Intra-articular therapy with recombinant human GDF5 arrests disease progression and stimulates cartilage repair in the rat medial meniscus transection (MMT) model of osteoarthritis.

OBJECTIVE: Investigation of osteoarthritis (OA) risk alleles suggests that reduced levels of growth and differentiation factor-5 (GDF5) may be a precipitating factor in OA. We hypothesized that intra-articular recombinant human GDF5 (rhGDF5) supplementation to the OA joint may alter disease progression. METHODS: A rat medial meniscus transection (MMT) joint instability OA model was used. Animals received either one intra-articular injection, or two or three bi-weekly intra-articular injections of either 30 µg or 100 µg of rhGDF5 beginning on day 21 post surgery after structural pathology had been established. Nine weeks after MMT surgery, joints were processed for histological analysis following staining with toluidine blue. Control groups received intra-articular vehicle injections, comprising a glycine-buffered trehalose solution. OA changes in the joint were evaluated using histopathological end points that were collected by a pathologist who was blinded to treatment. RESULTS: Intra-articular rhGDF5 supplementation reduced cartilage lesions on the medial tibial plateau in a dose-dependent manner when administered therapeutically to intercept OA disease progression. A single 100 µg rhGDF5 injection on day 21 slowed disease progression at day 63. A similar effect was achieved with two bi-weekly injections of 30 µg. Two bi-weekly injections of 100 µg or three bi-weekly injections of 30 µg stopped progression of cartilage lesions. Importantly, three biweekly injections of 100 µg rhGDF5 stimulated significant cartilage repair. CONCLUSIONS: Intra-articular rhGDF5 supplementation can prevent and even reverse OA disease progression in the rat MMT OA model. Collectively, these results support rhGDF5 supplementation as an intra-articular disease modifying OA therapy.

OARSI Clinical Trials Recommendations: Design and conduct of clinical trials for primary prevention of osteoarthritis by joint injury prevention in sport and recreation.

The risk of post-traumatic osteoarthritis (PTOA) substantially increases following joint injury. Research efforts should focus on investigating the efficacy of preventative strategies in high quality randomized controlled trials (RCT). The objective of these OARSI RCT recommendations is to inform the design, conduct and analytical approaches to RCTs evaluating the preventative effect of joint injury prevention strategies. Recommendations regarding the design, conduct, and reporting of RCTs evaluating injury prevention interventions were established based on the consensus of nine researchers internationally with expertise in epidemiology, injury prevention and/or osteoarthritis (OA). Input and resultant consensus was established through teleconference, face to face and email correspondence over a 1 year period. Recommendations for injury prevention RCTs include context specific considerations regarding the research question, research design, study participants, randomization, baseline characteristics, intervention, outcome measurement, analysis, implementation, cost evaluation, reporting and future considerations including the impact on development of PTOA. Methodological recommendations for injury prevention RCTs are critical to informing evidence-based practice and policy decisions in health care, public health and the community. Recommendations regarding the interpretation and conduct of injury prevention RCTs will inform the highest level of evidence in the field. These recommendations will facilitate between study comparisons to inform best practice in injury prevention that will have the greatest public health impact.

Interleukin-6 enhances hypercalcemia and bone resorption mediated by parathyroid hormone-related protein in vivo.

