Eric J. Vanderploeg

Mechanical injury potentiates proteoglycan catabolism induced by interleukin‐6 with soluble interleukin‐6 receptor and tumor necrosis factor α in immature bovine and adult human articular cartilage

OBJECTIVE Traumatic joint injury can damage cartilage and release inflammatory cytokines from adjacent joint tissue. The present study was undertaken to study the combined effects of compression injury, tumor necrosis factor alpha (TNFalpha), and interleukin-6 (IL-6) and its soluble receptor (sIL-6R) on immature bovine and adult human knee and ankle cartilage, using an in vitro model, and to test the hypothesis that endogenous IL-6 plays a role in proteoglycan loss caused by a combination of injury and TNFalpha. METHODS Injured or uninjured cartilage disks were incubated with or without TNFalpha and/or IL-6/sIL-6R. Additional samples were preincubated with an IL-6-blocking antibody Fab fragment and subjected to injury and TNFalpha treatment. Treatment effects were assessed by histologic analysis, measurement of glycosaminoglycan (GAG) loss, Western blot to determine proteoglycan degradation, zymography, radiolabeling to determine chondrocyte biosynthesis, and Western blot and enzyme-linked immunosorbent assay to determine chondrocyte production of IL-6. RESULTS In bovine cartilage samples, injury combined with TNFalpha and IL-6/sIL-6R exposure caused the most severe GAG loss. Findings in human knee and ankle cartilage were strikingly similar to those in bovine samples, although in human ankle tissue, the GAG loss was less severe than that observed in human knee tissue. Without exogenous IL-6/sIL-6R, injury plus TNFalpha exposure up-regulated chondrocyte production of IL-6, but incubation with the IL-6-blocking Fab significantly reduced proteoglycan degradation. CONCLUSION Our findings indicate that mechanical injury potentiates the catabolic effects of TNFalpha and IL-6/sIL-6R in causing proteoglycan degradation in human and bovine cartilage. The temporal and spatial evolution of degradation suggests the importance of transport of biomolecules, which may be altered by overload injury. The catabolic effects of injury plus TNFalpha appeared partly due to endogenous IL-6, since GAG loss was partially abrogated by an IL-6-blocking Fab.

Effect of Self-assembling Peptide, Chondrogenic Factors, and Bone Marrow Derived Stromal Cells on Osteochondral Repair

OBJECTIVE The goal of this study was to test the ability of an injectable self-assembling peptide (KLD) hydrogel with or without chondrogenic factors (CF) and allogeneic bone marrow stromal cells (BMSCs) to stimulate cartilage regeneration in a full-thickness, critically-sized, rabbit cartilage defect model in vivo. We used CF treatments to test the hypotheses that CF would stimulate chondrogenesis and matrix production by cells migrating into acellular KLD (KLD+CF) or by BMSCs delivered in KLD (KLD+CF+BMSCs). DESIGN Three groups were tested against contralateral untreated controls: KLD, KLD+CF, and KLD+CF+BMSCs, n=6-7. Transforming growth factor-β1 (TGF-β1), dexamethasone, and insulin-like growth factor-1 (IGF-1) were used as CF pre-mixed with KLD and BMSCs before injection. Evaluations included gross, histological, immunohistochemical and radiographic analyses. RESULTS KLD without CF or BMSCs showed the greatest repair after 12 weeks with significantly higher Safranin-O, collagen II immunostaining, and cumulative histology scores than untreated contralateral controls. KLD+CF resulted in significantly higher aggrecan immunostaining than untreated contralateral controls. Including allogeneic BMSCs+CF markedly reduced the quality of repair and increased osteophyte formation compared to KLD-alone. CONCLUSIONS These data show that KLD can fill full-thickness osteochondral defects in situ and improve cartilage repair as shown by Safranin-O, collagen II immunostaining, and cumulative histology. In this small animal model, the full-thickness critically-sized defect provided access to the marrow, similar in concept to abrasion arthroplasty or spongialization in large animal models, and suggests that combining KLD with these techniques may improve current practice.

Articular Chondrocytes Derived from Distinct Tissue Zones Differentially Respond to In Vitro Oscillatory Tensile Loading

OBJECTIVE The cell morphology, gene expression, and matrix synthesis of articular chondrocytes are known to vary with depth from the tissue surface. The objective of this study was to investigate if chondrocytes from different zones respond to in vitro oscillatory tensile loading in distinct ways and whether tensile strain, which is most prevalent near the articular surface, would preferentially stimulate superficial zone chondrocytes. DESIGN Chondrocytes were separately isolated from the superficial, middle, and deep zones of articular cartilage and seeded into three-dimensional fibrin hydrogel constructs. An intermittent protocol of oscillatory tensile loading was applied for 3 days, and the effects on extracellular matrix (ECM) synthesis were assessed by measuring the incorporation of radiolabed precursors, size exclusion gel chromatography, and western blotting. RESULTS Tensile loading was found to be a potent stimulus for proteoglycan synthesis only in superficial zone chondrocytes. Although overall biosynthesis rates by deep zone chondrocytes were unaffected by tensile loading, the molecular characteristics of proteins and proteoglycans released to the culture medium were significantly altered so as to resemble those of superficial zone chondrocytes. CONCLUSIONS Oscillatory tensile loading differentially affected subpopulations of articular chondrocytes in three-dimensional fibrin hydrogel constructs. Cells isolated from deeper regions of the tissue developed some characteristics of superficial zone chondrocytes after exposure to tensile loading, which may indicate an adaptive response to the new mechanical environment. Understanding how exogenous mechanical stimuli can differentially influence chondrocytes from distinct tissue zones will yield important insights into mechanobiological processes involved in cartilage tissue development, maintenance, disease, and repair.

