Michael Leunig

Fluorescence photobleaching with spatial Fourier analysis: measurement of diffusion in light-scattering media.

A new method for the measurement of diffusion in thick samples is introduced, based upon the spatial Fourier analysis of Tsay and Jacobson (Biophys. J. 60: 360-368, 1991) for the video image analysis of fluorescence recovery after photobleaching (FRAP). In this approach, the diffusion coefficient is calculated from the decay of Fourier transform coefficients in successive fluorescence images. Previously, the application of FRAP in thick samples has been confounded by the optical effects of out-of-focus light and scattering and absorption by the sample. The theory of image formation is invoked to show that the decay rate is the same for both the observed fluorescence intensity and the true concentration distribution in the tissue. The method was tested in a series of macromolecular diffusion measurements in aqueous solution, in agarose gel, and in simulated tissue consisting of tumor cells (45% v/v) and blood cells (5% v/v) in an agarose gel. For a range of fluorescently labeled proteins (MW = 14 to 600 kD) and dextrans (MW = 4.4 to 147.8 kD), the diffusion coefficients in aqueous solution were comparable to previously published values. A comparison of the spatial Fourier analysis with a conventional direct photometric method revealed that even for the weakly scattering agarose sample, the conventional method gives a result that is inaccurate and dependent on sample thickness whereas the diffusion coefficient calculated by the spatial Fourier method agreed with published values and was independent of sample thickness. The diffusion coefficient of albumin in the simulated tissue samples, as determined by the spatial Fourier analysis, varied slightly with sample thickness. In contrast, when the same video images were analyzed by direct photometric analysis, the calculated diffusion coefficients were grossly inaccurate and highly dependent on sample thickness. No simple correction could be devised to ensure the accuracy of the direct photometric method of analysis.These in vitro experiments demonstrate the advantage of our new analysis for obtaining an accurate measure of the local diffusion coefficient in microscopic samples that are thick (thickness greater than the microscope depth of focus) and scatter light.

Fractal Characteristics of Tumor Vascular Architecture During Tumor Growth and Regression

Objective: Tumor vascular networks are different from normal vascular networks, but the mechanisms underlying these differences are not known. Understanding these mechanisms may be the key to improving the efficacy of treatment of solid tumors.

The 12-item Oxford Knee Score: cross-cultural adaptation into German and assessment of its psychometric properties in patients with osteoarthritis of the knee.

OBJECTIVE To cross-culturally adapt and validate the Oxford Knee Score (OKS) for use in German-speaking patients with osteoarthritis of the knee. METHODS After the cross-cultural adaptation (OKS-D), the following metric properties of the questionnaire were assessed in 100 consecutive patients (mean age 66.5 years, 61 women) undergoing total knee replacement: feasibility (percentage of fully completed questionnaires), reliability (Intraclass Correlation Coefficients [ICC] and Bland and Altmans limits of agreement), and construct validity (correlation with the Western Ontario and McMaster Universities Index [WOMAC], Knee Society Score [KSS], Activities of Daily Living Scale [ADLS], and Short Form 12 [SF-12]), floor and ceiling effects, and internal consistency (Cronbachs Alpha, CA). RESULTS We received 91.9% fully completed questionnaires. Reliability of the OKS-D was excellent (ICC 0.91). Bland and Altmans limits of agreement revealed no significant bias (-0.2) and a random error of 6.2. Correlation coefficients with the other questionnaires ranged from -0.22 (SF-12 Mental Component Scale [MCS]) to -0.77 (ADLS). We observed no floor or ceiling effects. The CA was 0.83. CONCLUSIONS The German version of the OKS is a reliable and valid measure for the self-assessment of pain and function in German-speaking patients with osteoarthritis of the knee.

