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.

Studies of craniofacial development in rotating bioreactors.

Several studies in our laboratory assessed the effect of 3-D culture in various rotating bioreactors on craniofacial development. Initially, mouse first branchial arches were cultured. Molar and incisor development occurred in both upper and lower jaws, but maxilla development was deficient because no brain was present. In a second study using excised whole heads, the oral epithelia fused and teeth did not develop. External structure of the face was obliterated, although internally, eye development was excellent. To preserve both internal spaces and external face structure, subsequent experiments used heads encapsulated in alginate. Teeth developed in these heads, though some interior components were necrotic. Additional experiments used older embryos, with already initiated structures, and less concentrated alginate. Orientation and unreserved identification of structures remain unresolved issues. Future studies will identify structures of interest using transcription factors unique to these structures at particular stages of fetal development.

Spatial and temporal localization of FGF-2 and VEGF in healing tooth extraction sockets in a rabbit model.

PURPOSE: The purpose of this study was to test the hypothesis that spatial and temporal localization of growth factors FGF-2 and VEGF in a rabbit tooth extraction socket model correlate with the histologic events of healing. MATERIALS AND METHODS: Twenty-four male New Zealand white rabbits divided into 8 groups of 3 were used in the study. Incisor teeth were extracted from both jaws and the healing extraction socket with surrounding jaw bone was harvested at 48 hours, 4 days, 1, 2, 4, 8, 12, and 16 weeks. Tissues were fixed, decalcified, and processed for hematoxylin-eosin and immunohistochemical staining. The sections were stained to detect FGF-2 and VEGF. The stained sections were then imaged and an automated computer program was used to detect the brown diaminobenzidine stain that represented the growth factors of interest. Data was obtained in the form of percentage area and intensity of stain and analyzed using the analysis of variance (ANOVA - Tukey Kramer and Scheffe's post-test). RESULTS: Spatial and temporal differences in localization of FGF-2 and VEGF were observed across all time frames in both jaws. Statistically significant differences in percentage area and intensity of brown diaminobenzidine stain were seen temporally between FGF-2 and VEGF (P < .05). CONCLUSION: The results of this study showed positive correlation of histologic events to spatial and temporal localization of FGF-2 and VEGF in a rabbit tooth extraction model.

Spatial and temporal localization of secretory IgA in healing tooth extraction sockets in a rabbit model.

PURPOSE: The purpose of this study was to look at the spatial and temporal localization of secretory IgA in healing tooth extraction sockets in a rabbit model. MATERIALS AND METHODS: Twenty-four male New Zealand White rabbits were used in the study. Incisor teeth were extracted from both jaws, and the healing extraction socket with the surrounding jaw bone was harvested at 48 hours, 4 days, and 1, 2, 4, 8, 12, and 16 weeks. Tissues were fixed, decalcified, and processed for hematoxylin and eosin and immunohistochemical staining. The sections were stained to detect secretory IgA. The stained sections were then imaged, and an automated computer program was used to detect the brown 3,3'-diaminobenzidine stain that represented the secretory IgA. The data were obtained in the form of percentage area and intensity of stain and analyzed using analysis of variance (Tukey-Kramer and Scheffé's tests). RESULTS: Spatial and temporal differences in localization of secretory IgA were observed across time frames in both jaws. CONCLUSION: The results of this study showed definite trends in the spatial and temporal localization of secretory IgA in healing tooth extraction sockets in a rabbit model.

Using cartilage to repair bone: an alternative approach in tissue engineering.

