Detailed comparison of retroviral vectors and promoter configurations for stable and high transgene expression in human induced pluripotent stem cells.

Correction of patient-specific induced pluripotent stem cells (iPSC) upon gene delivery through retroviral vectors offers new treatment perspectives for monogenetic diseases. Gene-modified iPSC clones can be screened for safe integration sites and differentiated into transplantable cells of interest. However, the current bottleneck is epigenetic vector silencing. In order to identify the most suitable retroviral expression system in iPSC, we systematically compared vectors from different retroviral genera, different promoters and their combination with ubiquitous chromatin opening elements (UCOE), and several envelope pseudotypes. Lentiviral vectors (LV) pseudotyped with vesicular stomatitis virus glycoprotein were superior to gammaretroviral and alpharetroviral vectors and other envelopes tested. The elongation factor 1α short (EFS) promoter mediated the most robust expression, whereas expression levels were lower from the potent but more silencing-prone spleen focus forming virus (SFFV) promoter. Both full-length (A2UCOE) and minimal (CBX3) UCOE juxtaposed to two physiological and one viral promoter reduced transgene silencing with equal efficiency. However, a promoter-specific decline in expression levels was not entirely prevented. Upon differentiation of transgene-positive iPSC into endothelial cells, A2UCOE.EFS and CBX3.EFS vectors maintained highest transgene expression in a larger fraction of cells as compared with all other constructs tested here. The function of UCOE diminished, but did not fully counteract, vector silencing and possibilities for improvements remain. Nevertheless, the CBX3.EFS in a LV background exhibited the most promising promoter and vector configuration for both high titer production and long-term genetic modification of human iPSC and their progeny.

Improved retroviral episome transfer of transcription factors enables sustained cell fate modification.

Retroviral vectors are versatile gene transfer vehicles widely used in basic research and gene therapy. Mutation of retroviral integrase converts these vectors into transient, integration-deficient gene delivery vehicles associated with a high degree of biosafety. We explored the option to use integration-deficient retroviral vectors to achieve transient ectopic expression of transcription factors, which is considered an important tool for induced cell fate conversion. Stepwise optimization of the retroviral episome transfer as exemplified for the transcription factor Oct4 enabled to improve both expression magnitude and endurance. Long terminal repeat-driven γ-retroviral vectors were identified as the most suitable vector architecture. Episomal expression was enhanced by epigenetic modifiers, and Oct4 activity was increased following fusion to a minimal transactivation motif of herpes simplex virus VP16. Based on kinetic analyses, we identified optimal time intervals for repeated vector administration and established prolonged expression windows of choice. Providing proof-of-concept, episomal transfer of Oct4 was potent to mediate conversion of human fibroblasts stably expressing Klf4, Sox2 and c-Myc into induced pluripotent stem cells, which were mainly free of residual Oct4 vector integration. This study provides evidence for suitability of retroviral episome transfer of transcription factors for cell fate conversion, allowing the generation of distinct patient- or disease-specific cell types.

Abnormal neuronal differentiation and mitochondrial dysfunction in hair follicle-derived induced pluripotent stem cells of schizophrenia patients.

One of the prevailing hypotheses suggests schizophrenia as a neurodevelopmental disorder, involving dysfunction of dopaminergic and glutamatergic systems. Accumulating evidence suggests mitochondria as an additional pathological factor in schizophrenia. An attractive model to study processes related to neurodevelopment in schizophrenia is reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) and differentiating them into different neuronal lineages. iPSCs from three schizophrenia patients and from two controls were reprogrammed from hair follicle keratinocytes, because of their accessibility and common ectodermal origin with neurons. iPSCs were differentiated into Pax6(+)/Nestin(+) neural precursors and then further differentiated into ß3-Tubulin(+)/tyrosine hydroxylase(+)/DAT(+) dopaminergic neurons. In addition, iPSCs were differentiated through embryonic bodies into ß3-Tubulin(+)/Tbox brain1(+) glutamatergic neurons. Schizophrenia-derived dopaminergic cells showed severely impaired ability to differentiate, whereas glutamatergic cells were unable to maturate. Mitochondrial respiration and its sensitivity to dopamine-induced inhibition were impaired in schizophrenia-derived keratinocytes and iPSCs. Moreover, we observed dissipation of mitochondrial membrane potential (Δψm) and perturbations in mitochondrial network structure and connectivity in dopaminergic along the differentiation process and in glutamatergic cells. Our data unravel perturbations in neural differentiation and mitochondrial function, which may be interconnected, and of relevance to dysfunctional neurodevelopmental processes in schizophrenia.