Quantitative analysis of angiogenesis and growth of bone: effect of indomethacin exposure in a combined in vitro-in vivo approach

Nonsteroidal anti-inflammatory agents have been used experimentally and clinically to suppress a variety of physiological events, including angiogenesis and formation of bone. The exact mechanisms by which indomethacin alters skeletal tissue generation are unknown, due in part to methodological limitations. By the use of an organ culture assay and an animal model using intravital microscopy in mice bearing dorsal skinfold chambers, the effect of indomethacin on growth and angiogenesis of neonatal femora was characterized over 16 days. In both assays, femora significantly elongated with time (P<0.05). The in vitro growth rate was more rapid than in vivo and dependent on the serum concentration, culture medium and age of mice. Although enthancing the serum content promoted cellular proliferation in organ culture, it dose-dependently suppressed femoral elongation, leading at 20% fetal calf serum to growth rates identical to those observed in vivo. Indomethacin supplementation (2 and 10 mg l−1) significantly accelerated longitudinal femoral growth in organ culture (P<0.05), whereas in vivo indomethacin (2 mg kg−1) did not modulate either angiogenesis or elongation of bone. Our in vitro data propose a central role of serum in the regulation of bone formation. Although indomethacin altered femoral gowth in vitro, our findings do not suggest that indomethacin suppresses angiogenesis or growth of bone in vivo. The complexity of physiological events in vivo may be obscuring a detectable effect.

Heating or freezing bone: Effects on angiogenesis induction and growth potential in mice

We have characterized the effect of bone graft treatment by heating or freezing (with or without dimethyl sulfoxide (DMSO)). Tissue culture and dorsal skin-fold chambers in mice were used as sites to quantify the effect on angiogenesis, growth and calcification of neonatal femora. Fresh femora increased in both length and cartilage diameter (calcification in vivo only), but cryopreservation or heating abolished the increase in femoral dimensions. In vivo, femora of all experimental groups elicited an angiogenic response from the host tissue, which was most pronounced for fresh femora, weaker for DMSO-preserved frozen bone and poor for unprotected frozen bone and boiled femora. Freezing in the presence of a cryopreservative (DMSO) was found to preserve the angiogenic potential of frozen bone, whereas unprotected heating or freezing significantly impaired angiogenesis induction and growth potential.

Effect of Basic Fibroblast Growth Factor on Angiogenesis and Growth of Isografted Bone: Quantitative in vitro-in vivo Analysis in Mice

Basic fibroblast growth factor (bFGF), a constituent of bone and cartilage matrix, has been shown to be a potent mitogen for osteoblasts and chondrocytes and yet an inhibitor of chondrocyte terminal differentiation in cell culture. To characterize the effect of bFGF on bone formation, whole neonatal murine femora were cultured in the presence or absence of bFGF and a neutralizing antibody against bFGF. In vitro, femoral elongation was provided by cartilage growth only; the calcified diaphyseal zone stained by oxytetracycline did not increase. When bFGF was added to the culture medium, longitudinal growth of the proximal and distal cartilage was inhibited in a dose-dependent manner (p < 0.05), and the number of hypertrophic chondrocytes in the growth plate was reduced. This phenomenon was absent in the presence of a neutralizing antibody, which when given alone significantly promoted femoral elongation. In contrast, in vivo after transplantation into adult mice bearing dorsal skin fold chambers, femora rapidly calcified after revascularization. This observation supports the notion that bone formation largely depends on angiogenesis-mediated events. To verify this hypothesis, angiogenesis and bone formation were quantified using bFGF known to be a stimulator of angiogenesis. Calcification of grafted femora was accelerated by bFGF given intraperitoneally. The neutralizing antibody slightly suppressed angiogenesis and femoral elongation (not statistically significant), whereas intravenous injections of both substances did not reveal a significant modulatory effect. In vivo the effect of systemically administered bFGF was inhomogeneous, but there was a strong correlation between angiogenesis and endochondral calcification (p < 0.001). These results suggest that exogenous bFGF modulates bone formation in vitro by inhibition of terminal differentiation of chondrocytes in the growth plate, and angiogenesis and concomitant in vivo events are pivotal in the promotion of rapid bone formation.

Twenty Years of Experience with the Bernese Periacetabular Osteotomy for Residual Acetabular Dysplasia

Residual acetabular dysplasia is known as the most frequent cause of early osteoarthritis of the hip. The degeneration starts with overload of the rim, leading to a variety of pathologies. This change may cause the femoral head to migrate further out of the socket, resulting in a loss of congruity and generating even higher pressure point loading, which finally leads to rapid destruction of the joint. It is well accepted today that the surgical increase of the load transmission area can slow down this process of destruction and postpone total hip replacement (THR) substantially. Among the different techniques available, reorientation procedures allow for the most physiological correction of the joint mechanics. Our proposition is a reorientation procedure, which was first executed in 1984. Techniques and results have been published on several occasions. Under the name of the Bernese periacetabular osteotomy, the technique has gained popularity, especially in North America. Our 20 years’ experience performing this osteotomy through a modified Smith-Peterson approach without dissection of the abductors has clearly shown that confound appreciation of joint mechanics is the key to a successful result. Addressing acetabular retroversion and an insufficient femoral head/neck offset has helped to avoid postosteotomy impingement and significantly improved our results. Today, in our armentarium of surgical techniques to preserve the natural hip joint, the periacetabular osteotomy leads to the most predictable results.

