Superficial Zone Of Articular Cartilage Research Articles

Prg4-Expressing Chondroprogenitor Cells in the Superficial Zone of Articular Cartilage

Prg4-Expressing Chondroprogenitor Cells in the Superficial Zone of Articular Cartilage

KARTOGENIN-ENCAPSULATED COAXIAL PGS/PCL-ALIGNED NANOFIBRES FOR ARTICULAR CARTILAGE REGENERATION

Electrospinning is an advantageous technique for cartilage tissue engineering (CTE) applications due to its ability to produce nanofibers recapitulating the size and alignment of the collagen fibers present within the articular cartilage superficial zone. Moreover, coaxial electrospinning allows the fabrication of core-shell fibers able to encapsulate and release bioactive molecules in a sustained manner. Kartogenin (KTG) is a small heterocyclic molecule, which was demonstrated to promote the chondrogenic differentiation of human bone marrow-derived mesenchymal stem/stromal cells(hBMSCs)[1].In this work, we developed and evaluated the biological performance of core-shell poly(glycerol sebacate)(PGS)/poly(caprolactone)(PCL) aligned nanofibers (core:PGS/shell:PCL) mimicking the native articular cartilage extracellular matrix(ECM) and able to promote the sustained release of the chondroinductive drug KTG[2].The produced coaxial aligned PGS/PCL scaffolds were characterized in terms of their structure and fiber diameter, chemical composition, thermal properties, mechanical performance under tensile testing and in vitro degradation kinetics, in comparison to monoaxial PCL aligned fibers and respective non-aligned controls. KTG was incorporated into the core PGS solution to generate core-shell PGS-KTG/PCL nanofibers and its release kinetics was studied by HPLC analysis. KTG-loaded electrospun aligned scaffolds capacity to promote hBMSCs chondrogenic differentiation was evaluated by assessing cell proliferation, typical cartilage-ECM production (sulfated glycosaminiglycans(sGAG)) and chondrogenic marker genes expression in comparison to non-loaded controls. All the scaffolds fabricated showed average fiber diameters within the nanometer-scale and the core-shell structure of the fibers was clearly confirmed by TEM. The coaxial PGS-KTG/PCL nanofibers evidenced a more sustained drug release over 21 days. Remarkably, in the absence of the chondrogenic cytokine TGF-β3, KTG-loaded nanofibers promoted significantly the proliferation and chondrogenic differentiation of hBMSCs, as suggested by the increased cell numbers, higher sGAG amounts and up-regulation of the chondrogenic genes COL2A1, Sox9, ACAN and PRG4 expression. Overall, our results highlight the potential of core-shell PGS-KTG/PCL aligned nanofibers for the development of novel MSC-based CTE strategies.Acknowledgements: The authors thank FCT for funding through the project InSilico4OCReg (PTDC/EME-SIS/0838/2021) and to institutions iBB (UID/BIO/04565/2020) and Associate Laboratory I4HB (LA/P/0140/2020).

Endogenus chondrocytes immobilized by G-CSF in nanoporous gels enable repair of critical-size osteochondral defects

Injured articular cartilage is a leading cause for osteoarthritis. We recently discovered that endogenous stem/progenitor cells not only reside in the superficial zone of mouse articular cartilage, but also regenerated heterotopic bone and cartilage in vivo. However, whether critical-size osteochondral defects can be repaired by pure induced chemotatic cell homing of these endogenous stem/progenitor cells remains elusive. Here, we first found that cells in the superficial zone of articular cartilage surrounding surgically created 3 × 1 mm defects in explant culture of adult goat and rabbit knee joints migrated into defect-filled fibrin/hylaro1nate gel, and this migration was significantly more robust upon delivery of exogenous granulocyte-colony stimulating factor (G-CSF). Remarkably, G–CSF–recruited chondrogenic progenitor cells (CPCs) showed significantly stronger migration ability than donor-matched chondrocytes and osteoblasts. G–CSF–recruited CPCs robustly differentiated into chondrocytes, modestly into osteoblasts, and barely into adipocytes. In vivo, critical-size osteochondral defects were repaired by G–CSF–recruited endogenous cells postoperatively at 6 and 12 weeks in comparison to poor healing by gel-only group or defect-only group. ICRS and O'Driscoll scores of articular cartilage were significantly higher for both 6- and 12-week G-CSF samples than corresponding gel-only and defect-only groups. Thus, endogenous stem/progenitor cells may be activated by G-CSF, a Food and Drug Administration (FDA)-cleared bone-marrow stimulating factor, to repair osteochondral defects.

Effect of Moderate Exercise on the Superficial Zone of Articular Cartilage in Age-Related Osteoarthritis.

