Microcomputed tomography imaging in a rat model of delayed union/non-union fracture.

We aimed to develop a clinically relevant delayed union/non-union fracture model to evaluate a cell therapy intervention repair strategy. Histology, three-dimensional (3D) microcomputed tomography (micro-CT) imaging and mechanical testing were utilized to develop an analytical protocol for qualitative and quantitative assessment of fracture repair. An open femoral diaphyseal osteotomy, combined with periosteal diathermy and endosteal excision, was held in compression by a four pin unilateral external fixator. Three delayed union/non-union fracture groups established at 6 weeks--(a) a control group, (b) a cell therapy group, and (c) a group receiving phosphate-buffered saline (PBS) injection alone--were examined subsequently at 8 and 14 weeks. The histological response was combined fibrous and cartilaginous non-unions in groups A and B with fibrous non-unions in group C. Mineralized callus volume/total volume percentage showed no statistically significant differences between groups. Endosteal calcified tissue volume/endosteal tissue volume, at the center of the fracture site, displayed statistically significant differences between 8 and 14 weeks for cell and PBS intervention groups but not for the control group. The percentage load to failure was significantly lower in the control and cell treatment groups than in the PBS alone group. High-resolution micro-CT imaging provides a powerful tool to augment characterization of repair in delayed union/non-union fractures together with outcomes such as histology and mechanical strength measurement. Accurate, nondestructive, 3D identification of mineralization progression in repairing fractures is enabled in the presence or absence of intervention strategies.

Degradation of poly-L-lactide. Part 1: in vitro and in vivo physiological temperature degradation.

Poly-L-lactide (PLLA) is one of the most significant members of a group of polymers regarded as bioresorbable. The degradation of PLLA proceeds through hydrolysis of the ester linkage in the polymer's backbone and is influenced by the polymer's initial molecular weight and degree of crystallinity. To evaluate its degradation PLLA pellets were processed by compression moulding into tensile test specimens and by extrusion into 2 mm diameter lengths of rod, prior to being sterilized by ethylene oxide gas (EtO) and degraded in both in vitro and in vivo environments. On retrieval at predetermined time intervals, procedures were used to evaluate the material's molecular weight, crystallinity, mechanical strength, and thermal properties. Additionally, the in vivo host tissue's biological response was analysed. The results from this study suggest that in both the in vitro and in vivo environments, degradation proceeded at the same rate and followed the general sequence of aliphatic polyester degradation, ruling out enzymes contributing and accelerating the degradation rate in vivo. Additionally, the absence of cells marking an inflammatory response suggests that the PLLA rods investigated in vivo were biocompatible throughout the 44 weeks duration of the study, before any mass loss was observed.

Degradation of poly-L-lactide. Part 2: increased temperature accelerated degradation.

Poly-L-lactide (PLLA) is one of the most significant members of a group of polymers regarded as bioresorbable. The degradation of PLLA proceeds through hydrolysis of the ester linkages in the polymer's backbone; however, the time for the complete resorption of orthopaedic devices manufactured from PLLA is known to be in excess of five years in a normal physiological environment. To evaluate the degradation of PLLA in an accelerated time period, PLLA pellets were processed by compression moulding into tensile test specimens, prior to being sterilized by ethylene oxide gas (EtO) and degraded in a phosphate-buffered solution (PBS) at both 50 degrees C and 70 degrees C. On retrieval, at predetermined time intervals, procedures were used to evaluate the material's molecular weight, crystallinity, mechanical strength, and thermal properties. The results from this study suggest that at both 50 degrees C and 70 degrees C, degradation proceeds by a very similar mechanism to that observed at 37 degrees C in vitro and in vivo. The degradation models developed also confirmed the dependence of mass loss, melting temperature, and glass transition temperature (Tg) on the polymer's molecular weight throughout degradation. Although increased temperature appears to be a suitable method for accelerating the degradation of PLLA, relative to its physiological degradation rate, concerns still remain over the validity of testing above the polymer's Tg and the significance of autocatalysis at increased temperatures.

Increased femoral neck cancellous bone and connectivity in coxarthrosis (hip osteoarthritis).

