PEDF Is Associated with the Termination of Chondrocyte Phenotype and Catabolism of Cartilage Tissue

Objective.To investigate the expression and target genes of pigment epithelium-derived factor (PEDF) in cartilage and chondrocytes, respectively.Methods.We analyzed the expression pattern of PEDF in different human cartilaginous tissues including articular cartilage, osteophytic cartilage, and fetal epiphyseal and growth plate cartilage, by immunohistochemistry and quantitative real-time (qRT) PCR. Transcriptome analysis after stimulation of human articular chondrocytes with rhPEDF was performed by RNA sequencing (RNA-Seq) and confirmed by qRT-PCR.Results.Immunohistochemically, PEDF could be detected in transient cartilaginous tissue that is prone to undergo endochondral ossification, including epiphyseal cartilage, growth plate cartilage, and osteophytic cartilage. In contrast, PEDF was hardly detected in healthy articular cartilage and in the superficial zone of epiphyses, regions that are characterized by a permanent stable chondrocyte phenotype. RNA-Seq analysis and qRT-PCR demonstrated that rhPEDF significantly induced the expression of a number of matrix-degrading factors including SAA1, MMP1, MMP3, and MMP13. Simultaneously, a number of cartilage-specific genes including COL2A1, COL9A2, COMP, and LECT were among the most significantly downregulated genes.Conclusions.PEDF represents a marker for transient cartilage during all neonatal and postnatal developmental stages and promotes the termination of cartilage tissue by upregulation of matrix-degrading factors and downregulation of cartilage-specific genes. These data provide the basis for novel strategies to stabilize the phenotype of articular cartilage and prevent its degradation.

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Small RNA sequencing evaluation of renal microRNA biomarkers in dogs with X-linked hereditary nephropathy

Scientific Reports ◽

10.1038/s41598-021-96870-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Candice P. Chu ◽

Shiguang Liu ◽

Wenping Song ◽

Ethan Y. Xu ◽

Mary B. Nabity

Keyword(s):

Small Rna ◽

Target Genes ◽

Differentially Expressed ◽

Small Rna Sequencing ◽

Rna Seq ◽

Ckd Progression ◽

Critical Pathways ◽

Qrt Pcr ◽

Hereditary Nephropathy ◽

Differentially Expressed Mirnas

AbstractDogs with X-linked hereditary nephropathy (XLHN) are an animal model for Alport syndrome in humans and progressive chronic kidney disease (CKD). Using mRNA sequencing (mRNA-seq), we have characterized the gene expression profile affecting the progression of XLHN; however, the microRNA (miRNA, miR) expression remains unknown. With small RNA-seq and quantitative RT-PCR (qRT-PCR), we used 3 small RNA-seq analysis tools (QIAGEN OmicSoft Studio, miRDeep2, and CPSS 2.0) to profile differentially expressed renal miRNAs, top-ranked miRNA target genes, and enriched biological processes and pathways in CKD progression. Twenty-three kidney biopsies were collected from 5 dogs with XLHN and 4 age-matched, unaffected littermates at 3 clinical time points (T1: onset of proteinuria, T2: onset of azotemia, and T3: advanced azotemia). We identified up to 23 differentially expressed miRNAs at each clinical time point. Five miRNAs (miR-21, miR-146b, miR-802, miR-142, miR-147) were consistently upregulated in affected dogs. We identified miR-186 and miR-26b as effective reference miRNAs for qRT-PCR. This study applied small RNA-seq to identify differentially expressed miRNAs that might regulate critical pathways contributing to CKD progression in dogs with XLHN.

