Cluster analysis of infrared spectra can differentiate intact and repaired articular cartilage.

OBJECTIVE: Successful repair of articular cartilage (AC) defects would be a major advantage due to the low ability of AC to heal spontaneously. Sensitive methods to determine changes in AC composition and structure are required to monitor the success of repair. This study evaluates the ability of unsupervised cluster analysis applied to Fourier transform infrared (FTIR) microspectroscopy to discriminate between healthy and repaired AC. METHODS: Osteochondral lesions (3 mm in depth) were surgically created in patellar grooves of rabbit femurs and were either left to heal spontaneously (n = 6) or surgically repaired with autologous chondrocytes in type II collagen gel (n = 6). After 6 months, tissues were harvested, FTIR microspectroscopy was conducted and Fuzzy c-means (FCM) cluster analysis applied to spectra of pairs of intact and repaired AC samples from each rabbit. Two spectral regions [amide I and carbohydrate (CHO)] were analyzed and the results from the two types of repair were compared. RESULTS: Two separate regions of repair were detected with FCM. The estimated proteoglycan content (from CHO region) in the repaired AC was significantly lower than that in intact AC. The spontaneously repaired AC was better distinguished from the intact AC than the collagen II gel repaired AC. The most distinct clustering was observed for spontaneously repaired samples using CHO region. CONCLUSIONS: This study revealed that unsupervised cluster analysis applied to FTIR microspectroscopy can detect subtle differences in infrared spectra between normal and repaired AC. The method may help in evaluation and optimization of future AC repair strategies.

Repair of osteochondral defects with recombinant human type II collagen gel and autologous chondrocytes in rabbit.

OBJECTIVE: Recombinant human type II collagen (rhCII) gels combined with autologous chondrocytes were tested as a scaffold for cartilage repair in rabbits in vivo. METHOD: Autologous chondrocytes were harvested, expanded and combined with rhCII-gel and further pre-cultivated for 2 weeks prior to transplantation into a 4 mm diameter lesion created into the rabbit's femoral trochlea (n = 8). Rabbits with similar untreated lesions (n = 7) served as a control group. RESULTS: Six months after the transplantation the repair tissue in both groups filled the lesion site, but in the rhCII-repair the filling was more complete. Both repair groups also had high proteoglycan and type II collagen contents, except in the fibrous superficial layer. However, the integration to the adjacent cartilage was incomplete. The O'Driscoll grading showed no significant differences between the rhCII-repair and spontaneous repair, both representing lower quality than intact cartilage. In the repair tissues the collagen fibers were abnormally organized and oriented. No dramatic changes were detected in the subchondral bone structure. The repair cartilage was mechanically softer than the intact tissue. Spontaneously repaired tissue showed lower values of equilibrium and dynamic modulus than the rhCII-repair. However, the differences in the mechanical properties between all three groups were insignificant. CONCLUSION: When rhCII was used to repair cartilage defects, the repair quality was histologically incomplete, but still the rhCII-repairs showed moderate mechanical characteristics and a slight improvement over those in spontaneous repair. Therefore, further studies using rhCII for cartilage repair with emphasis on improving integration and surface protection are required.

Engineering of cartilage in recombinant human type II collagen gel in nude mouse model in vivo.

OBJECTIVE: Our goal was to test the recombinant human type II collagen (rhCII) material as a gel-like scaffold for chondrocytes in a nude mouse model in vivo. DESIGN: Isolated bovine chondrocytes (6x10(6)) were seeded into rhCII gels (rhCII-cell) and injected subcutaneously into the backs of nude mice. For comparison, chondrocytes (6x10(6)) in culture medium (Med-cell) and cell-free rhCII gels (rhCII-gel) were similarly injected (n=24 animals, total of three injections/animal). After 6 weeks, the tissue constructs were harvested and analyzed. RESULTS: Chondrocytes with or without rhCII-gel produced white resilient tissue, which in histological sections had chondrocytes in lacunae-like structures. Extracellular matrix stained heavily with toluidine blue stain and had strongly positive collagen type II immunostaining. The tissue did not show any evidence of vascular invasion or mineralization. The cell-free rhCII-gel constructs showed no signs of cartilage tissue formation. Cartilage tissue produced by Med-cell was thin and macroscopically uneven, while the rhCII-cell construct was smooth and rounded piece of neotissue. RhCII-cell constructs were statistically thicker than Med-cell ones. However, no statistical differences were found between the groups in terms of glycosaminoglycan (GAG) content or biomechanical properties. CONCLUSIONS: These results show that rhCII-gel provides good expansion and mechanical support for the formation of cartilage neotissue. RhCII material may allow favorable conditions in the repair of chondral lesions.

Bioreactor improves the growth and viability of chondrocytes in the knitted poly-L,D-lactide scaffold.

In the present study bovine chondrocytes were cultured in two different environments (static flasks and bioreactor) in knitted poly-L,D-lactide (PLDLA) scaffolds up to 4 weeks. Chondrocyte viability was assessed by employing cell viability fluorescence markers. The cells were visualized using confocal laser scanning microscopy and scanning electron microscopy. The mechanical properties and uronic acid contents of the scaffolds were tested. Our results showed that cultivation in a bioreactor improved the growth and viability of the chondrocytes in the PLDLA scaffolds. Cells were observed both on and in between the fibrils of scaffold. Furthermore, chondrocytes cultured in the bioreactor, regained their original round phenotypes, whereas those in the static flask culture were flattened in shape. Confocal microscopy revealed that chondrocytes from the bioreactor were attached on both sides of the scaffold and sustained viability better during the culture period. Uronic acid contents of the scaffolds, cultured in bioreactor, were significantly higher than in those cultured in static flasks for 4 weeks. In summary, our data suggests that the bioreactor is superior over the static flask culture when culturing chondrocytes in knitted PLDLA scaffold.