Tumors frequently induce the multifunctional cytokine IL-6, which has been linked to several paraneoplastic syndromes, most notably cachexia. IL-6 stimulates osteoclast formation, causes mild hypercalcemia, and is produced by bone cells in vitro upon exposure to systemic hormones. Since IL-6 is produced together with parathyroid hormone-related protein (PTH-rP) in some patients with cancer, we tested the hypothesis that production of IL-6 potentiates the effects of PTH-rP on Ca2+ homeostasis and osteoclastic bone resorption and examined potential mechanisms for these interactions in vivo. Chinese hamster ovarian (CHO) cells stably transfected with cDNAs for IL-6 (CHO/IL-6) and PTH-rP sense (CHO/PTH-rP) or antisense (CHO/PTH-rP AS) were inoculated intramuscularly into nude mice. Experimental groups included CHO/IL-6 plus CHO/PTH-rP; CHO/IL-6 plus CHO/PTH-rP AS; CHO/IL-6 alone; and CHO/PTH-rP alone. Blood ionized Ca2+ was measured on days 0, 7, 10, 12, and 13. Three different developmental stages in the osteoclast lineage were examined at day 13: the early multipotential precursor, granulocyte macrophage colony-forming units (CFU-GM); more mature mononuclear osteoclast precursors, assessed by their capacity to form tartrate-resistant acid phosphatase-positive multinucleated cells in marrow cultures; and mature osteoclasts, assessed by histomorphometry. IL-6 increased CFU-GM but not bone resorption or Ca2+. In contrast, PTH-rP induced hypercalcemia and bone resorption and increased multinucleated osteoclasts and more mature precursors cells, but not CFU-GM. However, mice treated with both IL-6 and PTH-rP had very marked hypercalcemia and osteoclastosis as well as an increase in the number of both CFU-GM and mature osteoclast precursors. These data demonstrate that IL-6 enhances PTH-rP-mediated hypercalcemia and bone resorption, most likely by increasing the pool of early osteoclast precursors that in turn can differentiate to mature osteoclasts. We conclude that IL-6 stimulatory effects on osteoclast precursors may enhance the effects of other bone resorption factors that act at later stages in the osteoclast lineage.

Bisphosphonate risedronate reduces metastatic human breast cancer burden in bone in nude mice.

Human breast cancer frequently metastasizes to the skeleton to cause osteolysis and subsequent pain, pathological fracture, and hypercalcemia. Because bone continuously releases growth factors stored in bone matrix by bone resorption during physiological remodeling and, thus, possibly provides a favorable microenvironment for metastatic breast cancer cells to proliferate, inhibitors of bone resorption used either prophylactically or in patients with established disease, therefore, would seem likely to be useful adjuvant therapy in patients with breast cancer. However, the parameters for monitoring progressive osteolytic bone disease in humans are imprecise. We examined the effects of the third generation bisphosphonate, risedronate, which is a specific inhibitor of osteoclastic bone resorption, in a bone metastasis model in nude mice in which intracardiac injection of the human breast cancer cell line MDA-231 leads to osteolytic bone metastases. Risedronate (4 micrograms/animal/day) was given s.c. to animals (a) after radiologically small but defined osteolytic metastases were observed; (b) simultaneously with MDA-231 cell inoculation through the entire experimental period; or (c) by short-term prophylactic administration before inoculation of MDA-231 cells. In all experiments, risedronate either slowed progression or inhibited the development of bone metastases assessed radiographically. Furthermore, mice treated continuously with risedronate showed significantly longer survival than did control mice. Histomorphometrical analysis revealed that osteoclast numbers were diminished at metastatic tumor sites. Unexpectedly, there was also a marked decrease in tumor burden in bone in risedronate-treated animals. In contrast, the growth of metastatic breast cancer in soft tissues surrounding bones was not affected by risedronate. Moreover, risedronate had no effects on the local growth of s.c. implanted MDA-231 breast cancers in nude mice or on MDA-231 cell proliferation in culture. These data demonstrate that risedronate decreases metastatic MDA-231 breast cancer burden selectively in bone, as well as suppresses progression of established osteolytic lesions and prevents the development of new osteolytic lesions; thus, the data suggest that inhibition of osteoclastic bone resorption may be a useful adjunctive therapy for the treatment of cancers that have colonized in bone.

Tumor necrosis factor enhances parathyroid hormone-related protein-induced hypercalcemia and bone resorption without inhibiting bone formation in vivo.