Materials and techniques currently used for bone replacement/repair conform to the current paradigm, relying on bone or bone products to produce bone or induce bone formation. Yet, nature forms and heals most of the skeleton by ossification of a cartilaginous model. In this study, we cultured aggregates of E10.5 or E12 mouse embryonic limb cells in the bioreactor for 3 weeks, determined the stages of cartilage differentiation attained, and assessed the ossification and bone healing potential of the spheroids by implantation adjacent to, or directly in, a skull defect. Cultured spheroids had large cartilaginous areas, sometimes with cellular arrangements characteristic of growth plate zones. Aggregates implanted for 2 weeks adjacent to a defect mineralized and ossified (histology, micro-CT). Defects with implants had a central mass of differentiated and differentiating bone, with osteoclast activity, filling the defect. Controls had considerable remodeling on the bone edges demarcating the still present defect. This study shows that cartilage, grown in the bioreactor for 3 weeks, ossified when implanted adjacent to a bone defect, and when implanted directly in a defect, contributed to its healing. Our ability to grow differentiated bone-forming cartilage for implantation is an alternative approach in the field of bone repair.

Spatial and temporal localization of transforming growth factor-beta1, bone morphogenetic protein-2, and platelet-derived growth factor-A in healing tooth extraction sockets in a rabbit model.

PURPOSE: The purpose of this study was to test the hypothesis that spatial and temporal localization of growth factors transforming growth factor (TGF)-beta1, bone morphogenetic protein (BMP)-2, and platelet derived growth factor (PDGF)-A in a rabbit tooth extraction model correlate with the histologic events contributing toward healing. MATERIALS AND METHODS: Twenty-four male New Zealand White rabbits were used in the study. Incisor teeth were extracted from both jaws, and the healing extraction socket with the surrounding jaw bone was harvested at 48 hours, 4 days, and 1, 2, 4, 8, 12, and 16 weeks. Tissues were fixed, decalcified, and processed for hematoxylin and eosin and immunohistochemical staining. The sections were stained to detect TGF-beta1, BMP-2, and PDGF-A. Stained sections were then imaged, and an automated computer program was used to detect the brown 3,3'-diaminobenzidine regions representing the location of growth factors of interest. The data were collected in terms of percentage area and intensity of stain, and an analysis of variance was conducted (Tukey-Kramer and Scheffe's test) for statistical comparison between different time points, jaws, and growth factors. These results were also compared with hematoxylin and eosin-stained histologic specimens obtained at similar time points. RESULTS: Spatial and temporal differences in localization of TGF-beta1, BMP-2, and PDGF-A were observed across all time frames in both jaws. Statistically significant differences in percentage area and intensity of brown diaminobenzidine stain were detected temporally between TGF-beta1, BMP-2, and PDGF-A (P < or =.0001). CONCLUSION: The results of this study showed positive correlation between histologic events and the spatial and temporal localization of TGF-beta1, BMP-2, and PDGF-A in a rabbit tooth extraction model.

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.

Galacturonomannan and Golgi-derived membrane linked to growth and shaping of biogenic calcite.

The coccolithophores are valuable models for the design and synthesis of composite materials, because the cellular machinery controlling the nucleation, growth, and patterning of their calcitic scales (coccoliths) can be examined genetically. The coccoliths are formed within the Golgi complex and are the major CaCO(3) component in limestone sediments-particularly those of the Cretaceous period. In this study, we describe mutants lacking a sulfated galacturonomannan and show that this polysaccharide in conjunction with the Golgi-derived membrane is directly linked to the growth and shaping of coccolith calcite but not to the initial orientated nucleation of the mineral phase.

Comorbid non-alcohol substance misuse among people with schizophrenia: epidemiological study in central London.

BACKGROUND: Few epidemiological studies have assessed the extent and nature of comorbid non-alcohol substance misuse in people with schizophrenia in the community in the UK. AIMS: To study the extent and nature of comorbid non-alcohol substance misuse in people with schizophrenia in central London. METHOD: Subjects were identified in an epidemiological census survey of South Westminster. Standardised assessment of each subject included demographic data, ratings of mental state and movement disorder and questioning about drug and alcohol misuse. RESULTS: Individuals with schizophrenia or related psychoses were identified (n=352) and 57 (16%) reported a lifetime history of non-alcohol substance misuse. Age and gender were the main variables relevant to the extent and pattern of misuse. Self-reported non-alcohol substance misuse showed no significant relationship with a range of outcome measures. CONCLUSIONS: The high proportion of subjects reporting non-alcohol substance misuse is comparable with figures from the USA. The reports of lifetime misuse most commonly referred to cannabis, psychostimulants, LSD, opiates and anticholinergics. Misuse was concentrated in those younger than 36 years and was reported more often by males.

