Blocking vascular endothelial growth factor with soluble Flt‐1 improves the chondrogenic potential of mouse skeletal muscle–derived stem cells

Objective

To investigate the effect of vascular endothelial growth factor (VEGF) stimulation and the effect of blocking VEGF with its antagonist, soluble Flt‐1 (sFlt‐1), on chondrogenesis, using muscle‐derived stem cells (MDSCs) isolated from mouse skeletal muscle.

Methods

The direct effect of VEGF on the in vitro chondrogenic ability of mouse MDSCs was tested using a pellet culture system, followed by real‐time quantitative polymerase chain reaction (PCR) and histologic analyses. Next, the effect of VEGF on chondrogenesis within the synovial joint was tested, using genetically engineered MDSCs implanted into rat osteochondral defects. In this model, MDSCs transduced with a retroviral vector to express bone morphogenetic protein 4 (BMP‐4) were coimplanted with MDSCs transduced to express either VEGF or sFlt‐1 (a VEGF antagonist) to provide a gain‐ and loss‐of‐function experimental design. Histologic scoring was used to compare cartilage formation among the treatment groups.

Results

Hyaline‐like cartilage matrix production was observed in both VEGF‐treated and VEGF‐blocked (sFlt‐1–treated) pellet cultures, but quantitative PCR revealed that sFlt‐1 treatment improved the expression of chondrogenic genes in MDSCs that were stimulated to undergo chondrogenic differentiation with BMP‐4 and transforming growth factor β3 (TGFβ3). In vivo testing of articular cartilage repair showed that VEGF‐transduced MDSCs caused an arthritic change in the knee joint, and sFlt‐1 improved the MDSC‐mediated repair of articular cartilage, compared with BMP‐4 alone.

Conclusion

Soluble Flt‐1 gene therapy improved the BMP‐4– and TGFβ3‐induced chondrogenic gene expression of MDSCs in vitro and improved the persistence of articular cartilage repair by preventing vascularization and bone invasion into the repaired articular cartilage.

     

Cartilage repair using bone morphogenetic protein 4 and muscle‐derived stem cells

Objective

Muscle‐derived stem cells (MDSCs) isolated from mouse skeletal muscle exhibit long‐time proliferation, high self‐renewal, and multipotent differentiation. This study was undertaken to investigate the ability of MDSCs that were retrovirally transduced to express bone morphogenetic protein 4 (BMP‐4) to differentiate into chondrocytes in vitro and in vivo and enhance articular cartilage repair.

Methods

Using monolayer and micromass pellet culture systems, we evaluated the in vitro chondrogenic differentiation of LacZ‐ and BMP‐4–transduced MDSCs with or without transforming growth factor β1 (TGFβ1) stimulation. We used a nude rat model of a full‐thickness articular cartilage defect to assess the duration of LacZ transgene expression and evaluate the ability of transplanted cells to acquire a chondrocytic phenotype. We evaluated cartilage repair macroscopically and histologically 4, 8, 12, and 24 weeks after surgery, and performed histologic grading of the repaired tissues.

Results

BMP‐4–expressing MDSCs acquired a chondrocytic phenotype in vitro more effectively than did MDSCs expressing only LacZ; the addition of TGFβ1 did not alter chondrogenic differentiation of the BMP‐4–transduced MDSCs. LacZ expression within the repaired tissue continued for up to 12 weeks. Four weeks after surgery, we detected donor cells that coexpressed β‐galactosidase and type II collagen. Histologic scoring of the defect sites 24 weeks after transplantation revealed significantly better cartilage repair in animals that received BMP‐4–transduced MDSCs than in those that received MDSCs expressing only LacZ.

Conclusion

Local delivery of BMP‐4 by genetically engineered MDSCs enhanced chondrogenesis and significantly improved articular cartilage repair in rats.

