CORRELATION BETWEEN MICRO-CT SECTIONS AND HISTOLOGICAL SECTIONS OF MOUSE SKULL DEFECTS IMPLANTED WITH ENGINEERED CARTILAGE.

One advantage of using cartilage to replace/repair bone is that the implant disappears as bone is formed by endochondral ossification. Previously, we showed that cartilage spheroids, grown in a rotating bioreactor (Synthecon, Inc.) and implanted into a 2 mm skull defect, contributed to healing of the defect. Skulls with or without implants were subjected to microCT scans. Mineralized regions from microCT sections correlated with regions of bone in histological sections of the defect region of demineralized skulls. Recently, sections from microCT scans of live mice were compared to histological sections from the same mice. The area of the defect staining for bone in histological sections of demineralized skulls was the same region shown as mineralized in microCT sections. Defects without implants were not healed. This study demonstrates that microCT scans are an important corollary to histological studies evaluating the use of implants in healing of bony defects.

Cytoskeletal abnormalities in chondrocytes with EXT1 and EXT2 mutations.

The EXT genes are a group of putative tumor suppressor genes that previously have been shown to participate in the development of hereditary multiple exostoses (HME), HME-associated and isolated chondrosarcomas. Two HME disease genes, EXT1 and EXT2, have been identified and are expressed ubiquitously. However, the only known effect of mutations in the EXT genes is on chondrocyte function as evidenced by aberrant proliferation of chondrocytes leading to formation of bony, cartilage-capped projections (exostoses). In this study, we have characterized exostosis chondrocytes from three patients with HME (one with EXT1 and two with EXT2 germline mutations) and from one individual with a non-HME, isolated exostosis. At the light microscopic level, exostosis chondrocytes have a stellate appearance with elongated inclusions in the cytoplasm. Confocal and immunofluorescence of in vitro and in vivo chondrocytes showed that these massive accumulations are composed of actin bundled by 1.5-microm repeat cross-bridges of alpha-actinin. Western blot analysis shows that exostosis chondrocytes from two out of three patients aberrantly produce high levels of muscle-specific alpha-actin, whereas beta-actin levels are similar to normal chondrocytes. These findings suggest that mutations in the EXT genes cause abnormal processing of cytoskeleton proteins in chondrocytes.

Retention of cartilage oligomeric matrix protein (COMP) and cell death in redifferentiated pseudoachondroplasia chondrocytes.

Cartilage oligomeric matrix protein (COMP) is a large extracellular glycoprotein that is found in the territorial matrix surrounding chondrocytes. Two skeletal dysplasias, pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (EDM1) are caused by mutations in the calcium binding domains of COMP. In this study, we identified two PSACH mutations and assessed the effect of these mutations on redifferentiated chondrocyte structure and function. We confirmed, in vitro, that COMP is retained in enormous cisternae of the rough endoplasmic reticulum (rER) and relatively absent in the PSACH matrix. The rER accumulation may compromise chondrocyte function, leading to chondrocyte death. Moreover, while COMP appears to be deficient in the PSACH matrix, the matrix appeared to be normal but the over-all quantity was reduced. These results suggest that the abnormality in linear growth in PSACH may result from decreased chondrocyte numbers which would also affect the amount of matrix produced.

Calreticulin, PDI, Grp94 and BiP chaperone proteins are associated with retained COMP in pseudoachondroplasia chondrocytes.

Cartilage oligomeric matrix protein (COMP), a large pentameric glycoprotein and member of the thrombospondin (TSP) group of extracellular proteins, is found in the territorial matrix surrounding chondrocytes. More than 50 unique COMP mutations have been identified as causing two skeletal dysplasias: pseudoachondroplasia (PSACH); and multiple epiphyseal dysplasia (EDM1). Recent studies suggest that calcium-binding and calcium-induced protein folding differ between wild type and mutant proteins, and abnormal processing of the mutant COMP protein contributes to the characteristic enlarged lamellar appearing rER cisternae in PSACH and EDMI chondrocytes in vivo and in vitro. Towards the goal of delineating the pathogenesis of PSACH and EDM1, in-vivo PSACH growth plate and in-vitro PSACH chondrocytes cultured in alginate beads were examined to identify and localize the chaperone proteins participating in the processing of the retained extracellular matrix proteins in the PSACH rER. Aggrecan was localized to both the rER cisternae and matrix while COMP and type IX collagen were only found in the rER. Type II collagen was solely found in the ECM suggesting that it is processed and transported differently from other retained ECM proteins. Five chaperone proteins: BiP (Grp78); calreticulin (CRT); protein disulfide (PDI); ERp72; and Grp94, demonstrated immunoreactivity in the enlarged PSACH cisternae and the short rER channels of chondrocytes from both in-vivo and in-vitro samples. The chaperone proteins cluster around the electron dense material within the enlarged rER cisternae. CRT, PDI and GRP94 AB-gold particles appear to be closely associated with COMP. Immunoprecipitation and Western blot, and Fluorescence Resonance Energy Transfer (FRET) analyses indicate that CRT, PDI and GRP94 are in close proximity to normal and mutant COMP and BiP to mutant COMP. These results suggest that these proteins play a role in the processing and transport of wild type COMP in normal chondrocytes and in the retention of mutant COMP in PSACH chondrocytes.

