Additive enhancement of implant fixation following combined treatment with rhTGF-beta2 and rhBMP-2 in a canine model.

BACKGROUND: Gaps at the interface between implant and bone increase the risk of diminished implant fixation and eventual loosening. The purpose of the present study was to determine if combined use of recombinant human transforming growth factor-beta 2 (rhTGF-beta2) and bone morphogenetic protein 2 (rhBMP-2) led to greater implant fixation strength in the presence of interface gaps than the use of either growth factor alone. METHODS: Twenty-eight skeletally mature adult male dogs received one porous-coated titanium implant in the proximal part of each humerus, for a total of fifty-six implantation sites. Spacers were used to establish an initial 3-mm gap between the implant and the host bone at all fifty-six sites. Forty-two implants were coated with hydroxyapatite-tricalcium phosphate and were used in three growth-factor-treatment groups in which the implants placed in the left humerus were loaded with 12 microg of rhTGF-beta2 (Group 1, seven animals), 25 microg of rhBMP-2 (Group 2, seven animals), or 12 microg of rhTGF-beta2 combined with 25 microg of rhBMP-2 (Group 3, seven animals). In these animals, the twenty-one implants that were placed in the right humerus were loaded with buffer only to serve as contralateral controls. In Group 4 (seven animals), the implants were not coated with hydroxyapatite-tricalcium phosphate, the gap in the left humerus was lightly packed with autogenous bone graft, and the gap in the right humerus was left empty to serve as a contralateral control. All animals were killed at twenty-eight days. The primary end points included three mechanical variables: fixation strength, interface stiffness, and energy to failure. Secondary end points included bone ingrowth and bone volume and trabecular architecture in the gap and in a region located 2 mm medial to the implantation site. RESULTS: The hydroxyapatite-tricalcium phosphate coating had no effect on implant fixation, bone ingrowth, or bone formation in the 3-mm gap. Individual growth factor treatments led to 2.3 to 3.2-fold increases in fixation strength and stiffness as compared with the values for the contralateral controls (p < 0.05). The combined growth factor treatment led to 5.7-fold increases in fixation strength and stiffness compared with the values for the contralateral controls (p < 0.01). Autogenous bone graft treatment was associated with 4.5 to 6.4-fold increases in implant fixation strength and stiffness as compared with the values for the contralateral controls (p < 0.01). Compared with the relevant contralateral controls, energy to failure was increased 3.5-fold in association with TGF-beta2 alone (p < 0.05), 4.5-fold in association with TGF-beta2 combined with BMP-2 (p < 0.01), and 2.5-fold in association with autogenous bone-grafting. As much as 63% of the variance in the mechanical end points was associated with variance in bone volume and architecture in the 3-mm gap and in the region of interest located 2 mm medial to the implantation site (p < 0.01). CONCLUSIONS: In this animal model, the combined use of TGF-beta2 and BMP-2 led to more secure mechanical fixation of the implant than did the use of either growth factor alone and demonstrated results that were similar to those associated with the use of autogenous bone graft.

Depressed bone mineralization following high dose TGF-beta1 application in an orthopedic implant model.

Several previous studies of bone repair have shown 2- to 4-fold increases in bone formation following local delivery of exogenous transforming growth factor-beta (TGF-beta). Here, we use quantitative backscatter electron microscopy to test the effect of TGF-beta1 on mineralization of regenerated bone by examining tissue samples from a previously published canine study in which we found increased bone formation. In the experiment, the proximal humeri of 10 male canines were implanted bilaterally for 28 days with porous-coated implants in the presence of a 3 mm gap between the surface of the implant and the host bone. Implants placed in the left humeri were treated with TGF-beta1 at a dose of either 120 microg (n = 5) or 335 microg (n = 5), and the implants placed in the contralateral humeri served as untreated controls. Quantitative backscatter scanning electron microscopy was used to assess the volume fraction of bone and its degree of mineralization in the 3 mm gaps. The calibrated grayscale mean and median values were depressed compared to the controls in the high dose group (p = 0.048 and p = 0.041, respectively), suggesting that high dose TGF-beta delayed or inhibited mineralization of newly formed osteoid.

Locally delivered rhBMP-2 enhances bone ingrowth and gap healing in a canine model.

