Spine fractures usually occur less commonly in men than in women. To identify the structural basis for this gender difference in vertebral fragility, we studied 1013 healthy subjects (327 men and 686 women) and 76 patients with spine fractures (26 men and 50 women). Bone mineral content (BMC), cross-sectional area (CSA), and volumetric bone mineral density (vBMD) of the third lumbar vertebral body (L3) were measured by posteroanterior (PA) and lateral scanning using dual-energy X-ray absorptiometry (DXA). In this cross-sectional study, the diminution in peak vertebral body BMC from young adulthood to old age was less in men than in women (6% vs. 27%). This diminution was the net result of two opposing changes occurring concurrently throughout adult life: the removal of bone adjacent to marrow on the inner (endosteal) surface by bone resorption and the deposition of bone on the outer (periosteal) surface by bone formation. For L3, we estimated that men resorbed 3.7 g and deposited 3.1 g, producing a net loss of 0.6 g from young adulthood to old age and women resorbed 3.1 g and deposited only 1.2 g, producing a net loss of 1.9 g. Thus, based on our indirect estimates of periosteal gain and endosteal loss across life, the observed net diminution in BMC during aging was less in men than women because absolute periosteal bone formation was greater in men than women (3.1 g vs. 1.2 g) not because absolute bone resorption was less in men. On the contrary, the absolute amount of bone resorbed was greater in men than women (3.7 g vs. 3.1 g). Periosteal bone formation also increased vertebral body CSA 3-fold more in men than in women, distributing loads onto a larger CSA, so that the load imposed per unit CSA decreased twice as much in men than in women (13% vs. 5%). In men and women with spine fractures, CSA and vBMD were reduced relative to age-matched controls. However, vBMD was no different to the adjusted vBMD in age-matched controls derived assuming controls had no periosteal bone formation during aging. Thus, large amounts of bone are resorbed in men as well as in women, accounting for the age-related increase in spine fractures in both genders. Periosteal bone formation increases CSA and offsets bone loss in both genders but more greatly in men, accounting for the lower incidence of spine fractures in men than in women. We speculate that reduced periosteal bone formation, during growth or aging, may be in part responsible for both reduced vertebral size and reduced vBMD in men and women with spine fractures. Sexual dimorphism in vertebral fragility is more the result of gender differences in age-related bone gain than age-related bone loss.
THE INCIDENCE of spine fractures increases with advancing age in men but is lower than in women while the prevalence of spine fractures has been reported to be similar, lower, or higher in men than in women.1-8) Thus, there is growing awareness that spine fractures due to osteoporosis are a public health problem in men as well as women.(9, 10) Despite this, little information is available defining the age-related changes in skeletal structure that may contribute to vertebral fragility in men and why men have fewer fractures than women.(10)
During aging in men and women, bone resorption occurs on the endocortical and trabecular surfaces of the mineralized skeleton adjacent to marrow producing bone loss and cortical and trabecular thinning.(11) Concurrently, and in both genders, bone is laid down on the outer (periosteal) surface, increasing bone size and partly offsetting the bone lost “inside” the bone.12-15) Thus, the net amount of bone lost during aging is determined by the sum of the absolute amount of bone lost inside bone plus the absolute amount of bone gained on the “outside” of the bone (Fig. 1).
It is commonly believed that men lose less bone than women. This notion is largely based on studies using bone densitometry, a technique that measures net bone loss.(16) Net bone loss is indeed less in men than women. However, this may be because bone resorption inside bone is less in men than in women or because periosteal bone formation is greater in men than in women.
Contrary to the prevailing view, close examination of the literature suggests that the amount of bone lost on the inner (endocortical and trabecular) surfaces of the vertebra during aging, measured using quantitative computed tomography, is similar in men and women.17-19) Likewise, trabecular bone loss at the iliac crest, measured using quantitative histomorphometry, is similar in men and women.20-22) In addition, the decrease in ash density of vertebral trabecular bone is similar in men and women.(23, 24) By contrast, studies in long bones such as the tibia or femur do confirm that cortical bone loss is less in men than in women. However, this lesser fall in cortical bone mass across age in men, at least in the appendicular skeleton, is due to greater periosteal bone formation in men, not due to less resorptive removal of bone on the inner (endocortical) surface of the cortical shell in men than women.(14, 15)
Thus, the periosteal and endosteal surfaces of the skeleton behave differently during aging, with bone formation occurring on the former and bone resorption occurring on the latter. These surfaces are independently regulated, and changes on each surface are gender specific, site specific, and have different biomechanical effects.
