Orally absorbable cephalosporin antibiotics. 1. Structure-activity relationships of benzothienyl- and naphthylglycine derivatives of 7-aminodeacetoxycephalosporanic acid.

A structure-activity relationship study of a number of orally absorbed cephalosporins together with their syntheses is described. These new cephalosporins are benzothienyl- and naphthylglycine derivatives of 7-aminodeacetoxycephalosporanic acid. Several different synthetic methods for the glycine side chains, their protection, and the final acylations are reported. Several of these analogues were more active than cephalexin both in vitro and in vivo against commonly encountered Gram-positive bacteria. (R)-7-(3-Benzothienylglycylamido)-3-methyl-3-cephem-4-carboxylic acid (1R) has emerged as a potent antibacterial agent and is currently undergoing preclinical evaluation.

Coralline hydroxyapatite bone-graft substitutes in a canine metaphyseal defect model. Radiographic-histometric correlation.

Radiographic and histometric evaluation of a new form of bone-graft substitute derived from reef-building sea coral was performed in a canine metaphyseal defect model. Blocks of this material were implanted into the proximal tibial metaphyses of eight dogs, with radiographic densitometry and harvesting performed at two, four, six, and 12 months. Histometric analysis demonstrated progressive apposition of host compact bone at the margins and trabecular bone at the interior of the implants with time following surgery. Corrected transmission density determinations correlated significantly with degree of osseous ingrowth (R = -0.78), void volume fraction (R = 0.88), and postoperative interval (R = 0.88). These results support the successful early application of coralline hydroxyapatite bone-graft substitutes as an alternative to autogenous grafting in the clinical setting, and indicate that the course of incorporation into host bone can be noninvasively monitored using densitometric techniques.

Coralline hydroxyapatite bone-graft substitutes in a canine diaphyseal defect model. Radiographic-histometric correlation.

Radiographic and histometric evaluation of a new form of bonegraft substitute derived from reef-building sea coral was performed in a canine diaphyseal defect model. Comparably sized blocks of this material and iliac crest autograft were placed into bilateral surgically created cortical windows in the distal radial diaphyses of 14 dogs. Representative graft specimens of both types underwent densitometric radiography and harvesting at 3, 6, 12, 24, and 48 months. Histometric analysis of implant specimens revealed satisfactory union and native osseous ingrowth at all time intervals. Graft specimens exhibited adequate union with scant host bone ingrowth at three months, but subsequent progressive appositional closure of cancellous spaces during the ensuing 45 months. Volume fraction and mean width of host bone remained stable in the implants but increased in the grafts due to neocortex formation. Stereologic distribution of bone was homogeneous in both implant and graft specimens, and no evidence for biodegradation of the former was observed. Corrected transmission density determinations accurately reflected these differences but correlated significantly with volume fractions and tissue widths of bone and soft tissue only in the autografts. These results support the successful early application of coralline hydroxyapatite bone graft substitutes as an alternative to autogenous grafting in the clinical setting and emphasize the potential role of noninvasive densitometric techniques in monitoring the incorporation of bone-graft materials.

Coralline hydroxyapatite bone graft substitutes. Evaluation of bone density with dual energy x-ray absorptiometry.

RATIONALE AND OBJECTIVES: The authors evaluate whether dual-energy x-ray absorptiometry (DXA) is a reliable method to determine the density of natural coralline hydroxyapatite (HA) blocks used as bone graft substitutes. METHODS: To evaluate the basic density of HA blocks from the same coral heads with and without titanium meshes, densitometry of 12 HA-500 blocks (genus Goniopora) and 12 HA-200 blocks (genus Porites) was performed. In addition, density measurements of 30 HA blocks (HA-500, n = 15; HA-200, n = 15) from different coral heads were obtained to assess if the originating coral head influences the basic density of blocks within one coral genera. To assess standard deviation serial measurements on eight coralline HA blocks, four with titanium meshes and four without were performed. In the ex vivo study, densitometry of 12 HA blocks (HA-500, n = 4; HA-200, n = 8) used as bone graft substitutes in the mandibles and craniums of adult mongrel dogs was performed. Densities were measured after bone ingrowth for 2 and 4 months, respectively. All measurements were obtained with a Lunar DPX with scan mode "slow 750" in the spine program with the regions-of-interests selected manually. Bone ingrowth was assessed by computer-assisted histomorphometry, which was considered the gold standard. Statistical analysis was performed to correlate the densities of plain HA blocks with and without meshes to the specific weights of the blocks. RESULTS: Significant positive correlation was found between the density of each HA block (both coral species) with and without meshes and the calculated specific weights. Densitometry values showed no significant differences depending on the originating coral heads. Standard deviation ranged between +/- 3.8% and +/- 4.1% (HA-500) and between +/- 3.0% and +/- 3.8% (HA-200). Hydroxyapatite-500 blocks showed marked increased densities between 15% and 34% after 4 months in three specimens in which bone ingrowth between 16.9% and 21.1% was revealed by histomorphometry; no increase of density was observed in one specimen, which presented only minimal bone ingrowth and signs of infection. Despite bone invasion between 12% and 25.8%, no increased densities were observed for HA-200 implants. CONCLUSIONS: Dual-energy x-ray absorptiometry is an accurate and reproducible modality to assess the densities of plain coralline HA blocks and to monitor bone ingrowth into coralline HA-500 but not into HA-200 block implants.