Adult equine bone marrow stromal cells produce a cartilage-like ECM mechanically superior to animal-matched adult chondrocytes.

Our objective was to evaluate the age-dependent mechanical phenotype of bone marrow stromal cell- (BMSC-) and chondrocyte-produced cartilage-like neo-tissue and to elucidate the matrix-associated mechanisms which generate this phenotype. Cells from both immature (2-4 month-old foals) and skeletally-mature (2-5 year-old adults) mixed-breed horses were isolated from animal-matched bone marrow and cartilage tissue, encapsulated in self-assembling-peptide hydrogels, and cultured with and without TGF-beta1 supplementation. BMSCs and chondrocytes from both donor ages were encapsulated with high viability. BMSCs from both ages produced neo-tissue with higher mechanical stiffness than that produced by either young or adult chondrocytes. Young, but not adult, chondrocytes proliferated in response to TGF-beta1 while BMSCs from both age groups proliferated with TGF-beta1. Young chondrocytes stimulated by TGF-beta1 accumulated ECM with 10-fold higher sulfated-glycosaminoglycan content than adult chondrocytes and 2-3-fold higher than BMSCs of either age. The opposite trend was observed for hydroxyproline content, with BMSCs accumulating 2-3-fold more than chondrocytes, independent of age. Size-exclusion chromatography of extracted proteoglycans showed that an aggrecan-like peak was the predominant sulfated proteoglycan for all cell types. Direct measurement of aggrecan core protein length and chondroitin sulfate chain length by single molecule atomic force microscopy imaging revealed that, independent of age, BMSCs produced longer core protein and longer chondroitin sulfate chains, and fewer short core protein molecules than chondrocytes, suggesting that the BMSC-produced aggrecan has a phenotype more characteristic of young tissue than chondrocyte-produced aggrecan. Aggrecan ultrastructure, ECM composition, and cellular proliferation combine to suggest a mechanism by which BMSCs produce a superior cartilage-like neo-tissue than either young or adult chondrocytes.

Sustained delivery of bioactive TGF-β1 from self-assembling peptide hydrogels induces chondrogenesis of encapsulated bone marrow stromal cells

Tissue engineering strategies for cartilage defect repair require technology for local targeted delivery of chondrogenic and anti-inflammatory factors. The objective of this study was to determine the release kinetics of transforming growth factor β1 (TGF-β1) from self-assembling peptide hydrogels, a candidate scaffold for cell transplant therapies, and stimulate chondrogenesis of encapsulated young equine bone marrow stromal cells (BMSCs). Although both peptide and agarose hydrogels retained TGF-β1, fivefold higher retention was found in peptide. Excess unlabeled TGF-β1 minimally displaced retained radiolabeled TGF-β1, demonstrating biologically relevant loading capacity for peptide hydrogels. The initial release from acellular peptide hydrogels was nearly threefold lower than agarose hydrogels, at 18% of loaded TGF-β1 through 3 days as compared to 48% for agarose. At day 21, cumulative release of TGF-β1 was 32-44% from acellular peptide hydrogels, but was 62% from peptide hydrogels with encapsulated BMSCs, likely due to cell-mediated TGF-β1 degradation and release of small labeled species. TGF-β1 loaded peptide hydrogels stimulated chondrogenesis of young equine BMSCs, a relevant preclinical model for treating injuries in young human cohorts. Self-assembling peptide hydrogels can be used to deliver chondrogenic factors to encapsulated cells making them a promising technology for in vivo, cell-based regenerative medicine.

Regional variations in the distribution and colocalization of extracellular matrix proteins in the juvenile bovine meniscus

A deeper understanding of the composition and organization of extracellular matrix molecules in native, healthy meniscus tissue is required to fully appreciate the degeneration that occurs in joint disease and the intricate environment in which an engineered meniscal graft would need to function. In this study, regional variations in the tissue‐level and pericellular distributions of collagen types I, II and VI and the proteoglycans aggrecan, biglycan and decorin were examined in the juvenile bovine meniscus. The collagen networks were extensively, but not completely, colocalized, with tissue‐level organization that varied with radial position across the meniscus. Type VI collagen exhibited close association with large bundles composed of type I and II collagen and, in contrast to type I and II collagen, was further concentrated in the pericellular matrix. Aggrecan was detected throughout the inner region of the meniscus but was restricted to the pericellular matrix and sheaths of collagen bundles in the middle and outer regions. The small proteoglycans biglycan and decorin exhibited regional variations in staining intensity but were consistently localized in the intra‐ and/or peri‐cellular compartments. These results provide insight into the complex hierarchy of extracellular matrix organization in the meniscus and provide a framework for better understanding meniscal degeneration and disease progression and evaluating potential repair and regeneration strategies.