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Properties of Cartilage–Subchondral Bone Junctions: A Narrative Review with Specific Focus on the Growth Plate

Cartilage ◽

10.1177/1947603520924776 ◽

2020 ◽

pp. 194760352092477

Author(s):

Masumeh Kazemi ◽

John Leicester Williams

Keyword(s):

Mechanical Properties ◽

Articular Cartilage ◽

Growth Plate ◽

Subchondral Bone ◽

Bone Growth ◽

Narrative Review ◽

Bimaterial Interface ◽

Bone Interface ◽

Growth Plate Cartilage ◽

Specific Focus

Objective The purpose of this narrative review is to summarize what is currently known about the structural, chemical, and mechanical properties of cartilage-bone interfaces, which provide tissue integrity across a bimaterial interface of 2 very different structural materials. Maintaining these mechanical interfaces is a key factor for normal bone growth and articular cartilage function and maintenance. Materials and Methods A comprehensive search was conducted using Google Scholar and PubMed/Medline with a specific focus on the growth plate cartilage–subchondral bone interface. All original articles, reviews in journals, and book chapters were considered. Following a review of the overall structural and functional characteristics of the physis, the literature on histological studies of both articular and growth plate chondro-osseous junctions is briefly reviewed. Next the literature on biochemical properties of these interfaces is reviewed, specifically the literature on elemental analyses across the cartilage–subchondral bone junctions. The literature on biomechanical studies of these junctions at the articular and physeal interfaces is also reviewed and compared. Results Unlike the interface between articular cartilage and bone, growth plate cartilage has 2 chondro-osseous junctions. The reserve zone of the mature growth plate is intimately connected to a plate of subchondral bone on the epiphyseal side. This interface resembles that between the subchondral bone and articular cartilage, although much less is known about its makeup and formation. Conclusion There is a notably paucity of information available on the structural and mechanical properties of reserve zone–subchondral epiphyseal bone interface. This review reveals that further studies are needed on the microstructural and mechanical properties of chondro-osseous junction with the reserve zone.

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RECENT RESEARCH ON THE GROWTH PLATE: Mechanisms for growth plate injury repair and potential cell-based therapies for regeneration

Journal of Molecular Endocrinology ◽

10.1530/jme-14-0062 ◽

2014 ◽

Vol 53 (1) ◽

pp. T45-T61 ◽

Cited By ~ 26

Author(s):

Rosa Chung ◽

Cory J Xian

Keyword(s):

Growth Factor ◽

Growth Plate ◽

Cartilage Regeneration ◽

Cartilage Tissue ◽

Injury Repair ◽

Growth Plate Cartilage ◽

New Findings ◽

Osteogenic Response ◽

Growth Plate Injury

Injuries to the growth plate cartilage often lead to bony repair, resulting in bone growth defects such as limb length discrepancy and angulation deformity in children. Currently utilised corrective surgeries are highly invasive and limited in their effectiveness, and there are no known biological therapies to induce cartilage regeneration and prevent the undesirable bony repair. In the last 2 decades, studies have investigated the cellular and molecular events that lead to bony repair at the injured growth plate including the identification of the four phases of injury repair responses (inflammatory, fibrogenic, osteogenic and remodelling), the important role of inflammatory cytokine tumour necrosis factor alpha in regulating downstream repair responses, the role of chemotactic and mitogenic platelet-derived growth factor in the fibrogenic response, the involvement and roles of bone morphogenic protein and Wnt/B-catenin signalling pathways, as well as vascular endothelial growth factor-based angiogenesis during the osteogenic response. These new findings could potentially lead to identification of new targets for developing a future biological therapy. In addition, recent advances in cartilage tissue engineering highlight the promising potential for utilising multipotent mesenchymal stem cells (MSCs) for inducing regeneration of injured growth plate cartilage. This review aims to summarise current understanding of the mechanisms for growth plate injury repair and discuss some progress, potential and challenges of MSC-based therapies to induce growth plate cartilage regeneration in combination with chemotactic and chondrogenic growth factors and supporting scaffolds.

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Chondrocyte heterogeneity: immunohistologically defined variation of integrin expression at different sites in human fetal knees.

