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.

     

G1 domain of aggrecan cointernalizes with hyaluronan via a CD44‐mediated mechanism in bovine articular chondrocytes

Objective

To determine whether aggrecan fragments bound to hyaluronan (HA) can be retained and internalized by articular chondrocytes and whether these events are dependent on HA and its receptor, CD44. An additional objective was to determine whether partial degradation of aggrecan is a prerequisite for internalization.

Methods

Binding and internalization of a variety of fluorescein isothiocyanate (FITC)– or biotin‐labeled HA/proteoglycan probes were investigated on normal bovine articular cartilage chondrocytes, bovine articular chondrocytes transfected with a dominant‐negative construct of CD44, or COS‐7 cells transfected with wild‐type CD44. The probes were defined as being internalized by the presence of label associated with the cells following extensive trypsinization of the cell surface.

Results

Biotinylated aggrecan fragments bound to FITC‐HA were cointernalized in bovine articular chondrocytes or COS‐7 cells transfected with CD44. Intracellular vesicles containing FITC‐HA colocalized with a fluorescent probe for lysosomes. The internalization of the aggrecan fragments was dependent on the presence of HA as well as the presence of functional CD44. Intact aggrecan/FITC‐HA complexes bound to the cell surface but were not internalized. However, following brief trypsin digestion of the aggrecan/HA complex, the remaining proteoglycan fragments were bound and internalized.

Conclusion

Partially degraded aggrecan fragments (e.g., aggrecan G1 domains bound to HA) can be internalized by articular chondrocytes via a mechanism involving HA/CD44‐mediated endocytosis. Further, the presence of an intact aggrecan monomer bound to HA inhibits the internalization of HA as well as HA‐bound fragments.

     

Cell‐based resurfacing of large cartilage defects: Long‐term evaluation of grafts from autologous transgene‐activated periosteal cells in a porcine model of osteoarthritis

Objective

To investigate the potential of transgene‐activated periosteal cells for permanently resurfacing large partial‐thickness cartilage defects.

Methods

In miniature pigs, autologous periosteal cells stimulated ex vivo by bone morphogenetic protein 2 gene transfer, using liposomes or a combination of adeno‐associated virus (AAV) and adenovirus (Ad) vectors, were applied on a bioresorbable scaffold to chondral lesions comprising the entire medial half of the patella. The resulting repair tissue was assessed, 6 and 26 weeks after transplantation, by histochemical and immunohistochemical methods. The biomechanical properties of the repair tissue were characterized by nanoindentation measurements. Implants of unstimulated cells and untreated lesions served as controls.

Results

All grafts showed satisfactory integration into the preexisting cartilage. Six weeks after transplantation, AAV/Ad‐stimulated periosteal cells had adopted a chondrocyte‐like phenotype in all layers; the newly formed matrix was rich in proteoglycans and type II collagen, and its contact stiffness was close to that of healthy hyaline cartilage. Unstimulated periosteal cells and cells activated by liposomal gene transfer formed only fibrocartilaginous repair tissue with minor contact stiffness. However, within 6 months following transplantation, the AAV/Ad‐stimulated cells in the superficial zone tended to dedifferentiate, as indicated by a switch from type II to type I collagen synthesis and reduced contact stiffness. In deeper zones, these cells retained their chondrocytic phenotype, coinciding with positive staining for type II collagen in the matrix.

Conclusion

Large partial‐thickness cartilage defects can be resurfaced efficiently with hyaline‐like cartilage formed by transgene‐activated periosteal cells. The long‐term stability of the cartilage seems to depend on physicobiochemical factors that are active only in deeper zones of the cartilaginous tissue.

     

Molecular phenotyping of human chondrocyte cell lines T/C‐28a2, T/C‐28a4, and C‐28/I2

Objective

Because the immortalized chondrocyte cell lines C‐28/I2, T/C‐28a2, and T/C‐28a4 have become a common tool in cartilage research, permitting investigations in a largely unlimited and standardized manner, we investigated the molecular phenotype of these cell lines by gene expression profiling.

Methods

Complementary DNA–array analysis as well as online quantitative polymerase chain reaction were used to identify the gene expression profiles of the 3 cell lines cultured in monolayer and alginate beads, as compared with the expression profiles of cultured human adult primary chondrocytes.

Results

A similar, but not identical, gene expression profile was established for all 3 cell lines. SOX9 was expressed at a significant level in all 3 cell lines. Extracellular matrix proteins and matrix‐degrading proteases were rarely expressed. In contrast, genes involved in the cell cycle were strongly up‐regulated, as compared with the expression levels in physiologic chondrocytes.