The purpose of the present study was to determine if recombinant human bone morphogenetic protein-2 (rhBMP-2) enhances bone ingrowth into porous-coated implants and gap healing around the implants. In the presence of a 3-mm gap between the implant and host bone, porous-coated implants were placed bilaterally for four weeks in the proximal humeri of skeletally mature, adult male dogs. In three treatment groups, the test implant was treated with HA/TCP and rhBMP-2 in buffer at a dose of 100 microg/implant (n=5), 400 microg/implant (n=6), or 800 microg/implant (n=5) and placed in the left humerus. In these same animals, an internal control implant was treated only with HA/TCP and buffer and placed in the right humerus. These groups were compared with a previously reported external control group of seven animals in which no growth factor was delivered [J. Orthop. Res. 19 (2001) 85]. The BMP treated implants in the two lower dose groups had significantly more bone ingrowth than the external controls with the greatest effect in the 100 g/implant group (a 3.5-fold increase over the external control, p=0.008). All three dose groups had significantly more bone formation in the 3-mm gap surrounding the BMP treated implants than the external controls with the greatest effect in the 800 microg group (2.9-fold increase, p<0.001). Thus, application of rhBMP-2 to a porous-coated implant stimulated local bone ingrowth and gap healing. The enhancement of bone formation within the implant (bone ingrowth) was inversely related to dose.

Aging does not lessen the effectiveness of TGFbeta2-enhanced bone regeneration.

Controversy exists over the potency of bone healing in the aged skeleton, and there is concern that enhancement of bone regeneration after use of bone-stimulating growth factors may not be effective in the aged. In this study, 30 skeletally mature beagles (1-2 or 10-12 years old) had titanium implants placed bilaterally in the proximal humerus for a period of 4 weeks in a model of intramembranous bone regeneration. A bony defect made at the time of surgery created a 3-mm gap between the implant surface and the host bone. Some of the implants were treated with recombinant human TGFbeta2 (rhTGFbeta2) at various does (0.32-35 microg per implant), and some served as paired controls. The dose response was similar in young and old animals. The most effective dose, 35 microg, led to a 3-fold increase in the volume fraction of new bone within the gap in both the young (p = 0.001) and old (p = 0.002) animals. At this dose, there was a 5-fold increase in osteoblast surface. While age did not significantly affect the quantity of new bone formed as assessed by backscatter scanning electron microscopy, the older animals had thinner regenerated trabeculae that tended to be spaced more closely than the younger animals. Coupled with the finding that the increase in osteoid was greater in the old animals compared with the young animals, these qualitative differences suggest that there may have been a slight delay in the rate or a defect of mineralization in the old animals.

Carcinoid tumor development in an intramedullary spinal cord mature teratoma.

The case of a 43-year-old woman with a several month history of severe back pain is reported. CT and MR imaging revealed an intramedullary cystic tumor, which was considered a dermoid cyst or teratoma. During surgery, the tumor was found within the base of the filum terminale and completely resected. Microscopic studies revealed a mature teratoma with an intramural carcinoid nodule. Thirteen-month follow-up after surgical resection showed no evidence of tumor recurrence or neoplasms elsewhere.

Locally delivered rhTGF-beta2 enhances bone ingrowth and bone regeneration at local and remote sites of skeletal injury.

The purposes of the present study were to determine if recombinant human transforming growth factor-beta-2 (rhTGF-beta2) enhances bone ingrowth into porous-coated implants and bone regeneration in gaps between the implant and surrounding host bone. The implants were placed bilaterally for four weeks in the proximal humeri of skeletally mature, adult male dogs in the presence of a 3-mm gap. In three treatment groups of animals, the test implant was treated with hydroxyapatite/tricalcium phosphate (HA/TCP) and rhTGF-beta2 in buffer at a dose per implant of 1.2 microg (n = 6), 12 microg (n = 7), or 120 microg (n = 7) and placed in the left humerus. In these same animals, an internal control implant treated only with HA/TCP and buffer was placed in the right humerus. In a non-TGF-beta treated external control group of animals (n = 7), one implant was treated with HA/TCP while the contralateral implant was not treated with the ceramic. In vitro analyses showed that approximately 15%, of the applied dose was released within 120 h with most of the release occurring in the first 24 h. The TGF-beta treated implants had significantly more bone ingrowth than the controls with the greatest effect in the 12 microg/implant group (a 2.2-fold increase over the paired internal control (P = 0.004) and a 4-fold increase over the external control (P < 0.001)). The TGF-beta treated implants had significantly more bone formation in the gap than the controls with the greatest effect in the 12 and 120 microg groups (1.8-fold increases over the paired internal controls (P = 0.003 and P = 0.012, respectively) and 2.8-fold increases over the external controls (P < 0.001 and P = 0.001, respectively)). Compared to the external controls, the internal control implants tended to have more bone ingrowth (1.9-fold increase, P = 0.066) and had significantly more bone formation in the gap (1.7-fold increase. P = 0.008). Thus, application of rhTGF-beta2 to a porous-coated implant-stimulated local bone ingrowth and gap healing in a weakly dose-dependent manner and stimulated bone regeneration in the 3-mm gap surrounding the contralateral control implant, a site remote from the local treatment with the growth factor.