Journal of Histochemistry & Cytochemistry ◽

10.1177/43.4.7897185 ◽

1995 ◽

Vol 43 (4) ◽

pp. 447-457 ◽

Cited By ~ 57

Author(s):

D M Salter ◽

J L Godolphin ◽

M S Gourlay

Keyword(s):

Articular Cartilage ◽

Growth Plate ◽

Articular Chondrocytes ◽

Knee Joints ◽

Matrix Composition ◽

Epiphyseal Growth Plate ◽

Surface Receptors ◽

Cartilage Differentiation ◽

Health And Disease ◽

And Function

During development and at maturity different forms of cartilage vary in morphology and macromolecular content. This reflects heterogeneity of chondrocyte activity, in part involving differential interactions with the adjacent extracellular matrix via specialized cell surface receptors such as integrins. We undertook an immunohistological study on a series of human fetal knee joints to assess variation in the expression of integrins by chondrocytes and potential matrix ligands in articular, epiphyseal, growth plate, and meniscal cartilage. The results show that articular chondrocytes (beta 1+, beta 5 alpha V+, alpha 1+, alpha 2+/-, alpha 5+, weakly alpha 6+, alpha V+) differed from epiphyseal (beta 1+, beta 5 alpha V+, alpha 1+/-, alpha 2+/-, alpha 5+, alpha 6+, alpha V+) growth plate (beta 1+, beta 5 alpha V+, alpha 1-, alpha 2-, alpha 5+, alpha 6+, alpha V+), and meniscal cells (beta 1+, beta 5 alpha V+, alpha 1+, strongly alpha 2+, alpha 5+, alpha 6+, alpha V+ in expression of integrin subunits. There was no expression of beta 3, beta 4, beta 6, or alpha 3 by chondrocytes. These results differ from previous reports on the expression of integrins by adult articular cartilage, where alpha 2 and alpha 6 are not seen. Variation in distribution of matrix ligands was also seen. Fibronectin, laminin and Type VI collagen were expressed in all cartilages but there was restricted expression of tenascin, ED-A and ED-B fibronectin isoforms (articular cartilage and meniscus), and vitronectin (absent from growth plate cartilage). Regulated expression of integrins by chondrocytes, associated with changes in the pericellular matrix composition, is of potential importance in control of cartilage differentiation and function in health and disease.

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Expression of a Truncated, Kinase-Defective TGF-β Type II Receptor in Mouse Skeletal Tissue Promotes Terminal Chondrocyte Differentiation and Osteoarthritis

The Journal of Cell Biology ◽

10.1083/jcb.139.2.541 ◽

1997 ◽

Vol 139 (2) ◽

pp. 541-552 ◽

Cited By ~ 355

Author(s):

Rosa Serra ◽

Mahlon Johnson ◽

Ellen H. Filvaroff ◽

James LaBorde ◽

Daniel M. Sheehan ◽

...

Keyword(s):

Articular Cartilage ◽

Transgenic Mice ◽

Growth Plate ◽

Dominant Negative Mutant ◽

Type I ◽

Type Ii ◽

Type X Collagen ◽

Skeletal Tissue ◽

Hypertrophic Cartilage ◽

Growth Plate Cartilage

Members of the TGF-β superfamily are important regulators of skeletal development. TGF-βs signal through heteromeric type I and type II receptor serine/threonine kinases. When over-expressed, a cytoplasmically truncated type II receptor can compete with the endogenous receptors for complex formation, thereby acting as a dominant-negative mutant (DNIIR). To determine the role of TGF-βs in the development and maintenance of the skeleton, we have generated transgenic mice (MT-DNIIR-4 and -27) that express the DNIIR in skeletal tissue. DNIIR mRNA expression was localized to the periosteum/perichondrium, syno-vium, and articular cartilage. Lower levels of DNIIR mRNA were detected in growth plate cartilage. Transgenic mice frequently showed bifurcation of the xiphoid process and sternum. They also developed progressive skeletal degeneration, resulting by 4 to 8 mo of age in kyphoscoliosis and stiff and torqued joints. The histology of affected joints strongly resembled human osteo-arthritis. The articular surface was replaced by bone or hypertrophic cartilage as judged by the expression of type X collagen, a marker of hypertrophic cartilage normally absent from articular cartilage. The synovium was hyperplastic, and cartilaginous metaplasia was observed in the joint space. We then tested the hypothesis that TGF-β is required for normal differentiation of cartilage in vivo. By 4 and 8 wk of age, the level of type X collagen was increased in growth plate cartilage of transgenic mice relative to wild-type controls. Less proteoglycan staining was detected in the growth plate and articular cartilage matrix of transgenic mice. Mice that express DNIIR in skeletal tissue also demonstrated increased Indian hedgehog (IHH) expression. IHH is a secreted protein that is expressed in chondrocytes that are committed to becoming hypertrophic. It is thought to be involved in a feedback loop that signals through the periosteum/ perichondrium to inhibit cartilage differentiation. The data suggest that TGF-β may be critical for multifaceted maintenance of synovial joints. Loss of responsiveness to TGF-β promotes chondrocyte terminal differentiation and results in development of degenerative joint disease resembling osteoarthritis in humans.

