that no hypertrophic differentiation occurred in the growth plate cartilage ectopically placed at the joint surface. Instead, the transplanted growth plate cartilage, with time, remodeled into articular cartilage. This finding suggests that the microenvironment at the articular surface inhibits hypertrophic differentiation and supports articular cartilage formation. To explore this hypothesis, rat chondrocyte pellets were cultured with and without synoviocyte-conditioned media. Consistent with the hypothesis, hypertrophic differentiation was inhibited and expression of the articular surface marker lubricin (Prg4) was dramatically induced when chondrocyte pellets were exposed to synovium- or synoviocyte-conditioned media, but not to chondrocyte- or osteoblast-conditioned media. Taken together, we present evidence for a novel mechanism by which synoviocytes, through the secretion of a factor or factors, act directly on chondrocytes to inhibit hypertrophic differentiation and endochondral bone formation and promote articular cartilage formation. This mechanism may have important implications for articular cartilage development, maintenance, and regeneration.

Arthroscopic isolated long head of biceps tenotomy in patients with degenerative rotator cuff tears: mid-term clinical results and prognostic factors.

INTRODUCTION: The long head of biceps tendon is frequently involved in degenerative rotator cuff tears. Therefore, this study explored the clinical results of an isolated biceps tenotomy and identified prognostic factors for improvement in pain and function. MATERIALS AND METHODS: Between 2008 and 2017, an arthroscopic isolated biceps tenotomy was performed on 64 patients with a degenerative rotator cuff tear (> 65 years). Primary outcome was patient-perceived improvement in pain and function. Potential prognostic factors for improvement in pain and function were identified. RESULTS: A perceived improvement in pain was reported in 78% of the patients at three months after surgery and in 75% at a mean follow-up of 4.2 years (1-7 years; n = 55). A perceived improvement in function was observed in 49% of patients at three months and in 76% of patients at follow-up. Patients with a preoperatively normal acromiohumeral distance (> 10 mm) reported an improvement in pain and function significantly more often. Retraction of the supraspinatus tendon Patte 3 was significantly associated with worse functional outcome. CONCLUSIONS: A biceps tenotomy can be a reliable treatment option for patients with symptomatic degenerative cuff tears who fail conservative treatment and have a normal acromiohumeral distance (> 10 mm).

The results of using a tendon autograft as a new rotator cable for patients with a massive rotator cuff tear: a technical note and comparative outcome analysis.

BACKGROUND: Several surgical reconstructive options are available to treat massive rotator cuff tears (MRCTs). The rotator cable has an important function and we evaluated the clinical result after arthroscopic reconstruction of the rotator cable with an autograft tendon. METHODS: A prospective pilot study was performed with inclusion of four patients, average age of 64 years, with an irreparable MRCT. The patients underwent an arthroscopic reconstruction of the rotator cable with the use of the long head of biceps tendon autograft, except for one which was reconstructed with a hamstring tendon. Pre- and postsurgically, the Constant-Murley Score (CMS), Western Ontario Rotator Cuff Index (WORC), Simple Shoulder Test (SST), visual analog scale (VAS) scores, and an MRI was performed. Clinical results of the study group were compared with clinical results of comparable cohort of patients with a MRCT, treated non-operatively with physiotherapy. RESULTS: The CMS score increased after surgery in three of the four patients. The improvement of CMS score was comparable to the improvement of the CMS score encountered in a comparable cohort. The MRI at 12 months follow-up showed that the reconstructed rotator cable was disintegrated in all patients and the rotator cuff was detached and retracted. CONCLUSIONS: In our pilot study, arthroscopic reconstruction of the rotator cable using a tendon autograft failed over time and showed no clinical benefit in comparison to the non-operative treatment with physiotherapy. TRIAL REGISTRATION: The regional Medical Ethical Committee (Zwolle) gave approval at 14th of October 2016 and assigned no. 16.06100.

Prospective, observational study to evaluate the relation between patient-reported outcomes and component rotation in total knee replacement: protocol for the CRACK study.