Animal models relevant to cementless joint replacement.

This review focuses on animal models used to study certain aspects of 'cementless' joint replacement. Implants used in this application are designed to become attached to the host skeleton through either bone ingrowth into porous surfaces or bone apposition (ongrowth) onto other types of surfaces. Biological fixation of cementless joint replacement implants relies on intramembranous bone regeneration. We describe a framework for understanding research design in light of the type of research questions now being asked. In particular, species choice, implant design and placement, and experimental endpoints are described in some detail. We provide a summary of recent studies specifically focused on implant fixation, demonstrating that most work is still at the morphological and biomechanical levels with little understanding at the molecular level. We also provide a more comprehensive listing of studies using hip and knee replacement models, demonstrating that most work is focused on the interface, and responses of the immediately adjacent trabecular bone and the more distant cortical bone. We conclude by encouraging investigators to design their experiments so that there is enough power to answer a limited number of questions as opposed to providing limited data on a broader number of issues.

Synthesis and preliminary analysis of a P-glycoprotein-specific [3H]-benzophenone photoaffinity label based on (-)-stipiamide.

A benzophenone photoaffinity label 9 based on the polyene natural product (-)-stipiamide has been constructed using a diaminoethane spacer and the radioactive agent [3H]-BZDC (N-succinimidyl p-benzoyl-(2,3-3H)-dehydrocinnamate). Photoaffinity experiments show specific binding to human P-glycoprotein (Pgp) in the presence of cis-flupentixol but not with cyclosporin A.

The synthesis and evaluation of a solution phase indexed combinatorial library of non-natural polyenes for reversal of P-glycoprotein mediated multidrug resistance.

A combinatorial library of polyenes, based on (-)-stipiamide, has been constructed and evaluated for the discovery of new multidrug resistance reversal agents. A palladium coupling was used to react each individual vinyl iodide with a mixture of the seven acetylenes at near 1:1 stoichiometry. The coupling was also used to react each individual acetylene with the mixture of six vinyl iodides to create 13 pools indexed in two dimensions for a total of 42 compounds. Individual compounds were detected at equimolar concentration. The vinyl iodides, made initially using a crotylborane addition to generate the anti1,2-hydroxylmethyl products, were now made using a more efficient norephedrine propionate boron enolate aldol reaction. The indexed approach, ideally suited for cellular assays that involve membrane-bound targets, allowed for the rapid identification of reversal agents using assays with drug-resistant human breast cancer MCF7-adrR cells. Intersections of potent pools identified new compounds with promising activity. Aryl dimension pools showed R = ph and naphthyl as the most potent. The acetylene dimension had R' = phenylalaninol and alaninol as the most potent. Isolated individual compounds, both active and nonpotent, were assayed to confirm the library results. The most potent new compound was 4ek (R = naphthyl, R' = phenylaninol) at 1.45 microM. Other nonnatural individual naphthyl-amide compounds showed potent MDR reversal including the morpholino-amide 4ej (1.69 microM). Synergistic activities attributed to the two ends of the molecule were also identified. Direct interaction with Pgp was established by ATPase and photoaffinity displacement assays. The results indicate that both ends of the polyene reversal agent are involved in Pgp interaction and can be further modified for increased potency.

Radiographic, biomechanical, and pathologic effects of hemoglobin glutamer-200 in dogs undergoing cemented total hip arthroplasty.