Effects of bone ingrowth on the strength and non-invasive assessment of a coralline hydroxyapatite material.

The dependence of strength on the amount of bone growth into a hydroxyapatite material made from coral was investigated. Block and granular forms of the material were implanted into cortical and trabecular regions of the skeletons of 16 dogs. The results were examined after 4, 8, 12 and 16 wk, with four dogs in each experimental group. When implanted into cortical bone, the bending strength of the implant material was found to be highly correlated with the amount of pore space which had become occupied by bone (r = 0.92, P less than 0.005 for the block form; r = 0.84, P less than 0.005 for the granular form). Multiple regression analysis showed that six histomorphometric measures of ingrowth accounted for 96% of the variability in bending strength of the block material, and there were no significant differences between block and granular forms of the material. On the other hand, when implanted into trabecular bone, the block form of the material achieved greater compressive strength than the granular form. While both strength and ingrowth increased with time, there were poor correlations between these two variables. Finally, when the material is implanted into trabecular bone, it becomes stronger in compression than the surrounding bone; when implanted in cortical bone, linear modelling suggests that resorption and replacement of the implant would be required to approximate the bending strength of the surrounding bone.

Porous hydroxyapatite as a bone graft substitute in diaphyseal defects: a histometric study.

Porous hydroxyapatite (IP200), formed by conversion of the Poritidae porites exoskeleton, has pores averaging 230 microns and pore interconnections averaging 190 microns in diameter. In the distal radial diaphyses of 14 dogs, bilateral 7.5 X 20 mm cortical windows were created and fitted with 5 X 7.5 X 20 mm blocks of IP200 implants and iliac autografts. Both implanted and contralateral grafted radius specimens were retrieved at 3, 6, 12, 24, and 48 months. Unstained undecalcified sections were examined by microradiography and UV epi-illumination. Stained undecalcified sections were examined by light microscopy and quantitated by histometric methods. Implant specimens demonstrated good union and bone ingrowth at all time intervals. The implant specimens were composed of (mean +/- SE) 10.6% +/- 1.0% soft tissue, 51.2% +/- 1.3% bone, and 38.2% +/- 1.0% IP200. The graft specimens showed good union with little apparent ingrowth at 3 months, followed by progressive appositional closure of cancellous spaces. The graft specimens contained 21.9% +/- 0.9% bone at 3 months with increases at each time interval to 73.1% +/- 8.7% at 48 months. The volume fraction and mean width of IP200 did not change with time, confirming the absence of implant biodegradation. The volume fraction and mean width of bone remained stable in the implant but increased in the graft specimens, corresponding to graft neocortex formation. It is concluded that implants initially filled in with bone while grafts initially replaced much of their spongiosa and subsequently filled in with bone. Histometry of untreated defects and measurement of mechanical properties are suggested for further study.

Bone ingrowth into porous calcium phosphate ceramics: influence of pulsing electromagnetic field.

