A polarized light microscopy method for accurate and reliable grading of collagen organization in cartilage repair.

OBJECTIVES: Collagen organization, a feature that is critical for cartilage load bearing and durability, is not adequately assessed in cartilage repair tissue by present histological scoring systems. Our objectives were to develop a new polarized light microscopy (PLM) score for collagen organization and to test its reliability. DESIGN: This PLM score uses an ordinal scale of 0-5 to rate the extent that collagen network organization resembles that of young adult hyaline articular cartilage (score of 5) vs a totally disorganized tissue (score of 0). Inter-reader reliability was assessed using Intraclass Correlation Coefficients (ICC) for Agreement, calculated from scores of three trained readers who independently evaluated blinded sections obtained from normal (n=4), degraded (n=2) and repair (n=22) human cartilage biopsies. RESULTS: The PLM score succeeded in distinguishing normal, degraded and repair cartilages, where the latter displayed greater complexity in collagen structure. Excellent inter-reader reproducibility was found with ICCs for Agreement of 0.90 [ICC(2,1)] (lower boundary of the 95% confidence interval is 0.83) and 0.96 [ICC(2,3)] (lower boundary of the 95% confidence interval is 0.94), indicating the reliability of a single reader's scores and the mean of all three readers' scores, respectively. CONCLUSION: This PLM method offers a novel means for systematically evaluating collagen organization in repair cartilage. We propose that it be used to supplement current gold standard histological scoring systems for a more complete assessment of repair tissue quality.

Structural characteristics of the collagen network in human normal, degraded and repair articular cartilages observed in polarized light and scanning electron microscopies.

OBJECTIVE: This study characterizes collagen organization (CO) in human normal (n = 6), degraded (n = 6) and repair (n = 22) cartilages, using polarized light (PLM) and scanning electron (SEM) microscopies. DESIGN: CO was assessed using a recently developed PLM-CO score (Changoor et al. Osteoarthritis Cartilage 2011;19:126-35), and zonal proportions measured. SEM images were captured from locations matched to PLM. Fibre orientations were assessed in SEM and compared to those observed in PLM. CO was also assessed in individual SEM images and combined to generate a SEM-CO score for overall CO analogous to PLM-CO. Fibre diameters were measured in SEM. RESULTS: PLM-CO and SEM-CO scores were correlated, r = 0.786 (P < 0.00001, n = 32), after excluding two outliers. Orientation observed in PLM was validated by SEM since PLM/SEM correspondence occurred in 91.6% of samples. Proportions of the deep (DZ), transitional (TZ) and superficial (SZ) zones averaged 74.0 ± 9.1%, 18.6 ± 7.0%, and 7.3 ± 1.2% in normal, and 45.6 ± 10.7%, 47.2 ± 10.1% and 9.5 ± 3.4% in degraded cartilage, respectively. Fibre diameters in normal cartilage increased with depth from the articular surface [55.8 ± 9.4 nm (SZ), 87.5 ± 1.8 nm (TZ) and 108.2 ± 1.8 nm (DZ)]. Fibre diameters were smaller in repair biopsies [60.4 ± 0.7 nm (SZ), 63.2 ± 0.6 nm (TZ) and 67.2 ± 0.8 nm (DZ)]. Degraded cartilage had wider fibre diameter ranges and bimodal distributions, possibly reflecting new collagen synthesis and remodelling or collagen fibre unravelling. Repair tissues revealed the potential of microfracture-based repair procedures to produce zonal CO resembling native articular cartilage structure. Values are reported as mean ± 95% confidence interval. CONCLUSION: This detailed assessment of collagen architecture could benefit the development of cartilage repair strategies intended to recreate functional collagen architecture.

Frontal ostium restenosis after the endoscopic modified Lothrop procedure.

OBJECTIVES: To evaluate the time course and extent of the restenosis that occurs after an endoscopic modified Lothrop procedure (EMLP) and to identify factors that contribute to this process. STUDY DESIGN: : Retrospective study of prospectively collected data. METHODS: Seventy-seven consecutive patients undergoing EMLP between November 1998 and June 2005 with frontal ostium measurements documented intraoperatively and at follow-up visits for a minimum of 12 months were included. Data on patient demographics, medical history, comorbidities, and computed tomography scans were collected. RESULTS: Significant restenosis of the frontal sinus neo-ostia was defined as a loss of more than 60% of the original intraoperative area. The mean follow-up in our cohort was 29.2 months. Twenty-two patients were identified with increased stenosis, all of which arose within the first 12 months postsurgery. Nine of these 22 patients required revision EMLP. In all patients, the new frontal ostium narrowed at 1 year by an average of 33% (from 290 to 191 mm2, confidence interval 159-223 mm2). Restenosis and revision surgery are partly predicted by the presence of eosinophilic mucin chronic rhinosinusitis as demonstrated by logistic regression analysis. Linear regression analysis confirmed that the intraoperative frontal ostium size determines the frontal ostium area at 1 year. CONCLUSION: This study provides benchmarking values for the time course of the frontal ostium area after EMLP for normal wound healing and increased stenosis. On the basis of our results, patients with a higher risk for developing restenosis and of having revision surgery can be identified preoperatively and during the early postoperative period to facilitate special postoperative care.