The purpose of this study was to compare and contrast the structural changes that occur in the vertebral body with advancing age in healthy men and women and in men and women with spine fractures to gain insight into the structural basis of vertebral fragility. We asked the following questions. (i) Is vertebral body periosteal bone formation greater in men than women? If so, to what extent does periosteal bone formation offset endosteal bone loss and increase the cross-sectional area (CSA) of the vertebral body in men and women? (ii) What are the biomechanical effects of the changes in bone mass and vertebral body CSA with age in men and women? (iii) What structural and biomechanical abnormalities are found in men and women with spine fractures?
MATERIALS AND METHODS
We studied 327 healthy men and 686 healthy women aged 18-92 years: 85 young men and 282 premenopausal women aged between 18 and 43 years, 85 men and 209 women aged between 44 and 59 years, and 157 elderly men and 195 elderly women aged between 60 and 92 years. All subjects were ambulatory and were recruited from the local community. They had no illnesses, received no medication known to affect bone mass or bone size, and had no history of spine and hip fractures.
We also studied 26 men aged 41-84 years and 50 postmenopausal women aged 46-85 years with one or more vertebral fractures based on a reduction of vertebral body height by >20% on a lateral radiograph. The patients were recruited from the Metabolic Bone Clinic of the Austin and Repatriation Medical Center in Melbourne, a referral center for the investigation and management of patients with fragility fractures. We excluded patients with secondary osteoporosis due to thyroid diseases, primary hyperparathyroidism, multiple myeloma, corticosteroid therapy, and chronic liver or renal disease. We also excluded patients with a fracture of the third lumbar vertebra (the measurement site) and those taking medication known to affect bone at the time of bone mineral density (BMD) measurement. All volunteers signed a general consent form for bone densitometry. The study was approved by the Human Research Ethics Committee of the Austin and Repatriation Medical Center.
Vertebral body dimensions, bone mineral content, and areal and volumetric BMD
The bone mineral content (BMC; g), areal BMD (aBMD, g/cm2), and dimensions of the third lumbar vertebra were measured by posteroanterior (PA) and lateral scanning using dual-energy X-ray absorptiometry (DXA; DPX-L, Version 1.3z; Lunar Corp., Madison, WI, USA).(25) Lateral scanning allowed measurement of the vertebral body BMC free of the posterior processes. Vertebral body average width in the frontal view was measured from the PA scan. Vertebral body height and depth were derived from the lateral scan. Height was the average of anterior, middle, and posterior heights using the DXA ruler function. The average depth was derived by dividing the vertebral body area in the lateral scan by its height. Vertebral body CSA = π × width/2 × depth/2. Vertebral body volume (V) = π × width/2 × depth/2 × height,(26) assuming the vertebral body is an ellipsoid cylinder, an accurate approximation compared with submersion.(27) Vertebral body volumetric BMD (vBMD) was calculated as BMC/volume given the that volume is estimated from the formula provided.
The measures of the vertebral body BMC, area, and depth also were done in its midportion in the lateral scan to avoid end plate sclerosis. The region of interest was the middle one-third of the vertebral body. Depth was measured in the lateral scan at the middle of the vertebral body, and height was derived by dividing the area of the middle one-third of the vertebral body by its depth. Middle vertebral body width in the PA view could not be measured directly and therefore it was derived from the regression between the average width (provided by the printout of the projected area in the PA direction) versus the average depth. vBMD of the middle one-third vertebral body was calculated as BMC/volume, where volume = π × width/2 × depth/2 × height. All the manual measurements were made by one investigator (Y.D.) blinded to age, gender, and fracture status. The CV for these measurements ranged between 1.5 and 5.7% based on scanning 15 subjects twice within 3 months.