     

Resemblance of osteophytes in experimental osteoarthritis to transforming growth factor β–induced osteophytes: Limited role of bone morphogenetic protein in early osteoarthritic osteophyte formation

Objective

Osteoarthritis (OA) is characterized by cartilage damage, synovial fibrosis, and osteophyte formation. Both transforming growth factor β (TGFβ) and bone morphogenetic protein 2 (BMP‐2) can induce the formation of osteophytes during OA, but their specific role in this process is unclear. The purpose of this study was to investigate the respective contributions of TGFβ and BMP‐2 to OA.

Methods

Mouse knee joints injected with adenovirus (Ad‐TGFβ or Ad‐BMP‐2) were compared histologically with knee joints from murine models of OA (joints injected with collagenase and joints from STR/Ort mice with spontaneous OA). To further investigate the role of BMP during osteophyte formation, adenovirus Ad‐Gremlin was injected into knee joints that had previously been injected with Ad‐TGFβ or collagenase.

Results

BMP‐2 induced early osteophytes, which bulged from the growth plates on the femur and grew on top of the patella, whereas TGFβ induced early osteophyte formation on the bone shaft beneath the collateral ligament on the femur as well as on top of the patella. The pattern of osteophyte formation during experimental OA closely resembled that of TGFβ‐induced osteophyte formation, but differed from the pattern induced by BMP‐2. Ad‐Gremlin proved to be able to totally block BMP‐2–induced osteophyte formation. However, blocking BMP activity inhibited neither TGFβ‐induced nor experimental OA–associated osteophyte formation.

Conclusion

Our findings demonstrate that the role of BMP during the onset of TGFβ‐induced and experimental OA–induced osteophyte formation is limited. The latter finding does not rule out a role of BMP during osteophyte maturation.

     

Repair of articular cartilage defect by autologous transplantation of basic fibroblast growth factor gene–transduced chondrocytes with adeno‐associated virus vector

Objective

To examine the effects of basic fibroblast growth factor (bFGF) gene–transduced chondrocytes on the repair of articular cartilage defects.

Methods

LacZ gene or bFGF gene was transduced into primary isolated rabbit chondrocytes with the use of a recombinant adeno‐associated virus (AAV) vector. These gene‐transduced chondrocytes were embedded in collagen gel and transplanted into a full‐thickness defect in the articular cartilage of the patellar groove of a rabbit. The efficiency of gene transduction was assessed according to the percentage of LacZ‐positive cells among the total number of living cells. The concentration of bFGF in the culture supernatant was measured by enzyme‐linked immunosorbent assay to confirm the production by bFGF gene–transduced chondrocytes. At 4, 8, and 12 weeks after transplantation, cartilage repair was evaluated histologically and graded semiquantitatively using a histologic scoring system ranging from 0 (complete regeneration) to 14 (no regeneration) points.

Results

LacZ gene expression by chondrocytes was maintained until 8 weeks in >85% of the in vitro population. LacZ‐positive cells were found at the transplant sites for at least 4 weeks after surgery. The mean concentration of bFGF was significantly increased in bFGF gene–transduced cells compared with control cells (P < 0.01). Semiquantitative histologic scoring indicated that the total score was significantly lower in the bFGF‐transduced group than in the control group throughout the observation period.

Conclusion

These results demonstrated that gene transfer to chondrocytes by an ex vivo method was established with the AAV vector, and transplantation of bFGF gene–transduced chondrocytes had a clear beneficial effect on the repair of rabbit articular cartilage defects.

     

Matrix‐assisted laser desorption ionization–imaging mass spectrometry: A new methodology to study human osteoarthritic cartilage

Objective

Information about the distribution of proteins and the modulation that they undergo in the different phases of rheumatic pathologies is essential to understanding the development of these diseases. We undertook this study to demonstrate the utility of mass spectrometry (MS)–based molecular imaging for studying the spatial distribution of different components in human articular cartilage sections.

