A Novel Approach to Stimulate Cartilage Repair: Targeting Collagen Turnover

OA is a complex disease of which the ethiopathology is not completely known and therapies to repair cartilage are still under investigation. The increase of collagen type II expression in osteoarthritic cartilage suggests an activated repair mechanism that is however ineffective in repairing or maintaining the ECM homeostasis. We therefore investigated the ability to modulate the formation of a functional collagen type II network that can ultimately contribute to innovation of cartilage repair in OA. To do so we used different approaches: addition of growth factors, inhibition of collagen cross-links, inhibition of proteoglycan formation, overexpression of cartilage oligomeric matrix protein (COMP) and knock-down of COMP and collagen IX, Of the growth factors used in this thesis, IGF1 had positive effects on the parameters in our chondrocyte alginate cultures. It stimulated chondrocytes to deposit more collagen and proteoglycans without affecting collagen cross-linking, it increased mechanical functioning and decreased MMP-1 gene expression. FGF2 on the other hand had no positive effects in our culture system: It lowered the collagen deposition and did not affect the proteoglycan deposition. The effect TGFb had on chondrocytes in culture was more complex. Addition of TGFb to chondrocytes in alginate had no large effect on proteoglycan and collagen deposition but did lower the number of cross-links per collagen molecule and reduced the fibril thickness. The effect of TGF-beta appeared to depend on the physical environment of the cell as concluded from our comparison between chondrocytes cultured in monolayer on plastic or in alginate beads. We inhibited crosslink formation by addition of beta-aminopropionitrile (BAPN), which inhibits one of the key enzymes for crosslink formation, lysyloxidase, via covalent binding. As a result, the chondrocyte produced more collagen (as shown by increased collagen type II gene expression) and also more collagen was deposited. Furthermore we were also interested in the effect of other extracellular matrix components on collagen network formation. Addition of FGF2 decreased and addition of TGF-beta increased COMP deposition. We overexpressed COMP in chondrocytes and found no effect on matrix deposition. However, COMP overexpression also resulted in collagen fibrils with a smaller diameter, like we saw after addition of TGFb. By inhibiting the formation of a proteoglycan network using para-nitrophenyl-beta-D-xyloside (PNPX) and therefore proteoglycan deposition, collagen deposition was decreased. The absence of an intact proteoglycan network lowered the collagen retention in our culture system. Cultures of collagen type IX deficient chondrocytes also had altered collagen retention. Since collagen type IX forms a network together with collagen type II and other matrix molecules, the absence of this network probably also reduced collagen retention in the alginate bead. However, GAG distribution was also altered when collagen type IX was absent in the newly formed matrix. Which in turn could also have contributed to altered collagen retention. Interestingly, the absence of COMP in addition to collagen type IX deficiency did not alter matrix production and distribution. This leads to the conclusion that collagen type IX is more important in the matrix interaction than COMP. Taken together, it seems that for the production of collagen in cartilage, focussing on the formation of an intact cartilage network is as important as stimulating collagen type II production. This network formation is important to retain the collagen type II in the newly formed matrix. Growth factors can influence collagen network formation, either directly or via their effect on other extracellular matrix components

Proteoglycan production is required in initial stages of new cartilage matrix formation but inhibits integrative cartilage repair

The optimal stimulus to repair or regenerate cartilage is not known. We therefore modulated collagen deposition, collagen crosslinking and GAG deposition simultaneously during cartilage matrix production and integrative repair, creating more insight into their role in cartilage repair processes. Insulin-like growth factor 1 (IGF-1; increases proteoglycan and collagen synthesis), beta-aminopropionitrile (BAPN; a reversible inhibitor of collagen crosslinking) and para-nitrophenyl-beta-D-xyloside (PNPX; interferes with proteoglycan production) were used. Bovine articular chondrocytes were cultured in alginate beads for 3 weeks with or without IGF-1, BAPN or PNPX alone and in all possible combinations, followed by 3 weeks in control medium. DNA content, GAG and collagen deposition and collagen crosslinks were determined. Cartilage constructs were cultured under the same conditions and histologically analysed for integration of two opposing cartilage matrices. In alginate cultures, inhibition of collagen crosslinking with BAPN, in combination with promotion of matrix synthesis using IGF1, was most beneficial for matrix deposition. Addition of PNPX was always detrimental for matrix deposition. For integration of opposing cartilage constructs, the combination of BAPN, IGF1 and temporary prevention of proteoglycan formation with PNPX was most beneficial. When a new matrix is produced, proteoglycans are important to retain collagen in the matrix. When two already formed cartilage matrices have to integrate, a temporary absence of proteoglycans and temporary inhibition of collagen crosslinking might be more beneficial in combination with stimulation of collagen production, e.g. by IGF1. Therefore, the choice of soluble factors to promote cartilage regeneration depends on the type of therapy that will be used