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Bioprinting of a Zonal-Specific Cell Density Scaffold: A Biomimetic Approach for Cartilage Tissue Engineering

Applied Sciences ◽

10.3390/app11177821 ◽

2021 ◽

Vol 11 (17) ◽

pp. 7821

Author(s):

Angeliki Dimaraki ◽

Pedro J. Díaz-Payno ◽

Michelle Minneboo ◽

Mahdiyeh Nouri-Goushki ◽

Maryam Hosseini ◽

...

Keyword(s):

Tissue Engineering ◽

Articular Cartilage ◽

Cell Density ◽

Articular Chondrocytes ◽

Cartilage Tissue Engineering ◽

Smooth Transition ◽

Cartilage Tissue ◽

Cartilage Defects ◽

Specific Cell ◽

Cell Densities

The treatment of articular cartilage defects remains a significant clinical challenge. This is partially due to current tissue engineering strategies failing to recapitulate native organization. Articular cartilage is a graded tissue with three layers exhibiting different cell densities: the superficial zone having the highest density and the deep zone having the lowest density. However, the introduction of cell gradients for cartilage tissue engineering, which could promote a more biomimetic environment, has not been widely explored. Here, we aimed to bioprint a scaffold with different zonal cell densities to mimic the organization of articular cartilage. The scaffold was bioprinted using an alginate-based bioink containing human articular chondrocytes. The scaffold design included three cell densities, one per zone: 20 × 106 (superficial), 10 × 106 (middle), and 5 × 106 (deep) cells/mL. The scaffold was cultured in a chondrogenic medium for 25 days and analyzed by live/dead assay and histology. The live/dead analysis showed the ability to generate a zonal cell density with high viability. Histological analysis revealed a smooth transition between the zones in terms of cell distribution and a higher sulphated glycosaminoglycan deposition in the highest cell density zone. These findings pave the way toward bioprinting complex zonal cartilage scaffolds as single units, thereby advancing the translation of cartilage tissue engineering into clinical practice.

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Harnessing Biomechanics to Develop Cartilage Regeneration Strategies

Journal of Biomechanical Engineering ◽

10.1115/1.4028825 ◽

2015 ◽

Vol 137 (2) ◽

Cited By ~ 23

Author(s):

Kyriacos A. Athanasiou ◽

Donald J. Responte ◽

Wendy E. Brown ◽

Jerry C. Hu

Keyword(s):