Humoral hypercalcemia of malignancy results from the effects of tumor-produced factors on bone, kidney, and intestine that disrupt normal calcium homeostasis. Although parathyroid hormone-related protein (PTHrP) is a major mediator of the syndrome, tumors also produce other hypercalcemic factors, such as tumor necrosis factor (TNF), which may modulate the effects of PTHrP. It has been postulated that TNF may counteract the stimulatory effects of PTHrP on bone formation. To examine the effects of TNF on PTHrP-induced changes in calcium and bone metabolism, a murine tumor model of hypercalcemia was used. Nude mice were inoculated with Chinese hamster ovarian (CHO) cells expressing human TNF (CHO/TNF) or nontransfected CHO cells (CHO/-) and further treated with injections of human PTHrP(1-34) or vehicle. The effects of TNF, PTHrP, and the combination of the two factors on blood ionized calcium, osteoclast recruitment, and bone histomorphometry were evaluated. Mice bearing CHO/TNF tumors that were injected with PTHrP had significantly higher calcium concentrations, increased committed osteoclast progenitors, and mature osteoclasts as well as enhanced bone resorption compared with mice bearing CHO/TNF tumors injected with vehicle or those bearing CHO/- tumors injected with PTHrP or vehicle. A 2-fold increase in new woven bone formed in the calvaria at sites of previous bone resorption was observed in CHO/TNF mice treated with PTHrP. Bone formation rates in the vertebrae were similar in both CHO/- and CHO/TNF mice treated with PTHrP. These data demonstrate that the hypercalcemic effects of PTHrP are enhanced by TNF and that this effect is due to the increased production of committed osteoclast precursors with a subsequent increase in osteoclastic bone resorption. Furthermore, PTHrP caused a coupled increase in osteoclastic bone resorption and new bone formation that was not inhibited by TNF. These findings highlight the complex interactions that may occur between tumor-produced factors on bone that result in malignancy-associated hypercalcemia and suggest that TNF may not be responsible for the decreased bone formation seen in some patients with this condition.

Targeting simian virus 40 T antigen to the osteoclast in transgenic mice causes osteoclast tumors and transformation and apoptosis of osteoclasts.

Osteoclasts are terminally differentiated cells that express tartrate-resistant acid phosphatase (TRAP) at a higher level than other normal cells. Therefore, in an attempt to develop immortalized osteoclasts, we produced two lines of transgenic mice in which expression of the simian virus 40 T antigen oncogene was targeted to osteoclasts using the TRAP gene promoter. Osteoclasts were increased in number in bones from both lines. More than 50% of them appeared morphologically transformed, 2-5% were mitotic, but, unexpectedly, 5% were apoptotic. Osteoclast tumors were observed occasionally in one line of mice (line 4), and sheets of TRAP-positive cells (tumorlets) developed in most mice in both lines. Although cells isolated from these tumorlets formed multinucleated TRAP-positive cells that resorbed bone in vitro, to date we have been unable to develop an immortalized osteoclast cell line from them. Osteoclasts from one line (line 5) had reduced ruffled border formation and a higher level of T-antigen expression than osteoclasts in the other line (line 4), and these features were associated with the presence of osteopetrosis. However, osteoclasts from these osteopetrotic mice and from line 4 mice resorbed bone normally when the mice were treated with interleukin-1. These findings indicate that T antigen can be targeted to osteoclasts in transgenic mice and causes osteoclast transformation, tumors, mitosis, and apoptosis. When T antigen is expressed at high levels, functional impairment of osteoclasts can be detected. Furthermore, these results suggest that T antigen is insufficient on its own to immortalize cells in the osteoclast lineage.

Effects of parathyroid hormone (PTH)-related protein and PTH on osteoclasts and osteoclast precursors in vivo.

Increased production of PTH-related protein (PTHrP) and PTH is frequently responsible for hypercalcemia and its associated morbidity. However, it is unclear whether these peptides produce identical effects on cells in the osteoclast lineage in vivo. To examine the effects of continuous in vivo exposure to these factors on both the osteoclast precursors and mature osteoclasts, we inoculated Chinese hamster ovarian cells expressing PTH-(1-84), PTHrP-(1-141), or nontransfected Chinese hamster ovarian cells into nude mice. The effects of these tumors on blood ionized calcium, plasma PTH and PTHrP concentrations, and osteoclast formation were then determined. PTH and PTHrP tumor-bearing mice became hypercalcemic (1.90 +/- 0.04 and 1.97 +/- 0.16 mmol/liter, respectively) compared with control mice (1.29 +/- 0.015 mmol/liter). After 4 days of hypercalcemia, mice were killed, and bone marrow cells were harvested to examine cells at three discrete stages of osteoclast development: multipotent osteoclast precursors, the granulocyte/macrophage colony-forming unit; more committed marrow mononuclear osteoclast precursors; and mature osteoclasts. Neither PTH nor PTHrP had an effect on granulocyte/macrophage colony-forming unit, but similarly increased the number of more committed mononuclear osteoclast progenitors as well as mature osteoclasts in the calvaria. No differences were detected between the effects of PTH and PTHrP on cells in the osteoclast lineage in vivo. Thus, PTH and PTHrP appear to affect only more differentiated cells in the osteoclast lineage, and the differences in osteoclastic bone resorption between primary hyperparathyroidism and humoral hypercalcemia of malignancy are probably due to mechanisms other than effects on osteoclast precursor cells in vivo.