The effect of a pulsing electromagnetic field (PEMF) on bone ingrowth into porous hydroxyapatite (HA) and porous tricalcium phosphate (TCP) implanted in rabbit tibiae was studied. To quantitate the biological response, a recently developed method of surface measurement using a scanning electron microscope was used. The morphometrical findings in the HA pores demonstrated a significantly greater amount of bone and thicker bone trabeculae in the PEMF group as compared with the nonpulsed control group at 3 to 4 weeks postimplantation. No significant differences for these parameters were found in the TCP pores. Histologically, more bone and wider bone trabeculae were observed in the HA implants for the PEMF-treated animals at the early time periods when compared with those of the control animals. Alternatively, the histological findings of the TCP implants were similar between these two groups. These histological results tended to correlate with the morphometrical data. Together, these results suggest that accelerated bone formation and bone maturation occurred in response to PEMF in the HA pores but was without effect in the TCP pores. This stimulatory effect is most significant after 3-4 weeks of PEMF stimulation.

Porous hydroxyapatite as a bone-graft substitute in metaphyseal defects. A histometric study.

Porous hydroxyapatite (Interpore 500) formed by conversion of the Porites goniopora coral exoskeleton has pores averaging 600 micrometers and pore interconnections averaging 260 micrometers in diameter. In the proximal tibial metaphysis of eight dogs, a defect one cubic centimeter in size was created unilaterally and was fitted with a block of Interpore 500. Both proximal tibial metaphyses were retrieved at two, four, six, and twelve months. Stained undecalcified sections were examined by light microscopy and quantitated by histometric methods. The implant-side specimens contained compact bone along the external surface and trabecular bone interiorly. The interior of these specimens was composed of 51.9 +/- 1.3 per cent soft tissue, 13.0 +/- 1.2 per cent bone, and 35.1 +/- 1.2 per cent Interpore 500 (mean and standard error). The interior of the normal specimens was composed of 79.7 +/- 1.4 per cent soft tissue and 20.2 +/- 1.4 per cent bone. The allocation of implant pore space between bone and soft tissue was proportional to that of bone and soft tissue in the normal tibiae. The stereological distribution of regenerated bone in the porous hydroxyapatite was also the same as in the normal tibiae. The appositional process of incorporation of the implant was confirmed by the finding that 66.5 per cent of the surface of the Interpore 500 was covered with bone ingrowth at twelve months.

Six-year clinical and histologic study of sinus-lift grafts.

This study retrospectively evaluated the results of 36 sinus-lift graft augmentations performed to place 66 implant cylinders in the posterior maxilla where vertical bone height was less than ideal. Grafts were accomplished with porous hydroxyapatite (HA) alone, porous HA mixed with autogenous bone removed from the iliac crest, and HA combined with autogenous bone removed from within the mouth. Nineteen core biopsy specimens were taken from different grafts at time intervals ranging from 4 to 36 months from the time of grafting. These cores were evaluated using histomorphometric analysis to yield data comparing volume fractions of bone formation, allowing comparison of different graft materials and healing times. Grafts using porous HA alone on mean produced 16.38% bone by volume after 6 months. After the same healing time, grafts using HA and hip bone on mean produced 19.30% bone by volume compared with an 11.30% bone volume when using HA and bone harvested intraorally. Evaluation of cores taken at 19 and 36 months revealed greater volumes of bone formation. Recommendations based on clinical and histologic results are discussed in detail.

Quantification of bone in dental implant sites after composite grafting of the mandible: report of a case.

The amount of viable bone in a composite graft that consisted of autogenous iliac bone marrow, allogenic rib, and porous hydroxyapatite was quantified using standard histologic techniques and an optical insert disk prior to placement of endosteal implants in an augmented mandible. It is suggested that this technique could be used in such cases before deciding whether to proceed with implant insertion.

Maxillary sinus augmentation prior to placement of endosseous implants: A histomorphometric analysis.