Induced pluripotent stem cells from hair follicles as a cellular model for neurodevelopmental disorders.

Disease-specific induced pluripotent stem cells (iPSC) allow unprecedented experimental platforms for basic research as well as high-throughput screening. This may be particularly relevant for neuropsychiatric disorders, in which the affected neuronal cells are not accessible. Keratinocytes isolated from hair follicles are an ideal source of patients' cells for reprogramming, due to their non-invasive accessibility and their common neuroectodermal origin with neurons, which can be important for potential epigenetic memory. From a small number of plucked human hair follicles obtained from two healthy donors we reprogrammed keratinocytes to pluripotent iPSC. We further differentiated these hair follicle-derived iPSC to neural progenitors, forebrain neurons and functional dopaminergic neurons. This study shows that human hair follicle-derived iPSC can be differentiated into various neural lineages, suggesting this experimental system as a promising in vitro model to study normal and pathological neural developments, avoiding the invasiveness of commonly used skin biopsies.

Tibial pilon fractures.

Tibial pilon fractures are severe injuries to the distal articular surface of the tibia. Such injuries frequently result from high-energy axial impact and are often associated with extended soft tissue injury. Various treatment methods are available, depending not only on the fracture type but mostly on the extent of the soft tissue injury; one of the most frequent procedures is a two-stage surgery: the initial closed reduction of the fracture via primary placement of an ankle joint-spanning external fixator, if possible in conjunction with open reduction and internal fixation of the fractured fibula followed by a secondary procedure after soft tissue recovery by open reduction and internal fixation of the tibial plafond. By now, new types of low-profile and locking plates are available for internal fixation allowing the anatomical reconstruction of the fractured articular surface while sparing the soft tissue. Nonetheless, the treatment of tibial pilon fractures is technically demanding because of their potential for severe complications.

Focal lesions of human hippocampal CA1 neurons in transient global amnesia impair place memory.

A critical role in place learning has been attributed to place cells within the cornu ammonis 1 (CA1) sector of the hippocampus in rodents. The role of CA1 cells in the human hippocampus with regard to place learning remains elusive. Using a virtual Morris water maze, we investigated patients with acute transient global amnesia (TGA), a rare self-limiting dysfunction of the hippocampal system. Fourteen individuals with selective and focal lesions in the CA1 sector of the hippocampus showed a profound impairment in place learning. The size of the lesions and the duration of the TGA correlated with the deficit in the performance.

Factors associated with concentrations of select cytokine and acute phase proteins in dairy cows with naturally occurring clinical mastitis.

The objective of the current observational study was to determine the potential associations between cow factors, clinical mastitis (CM) etiology, and concentrations of select acute phase proteins and cytokines in milk from affected quarters of cows with CM. Cows with CM (n=197) were grouped based on systemic disease severity, milk culture result, parity, days in milk (DIM), previous CM occurrence, and season of the year when CM occurred. Concentrations of lipopolysaccharide-binding protein (LBP), haptoglobin (Hp), BSA, IFN-gamma, tumor necrosis factor-alpha (TNF-alpha), IL-1beta, IL-8, IL-10, IL-12, transforming growth factor (TGF)-alpha, and TGF-beta and activity of lactate dehydrogenase (LDH) were evaluated. Differences in the least squares means log(10) transformed concentrations of these proteins were compared using multiple linear regression mixed models. The milk concentrations of LBP, Hp, IL-1beta, IL-10, and IL-12, and activity of LDH in milk were higher in cows with moderate to severe versus mild systemic disease. The concentrations of Hp, BSA, IL-1beta, and IL-10 in milk were higher in cows with a gram-negative versus gram-positive milk culture result. Season of the year when CM occurred was associated with the concentration of all proteins evaluated except for IL-1beta and IL-12. Concentrations were higher in the winter versus summer except for Hp and TGF-beta, for which the opposite was true. Concentrations of LBP, IL-10, and IL-12, and LDH activity in milk were associated with DIM group. Except for LBP, these proteins were lower in cows with CM during the first 60 DIM versus those in mid or later lactation. Interferon-gamma, TNF-alpha, and IL-8 were undetectable in 67, 31, and 20% of samples, respectively. Detection of IFN-gamma and IL-8 was associated with season, and detection of TNF-alpha and IL-8 was associated with systemic disease severity. The current study provides the most comprehensive report of milk concentrations of innate immune response proteins in cows with naturally occurring CM and identifies factors that potentially influence those concentrations. Further investigation into the seasonal variation of cytokine production and its potential effect on the outcome of CM is warranted. Furthermore, the results of this study provide useful data for planning future studies examining the role of the innate immune response in CM.