Patients with coxarthrosis (cOA) have a reduced incidence of intracapsular femoral neck fracture, suggesting that cOA offers protection. The distribution of bone in the femoral neck was compared in cases of coxarthrosis and postmortem controls to assess the possibility that disease-associated changes might contribute to reduced fragility. Whole cross-section femoral neck biopsies were obtained from 17 patients with cOA and 22 age- and sex-matched cadaveric controls. Densitometry was performed using peripheral quantitated computed tomography (pQCT) and histomorphometry on 10-microm plastic-embedded sections. Cortical bone mass was not different between cases and controls (P > 0.23), but cancellous bone mass was increased by 75% in cOA (P = 0.014) and histomorphometric cancellous bone area by 71% (P < 0.0001). This was principally the result of an increase of apparent density (mass/vol) of cancellous bone (+45%, P = 0.001). Whereas cortical porosity was increased in the cases (P < 0.0001), trabecular width was also increased overall in the cases by 52% (P < 0.001), as was cancellous connectivity measured by strut analysis (P < 0.01). Where osteophytic bone was present (n = 9) there was a positive relationship between the amount of osteophyte and the percentage of cancellous area (P < 0.05). Since cancellous bone buttresses and stiffens the cortex so reducing the risk of buckling, the increased cancellous bone mass and connectivity seen in cases of cOA probably explain, at least in part, the ability of patients with cOA to resist intracapsular fracture of the femoral neck during a fall.

Evaluation of locally induced osteoarthritis by the complete and incomplete Freund's adjuvant in mice. The application of DEXA measurements.

The inflammatory reactions elicited in mice by subcutaneous injections of IFA and CFA had opposite effects when tested on local metacarpal shank bones and the distal epiphysis of shank bones. Although the intensity of the immune reactions was similar, IFA induced bone loss, while CFA induced bone formation, which was mostly periosteal in nature. BMC and BMD measurements were assessed by means of high resolution DEXA, using a hologic 4500A bone scanner with software dedicated for the analysis of small animal bones. DEXA scans were evaluated and related to histological and bone ash content analyses. The morphological and quantitative ash weight analyses of bones exposed to the adjuvants were consistent with DEXA bone density scan measurements.

Three-dimensional cultures of normal human osteoblasts: proliferation and differentiation potential in vitro and upon ectopic implantation in nude mice.

We report the establishment in vitro of three-dimensional (3D) cultures of human osteoblasts (hOB) derived from normal adults and supported uniquely by the extracellular matrix (ECM) they deposit. Osteoblasts were cultured in 3D cultures in vitro for up to 120 days. The 3D cultures, examined at 25, 31, and 48 days, expressed protein markers of osteoblastic cells, namely osteonectin, collagen type I, fibronectin, osteopontin, bone sialoprotein, biglycan, and decorin. Sequentially, alkaline phosphatase (AP) and then Ca incorporation, mineralization of matrix (monitored by histochemistry and transmission electron microscopy), and finally osteocalcin expression, were detected in the 3D cultures. Ultrastructurally, morphology progressed from early to mature osteoblast and to osteocyte-like. Cells were embedded in a matrix with organized collagen type I fibers containing, increasingly with time of culture, needle-shaped crystals, often associated with matrix vesicles, characteristic of those in bone. During the culture (up to 120 days) there was an outgrowth of proliferating osteogenic cells from the 3D structure. Subcutaneous implantation in nude mice for 20 days of osteoblasts cultured in 3D culture for different lengths of time in vitro, showed progression of mineralization from the inner region of the implant outward, with peripheral cells being embedded in nonmineralized, collagen-rich matrix. The 3D implants were invaded by vessels derived from the host.

A negative search for a paramyxoviral etiology of Paget's disease of bone: molecular, immunological, and ultrastructural studies in UK patients.