Cartilage inflammation and degeneration is enhanced by pro-inflammatory (M1) macrophages in vitro, but not inhibited directly by anti-inflammatory (M2) macrophages

Objective Macrophages play a crucial role in the progression of osteoarthritis (OA). Their phenotype may range from pro-inflammatory to anti-inflammatory. The aim of this study was to evaluate the direct effects of macrophage subtypes on cartilage by culturing macrophage conditioned medium (MCM) on human articular cartilage. Design Human OA cartilage explants were cultured with MCM of pro-inflammatory M(IFNγ+TNFα), or anti-inflammatory M(IL-4) or M(IL-10) human monocyte-derived macrophages. To assess effects of anti-inflammatory macrophages, the cartilage was cultured with a combination of MCM phenotypes as well as pre-stimulated with IFNγ+TNFα cartilage before culture with MCM. The reactions of the explants were assessed by gene expression, nitric oxide (NO) production and release of glycosaminoglycans (GAGs). Results M(IFNγ+TNFα) MCM affected OA cartilage by upregulation of IL1B (Interleukin 1β), IL6, MMP13 (Matrix Metalloproteinase-13) and ADAMTS5 (A Disintegrin And Metalloproteinase with Thrombospondin Motifs-5), while inhib

Improved Understanding of the Inflammatory Response in Synovial Fluid and Serum after Traumatic Knee Injury, Excluding Fractures of the Knee:A Systematic Review

Background: Traumatic knee injury results in a 4- to 10-fold increased risk of post-traumatic osteoarthritis (PTOA). Currently, there are no successful interventions for preventing PTOA after knee injury. The aim of this study is to identify inflammatory proteins that are increased in serum and synovial fluid after acute knee injury, excluding intra-articular fractures. Methods: A literature search was done according to the PRISMA guidelines. Articles reporting about inflammatory proteins after knee injury, except fractures, up to December 8, 2021 were collected. Inclusion criteria were as follows: patients younger than 45 years, no radiographic signs of knee osteoarthritis at baseline, and inflammatory protein measurement within 1 year after trauma. Risk of bias was assessed of the included studies. The level of evidence was determined by the Strength of Recommendation Taxonomy. Results: Ten studies were included. All included studies used a healthy control group or the contralateral knee as healthy control. Strong evidence for interleukin 6 (IL-6) and limited evidence for CCL4 show elevated concentrations of these proteins in synovial fluid (SF) after acute knee injury; no upregulation in SF for IL-2, IL-10, CCL3, CCL5, CCL11, granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF) was found. Limited evidence was found for no difference in serum concentration of IL-1β, IL-6, IL-10, CCL2, and tumor necrosis factor alpha (TNF-α) after knee injury. Conclusion: Interleukin 6 and CCL4 are elevated in SF after acute knee injury. Included studies failed to demonstrate increased concentration of inflammatory proteins in SF samples taken 6 weeks after trauma. Future research should focus on SF inflammatory protein measurements taken less than 6 weeks after injury.</p

Guiding synovial inflammation by macrophage phenotype modulation: An in vitro study towards a therapy for osteoarthritis

Objective: The aims of this study were to modulate inflammation in synovial explants with the compounds: dexamethasone, rapamycin, bone morphogenetic protein 7 (BMP-7) and pravastatin, and to investigate the mod

The Effect of Biomaterials Used for Tissue Regeneration Purposes on Polarization of Macrophages

Activation of macrophages is critical in the acute phase of wound healing after implantation of surgical biomaterials. To understand the response of macrophages, they are often cultured in vitro on biomaterials. Since a wide range of biomaterials is currently used in the clinics, we undertook a systematic review of the macrophage polarization in response to these different surgical biomaterials in vitro. Beside the chemistry, material characteristics such as dimension, pore size, and surface topography are of great influence on the response of macrophages. The macrophage response also appears to depend on the differences in sterilization techniques that induce lasting biochemical changes or residues of chemicals and their byproducts used for sterilization. Regarding tissue-based biomaterials, macrophages on human or porcine dermis, strongly cross-linked by chemicals elicit in general a proinflammatory response with higher amounts of proinflammatory cytokines. Synthetic biomaterials such as polyethylene, polyethylene terephthalate (PET) + polyacrylamide (PAAm), PET + sodium salt of poly(acrylic acid) (PAANa), perfluoropolyether (PFPE) with large posts, PEG-g-PA, and polydioxanone (PDO) always appear to elicit an anti-inflammatory response in macrophages, irrespective of origin of the macrophages, for example, buffy coats or full blood. In conclusion, in general in vitro models contribute to evaluate the foreign body reaction on surgical biomaterials. Although it is difficult to simulate complexity of host response elicited by biomaterials, after their surgical implantation, an in vitro model gives indications of the initial foreign body response and allows the comparison of this response between biomaterials