The Management of Chondral Defects in the Hip

Prior to the description of the surgical hip dislocation, the results following treatment of focal cartilage lesions in the hip were generally poor and confounded by the risk of avascular necrosis associated with the surgical approach. The successful treatment of focal cartilage defects in the hip is relatively new and has been facilitated by advancements in open and arthroscopic surgical techniques. Some, but not all, of the cartilage basic science and treatments developed for the knee are applicable in the hip. The treatment goals for patients with focal defects are: resolution of pain, restoration of function, and return to activity. Although it has yet to be definitively proven, early treatment of a focal cartilage lesion may also help to prevent the progression of cartilage degeneration and osteoarthrosis. A better understanding of the cartilage biomechanics specific to the hip as well as more biomechanical and animal models of hip cartilage lesions will help to advance these treatments. In addition, all of the current clinical literature consists of case series and small case reports, so more prospectively collected data and longer follow up is necessary. Nonetheless, the recent experience in treating these lesions is encouraging and appears to be of significant benefit to young and active adults with cartilage defects.

Anterior Approach for Total Hip Arthroplasty: Technique Without Fracture Table

Objective. To describe the minimally invasive anterior approach for total hip arthroplasty using a standard operating room table and report the short-term outcomes in a series of 128 patients. Indications. So-called primary osteoarthritis, rheumatoid arthritis, and degenerative arthritis of the hip. Contraindications. Complex primary hips might be avoided, such as hips after prior hip surgery, revision total hip arthroplasty, posterior acetabular deficiency, proximal femoral deformities, or difficult dysplastic cases as a Crowe type 4 deformity. Surgical Technique. Through a straight 8–10 cm incision starting 2 cm lateral and distal to the ASIS the fascia of the TFL is opened anteriorly. After obtaining hemostasis, the rectus femoris is identified and retracted medially with or without transecting the indirect head. The gluteus medius and minimus and TFL are retracted laterally to expose the hip capsule. After capsulectomy and femoral neck osteotomy, the acetabulum is exposed. The patients’ legs are placed in the figure-of-four position, with the operative hip extended and the femur externally rotated to expose the femoral canal. A press fit or cemented femoral component can be used with this approach. Postoperative Management. Postoperatively, hip flexion is limited to 90° for 4 weeks. Patients are encouraged to ambulate on postoperative day 1 and are usually ready for discharge to home by postoperative day 4. Results. One hundred and 141 hips were operated on in 128 patients during a 1-year period (2007). There were 26 cemented femoral stems implanted, and 115 were press fit. All acetabular components were press fit. The mean patient age was 68 years, of which 84 were females and 57 were males. The average operative time ranged from 60 to 75 min. There were three complications: one dislocation (0.7 %) which did not require treatment and two revisions (1.4 %) for a socket fracture after a low velocity trauma and a cup revision for persistent iliopsoas pain. Radiographic evaluation of acetabular cup position demonstrated the median abduction angle of 44° and anteversion of 23°.

Slipped Capital Femoral Epiphysis and Its Variants

Although SCFE has been recognized and treated by orthopaedists for over a century, significant advances in the understanding and management have occurred in the past decade. Classically, the goals of treatment have been to stabilize the physis and prevent the iatrogenic complications of osteonecrosis and chondrolysis. This schema is currently undergoing re-evaluation due to the recognition that even mild stable SCFE can cause femoroacetabular impingement (FAI). Although the potential for impingement in SCFE has been recognized for some time, preventing impingement and the resultant damage to the cartilage and labrum is becoming a more important principle of SCFE treatment. Improved knowledge of the vascular anatomy responsible for femoral head perfusion allowed the development of a safe technique for open reduction and internal fixation of the displaced epiphysis. Although the surgical dislocation and open reduction are technically demanding, safe correction of the physis is now possible and the short to mid-term results are good. Long-term results should be similar, but may be influenced by the amount of cartilage and labral damage at the time of surgery.