Paget's disease of bone is a common bone disease characterized by increased and disorganized bone remodeling at focal sites throughout the skeleton. The etiology of the disease is unresolved. A persistent viral infection has long been suggested to cause the disease. Antigen and/or nucleic acid sequences of paramyxoviruses (in particular measles virus [MV], canine distemper virus [CDV], and respiratory syncytial virus [RSV]) have been reported in pagetic bone by a number of groups; however, others have been unable to confirm this and so far no virus has been isolated from patients. Here, we reexamined the question of viral involvement in Paget's disease in a study involving 53 patients with established disease recruited from seven centers throughout the United Kingdom. Thirty-seven patients showed clear signs of active disease by bone scan and/or histological assessment of the bone biopsy specimens and 12 of these had not received any therapy before samples were taken. Presence of paramyxovirus nucleic acid sequences was sought in bone biopsy specimens, bone marrow, or peripheral blood mononuclear cells using reverse-transcription polymerase chain reaction (RT-PCR) with a total of 18 primer sets (7 of which were nested), including 10 primer sets (including 3 nested sets) specifically for MV or CDV. For each patient at least one sample was tested with all primer sets by RT-PCR and no evidence for the presence of paramyxovirus RNA was found in any patient. In 6 patients, bone biopsy specimens with clear histological evidence of active disease tested negative for presence of measles and CDV using immunocytochemistry (ICC) and in situ hybridization (ISH). Intranuclear inclusion bodies, similar to those described by others previously, were seen in pagetic osteoclasts. The pagetic inclusions were straight, smooth tubular structures packed tightly in parallel bundles and differed from nuclear inclusions, known to represent MV nucleocapsids, in a patient with subacute sclerosing panencephalitis (SSPE) in which undulating, diffuse structures were found, arranged loosely in a nonparallel fashion. In the absence of amplification of viral sequences from tissues that contain frequent nuclear inclusions and given that identical inclusions are found in other bone diseases with a proven genetic, rather than environmental, etiology, it is doubtful whether the inclusions in pagetic osteoclasts indeed represent viral nucleocapsids. Our findings in this large group of patients recruited from throughout the United Kingdom do not support a role for paramyxovirus in the etiology of Paget's disease.

Visualisation of three-dimensional microcracks in compact bone.

Microdamage in bone contributes to the loss of bone quality in osteoporosis and is thought to play a major role in both fragility and stress fractures (Schaffler et al. 1995). In this study, in vivo microcracks in human ribs were bulk-stained in basic fuchsin and viewed in longitudinal section and in 3 dimensions using 2 different computer-based methods of reconstruction: (1) serial sectioning of methylmethacrylate embedded sections using a sledge macrotome and identification of microcracks using UV epifluorescence followed by computerised reconstruction of microcracks using software and (2) laser scanning confocal microscopy of thick sections followed by reconstruction of microcracks into a 3-D image. The size and shape of microcracks were found to be similar using both techniques. Both techniques of reconstruction showed microcracks to be approximately elliptical in shape. From the serial sectioning reconstructions (n = 9), microcracks were found to have a mean length of 404 +/- 145 microm (mean +/- S.D.) (in the longitudinal direction) and mean width of 97 +/- 38 microm (in the transverse direction). Using epifluorescence microscopy, 92 microcracks were identified; mean microcrack length was 349 +/- 100 microm in the longitudinal direction. This was consistent with other results (Burr & Martin, 1993) and with the theoretical prediction of an elliptical crack shape with aspect ratio (longitudinal: transverse) of 5:1 deduced from analysis of random 2-D sections (Taylor & Lee, 1998). The results obtained provide new data on the nature of microcracks in bone and the method has the potential to become a useful tool in the calculation of stress intensity values which indicate the probability of an individual microcrack propagating to cause a stress or fragility fracture.

The expression of metalloproteinase-2, -9, and -14 and of tissue inhibitors-1 and -2 is developmentally modulated during osteogenesis in vitro, the mature osteoblastic phenotype expressing metalloproteinase-14.

