UDP-glucose dehydrogenase (UGDH) activity is suppressed by peroxide and promoted by PDGF in fibroblast-like synoviocytes: Evidence of a redox control mechanism.

UDP-glucose dehydrogenase (UGDH) generates essential precursors of hyaluronic acid (HA) synthesis, however mechanisms regulating its activity are unclear. We used enzyme histostaining and quantitative image analysis to test whether cytokines that stimulate HA synthesis upregulate UGDH activity. Fibroblast-like synoviocytes (FLS, from N = 6 human donors with knee pain) were cultured, freeze-thawed, and incubated for 1 hour with UDP-glucose, NAD+ and nitroblue tetrazolium (NBT) which allows UGDH to generate NADH, and NADH to reduce NBT to a blue stain. Compared to serum-free medium, FLS treated with PDGF showed 3-fold higher UGDH activity and 6-fold higher HA release, but IL-1beta/TGF-beta1 induced 27-fold higher HA release without enhancing UGDH activity. In selected proliferating cells, UGDH activity was lost in the cytosol, but preserved in the nucleus. Cell-free assays led us to discover that diaphorase, a cytosolic enzyme, or glutathione reductase, a nuclear enzyme, was necessary and sufficient for NADH to reduce NBT to a blue formazan dye in a 1-hour timeframe. Primary synovial fibroblasts and transformed A549 fibroblasts showed constitutive diaphorase/GR staining activity that varied according to supplied NADH levels, with relatively stronger UGDH and diaphorase activity in A549 cells. Unilateral knee injury in New Zealand White rabbits (N = 3) stimulated a coordinated increase in synovial membrane UGDH and diaphorase activity, but higher synovial fluid HA in only 2 out of 3 injured joints. UGDH activity (but not diaphorase) was abolished by N-ethyl maleimide, and inhibited by peroxide or UDP-xylose. Our results do not support the hypothesis that UGDH is a rate-liming enzyme for HA synthesis under catabolic inflammatory conditions that can oxidize and inactivate the UGDH active site cysteine. Our novel data suggest a model where UGDH activity is controlled by a redox switch, where intracellular peroxide inactivates, and high glutathione and diaphorase promote UGDH activity by maintaining the active site cysteine in a reduced state, and by recycling NAD+ from NADH.

Bone-repair properties of biodegradable hydroxyapatite nano-rod superstructures.

Nano-hydroxyapatite (nano-HAp) materials show an analogous chemical composition to the biogenic mineral components of calcified tissues and depending on their topography they may mimic the specific arrangement of the crystals in bone. In this work, we have evaluated the potential of four synthesized nano-HAp superstructures for the in vitro conditions of bone-repair. Experiments are underway to investigate the effects of the material microstructure, surface roughness and hydrophilicity on their osseo-integration, osteo-conduction and osteo-induction abilities. Materials were tested in the presence of both, rat primary osteoblasts and rabbit mesenchymal stem cells. The following aspects are discussed: (i) cytotoxicity and material degradation; (ii) rat osteoblast spreading, proliferation and differentiation; and (iii) rabbit mesenchymal stem cell adhesion on nano-HAp and nano-HAp/collagen type I coatings. We effectively prepared a material based on biomimetic HAp nano-rods displaying the appropriate surface topography, hydrophilicity and degradation properties to induce the in vitro desired cellular responses for bone bonding and healing. Cells seeded on the selected material readily attached, proliferated and differentiated, as confirmed by cell viability, mitochondrial metabolic activity, alkaline phosphatase (ALP) activity and cytoskeletal integrity analysis by immunofluorescence localization of alpha-smooth muscle actin (α-SMA) protein. These results highlight the influence of material's surface characteristics to determine their tissue regeneration potential and their future use in engineering osteogenic scaffolds for orthopedic implants.

Chondroinduction Is the Main Cartilage Repair Response to Microfracture and Microfracture With BST-CarGel: Results as Shown by ICRS-II Histological Scoring and a Novel Zonal Collagen Type Scoring Method of Human Clinical Biopsy Specimens.

BACKGROUND: Current cartilage repair histological scoring systems are unable to explain the relationship between collagen type II deposition and overall repair quality. PURPOSE/HYPOTHESIS: The purpose of this study was to develop a novel zonal collagen type (ZCT) 5-point scoring system to measure chondroinduction in human clinical biopsy specimens collected after marrow stimulation. The hypothesis was that the ZCT scores would correlate with the International Cartilage Repair Society-II (ICRS-II) overall histological repair assessment score and glycosaminoglycan (GAG) content. STUDY DESIGN: Descriptive laboratory study. METHODS: After optimizing safranin O staining for GAG and immunostaining for human collagen type II and type I (Col2 and Col1, respectively), serial sections from clinical osteochondral repair biopsy specimens (13 months after microfracture or microfracture with BST-CarGel; n = 39 patients) were stained and 3 blinded readers performed histomorphometry for percentage of staining, ICRS-II histological scoring, polarized light microscopy (PLM) scoring, and 5-point ZCT scoring based on tidemark morphology, zonal distribution of Col2 and Col1, and Col1 percentage stain. Because 1 biopsy specimen was missing bone, 38 biopsy specimens were evaluated for ICRS-II, PLM, and ZCT scores. RESULTS: Chondroinduction was identified in 21 biopsy specimens as a Col2 matrix fused to bone that spanned the deep-middle-superficial zones ("full-thickness hyaline repair"), deep-middle zones, or deep zone ("stalled hyaline") that was covered with a variable-thickness Col1-positive matrix, and was scored, respectively, as ZCT = 1 (n = 4 biopsy specimens), ZCT = 2 (n = 6) and ZCT = 3 (n = 11). Other biopsy specimens (n = 17) were fibrocartilage (n = 9; ZCT = 4), fibrous tissue (n = 4, ZCT = 5), or non-marrow derived (n = 4; ZCT = 0). Non-marrow derived tissue had a mean mature tidemark score of 84 out of 100 versus a regenerating tidemark score of 24 for all other biopsy specimens (P = .005). Both "stalled hyaline" repair and fibrocartilage had the same mean Col2 percentage stain; however, fibrocartilage was distinguished by heavy Col1 deposits in the deep zone, a 2-fold higher mean Col1 percentage stain (P = .001), and lower surface integrity (P = .03). ZCT scores correlated with GAG content and the ICRS-II overall assessment score, especially when combined with the PLM score for collagen organization (R = 0.82). Histological scores of the deep zone strongly predicted the ICRS-II overall assessment score (R = 0.99). CONCLUSION: The ICRS-II overall repair assessment score and GAG content correlated with the extent of Col2 deposition free of fibrosis in the deep/middle zone rather than bulk accumulation of Col2. CLINICAL RELEVANCE: Biopsy tissue from the BST-CarGel randomized clinical trial (microfracture without and with BST-CarGel, as treatment groups were not unblinded) showed regenerated tissue consistent with a chondroinduction mechanism in at least half of the treated lesions.