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.

The Spacelab 3 simulation: basis for a model of growth plate response in microgravity in the rat.

Data from Spacelab 3 (SL3) suggested that spaceflight significantly reduces the activity of the rat tibial growth plate. Animal processing after SL3 began twelve hours post-landing, so data reflect post-flight re-adaptation in addition to spaceflight effects. To determine if a twelve-hour period of weight bearing after seven days of unloading could affect the physes of spaceflown rats, the present study assessed the growth plate response to unloading with or without a reloading period. Rats were subjected to hind-limb suspension for seven days and then euthanized, with or without twelve hours of reloading. Activity of the growth plate was assessed by morphometric analysis. Rats suspended without reloading had reserve zone (RZ) height greater than controls, and shorter hypertrophy/calcification zone (HCZ) with fewer cells. The greater RZ was associated with a larger cell area, indicating a possible mitotic delay or secretion defect. Twelve hours of reloading decreased RZ height and cell number, and restored the number of cells in HCZ to control values, but the number of cells in the proliferative zone and height in HCZ were reduced. These results suggest the rebound response to preserve/restore skeletal function after a period of unloading involves an acceleration of growth associated with a decreased cell cycle time in PZ. Changes during the reloading period in this simulation support our hypothesis that the effects of spaceflight on SL3 growth plates were altered by changes that occurred post-landing. The similarities in response to unloading by suspension or during spaceflight are used to propose a model of growth plate response during spaceflight.

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.

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.

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.

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.

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.

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.

Effects of long-term hypergravity on muscle, heart and lung structure of mice.

Quantitative changes in lung, heart and muscle structure were assessed in mice exposed for 14 weeks to a gravitational field of 3 G since the age of 4 weeks; matched controls were kept at normal gravity (1 G). The body mass of 3-G-exposed mice was significantly reduced by 9%, while total skeletal muscle mass remained the same fraction of body mass. The mass of the soleus muscle was found to be significantly larger in 3-G-exposed mice both in absolute (+27%) and body mass specific terms (+42%). Capillary density was significantly reduced by 22% because of a relatively larger increase of fiber cross-sectional area (+47%) than of capillary to fiber ratio (+16%). Other morphometric variables remained unchanged with hypergravity. Heart mass and mitochondrial volume were both larger in 3-G-exposed mice (+15% and +27%, respectively). This difference reached statistical significance when normalized to body mass. The only significant difference in lung structure detectable by morphometric methods were a smaller volume (-9%), that paralleled lower body mass, and thinner alveolar septa (-12%). From these results it is concluded that the lung's support structures in mice are sufficiently strong to withstand the stress of long-term hypergravity; however, 3-G exposure leads to a selective hypertrophy of soleus muscle fibers while absolute capillary length in this muscle remains unaltered.

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.

Cognitive function and duration of rooflessness in entrants to a hostel for homeless men.

BACKGROUND: Four previous studies of homeless adults have yielded conflicting results regarding the presence of cognitive impairment. METHOD: A consecutive series of 80 roofless entrants to a hostel for homeless men were sampled and 62 (76%) completed a range of assessments, including measures of mental state, cognitive functions and substance use. RESULTS: Estimated premorbid IQ (mean = 96), current IQ (mean = 84) and cognitive speed were significantly lower than the norm. There was a significant IQ drop in all diagnostic groups. IQ drop, but not current IQ, correlated with duration of rooflessness. Those with schizophrenia or alcohol problems were roofless for longest. Alcohol misuse did not correlate with IQ drop, excepting alcohol withdrawal symptoms in those with schizophrenia. CONCLUSION: The hypothesis that low IQ is a risk factor for rooflessness is supported. However, length of rooflessness was more closely related to IQ drop than to current IQ, suggesting that some third factor may be affecting both rooflessness and intellectual functioning. Roofless men with schizophrenia or alcohol problems may be especially at risk of long-term rooflessness.