Four behavioural syndromes of schizophrenia: a replication in a second inner-London epidemiological sample.

In a previous large epidemiological survey of patients with strictly defined schizophrenia in the London borough of Camden, we extracted four behavioural syndromes (Social withdrawal, Thought disturbance, Anti-social behaviour and Depressed behaviour) by factor analysis of MRC Social Behaviour Schedule (SBS) data. These syndromes had significant differential relationships to symptoms assessed using the Manchester Scale (MS), symptom-derived syndromes, and social functioning variables. A second inner-London epidemiological survey of schizophrenia in South Westminster using identical methodology found the same four behavioural syndromes with identical core component items. The same four behavioural syndromes were extracted, whether applying strict Feighner diagnostic criteria (n=112) or broader DSM-III-R criteria (n=198). The four syndromes extracted from the Feighner positive sample showed relationships to symptoms and social functioning variables similar to those found in the original Camden study. However, the symptom-derived factors were not the same and did not conform to the three recognised symptom-based syndromes of schizophrenia. This successful replication suggests that assessment of the four behavioural syndromes of schizophrenia offers a different perspective on disability and a potentially relevant measure in clinical practice, clinical trials and studies of the neuropsychology and pathophysiology of schizophrenia.

Detecting comorbid substance misuse among people with schizophrenia in the community: a study comparing the results of questionnaires with analysis of hair and urine.

Substance misuse among people with schizophrenia is thought to be common and to adversely affect the outcome of the illness. The shortcomings of studies in this area include patient samples that are not epidemiologically-based, and methods for detecting substance misuse that have serious limitations. We investigated the frequency and severity of substance misuse among people with schizophrenia living in the community in London. Interviews were conducted with a community-based sample of 39 people with schizophrenia aged 35 years or less, living in Inner London. The assessments included ratings of psychopathology, movement disorders and substance misuse, and co-informant histories. Urine and hair specimens were analysed for a range of substances. Urine samples were collected from 37 patients and hair samples were provided by 36 patients. Comorbid substance misuse was reported or detected in 63% of the sample. The information elicited using a structured questionnaire for both informants and subjects represented an under-estimate of psychostimulant misuse and opiate misuse compared with the results obtained by hair or urine analysis. Hair analysis revealed that 12 (33%) of those patients providing samples had covertly abused amphetamines, opiates or cocaine in the previous 3 months. The study demonstrated that hair analysis is a well-tolerated, sensitive test for substance misuse. The technique has several advantages over questionnaires and urine analysis for clinical and research purposes. Further applications include the assessment of comorbid substance use in particular groups of patients with schizophrenia, such as during first-episode or psychotic relapse, or those with forensic problems or apparent resistance to treatment.

Spaceflight and age affect tibial epiphyseal growth plate histomorphometry.

Growth plate histomorphometry of rats flown aboard the Soviet biosatellite COSMOS 2044, a 14-day spaceflight, was compared with that of control groups. In growth plates of flight animals, there was a significant increase in cell number per column and height of the proliferative zone and a reduction in height and cell number in the hypertrophy/calcification zone. No significant differences were found in matrix organization at the ultrastructural level of flight animals, indicating that although spaceflight continues to affect bone growth of 15-wk-old rats, extracellular matrix is not altered in the same manner as seen previously in younger animals. All groups showed growth plate characteristics attributed to aging: lack of calcification zone, reduced hypertrophy zone, and unraveling of collagen fibrils. Tail-suspended controls did not differ from other controls in any of the parameters measured. Our results suggest that growth plates of older rats are less responsive to unloading by spaceflight or suspension than those of younger rats and provide new evidence about the modifying effect of spaceflight on the growth plate.

Stereochemistry of iron in deoxyhaemoglobin.