During osteogenesis, in vitro, of tibial-derived rat osteoblasts (ROB) and derived clones, changes occur in the interactions of mature osteoblasts with the endogenous extracellular matrix (ECM) and these culminate in the formation of tridimensional nodules, which become sites of mineral deposition. We investigated if these changes might be mediated by remodeling of ECM, and we focused our study on the neutral metalloproteinases (MMPs), known agents of matrix remodeling, and on their tissue inhibitors (TIMPs). We report that during in vitro differentiation, osteoblasts express the secreted MMP-2 and -9 and the membrane gelatinase MMP-14. These, along with the tissue inhibitors TIMP-1 and -2, are developmentally regulated according to the maturation stage of osteoblasts. Their levels change in a similar association with osteoblast phenotypic maturation in different populations of ROB, which take different times to complete osteogenesis in vitro. MMP-14 expression coincides in both cell populations with the mature osteoblastic phenotype and is localized in the cells forming nodules. MMP-2 and -9 are expressed diffusely in the osteoblast population. Developmentally associated changes in the activation of MMP-2 are detected, associated in their timing with the expression of MMP-14 in both populations of ROB, and MMP-14 activates pro-MMP-2 in vitro. Expression of messenger RNAs (mRNAs) for the three MMPs increases up to the time of nodule formation. At this stage, TIMP-1 mRNA levels are lowest. TIMP-2 mRNA decreases throughout osteogenesis. In situ hybridization in 7-day-old rat tibias shows the strongest expression of MMP-14 among osteogenic cells, in lining osteoblasts on the newly formed trabeculae under the growth plate, and on the endosteal surface of cortical bone. Our data support the concept that the developmentally regulated expression of MMP-14 triggers localized proteolysis within the osteogenic population, concomitant in vitro to nodule formation.

Uptake of yeast (Saccharomyces boulardii) in normal and rotavirus treated intestine.

BACKGROUND: There has recently been a growing interest in the use of the non-pathogenic yeast Saccharomyces boulardii, in the treatment of gastrointestinal disorders, including diarrhoea. The full effects of administration of the yeast are not fully understood. AIMS: To investigate the morphological effects of inoculated S boulardii on mouse intestinal villi, both in control animals and those treated with rotavirus. METHODS: Seven day old BALB/c seronegative mice were intubated with either rotavirus (30 microliters orally) or S boulardii (1.5 g/kg) or both rotavirus and S boulardii administered together. Control animals were given saline only. Animals were killed by decapitation 48 hours post-treatment. The middle region of the small intestine was studied using light microscopy and transmission and scanning electron microscopy, including backscattered electron imaging. RESULTS: Animals treated with rotavirus with or without S boulardii developed severe diarrhoea and showed morphological villous changes such as stromal separation and increased epithelial vacuolation. Specimens treated with S boulardii contained yeast particles within the mucosal tissues. CONCLUSION: The administration of S boulardii did not influence the changes produced by rotavirus, but yeast particles appeared to be taken up by the villous mucosa, with the predominant route apparently being uptake between adjacent epithelial cells.

The effect of graft-bed irradiation on the healing of rat skin grafts.

This study explores the possible side effects on healing skin grafts of irradiation, commonly used intraoperatively following surgical tumor removal. The experimental model involved the delivery of a single 10-Gy dose of electron radiation to the recipient bed of a skin wound, followed by attachment of a full thickness rat skin autograft. Skin graft repair was assessed by light microscopy, immunohistochemistry, and transmission electron microscopy over a 3-week period for grafted and grafted-irradiated groups. Graft-bed irradiation reduced fibrinogen, fibrin, and fibronectin deposition in the wound. It also produced brief changes in the extent of both re-epithelialization and granulation tissue formation, and reduced the diameter of collagen fibrils in the granulation tissue. Despite these changes, the results suggest that graft-bed irradiation only delays the healing process, producing no serious clinical complications at the time points studied.

Audit of intensive care burn patients: 1982-92.

This study attempts to measure and quantify changes in workload and outcome in clinically ill burn patients admitted to the intensive care unit at this institution over the 11-year period 1982-92. The case notes were studied for all patients admitted to the intensive care unit, 163 cases in total, but information was incomplete in 14. Mortality over the period is compared, using Chi squared analysis with Yates correction, with mortality probability from Bull's chart relating age and body surface area of burn (1971). The trends show increasing admission rates and referral rates to ICU from other hospitals in the region, despite declining admission rates to the regional burn unit as a whole. The duration of stay for admitted patients also shows an increase, the combination of these factors suggesting an increasing workload. There has been no change in outcome over the period. The figures provide a baseline for comparison of outcome in critically ill burn patients and are an important means by which to measure future change.