This study aimed to evaluate the effect of exercise on the superficial zone of the osteoarticular cartilage during osteoarthritis progression. Three-month-old, nine-month-old, and eighteen-month-old Sprague Dawley rats were randomly divided into two groups, moderate exercise and no exercise, for 10 weeks. Histological staining, immunostaining, and nanoindentation measurements were conducted to detect changes in the superficial zone. X-ray and micro-CT were quantitated to detect alterations in the microarchitecture of the tibial subchondral bone. Cells were extracted from the superficial zone of the cartilage under fluid-flow shear stress conditions to further verify changes in vitro. The number of cells and proteoglycan content in the superficial zone increased more in the exercise group than in the control group. Exercise can change the content and distribution of collagen types I and III in the superficial layer. In addition, TGFβ/pSmad2/3 and Prg4 expression levels increased under the intervention of exercise on the superficial zone. Exercise can improve the Young's modulus of the cartilage and reduce the abnormal subchondral bone remodeling which occurs after superficial zone changes. Moderate exercise delays the degeneration of the articular cartilage by its effect on the superficial zone, and the TGFβ/pSmad2/3 signaling pathways and Prg4 play an important role.

Development of a computational-experimental framework for enhanced mechanical characterization and cross-species comparison of the articular cartilage superficial zone

To provide a better understanding of the contribution of specific constituents (i.e. proteoglycan, collagen, fluid) to the mechanical behavior of the superficial zone of articular cartilage, a complex biological tissue with several time-dependent properties, a finite element model was developed. Optimization was then used to fit the model to microindentation experiments. We used this model to compare superficial zone material properties of mature human vs. immature bovine articular cartilage. Non-linearity and stiffness of the fiber-reinforced component of the model differed between human and bovine tissue. This may be due to the more complex collagen architecture in mature tissue and is of interest to investigate in future work.

Single-cell transcriptomic analysis identifies a highly replicating Cd168+ skeletal stem/progenitor cell population in mouse long bones

Skeletal stem/progenitor cells (SSPCs) are tissue-specific stem/progenitor cells localized within skeletons and contribute to bone development, homeostasis, and regeneration. However, the heterogeneity of SSPC populations in mouse long bones and their respective regenerative capacity remain to be further clarified. In this study, we perform integrated analysis using single-cell RNA sequencing (scRNA-seq) datasets of mouse hindlimb buds, postnatal long bones, and fractured long bones. Our analyses reveal the heterogeneity of osteochondrogenic lineage cells and recapitulate the developmental trajectories during mouse long bone growth. In addition, we identify a novel Cd168+ SSPC population with highly replicating capacity and osteochondrogenic potential in embryonic and postnatal long bones. Moreover, the Cd168+ SSPCs can contribute to newly formed skeletal tissues during fracture healing. Furthermore, the results of multicolor immunofluorescence show that Cd168+ SSPCs reside in the superficial zone of articular cartilage as well as in growth plates of postnatal mouse long bones. In summary, we identify a novel Cd168+ SSPC population with regenerative potential in mouse long bones, which adds to the knowledge of the tissue-specific stem cells in skeletons.

Early patellofemoral cartilage and bone pathology in a rat model of noninvasive anterior cruciate ligament rupture

ABSTRACT Objective Anterior cruciate ligament rupture (ACLR) is a risk factor for the development of post-traumatic osteoarthritis (PTOA). While PTOA in the tibiofemoral joint compartment is well-characterized, very little is known about pathology in the patellofemoral compartment after ACL injury. Here, we evaluated the extent to which ACLR induces early patellofemoral joint damage in a rat model. Methods Adult female Lewis rats were randomized to noninvasive ACLR or Sham. Two weeks post-injury, contrast-enhanced micro-computed tomography (µCT) of femoral and patellar cartilage was performed using 20% v/v ioxaglate. Morphometric parameters of femoral trochlear and patellar cartilage, subchondral bone, and trabecular bone were derived from µCT. Sagittal Safranin-O/Fast-Green-stained histologic sections were graded using the OARSI score in a blinded fashion. Results Cartilage and bone remodelling consistent with an early PTOA phenotype were observed in both femoral trochleas and patellae. ACLR caused osteophyte formation of the patella and pathology in the superficial zone of articular cartilage, including surface fibrillation, fissures, increased cellularity, and abnormal chondrocyte clustering. There were significant increases in thickness of patellar and trochlear cartilage. Loss of subchondral bone thickness, bone volume fraction, and tissue mineral density, as well as changes to patellar and trochlear trabecular microarchitecture, were indicative of catabolic bone remodelling. Several injury-induced changes, including increased cartilage thickness and trabecular spacing and decreased trabecular number were more severe in the patella compared to the trochlea. Conclusion The patellofemoral joint develops mild but evident pathology in the early period following ACL rupture, extending the utility of this model to the study of patellofemoral PTOA.