Effect of Cell Seeding Density and Inflammatory Cytokines on Adipose Tissue-Derived Stem Cells: an in Vitro Study

Adipose tissue-derived stem cells (ASCs) are known to be able to promote repair of injured tissue via paracrine factors. However, the effect of cell density and inflammatory cytokines on the paracrine ability of ASCs remains largely unknown. To investigate these effects, ASCs were cultured in 8000 cells/cm2, 20,000 cells/cm2, 50,000 cells/cm2, and 400,000 cells/cm2 with and without 10 or 20 ng/ml tumor necrosis factor alpha (TNFα) and 25 or 50 ng/ml interferon gamma (IFNγ). ASC-sheets formed at 400,000 cells/cm2 after 48 h of culture. With increasing concentrations of TNFα and IFNγ, ASC-sheets with 400,000 cells/cm2 had increased production of angiogenic factors Vascular Endothelial Growth Factor and Fibroblast Growth Factor and decreased expression of pro-inflammatory genes TNFA and Prostaglandin Synthase 2 (PTGS2) compared to lower density ASCs. Moreover, the conditioned medium of ASC-sheets with 400,000 cells/cm2 stimulated with the low concentration of TNFα and IFN

Biomaterials Influence Macrophage-Mesenchymal Stem Cell Interaction In Vitro

Background: Macrophages and mesenchymal stem cells (MSCs) are important cells in wound healing. We hypothesized that the cross-talk between macrophages and adipose tissue-derived MSCs (ASCs) is biomaterial dependent, thereby influencing processes involved in wound healing. Materials and Methods: The effect of macrophages cultured on polypropylene (PP) or polyethylene terephthalate coated with a collagen film (PET/Col) on ASCs in monolayer or on the same material was examined either through conditioned medium (CM) or in a direct coculture. ASC proliferation, collagen production, and gene expression were examined. As comparison, the effect of macrophages stimulated with lipopolysaccharide (LPS) and interferon gamma (IFNγ) [M(LPS/IFNγ)] or interleukin (IL) 4 [M(IL-4)] on ASCs was examined. Results: Macrophage-CM increased collagen deposition, proliferation, and gene expression of MMP1, PLOD2, and PTGS2 in ASCs, irrespective of the material. Culturing ASCs and macrophages in coculture when only macrophages were on the materials induced the same effects on gene expression. When both ASCs and macrophages were cultured on biomaterials, PP induced COL1A1 and MMP1 more than PET/Col. M(LPS/IFNγ) CM increased PLOD2, MMP1, and PTGS2 and decreased TGFB in ASCs more than the M(IL-4) CM. Conclusion: Biomaterials influence wound healing by influencing the interaction between macrophages and ASCs. We provided more insight into the behavior of different cell types during wound healing. This behavior appears to be biomaterial specific depending on which cell type interacts with the biomaterial. As such, the biomaterial will influence tissue regeneration

Correcting for the Effects of Interstellar Extinction

This paper addresses the issue of how best to correct astronomical data for the wavelength-dependent effects of Galactic interstellar extinction. The main general features of extinction from the IR through the UV are reviewed, along with the nature of observed spatial variations. The enormous range of extinction properties found in the Galaxy, particularly in the UV spectral region, is illustrated. Fortunately, there are some tight constraints on the wavelength dependence of extinction and some general correlations between extinction curve shape and interstellar environment. These relationships provide some guidance for correcting data for the effects of extinction. Several strategies for dereddening are discussed along with estimates of the uncertainties inherent in each method. In the Appendix, a new derivation of the wavelength dependence of an average Galactic extinction curve from the IR through the UV is presented, along with a new estimate of how this extinction law varies with the parameter R = A(V)/E(B-V). These curves represent the true monochromatic wavelength dependence of extinction and, as such, are suitable for dereddening IR--UV spectrophotometric data of any resolution, and can be used to derive extinction relations for any photometry system.Comment: To appear in PASP (January 1999) 14 pages including 4 pages of figures Uses emulateapj style. PASP, in press (January 1999