INTRODUCTION: Total knee replacement (TKR) for osteoarthritis results in a satisfactory outcome in the majority of patients, although up to one in five patients may be dissatisfied with the outcome. Persistent pain is a main contributor to patient dissatisfaction, and femoral and tibial component malrotation have been identified as a potential cause for both persistent pain and patellofemoral problems. Based on the assumption that component malrotation is the causative factor for persistent pain, early revision for patients with symptomatic malrotated components has been advocated in the literature. However, convincing evidence that component malrotation indeed results in less than optimal outcomes is lacking. This study aims to assess the relation between knee prosthesis component rotation and patient-reported outcomes in a large group of patients and to determine the range of femoral, tibial and combined rotation that results in the best clinical outcomes. METHODS AND ANALYSIS: In this single-centre, prospective observational cohort study, a total of 500 patients will undergo TKR. All patients will have a 3D-CT assessment of femoral and tibial component rotation within 8 weeks after surgery. Outcome measures will include the Oxford Knee Score, the Knee Injury and Osteoarthritis Outcome Score, EQ-5D, visual analogue scale for pain, the American Knee Society Score and the knee joint range of motion. We will assess the relation between femoral, tibial and combined component rotation and patient-reported outcome measures at 8 weeks and 1 year of follow-up, and we will determine the range of femoral, tibial and combined rotation that results in the best clinical outcomes. ETHICS AND DISSEMINATION: Ethical approval for this study has been granted by the Isala Hospital ethics committee. The results will be published in a peer-reviewed journal and presented at relevant meetings. TRIAL REGISTRATION NUMBER: NL7635.

Patellar tendon shortening following patellofemoral joint replacement.

PURPOSE: Patellar tendon shortening may occur following patellofemoral joint replacement (PFJR). We hypothesized that patellar tendon shortening results in unfavourable patient-reported outcomes (PROs). The aim of this study was to determine the effect of patellar tendon shortening following PFJR on PROs. METHODS: In this substudy of a prospective cohort study, a total of 108 patients with isolated patellofemoral osteoarthritis underwent 124 patellofemoral joint replacements. We measured both patellar tendon length and length of the patella on pre-operative radiographs, and on radiographs acquired at eight weeks and at one year post-operative. More than 10% decrease in patellar tendon length relative to the pre-operative patellar tendon length was defined as patellar tendon shortening. Clinical outcomes were assessed using the knee-specific KOOS questionnaire (Knee Injury and Osteoarthritis Outcome Score). Repeated measures ANOVA was used to analyze for differences in change from baseline KOOS subscales between patients with and patients without patellar tendon shortening. RESULTS: A complete series of standardized pre-operative, eight weeks and one year post-operative radiographs was available for 87 knees in 82 patients. At eight weeks, 16 of 87 knees (18%) showed patellar tendon length shortening, and 27 of 87 knees (31%) at one year. We found no statistically significant relation between patellar tendon length shortening and change from baseline KOOS subscales at one year follow-up (pain p = 0.29, symptoms p = 0.56, ADL p = 0.23, sport or recreation p = 0.22, knee-related quality of life (QOL) p = 0.15). CONCLUSIONS: Patellar tendon length shortening following PFJR occurs in 31% of knees at one year, and does not result in inferior PROs.

Long-term clinical outcome of arthroscopic Bankart repair with suture anchors.

