Alignment Of Chondrocytes Research Articles

Biomimetic approach to articular cartilage tissue engineering using carbon nanotube-coated and textured polydimethylsiloxane scaffolds.

There is a significant need to understand the complexity and heterogeneity of articular cartilage to develop more effective therapeutic strategies for diseases such as osteoarthritis. Here, we show that carbon nanotubes (CNTs) are excellent candidates as a material for synthetic scaffolds to support the growth of chondrocytes—the cells that produce and maintain cartilage. Chondrocyte morphology, proliferation, and alignment were investigated as nanoscale CNT networks were applied to macroscopically textured polydimethylsiloxane (PDMS) scaffolds. The application of CNTs to the surface of PDMS‐based scaffolds resulted in an up to 10‐fold increase in cell adherence and 240% increase in proliferation, which is attributable to increased nanoscale roughness and hydrophilicity. The introduction of macroscale features to PDMS induced alignment of chondrocytes, successfully mimicking the cell behavior observed in the superficial layer of cartilage. Raman spectroscopy was used as a noninvasive, label‐free method to monitor extracellular matrix production and chondrocyte phenotype. Chondrocytes on these scaffolds successfully produced collagen, glycosaminoglycan, and aggrecan. This study demonstrates that introducing physical features at different length scales allows for a high level of control over tissue scaffold design and, thus, cell behavior. Ultimately, these textured scaffolds can serve as platforms to improve the understanding of osteoarthritis and for early‐stage therapeutic testing.

Silencing of Vangl2 attenuates the inflammation promoted by Wnt5a via MAPK and NF-\u03baB pathway in chondrocytes

BackgroundThe Wnt planar cell polarity (PCP) pathway is implicated in osteoarthritis (OA) both in animals and in humans. Van Gogh-like 2 (Vangl2) is a key PCP protein that is required for the orientation and alignment of chondrocytes in the growth plate. However, its functional roles in OA still remain undefined. Here, we explored the effects of Vangl2 on OA chondrocyte in vitro and further elucidated the molecular mechanism of silencing Vangl2 in Wnt5a-overexpressing OA chondrocytes.MethodsChondrocytes were treated with IL-1β (10 ng/mL) to simulate the inflammatory microenvironment of OA. The expression levels of Vangl2, Wnt5a, MMPs, and related proinflammatory cytokines were measured by RT-qPCR. Small interfering RNA (siRNA) of Vangl2 and the plasmid targeting Wnt5a were constructed and transfected into ATDC5 cells. Then, the functional roles of silencing Vangl2 in the OA chondrocytes were investigated by Western blotting, RT-qPCR, and immunocytochemistry (ICC). Transfected OA chondrocytes were subjected to Western blotting to analyze the relationship between Vangl2 and related signaling pathways.ResultsIL-1β induced the production of Vangl2, Wnt5a, and MMPs in a time-dependent manner and the significantly increased expression of Vangl2. Vangl2 silencing effectively suppressed the expression of MMP3, MMP9, MMP13, and IL-6 at both gene and protein levels and upregulated the expression of type II collagen and aggrecan. Moreover, knockdown of Vangl2 inhibited the phosphorylation of MAPK signaling molecules (P38, ERK, and JNK) and P65 in Wnt5a-overexpressing OA chondrocytes.ConclusionsFor the first time, we demonstrate that Vangl2 is involved in the OA process. Vangl2 silencing can notably alleviate OA progression in vitro by inhibiting the expression of MMPs and increasing the formation of the cartilage matrix and can inhibit the proinflammatory effects of Wnt5a via MAPK and NF-κB pathway. This study provides new insight into the mechanism of cartilage inflammation.

Articular chondrocyte alignment in the rat after surgically induced osteoarthritis.