OBJECTIVE: To determine whether use of hemoglobin glutamer-200 (bovine) as a partial blood volume replacement in dogs undergoing cemented total hip replacement caused any deleterious effects on the bone-cement or cement-prosthesis interface, exerted any deleterious effects on body organs, or caused any complications during the anesthetic, immediate recovery, or long-term recovery period. ANIMALS: 9 adult dogs. METHODS: Dogs were anesthetized, and 15% of the blood volume was removed. Simultaneously, lactated Ringer's solution was infused, and 6 dogs were given hemoglobin glutamer (1 g/kg of body weight, IV). Unilateral total hip replacement was performed. Limb use was assessed visually, and force-plate and radiographic evaluations were performed before, and 8 weeks after, surgery. Eight weeks after surgery, dogs were euthanatized, necropsies were performed, and prosthetic component pullout forces were determined. RESULTS: There were no significant differences between treated and control dogs in regard to biomechanical (visual assessment of gait, force-plate analysis, femoral and acetabular component pullout forces) and pathologic evaluations (physical examination, CBC, serum biochemical analyses, necropsy, and histologic evaluations). Radiographic signs of loosening of the femoral component were seen in 4 dogs treated with hemoglobin glutamer. CONCLUSIONS AND CLINICAL RELEVANCE: Administration of hemoglobin glutamer as a blood substitute did not appear to have any deleterious effects in dogs undergoing total hip arthroplasty. The radiographic findings, which were discordant with the biomechanical results, merit further investigation.

Functional adaptation and ingrowth of bone vary as a function of hip implant stiffness.

The purpose of the present study was to test the hypothesis that cortical bone loss, trabecular bone density and the amount of bone ingrowth vary as a function of stem stiffness in a canine cementless hip replacement model. The study was motivated by the problem of cortical bone atrophy in the proximal femur following cementless total hip replacement. Two stem stiffnesses were used and both designs were identical in external geometry and porous coating placement. The high stiffness stem caused approximately 26% cortical bone stress-shielding and the low stiffness stem caused approximately 7.5% stress-shielding, as assessed by beam theory. Each group included nine adult, male canines who received unilateral arthroplasties for a period of six months. The animals with the low stiffness stems tended to lose less proximal cortical bone than the animals with high stiffness stems (4% +/- 9 as opposed to 11% +/- 14), but the difference was not statistically significant (p = 0.251). However, the patterns of bone ingrowth into the implant and change in medullary bone density adjacent to the implant were fundamentally different as a function of stem stiffness (p < 0.01). Most importantly, while the high stiffness group had peaks in these variables at the distal end of the stem, the low stiffness group had peak values proximally. These different patterns of functional adaptation are consistent with the idea that reduced stem stiffness enhances proximal load transfer.

Fluconazole disk diffusion susceptibility testing of Candida species.

We describe a simple procedure for detecting fluconazole-resistant yeasts by a disk diffusion method. Forty clinical Candida sp. isolates were tested on RPMI-glucose agar with either 25- or 50-microgram fluconazole disks. With 25-microgram disks, zones of inhibition of >/=20 mm at 24 h accurately identified 29 of 29 isolates for which MICs were /=27 mm identified 28 of 29 such isolates. All 11 isolates for which MICs were >8 microgram/ml were identified by using either disk. Disk diffusion may be a useful screening method for clinical microbiology laboratories.

Maintenance of proximal cortical bone with use of a less stiff femoral component in hemiarthroplasty of the hip without cement. An investigation in a canine model at six months and two years.

A canine model of hemiarthroplasty of the hip was used to determine if the use of a less stiff femoral stem can reduce the amount of bone loss induced by stress-shielding. Two types of stem were used: the stiffer stems were made of a titanium alloy, and the less stiff stems were composed of a cobalt-chromium-alloy core with an outer polymer layer. The stems were identical in shape, and both types were circumferentially coated along their entire length (except for the distal five millimeters) with commercially pure titanium fiber metal. Ten dogs with each type of stem were followed for six months, and twelve dogs with each type of stem were followed for two years. Loss of cortical bone from the proximal part of the femur was associated with both types of stem, but typically 50 per cent less bone was lost with the less stiff implants. Most of the cortical loss occurred at the subperiosteal surface. The amount of medullary bone adjacent to the proximal and distal aspects of both types of stem increased; the less stiff stems were associated with a greater increase in the proximal region, and the stiffer stems were associated with a greater increase in the distal region. Similarly, there were peaks in the amount of bone growth into the proximal and distal portions of both types of stem, with a greater peak in proximal bone growth into the less stiff stems and a greater peak in distal bone growth into the stiffer stems.