A review of the species of Mesopolobus (Chalcidoidea: Pteromalidae) associated with Ceutorhynchus (Coleoptera: Curculionidae) host-species of European origin.

Four species of Mesopolobus Westwood were reared as parasitoids of Ceutorhynchinae hosts in Europe during surveys in 2000-2004. An illustrated key is given to differentiate the four species, M. gemellus Baur & Muller sp. n., M. incultus (Walker), M. morys (Walker) and M. trasullus (Walker), plus M. moryoides Gibson, a parasitoid of the cabbage seedpod weevil, Ceutorhynchus obstrictus (Marsham), in North America. Pteromalus clavicornis Walker is recognized as a junior synonym of M. incultus syn. n., and Pteromalus berecynthos Walker (also a junior synonym of M. incultus) is considered a correct original spelling. For Disema pallipes Förster (a junior synonym of Mesopolobus morys), a lectotype is designated. Mesopolobus morys is for the first time accurately associated with the seed weevil Ceutorhynchus turbatus (Schultze), a potential agent for classical biological control, of hoary cress, Lepidium draba L. (Brassicaceae), in North America. Mesopolobus gemellus is associated with another seed weevil, Ceutorhynchus typhae (=C. floralis) (Herbst), in pods of shepherd's purse, Capsella bursa-pastoris (L.) Medik. (Brassicaceae). Implications of the host-parasitoid associations are discussed relative to the introduction of species to North America for classical biological control of the cabbage seedpod weevil.

Tuberculosis at Edendale Hospital in Pietermaritzburg, Kwazulu Natal, South Africa.

SETTING: Edendale Hospital, Pietermaritzburg, KwaZulu Natal, South Africa, a 1275-bed hospital that serves a mainly ethnic African population of 1.6 million. OBJECTIVE: To describe the demographic and clinical characteristics of hospitalised active tuberculosis (TB) cases, and correlates of their in-hospital survival. METHODS: A retrospective cohort study of adult TB cases admitted to the medical wards, 16 November to 13 December 2001. RESULTS: Of 760 (28%) admissions, 215 had active TB, of whom 26.5% died in hospital. Patients were mostly young, first diagnosed on admission, and had pulmonary TB. Human immunodeficiency virus (HIV) co-infection was common and predicted by lower absolute lymphocyte count (OR 1.2, 95% CI 1.05-1.38). Extra-pulmonary TB, including pleural and pericardial, was significantly associated with not having HIV infection. In-hospital death was predicted by TB diagnosed prior to admission (OR 3.18, 95% CI 1.67-6.07), acquired immune-deficiency syndrome (AIDS) associated disease, and higher total leukocyte count--by higher leukocytes only in patients without AIDS (OR 8.52, 95% CI 2.67-27.13). CONCLUSION: Active TB was common in in-patients at an acute care hospital. TB patients presented late in disease and had high in-hospital mortality. Early detection and effective treatment of active TB in the community is likely to reduce hospitalisation and improve survival.

Surgical management of symptomatic spinal metastases. Postoperative outcome and quality of life.