Regulatory Mechanisms of Prg4 and Gdf5 Expression in Articular Cartilage and Functions in Osteoarthritis.

Owing to the rapid aging of society, the numbers of patients with joint disease continue to increase. Accordingly, a large number of patients require appropriate treatment for osteoarthritis (OA), the most frequent bone and joint disease. Thought to be caused by the degeneration and destruction of articular cartilage following persistent and excessive mechanical stimulation of the joints, OA can significantly impair patient quality of life with symptoms such as knee pain, lower limb muscle weakness, or difficulty walking. Because articular cartilage has a low self-repair ability and an extremely low proliferative capacity, healing of damaged articular cartilage has not been achieved to date. The current pharmaceutical treatment of OA is limited to the slight alleviation of symptoms (e.g., local injection of hyaluronic acid or non-steroidal anti-inflammatory drugs); hence, the development of effective drugs and regenerative therapies for OA is highly desirable. This review article summarizes findings indicating that proteoglycan 4 (Prg4)/lubricin, which is specifically expressed in the superficial zone of articular cartilage and synovium, functions in a protective manner against OA, and covers the transcriptional regulation of Prg4 in articular chondrocytes. We also focused on growth differentiation factor 5 (Gdf5), which is specifically expressed on the surface layer of articular cartilage, particularly in the developmental stage, describing its regulatory mechanisms and functions in joint formation and OA pathogenesis. Because several genetic studies in humans and mice indicate the involvement of these genes in the maintenance of articular cartilage homeostasis and the presentation of OA, molecular targeting of Prg4 and Gdf5 is expected to provide new insights into the aetiology, pathogenesis, and potential treatment of OA.

Role of WNT Agonists, BMP and VEGF Antagonists in Rescuing Osteoarthritic Knee Cartilage in a Rat Model.

The superficial zone of articular cartilage (AC) is vital for its function and biomechanics. The damaged AC gets vascularized and undergoes hypertrophy and ossification. Studies have highlighted these two as the major causative factors in osteoarthritis (OA). We aimed at preventing the OA progression in a rat knee instability model by inhibiting the vascular ingrowth and ossification using VEGF and BMP antagonist. A WNT agonist was also used to promote AC regeneration because of its protective effect on the superficial layer. Rat knee OA was created by surgical excision of the medial meniscus and medial collateral ligament. Forty rats were divided into two groups of twenty each for surgical control and tests (surgery + intra-articular injection of drugs every two weeks). Ten animals from each group were sacrificed at four and eight weeks. Histology was mainly used to evaluate the outcome. A surgical OA model was successfully created with higher histological scores for operated knees, both in short- (P = 0.0001) and long-term (P = 0.001). Modified Mankin score was lesser in the test animals as compared to control (P = 0.17) in the short-term, but the trend was reversed in the long-term (P = 0.13). Subgroup analysis revealed that repeated injections in the anterolateral compartment contributed to higher scores in the lateral (P = 0.03) and anterior (P = 0.03) compartment of the knee in the long-term. The combinatorial approach was effective in controlling the OA in short-term. Further studies are needed to test the sustained drug delivery system to improve the outcome.

Creb5 establishes the competence for Prg4 expression in articular cartilage

A hallmark of cells comprising the superficial zone of articular cartilage is their expression of lubricin, encoded by the Prg4 gene, that lubricates the joint and protects against the development of arthritis. Here, we identify Creb5 as a transcription factor that is specifically expressed in superficial zone articular chondrocytes and is required for TGF-β and EGFR signaling to induce Prg4 expression. Notably, forced expression of Creb5 in chondrocytes derived from the deep zone of the articular cartilage confers the competence for TGF-β and EGFR signals to induce Prg4 expression. Chromatin-IP and ATAC-Seq analyses have revealed that Creb5 directly binds to two Prg4 promoter-proximal regulatory elements, that display an open chromatin conformation specifically in superficial zone articular chondrocytes; and which work in combination with a more distal regulatory element to drive induction of Prg4 by TGF-β. Our results indicate that Creb5 is a critical regulator of Prg4/lubricin expression in the articular cartilage.