BACKGROUND: The most common surgical technique in traumatic anterior shoulder instability is the arthroscopic Bankart repair, which has excellent short-term results. The long-term results of the arthroscopic Bankart repair are less frequently studied, with a high recurrence rate of 23% to 35%. The aim of this study was to evaluate the medium-term to long-term results of arthroscopic Bankart repair using suture anchors and to identify specific risk factors for recurrent instability. METHODS: Included were 147 patients after traumatic anterior shoulder dislocation who underwent an arthroscopic Bankart repair. The primary outcome was recurrent instability, defined as dislocation or subluxation as perceived by the patients. The secondary outcome was subjective shoulder stability and function as well as quality of life, evaluated using the Western Ontario Shoulder Instability Index, the Simple Shoulder Test, and the 12-Item Short Form Health Survey. Prognostic factors for recurrent instability were analyzed. RESULTS: Recurrent instability occurred in 22% of patients with a mean follow-up of 6.3 years. Survival at 5 and 10 years without recurrent instability was 79% and 78%, respectively (95% confidence interval, 72%-85% and 71%-85%, respectively). The Western Ontario Shoulder Instability Index score, the Simple Shoulder Test score, and the 12-item Short Form Physical Component Summary improved significantly in the nonrecurrence group (P < .001, P = .004, and P = .002, respectively). Younger age and use of fewer than 3 anchors were associated with a higher risk of recurrent dislocation (P = .008 and P = .039, respectively). CONCLUSION: We found an overall recurrent instability rate of 22% (dislocation or subluxation). Good long-term results were observed after arthroscopic Bankart repair in patients older than 20 years with 3 or more suture anchors used.

Psychological symptoms and the MCID of the DASH score in shoulder surgery.

BACKGROUND: Psychological symptoms are frequently present in patients scheduled for shoulder surgery. The perception of functional disability, activity level and pain in the shoulder is negatively influenced by psychological symptoms, which leads to higher scores of the Disabilities of the Arm, Shoulder and Hand (DASH) questionnaire. The aim of this study was to determine the influence of psychological symptoms on the minimal clinically important difference (MCID) of the DASH score in patients after shoulder surgery. METHODS: In this prospective longitudinal cohort study, 176 patients were included. Group 1 (32 patients) had symptoms of psychological disorders before and after surgery; group 2 had no symptoms of psychological disorders (110 patients). In the remaining patients (34 patients), psychological disorders changed after surgery. Clinical outcome was measured with the change of DASH score and anchor questions for perceived improvement of pain and function after surgery. Symptoms of psychological disorders were identified with the Four-Dimensional Symptom Questionnaire. An anchor-based mean change score technique was used to determine the MCID of the DASH score. RESULTS: DASH scores before and 12 months after shoulder surgery were significantly higher in patients with symptoms of psychological disorders; change of DASH score was not different between the two groups. The MCID of the DASH score was 13.0 [SD 20.7] in the group with symptoms of psychological disorders and 12.7 [SD 17.6] in the group with no symptoms of psychological disorders. We observed no difference (p = 0.559) in the MCID between the group with and the group without symptoms of psychological disorders. CONCLUSION: Symptoms of psychological disorders had a negative effect on the DASH score but no influence on the MCID of the DASH score. The DASH score could be used in future studies to assess the influence of psychological factors on the clinical outcome of treatment.

Hydrolytically Labile Linkers Regulate Release and Activity of Human Bone Morphogenetic Protein-6.

Release of growth factors while simultaneously maintaining their full biological activity over a period of days to weeks is an important issue in controlled drug delivery and in tissue engineering. In addition, the selected strategy to immobilize growth factors largely determines their biological activity. Silica surfaces derivatized with glycidyloxy propyl trimethoxysilane and poly(glycidyl methacrylate) brushes yielded epoxide-functionalized surfaces onto which human bone morphogenetic protein-6 (hBMP-6) was immobilized giving stable secondary amine bonds. The biological activity of hBMP-6 was unleashed by hydrolysis of the surface siloxane and ester bonds. We demonstrate that this type of labile bonding strategy can be applied to biomaterial surfaces with relatively simple and biocompatible chemistry, such as siloxane, ester, and imine bonds. Our data indicates that the use of differential hydrolytically labile linkers is a versatile method for functionalization of biomaterials with a variety of growth factors providing control over their biological activity.

Predictive findings on magnetic resonance imaging in patients with symptomatic acromioclavicular osteoarthritis.