STUDY DESIGN: Eighty-six surgical interventions in 76 consecutive patients with symptomatic spinal metastases were reviewed retrospectively. OBJECTIVES: To evaluate the postoperative outcome and quality of life of patients surgically treated for symptomatic spinal metastases. SUMMARY OF BACKGROUND DATA: The standard surgical treatment for patients with symptomatic spinal metastases is anterior spinal cord decompression with stabilization. However, because therapy is only palliative, satisfactory quality of life and high patient acceptance are essential. METHODS: The medical records of all patients were reviewed retrospectively. Furthermore, all surviving patients or the next of kin of deceased patients were interviewed by telephone, and the family doctors or the care-providing physicians of external institutions were contacted. RESULTS: First-choice surgical treatment was anterior spinal cord decompression with stabilization. Postoperative mean survival was 13.1 months, and mean time at home after spinal surgery was 11.1 months. Neurologic improvement with regard to Frankel classification was observed in 58% of the patients, and 93% were able to walk postoperatively. Pain relief was noted in 89%. Overall, 67% of the patients achieved moderate or good general health as shown by the Karnofsky Index, and 80% were satisfied or very satisfied with the surgical intervention. Moreover, 19% of the surgical interventions were associated with complications, local tumor recurrence developed in 22% of the patients, and paraplegia ultimately developed in 18% of patients. CONCLUSIONS: Surgical management of symptomatic spinal metastases, in particular anterior decompression, is of benefit in most metastatic lesions in terms of satisfactory postoperative outcome and quality of life. However, in patients with melanoma or lung carcinoma, the authors advocate spinal surgery only in very exceptional cases.

Mechanical behavior and biochemical composition of canine knee cartilage following periods of joint disuse and disuse with remobilization.

The mechanical behavior and biochemical composition of articular cartilage were studied in an experimental model of joint disuse, in which the canine knee was immobilized in a sling at 90 degrees of flexion. Articular cartilage from the surface zone of the femur was tested in an isometric tensile test and full-thickness cartilage on the tibial plateau was tested in a compressive indentation test. Water, proteoglycan and collagen contents were measured in site-matched samples. Site-specific increases in the tensile moduli (approximately 88% above control values in distal femoral groove) were observed in cartilage after 8 weeks of joint disuse, and after 3 weeks of remobilization following either 4 (approximately 140%, distal and proximal femoral groove) or 8 weeks (approximately 140%, distal femoral groove) of joint disuse. In contrast, the compressive properties of cartilage determined in the indentation test exhibited no change from control values with joint disuse or disuse followed by remobilization. Water contents increased at some sites on the tibia after 8 weeks of joint disuse (approximately 6% of tissue wet weight, posterior site), but not in the surface zone tissue of the femur. Proteoglycan/collagen and cartilage thickness were not found to change with disuse or disuse followed by remobilization. Reduced values for the ratio of proteoglycan:water were observed in the surface zone tissue of the femur (approximately 23%, distal femoral groove) and in the full-thickness tissue of the tibia (approximately 21%, anterior and posterior sites) after periods of joint disuse. In this study, the measured material properties suggest that the articular surface remains intact following periods of disuse or disuse with remobilization. This finding suggests one important difference between this model of joint disuse and other experimental models in which cartilage changes are both progressive and degenerative, such as surgically-induced joint instability.

Cartilage metalloproteases in disuse atrophy.

A canine knee model of disuse atrophy produced by nonrigid fixation (sling) was characterized in respect to variables of proteoglycan size distribution, as well as biomechanical properties versus controls. Using this model, we found, in addition to the accepted dogma attributing changes to reduced protein synthesis by chondrocytes, that there is elevation of proteases and depression of tissue inhibitor of metalloproteases (TIMP) in atrophic knee cartilage. The findings are suggestive of cartilage remodelling reminiscent of bone remodelling in disuse atrophy reported by others. Whether the abnormal changes of protease-TIMP balance in knee cartilage can be retarded prophylactically by concurrent treatment with pentosan polysulfate and insulin like growth factor 1 remains uncertain.

Mechanical properties of canine articular cartilage are significantly altered following transection of the anterior cruciate ligament.