[Purpose] Chondrocytes in articular cartilage are aligned as columns from the joint surface. Notably, loss of chondrocyte and abnormalities of differentiation factors give rise to osteoarthritis (OA). However, the relationship between chondrocyte alignment and OA progression remains unclear. This study was performed to investigate temporal alterations in surgically-induced OA rats. [Subjects and Methods] Thirteen-week-old Wistar rats (n=30) underwent destabilized medial meniscus surgery in their right knee and sham surgery in their left knee. Specimens (n=5) were collected at 0, 1, 2, 4 and 8 weeks after surgery. Histological analysis with Osteoarthritis Research Society International (OARSI) scores, cell density ratios, cell alignments and correlation between OARSI scores and cell density/alignment was performed. [Results] OARSI scores were significantly higher at 1, 2, 4 and 8 weeks in the DMM group than in the control. Cell density ratios were decreased significantly in the DMM group at 2, 4 and 8 weeks compared with the control. Chondrocyte alignment was decreased significantly in the DMM group at 4 and 8 weeks. There were negative correlations between OA severity and cell density / cell alignment. [Conclusion] The results suggest a relationship between chondrocyte alignment and cartilage homeostasis, which plays an important role in OA progression.

SOXC Transcription Factors Induce Cartilage Growth Plate Formation in Mouse Embryos by Promoting Noncanonical WNT Signaling.

Growth plates are specialized cartilage structures that ensure the elongation of most skeletal primordia during vertebrate development. They are made by chondrocytes that proliferate in longitudinal columns and then progress in a staggered manner towards prehypertrophic, hypertrophic and terminal maturation. Complex molecular networks control the formation and activity of growth plates, but remain incompletely understood. We investigated here the importance of the SoxC genes, which encode the SOX4, SOX11 and SOX12 transcription factors, in growth plates. We show that the three genes are expressed robustly in perichondrocytes and weakly in growth plate chondrocytes. SoxC(Prx1Cre) mice, which deleted SoxC genes in limb bud skeletogenic mesenchyme, were born with tiny appendicular cartilage primordia because of failure to form growth plates. In contrast, SoxC(Col2Cre) and SoxC(ATC) mice, which deleted SoxC genes primarily in chondrocytes, were born with mild dwarfism and fair growth plates. Chondrocytes in the latter mutants matured normally, but formed irregular columns, proliferated slowly and died ectopically. Asymmetric distribution of VANGL2 was defective in both SoxC(Prx1Cre) and SoxC(ATC) chondrocytes, indicating impairment of planar cell polarity, a noncanonical WNT signaling pathway that controls growth plate chondrocyte alignment, proliferation and survival. Accordingly, SoxC genes were necessary in perichondrocytes for expression of Wnt5a, which encodes a noncanonical WNT ligand required for growth plate formation, and in chondrocytes and perichondrocytes for expression of Fzd3 and Csnk1e, which encode a WNT receptor and casein kinase-1 subunit mediating planar cell polarity, respectively. Reflecting the differential strengths of the SOXC protein transactivation domains, SOX11 was more powerful than SOX4, and SOX12 interfered with the activity of SOX4 and SOX11. Altogether, these findings provide novel insights into the molecular regulation of skeletal growth by proposing that SOXC proteins act cell- and non-cell-autonomously in perichondrocytes and chondrocytes to establish noncanonical WNT signaling crosstalk essential for growth plate induction and control.

Schmitt JF, See KH, Yang Z, Hui JHP, Lee EH. Sequential differentiation of mesenchymal stem cells in an agarose scaffold promotes a physis‐like zonal alignment of chondrocytes. J Orthop Res 30:1753–1759, 10.1002/jor.22123 2012

Schmitt JF, See KH, Yang Z, Hui JHP, Lee EH. Sequential differentiation of mesenchymal stem cells in an agarose scaffold promotes a physis‐like zonal alignment of chondrocytes. J Orthop Res 30:1753–1759, 10.1002/jor.22123 2012

Sequential differentiation of mesenchymal stem cells in an agarose scaffold promotes a physis‐like zonal alignment of chondrocytes