Methods

We compared the distribution of peptides and proteins in human control and osteoarthritic (OA) cartilage. Human control and OA cartilage slices were cut and deposited on conductive slides. After tryptic digestion, we performed matrix‐assisted laser desorption ionization–imaging MS (MALDI‐IMS) experiments in a MALDI–quadrupole time‐of‐flight mass spectrometer. Protein identification was undertaken with a combination of multivariate statistical methods and Mascot protein database queries. Hematoxylin and eosin staining and immunohistochemistry were performed to validate the results.

Results

We created maps of peptide distributions at 150‐μm raster size from control and OA human cartilage. Proteins such as biglycan, prolargin, decorin, and aggrecan core protein were identified and localized. Specific protein markers for cartilage oligomeric matrix protein and fibronectin were found exclusively in OA cartilage samples. Their distribution displayed a stronger intensity in the deep area than in the superficial area. New tentative OA markers were found in the deep area of the OA cartilage.

Conclusion

MALDI‐IMS identifies and localizes disease‐specific peptides and proteins in cartilage. All the OA‐related peptides and proteins detected display a stronger intensity in the deep cartilage. MS‐based molecular imaging is demonstrated to be an innovative method for studying OA pathology.

     

Role of interleukin‐8 in PiT‐1 expression and CXCR1‐mediated inorganic phosphate uptake in chondrocytes

Objective

The proinflammatory chemokine interleukin‐8 (IL‐8) induces chondrocyte hypertrophy. Moreover, chondrocyte hypertrophy develops in situ in osteoarthritic (OA) articular cartilage and promotes dysregulated matrix repair and calcification. Growth plate chondrocyte hypertrophy is associated with expression of the type III sodium‐dependent inorganic phosphate (Pi) cotransporter phosphate transporter/retrovirus receptor 1 (PiT‐1). This study was undertaken to test the hypothesis that IL‐8 promotes chondrocyte hypertrophy by modulating chondrocyte PiT‐1 expression and sodium‐dependent Pi uptake, and to assess differential roles in this activity.

Methods

The selective IL‐8 receptor CXCR1 and the promiscuous chemokine receptor CXCR2 were used. Human knee OA cartilage, cultured normal bovine knee chondrocytes, and immortalized human articular chondrocytic CH‐8 cells were transfected with CXCR1/CXCR2 chimeric receptors in which the 40–amino acid C‐terminal cytosolic tail domains were swapped and site mutants of a CXCR1‐specific region were generated.

Results

Up‐regulated PiT‐1 expression was detected in OA cartilage. IL‐8, but not IL‐1 or the CXCR2 ligand growth‐related oncogene α, induced PiT‐1 expression and increased sodium‐dependent Pi uptake by >40% in chondrocytes. The sodium/phosphate cotransport inhibitor phosphonoformic acid blocked IL‐8–induced chondrocyte hypertrophic differentiation. Signaling mediated by kinase Pyk‐2 was essential for IL‐8 induction of PitT‐1 expression and Pi uptake. Signaling through the TSYT^346–349 region of the CXCR1 cytosolic tail, a region divergent from the CXCR2 cytosolic tail, was essential for IL‐8 to induce Pi uptake.

Conclusion

Our results link low‐grade IL‐8–mediated cartilaginous inflammation in OA to altered chondrocyte differentiation and disease progression through PiT‐1 expression and sodium‐dependent Pi uptake mediated by CXCR1 signaling.

     

Accelerated,aging‐dependent development of osteoarthritis in α1 integrin–deficient mice

Objective

Cell–matrix interactions regulate chondrocyte differentiation and survival. The α1β1 integrin is a major collagen receptor that is expressed on chondrocytes. Mice with targeted inactivation of the integrin α1 gene (α1‐KO mice) provide a model that can be used to address the role of cell–matrix interactions in cartilage homeostasis and osteoarthritis (OA) pathogenesis.

Methods

Knee joints from α1‐KO and wild‐type (WT) BALB/c mice were harvested at ages 4–15 months. Knee joint sections were examined for inflammation, cartilage degradation, and loss of glycosaminoglycans (by Safranin O staining). Immunohistochemistry was performed to detect the distribution of α1 integrin, matrix metalloproteinases (MMPs), and chondrocyte apoptosis.