Evaluating procedural skills competence: inter-rater reliability of expert and non-expert observers.

PURPOSE: To examine the inter-rater reliability of expert and non-expert observers when they used objective structured checklists to evaluate candidates' performances on three simulated medical procedures. METHOD: Simulations and structured checklists were developed for three medical procedures: endotracheal intubation, application of a forearm cast, and suturing a simple skin laceration. Groups comprised of two expert and two non-expert observers scored the performances of 101 procedures by 38 medical trainees and practitioners of varying skill levels. Inter-rater reliability was assessed using Pearson correlation coefficients. RESULTS: Inter-rater reliability was good for expert/expert, expert/non-expert, and non-expert/non-expert pairings in all three skills simulations. CONCLUSION: Both expert and non-expert observers demonstrated good inter-rater reliability when using structured checklists to assess procedural skills. Further study is required to determine whether this conclusion may be extrapolated to other study groups or procedures.

In vivo performance of a modified CSTi dental implant coating.

Cylindrical dental implants coated with cancellous structured titanium (CSTi) were studied in a dog model. CSTi-2-coated and hydroxyapatite-coated (HA) implants were placed in 8 mongrel dogs. The porosity of the CSTi-2 coating was 9% less than that of the previously studied CSTi-1, resulting in greatly improved mechanical strength and cosmetic appearance. A slightly lower level of bone ingrowth was observed for CSTi-2 than for CSTi-1. However, the in vivo attachment strength of the CSTi-2 coating was comparable both to CSTi-1 and to an HA-coated control after 8 weeks. Measured porosity is technique dependent; digital analysis of in vitro samples yielded higher porosity values than in vivo histology cross sections.

Acoustic impedance of an artificially lengthened and constricted vocal tract.

Voice training techniques often make use of exercises involving partial occlusion of the vocal tract, typically at the anterior part of the oral cavity or at the lips. In this study two techniques are investigated: a bilabial fricative and a small diameter hard-walled tube placed between the lips. Because the input acoustic impedance of the vocal tract is known to affect both the shaping of the glottal flow pulse and the vibrational pattern of the vocal folds, a study of the input impedance is an essential step in understanding the benefits of these two techniques. The input acoustic impedance of the vocal tract was investigated theoretically for cases of a vowel, bilabial occlusion (fully closed lips), a bilabial fricative, and artificially lengthening the tract with small diameter tubes. The results indicate that the tubes increase the input impedance in the range of the fundamental frequency of phonation by lowering the first formant frequency to nearly that of the bilabial occlusion (the lower bound on the first formant) while still allowing a continuous airflow. The bilabial fricative also has the effect of lowering the first formant frequency and increasing the low-frequency impedance, but not as effectively as the extension tubes.

Bifurcations in excised larynx experiments.

Bifurcation analysis was applied to vocal fold vibration in excised larynx experiments. Phonation onset and vocal instabilities were studied in a parameter plane spanned by subglottal pressure and asymmetry of either vocal fold adduction or elongation. Various phonatory regimes were observed, including single vocal fold oscillations. Selected spectra demonstrated correspondence between these regimes and vocal registers noted in the literature. To illustrate the regions spanned by the various phonatory regimes, two-dimensional bifurcation diagrams were generated. Many instabilities or bifurcations were noted in the regions of coexistence, i.e., regions in which the phonatory regimes overlap. Bifurcations were illustrated with spectrograms and fundamental frequency contours. Where possible, results from these studies were related to clinical observations.

Voice simulation with a body-cover model of the vocal folds.