Chondrocytes respond both anabolically and catabolically to impact loading generally considered non-injurious

We aimed to determine the longitudinal effects of low-energy (generally considered non-injurious) impact loading on (1) chondrocyte proliferation, (2) chondroprogenitor cell activity, and (3) EGFR signaling. In an in vitro study, we assessed 127 full-thickness, cylindrical osteochondral plugs of bovine cartilage undergoing either single, uniaxial unconfined impact loads with energy densities in the range of 1.5–3.2mJ/mm3 or no impact (controls). We quantified cell responses at two, 24, 48, and 72 h via immunohistochemical labeling of Ki67, Sox9, and pEGFR antibodies. We compared strain, stress, and impact energy density as predictors for mechanotransductive responses from cells, and fit significant correlations using linear regressions. Our study demonstrates that low-energy mechanical impacts (1.5–3.2mJ/mm3) generally stimulate time-dependent anabolic responses in the superficial zone of articular cartilage and catabolic responses in the middle and deep zones. We also found that impact energy density is the most consistent predictor of cell responses to low-energy impact loading. These spatial and temporal changes in chondrocyte behavior result directly from low-energy mechanical impacts, revealing a new level of mechanotransductive sensitivity in chondrocytes not previously appreciated.

EGFR Signaling Is Required for Maintaining Adult Cartilage Homeostasis and Attenuating Osteoarthritis Progression.

The uppermost superficial zone of articular cartilage is the first line of defense against the initiation of osteoarthritis (OA). We previously used Col2-Cre to demonstrate that epidermal growth factor receptor (EGFR), a tyrosine kinase receptor, plays an essential role in maintaining superficial chondrocytes during articular cartilage development. Here, we showed that EGFR activity in the articular cartilage decreased as mice age. In mouse and human OA samples, EGFR activity was initially reduced at the superficial layer and then resurged in cell clusters within the middle and deep zone in late OA. To investigate the role of EGFR signaling in postnatal and adult cartilage, we constructed an inducible mouse model with cartilage-specific EGFR inactivation (Aggrecan-CreER EgfrWa5/flox , Egfr iCKO). EdU incorporation revealed that postnatal Egfr iCKO mice contained fewer slow-cycling cells than controls. EGFR deficiency induced at 3months of age reduced cartilage thickness and diminished superficial chondrocytes, in parallel to alterations in lubricin production, cell proliferation, and survival. Furthermore, male Egfr iCKO mice developed much more severe OA phenotypes, including cartilage erosion, subchondral bone plate thickening, cartilage degeneration at the lateral site, and mechanical allodynia, after receiving destabilization of the medial meniscus (DMM) surgery. Similar OA phenotypes were also observed in female iCKO mice. Moreover, tamoxifen injections of iCKO mice at 1 month post-surgery accelerated OA development 2 months later. In summary, our data demonstrated that chondrogenic EGFR signaling maintains postnatal slow-cycling cells and plays a critical role in adult cartilage homeostasis and OA progression. © 2022 American Society for Bone and Mineral Research (ASBMR).

Time- and depth-dependent changes to articular cartilage mitochondrial content occur during the 48 hours after injury