Results

In WT mice, the α1 integrin subunit was detected in hypertrophic chondrocytes in the growth plate and in a subpopulation of cells in the deep zone of articular cartilage. There was a marked increase in α1‐positive chondrocytes in the superficial and upper mid‐zones in OA‐affected areas in joints from old WT mice. The α1‐KO mice showed more severe cartilage degradation, glycosaminoglycan depletion, and synovial hyperplasia as compared with the WT mice. MMP‐2 and MMP‐3 expression was increased in the OA‐affected areas. In cartilage from α1‐KO mice, the cellularity was reduced and the frequency of apoptotic cells was increased. These results suggest that the α1 integrin subunit is involved in the early remodeling process in OA cartilage.

Conclusion

Deficiency in the α1 integrin subunit is associated with an earlier deregulation of cartilage homeostasis and an accelerated, aging‐dependent development of OA.

     

Reduction of osteophyte formation and synovial thickening by adenoviral overexpression of transforming growth factor β/bone morphogenetic protein inhibitors during experimental osteoarthritis

Objective

Osteoarthritis (OA) is a joint disease characterized by osteophyte development, fibrosis, and articular cartilage damage. Effects of exogenous transforming growth factor β (TGFβ) isoforms and bone morphogenetic proteins (BMPs) suggest a role for these growth factors in the pathogenesis of OA. The aim of this study was to elucidate the role of endogenous TGFβ and BMP during papain‐induced OA‐like changes in mice.

Methods

We used adenoviral overexpression of TGFβ and BMP antagonists to block growth factor signaling. An adenovirus expressing a secreted, pan–specific TGFβ antagonist called murine latency‐associated peptide 1 (mLAP‐1) was used. In addition, we used intracellular inhibitory Smad6 as a BMP antagonist and Smad7 as a TGFβ/BMP inhibitor. Papain was injected into the knee joints of C57BL/6 mice to induce osteophyte development, synovial thickening, and articular cartilage proteoglycan (PG) loss.

Results

Intraarticular injection of papain caused increased protein expression of several TGFβ and BMP isoforms in synovium and cartilage. Adenovirus transfection into the joint resulted in a strong expression of the transgenes in the synovial lining. Overexpression of mLAP‐1, Smad6, and Smad7 led to a significant reduction in osteophyte formation compared with that in controls. Smad6 and Smad7 overexpression also significantly decreased synovial thickening. Furthermore, the secreted TGFβ inhibitor mLAP‐1 increased articular cartilage PG loss.

Conclusion

Our results indicate a pivotal role of endogenous TGFβ in the development of osteophytes and synovial thickening, implicating endogenous TGFβ in the pathogenesis of OA. In contrast, the prevention of cartilage damage by endogenous TGFβ signifies the protective role of TGFβ in articular cartilage. This is the first study to demonstrate that endogenous BMPs are involved in osteophyte formation and synovial thickening in experimental OA.

     

Bone morphogenetic protein and transforming growth factor β inhibitory Smads 6 and 7 are expressed in human adult normal and osteoarthritic cartilage in vivo and are differentially regulated in vitro by interleukin‐1β

Objective

Bone morphogenetic protein (BMP) and transforming growth factor β (TGFβ) are potent anabolic factors in adult articular chondrocytes. In this study, we investigated whether intracellular inhibitors of BMP and TGFβ signaling, inhibitory Smad6 (I‐Smad6) and I‐Smad7, are expressed in articular chondrocytes in normal and osteoarthritic (OA) cartilage, and whether their expression shows a correlation with the anabolic activity of OA chondrocytes in vivo and after interleukin‐1β (IL‐1β) stimulation in vitro.

Methods

RNA isolated directly from normal and OA human knee cartilage as well as from cultured articular chondrocytes was analyzed by (quantitative) polymerase chain reaction technology. Immunolocalization of the I‐Smads was performed on tissue sections and compared with the anabolic cellular activity as documented by in situ hybridization experiments for aggrecan and type II collagen.