Chondrocytes of the epiphyseal growth plate (physis) differentiate and mature in defined linear zones. The current study examines the differentiation of human bone marrow derived mesenchymal stem cells (hBMSCs) into zonal physeal cartilage. hBMSCs were embedded in an agarose scaffold with only the surface of the scaffold in direct contact with the culture medium. The cells were differentiated using a two-step system involving the sequential addition of TGFβ followed by BMP2. The resultant samples displayed a heterogenic population of physis-like collagen type 2 positive cells including proliferating chondrocytes and mature chondrocytes showing hypertrophy, expression of early bone markers and matrix mineralization. Histological analysis revealed a physis-like linear zonal alignment of chondrocytes in varying stages of differentiation. The less mature chondrocytes were seen at the base of the construct while hypertrophic chondrocytes and matrix mineralization was observed closer to the surface of the construct. The described differentiation protocol using hBMSCs in an agarose scaffold can be used to study the factors and conditions that influence the differentiation, proliferation, maturation, and zonal alignment of physeal chondrocytes.

Characterization of Osteoarthritic and Normal Human Patella Cartilage by Computed Tomography X-ray Phase-Contrast Imaging

Early research in phase-contrast imaging indicates that substantial higher soft-tissue contrast resolution can be obtained compared with conventional absorption radiography. In the present feasibility study, we used the phase contrast analyzer-based technique in tomographic mode to investigate whether structural cartilage matrix properties can be depicted in an ex vivo set-up and whether high resolution CT-phase contrast imaging may enable differentiation of osteoarthritic and intact cartilage matrixes. Four postmortem osteochondral cylinders (7 mm diameter, 2 osteoarthritic, 2 healthy control samples from 4 human patellae) underwent tomographic phase-contrast analyzer-based imaging at high resolution (voxel size: 8(3) micron3) at 26 keV (European Synchrotron Radiation Facility, Grenoble, France). From the acquired data volumes, sets of reconstructed sagittal slices were selected at 0.5 mm increments from osteoarthritic and control specimens. Two independent, blinded observers assessed structural characteristics (cartilage thickness, topographic chondrocyte distribution homogeneity, zonal height, and surface damage) and differences between the 2 groups were determined. Phase contrast analyzer-based CT showed excellent depiction of the complete volume and of the 3D architecture of the cartilage in all samples. A distinct zonal pattern in the cartilage matrix could consistently be visualized. The osteoarthritic samples showed significantly lower chondrocyte distribution homogeneity (0% vs. 76% homogeneous, P < 0.001), less chondrocyte alignment (0% vs. 59% fully aligned, P < 0.001), lower height of tangential, transitional, and radial zones (all P < 0.001) and a higher prevalence of superficial cartilage damage (84% vs. 10%, P < 0.001). This first proof-of-concept study demonstrates that high resolution phase contrast CT visualizes structural details in relatively thick ex vivo cartilage samples. Our results suggest that the technique permits differentiation of osteoarthritic and healthy cartilage by enabling assessment of histologic characteristics of cartilage structures.

A scaffold-bioreactor system for a tissue-engineered trachea

A scaffold-bioreactor system was developed for growing tissue-engineered trachea and the effect of fluid flow on producing trachea-like neotissue was investigated. Chondrocytes were seeded in the poly(ɛ-caprolactone)-type II collagen scaffold and grown in the bioreactor operated under continuous flow at a rotational speed from 5 to 20 rpm. Flow analysis showed that the maximal and minimal shear stress in the bioreactor was 0.189–0.752 dyne/cm 2 and 30.3 × 10 −5–104 × 10 −5 dyne/cm 2, respectively. After 4 and 8 weeks, the constructs were harvested from the bioreactor and analyzed. The application of rotation increased cell proliferation, GAG and collagen content in the constructs. Especially at 15 rpm, a two-fold increase in cell number, 170% increase in GAG, and 240% increase in collagen were found compared to static culture at 8 weeks. H&E staining showed the formation of neocartilage and the alignment of chondrocytes along the flow direction. The constructs grown under 15 rpm was selected for implantation into tracheal defects of rabbits. The mean survival of six animals was 52 days. The re-epithelialization of respiratory epithelium from the anastomotic sites was observed, with granulation tissue overgrowth. This successful initial step would allow us to make further improvement in applying tissue-engineering techniques to regenerate tracheas for practical use.