Purpose: Posttraumatic osteoarthritis (PTOA) is a degenerative joint disease after injury with no viable long-term treatments beyond joint arthroplasty and analgesia. It has recently been shown that impact injury and subsequent electron transport initiate a decrease in mitochondrial content in the superficial zone of articular cartilage, suggesting mitochondria play a role early in the development of PTOA. Electron transport inhibition after trauma has shown some promise; however, in published studies this approach is only effective if applied less than 24 hours after injury. Given that dysfunctional mitochondria have been observed as late as 4 weeks after injury and it has been reported several times that end-stage OA chondrocytes have mitochondrial dysfunction, we wanted to better understand the development and extent of mitochondrial dysfunction throughout cartilage after injury. We hypothesized injured areas deeper than the superficial zone would show decreases in mitochondrial content between 24 and 48 h after injury. Methods: Healthy bovine stifle (knee) joints with no signs of osteoarthritis were obtained from a local abattoir (Bud’s Custom Meats Riverside, IA). Matched 10 mm diameter osteochondral plugs were taken from the loaded portion of the femoral condyle. Once removed, the plugs were washed twice in HBSS with antibiotics then cultured in normal media (45% DMEM, 45% F12, 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2.5 μm/mL amphotericin B) and maintained at 37°C, 5% CO2, and 5% O2. After 24 h equilibration to culture conditions, the plugs were subject to either a sham (0 J/cm2) or a 2 J/cm2 impact delivered via a custom-built drop tower utilizing a 6 mm flat, beveled, stainless steel platen. After impact, the samples were placed in fresh porcine media and returned to the incubator. Prior studies had viewed plugs from above using confocal microscopy; in this case, to view the deeper zones of cartilage plugs were bisected slightly off center of the impact into a 5.9 mm section with an Isomet 1000 (Buehler, Lake Bluff, IL), then faced by trimming 0.5 mm of the cartilage with a single straight scalpel cut. These measured cuts (the combination of the isomet blade with and the cartilage removed during facing) provided a sagittal cross-section of the plug at the center of the impact site. The plugs were washed in phenol red free DMEM/F12 media and co-stained for 30 min in the incubator with 1 mg/mL Calcein Green AM and 200 nM MitoTracker Deep Red (both Life Technologies, Waltham, MA). The samples were then washed and imaged on an Olympus FV1000 (Shinjuku, Japan) confocal microscope. Images were taken at the middle of the impact site with a 10X objective lens and images were manually segmented according to either specific zones of the cartilage (superficial, transitional, radial, or deep zone) or by 50 μm increments in depth starting from the superficial zone (Figure 1). Within each zone or depth, a custom watershed-based 3D image analysis algorithm was used to segment live cells according to Calcein Green AM. Per-cell MitoTracker staining intensity for live cells was extracted and averaged by zone depth. Intensities of cells in injured plugs were normalized to their respective sham control. Two-way ANOVA with Tukey multiple comparisons was performed to determine significance between groups. Results: Live cell mitochondrial staining intensity in the superficial and transitional zones of the cartilage significantly increased in the first 24 h after injury compared to sham (Figure 2, n = 4, p < 0.05). However, 48 h after impact, live cell mitochondrial staining decreased significantly both in the superficial zone and in the transitional zone compared to 24 h after injury (p < 0.05). Mitochondrial content at 48 h decreased below control levels in the superficial and transitional zones of three out of four specimens. Analysis by either measured depth or cartilage zone each revealed this significant change from 24 to 48 h after injury. Conclusions: We observed a significant increase in live cell mitochondrial staining 24 h after impact compared to sham; however, this time point was marked by significant inter- and intra-specimen variation as shown in the variance in Figure 2. This acute increase in staining may indicate stimulation of the chondrocytes by the mechanical injury. However, we observed a substantial and significant decrease in live chondrocyte mitochondrial staining in the superficial and transitional zones by 48 h after injury. These changes must be verified via immunohistochemistry, but when compared to prior results they suggest dynamic changes in mitochondrial dysfunction through the depth of articular cartilage after injury.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Senescent Cell-Secreted Netrin-1 Modulates Ageing-Related Disorders by Recruiting Sympathetic Fibres

Cellular senescence is implicated in several lines of ageing-related disorders. However, the potential molecular mechanisms by which cellular senescence modulates age-related pathologies remain largely unexplored. Herein, we report that the density of sympathetic fibres (SFs) is significantly elevated in naturally aged mouse tissues and human colon adenoma tissues compared to the SF densities in the corresponding young mouse tissues and human non-lesion colon tissues. A dorsal root ganglion (DRG)-human diploid fibroblast coculture assay revealed that senescent cells promote the outgrowth of SFs, indicating that the senescent cell-induced recruitment of SFs contributes to an elevation in SFs density. Clearing senescent cells by senolytics (Dasatinib(D)+ Quercetin (Q)) improved osteoarthritis (OA), thereby reducing the SF infiltration in the superficial zone of articular cartilage. Mechanistically, senescent cells recruited SFs by secreting the axon guidance cue netrin-1. Notably, the elevated level of SFs contributes to impaired cognitive function in naturally aged mice, which can be reversed by treatment with propranolol hydrochloride, a non-selective β receptor blocker that inhibits sympathetic nerve activity (SNA) by blocking non-selective β receptors. Additionally, 6-hydroxydopamine (6-OHDA)-induced sympathectomy improved hepatic sympathetic overactivity mediated hepatic steatosis in high fat diet (HFD)-fed APOE knockout mice (APOE-/-mice) by reducing hepatic sympathetic nerve activity (SNA). We conclude that senescent cell-secreted netrin-1 mediates SFs outgrowth and leads to elevated SNA, which contributes to ageing-related disorders. This suggests that clearing senescent cells is a promising therapeutic strategy for improving sympathetic nervous system (SNS) hyperactivity-induced ageing-related pathologies. Funding Statement: Our work was supported by the National Natural Science Foundation of China (grant number 81771505 and 81773079), and the Fundamental Research Funds for the Higher Education of Tianjin (Grant Number 2018KJ079). Declaration of Interests: The authors stated that no potential conflicts of interest were disclosed. Ethics Approval Statement: All experiments involving the handling of mice were approved by the animal ethics committee of Peking University Health Science Centre. The human tissue samples were obtained with informed consent, and the study was approved by the Clinical Research Ethics Committee.

Kartogenin-loaded coaxial PGS/PCL aligned nanofibers for cartilage tissue engineering.