Results

Both Smad6 and Smad7 were expressed in all samples of normal and OA cartilage. Immunostaining (including confocal microscopy) confirmed the presence of Smad6 and Smad7 in the majority of normal and degenerated articular chondrocytes; localization was mostly cytoplasmic. No correlation between expression of the main anabolic genes and expression of the I‐Smads was found. In cultured articular chondrocytes, stimulation with IL‐1β showed up‐regulation of Smad7, whereas Smad6 was down‐regulated.

Conclusion

Both Smad6 and Smad7 are expressed in adult human articular chondrocytes. The primarily cytoplasmic localization suggests permanent activation of the I‐Smads in articular cartilage in vivo. No evidence was found that up‐regulation or down‐regulation of I‐Smads in OA cartilage correlates directly with the anabolic (or catabolic) activity of articular chondrocytes. The regulation in chondrocytes of Smad6 and Smad7 expression by IL‐1β suggests a potentially important role of IL‐1β signaling in chondrocytes, via indirect influencing of the BMP/TGFβ signaling cascade.

     

Quantitative imaging of murine osteoarthritic cartilage by phase‐contrast micro–computed tomography

Objective

The mouse is an optimal model organism in which gene–environment interactions can be used to study the pathogenesis of osteoarthritis (OA). The gold standard for arthritis research in mice is based on histopathology and immunohistochemistry, which are labor‐intensive, prone to sampling bias and technical variability, and limited in throughput. The aim of this study was to develop a new technique that assesses mouse cartilage by integrating quantitative volumetric imaging techniques.

Methods

A novel mouse model of OA was generated by cruciate ligament transection (CLT) and evaluated by histopathology and immunohistochemistry. Knee joint specimens were then imaged using a new technique that combines high‐resolution micro–computed tomography (micro‐CT) and phase‐contrast optics followed by quantitative analyses. A comparative analysis was also performed in a previously established mouse model of OA generated by destabilization of the medial meniscus (DMM).

Results

Phase‐contrast micro‐CT achieved cellular resolution of chondrocytes and quantitative assessment of parameters such as articular cartilage volume and surface area. In mouse models of OA generated by either CLT or DMM, we showed that phase‐contrast micro‐CT distinguished control and OA cartilage by providing quantitative measures with high reproducibility and minimal variability. Features of OA at the cellular or tissue level could also be observed in images generated by phase‐contrast micro‐CT.

Conclusion

We established an imaging technology that comprehensively assessed and quantified the 2‐dimensional and 3‐dimensional changes of articular cartilage. Application of this technology will facilitate the rapid and high‐throughput assessment of genetic and therapeutic models of OA in mice.

     

One of two chondrocyte‐expressed isoforms of cartilage intermediate‐layer protein functions as an insulin‐like growth factor 1 antagonist

Objective

Aging and osteoarthritic (OA) cartilage commonly demonstrate enhanced expression of the large, transforming growth factor β (TGFβ)–inducible glycoprotein cartilage intermediate‐layer protein (CILP) as well as enhanced extracellular inorganic pyrophosphate (PPi) that promotes the deposition of calcium pyrophosphate dihydrate crystals. In normal chondrocytes, TGFβ induces elevated chondrocyte extracellular PPi. Insulin‐like growth factor 1 (IGF‐1) normally blocks this response and reduces extracellular PPi. However, chondrocyte resistance to IGF‐1 is observed in OA and aging. Because CILP was reported to chromatographically fractionate with PPi‐generating nucleotide pyrophosphatase phosphodiesterase (NPP) activity, it has been broadly assumed that CILP itself has NPP activity. Our objective was to directly define CILP functions and their relationship to IGF‐1 in chondrocytes.

Methods

Using primary cultures of articular chondrocytes from the knee, we defined the function of the previously described CILP (CILP‐1) and of a recently described 50.6% identical protein that we designated the CILP‐2 isoform.