Transduction of Anti-Cell Death Protein FNK Suppresses Graft Degeneration After Autologous Cylindrical Osteochondral Transplantation

This study shows that artificial super antiapoptotic FNK protein fused with a protein transduction domain (PTD-FNK) maintains the quality of osteochondral transplant by preventing chondrocyte death. Cylindrical osteochondral grafts were obtained from enhanced green fluorescent protein (EGFP)-expressing transgenic rats, in which living chondrocytes express green fluorescence, and submerged into medium containing PTD-FNK, followed by transplantation into cartilage defects of wild-type rats by impact insertion simulating autologous transplantation. The tissues were histologically evaluated by hematoxylin-eosin and Safranin-O staining. At 1 week, chondrocyte alignment was normal in the PTD-FNK treatment group, whereas all grafts without PTD-FNK treatment showed mixed cluster cell distribution. At 4 weeks, all grafts with PTD-FNK treatment showed almost normal matrix, whereas two grafts without PTD-FNK treatment showed fibrocartilage. Notably, all grafts with PTD-FNK retained high intensity of Safranin-O staining, but all grafts without PTD-FNK largely lost Safranin-O staining. PTD-FNK significantly suppressed a decrease in the survival rate and the density of EGFP-positive cells at 1 and 2 weeks, and this tendency continued at 4 weeks. The results of terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP)-nick end-labeling staining showed that PTD-FNK inhibited cell death, indicating that PTD-FNK protects chondrocyte death and suppresses graft degeneration.

Delay of supraspinatus repair by up to 12 weeks does not impair enthesis formation: A quantitative histologic study in rabbits

The purpose of this study was to find out whether supraspinatus repair delayed by up to12 weeks affects the formation of a new enthesis when compared to an immediate repair. In 67 rabbits, the supraspinatus fibrocartilaginous enthesis of one shoulder was resected. The tendon was attached to the greater tuberosity either immediately, after a 6-week, or after a 12-week delay. Five histologic variables were used to assess enthesis formation: number of non-chondrocytes, number of chondrocytes, alignment of chondrocytes in rows, area of metachromasia on toluidine blue (TB)-stained sections indicating proteoglycan content, and area of diffracted polarized light indicating spatial alignment of collagen fibers. For every variable, progressive enthesis formation was observed. Again, for every variable and at every time point studied, no statistically significant difference was observed between tendons repaired immediately, after 6, or after 12 weeks (all p>0.05). Supraspinatus tendon repairs delayed by 6 and 12 weeks constituted an enthesis which proceeded identically to one immediately repaired. Formation of a fibrocartilaginous enthesis depended on the elapsed time after repair and not on the duration between detachment and repair. Despite stated limitations, these results support both a trial of conservative treatment after a rotator cuff tear and a positive outcome of rotator cuff repair even if delayed by up to 12 weeks.

Formation of a new enthesis after attachment of the supraspinatus tendon: A quantitative histologic study in rabbits