Electrospinning is a valuable technology for cartilage tissue engineering (CTE) due to its ability to produce fibrous scaffolds mimicking the nanoscale and alignment of collagen fibers present within the superficial zone of articular cartilage. Coaxial electrospinning allows the fabrication of core-shell fibers able to incorporate and release bioactive molecules (e.g., drugs or growth factors) in a controlled manner. Herein, we used coaxial electrospinning to produce coaxial poly(glycerol sebacate) (PGS)/poly(caprolactone) (PCL) aligned nanofibers (core:PGS/shell:PCL). The obtained scaffolds were characterized in terms of their structure, chemical composition, thermal properties, mechanical performance and in vitro degradation kinetics, in comparison to monoaxial PCL aligned fibers and respective non-aligned controls. All the electrospun scaffolds produced presented average fiber diameters within the nanometer-scale and the core-shell structure of the composite fibers was clearly confirmed by TEM. Additionally, fiber alignment significantly increased (>2-fold) the elastic modulus of both coaxial and monoxial scaffolds. Kartogenin (KGN), a small molecule known to promote mesenchymal stem/stromal cells (MSC) chondrogenesis, was loaded into the core PGS solution to generate coaxial PGS-KGN/PCL nanofibers. The KGN release kinetics and scaffold biological performance were evaluated in comparison to KGN-loaded monoaxial fibers and respective non-loaded controls. Coaxial PGS-KGN/PCL nanofibers showed a more controlled and sustained KGN release over 21 days than monoaxial PCL-KGN nanofibers. When cultured with human bone marrow MSC in incomplete chondrogenic medium (without TGF-β3), KGN-loaded scaffolds enhanced significantly cell proliferation and chondrogenic differentiation, as suggested by the increased sGAG amounts and chondrogenic markers gene expression levels. Overall, these findings highlight the potential of using coaxial PGS-KGN/PCL aligned nanofibers as a bioactive scaffold for CTE applications.

IL-1β regulates cell shape and expression of prg4 independent of actin polymerization in superficial zone chondrocytes

Purpose: The superficial zone (SZ) of articular cartilage plays a multifaceted role in joint homeostasis contributing the biomechanical integrity and metabolic homeostasis of the tissue. In the early stages of osteoarthritis (OA), one of the first observable changes is loss of the SZ. Although the pathogenesis of OA is multifactorial, elevated levels of inflammatory cytokines are one contributing factor to disease progression. Interleukin-1 is an inflammatory cytokine that contributes to joint remodeling by regulating the expression of collagenases, aggrecanases, and nitric oxide (NO). Additionally, decreased expression of the chondroprotective molecule PRG4 is observed in SZ chondrocytes (SZC) in vitro and in the SZ of cartilage explant culture with IL-1 treatment. Studies using chondrocytes isolated from full-thickness cartilage observed changes in cellular mechanics with IL-1β treatment including increased filamentous actin and cellular stiffness. Unlike full-thickness chondrocytes, where actin polymerization leads to loss of the chondrogenic phenotype, the polymerized form of actin is important for the maintenance of the SZ phenotype and expression of PRG4. Although a decrease in PRG4 expression has previously been observed with IL-1β treatment in SZC the mechanism has not yet been fully elucidated. The purpose of this study is to determine whether IL-1β regulates PRG4 expression through an actin-dependent mechanism. Methods: The SZ of articular cartilage was dissected from the metacarpal phalangeal joint of calves aged 6-9 months. SZC were isolated by sequential digestion in 0.5% protease for 45 minutes, followed by 0.1% collagenase for 14-17 hours. SZC were seeded in monolayer culture (52,000/cm2) in high glucose DMEM supplemented with ITS, 100nM dexamethasone, 40μg/mL proline, 100mM pyruvate, 100μg/mL ascorbic acid. After 24 hours in culture, SZC were treated with IL-1β (10ng/mL) and maintained for 24 hours prior to harvesting for analysis. The effect of IL-1β on cell morphology and the cytoskeleton was determined. Cellular morphology was assessed using Image J analysis software to calculate cell area and circularity. Gene and protein expression was determined by RT-PCR and Western blotting, respectively. The cytoskeleton polymerization status was determined by performing differential triton extraction containing the soluble portion (G-actin and M-tubulin) and insoluble portion (F-actin and P-tubulin). The fractions were separated by SDS-PAGE and the resultant bands were semi-quantified using Image J analysis software to determine the G-/F-actin ratio and M-/P-tubulin ratio. Confocal imaging of the actin cytoskeleton visualizing the G- and F-actin was performed by confocal microscopy. Experiments were performed with SZC from independent cell isolations. Each experiment was repeated 2-4 times; experiments were performed in triplicate, where appropriate. Student’s T-test was used to evaluate the results and significance assigned at p <0.05. Results: To confirm observations previously reported SZC treated with IL-1β were assessed by RT-PCR to determined PRG4 expression levels. PRG4 mRNA levels significantly decreased with treatment at 24 hours (p<0.05; N=4). The effect of IL-1β treatment on cellular morphology and actin organization was determined. Treatment with IL-1β resulted in no significant changes in cell area, but did result in a significant increase in cell circularity (p<0.001; N=2). Visualization of the G- and F-actin by confocal microscopy yielded no observable differences in the actin structure (N=2). To confirm this, the polymerization status of the actin cytoskeleton was determined by calculating the G-/F-actin ratio. Despite changes in cell circularity, no significant changes in the G-/F-actin ratio were observed (p=0.8; N=4). Changes in the microtubule structure can also induce changes in cellular morphology, thus we investigated whether the microtubule polymerization status was altered with IL-1β treatment, and observed no significant difference in the M-/P-tubulin ratio (p=0.3; N=3), suggesting another regulator of cell shape may be modulating PRG4 expression in SZC. Conclusions: The data suggests that the elongated morphology is an important regulator of PRG4 expression. Despite changes in cell circularity and reduced expression of PRG4, no changes in actin polymerization or tubulin polymerization were observed. Although actin polymerization has previously been shown to regulate PRG4 expression, additional regulators of cellular morphology may also be important for the maintenance of the SZ phenotype.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