BACKGROUND: A magnetic resonance imaging (MRI) scan of the shoulder can have added value in diagnosing symptomatic osteoarthritis of the acromioclavicular (AC) joint. Specific MRI signs have been recognized but not analyzed extensively before. This study aims to identify predictive MRI signs in patients with symptomatic AC osteoarthritis. METHODS: The MRI scans of 70 patients with symptomatic AC osteoarthritis were compared with those of 70 patients with subacromial pain syndrome and no clinical signs of symptomatic AC osteoarthritis. Seven variables were evaluated on the MRI scans of the AC joint: joint space narrowing, inferior osteophytes, joint effusion, osteolysis, bone marrow edema, impression on the supraspinatus, and inferior joint distension. Logistic regression analysis of these variables was performed. RESULTS: The presence of inferior osteophytes, bone marrow edema, impression on the supraspinatus, and inferior joint distension was individually associated with symptomatic AC osteoarthritis. Bone marrow edema was observed only in patients with symptomatic AC osteoarthritis. Multivariate analysis showed a significant association between inferior joint distension, as well as impression on the supraspinatus muscle, and symptomatic AC osteoarthritis. The area under the receiver operating characteristic curve in the multivariate logistic model was 0.839 (95% confidence interval, 0.771 to 0.907). Interobserver and intraobserver variability showed good to excellent κ values (range, 0.68 to 0.88). CONCLUSION: We identified predictive MRI signs in patients with symptomatic AC osteoarthritis. These findings, including bone marrow edema, inferior joint distension, and impression on the supraspinatus muscle, showed good discriminative ability. They are practical and easy to use and can assist the physician in diagnosing symptomatic AC osteoarthritis.

O-Phenanthroline as modulator of the hypoxic and catabolic response in cartilage tissue-engineering models.

Hypoxia has been shown to be important for maintaining cartilage homeostasis as well as for inducing chondrogenic differentiation. Ensuring low oxygen levels during in vitro culture is difficult, therefore we assessed the chondro-inductive capabilities of the hypoxia-mimicking agent O-phenanthroline, which is also known as a non-specific matrix metalloproteinase (MMP) inhibitor. We found that O-phenanthroline reduced the expression of MMP3 and MMP13 mRNA levels during chondrogenic differentiation of human chondrocytes (hChs), as well as after TNFα/IL-1ß exposure in an explant model. Interestingly, O-phenanthroline significantly inhibited matrix degradation in a TNFα/IL-1ß-dependent model of cartilage degeneration when compared to control and natural hypoxia (2.5% O2 ). O-Phenanthroline had limited ability to improve the chondrogenic differentiation or matrix deposition in the chondrogenic pellet model. Additionally, O-phenanthroline alleviated MMP-induced cartilage degradation without affecting chondrogenesis in the explant culture. The data presented in this study indicate that the inhibitory effect of O-phenanthroline on MMP expression is dominant over the hypoxia-mimicking effect. Copyright © 2014 John Wiley & Sons, Ltd.

EZH1 and EZH2 promote skeletal growth by repressing inhibitors of chondrocyte proliferation and hypertrophy.

Histone methyltransferases EZH1 and EZH2 catalyse the trimethylation of histone H3 at lysine 27 (H3K27), which serves as an epigenetic signal for chromatin condensation and transcriptional repression. Genome-wide associated studies have implicated EZH2 in the control of height and mutations in EZH2 cause Weaver syndrome, which includes skeletal overgrowth. Here we show that the combined loss of Ezh1 and Ezh2 in chondrocytes severely impairs skeletal growth in mice. Both of the principal processes underlying growth plate chondrogenesis, chondrocyte proliferation and hypertrophy, are compromised. The decrease in chondrocyte proliferation is due in part to derepression of cyclin-dependent kinase inhibitors Ink4a/b, while ineffective chondrocyte hypertrophy is due to the suppression of IGF signalling by the increased expression of IGF-binding proteins. Collectively, our findings reveal a critical role for H3K27 methylation in the regulation of chondrocyte proliferation and hypertrophy in the growth plate, which are the central determinants of skeletal growth.