A simple, low-dimensional model of the body-cover vocal-fold structure is proposed as a research tool to study both normal and pathological vocal-fold vibration. It maintains the simplicity of a two-mass model but allows for physiologically relevant adjustments and separate vibration of the body and the cover. The classic two-mass model of the vocal folds [K. Ishizaka and J. L. Flanagan, Bell Syst. Tech. J. 51, 1233-1268 (1972)] has been extended to a three-mass model in order to more realistically represent the body-cover vocal-fold structure [M. Hirano, Folia Phoniar. 26, 89-94 (1974)]. The model consists of two "cover" masses coupled laterally to a "body" mass by nonlinear springs and viscous damping elements. The body mass, which represents muscle tissue, is further coupled laterally to a rigid wall (assumed to represent the thyroid cartilage) by a nonlinear spring and a damping element. The two cover springs are intended to represent the elastic properties of the epithelium and the lamina propria while the body spring simulates the tension produced by contraction of the thyroarytenoid muscle. Thus contractions of the cricothyroid and thyroarytenoid muscles are incorporated in the values used for the stiffness parameters of the body and cover springs. Additionally, the two cover masses are coupled to each other through a linear spring which can represent vertical mucosal wave propagation. Simulations show reasonable similarity to observed vocal-fold motion, measured vertical phase difference, and mucosal wave velocity, as well as experimentally obtained intraglottal pressure.

Acoustic interactions of the voice source with the lower vocal tract.

The linear source-filter theory of speech production assumes that vocal fold vibration is independent of the vocal tract. The justification is that the glottis often behaves as a high-impedance (constant flow) source. Recent imaging of the vocal tract has demonstrated, however, that the epilarynx tube is quite narrow, making the input impedance to the vocal tract comparable to the glottal impedance. Strong interactions can exist, therefore. In particular, the inertance of the vocal tract facilitates vocal fold vibration by lowering the oscillation threshold pressure. This has a significant impact on singing. Not only does the epilarynx tube produce the desirable singer's formant (vocal ring), but it acts like the mouthpiece of a trumpet to shape the flow and influence the mode of vibration. Effects of the piriform sinuses, pharynx expansion, and nasal coupling are also discussed.

Acoustics of the tenor high voice.

The spectra of six tenors were analyzed at high pitches, F4 to B4. Because of the wide separation between harmonics, formant frequencies could not be extracted in the traditional way. Rather, an analysis-by-synthesis technique was used to match the spectra of a model to the measured spectra, using parameter optimization. Results suggest that tenors maintain their first formant frequencies well above the fundamental for all vowels except [u]. The purpose of this seems to be to distribute the acoustic energy between harmonics 2, 3, and 4 rather than to boost the fundamental. Tuning the first formant to the fundamental is a technique used effectively by sopranos but seems to be deliberately avoided by tenors in order to preserve a male quality.

The relationship of vocal tract shape to three voice qualities.

Three-dimensional vocal tract shapes and consequent area functions representing the vowels [i, ae, a, u] have been obtained from one male and one female speaker using magnetic resonance imaging (MRI). The two speakers were trained vocal performers and both were adept at manipulation of vocal tract shape to alter voice quality. Each vowel was performed three times, each with one of the three voice qualities: normal, yawny, and twangy. The purpose of the study was to determine some ways in which the vocal tract shape can be manipulated to alter voice quality while retaining a desired phonetic quality. To summarize any overall tract shaping tendencies mean area functions were subsequently computed across the four vowels produced within each specific voice quality. Relative to normal speech, both the vowel area functions and mean area functions showed, in general, that the oral cavity is widened and tract length increased for the yawny productions. The twangy vowels were characterized by shortened tract length, widened lip opening, and a slightly constricted oral cavity. The resulting acoustic characteristics of these articulatory alterations consisted of the first two formants (F1 and F2) being close together for all yawny vowels and far apart for all the twangy vowels.

Vocal tract area functions for an adult female speaker based on volumetric imaging.

Magnetic resonance imaging (MRI) was used to acquire vocal tract shapes of ten vowels /i, I, [symbol: see text] a, [symbol: see text], o, [symbol: see text] u/ and two liquid approximants /3[symbol: see text], 1/ for a 27-year-old adult female. These images were complemented with additional images acquired with electron beam computed tomography (CT) of /i/ and /a/. Each 3-D shape was condensed into a set of cross-sectional areas of oblique sections perpendicular to the centerline of the vocal tract's long axis, resulting in an "area function." Formant frequencies computed for each area function showed reasonable similarity to those determined from the natural (recorded) speech of the imaged subject, but differences suggest that some of the imaged vocal tract shapes were articulated differently during imaging than during recording of natural speech, and also that imaging procedures may have compromised some accuracy for a few shapes. The formant calculations also confirmed the significant effect that the piriform sinus can have on lowering the formant frequencies. A comparison is made between area functions derived using both MRI and CT methods for the vowels /i/ and /a/. Additionally, the area functions reported in this study are compared with those from two previous studies and demonstrate general similarities in shape but also obvious differences that can be attributed to anatomical differences of the imaged subjects and to differences in imaging techniques and image processing methods.