The aim of this prospective study was to histomorphometrically evaluate at various time intervals the mineralization stage and process of an allogeneic-xenogeneic bone graft used in sinus augmentation procedures. One biopsy was taken from 20 patients at either 6, 8, 10, or 12 months after sinus augmentation. Immediately following the biopsy, an endosseous implant was placed into the biopsy site. This protocol provided 4 groups of 5 patients each, based on healing time following sinus augmentation. Using backscattered electron image analysis, the specimens were histomorphometrically analyzed to determine the volume fractions of residual cancellous bone, newly formed bone, soft tissue, bovine hydroxyapatite, and "remineralized" freeze-dried demineralized bone allograft (rDFDBA). "Remineralization" of DFDBA particles was observed in a few areas in all specimens. Polarized light microscopy showed that only the 12-month biopsies had a predominance of lamellar bone formation. The area within the biopsies that represented the residual alveolar ridge consisted of 32.6% +/- 8.6% (mean +/- SD) of bone. In the grafted area of the biopsies the volume fraction of newly formed bone at 12 months (20.7% +/- 8.3%) was significantly higher (P < .05, analysis of variance) than at 6 months (8.1% +/- 3.0%). There was no statistically significant difference between newly formed bone in the inferior, central, and superior grafted areas in all 4 time intervals. This prospective study indicates that the mineralization process of an allogeneic-xenogeneic sinus graft is incomplete 6 months after the sinus augmentation procedure. New bone formation increased up to 12 months postaugmentation; however, it remained lower than the volume of residual bone.

Comparison of hydroxyapatite granules to autogenous bone graft in fusion cages in a goat model.

BACKGROUND: With the increased use of fusion cages to achieve lumbar intervertebral fusion, the question arises as to the potential for bone ingrowth from the host bone through the entire cage. Is it even necessary to have an autogenous graft to achieve total bone incorporation? METHODS: Nine adult male goats had fusion cages implanted into three vertebral bodies. The design was Surgical Dynamics/Ray Fusion Cage, measuring 21 mm x 14 mm. In each animal, one fusion cage was filled with autogenous graft, one with hydroxyapatite, porous granules, and the other with nonporous granules. Amount of new bone formation was determined by backscatter electron microscopy at 3 months post implantation in all animals. RESULTS: The histologic section shows that there was total incorporation in all specimens at 3 months. There was slightly more new bone (43%) with the nonporous granules compared with the porous granules (35%). The amount of residual void space was about the same in all specimens, indicating that the amount of new bone formation was similar and not statistically different in cages filled with hydroxyapatite granules versus granules of autogenous bone. CONCLUSION: This study confirms that total incorporation by ingrowth of new bone can be expected in fusion cages. The amount of ingrowth is about the same for autogenous graft versus hydroxyapatite granules. Apparently, it is not necessary to use bone graft to achieve successful bone incorporation if an acceptable biocompatable lattice, such as hydroxyapatite granules, is used.

Bone regeneration within a coralline hydroxyapatite implant.

The hypothesis that incomplete resorption of osteons in an autogenous cortical bone graft may limit its replacement by new bone regeneration was explored by implanting a hydroxyapatite replica of a coral skeletal structure into bone gaps. This implant contained channels and interconnections similar to those in osteon-evacuated bone grafts. In 6 implanted mandibular defects in dogs, two of which were examined at two, 4, and 6 months, 11 percent, 46 percent, and 88 percent of the implant areas were filled with regenerated bone. The regenerated bone was a woven type at two months, but changed to a lamellar type by 6 months. In two implanted defects examined at 12 months, biodegradation of 29 percent of the implant had occurred. The bone regeneration was physiological, the implant was biocompatible, and the biodegradation began after the bone had regenerated.

Porous hydroxyapatite as a bone graft substitute in cranial reconstruction: a histometric study.

To assess the potential of a porous hydroxyapatite matrix to serve as a bone graft substitute, bilateral 15 X 20 mm craniectomy defects were reconstructed in 17 dogs with blocks of implant and split-rib autografts. Specimens were retrieved at 3, 6, 12, 24, and 48 months, and undecalcified sections were prepared for microscopy and histometry. The implant and graft cross-sectional areas did not change with time, documenting their equivalent ability to maintain cranial contour. Bone ingrowth extended across the implant from one cranial shelf to the other in 15 specimens. Little apparent bone ingrowth was seen in most graft specimens. Two implants and three grafts were nonunited, possibly due to lack of fixation or the orientation of the histology sections. The implant specimens were composed of 39.3 percent hydroxyapatite matrix, 17.2 percent bone ingrowth, and 43.5 percent soft-tissue ingrowth. The graft specimens were composed of 43.7 percent bone and 56.3 percent soft tissue. This study supported the thesis that a porous hydroxyapatite matrix may function in part as a bone graft substitute. The brittle hydroxyapatite matrix undoubtedly became stronger with bone ingrowth, but the degree of cranial protection achieved was not measured in this study. The size of the cranial defect used in this study did not permit estimation of the distance over which bone ingrowth may be reliably expected. There remains a need for greater understanding of the causes of nonunion, the extent of predictable ingrowth depth, and the strength of the resultant implant-bone composite.