To quantify in a longitudinal study non-chondrocytic cells and chondrocytes, tissular architecture as well as extracellular matrix restoration during the formation of an enthesis following supraspinatus tendon attachment to the humerus. In 89 rabbits, one supraspinatus fibrocartilaginous enthesis was resected and the tendon either attached to the greater tuberosity (n=75) or not attached (n=14). The animals were sacrificed after 2, 6, 8, 12 or 24 weeks. The operated and contralateral shoulders were processed for histologic sections. Number of non-chondrocytes, chondrocytes and alignment of chondrocytes in rows were assessed histologically. Extracellular matrix restoration was measured based on (1) area of toluidine blue metachromasia indicating proteoglycan content and (2) on area of diffracted polarized light indicating spatial collagen fiber alignment. In the attached tendon, the number of non-chondrocytic cells sharply increased at 2 weeks, progressively decreased thereafter but remained higher than controls at all time points. Chondrocytes appeared at 2 weeks and their number reached control levels by 6 weeks (136+/-14 vs 144+/-15 controls, p>.05). The percentage of chondrocytes aligned in rows increased from 19+/-4% at 2 weeks to reach near normal values at 24 weeks (71+/-3% vs 78+/-2% controls, p>.05). Area of metachromasia increased from 0.1+/-0.1 mm(2) at 2 weeks to 3.8+/-0.3 mm(2) at 24 weeks, still below contralateral enthesis levels (4.6+/-0.1 mm(2), p<.05). Area of diffracted polarized light enlarged from 12+/-2 x 10(3) microm(2) at 2 weeks to 151+/-19 x 10(3) microm(2) at 24 weeks, still significantly smaller than contralateral levels (177+/-13 x 10(3) microm(2), p<.05). Neither chondrocytes nor metachromasia were observed in the non-attached tendons. A new enthesis was formed after attachment of the supraspinatus tendon into bony trough. Histomorphometry allowed to document extensive non-chondrocytic proliferation that was followed by appearance of chondrocytes and their spatial organization, a process was complete by 24 weeks. Extracellular matrix formation as well as spatial alignment of collagen fibers were delayed and not complete by 24 weeks. This first longitudinal investigation on the formation of the supraspinatus enthesis using quantitative outcome measures cautions against too early and too aggressive a rehabilitation program.

Proliferative and re-defferentiative effects of photo-immobilized micro-patterned hyaluronan surfaces on chondrocyte cells

A photo-immobilisation procedure was utilised to create two different micro-patterned surfaces (tracks 25 and 5 μm wide) of hyaluronan (Hyal) on polyethylene-terephthalate (PET) previously plasma activated. Aim of the study was to investigate the proliferation and re-differentiation capacity of articular chondrocytes cultured on micro-patterned Hyal, compared to homogeneous Hyal and plain plasma-treated (pt-)PET substrates. Cytotoxicity, cell proliferation, activation and differentiation of articular knee cartilage chondrocytes (Mongrel sheep) were evaluated after 14 days of culture. It was found that micro-patterned Hyal surfaces induced the adhesion, migration and alignment of chondrocytes, as shown by light and scanning electron microscopy. Furthermore, the same surfaces induced chondrocyte differentiation, with a significant increase of aggrecan and collagen type II production, while homogeneous Hyal and pt-PET surfaces did not.

Formation of a new enthesis after attachment of the supraspinatus tendon: A quantitative histologic study in rabbits

Purpose. To quantify in a longitudinal study non-chondrocytic cells and chondrocytes, tissular architecture as well as extracellular matrix restoration during the formation of an enthesis following supraspinatus tendon attachment to the humerus. Methods. In 89 rabbits, one supraspinatus fibrocartilaginous enthesis was resected and the tendon either attached to the greater tuberosity ( n = 75) or not attached ( n = 14). The animals were sacrificed after 2, 6, 8, 12 or 24 weeks. The operated and contralateral shoulders were processed for histologic sections. Number of non-chondrocytes, chondrocytes and alignment of chondrocytes in rows were assessed histologically. Extracellular matrix restoration was measured based on (1) area of toluidine blue metachromasia indicating proteoglycan content and (2) on area of diffracted polarized light indicating spatial collagen fiber alignment. Results. In the attached tendon, the number of non-chondrocytic cells sharply increased at 2 weeks, progressively decreased thereafter but remained higher than controls at all time points. Chondrocytes appeared at 2 weeks and their number reached control levels by 6 weeks (136 ± 14 vs 144 ± 15 controls, p > .05). The percentage of chondrocytes aligned in rows increased from 19 ± 4% at 2 weeks to reach near normal values at 24 weeks (71 ± 3% vs 78 ± 2% controls, p > .05). Area of metachromasia increased from 0.1 ± 0.1 mm 2 at 2 weeks to 3.8 ± 0.3 mm 2 at 24 weeks, still below contralateral enthesis levels (4.6 ± 0.1 mm 2, p < .05). Area of diffracted polarized light enlarged from 12 ± 2 × 10 3 μm 2 at 2 weeks to 151 ± 19 × 10 3 μm 2 at 24 weeks, still significantly smaller than contralateral levels (177 ± 13 × 10 3 μm 2, p < .05). Neither chondrocytes nor metachromasia were observed in the non-attached tendons. Conclusion. A new enthesis was formed after attachment of the supraspinatus tendon into bony trough. Histomorphometry allowed to document extensive non-chondrocytic proliferation that was followed by appearance of chondrocytes and their spatial organization, a process was complete by 24 weeks. Extracellular matrix formation as well as spatial alignment of collagen fibers were delayed and not complete by 24 weeks. This first longitudinal investigation on the formation of the supraspinatus enthesis using quantitative outcome measures cautions against too early and too aggressive a rehabilitation program.

