Hongjun Zheng

Chondrogenic progenitor cells respond to cartilage injury.

OBJECTIVE Hypocellularity resulting from chondrocyte death in the aftermath of mechanical injury is thought to contribute to posttraumatic osteoarthritis. However, we observed that nonviable areas in cartilage injured by blunt impact were repopulated within 7-14 days by cells that appeared to migrate from the surrounding matrix. The aim of this study was to assess our hypothesis that the migrating cell population included chondrogenic progenitor cells that were drawn to injured cartilage by alarmins. METHODS Osteochondral explants obtained from mature cattle were injured by blunt impact or scratching, resulting in localized chondrocyte death. Injured sites were serially imaged by confocal microscopy, and migrating cells were evaluated for chondrogenic progenitor characteristics. Chemotaxis assays were used to measure the responses to chemokines, injury-conditioned medium, dead cell debris, and high mobility group box chromosomal protein 1 (HMGB-1). RESULTS Migrating cells were highly clonogenic and multipotent and expressed markers associated with chondrogenic progenitor cells. Compared with chondrocytes, these cells overexpressed genes involved in proliferation and migration and underexpressed cartilage matrix genes. They were more active than chondrocytes in chemotaxis assays and responded to cell lysates, conditioned medium, and HMGB-1. Glycyrrhizin, a chelator of HMGB-1 and a blocking antibody to receptor for advanced glycation end products (RAGE), inhibited responses to cell debris and conditioned medium and reduced the numbers of migrating cells on injured explants. CONCLUSION Injuries that caused chondrocyte death stimulated the emergence and homing of chondrogenic progenitor cells, in part via HMGB-1 release and RAGE-mediated chemotaxis. Their repopulation of the matrix could promote the repair of chondral damage that might otherwise contribute to progressive cartilage loss.

Use of Recombinant Human Stromal Cell–Derived Factor 1α–Loaded Fibrin/Hyaluronic Acid Hydrogel Networks to Achieve Functional Repair of Full-Thickness Bovine Articular Cartilage Via Homing of Chondrogenic Progenitor Cells

Articular cartilage damage after joint trauma seldom heals and often leads to osteoarthritis. We previously identified a migratory chondrogenic progenitor cell (CPC) population that responds chemotactically to cell death and rapidly repopulates the injured cartilage matrix, which suggests a potential approach for articular cartilage repair. This study was undertaken to determine whether recombinant human stromal cell–derived factor 1α (rhSDF‐1α), a potent CPC chemoattractant, would improve the quality of cartilage regeneration, hypothesizing that increased recruitment of CPCs by rhSDF‐1α would promote the formation of cartilage matrix upon chondrogenic induction.

Selection of reference genes for normalization of quantitative real-time PCR in organ culture of the rat and rabbit intervertebral disc

BackgroundThe accuracy of quantitative real-time RT-PCR (qRT-PCR) is often influenced by experimental artifacts, resulting in erroneous expression profiles of target genes. The practice of employing normalization using a reference gene significantly improves reliability and its applicability to molecular biology. However, selection of an ideal reference gene(s) is of critical importance to discern meaningful results. The aim of this study was to evaluate the stability of seven potential reference genes (Actb, GAPDH, 18S rRNA, CycA, Hprt1, Ywhaz, and Pgk1) and identify most stable gene(s) for application in tissue culture research using the rat and rabbit intervertebral disc (IVD).FindingsIn vitro, four genes (Hprt1, CycA, GAPDH, and 18S rRNA) in rat IVD tissue and five genes (CycA, Hprt1, Actb, Pgk1, and Ywhaz) in rabbit IVD tissue were determined as most stable for up to 14 days in culture. Pair-wise variation analysis indicated that combination of Hprt1 and CycA in rat and the combination of Hprt1, CycA, and Actb in rabbit may most stable reference gene candidates for IVD tissue culture.ConclusionsOur results indicate that Hprt1 and CycA are the most stable reference gene candidates for rat and rabbit IVD culture studies. In rabbit IVD, Actb could be an additional gene employed in conjunction with Hprt1 and CycA. Selection of optimal reference gene candidate(s) should be a pertinent exercise before employment of PCR outcome measures for biomedical research.

Single cell sorting identifies progenitor cell population from full thickness bovine articular cartilage

OBJECTIVE To date, no approved clinical intervention successfully prevents the progressive degradation of injured articular cartilage that leads to osteoarthritis (OA). Stem/progenitor cell populations within tissues of diarthrodial joint have shown their therapeutic potential in treating OA. However, this potential has not been fully realized due in part to the heterogeneity of these subpopulations. Characterization of clonal populations derived from a single cell may help identify more homogenous stem/progenitor populations within articular cartilage. Moreover, chondrogenic potential of clonal populations from different zones could be further examined to elucidate their differential roles in maintaining articular cartilage homeostasis. METHOD We combined Fluorescence-activated cell sorting (FACS) and clonogenicity screening to identify stem/progenitor cells cloned from single cells. High-efficiency colony-forming cells (HCCs) were isolated, and evaluated for stem/progenitor cell characteristics. HCCs were also isolated from different zones of articular cartilage. Their function was compared by lineage-specific gene expression, and differentiation potential. RESULTS A difference in colony-forming efficiency was observed in terms of colony sizes. HCCs were highly clonogenic and multipotent, and overexpressed stem/progenitor cell markers. Also, proliferation and migration associated genes were over-expressed in HCCs. HCCs showed zonal differences with deep HCCs more chondrogenic and osteogenic than superficial HCCs. CONCLUSION Our approach is a simple yet practical way to identify homogeneous stem/progenitor cell populations with clonal origin. The discovery of progenitor cells demonstrates the intrinsic self-repairing potential of articular cartilage. Differences in differentiation potential may represent the distinct roles of superficial and deep zone stem/progenitor cells in the maintenance of articular cartilage homeostasis.