Hydroxyapatite and tricalcium phosphate bone graft substitutes.

Autografts have consistently outperformed calcium phosphate implants in most experimental models and clinical applications. However, human trials in metaphyseal defects demonstrate comparable results with autograft, hydroxyapatite, and TCP. Diaphyseal fractures and segmental defects represent more challenging problems, with less predictable results achieved with the synthetic bone graft substitutes. Improvements in the pore configuration, mechanical properties, and osteoinductive capacity of these implants should widen their future clinical application.

A classification of facial wrinkles.

An increasing number of injectable filler materials for facial wrinkles and folds points to the need for objective measurements of their effectiveness. Patient satisfaction is the goal, but proof of the value of a particular product requires objective measurement. A wrinkle assessment scale was developed as a simple tool for use by plastic surgeons, dermatologists, and aesthetic surgeons who want to assess the changes resulting from injecting filler materials in their patients. By correlating the grade of the wrinkle in the reference photographs with the wrinkle in a patient's face, a classification of 0 to 5 is assigned. Reliability of the scale was assessed by "live" judgment of 76 wrinkles by nine observers. The same rating was given to 92.7 percent of all wrinkles. In a second trial, photographs from 130 wrinkles were presented to eight observers who rated 89.4 percent of all wrinkles equally. A significant correlation of 87 percent was found between subjective ratings and objective wrinkle depth measured by profilometry on 40 silicone impressions. Manufacturers, monitors of clinical trials, health authorities, and most important, patients will benefit from objective data on current and new injectable materials.

Bone-graft reconstruction of the monkey orbital floor with iliac grafts and titanium mesh plates: a histometric study.

Bone-graft reconstruction of large orbital defects has been difficult because of a lack of marginal support of the grafts and unpredictable resorption. A titanium mesh orbital plate has been developed to provide this marginal support for bone grafts. However, the problem of unpredictable bone-graft resorption remains. To determine if this plate has any effect on graft resorption, this study was designed to quantitate the dimensions and composition of bone autografts (1) with and without titanium plate support and (2) in the anterior and posterior orbit. Bilateral full-thickness large orbital floor defects were surgically created in five monkeys, and a titanium orbital floor plate was fixed with screws into the right orbit. Two iliac crest grafts were measured and placed transversely and without fixation in each orbit, one anterior and the other posterior to the axis of the globe. The orbits were retrieved 28 weeks after surgery and were analyzed histologically and histometrically. Comparison of the supported and nonsupported grafts revealed no differences in their histologic appearance. There were three significant histometric findings: (1) resorption of bone was similar for those grafts which spanned an orbital floor defect and those which were supported by a titanium plate; (2) resorption of grafts in the posterior orbit did not differ from that of grafts in the anterior orbit; and (3) resorption of approximately one-third of bone-graft thickness and width had taken place during the 28-week study interval. We conclude that the benefits of bone-graft support by a titanium mesh orbital floor plate are not offset by any alteration in bone-graft resorption.

The induction of bone by an osteogenic protein and the conduction of bone by porous hydroxyapatite: a laboratory study in the rabbit.

The influence of the addition of an osteoinductive protein, capable of inducing extraskeletal ossification, on bone ingrowth into coralline porous hydroxyapatite was evaluated in the rabbit using a calvarium onlay model. Twenty-three rabbits received hydroxyapatite implants (10 x 10 x 2 mm) prepared with and without osteoinductive protein. These were implanted on the frontal bone and secured by wire fixation after 0.25 mm of the cortical surface was abraded. The implants were harvested at 3 and 4 months and analyzed for percentage of bone ingrowth by histologic examination of decalcified H&E sections and by scanning electron microscope backscatter image analysis. The osteoinductive protein-treated implants demonstrated significantly greater amounts of bone ingrowth at both 3 (52.0 versus 10.3 percent; p less than 0.001) and 4 months (66.1 versus 39.2 percent; p less than 0.005) than the untreated implants. The type of bone found in all osteoinductive protein-treated implants was predominantly lamellar. Untreated implants contained mostly woven bone at 3 months, with increasing amounts of lamellar bone appearing at 4 months. These results suggest that the combination of a bone-inducing protein and a suitable osteoconductive matrix may provide an alternative to bone grafting.