Conclusion

The expression of SOX9, the master gene of chondrocytic cell differentiation, reflects the basically chondrocytic phenotype of these cells. However, the major issue appears to be that these cell lines mainly proliferate and show less expression of genes involved in matrix synthesis and turnover. In this respect, C‐28/I2 cells display the highest levels of matrix‐anabolic and matrix‐catabolic genes and thus are presumably preferable for use in investigating chondrocyte anabolic and catabolic activity and its regulation. None of the 3 cell lines appears to be a direct substitute for primary chondrocytes. A successful approach will have to validate the findings obtained with chondrocyte cell lines by using primary chondrocytes or cartilage‐tissue cultures. This would permit the establishment of reproducible in vitro models and subsequently allow investigators to relate the findings to the physiologic situation.

     

Insulin‐like growth factor 1–induced interleukin‐1 receptor II overrides the activity of interleukin‐1 and controls the homeostasis of the extracellular matrix of cartilage

Objective

We examined the effect of the insulin‐like growth factor 1 (IGF‐1)/IGF receptor I (IGFRI) autocrine/paracrine anabolic pathway on the extracellular matrix (ECM) of human chondrocytes and the mechanism by which IGF‐1 reverses the catabolic effects of interleukin‐1 (IL‐1).

Methods

Phenotypically stable human articular cartilage cells were obtained from normal cartilage and maintained in culture in alginate beads for 1 week to reach equilibrium of accumulated cell‐associated matrix (CAM) compounds. Levels of CAM components aggrecan and type II collagen (CII) and levels of intracellular IGF‐1, IL‐1α, and IL‐1β and their respective plasma membrane–bound receptors IGFRI, IL‐1 receptor I (IL‐1RI), and the decoy receptor IL‐1RII were assayed using flow cytometry to investigate the relationship between the autocrine/paracrine pathways and the homeostasis of ECM molecules in the CAM. The effects of IGF‐1 on the expression of IGF‐1, IL‐1α, and IL‐1β and their respective receptor systems, the aggrecan core protein, and CII were determined by flow cytometry.

Results

Cause–effect relationship experiments showed that IGF‐1 up‐regulates the levels of IGF‐1, IGFRI, aggrecan, and CII in the CAM. No effects on the expression of IL‐1α and IL‐1β and their signaling receptor IL‐1RI were observed. However, IGF‐1 was able to reverse IL‐1β–mediated degradation of aggrecan and the repression of the aggrecan synthesis rate. Interestingly, levels of aggrecan and CII in the CAM strongly correlated not only with IGF‐1, but also with IL‐1RII, which acts as a decoy receptor for IL‐1α and IL‐1β. This suggests that IGF‐1 and IL‐1RII may cooperate in regulating ECM homeostasis. Additional experiments demonstrated that IGF‐1 up‐regulated IL‐1RII, thereby overriding the catabolic effects of IL‐1.

Conclusion

These findings reveal a new paradigm by which IGF‐1 influences chondrocyte metabolism, by reversing the IL‐1–mediated catabolic pathway through up‐regulation of its decoy receptor.

     

Chondrocyte‐intrinsic Smad3 represses Runx2‐inducible matrix metalloproteinase 13 expression to maintain articular cartilage and prevent osteoarthritis

Objective

To identify mechanisms by which Smad3 maintains articular cartilage and prevents osteoarthritis.

Methods

A combination of in vivo and in vitro approaches was used to test the hypothesis that Smad3 represses Runx2‐inducible gene expression to prevent articular cartilage degeneration. Col2‐Cre;Smad3^fl/fl mice allowed study of the chondrocyte‐intrinsic role of Smad3 independently of its role in the perichondrium or other tissues. Primary articular cartilage chondrocytes from Smad3^fl/fl mice and ATDC5 chondroprogenitor cells were used to evaluate Smad3 and Runx2 regulation of matrix metalloproteinase 13 (MMP‐13) messenger RNA (mRNA) and protein expression.

Results

Chondrocyte‐specific reduction of Smad3 caused progressive articular cartilage degeneration due to imbalanced cartilage matrix synthesis and degradation. In addition to reduced type II collagen mRNA expression, articular cartilage from Col2‐Cre;Smad3^fl/fl mice was severely deficient in type II collagen and aggrecan protein due to excessive MMP‐13–mediated proteolysis of these key cartilage matrix constituents. Normally, transforming growth factor β (TGFβ) signals through Smad3 to confer a rapid and dynamic repression of Runx2‐inducible MMP‐13 expression. However, we found that in the absence of Smad3, TGFβ signals through p38 and Runx2 to induce MMP‐13 expression.

Conclusion

Our findings elucidate a mechanism by which Smad3 mutations in humans and mice cause cartilage degeneration and osteoarthritis. Specifically, Smad3 maintains the balance between cartilage matrix synthesis and degradation by inducing type II collagen expression and repressing Runx2‐inducible MMP‐13 expression. Selective activation of TGFβ signaling through Smad3, rather than p38, may help to restore the balance between matrix synthesis and proteolysis that is lost in osteoarthritis.