The superficial zone in cartilage adapts with stiffening when challenged tribologically

Purpose: In osteoarthritis, the superficial zone of articular cartilage is compromised by fissures and cracks and, thus, may lose its intended function. While there is agreement that the superficial zone is important for the load bearing capacity of the tissue, its mechanical response upon articulation is less investigated. In previous studies, using fully intact cartilage samples, we have shown that the cartilage surface undergoes a transient stiffening response upon articulation. Here, we hypothesize that this response is lost when the surface is removed. In order to address this question we used a tribological bioreactor and articulated on cartilage samples with and without superficial zone. Further, we started to study structural changes at the surface with an atomic force microscope (AFM) before and after the application of tribological stress. Methods: Twenty-four 14x20x3 mm oval bovine cartilage explants that had undergone one freeze-thaw cycle were distributed into four groups per animal: (1) intact surface and articular loading; (2) removed surface and articular loading; (3) intact free-swelling-control (FSC); 4) removed surface FSC. Thus, each group had n=6 samples. A vibratome was used to remove the superficial zone of the explants (at least 20% of full sample thickness). Microindentation stress-relaxation tests were performed in the center of explants using a Hysitron TI 950 with a 20 μm spherical indenter and an 8 μm indent depth. All indentation on explants was performed submerged in 1× PBS to maintain tissue hydration. Following initial characterization, cartilage explants were removed from the indentation sample holder and placed into the bioreactor. Cartilage explants were confined in porous polyethylene scaffold rings and fixed in a well. Using a 32-mm ceramic ball (Ra ∼10 nm), a 40 N static confined compression was applied that lead to approx. 2 MPa contact stress. The ball oscillated at 0.5 Hz, and was migrated at a speed of 1mm/s across the cartilage surface generating a wear scar length of 10 mm. Following 1-hr of bioreactor testing, microindentation was repeated in the center/wear scar. The reduced modulus was obtained using the Oliver-Pharr method on 80-95% of the unloading curve. Cartilage surfaces removed by vibratome were prepared for AFM analysis (Nanowizard Ultra, JPK Instruments, Germany). All measurements were conducted in 1× PBS. Using QI imaging, a force sampling mode, an initial image of 256 x 256 pixels (approx. 10 x 10 μm2) was obtained. The AFM was then put into contact mode, and an 80 nm silicon tip was repeatedly (>850 times) slid linearly across the cartilage surface. Following these sliding experiments, the tissue was reimaged in QI mode. For data analysis, the ratio of post- to pre-bioreactor reduced modulus was calculated and analyzed using Shapiro-Wilks tests for normality and Paired Student’s T-tests. Results: Our results show that intact surface groups exhibited significantly increased stiffening following articular loading compared to the FSC (p=0.006). For the removed surface groups, no significant difference between articulated and FSC explants was detected (p=0.927) (Fig 1). Also in the AFM the cartilage surface demonstrated topographical and stiffness related changes upon sliding that are shown in Figure 2. Conclusions: Surface stiffening following tribological loading may be an important biomechanical function of load transfer during articulation. Surface removal leads to a loss of this adaptive response. The stiffening response may be related to a reorganization of matrix molecules that are unique to the superficial zone. In the future we plan to investigate the influencing parameters of this stiffening response and identify the matrix molecules that are involved.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Influence of hydrodynamic pressure on chondrogenic differentiation of human bone marrow mesenchymal stem cells cultured in perfusion system