Aggrecanolysis and in vitro matrix degradation in the immature bovine meniscus: mechanisms and functional implications

IntroductionLittle is known about endogenous or cytokine-stimulated aggrecan catabolism in the meniscal fibrocartilage of the knee. The objectives of this study were to characterize the structure, distribution, and processing of aggrecan in menisci from immature bovines, and to identify mechanisms of extracellular matrix degradation that lead to changes in the mechanical properties of meniscal fibrocartilage.MethodsAggrecanase activity in the native immature bovine meniscus was examined by immunolocalization of the aggrecan NITEGE neoepitope. To investigate mechanisms of cytokine-induced aggrecan catabolism in this tissue, explants were treated with interleukin-1α (IL-1) in the absence or presence of selective or broad spectrum metalloproteinase inhibitors. The sulfated glycosaminoglycan (sGAG) and collagen contents of explants and culture media were quantified by biochemical methods, and aggrecan catabolism was examined by Western analysis of aggrecan fragments. The mechanical properties of explants were determined by dynamic compression and shear tests.ResultsThe aggrecanase-generated NITEGE neoepitope was preferentially localized in the middle and outer regions of freshly isolated immature bovine menisci, where sGAG density was lowest and blood vessels were present. In vitro treatment of explants with IL-1 triggered the accumulation of NITEGE in the inner and middle regions. Middle region explants stimulated with IL-1 exhibited substantial decreases in sGAG content, collagen content, and mechanical properties. A broad spectrum metalloproteinase inhibitor significantly reduced sGAG loss, abrogated collagen degradation, and preserved tissue mechanical properties. In contrast, an inhibitor selective for ADAMTS-4 and ADAMTS-5 was least effective at blocking IL-1-induced matrix catabolism and loss of mechanical properties.ConclusionsAggrecanase-mediated aggrecanolysis, typical of degenerative articular cartilage, may play a physiologic role in the development of the immature bovine meniscus. IL-1-induced release of sGAG and loss of mechanical properties can be ascribed primarily to the activity of MMPs or aggrecanases other than ADAMTS-4 and ADAMTS-5. These results may have implications for the clinical management of osteoarthritis.

Mechanical injury of explants from the articulating surface of the inner meniscus.

Knee osteoarthritis is accelerated by damage to the meniscus, a fibrocartilage tissue that assists in load transmission. However, little is known about the mechanical or cellular response of the meniscus to injurious overloading. Here, in vitro studies explored injury to meniscal explants using a compressive overloading protocol that has been well characterized for articular cartilage. Cartilage samples were processed in parallel as a reference to the extensive literature on cartilage injury. Injured meniscal explants showed extensive cell death at the articulating surface but no gross tissue damage, while similar conditions of peak stress and strain resulted in cartilage surface fissures and cell death consistent with moderate overloading. Post-injury gene expression in meniscal explants indicated a decrease in seven of the nine catabolic and pro-inflammatory molecules surveyed, while cartilage experienced a downregulation in ADAMTS-5 and TNF-alpha only. These data demonstrated a resiliency of the meniscus to injury, and that an acute increase in catabolic activities is not necessarily a consequence of mechanical overloading.

479 SELF-ASSEMBLING PEPTIDE HEALS RABBIT DEFECTS IN VIVO

479 – Figure 1. In vitro DNA and ECM Content and Biosynthesis. Figure 2. Representative Safranin-O staining for in vivo defect repair. KLD only treated had significantly higher staining than the bilateral empty control. full-thickness defect was created in the femoropatellar groove of each knee. GF and cells were pre-mixed with KLD and directly injected into left-knee defects with right-knee defects left empty as controls. There were 6-7 rabbits used for each condition. ACUC approval was given by CSU. Repair tissue was analyzed by gross and histological scoring. Results: In vitro testing confirmed that including IGF-1, along with TGF-β1 and dexamethasone, improved chondrogenesis of BMSCs in KLD as shown by higher sGAG content and PG synthesis (Fig. 1). However, in the rabbit model, the KLD group without any growth factors or cells showed the greatest repair after 12 weeks. KLD had a significantly higher total histology score and more Safranin-O staining (Fig. 2, 3). Including BMSCs with GF resulted Osteoarthritis and Cartilage Vol. 17, Supplement 1 S257 Abstract 479 – Figure 3. Histology Scores for in vivo defect repair. *vs. untreated KLD, M (treated + untreated) vs. KLD + GF + MSCs (treated + untreated), U (treated)479 – Figure 3. Histology Scores for in vivo defect repair. *vs. untreated KLD, M (treated + untreated) vs. KLD + GF + MSCs (treated + untreated), U (treated)