     

Formation of invadopodia‐like structures by synovial cells promotes cartilage breakdown in collagen‐induced arthritis: Involvement of the protein tyrosine kinase Src

Objective

Invasive synovial fibroblasts are suggested to be the major effectors of cartilage and bone destruction, and this aggressive phenotype can lead to irreversible damage. In cancer cells, invasion across tissue boundaries and metastasis have recently been shown to depend on the capacity of the cells to breach the basement membrane, a process that was linked to the formation of the actin‐rich cell protrusions called invadopodia. This study was undertaken to investigate whether arthritic synovial cells use invadopodia to invade and degrade cartilage components.

Methods

Fibroblast‐like synoviocytes (FLS) from control rats or rats with collagen‐induced arthritis (CIA) were cultured on fluorescent matrix in the presence of Src inhibitors or were transfected with wild‐type or variants of Src kinases. The in vivo effect of Src inhibition on cartilage degradation and invasion was studied in a rat model of CIA.

Results

FLS from rats with CIA produced more invadopodia‐like structures than did FLS from control rats, leading to increased extracellular matrix degradation. Furthermore, c‐Src activation was increased in synovial cells from rats with CIA, and Src activity was found to mediate the formation of invadopodia. Pharmacologic blockade of Src activity by PP2 or intraarticular expression of a c‐Src–specific short hairpin RNA in the CIA model reduced synovial membrane hyperplasia and cartilage degradation, an event linked to decreased invadopodia formation by synovial fibroblasts.

Conclusion

This study demonstrates that inhibition of invadopodia formation in arthritic synovial cells leads to a direct effect on extracellular matrix degradation in vitro and in vivo, making invadopodia a relevant therapeutic target for interfering with this process.

     

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.

     

Release of hyaluronan and hyaladherins (aggrecan G1 domain and link proteins) from articular cartilage exposed to ADAMTS‐4 (aggrecanase 1) or ADAMTS‐5 (aggrecanase 2)

Objective

To determine whether aggrecanase (ADAMTS) activities in articular cartilage can directly lead to the release of hyaluronan (HA) and hyaladherins (aggrecan G1 domain and link proteins), as may occur ex vivo during stimulation of cartilage explants with interleukin‐1 (IL‐1) or retinoic acid or in vivo in synovial joints during aging and joint pathology.

Methods

Bovine articular cartilage discs (live or freeze‐killed) were cultured in the presence of IL‐1 or were incubated in digestion buffer containing recombinant human ADAMTS‐4 (rHuADAMTS‐4; aggrecanase 1) or rHuADAMTS‐5 (aggrecanase 2). Culture media, digestion supernatants, and tissue extracts were assayed for sulfated glycosaminoglycan (sGAG) content and analyzed by Western blotting to detect aggrecanase‐generated G1 domain (using neoepitope monoclonal antibody AGG‐C1/anti‐NITEGE³⁷³) and link proteins (using monoclonal antibody 8‐A‐4), as well as by quantitative enzyme‐linked immunosorbent assays to detect aggrecanase‐generated G1 domain (G1‐NITEGE³⁷³) and HA.

Results

IL‐1 treatment of live cartilage explants induced a time‐dependent release of sGAG, aggrecanase‐generated G1 domain (G1‐NITEGE³⁷³), and HA into the culture media. Exposure of live or freeze‐killed articular cartilage discs to rHuADAMTS‐4 or rHuADAMTS‐5 resulted in a dose‐ and time‐dependent release of sGAG and hyaluronan from the tissue, accompanied by a concomitant release of functionally intact hyaladherins (aggrecan G1‐NITEGE³⁷³ and link proteins).

Conclusion

Coincident with aggrecanolysis, aggrecanase activities in articular cartilage may actuate the release of HA and associated hyaladherins, thereby further compromising the integrity of the cartilage matrix during degenerative joint diseases such as osteoarthritis.

     

Up‐regulated expression of the phosphodiesterase nucleotide pyrophosphatase family member PC‐1 is a marker and pathogenic factor for knee meniscal cartilage matrix calcification

Objective

Elevated cartilage inorganic pyrophosphate (PPi) production and PPi‐generating nucleoside triphosphate pyrophosphohydrolase (NTPPPH) activity are strongly linked with aging‐related cartilage calcification in meniscal and articular cartilages. We hypothesized that there were divergent relationships of 3 NTPPPH isozymes with cartilage matrix calcification and sought to identify them.

Methods

We studied knee medial meniscal expression in situ of 3 NTPPPH isozymes of the phosphodiesterase nucleotide pyrophosphatase (PDNP) family: plasma cell membrane glycoprotein 1 (PC‐1, or PDNP1), autotaxin (ATX, or PDNP2), and B10/PDNP3. We also used complementary DNA transfection to assess differential functions in matrix calcification of each NTPPPH isozyme in vitro in meniscal cells.