Recombinant human type II collagen as a material for cartilage tissue engineering.

PURPOSE: Collagen type II is the major component of cartilage and would be an optimal scaffold material for reconstruction of injured cartilage tissue. In this study, the feasibility of recombinant human type II collagen gel as a 3-dimensional culture system for bovine chondrocytes was evaluated in vitro. METHODS: Bovine chondrocytes (4x106 cells) were seeded within collagen gels and cultivated for up to 4 weeks. The gels were investigated with confocal microscopy, histology, and biochemical assays. RESULTS: Confocal microscopy revealed that the cells maintained their viability during the entire cultivation period. The chondrocytes were evenly distributed inside the gels, and the number of cells and the amount of the extracellular matrix increased during cultivation. The chondrocytes maintained their round phenotype during the 4-week cultivation period. The glycosaminoglycan levels of the tissue increased during the experiment. The relative levels of aggrecan and type II collagen mRNA measured with realtime polymerase chain reaction (PCR) showed an increase at 1 week. CONCLUSION: Our results imply that recombinant human type II collagen is a promising biomaterial for cartilage tissue engineering, allowing homogeneous distribution in the gel and biosynthesis of extracellular matrix components.

Use of psychotropic drugs and risk of myocardial infarction: a case-control study in Finnish farmers.

BACKGROUND: In 1992 Thorogood et al. reported an increased risk of myocardial infarction in women using psychotropic drugs. The aim of our study is to find out whether there is a link between the use of psychotropic drugs and subsequent myocardial infarction in males. METHOD: A cohort of 3172 male farmers was followed from 1 February 1980 to 31 December 1992. Those subjects who had myocardial infarction without any previous symptoms during the follow-up were considered as cases. For every case three matched controls were selected. The matched variables were age, smoking habits, social status and county. The final sample includes 83 cases and 249 controls. RESULTS: Those who had used psychotropic drugs had increased risk for myocardial infarction, odds ratio (OR) = 2.5, 95% confidence interval (CI):1.2-5.2. Most pronounced risk for myocardial infarction was found among users of anti-depressants, OR = 5.4 (CI:1.8-16.1). CONCLUSION: The use of psychotropic drugs, especially antidepressants, is associated with increased risk of myocardial infarction. Further attempts are needed to determine whether the relationship between use of psychotropic drugs and risk of myocardial infarction is causal or not.

Promoter polymorphism (-491A/T) in the APOE gene of Finnish Alzheimer's disease patients and control individuals.

Apolipoprotein E (APOE) epsilon4 allele is a major risk factor for the development of Alzheimer's disease (AD). It has been suggested that the quantitative expression of APOE alleles results from mutations in the promoter region of this gene. We studied the -491A/T promoter polymorphism and whether it is dependent on the APOE epsilon4 allele in clinic-based AD (n = 106) and community-based control (n = 123) samples. The -491A/T and APOE polymorphisms were analyzed using the polymerase chain reaction method and restriction fragment length polymorphism analysis. The APOE epsilon4 allele was strongly associated with AD when compared with controls, P < 0.001 (odds ratio 5.85, 95% CI 3.29- 10. 41). The genotype distribution of the -491A/T polymorphism did not significantly differ between the study groups (P = 0.063), and the -491A allele was not associated with any significant risk in the AD group when compared to controls (odds ratio 1.82, 95% CI 0.95-3.49). However, haplotype estimation analysis indicated linkage disequilibrium between APOE -491A/T polymorphism and the APOE epsilon4 allele. Our findings confirm APOE polymorphism still to be the most efficient predictor of risk in AD.

Water spin dynamics during apoptotic cell death in glioma gene therapy probed by T1rho and T2rho.

Longitudinal and transverse relaxations in the rotating frame, with characteristic time constants T1rho and T2rho, respectively, have potential to provide unique MRI contrast in vivo. On-resonance spin-lock T1rho with different spin-lock field strengths and adiabatic T2rho with different radiofrequency-modulation functions were measured in BT4C gliomas treated with Herpes Simplex Virus thymidine kinase (HVS-tk) gene therapy causing apoptotic cell death. These NMR tools were able to discriminate different treatment responses in tumor tissue from day 4 onward. An equilibrium two-site exchange model was used to calculate intrinsic parameters describing changes in water dynamics. Observed changes included increased correlation time of water associated with macromolecules and a decreased fractional population of this pool. These results are consistent with destructive intracellular processes associated with cell death and the increase of extracellular space during the treatment. Furthermore, association between longer exchange correlation time and decreased pH during apoptosis is discussed. In this study, we demonstrated that T1rho and T2rho MR imaging are useful tools to quantify early changes in water dynamics reflecting treatment response during gene therapy.

The risk of myocardial infarction among Finnish farmers seeking medical care for an infection.

OBJECTIVES: This study determined whether people visiting a doctor because of infection had an increased risk of myocardial infarction. METHODS: For 83 case patients and 249 matched control patients nested in a cohort of 3172 Finnish male farmers, comparisons were made on the basis of visits to a doctor because of infection. RESULTS: Infections of the upper respiratory tract were statistically significantly associated with coronary artery disease. The odds ratio was 3.2 (95% confidence interval = 1.2, 8.5) for patients visiting a doctor four or more times because of infection of upper respiratory tract compared with patients with three or fewer visits. CONCLUSION: Men in this sample with recurrent or chronic infections of the upper respiratory tract exhibited a pronounced risk for myocardial infarction.