gene expression profiles reveal that chondrogenic progenitor cells and synovial cells are closely related

We showed previously that chondrogenic progenitor cells (CPCs) from the superficial zone of articular cartilage respond vigorously to cartilage wounding by responding chemotactically to cell debris, but the physiologic functions of CPCs remain unclear. To help bridge this knowledge gap we undertook a comparative analysis of gene expression in bovine CPCs, chondrocytes, synovial fibroblasts (synoviocytes), and cells isolated from synovial fluid (SFCs). Analysis of microarrays parsed the four cell types into two distinct groups, one composed only of chondrocytes and the other of CPCs, synoviocytes, and SFCs. The groups differed with respect to metalloendopeptidase, collagen, and cytokine gene expression. Quantitative PCR showed that, relative to chondrocytes, all other cells under‐expressed cartilage matrix genes. CPCs significantly over‐expressed genes encoding the chemokines interleukin 8 (IL8), and C–C motif ligand 2, while synoviocytes over‐expressed the chemokine C–X–C motif Ligand 12. Sulfated glycosaminoglycan deposition in pellet cultures by CPCs was intermediate between chondrocytes and synoviocytes/SFCs. These results indicate that the CPC phenotype more closely resembles synoviocytes and SFCs than chondrocytes. CPCs show a tendency to over‐express chemokines that promote immune cell chemotaxis, suggesting they mediate inflammation in response to cartilage wounding.

Organ culture stability of the intervertebral disc: Rat versus rabbit

There is a need to develop mechanically active culture systems to better understand the role of mechanical stresses in intervertebral disc (IVD) degeneration. Motion segment cultures that preserve the native IVD structure and adjacent vertebral bodies are preferred as model systems, but rapid ex vivo tissue degeneration limits their usefulness. The stability of rat and rabbit IVDs is of particular interest, as their small size makes them otherwise suitable for motion segment culture. The goal of this study was to determine if there are substantial differences in the susceptibility of rat and rabbit IVDs to culture‐induced degeneration. Lumbar IVD motion segments were harvested from young adult male Sprague–Dawley rats and New Zealand White rabbits and cultured under standard conditions for 14 days. Biochemical assays and safranin‐O histology showed that while glycosaminoglycan (GAG) loss was minimal in rabbit IVDs, it was progressive and severe in rat IVDs. In the rat IVD, GAG loss was concomitant with the loss of notochordal cells and the migration of endplate (EP) cells into the nucleus pulposus (NP). None of these changes were evident in the rabbit IVDs. Compared to rabbit IVDs, rat IVDs also showed increased matrix metalloproteinase‐3 (MMP‐3) and sharply decreased collagen type I and II collagen expression. Together these data indicated that the rabbit IVD was dramatically more stable than the rat IVD, which showed culture‐related degenerative changes. Based on these findings we conclude that the rabbit motion segments are a superior model for mechanobiologic studies.

Nanog Maintains Human Chondrocyte Phenotype and Function In Vitro

Previous work showed that Nanog, a homeobox family transcription factor, maintains embryonic stem cell pluripotency, suggesting that it has a role in stabilizing cell phenotype. Human chondrocytes lose their phenotype and dedifferentiate after relatively few passages in culture, changes that may limit their value in restoring damaged articular cartilage. We hypothesized that Nanog could stabilize the phenotype of cultured human chondrocytes in long‐term monolayer cultures. To test this hypothesis, the human Nanog gene was stably transduced into human chondrocytes using a retroviral vector. Chondrocyte‐specific gene expression (collagen type II, aggrecan, cartilage link protein, and Sox9) was measured by reverse transcription‐polymerase chain reaction (RT‐PCR) and real‐time PCR in monolayer cultured chondrocytes transduced with Nanog and in control chondrocytes transduced with empty vector. In vitro cartilage matrix protein formation by Nanog‐transduced and control cells was compared using Safranin‐O and immunofluorescence stains. We found that after 25 passages, Nanog‐transduced chondrocytes maintained significantly higher expression of collagen type II, aggrecan, and cartilage link protein genes than controls. Under chondrogenic conditions, Nanog‐transduced cells produced significantly more cartilage‐specific matrix than control cells. These findings support the hypothesis that Nanog maintains the human chondrocyte phenotype and function after long‐term monolayer culture. Preservation of the chondrocyte phenotype may improve the ability of cultured chondrocytes to repair or restore articular cartilage.