Results

We observed diffuse cell‐associated ATX and B10/PDNP3 expression in central (chondrocytic) and, to a lesser degree, peripheral (fibroblastic) regions of normal, degenerative uncalcified, and degenerative calcified menisci. In contrast, PC‐1 expression was only robust at sites of apoptotic cells and calcification in central regions of degenerative menisci. Only PC‐1 was abundant at the perimeter of meniscal cells and in association with meniscal cell–derived matrix vesicles (MVs). Because each PDNP‐family isozyme was expressed by cells near calcifications, we transfected the isozymes in nonadherent knee meniscal cells cultured with ascorbic acid, β‐glycerophosphate, and dexamethasone supplementation to stimulate them to calcify the matrix. PC‐1, but not ATX or B10/PDNP3, consistently promoted increased MV NTPPPH, MV‐associated PPi, and extracellular PPi. PC‐1 also increased matrix calcification (with hydroxyapatite crystals) by meniscal cells. ATX uniquely induced alkaline phosphatase activity, but promoted only moderately increased matrix calcification.

Conclusion

We identified divergent effects of 3 PDNP‐family NTPPPH isozymes on meniscal cell matrix calcification. Increased expression of PC‐1 is both a marker and a potential pathogenic factor for knee meniscal cartilage matrix calcification.

     

In vitro stage‐specific chondrogenesis of mesenchymal stem cells committed to chondrocytes

Objective

Osteoarthritis is characterized by an imbalance in cartilage homeostasis, which could potentially be corrected by mesenchymal stem cell (MSC)–based therapies. However, in vivo implantation of undifferentiated MSCs has led to unexpected results. This study was undertaken to establish a model for preconditioning of MSCs toward chondrogenesis as a more effective clinical tool for cartilage regeneration.

Methods

A coculture preconditioning system was used to improve the chondrogenic potential of human MSCs and to study the detailed stages of chondrogenesis of MSCs, using a human MSC line, Kp‐hMSC, in commitment cocultures with a human chondrocyte line, hPi (labeled with green fluorescent protein [GFP]). In addition, committed MSCs were seeded into a collagen scaffold and analyzed for their neocartilage‐forming ability.

Results

Coculture of hPi‐GFP chondrocytes with Kp‐hMSCs induced chondrogenesis, as indicated by the increased expression of chondrogenic genes and accumulation of chondrogenic matrix, but with no effect on osteogenic markers. The chondrogenic process of committed MSCs was initiated with highly activated chondrogenic adhesion molecules and stimulated cartilage developmental growth factors, including members of the transforming growth factor β superfamily and their downstream regulators, the Smads, as well as endothelial growth factor, fibroblast growth factor, insulin‐like growth factor, and vascular endothelial growth factor. Furthermore, committed Kp‐hMSCs acquired neocartilage‐forming potential within the collagen scaffold.

Conclusion

These findings help define the molecular markers of chondrogenesis and more accurately delineate the stages of chondrogenesis during chondrocytic differentiation of human MSCs. The results indicate that human MSCs committed to the chondroprogenitor stage of chondrocytic differentiation undergo detailed chondrogenic changes. This model of in vitro chondrogenesis of human MSCs represents an advance in cell‐based transplantation for future clinical use.

     

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.

     

Zone‐specific gene expression patterns in articular cartilage

Objective

To identify novel genes and pathways specific to the superficial zone (SZ), middle zone (MZ), and deep zone (DZ) of normal articular cartilage.

Methods

Articular cartilage was obtained from the knees of 4 normal human donors. The cartilage zones were dissected on a microtome. RNA was analyzed on human genome arrays. The zone‐specific DNA array data obtained from human tissue were compared to array data obtained from bovine cartilage. Genes differentially expressed between zones were evaluated using direct annotation for structural or functional features, and by enrichment analysis for integrated pathways or functions.

Results

The greatest differences in genome‐wide RNA expression data were between the SZ and DZ in both human and bovine cartilage. The MZ, being a transitional zone between the SZ and DZ, thereby shared some of the same pathways as well as structural/functional features of the adjacent zones. Cellular functions and biologic processes that were enriched in the SZ relative to the DZ included, most prominently, extracellular matrix–receptor interactions, cell adhesion molecule functions, regulation of actin cytoskeleton, ribosome‐related functions, and signaling aspects such as the IFN, IL4, Cdc42/Rac, and JAK/STAT signaling pathways. Two pathways were enriched in the DZ relative to the SZ, including PPARG and EGFR/SMRTE.

Conclusion

These differences in cartilage zonal gene expression identify new markers and pathways that govern the unique differentiation status of chondrocyte subpopulations.