Tissue Engineering ◽

Articular Cartilage ◽

Biochemical Characterization ◽

Articular Chondrocytes ◽

Lysyl Oxidase ◽

Cartilage Tissue Engineering ◽

Cartilage Regeneration ◽

Cartilage Tissue ◽

Main Function ◽

Mechanical Stimuli

As this review was prepared specifically for the American Society of Mechanical Engineers H.R. Lissner Medal, it primarily discusses work toward cartilage regeneration performed in Dr. Kyriacos A. Athanasiou's laboratory over the past 25 years. The prevalence and severity of degeneration of articular cartilage, a tissue whose main function is largely biomechanical, have motivated the development of cartilage tissue engineering approaches informed by biomechanics. This article provides a review of important steps toward regeneration of articular cartilage with suitable biomechanical properties. As a first step, biomechanical and biochemical characterization studies at the tissue level were used to provide design criteria for engineering neotissues. Extending this work to the single cell and subcellular levels has helped to develop biochemical and mechanical stimuli for tissue engineering studies. This strong mechanobiological foundation guided studies on regenerating hyaline articular cartilage, the knee meniscus, and temporomandibular joint (TMJ) fibrocartilage. Initial tissue engineering efforts centered on developing biodegradable scaffolds for cartilage regeneration. After many years of studying scaffold-based cartilage engineering, scaffoldless approaches were developed to address deficiencies of scaffold-based systems, resulting in the self-assembling process. This process was further improved by employing exogenous stimuli, such as hydrostatic pressure, growth factors, and matrix-modifying and catabolic agents, both singly and in synergistic combination to enhance neocartilage functional properties. Due to the high cell needs for tissue engineering and the limited supply of native articular chondrocytes, costochondral cells are emerging as a suitable cell source. Looking forward, additional cell sources are investigated to render these technologies more translatable. For example, dermis isolated adult stem (DIAS) cells show potential as a source of chondrogenic cells. The challenging problem of enhanced integration of engineered cartilage with native cartilage is approached with both familiar and novel methods, such as lysyl oxidase (LOX). These diverse tissue engineering strategies all aim to build upon thorough biomechanical characterizations to produce functional neotissue that ultimately will help combat the pressing problem of cartilage degeneration. As our prior research is reviewed, we look to establish new pathways to comprehensively and effectively address the complex problems of musculoskeletal cartilage regeneration.

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Repair of full-thickness articular cartilage defects using IEIK13 self-assembling peptide hydrogel in a non-human primate model

Scientific Reports ◽

10.1038/s41598-021-83208-x ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Alexandre Dufour ◽

Jérôme E. Lafont ◽

Marie Buffier ◽

Michaël Verset ◽

Angéline Cohendet ◽

...

Keyword(s):

Articular Cartilage ◽

Articular Chondrocytes ◽

Cartilage Tissue ◽

Cartilage Defects ◽

Full Thickness ◽

Primate Model ◽

Self Assembling ◽

Articular Cartilage Defects ◽

Human Primate

AbstractArticular cartilage is built by chondrocytes which become less active with age. This declining function of the chondrocytes, together with the avascular nature of the cartilage, impedes the spontaneous healing of chondral injuries. These lesions can progress to more serious degenerative articular conditions as in the case of osteoarthritis. As no efficient cure for cartilage lesions exist yet, cartilage tissue engineering has emerged as a promising method aiming at repairing joint defects and restoring articular function. In the present work, we investigated if a new self-assembling peptide (referred as IEIK13), combined with articular chondrocytes treated with a chondrogenic cocktail (BMP-2, insulin and T3, designated BIT) could be efficient to restore full-thickness cartilage defects induced in the femoral condyles of a non-human primate model, the cynomolgus monkey. First, in vitro molecular studies indicated that IEIK13 was efficient to support production of cartilage by monkey articular chondrocytes treated with BIT. In vivo, cartilage implant integration was monitored non-invasively by contrast-enhanced micro-computed tomography, and then by post-mortem histological analysis and immunohistochemical staining of the condyles collected 3 months post-implantation. Our results revealed that the full-thickness cartilage injuries treated with either IEIK13 implants loaded with or devoid of chondrocytes showed similar cartilage-characteristic regeneration. This pilot study demonstrates that IEIK13 can be used as a valuable scaffold to support the in vitro activity of articular chondrocytes and the repair of articular cartilage defects, when implanted alone or with chondrocytes.

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SOX9 keeps growth plates and articular cartilage healthy by inhibiting chondrocyte dedifferentiation/osteoblastic redifferentiation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2019152118 ◽

2021 ◽

Vol 118 (8) ◽

pp. e2019152118

Author(s):

Abdul Haseeb ◽

Ranjan Kc ◽

Marco Angelozzi ◽

Charles de Charleroy ◽

Danielle Rux ◽

...