A supramolecular host-guest carrier system for growth factors employing V(H)H fragments.

A supramolecular strategy is presented for the assembly of growth factors employing His6-tagged single-domain antibodies (VHH). A combination of orthogonal supramolecular interactions of ß-cyclodextrin (ßCD)-adamantyl (Ad) host-guest and N-nitrilotriacetic acid (NTA)-histidine (His) interactions was employed to generate reversible and homogeneous layers of growth factors. A single-domain antibody V(H)H fragment was identified to bind to the human bone morphogenetic protein-6 (hBMP6) growth factor and could be recombinantly expressed in E. coli. The V(H)H fragment was equipped with a C-terminal hexahistidine (His6) tether to facilitate the assembly on ßCD surfaces using a linker that contains an Ad group to bind to the ßCD receptors and an NTA moiety to interact with the His6-tag upon cocomplexation of Ni(2+) ions. After exploring the thermodynamic and kinetic stability of the V(H)H assemblies on ßCD surfaces using a variety of experimental techniques including microcontact printing (µCP), surface plasmon resonance (SPR), microscale thermophoresis (MST), and theoretical models for determining the thermodynamic behavior of the system, hBMP6 was assembled onto the V(H)H-functionalized surfaces. After analyzing the immobilized hBMP6 using immunostaining, the biological activity of hBMP6 was demonstrated in cell differentiation experiments. Early osteogenic differentiation was analyzed in terms of alkaline phosphatase (ALP) activity of KS483-4C3 mouse progenitor cells, and the results indicated that the reversibly immobilized growth factors were functionally delivered to the cells. In conclusion, the supramolecular strategy used here offers the necessary affinity, reversibility, and temporal control to promote biological function of the growth factors that were delivered by this strategy.

Gene expression profiling reveals similarities between the spatial architectures of postnatal articular and growth plate cartilage.

Articular and growth plate cartilage are discrete tissues but arise from a common cartilaginous condensation and have comparable spatial architectures consisting of distinct layers of chondrocytes. To investigate similarities and differences between articular and growth plate cartilage and to explore transcriptional changes that occur during the onset of their divergence, we performed manual microdissection of 10-day-old rat proximal tibias, microarray analysis, bioinformatics, and real-time PCR to compare gene expression profiles in individual cartilage layers. We found that many genes that were spatially upregulated in the intermediate/deep zone of articular cartilage were also spatially upregulated in the resting zone of growth plate cartilage (overlap greater than expected by chance, P<0.001). Interestingly, the superficial zone of articular cartilage showed an expression profile with similarities to both the proliferative and hypertrophic zones of growth plate cartilage (P<0.001 each). Additionally, significant numbers of known proliferative zone markers (3 out of 6) and hypertrophic zone markers (27 out of 126) were spatially upregulated in the superficial zone (more than expected by chance, P<0.001 each). In conclusion, we provide evidence that the intermediate/deep zone of articular cartilage has a gene expression profile more similar to that of the resting zone of growth plate cartilage, whereas the superficial zone has a gene expression profile more similar to those of the proliferative and hypertrophic zones. These findings suggest that the superficial zone chondrocytes of articular cartilage differentiate according to a program that is not completely different from but instead has distinct similarities to the hypertrophic differentiation program of growth plate chondrocytes. We also present functional signaling pathways implicated by differential gene expression between articular and growth plate cartilage during their initial separation by the secondary ossification center.

Evidence that estrogen hastens epiphyseal fusion and cessation of longitudinal bone growth by irreversibly depleting the number of resting zone progenitor cells in female rabbits.

