Profile of circulating microRNAs in myalgic encephalomyelitis and their relation to symptom severity, and disease pathophysiology.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a complex chronic disease, rooted in multi-system dysfunctions characterized by unexplained debilitating fatigue. Post-exertional malaise (PEM), defined as the exacerbation of the patient's symptoms following minimal physical or mental stress, is a hallmark of ME/CFS. While multiple case definitions exist, there is currently no well-established biomarkers or laboratory tests to diagnose ME/CFS. Our study aimed to investigate circulating microRNA expression in severely ill ME/CFS patients before and after an innovative stress challenge that stimulates PEM. Our findings highlight the differential expression of eleven microRNAs associated with a physiological response to PEM. The present study uncovers specific microRNA expression signatures associated with ME/CFS in response to PEM induction and reports microRNA expression patterns associated to specific symptom severities. The identification of distinctive microRNA expression signatures for ME/CFS through a provocation challenge is essential for the elucidation of the ME/CFS pathophysiology, and lead to accurate diagnoses, prevention measures, and effective treatment options.

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.

Bone-Induced Chondroinduction in Sheep Jamshidi Biopsy Defects with and without Treatment by Subchondral Chitosan-Blood Implant: 1-Day, 3-Week, and 3-Month Repair.

OBJECTIVE: Delivery of chitosan to subchondral bone is a novel approach for augmented marrow stimulation. We evaluated the effect of 3 presolidified chitosan-blood implant formulations on osteochondral repair progression compared with untreated defects. DESIGN: In N = 5 adult sheep, six 2-mm diameter Jamshidi biopsy holes were created bilaterally in the medial femoral condyle and treated with presolidified chitosan-blood implant with fluorescent chitosan tracer (10 kDa, 40 kDa, or 150k Da chitosan, left knee) or left to bleed (untreated, right knee). Implant residency and osteochondral repair were assessed at 1 day (N = 1), 3 weeks (N = 2), or 3 months (N = 2) postoperative using fluorescence microscopy, histomorphometry, stereology, and micro-computed tomography. RESULTS: Chitosan implants were retained in 89% of treated Jamshidi holes up to 3 weeks postoperative. At 3 weeks, biopsy sites were variably covered by cartilage flow, and most bone holes contained cartilage flow fragments and heterogeneous granulation tissues with sparse leukocytes, stromal cells, and occasional adipocytes (volume density 1% to 3%). After 3 months of repair, most Jamshidi bone holes were deeper, remodeling at the edges, filled with angiogenic granulation tissue, and lined with variably sized chondrogenic foci fused to bone trabeculae or actively repairing bone plate. The 150-kDa chitosan implant elicited more subchondral cartilage formation compared with 40-kDa chitosan-treated and control defects (P < 0.05, N = 4). Treated defects contained more mineralized repair tissue than control defects at 3 months (P < 0.05, N = 12). CONCLUSION: Bone plate-induced chondroinduction is an articular cartilage repair mechanism. Jamshidi biopsy repair takes longer than 3 months and can be influenced by subchondral chitosan-blood implant.

The cartilage-bone interface.

In the knee joint, the purpose of the cartilage-bone interface is to maintain structural integrity of the osteochondral unit during walking, kneeling, pivoting, and jumping--during which tensile, compressive, and shear forces are transmitted from the viscoelastic articular cartilage layer to the much stiffer mineralized end of the long bone. Mature articular cartilage is integrated with subchondral bone through a approximately 20 to approximately 250 microm thick layer of calcified cartilage. Inside the calcified cartilage layer, perpendicular chondrocyte-derived collagen type II fibers become structurally cemented to collagen type I osteoid deposited by osteoblasts. The mature mineralization front is delineated by a thin approximately 5 microm undulating tidemark structure that forms at the base of articular cartilage. Growth plate cartilage is anchored to epiphyseal bone, sometimes via a thin layer of calcified cartilage and tidemark, while the hypertrophic edge does not form a tidemark and undergoes continual vascular invasion and endochondral ossification (EO) until skeletal maturity upon which the growth plates are fully resorbed and replaced by bone. In this review, the formation of the cartilage-bone interface during skeletal development and cartilage repair, and its structure and composition are presented. Animal models and human anatomical studies show that the tidemark is a dynamic structure that forms within a purely collagen type II-positive and collagen type I-negative hyaline cartilage matrix. Cartilage repair strategies that elicit fibrocartilage, a mixture of collagen type I and type II, are predicted to show little tidemark/calcified cartilage regeneration and to develop a less stable repair tissue-bone interface. The tidemark can be regenerated through a bone marrow-driven growth process of EO near the articular surface.

Young adult chondrocytes proliferate rapidly and produce a cartilaginous tissue at the gel-media interface in agarose cultures.

Primary chondrocytes cultured in agarose can escape the gel, accumulate at the interface between agarose and the culture medium, and form an outgrowing tissue. These outgrowths can appear as voluminous cartilage-like nodules that have never been previously investigated. In the present study, bovine articular chondrocytes from three age groups (fetal, young adult, aged) were seeded and cultured in agarose to test the hypothesis that hyaline-like cartilage outgrowths develop at the interface by appositional growth, in an age-dependant manner. Macroscopic appearance, cell content, cell division, cytoskeletal morphology, and extracellular matrix (ECM) composition were analyzed. Fetal chondrocytes produced a fibrous interfacial tissue while aged chondrocytes produced ECM-poor cell clusters. In contrast young adult chondrocytes produced large cartilaginous outgrowths, rich in proteoglycan and collagen II, where cells in the central region displayed a chondrocyte morphology. Cell proliferation was confined to the peripheral edge of these outgrowths, where elongated cell morphology, cell-cell contacts, and cell extensions toward the culture medium were seen. Thus these voluminous cartilaginous outgrowths formed in an appositional growth process and only for donor chondrocytes from young adult animals. This system offers an interesting ability to proliferate chondrocytes in a manner that results in a chondrocyte morphology and a cartilaginous ECM in central regions of the outgrowing tissue. It also provides an in vitro model system to study neocartilage appositional growth.

Drilling and microfracture lead to different bone structure and necrosis during bone-marrow stimulation for cartilage repair.

Bone marrow stimulation is performed using several surgical techniques that have not been systematically compared or optimized for a desired cartilage repair outcome. In this study, we investigated acute osteochondral characteristics following microfracture and comparing to drilling in a mature rabbit model of cartilage repair. Microfracture holes were made to a depth of 2 mm and drill holes to either 2 mm or 6 mm under cooled irrigation. Animals were sacrificed 1 day postoperatively and subchondral bone assessed by histology and micro-CT. We confirmed one hypothesis that microfracture produces fractured and compacted bone around holes, essentially sealing them off from viable bone marrow and potentially impeding repair. In contrast, drilling cleanly removed bone from the holes to provide access channels to marrow stroma. Our second hypothesis that drilling would cause greater osteocyte death than microfracture due to heat necrosis was not substantiated, because more empty osteocyte lacunae were associated with microfracture than drilling, probably due to shearing and crushing of adjacent bone. Drilling deeper to 6 mm versus 2 mm penetrated the epiphyseal scar in this model and led to greater subchondral hematoma. Our study revealed distinct differences between microfracture and drilling for acute subchondral bone structure and osteocyte necrosis. Additional ongoing studies suggest these differences significantly affect long-term cartilage repair outcome.