The compressive, tensile, and swelling properties of articular cartilage were studied at two time periods following transection of the anterior cruciate ligament in the knee of greyhound dogs. An experimental protocol was designed to quantify the essential equilibrium and biphasic material properties of cartilage in tension, compression, and shear, as well as the parameters of isometric swelling behavior. All properties were measured at several sites to elicit differences between sites of frequent and less frequent contact. Hydration was determined at each site and was compared with the material properties of cartilage from corresponding sites. There were extensive changes in all compressive, tensile, and swelling properties of cartilage after transection of the anterior cruciate ligament. Twelve weeks after surgery, the intrinsic moduli were reduced significantly in compression (approximately 24% of control values), tension (approximately 64%), and shear (approximately 24%), and the hydraulic permeability was elevated significantly (approximately 48%). Significant increases in hydration (approximately 9%) also were observed, as well as a strong correlation of hydration with hydraulic permeability. The pattern of these changes was not found to differ with site in the joint, but significant differences were observed in the magnitude of change for cartilage from the femoral groove and the femoral condyle. The pattern and extent of changes in the material properties following transection of the anterior cruciate ligament indicate that altered loading of the joint severely compromises the overall mechanical behavior of articular cartilage. The observed loss of matrix stiffness in compression, tension, and shear is associated with increases in the deformation of the solid matrix, a diminished ability to resist swelling, and the increase in hydration observed in this study. The increased swelling and elevated water content were related directly to the increase in hydraulic permeability; this suggests an associated loss of fluid pressurization as the load support mechanism in the degenerated cartilage. Without a successful mechanism for repair, damage to the solid matrix may progress and lead to further degenerative changes in the biochemistry, morphology, and mechanical behavior of articular cartilage.

Centrifugal and biochemical comparison of proteoglycan aggregates from articular cartilage in experimental joint disuse and joint instability.

Two models involving altered joint loading were compared with regard to their effects on the biochemical composition and proteoglycan aggregate structure of articular cartilage. Disuse atrophy was created in greyhound dogs by nonrigid immobilization of the right knee in 90 degrees of flexion, and joint instability was created by transection of the anterior cruciate ligament. Similarities and differences between the two experimental groups at two different time periods were examined to investigate why joint instability induces progressive and irreversible changes to the articular cartilage, whereas joint disuse induces changes that may be reversible when the joint is remobilized. The following studies were performed on the cartilage from all experimental and control groups: (a) compositional analyses to determine water, uronate, and hydroxyproline contents; (b) high performance liquid chromatography for detection of hyaluronan and chondroitin sulfates; and (c) centrifugation analyses of nondissociatively extracted and purified proteoglycans to isolate and quantify the populations of monomers and slow and fast-sedimenting families of aggregates. In general, all cartilage was found to have a decreased ratio of proteoglycan to collagen after 4 weeks of disuse, and this ratio returned to control values at 8 weeks. In contrast, cartilage had an elevated ratio of proteoglycan to collagen as well as increased hydration at 12 weeks after transection of the anterior cruciate ligament. The most striking contrast between the two models was the finding of an approximately 80% decrease in the content of hyaluronan at both time periods after transection of the anterior cruciate ligament, with no evidence of a change after disuse. The results of centrifugation analyses indicated a significant decrease in the quantity of proteoglycan aggregates in both models. However, this decrease was associated primarily with a loss of slow-sedimenting aggregates after disuse and a loss of both slow and fast-sedimenting aggregates after transection of the anterior cruciate ligament. Furthermore, the population of fast-sedimenting aggregates was depleted to a greater extent than that of the slow-sedimenting aggregates. The preservation of fast-sedimenting aggregates as well as hyaluronan after periods of joint disuse but not joint instability suggests a possible mechanism for the reversibility of cartilage changes. Although the proteoglycan aggregates were depleted after disuse atrophy, it is possible that an aggregate-depleted matrix could recover when normal proteoglycan synthesis is resumed. In contrast, although synthesis may be maintained or elevated after transection of the anterior cruciate ligament, the matrix may not be repopulated with aggregates because there is an insufficient amount of hyaluronan.

Structural differences between two populations of articular cartilage proteoglycan aggregates.

To determine if articular cartilage contains structurally distinct populations of proteoglycan aggregates, we extracted and purified proteoglycans from canine knee cartilage under associative conditions. Equilibrium density gradient centrifugation separated three proteoglycan populations, on the basis of differences in sedimentation velocity, into groups of 21, 106, and 270 S. Electron microscopic examination showed that the 21 S samples contained free aggrecan molecules and clusters of aggrecan molecules, with a mean of five aggrecan molecules per cluster. The 106 and 270 S samples contained proteoglycan aggregates consisting of central hyaluronan filaments with multiple attached aggrecan molecules. The two populations of aggregates did not differ in mean aggrecan length or in the spacing of aggrecan molecules along the hyaluronan filaments, but the slower sedimenting aggregates (106 S) had significantly shorter hyaluronan filaments as measured by electron microscopy (mean hyaluronan length, 400 compared with 1,162 nm) and one-third as many aggrecan molecules per aggregate (mean number of aggrecan molecules per aggregate, 15 compared with 44). This study shows that articular cartilage contains aggrecan clusters and two structurally distinct populations of proteoglycan aggregates. The differences between the two types of aggregate, in particular the number of aggrecan molecules per aggregate, may reflect differences in their assembly, stability, or turnover and give them different mechanical and biological properties.