With age, growth plate cartilage undergoes programmed senescence, eventually causing cessation of bone elongation and epiphyseal fusion. Estrogen accelerates this developmental process. We hypothesized that senescence occurs because progenitor cells in the resting zone are depleted in number and that estrogen acts by accelerating this depletion. To test this hypothesis, juvenile ovariectomized rabbits received injections of estradiol cypionate or vehicle for 5 weeks, and then were left untreated for an additional 5 weeks. Exposure to estrogen accelerated the normal decline in growth plate height and in the number of proliferative and hypertrophic chondrocytes. Five weeks after discontinuation of estrogen treatment, these structural parameters remained advanced, indicating an irreversible advancement in structural senescence. Similarly, transient estrogen exposure hastened epiphyseal fusion. Estrogen also caused a more rapid decline in functional parameters of growth plate senescence, including growth rate, proliferation rate, and hypertrophic cell size. However, in contrast to the structural parameters, once the estrogen treatment was discontinued, the growth rate, chondrocyte proliferation rate, and hypertrophic cell size all normalized, suggesting that estrogen has a reversible, suppressive effect on growth plate function. In addition, estrogen accelerated the normal loss of resting zone chondrocytes with age. This decrease in resting zone cell number did not appear to be due to apoptosis. However, it was maintained after the estrogen treatment stopped, suggesting that it represents irreversible depletion. The findings are consistent with the hypothesis that estrogen causes irreversible depletion of progenitor cells in the resting zone, thus irreversibly accelerating structural senescence and hastening epiphyseal fusion. In addition, estrogen reversibly suppresses growth plate function.

GREM1, FRZB and DKK1 mRNA levels correlate with osteoarthritis and are regulated by osteoarthritis-associated factors.

INTRODUCTION: Osteoarthritis is, at least in a subset of patients, associated with hypertrophic differentiation of articular chondrocytes. Recently, we identified the bone morphogenetic protein (BMP) and wingless-type MMTV integration site (WNT) signaling antagonists Gremlin 1 (GREM1), frizzled-related protein (FRZB) and dickkopf 1 homolog (Xenopus laevis) (DKK1) as articular cartilage's natural brakes of hypertrophic differentiation. In this study, we investigated whether factors implicated in osteoarthritis or regulation of chondrocyte hypertrophy influence GREM1, FRZB and DKK1 expression levels. METHODS: GREM1, FRZB and DKK1 mRNA levels were studied in articular cartilage from healthy preadolescents and healthy adults as well as in preserved and degrading osteoarthritic cartilage from the same osteoarthritic joint by quantitative PCR. Subsequently, we exposed human articular chondrocytes to WNT, BMP, IL-1ß, Indian hedgehog, parathyroid hormone-related peptide, mechanical loading, different medium tonicities or distinct oxygen levels and investigated GREM1, FRZB and DKK1 expression levels using a time-course analysis. RESULTS: GREM1, FRZB and DKK1 mRNA expression were strongly decreased in osteoarthritis. Moreover, this downregulation is stronger in degrading cartilage compared with macroscopically preserved cartilage from the same osteoarthritic joint. WNT, BMP, IL-1ß signaling and mechanical loading regulated GREM1, FRZB and DKK1 mRNA levels. Indian hedgehog, parathyroid hormone-related peptide and tonicity influenced the mRNA levels of at least one antagonist, while oxygen levels did not demonstrate any statistically significant effect. Interestingly, BMP and WNT signaling upregulated the expression of each other's antagonists. CONCLUSIONS: Together, the current study demonstrates an inverse correlation between osteoarthritis and GREM1, FRZB and DKK1 gene expression in cartilage and provides insight into the underlying transcriptional regulation. Furthermore, we show that BMP and WNT signaling are linked in a negative feedback loop, which might prove essential in articular cartilage homeostasis by balancing BMP and WNT activity.

Small molecule inhibitors of WNT/ß-catenin signaling block IL-1ß- and TNFα-induced cartilage degradation.