Morphological aspects of particle translocation in vivo following ingestion of the yeast Saccharomyces boulardii.

Particle translocation in vivo was studied in mice using the non-pathogenic yeast Saccharomyces boulardii (SB). Seven day old BALB/c mice were given either saline (control) or 1.5 g/Kg SB (every six hours, up to 48 hours), by intubation, and killed by decapitation 48 hours post-treatment. Light and scanning electron microscopy (SEM) examination of specimens prepared from the middle intestine revealed the presence of yeast inclusions localised in the cytoplasm of enterocytes.

A morphological and microanalytical investigation into the uptake of particulate iron across the gastrointestinal tract of rats.

The uptake and translocation of particulate iron across the gastrointestinal (GI) mucosa of young adult rats has been investigated using a range of morphological techniques and X-ray microanalysis (XRMA). In animals fed a suspension of iron powder constituted of metallic iron particles ranging in size from 6-9 microns down to 5-30 nm, light microscopic histochemistry has clearly revealed iron deposits within the tissues of the duodenum. Scanning electron microscopy of the duodenal tissue by back-scattered electron imaging has complemented the light microscopic observations and revealed a selective localization of iron in the villi with variation in levels of iron uptake by the mucosal cells. Ultrastructural and XRMA analysis of duodenum has established the presence of metallic iron nanoparticles within the brush border, lateral intercellular spaces of the mucosal cells, mitochondrial cristae and cytoplasm of both mucosal and stromal cells. The observations indicate that metallic iron particles, in the nano-size range, may be taken up by the GI mucosa and that the passage of such particles across the epithelial barrier may take place through both a paracellular as well as a transcytotic process.

Electron irradiation slows down wound repair in rat skin: a morphological investigation.

To date, there have been few morphological investigations of the effect of electron radiation on the healing of skin wounds in rats. The present morphological study examines the wound repair process in electron-irradiated rat skin by electron microscopy. Standardized, full-thickness, incisional wounds were made in the lower dorsal skin of animals which had been locally irradiated with 9.6 Gy electron radiation 7 days previously. The irradiation dose was maximal at 3 mm depth. Twenty-four rats were used in the investigation; 12 were irradiated and 12 sham-irradiated. Three rats from each experimental group were killed at 1, 3, 7 and 14-day time intervals after wounding. The morphological effect of electron irradiation on the repair of each wound was investigated by light microscopy (LM) and scanning electron microscopy (SEM). New granulation tissue visualized by SEM was quantified using computerized image analysis. The results suggest that a single, partial-body, controlled depth dose of electron irradiation delays wound repair. LM showed that there is a depression of the inflammatory cell and tissue exudate response, slowing of epithelial migration, and a decrease in fibroblast representation, together with a delay in the formation of collagen bundles. Granulation tissue formation was impaired up to 7 days post-wounding, but was restored to around control values by day 14, indicating that healing was delayed. However, as the healing of normal tissue was not prevented, this study supports a preoperative role for the use of low-dose electron irradiation therapy for the treatment of electron-sensitive superficial pathologies in surgical practice.

Influence of the fibula on growth of the rat tibia.

To investigate the effect of the fibula on growth of the tibia in the rat, (1) a sleeve of periosteum was removed from the middle third of the tibia, (2) a length of the fibula was excised, or (3) a sleeve of periosteum was removed from the middle third of the tibia and a length of fibula was also excised. Over a 14 wk experimental period subsequent tibial bone growth was measured on radiographs and compared with unoperated contralateral control tibiae. Procedure (1) had no effect on growth, (2) resulted in 4.2% overgrowth and (3) produced 19.7% overgrowth compared with control tibiae. The failure of overgrowth after periosteal resection from the middle third of the rat tibia argues against the vascular response theory in relation to bone overgrowth. The longitudinal overgrowth after procedure (2) and its further accentuation by procedure (3) suggests that the fibula influences tibial bone growth by exerting a mechanical restraint on it, which is reciprocal to the restraining influence of the tibial periosteum. Overgrowth appears to be facilitated by decompression of the cartilage growth plate of the rat tibia when a sleeve of the periosteum is removed from it, and this suggests a mechanical relationship between the fibrous periosteum and the cartilage growth plate of the tibia. It is concluded that the fibula plays a reciprocal role in regulating tibial bone growth in the rat.