Extracellular matrix mineralization in murine MC3T3-E1 osteoblast cultures: an ultrastructural, compositional and comparative analysis with mouse bone.

Bone cell culture systems are essential tools for the study of the molecular mechanisms regulating extracellular matrix mineralization. MC3T3-E1 osteoblast cell cultures are the most commonly used in vitro model of bone matrix mineralization. Despite the widespread use of this cell line to study biomineralization, there is as yet no systematic characterization of the mineral phase produced in these cultures. Here we provide a comprehensive, multi-technique biophysical characterization of this cell culture mineral and extracellular matrix, and compare it to mouse bone and synthetic apatite mineral standards, to determine the suitability of MC3T3-E1 cultures for biomineralization studies. Elemental compositional analysis by energy-dispersive X-ray spectroscopy (EDS) showed calcium and phosphorus, and trace amounts of sodium and magnesium, in both biological samples. X-ray diffraction (XRD) on resin-embedded intact cultures demonstrated that similar to 1-month-old mouse bone, apatite crystals grew with preferential orientations along the (100), (101) and (111) mineral planes indicative of guided biogenic growth as opposed to dystrophic calcification. XRD of crystals isolated from the cultures revealed that the mineral phase was poorly crystalline hydroxyapatite with 10 to 20nm-sized nanocrystallites. Consistent with the XRD observations, electron diffraction patterns indicated that culture mineral had low crystallinity typical of biological apatites. Fourier-transform infrared spectroscopy (FTIR) confirmed apatitic carbonate and phosphate within the biological samples. With all techniques utilized, cell culture mineral and mouse bone mineral were remarkably similar. Scanning (SEM) and transmission (TEM) electron microscopy showed that the cultures had a dense fibrillar collagen matrix with small, 100nm-sized, collagen fibril-associated mineralization foci which coalesced to form larger mineral aggregates, and where mineralized sites showed the accumulation of the mineral-binding protein osteopontin. Light microscopy, confocal microscopy and three-dimensional reconstructions showed that some cells had dendritic processes and became embedded within the mineral in an osteocyte-like manner. In conclusion, we have documented characteristics of the mineral and matrix phases of MC3T3-E1 osteoblast cultures, and have determined that the structural and compositional properties of the mineral are highly similar to that of mouse bone.

Defects in articular cartilage metabolism and early arthritis in fibroblast growth factor receptor 3 deficient mice.

Fibroblast growth factor (FGF) receptor 3 has been identified as a key regulator of endochondral bone development and of post-natal bone metabolism through its action on growth plate chondrocytes and osteoblasts, respectively. It has also been shown to promote chondrogenesis and cartilage production by cultured pre-chondrogenic cells in response to FGF18. In the current studies, we show that the absence of signaling through Fgfr3 in the joints of Fgfr3(-/-) mice leads to premature cartilage degeneration and early arthritis. Degenerative changes in cartilage matrix included excessive proteolysis of aggrecan core protein and type II collagen, as measured by neo-epitope immunoreactivity. These changes were accompanied by increased expression of metalloproteinase MMP13, type X collagen, cellular hypertrophy and loss of proteoglycan at the articular surface. Using a novel micro-mechanical indentation protocol, it was shown that articular cartilage in the humeral head of 4-month-old Fgfr3(-/-) mice was less resistant to compressive force and less stiff than that of littermate controls. These results identify Fgfr3 signaling as a potential target for intervention in degenerative disorders of cartilage metabolism.

Mature full-thickness articular cartilage explants attached to bone are physiologically stable over long-term culture in serum-free media.

Mature tissue explants containing the entire depth of articular cartilage, calcified and uncalcified, attached to a thin layer of subchondral bone were isolated from bovine humeral heads of 1-2-year-old steers. These explants were placed in defined serum-free culture medium for a period of 3 weeks to investigate their biological and mechanical stability and thus to determine their potential utility in studies of cartilage physiology. Tissue mass remained constant over the culture period and no evident tissue swelling or distortion was observed. Chondrocytes were viable in all zones at the time of tissue isolation and throughout the culture period, with the exception of a thin layer of cells at the articular surface and the cut radial edge of the disks. Proteoglycan metabolism attained a steady state after 5 days of culture when the rate of loss of proteoglycan to culture media was compensated by new synthesis to maintain a stable proteoglycan content. Collagen metabolism was also stable with a constant content of type II collagen and a constant content of denatured collagen II throughout culture; the content of the C-propeptide of type II procollagen as a measure of procollagen synthesis, dropped slightly during the first week to attain a steady state after that time. Dynamic and equilibrium mechanical properties of these explant disks were also stable confirming maintenance of these tissue properties during long-term culture. In addition, the disk geometry of the system, with the cut surface in the bone parallel to the intact articular surface, is well-suited to study tissue regulation by mechanical load. Taken together, the stability of these indicators of tissue physiology indicates the maintenance in serum-free conditions of normal metabolism for organ cultures containing full-depth mature articular cartilage attached to bone.