INTRODUCTION: In this study, we tested the ability of small molecule inhibitors of WNT/ß-catenin signaling to block interleukin 1ß (IL-1ß)- and tumor necrosis factor α (TNFα)-induced cartilage degradation. Proinflammatory cytokines such as IL-1ß and TNFα are potent inducers of cartilage degradation by upregulating matrix metalloproteinase (MMP) expression and activity. Because WNT/ß-catenin signaling was found to be involved in IL-1ß- and TNFα-induced upregulation of MMP activity, we hypothesized that inhibition of WNT/ß-catenin signaling might block IL-1ß- and TNFα-induced cartilage degradation. We tested the effect of small molecules that block the interaction between ß-catenin and TCF/Lef transcription factors on IL-1ß- and TNFα-induced cartilage degradation in mouse fetal metatarsals. METHODS: We used mouse fetal metatarsals treated with IL-1ß and TNFα as an ex vivo model for cytokine-induced cartilage degradation. Metatarsals were treated with IL-1ß and TNFα in combination with the small molecules PKF115-584, PKF118-310 and CGP049090 at different concentrations and then harvested them for histological and gene expression analysis. RESULTS: We found that IL-1ß- and TNFα-induced cartilage degradation in mouse fetal metatarsals was blocked by inhibiting WNT/ß-catenin signaling using small molecule PKF115-584 and partially using CGP049090 dose-dependently. In addition, we found that PKF115-584 blocked IL-1ß- and TNFα-induced MMP mRNA expression, but did not reverse the inhibitory effect of IL-1ß on the expression of cartilage anabolic genes. CONCLUSION: In this study, we show that inhibition of WNT/ß-catenin signaling by small molecules can effectively prevent IL-1ß- and TNFα-induced cartilage degradation by blocking MMP expression and activity. Furthermore, we elucidate the involvement of WNT/ß-catenin signaling in IL-1ß- and TNFα-induced cartilage degradation.

WNT signaling and cartilage: of mice and men.

In adult articular cartilage, the extracellular matrix is maintained by a balance between the degradation and the synthesis of matrix components. Chondrocytes that sparsely reside in the matrix and rarely proliferate are the key cellular mediators for cartilage homeostasis. There are indications for the involvement of the WNT signaling pathway in maintaining articular cartilage. Various WNTs are involved in the subsequent stages of chondrocyte differentiation during development, and deregulation of WNT signaling was observed in cartilage degeneration. Even though gene expression and protein synthesis can be activated upon injury, articular cartilage has a limited ability of self-repair and efforts to regenerate articular cartilage have so far not been successful. Because WNT signaling was found to be involved in the development and maintenance of cartilage as well as in the degeneration of cartilage, interfering with this pathway might contribute to improving cartilage regeneration. However, most of the studies on elucidating the role of WNT signaling in these processes were conducted using in vitro or in vivo animal models. Discrepancies have been found in the role of WNT signaling between chondrocytes of mouse and human origin, and extrapolation of results from mouse models to the human situation remains a challenge. Elucidation of detailed WNT signaling functions will provide knowledge to improve cartilage regeneration.

A fluorogenic monolayer to detect the co-immobilization of peptides that combine cartilage targeting and regeneration.

Strategies to generate platforms combining tissue targeting and regeneration properties are in great demand in the regenerative medicine field. Here we employ an approach to directly visualize the immobilization of cysteine-terminated peptides on a novel fluorogenic surface. Peptides with relevant biological properties, CLPLGNSH and CLRGRYW, were synthesized to function as peptide binders to transforming growth factor (TGF)-ß1 and collagen type II (CII). The selective immobilization of the peptides was directly detected using a fluorogenic surface. Adhered proteins were confined to patterns of these peptides matching with the fluorogenic areas. These results show that the fluorogenic signal can be used to detect the chemo-selective immobilization of non-fluorescent biomolecules and to correlate the cell response with the patterned peptides. After analyzing the sequence specificity and cross-reactivity of the binding of TGF-ß1 and CII to the respective peptide regions employing immunofluorescence assays, both peptides were co-immobilized in a step-wise process as detected by the fluorogenic surface. TGF-ß1 and CII could be self-sorted from a mixture in a regio-selective manner resulting in a bi-functional protein platform. Surfaces of CLPLGNSH pre-loaded with TGF-ß1 showed excellent bioactivity in combination with human articular chondrocytes (HACs) and stimulated expression of chondrogenic markers.