Keyword(s):

Articular Cartilage ◽

Growth Plate ◽

Molecular Mechanisms ◽

High Throughput Sequencing ◽

Articular Chondrocytes ◽

Treatment Options ◽

Cell Lineage ◽

Bmp Signaling ◽

Conditional Knockout ◽

Growth Plate Chondrocytes

Cartilage is essential throughout vertebrate life. It starts developing in embryos when osteochondroprogenitor cells commit to chondrogenesis, activate a pancartilaginous program to form cartilaginous skeletal primordia, and also embrace a growth-plate program to drive skeletal growth or an articular program to build permanent joint cartilage. Various forms of cartilage malformation and degeneration diseases afflict humans, but underlying mechanisms are still incompletely understood and treatment options suboptimal. The transcription factor SOX9 is required for embryonic chondrogenesis, but its postnatal roles remain unclear, despite evidence that it is down-regulated in osteoarthritis and heterozygously inactivated in campomelic dysplasia, a severe skeletal dysplasia characterized postnatally by small stature and kyphoscoliosis. Using conditional knockout mice and high-throughput sequencing assays, we show here that SOX9 is required postnatally to prevent growth-plate closure and preosteoarthritic deterioration of articular cartilage. Its deficiency prompts growth-plate chondrocytes at all stages to swiftly reach a terminal/dedifferentiated stage marked by expression of chondrocyte-specific (Mgp) and progenitor-specific (Nt5e and Sox4) genes. Up-regulation of osteogenic genes (Runx2, Sp7, and Postn) and overt osteoblastogenesis quickly ensue. SOX9 deficiency does not perturb the articular program, except in load-bearing regions, where it also provokes chondrocyte-to-osteoblast conversion via a progenitor stage. Pathway analyses support roles for SOX9 in controlling TGFβ and BMP signaling activities during this cell lineage transition. Altogether, these findings deepen our current understanding of the cellular and molecular mechanisms that specifically ensure lifelong growth-plate and articular cartilage vigor by identifying osteogenic plasticity of growth-plate and articular chondrocytes and a SOX9-countered chondrocyte dedifferentiation/osteoblast redifferentiation process.

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A scaffold-free approach to cartilage tissue generation using human embryonic stem cells

Scientific Reports ◽

10.1038/s41598-021-97934-9 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Lauren A. Griffith ◽

Katherine M. Arnold ◽

Bram G. Sengers ◽

Rahul S. Tare ◽

Franchesca D. Houghton

Keyword(s):

Stem Cells ◽

Articular Cartilage ◽

Embryonic Stem Cells ◽

Human Embryonic Stem Cells ◽

Articular Chondrocytes ◽

Embryonic Stem ◽

Cartilage Tissue ◽

Human Articular Cartilage ◽

3D Scaffold ◽

Tissue Constructs

AbstractArticular cartilage functions as a shock absorber and facilitates the free movement of joints. Currently, there are no therapeutic drugs that promote the healing of damaged articular cartilage. Limitations associated with the two clinically relevant cell populations, human articular chondrocytes and mesenchymal stem cells, necessitate finding an alternative cell source for cartilage repair. Human embryonic stem cells (hESCs) provide a readily accessible population of self-renewing, pluripotent cells with perceived immunoprivileged properties for cartilage generation. We have developed a robust method to generate 3D, scaffold-free, hyaline cartilage tissue constructs from hESCs that are composed of numerous chondrocytes in lacunae, embedded in an extracellular matrix containing Type II collagen, sulphated glycosaminoglycans and Aggrecan. The elastic (Young’s) modulus of the hESC-derived cartilage tissue constructs (0.91 ± 0.08 MPa) was comparable to full-thickness human articular cartilage (0.87 ± 0.09 MPa). Moreover, we have successfully scaled up the size of the scaffold-free, 3D hESC-derived cartilage tissue constructs to between 4.5 mm and 6 mm, thus enhancing their suitability for clinical application.

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