Results

Both CILP isoforms were constitutively expressed by primary cultured articular chondrocytes, but only CILP‐1 expression was detectable in cultured knee meniscal cartilage cells. Neither CILP isoform had intrinsic NPP activity. But CILP‐1 blocked the ability of IGF‐1 to decrease extracellular PPi, an activity specific for the CILP‐1 N‐terminal domain. The CILP‐1 N‐terminal domain also suppressed IGF‐1–induced (but not TGFβ‐induced) proliferation and sulfated proteoglycan synthesis, and it inhibited ligand‐induced IGF‐1 receptor autophosphorylation.

Conclusion

Two CILP isoforms are differentially expressed by chondrocytes. Neither CILP isoform exhibits PPi‐generating NPP activity. But, increased expression of CILP‐1, via N‐terminal domain–mediated inhibitory effects of CILP‐1 on chondrocyte IGF‐1 responsiveness, could impair chondrocyte growth and matrix repair and indirectly promote PPi supersaturation in aging and OA cartilage.

     

Tetracycline‐regulated bone morphogenetic protein 2 gene expression in lentivirally transduced primary rabbit chondrocytes for treatment of cartilage defects

Objective

Treatment of cartilage defects is still challenging, primarily because of the poor self‐healing capacity of articular cartilage. Gene therapy approaches have gained considerable attention, but, depending on the vector system used, they can lead to either limited or unrestrained gene expression, and therefore regulation of gene expression is necessary. This study was undertaken to construct an efficient tetracycline (Tet)–regulated, lentivirally mediated system for the expression of growth factor bone morphogenetic protein 2 (BMP‐2) in primary rabbit chondrocytes that will allow for the induction and termination of growth factor gene expression once cartilage regeneration is complete.

Methods

Chondrogenic ATDC5 cells and primary rabbit chondrocytes were lentivirally transduced with different tetracycline‐on (Tet‐On)–regulated, self‐inactivating vectors for the induction of expression of enhanced green fluorescent protein (eGFP) or BMP‐2, using either a 1‐vector system or a 2‐vector system.

Results

Expression of eGFP was induced on ATDC5 cells and chondrocytes. The highest induction rate and highest level of gene expression were reached when the spleen focus‐forming virus long terminal repeat promoter was used to drive the reverse transactivator expression, after the addition of doxycycline, in chondrocytes. An up to 20‐fold induction of Tet‐mediated BMP‐2 expression was observed on ATDC5 cells. The extent of induction and expression level of BMP‐2 in chondrocytes were similar between the 1‐vector system– and 2‐vector system–infected cells (mean ± SD 15.5 ± 1.1 ng/ml and 14.6 ± 0.4 ng/ml, respectively). In addition, prolonged induction and switching‐off of BMP‐2 expression, as well as repeated induction, were demonstrated. Production of proteoglycans, as shown by Alcian blue staining, demonstrated the functionality of the lentivirally expressed BMP‐2 under induced conditions.

Conclusion

The lentivirally mediated Tet‐On system is an effective strategy for efficient, repeatedly inducible expression of BMP‐2 in primary rabbit chondrocytes. Therefore, use of this system in in vivo experiments may be a promising approach as a treatment strategy for cartilage defects.

     

Involvement of the Wnt signaling pathway in experimental and human osteoarthritis: Prominent role of Wnt‐induced signaling protein 1

Objective

Wnt signaling pathway proteins are involved in embryonic development of cartilage and bone, and, interestingly, developmental processes appear to be recapitulated in osteoarthritic (OA) cartilage. The present study was undertaken to characterize the expression pattern of Wnt and Fz genes during experimental OA and to determine the function of selected genes in experimental and human OA.

Methods

Longitudinal expression analysis was performed in 2 models of OA. Levels of messenger RNA for genes from the Wnt/β‐catenin pathway were determined in synovium and cartilage, and the results were validated using immunohistochemistry. Effects of selected genes were assessed in vitro using recombinant protein, and in vivo by adenoviral overexpression.