Exercise affects the mechanical properties and histological appearance of equine articular cartilage.

Dorsal carpal osteochondral injury is a major cause of reduced performance in horses undergoing high-intensity training. It was hypothesised that the mechanical behaviour and histology of cartilage are influenced by the intensity of exercise and by location within a joint. Relationships between histology and mechanical behaviour were identified in 2-year-old horses undergoing 19 weeks of high-intensity treadmill training or low-intensity exercise and then compared between groups. Dorsal and palmar test sites were identified on radial, intermediate, and third carpal articular surfaces after euthanasia. The mechanical properties of cartilage were determined with an automated creep indentation apparatus as previously described for equine cartilage. Cartilage morphology was assessed with use of sections stained with haematoxylin and eosin and toluidine blue. Dorsal cartilage was less permeable, thinner, and had a loss of chondrocyte alignment compared with palmar cartilage. Cartilage from strenuously trained horses showed more fibrillation and chondrocyte clusters than did cartilage from gently exercised animals. Dorsal radial carpal cartilage and third carpal cartilage of strenuously trained animals were significantly less stiff than that from gently exercised animals, and the former had reduced superficial toluidine blue staining compared with that from the gently exercised group. These results indicate that topographical and exercise-related differences exist in the morphology and mechanical properties of carpal cartilage and suggest that strenuous training may lead to deterioration of cartilage at sites with a high clinical incidence of lesions.

Joint resurfacing using allograft chondrocytes and synthetic biodegradable polymer scaffolds.

Cartilage implants which could potentially be used to resurface damaged joints were created using rabbit articular chondrocytes and synthetic, biodegradable polymer scaffolds. Cells were serially passaged and then cultured in vitro on fibrous polyglycolic acid (PGA) scaffolds. Cell-PGA constructs were implanted in vivo as allografts to repair 3-mm diameter, full thickness defects in the knee joints of adult rabbits, and cartilage repair was assessed histologically over 6 months. In vitro, chondrocytes proliferated on PGA and regenerated cartilaginous matrix. Collagen and glycosaminoglycan (GAG) represented 20 to 8% of the implant dry weight (dw), respectively, at the time of in vivo implantation; the remainder was PGA and unspecified components. Implants based on passaged chondrocytes had 1.7-times as much GAG and 2.6-times as much collagen as those based on primary chondrocytes. In vivo, cartilaginous repair tissue was observed after implantation of PGA both with and without cultured chondrocytes. Six month repair was qualitatively better for cell-PGA allografts than for PGA alone, with respect to: 1) surface smoothness, 2) columnar alignment of chondrocytes, 3) spatially uniform GAG distribution, 4) reconstitution of the subchondral plate, and 5) bonding of the repair tissue to the underlying bone. These pilot studies demonstrate that it is feasible to use cell-polymer allografts for joint resurfacing in vivo.

Reversibility of joint changes produced by immobilization in rabbits.

Rabbits knee joints which had been immobilized for only 2 weeks regained motion during the first 2 months of remobilization. Histologically, joint tissues were normal. Only 9 of the 14 rabbits immobilized for 6 weeks regained normal or partial knee motion. Animals that regained knee motion generally did so during the first 4 months of remobilization. There are 2 types of repair of the damaged cartilage; replacement by fibrocartilage and proliferation of chondrocytes. Normal chondrocyte alignment and normal collagen fibril orientation was not restored. Repaired cartilage did not withstand normal joint stress.