The EXAFS of human deoxyhaemoglobin closely resembles that of a synthetic model in which the displacement of the iron from the mean porphyrin plane is 0.426 +/- 0.004 A, similar to the displacement of 0.56 +/- 0.03 A found by single crystal X-ray analysis of deoxyhaemoglobin. We find the same Fe-N of 2.06 +/- 0.01 A distance as Eisenberger et al., but show that the displacement of the iron from the nitrogen plane cannot be calculated from that distance.

Exposure to altered gravity affects all stages of endochondral cartilage differentiation.

Chondrogenesis has a number of well-defined steps: (1) condensation, which involves cell aggregation, adhesion and communication; (2) activation of cartilage genes, which is accompanied by rounding up of the cells and intracellular differentiation; and (3) production and secretion of cartilage specific matrix molecules. Our studies show that each of these steps is affected by exposure to gravitational changes. Clinorotation and centrifugation affected initial aggregation and condensation. In the CELLS experiment, where cells were exposed to microgravity after some condensation occurred preflight, intracellular differentiation and matrix production were delayed relative to controls. Once cartilage has developed, in rats, further differentiation (hypertrophy, matrix production) was also affected by spaceflight and hind limb suspension. For the process of chondrogenesis to proceed as we know it, loading and other factors present at 1g are required at each step of the process. This requirement means that not only will skeletal development and bone healing, processes involving chondrogenesis, be altered by long term exposure to microgravity, but that continuous intervention will be necessary to correct any defects produced by altered gravity environments.

Teratogenic effects of gravitational changes.

In investigating the effect of gravitational changes on development, it is instructive to think of altered gravity (delta g) as a teratogen--that is, an environmental factor influencing development. Observed effects on skeletal development include: suppression of morphogenesis in centrifuged mouse limb buds; advanced fusion stages in centrifuged mouse palates; smaller crown rump lengths (CRL) and decreased number of pregnancies in centrifuged rats and mice; altered differentiation of growth plates in young growing rats in space; and decreased length of calcified long bone regions in fetal rats exposed to microgravity in utero. These studies show that delta g is able to alter development in vivo and in vitro and suggest that delta g operates, at least in part, at the cellular level.

Differentiation of cartilaginous anlagen in entire embryonic mouse limbs cultured in a rotating bioreactor.

Mechanisms involved in development of the embryonic limb have remained the same throughout eons of genetic and environmental evolution under Earth gravity (1 g). During the spaceflight era it has been of interest to explore the ancient theory that form of the skeleton develops in response to gravity, and that changes in gravitational forces can change the developmental pattern of the limb. This has been shown in vivo and in vitro, allowing the hypergravity of centrifugation and microgravity of space to be used as tools to increase our knowledge of limb development. In recapitulations of spaceflight experiments, premetatarsals were cultured in suspension in a bioreactor, and found to be shorter and less differentiated than those cultured in standard culture dishes. This study only measured length of the metatarsals, and did not account for possible changes due to the skeletal elements having a more in vivo 3D shape while in suspension vs. flattened tissues compressed by their own weight. A culture system with an outcome closer to in vivo and that supports growth of younger limb buds than traditional systems will allow studies of early Hox gene expression, and contribute to the understanding of very early stages of development. The purpose of the current experiment was to determine if entire limb buds could be cultured in the bioreactor, and to compare the growth and differentiation with that of culturing in a culture dish system. Fore and hind limbs from E11-E13 ICR mouse embryos were cultured for six days, either in the bioreactor or in center-well organ culture dishes, fixed, and embedded for histology. E13 specimens grown in culture dishes were flat, while bioreactor culture specimens had a more in vivo-like 3D limb shape. Sections showed excellent cartilage differentiation in both culture systems, with more cell maturation, and hypertrophy in the specimens cultured in the bioreactor. Younger limb buds fused together during culture, so an additional set of E11.5 limb buds was cultured with and without encapsulation in alginate prior to culturing in the bioreactor. Encapsulated limbs grown in the bioreactor did not fuse together, but developed only the more proximal elements while limbs grown in culture dishes formed proximal and distal elements. Alginate encapsulation may have reduced oxygenation to the progress zone of the developing limb bud resulting in lack of development of the more distal elements. These results show that the bioreactor supports growth and differentiation of skeletal elements in entire E13 limb buds, and that a method to culture younger limb buds without fusing together needs to be developed if any morphometric analysis is to be performed.

In vivo and in vitro studies of cartilage differentiation in altered gravities.