Vocal tract area functions from magnetic resonance imaging.

There have been considerable research efforts in the area of vocal tract modeling but there is still a small body of information regarding direct 3-D measurements of the vocal tract shape. The purpose of this study was to acquire, using magnetic resonance imaging (MRI), an inventory of speaker-specific, three-dimensional, vocal tract air space shapes that correspond to a particular set of vowels and consonants. A set of 18 shapes was obtained for one male subject who vocalized while being scanned for 12 vowels, 3 nasals, and 3 plosives. The 3-D shapes were analyzed to find the cross-sectional areas evaluated within planes always chosen to be perpendicular to the centerline extending from the glottis to the mouth to produce an "area function." This paper provides a speaker-specific catalogue of area functions for 18 vocal tract shapes. Comparisons of formant locations extracted from the natural (recorded) speech of the imaged subject and from simulations using the newly acquired area functions show reasonable similarity but suggest that the imaged vocal tract shapes may be somewhat centralized. Additionally, comparisons of the area functions reported in this study are compared with those from four previous studies and demonstrate general similarities in shape but also obvious differences that can be attributed to differences in imaging techniques, image processing methods, and anatomical differences of the imaged subjects.

Acceptability of marketing in health services.

There is more to the operation of health care than patients and doctors. Alternative sources of income may be derived from taking a broader view of the marketing function. The overall success of a health facility's total operation may be affected by the administrator's view of the role and view of marketing.

Highly crystalline MP-1 hydroxylapatite coating. Part I: In vitro characterization and comparison to other plasma-sprayed hydroxylapatite coatings.

A novel pressurized hydrothermal post-plasma-spray process, referred to as MP-1, has been developed to convert the crystalline non-HA and amorphous components of plasma-sprayed hydroxylapatite coating back into crystalline HA. No detrimental effects are observed on the strength of either the base metal or the coating. X-ray diffraction (XRD) and FTIR analysis, surface roughness, shear adhesion strength and calcium solubility testing were conducted on Calcitite coated samples before and after treatment with this process. Other commercially available coatings were also studied using XRD and solubility testing. Quantitative XRD data show that the MP-1 treatment increases the average crystalline HA content of the Calcitite coating from 77% to 96%, while the amorphous content decreases from 21% to 4%. Other commercially available dental implant coatings ranged in crystalline HA content from 45% to 73%, with amorphous phase content ranging from 29% to 62%. FTIR spectra for treated coatings were significantly more well defined, and showed an increase in peak separation and intensity. Surface roughness and shear adhesion strength were not affected by the treatment. In vitro solubility testing revealed that for all coatings there is an initial introduction of calcium into solution over the first 2 h of testing; however, the amount of calcium dissolved was significantly lower for the MP-1 coating. Under a pH and temperature representative of normal physiologic conditions, the rate of calcium dissolution for the MP-1 coating was significantly lower than that of the other commercial HA coatings. In increasingly acidic conditions, the MP-1 coating was compared to the Calcitite coating and was found to have a significantly slower rate of calcium release. The MP-1 treatment enhances typical HA coatings by increasing the crystalline HA content at the expense of the plasma-spray-induced soluble phases without a reduction in the strength of the coating. The resulting coatings exhibit significantly decreased in vitro solubility over a wide range of pH. The results of this solubility testing suggest that the treated coating may show significantly enhanced in vivo stability, even under the extreme conditions encountered during periods of infection or rigorous detoxification procedures. The significant differences between plasma-sprayed HA coatings reported here underscore the need for industry and academic researchers to raise the level of discourse and understanding of HA coatings. By offering consistent and accurate descriptions of coating compositions and methods of analysis, meaningful comparisons between different HA coatings can be made.