Bony healing of large cranial and mandibular defects protected from soft-tissue interposition: A comparative study of spontaneous bone regeneration, osteoconduction, and cancellous autografting in dogs.

The purpose of this study was to compare spontaneous bone regeneration, osteoconduction, and bone autografting in critical size calvarial and mandibular defects (defects which do not heal spontaneously during the lifetime of the animal) that were protected from soft-tissue interposition. Eighteen adult mongrel dogs underwent osteotomies to create a unilateral 30-mm segmental defect in the midbody of the edentulated right mandible and bilateral 15-mm x 20-mm full-thickness window defects in the parietal bones. The defects were either left empty, implanted with coralline hydroxyapatite (HA) blocks, or autografted with iliac cancellous bone. All defects were protected with a macroporous titanium mesh and the segmental mandibular defects were additionally stabilized by internal plate fixation. Specimens were retrieved after 2 and 4 months and three undecalcified longitudinal central sections including the osteotomy interfaces were prepared from each specimen for histometry and histology. Sections were analyzed for volume fractions of bone, soft tissue, and implant using scanning electron microscopy, backscatter electron imaging and histometric computer software. In the mandibular model, the empty defects exhibited the greatest amount of bone formation after 4 months (47.3 percent), which was greater than the amount of bone in the autografted group (34.8 percent) and significantly greater than the amount of bone within the hydroxyapatite implants (19.0 percent, p < 0.05). In the cranial defects, the autografted specimens demonstrated the greatest volume fraction of bone after 4 months (27.3 percent), which was significantly greater than within both the empty defects (18.2 percent, p < 0.05) and the hydroxyapatite implants (18.2 percent, p < 0.05). New bone formation in the mandibular defects united the cut ends at 4 months regardless of treatment and originated predominantly from the periosteum which remained present only along the alveolar border after surgical closure. In the calvarial defects, periosteum was not preserved and bone regenerated centripetally, originating from the diploë without any evidence of dural osteogenesis. Bone bridging was incomplete in the empty cranial defects at 4 months. In both the mandibular and cranial specimens, new bone at 2 months was a mixture of woven and parallel fibered bone. At 4 months, the new bone had remodeled almost entirely into mature Haversian bone. This study demonstrated a remarkable ability of defect protection with a macroporous protective sheet to facilitate bone regeneration in critical size mandibular and cranial bone defects. When active osteogenic periosteum was present, as in our mandibular model, we concluded that defect protection alone was sufficient to allow for healing even of critical size defects. When periosteum was absent as in our cranial defects, the limited spontaneous bone formation benefited from the added contributions of cancellous grafting and osteoconductive implants, both of which promoted bone bridging across the defects. We suggest that in the future a resorbable macroporous protective sheet would be advantageous in comparison to a titanium mesh to facilitate bone regeneration by preventing soft-tissue prolapse and allowing the migration of mesenchymal cells and the proliferation of blood vessels from the adjacent soft tissues into the bone defect. Finally, this study identified the need to differentiate critical size defects into those with and without defect protection and periosteum.

Porous hydroxyapatite as a bone graft substitute in alveolar ridge augmentation: a histometric study.

Following bilateral alveolectomy, the ridge form was restored in 10 dogs with blocks of porous HA matrix and bicortical iliac autografts. The specimens were retrieved after 11 to 17 months and undecalcified sections were prepared for microscopy and histometry. Within the HA implants, bone ingrowth extended throughout the pores. This bone appeared mature and well vascularized. The autografts united to the mandible. However bone ingrowth into the cancellous spaces of the grafts was minimal or absent. Measurement of implant and graft cross-sectional areas showed the maintenance of alveolar ridge form to be equally permanent for the 2 materials over the 1.5 year duration of the study. The implant specimens were composed of 43.1% HA matrix, 45.2% bone and 11.7% soft tissue. The HA matrix had a surface area averaging 9.3 mm2/mm3 that was 91.1% covered with bone ingrowth. Although this study supported the thesis that a porous HA matrix can function as a bone graft substitute, it is noted that the unyielding nature of the implant blocks, compared to granules, requires a solution to the challenge of long-term denture support without ulceration before it can be used with clinical confidence.