The in vivo model our laboratory uses for studies of cartilage differentiation in space is the rat growth plate. Differences between missions, and in rat age and recovery times, provided differing results from each mission. However, in all missions, proliferation and differentiation of chondrocytes in the epiphyseal plate of spaceflown rats was altered as was matrix organization. In vitro systems, necessary complements to in vivo work, provide some advantages over the in vivo situation. In vitro, centrifugation of embryonic limb buds suppressed morphogenesis due to precocious differentiation, and changes in the developmental pattern suggest the involvement of Hox genes. In space, embryonic mouse limb mesenchyme cells differentiating in vitro on IML-1 had smoother membranes and lacked matrix seen in controls. Unusual formations, possibly highly ruffled membranes, were found in flight cultures. These results, coupled with in vivo centrifugation studies, show that in vivo or in vitro, the response of chondrocytes to gravitational changes follows Hert's curve as modified by Simon, i.e. decreased loading decreases differentiation, and increased loading speeds it up, but only to a point. After that, additional increases again slow down chondrogenesis.

Clinorotation reduces number, but not size, of cartilaginous nodules formed in micromass cultures of mouse limbbud cells.

In previous studies we used a ground based model to investigate the cellular responses to microgravity by exposing micromass cultures of embryonic limb cells to simulated weightlessness on a clinostat. Cultures set up in T-flasks and rotated at 30 rpm showed that clinostatted cultures had less chondrocyte differentiation than stationary or rotation controls, as assessed by number of nodules/culture stained with cartilage specific Alcian blue. In the current study, nodule size and shape of these nodules was assessed by interactive measurement of area, perimeter, circularity, and equivalent diameters, using the Optimas imaging software. Results show no significant difference in any of the measurements, indicating that clinorotation has no effect on expansion of the nodules either by differentiation of cells within the nodule, or by recruitment of cells into the nodule. The reduction in number of nodules without an alteration in size and shape indicates that the effect of simulated microgravity is to reduce the cell interactions required for the initial condensation of cells into a nodule, probably by interference with cell adhesion molecules.

Gravitational changes affect tibial growth plates according to Hert's curve.

BACKGROUND: Microgravity significantly affects chondrocyte differentiation within the tibial epiphyseal growth plate of space flown rats. The changes produced in height and number of cells in different zones of the plate are associated with ultrastructural changes in the extracellular matrix. Given the importance of the growth plate in endochondral ossification, we began to assess the response of the plate to hypergravity, and the countermeasure value of excess G. METHODS: Rats of the strain used in Cosmos biosatellite missions were housed under conditions similar to Cosmos flights and subjected to continuous hypergravity (2 G) for 14 d, in a 12-ft radius centrifuge. RESULTS: Histomorphometrical analyses of tibial growth plates from these rats found the hypertrophic/calcification zone to be significantly reduced in both height and cell number, and the proliferation zone in cell number. CONCLUSIONS: These results, along with those of spaceflight and of studies using suspension-centrifugation, indicate that rat growth plate responds to gravitational changes according to Hert's curve: i.e., a) an increased baseline (minimal) loading reduces cartilage differentiation; and b) a reduced baseline loading may lead to increased cartilage differentiation but only within a range, beyond which lack of differentiation results. The plasticity of the plate, i.e., its ability to increase or decrease its activity in response to changes in gravity suggests the possibility of a range of G that will produce the load necessary to maintain normal growth of the plate, i.e., possible countermeasures to the effects of either hypo- or hyper-gravity.

The effects of gravitational forces on reproduction and development.

It is clear that increased gravitational forces can affect biologic reproduction and development, although exact mechanisms of action have not been established. Except for three studies of male pilot and astronaut fertility, human reproduction and development studies of chronic or acute exposure to micro- or hypergravity environments have not been carried out. Only two reproduction and development studies (both Soviet) have been done under chronic or acute exposure to microgravity. It is unlikely that chronic human exposure to hypergravity will occur except when planets much larger than Earth are colonized. Colonies dependent on reproduction for continued existence probably could not be established on planets with masses significantly dissimilar to Earth because hypergravity or hypogravity are likely to affect reproduction adversely and cause abnormal growth and development. Acute exposures to gravitational changes in the later stages of development appear to be less detrimental, but much remains to be discovered about how gravity affects reproduction and development of mammals, humans in particular. Earth's gravitational force has helped to shape human reproduction and development. Only when the limits of mammalian biology on both sides of 1 g are explored will we be able to determine the extent to which gravity determines biologic structure and function.