Altered structure-function relationships for articular cartilage in human osteoarthritis and an experimental canine model.

A review of the structure-function relationships for normal articular cartilage is provided. This provides the foundation for understanding the roles played by collagen, proteoglycan and water in determining the material properties of the tissue. A summary of biomechanical and compositional changes in human osteoarthritic cartilage is also presented. Finally, the results from our recent interdisciplinary study on an experimental osteoarthritis model is described, and new hypotheses are proposed on the initiating factors responsible for the increase of tissue hydration. At present, it appears that microstructural alterations, rather than compositional changes, of the collagen-proteoglycan solid matrix are responsible for the early increase of hydration and the deterioration of biomechanical properties of articular cartilage.

Treatment of osteoarthritis with tiaprofenic acid: biochemical and histological protection against cartilage breakdown in the Pond-Nuki canine model.

Experimental and cage matched control animals were sacrificed 12 weeks after production of ligamentous instability in the right knee, and biochemical studies were performed on eroded OA and normal articular cartilage. Significant protection was afforded by tiaprofenic acid administered orally at 15 mg/kg body weight. Chondroprotection was manifested by reduction of fast sedimenting proteoglycan aggregates, as well as retention of hyaluronate content, and favorable proteoglycan aggregate S value levels. This agent showed significant chondroprotective action under the conditions of these studies.

Macro and micro rate zonal analytical centrifugation of polydisperse and slowly diffusing sedimenting systems in isovolumetric density gradients. Application to cartilage proteoglycans.

A method to study the polydispersity of zonally sedimenting and slowly diffusing macromolecules or particles in isokinetic or isovolumetric density gradients is presented. First, a brief theory is given for predicting the zonal profile after a "triangular" (or "inverse") zone is centrifuged. This type of zone is essential to preserve hydrodynamic stability of the very slowly diffusing polydisperse solutes. It is proven, both by semitheoretical considerations and by computer calculations, that the resulting concentration profile of macrosolute is almost identical with that obtainable with a rectangular zone coextensive with the triangular one and carrying the same total mass. Next, practical procedures are described for the convectionless layering of very small triangular zones (50 microL or less). The linearity and stability of the zones are experimentally tested and verified. Finally, the method is applied to cartilage proteoglycan preparations that included either the monomeric molecules only or both the monomeric and the aggregated ones. The zonal results are compared with those obtained by using conventional boundary sedimentation. The two sets of results are seen to coincide fairly well, thus proving that the present technique can add to preparative zonal centrifugation the analytical precision of boundary sedimentation. A multimodal polydisperse system is suggested to describe the aggregated proteoglycan macromolecules.

Centrifugal characterization of proteoglycans from various depth layers and weight-bearing areas of normal and abnormal human articular cartilage.

Ultracentrifugal polydispersity differential [g(S)] distributions were determined for the proteoglycans of various postmortem human articular cartilage samples extracted from six lateral patellar grooves in nondissociative conditions after mild collagenase digestion of the tissue. The samples consisted of 53 slices (250 microns thick), from normal, mildly fibrillated, and extensively ulcerated knee joints. When statistically analyzed in various subgroupings, the obtained average sedimentation coefficients and polydispersity profiles supported the following conclusions: (a) loss of proteoglycan aggregation and sedimentability is confirmed to be a primary sign of cartilage matrix degradation; (b) higher S values for proteoglycans of the high weight (HW)-bearing areas and lower values for those of the low weight (LW)-bearing areas were a typical finding in normal cartilage samples; (c) inversion of this pattern was indicative of matrix degradation, suggesting that the HW regions are more affected than the LW-bearing areas; (d) the average S value distribution across cartilage thickness tended to resemble the corresponding proteoglycan content versus distance from articular surface; and (e) the deepest cartilage layer had, in most cases, the smallest amount of aggregates while the highest average sedimentability was observed at the middle zone of the normal samples. In the discussion, a role of proteoglycan aggregation for providing a means to "pack" more proteoglycans within the collagen meshwork and to control the generation of osmotic pressure gradients is suggested.