Results

Wnt‐induced signaling protein 1 (WISP‐1) expression was strongly increased in the synovium and cartilage of mice with experimental OA. Wnt‐16 and Wnt‐2B were also markedly up‐regulated during the course of disease. Interestingly, increased WISP‐1 expression was also found in human OA cartilage and synovium. Stimulation of macrophages and chondrocytes with recombinant WISP‐1 resulted in interleukin‐1–independent induction of several matrix metalloproteinases (MMPs) and aggrecanase. Adenoviral overexpression of WISP‐1 in murine knee joints induced MMP and aggrecanase expression and resulted in cartilage damage.

Conclusion

This study included a comprehensive characterization of Wnt and Frizzled gene expression in experimental and human OA articular joint tissue. The data demonstrate, for the first time, that WISP‐1 expression is a feature of experimental and human OA and that WISP‐1 regulates chondrocyte and macrophage MMP and aggrecanase expression and is capable of inducing articular cartilage damage in models of OA.

     

Osteoarthritic chondrocyte–secreted morphogens induce chondrogenic differentiation of human mesenchymal stem cells

Objective

The potential of stem cells to repair compromised cartilage tissue, such as in osteoarthritis (OA), depends strongly on how transplanted cells respond to factors secreted from the residing OA chondrocytes. This study was undertaken to determine the effect of morphogenetic signals from OA chondrocytes on chondrogenic differentiation of human mesenchymal stem cells (MSCs).

Methods

The effect of OA chondrocyte–secreted morphogens on chondrogenic differentiation of human MSCs was evaluated using a coculture system involving both primary and passaged OA chondrocytes. The findings were compared against findings for human MSCs cultured in OA chondrocyte–conditioned medium. Gene expression analysis, biochemical assays, and immunofluorescence staining were used to characterize the chondrogenic differentiation of human MSCs. Mass spectrometry analysis was used to identify the soluble factors. Numerical analysis was carried out to model the concentration profile of soluble factors within the human MSC–laden hydrogels.

Results

The human MSCs cocultured with primary OA chondrocytes underwent chondrogenic differentiation even in the absence of growth factors; however, the same effect could not be mimicked using OA chondrocyte–conditioned medium or expanded cells. Additionally, the cocultured environment down‐regulated hypertrophic differentiation of human MSCs. Mass spectrometry analysis demonstrated cell–cell communication and chondrocyte phenotype–dependent effects on cell‐secreted morphogens.

Conclusion

The experimental findings, along with the results of the numerical analysis, suggest a crucial role of soluble morphogens and their local concentrations in the differentiation pattern of human MSCs in a 3‐dimensional environment. The concept of using a small number of chondrocytes to promote chondrogenic differentiation of human MSCs while preventing their hypertrophic differentiation could be of great importance in formulating effective stem cell–based cartilage repair.

     

Expression of hypoxia‐inducible factor 1α by macrophages in the rheumatoid synovium: Implications for targeting of therapeutic genes to the inflamed joint

Objective

To determine if the rheumatoid synovium is a suitable target for hypoxia‐regulated gene therapy.

Methods

Sequential sections of wax‐embedded synovial membrane samples were obtained from 10 patients with rheumatoid arthritis (RA), 10 with primary osteoarthritis (OA), and from 6 healthy controls. Membrane sections from each patient were immunostained for hypoxia‐inducible factor 1α (HIF‐1α) and CD68 (a pan–macrophage marker).

Results

HIF‐1α was expressed abundantly by macrophages in most rheumatoid synovia, predominantly close to the intimal layer but also in the subintimal zone. There was markedly lower expression of HIF‐1α in OA synovia, and it was absent from all of the healthy synovia.

Conclusion

These observations indicate that macrophages transduced with a therapeutic gene under the control of a hypoxia‐inducible promoter could be administered to RA patients systemically. Migration of these cells to synovial tissue would result in the transgene being switched on in diseased joints but not in healthy tissues.