Unilateral hip replacement causes bilateral changes in tibial bone mineral content in a canine model.

The presence of asymmetry in tibial bone mineral content (BMC) of the operated and control limbs at the end of the experimental period following unilateral hip replacement surgery is used as a marker of limb function. The goal of the present study was to determine the contribution of ipsilateral and contralateral bone gain and loss to control-treated side differences in BMC of the tibia in dogs following unilateral hip replacement surgery. Seven animals were followed longitudinally with single beam photon absorptiometry for 6 months after unilateral hip hemiarthroplasty. Bone loss, compared with preoperative baseline values, was observed in both limbs, with recovery in bone mass beginning 1 month after surgery in the contralateral tibia and 3 months after surgery in the ipsilateral tibia. Thus, the asymmetry in tibial BMC frequently seen after unilateral experimental hip replacement in the canine appears to be caused by differential timing of recovery of bone mass following a transient loss in both limbs. The mechanism defined in this study is in contrast to an alternative mechanism involving bone loss in the treated limb coupled with bone gain in the control limb.

Enhancement of bone ingrowth by transforming growth factor-beta.

Enhancement of bone ingrowth with transforming growth factor-beta was evaluated in a canine model. Ten dogs had bilateral implantation of a titanium-fiber-metal-coated rod in the proximal part of the humerus. A three-millimeter gap between the outer surface of the porous coating and the surrounding cancellous bone was created to impair bone ingrowth. All of the implants were plasma-flame-sprayed with hydroxyapatite and tricalcium phosphate. In each animal, one implant was also treated with recombinant transforming growth factor-beta 1 while the other implant, which was not so treated, served as a paired control. Two doses of transforming growth factor-beta 1 were used: 335 micrograms in five animals and 120 micrograms in the other five. At four weeks, the amount of bone ingrowth in the implants that had been treated with 120 micrograms of transforming growth factor-beta 1 was threefold higher than that in the paired controls (p = 0.009), but with the numbers available there was no significant increase in bone ingrowth with the higher dose. The amount of new-bone formation in the three-millimeter gaps adjacent to the treated implants was twice that in the gaps of the paired controls, regardless of the dose. The differences between the treated and control implants with regard to the architecture of the new bone in the gap indicate that the mechanism of action of transforming growth factor-beta 1 may include both proliferation of osteoprogenitor cells and production of matrix by committed osteoblasts. Compared with the findings in a previous study in which this canine model was used, the data from the present investigation indicate that enhancement of bone ingrowth in implants that have been treated with a combination of a hydroxyapatite-tricalcium phosphate coating and transforming growth factor-beta 1 may exceed that obtainable with grafting of the gap with autogenous cancellous bone.

Bone ingrowth and wear debris in well-fixed cementless porous-coated tibial components removed from patients.

Bone ingrowth and the distribution of wear debris within the porous coating of 13 primary cementless porous-coated tibial components removed for reasons unrelated to fixation or infection were quantitatively described. The average length of implantation was 15.3 months (range, 3-30 months). The implants were all of the same design, made for Ti6A14V with a commercially pure titanium fiber-metal porous coating, which covered the undersurface of the tray and the four fixation pegs. In all but one component, supplemental screw fixation was used. The average extent of bone ingrowth within the tray was 27.1 +/- 16.1%, and the average volume fraction was 9.5 +/- 7.5%. There was significantly more bone ingrowth within the fixation pegs than within the tray and also more bone ingrowth in the anterior half of the tray than posteriorly. There was no correlation between the amount of bone ingrowth and the length of implantation, age, or sex of the patient; however, the depth and orientation of the resection plane were found to correlate with the topographic distribution of bone ingrowth. Particulate debris appeared to gain access to the interface via soft tissue pathways both at the periphery and through the holes for adjuvant screw fixation.

The susceptibility of smooth implant surfaces to periimplant fibrosis and migration of polyethylene wear debris.