The natural conditions of chondrocytes in native cartilage including mechanical forces and surface topology could be simulated to enhance chondrogenesis. A perfusion system recapitulating the hydrodynamic pressure of cartilage tissue is designed. Mesenchymal stem cells (MSCs) are isolated and seeded on aligned nanofibrous PCL/PLGA scaffolds that mimic the structure of superficial zone of articular cartilage. The cell-seeded scaffolds are placed into the perfusion bioreactor and exposed to chondrogenic differentiating medium. The chondrogenesis is then investigated by histological analysis and real time PCR for cartilage-specific genes. The highest expression levels of aggrecan and type II collagen are observed in the cells cultured in the presence of differentiating medium and mechanical stimulation. The expression level of type II collagen is higher than aggrecan in presence of differentiating medium and absence of mechanical stimulation. On the contrary, the expression ratio of aggrecan is higher than type II collagen in presence of mechanical stimulation and absence of differentiating medium. These results show the dominant role of mechanical stimulation and differentiating medium on upregulated expression of aggrecan and type II collagen, respectively. The application of mechanical stimulation upon cells-seeded scaffolds could mimic superficial zone of articular cartilage tissue and increase derivation of chondrocytes from MSCs.

Site-specific glycosaminoglycan content is better maintained in the pericellular matrix than the extracellular matrix in early post-traumatic osteoarthritis.

IntroductionOne of the characteristics of early osteoarthritis (OA) is the loss of fixed charged density (FCD) of glycosaminoglycans in the superficial zone of articular cartilage. However, possible local changes in the FCD content of the pericellular matrix (PCM) are not fully understood. Hence, our aim was to investigate the effect of unilateral anterior cruciate ligament transection (ACLT) in rabbit knees on estimated FCD in the PCM compared to that in the ECM, and relate these results with cell morphology.MethodsArticular cartilage samples were collected from ACLT, contralateral and intact control knee joints from lateral and medial femoral condyles and tibial plateaus, and from the femoral groove and patella. Histological samples were prepared and stained with Safranin-O to estimate the FCD content around the chondrocytes in the PCM and the ECM with digital densitometry.ResultsAs a result of ACLT, the greatest decreases in the FCD content of the PCM were observed in the superficial zone of the lateral femoral condyle (p = 0.02), medial tibial plateau (p = 0.002) and patellar (p < 0.001) cartilage. The normalized FCD content of the PCM compared to the surrounding ECM was increased most in the femoral condyles (p < 0.01) and medial tibial plateau (p = 0.02) cartilage. The high normalized FCD content of the PCM in the superficial zone of lateral femoral condyle cartilage was consistent with the round cell morphology in that location.ConclusionsIn conclusion, we suggest that certain sites in the knee joint, particularly the lateral femoral condyle cartilage, experience less FCD loss in the PCM than in the ECM in early post-traumatic OA, which could lead to altered cell shape.

HMGB proteins and arthritis.

The high-mobility group box (HMGB) family includes four members: HMGB1, 2, 3 and 4. HMGB proteins have two functions. In the nucleus, HMGB proteins bind to DNA in a DNA structure-dependent but nucleotide sequence-independent manner to function in chromatin remodeling. Extracellularly, HMGB proteins function as alarmins, which are endogenous molecules released upon tissue damage to activate the immune system. HMGB1 acts as a late mediator of inflammation and contributes to prolonged and sustained systemic inflammation in subjects with rheumatoid arthritis. By contrast, Hmgb2 -/- mice represent a relevant model of aging-related osteoarthritis (OA), which is associated with the suppression of HMGB2 expression in cartilage. Hmgb2 mutant mice not only develop early-onset OA but also exhibit a specific phenotype in the superficial zone (SZ) of articular cartilage. Given the similar expression and activation patterns of HMGB2 and β-catenin in articular cartilage, the loss of these pathways in the SZ of articular cartilage may lead to altered gene expression, cell death and OA-like pathogenesis. Moreover, HMGB2 regulates chondrocyte hypertrophy by mediating Runt-related transcription factor 2 expression and Wnt signaling. Therefore, one possible mechanism explaining the modulation of lymphoid enhancer binding factor 1 (LEF1)-dependent transactivation by HMGB2 is that a differential interaction between HMGB2 and nuclear factors affects the transcription of genes containing LEF1-responsive elements. The multiple functions of HMGB proteins reveal the complex roles of these proteins as innate and endogenous regulators of inflammation in joints and their cooperative roles in cartilage hypertrophy as well as in the maintenance of joint tissue homeostasis.