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.

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.

Three-dimensional vocal tract imaging and formant structure: varying vocal register, pitch, and loudness.

Although advances in techniques for image acquisition and analysis have facilitated the direct measurement of three-dimensional vocal tract air space shapes associated with specific speech phonemes, little information is available with regard to changes in three-dimensional (3-D) vocal tract shape as a function of vocal register, pitch, and loudness. In this study, 3-D images of the vocal tract during falsetto and chest register phonations at various pitch and loudness conditions were obtained using electron beam computed tomography (EBCT). Detailed measurements and differences in vocal tract configuration and formant characteristics derived from the eight measured vocal tract shapes are reported.

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.

Comparison between electroglottography and electromagnetic glottography.

Newly developed glottographic sensors, utilizing high-frequency propagating electromagnetic waves, were compared to a well-established electroglottographic device. The comparison was made on four male subjects under different phonation conditions, including three levels of vocal fold adduction (normal, breathy, and pressed), three different registers (falsetto, chest, and fry), and two different pitches. Agreement between the sensors was always found for the glottal closure event, but for the general wave shape the agreement was better for falsetto and breathy voice than for pressed voice and vocal fry. Differences are attributed to the field patterns of the devices. Whereas the electroglottographic device can operate only in a conduction mode, the electromagnetic device can operate in either the forward scattering (diffraction) mode or in the backward scattering (reflection) mode. Results of our tests favor the diffraction mode because a more favorable angle imposed on receiving the scattered (reflected) signal did not improve the signal strength. Several observations are made on the uses of the electromagnetic sensors for operation without skin contact and possibly in an array configuration for improved spatial resolution within the glottis.

In vitro stability of a highly crystalline hydroxylapatite coating in a saturated citric acid solution.

A novel pressurized hydrothermal post-plasma-spray process has been developed to convert the crystalline non-HA and amorphous components of plasma-sprayed hydroxylapatite coatings back into crystalline HA. The process, known commercially as MP-1, was used to produce coatings comprising approximately 96% crystalline HA. The in vitro solubility of the coating in saturated citric acid solution has been measured to simulate the effect of implant detoxification procedures, which use citric acid as a cleaning medium. The MP-1 coating solubility in saturated citric acid solution (pH = 1) was compared to that of coatings with crystalline HA contents ranging from 37.5-82%. All coatings showed an initial sharp rise in coating dissolution, which correlated with crystalline HA content, followed by a steady state dissolution rate. After 60 s at 25 degrees C, the MP-1 coating showed a 65% decrease in solubility compared to a highly amorphous coating (AM-2). All coatings showed very similar steady state dissolution rates, except for AM-2, which was significantly higher. SEM analysis showed that the AM-2 coating surface was degraded substantially more than the other coatings, resulting in partial coating exfoliation. A mechanism of coating dissolution is proposed, in which the initial rapid leaching of soluble phases from the coating leaves behind a porous layer of highly crystalline HA at the coating surface. The stability of this porous crystalline layer leads to steady state, diffusion-limited dissolution of the remainder of the coating. The observed two-regime dissolution profile can be accurately represented by a 2-parameter model, which predicts the initial sharp rise in coating dissolution followed by a slower, steady state loss in coating mass. Model parameters were determined from experimental solubility data, and were shown to correlate with the percentage of crystalline HA in the coatings. The present data suggest that the treated coating is significantly more resistant to degradation from aggressive detoxification procedures such as citric acid burnishing.

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.

Highly crystalline MP-1 hydroxylapatite coating. Part II: In vivo performance on endosseous root implants in dogs.

The in vivo integration strength and degree of bone apposition were compared for oral endosseous implants with different plasma-sprayed hydroxylapatite (HA) coatings. Pullout strength measurements and histological analysis were used to compare two different commercially available coating from the same manufacturer. One coating does not receive a post-plasma-spray treatment and contains about 75% crystalline HA. The other coating is treated with the MP-1 process, a pressurized hydrothermal post-plasma-spray process, which increases the coating composition to approximately 95% crystalline HA without changing the coating's adhesive or cohesive strength. Comparisons were made in dogs after healing times of 3 and 15 weeks in the mandible. No significant differences were found in either case between the two coatings. Two different methods were used to determine the degree of bone apposition at 15 weeks. Both methods confirmed that the MP-1 process does not affect the osseointegration rate of plasma-sprayed HA coatings. Qualitative histology data suggest that the treated coating is more stable than the control coating, especially in cases of direct soft tissue attachment to the implant. The present data suggest that extensive dissolution of calcium phosphate components into surrounding tissue is not a necessary precursor for direct apposition of bone to HA-coated implants.

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.

Bone resorption caused by three periodontal pathogens in vivo in mice is mediated in part by prostaglandin.

Gingival inflammation, bacterial infection, alveolar bone destruction, and subsequent tooth loss are characteristic features of periodontal disease, but the precise mechanisms of bone loss are poorly understood. Most animal models of the disease require injury to gingival tissues or teeth, and the effects of microorganisms are thus complicated by host responses to tissue destruction. To determine whether three putative periodontal pathogens, Porphyromonas gingivalis, Campylobacter rectus, and Fusobacterium nucleatum, could cause localized bone resorption in vivo in the absence of tissue injury, we injected live or heat-killed preparations of these microorganisms into the subcutaneous tissues overlying the calvaria of normal mice once daily for 6 days and then examined the bones histologically. We found that all three microorganisms (both live and heat killed) stimulated bone resorption and that the strain of F. nucleatum used appeared to be the strongest inducer of osteoclast activity. Treatment of the mice concomitantly with indomethacin reduced but did not completely inhibit bone resorption by these microorganisms, suggesting that their effects were mediated, in part, by arachidonic acid metabolites (e.g., prostaglandins). Our findings indicate that these potential pathogens can stimulate bone resorption locally when placed beside a bone surface in vivo in the absence of prior tissue injury and support a role for them in the pathogenesis of bone loss around teeth in periodontitis.

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.

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.

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.

Increased bone formation in osteocalcin-deficient mice.

Vertebrates constantly remodel bone. The resorption of preexisting bone by osteoclasts and the formation of new bone by osteoblasts is strictly coordinated to maintain bone mass within defined limits. A few molecular determinants of bone remodelling that affect osteoclast activity have been characterized, but the molecular determinants of osteoblast activity are unknown. To investigate the role of osteocalcin, the most abundant osteoblast-specific non-collagenous protein, we have generated osteocalcin-deficient mice. These mice develop a phenotype marked by higher bone mass and bones of improved functional quality. Histomorphometric studies done before and after ovariectomy showed that the absence of osteocalcin leads to an increase in bone formation without impairing bone resorption. To our knowledge, this study provides the first evidence that osteocalcin is a determinant of bone formation.

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.

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.

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.

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.