Rabbit and human non-keratinising stratified squamous oesophageal epithelium displays similar microridge structure by scanning electron microscopy.

Since the oesophageal epithelium of common laboratory animals, rats and mice, is keratinized it is unsuitable for comparison with typical non-keratinized stratified squamous human epithelium. It is thus important to find a suitable animal model for the study of human oesophageal tissue changes. This study investigated the microridge structure of immature and adult rabbit specimens, and adult human biopsies by scanning electron microscopy and morphometry. The investigation revealed a similarity between typical squamous human and adult rabbit oesophageal mucosal epithelium. While human epithelium specimens subdivided into two other groups (non-typical squamous and non-squamous); all typical squamous human biopsies were from patients who had normal endoscopy reports and no reflux symptoms. The surface cells of typical squamous human epithelium displayed complex microridge patterns (64% of cell surface) but patterns in non-typical squamous specimens were more variable (38%) (P < 0.001) and cell boundaries less obvious. Rabbit squames displayed clear microridge patterns with an elevation in the percentage of cell surface covered by microridges, with increasing age, from immature to adult specimens (P < 0.001). There was no statistically significant differences between adult rabbit, and 'typical squamous' human biopsies (range 51-65%), results which suggest potential use of a rabbit model to study changes in human oesophageal tissue.

Early effects on the morphology of mouse small intestine of single or combined modality treatment with hyperthermia and X-irradiation.

This study describes the effects of hyperthermia and X-irradiation on the morphological appearance of normal, at risk tissues in the ileum of the mouse. The early morphological effects 1 day after a combined modality treatment are compared with those due to either hyperthermia or X-irradiation given alone. The response was assessed qualitatively and semiquantitatively using scanning electron microscopy and a villous scoring technique. Early post-irradiation effects on topography did not differ significantly from those observed after small intestine exteriorisation without treatment. The villous scores for the combined modality treatments reflected greater damage than would be expected from the sum of villous scores for each modality treatment on its own. This suggests that the combined modality treatment had a synergistic or enhancing effect. A 4 hour time interval between the two treatments did not seem to reduce the enhancing effect. Further studies are required to investigate the effects of fractionated combined treatment.

Serum c-reactive protein in patients with serious trauma.

Daily serum c-reactive protein (CRP) concentration was monitored in 98 patients (26 female) admitted to the Major Injuries Unit (MIU) at Birmingham Accident Hospital following serious trauma. The mean (SD) increase in CRP concentration for 79 survivors and 19 non-survivors between days 1 and 2 after trauma were 69.5 (74.6) and 111.8 (59.0) mg/l/24 h, respectively (P = < 0.001). By day 4 after trauma the mean serum CRP concentrations for survivors and non-survivors were 150.9 (76.9) and 233.4 (100.8) mg/l (P < 0.001), respectively. Injury severity data were available for 50 patients. The mean (range) injury severity score was 25.2 (4-50), Glasgow coma scale 10.4 (3-15), revised trauma score 6.5 (3.39-7.8) and predicted survival 0.78 (0.02-0.99). Univariate regression analysis of serum CRP on days 1-5 after injury against revised trauma score and injury severity score, revealed an inverse correlation between day 1 serum CRP and Glasgow Coma Score (r = -0.306, P < 0.05), but no correlation with injury severity score or predicted survival on any of the study days. The lack of correlation between serum CRP and injury severity or predicted survival, and the strong association with actual survival, suggests that the acute inflammatory response to serious trauma and subsequent complications, is an important determinant of outcome.