The purpose of this investigation was to establish whether the tissue response and migration of polyethylene debris differed at noncemented smooth and porous implant surfaces. This was accomplished through 3 separate but closely related studies: (1) a canine cylindrical implant model with smooth and porous surfaces exposed to polyethylene debris; (2) a canine total hip arthroplasty model analyzing the interface between bone and femoral implants with various porous-coating configurations; and (3) a histologic analysis of autopsy-retrieved, human, noncemented hip prostheses with noncircumferential porous coating. The cylindrical implant model involved the placement of split cylinders, 1/2 porous and 1/2 smooth, into the distal femur and proximal tibia of 4 dogs. Four control implants and 10 test implants (chronically exposed to simulated polyethylene debris with a mean size of 4.7 microns) were examined histologically as long as 30 weeks after surgery. The canine hip study involved the study of 54 noncemented hip prostheses at periods of 1, 6, and 24 months. The prostheses possessed 4 different porous surface configurations: 1 with circumferential porous coating, 2 with noncircumferential coating, and 1 without porous coating. The human retrieval analysis involved the study of 7 cadaveric femora (age, 6 months-5 years) implanted with a straight titanium-alloy prosthesis possessing proximal pads of titanium fiber metal on the anterior, posterior, and medial aspects. With all implants in all 3 studies, there was the common finding of bone ingrowth at the porous implant surface and a fibrous interface or periprosthetic cavity around the portion of the implant that was smooth surfaced. The periprosthetic cavity typically was encapsulated by a thin continuous shell of trabecular bone. In addition, polyethylene debris was found to have preferentially migrated along the smooth implant surfaces. In the longer-term canine and human hip retrievals, polyethylene particles in the micron size range were present within histiocytes, whereas larger particles as much as 100 microns were found within foreign-body giant cells. Of importance for the implants from all 3 studies, with the exception of some pronounced cavities on the lateral aspect of the human hip prostheses, the periimplant cavities around the smooth surfaces were not detectable radiographically. This study clearly established a fundamental principle of relative barriers to particulate debris migration. Smooth implant surfaces are more susceptible than porous surfaces to the development of a fibrous tissue filled periimplant cavity and the subsequent migration of polyethylene wear debris.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of pegs and screws on bone ingrowth in cementless total knee arthroplasty.

The purpose of the present study was to assess the effects of pegs and screws on bone ingrowth into the tibial component in cementless total knee replacement. Left total knee replacements were performed in 21 mature male dogs with 3 cementless porous-coated tibial tray configurations (7 animals per group): (1) 4-peg design implanted with cortical screws passing through the pegs; (2) 4-peg design implanted without screws; and (3) pegless design secured by 4 cortical screws. The animals were allowed unrestricted activity and were euthanized 6 months postoperatively. The pegless components (Group 3) had the highest extent of bone ingrowth into the tray (90.3% +/- 9.4%), followed by the components with 4 pegs only (Group 2, 82.8% +/- 9.2%), and the components with 4 pegs and 4 screws (Group 1, 75.9% +/- 11.8%). The difference between Groups 1 and 3 was statistically significant (p < 0.05). The volume fraction of bone ingrowth in the tray did not differ among the 3 groups, with an overall mean of 22.5% (+/- 4.6%). At the posterolateral quadrant, Group 1 had significantly less bone ingrowth than Group 3 within the tray whether measured as the extent (63.6% +/- 20.5% versus 91.0% +/- 10.6%, p < 0.05) or volume fraction (19.1% +/- 8.8% versus 32.9% +/- 10.5%, p < 0.05). There were no between-group differences at the other quadrants. This study indicated that pegs provided no added benefit in a circumstance where sufficient initial fixation was obtained with screws.

Distribution of Young's modulus in the cancellous bone of the proximal canine tibia.

Canine cancellous bone is used as a model for human bone in experimental orthopedic research, including models of total knee arthroplasty. Depth-force measurements produced by small-diameter indentation testing were used to derive the variation of Young's modulus over the transverse cross-sectional surface at three levels within the proximal canine tibia. At the most proximal section the presence of lateral and medial peaks of equal modulus (approximately 1100 MPa) was found. Modulus averages for the three resection levels revealed a trend of distally decreasing values, from 692 MPa proximally to 417 MPa distally. Average regional modulus values for the canine tibia were 50-75% higher than previously reported for the tibia of healthy young adult humans, although the local maxima were only 5-20% greater in canines.