Calculation of absolute amount of periosteal bone formation and endosteal bone resorption
The change in BMC with aging is the result of two processes, periosteal apposition that takes place on the outside of the bone and endosteal resorption that takes place on the inside of the bone, by bone resorption on its trabecular, endocortical, and intracortical surfaces adjacent to the marrow (Fig. 1). We estimated the amount of periosteal apposition that occurred during aging by assuming that the increase in volume of the vertebral body with age was caused by only periosteal apposition. Age-related periosteal bone formation increases vertebral body volume by increasing vertebral body width and depth, not height. To avoid confounding factors like change in vertebral height with age and osteophyte formation, analyses also were confined to the middle one-third of the vertebral body. The difference between the observed BMC in old age and the amount of bone deposited on the periosteum gives us the estimated BMC that would have been observed had there not been periosteal apposition during aging. That is, the BMC remaining if the only process occurring was bone loss inside the bone. To estimate bone loss from the endocortical and trabecular surfaces, we subtracted the adjusted BMC in old age (the BMC after removing the estimated periosteal apposition during aging) from the observed peak BMC in young adulthood. In mathematical terms, the calculation was where BMCadded is the estimated amount of BMC added by periosteal apposition, Volo is the volume of the vertebral body in older subjects, Voly is the volume of the vertebral body in younger subjects, 1.2 g/cm3 is the mineral density of cortical bone, BMCadj is the BMC adjusted to eliminate the effects of periosteal apposition, BMCo is the BMC of older subjects, BMCy is the BMC of younger subjects, and BMClost is the estimated amount of BMC lost from endocortical and trabecular surfaces.
The new bone deposited on the external surface of the vertebral body during aging was assumed to be cortical bone, because periosteal osteoblasts produce lamellar cortical bone at most skeletal sites in the adult human, including the vertebral bodies. The mineralized matrix density of this periosteal bone was assumed to be 1.2 g/cm3.(28, 29) The mineralized matrix density (sometimes called true mineral density(29)) represents the mass of the bone mineral divided by the bone tissue volume. Laval-Jeantet et al. found mineralized matrix density of cortical bone to vary from 1.244 g/cm3 in middle aged (42-49 years) individuals to 1.190 g/cm3 in elderly (80-89 years) individuals.(29) They found the average mineralized matrix density for subjects ranging from 42 to 90 years to be 1.201 g/cm3. Consequently, 1.2 g/cm3 was used for the calculations in this study. However, we recognize that the use of a single value for mineralized matrix density represents a simplification because mineralized matrix density varies slightly with age.
It is important to note that mineralized matrix density (1.2 g/cm3) is different than the tissue density (or specific mass) of bone, which is about 1.85 g/cm3.(29) This difference arises because mineral density is mass of mineral divided by tissue volume, whereas tissue density is the mass of mineral, collagen, and water divided by tissue volume. X-ray absorptiometry does not “see” water or collagen. Instead, it is calibrated to register only mineral. Therefore, the vBMD derived from DXA is equivalent to the mineralized matrix density rather than tissue density. Dividing the BMCadj in old age by the observed vertebral body volume in the young adulthood gives the vBMDadj in old age that would have occurred had there been no periosteal bone formation during aging.
Structural failure occurs when the load per unit area on the vertebral body exceeds its strength. The load is determined by body weight, height, and the extensor muscle moment arm. During the sit-to-stand movement, the torso flexes and extends upward until an upright posture is achieved. The maximal load per unit area occurs when flexion is 50° from vertical at which point the upper body weight creates a forward bending moment supported by the extensors of the lower back (Fig. 2).(30) where 0.455 BW is the proportion of body weight in newtons (n * kg9.81) above the third lumbar (L3) vertebral body,(31)H is body height (cm) and 0.186 H is the distance from the L3 vertebral body to the center of mass of the upper body, 50° is the angle of flexion, and d is the extensor muscle moment arm.(32)
To derive d, we identified two posterior landmarks: (i) the posterior edge of the higher density region (the transverse process), and (ii) the anterior edge of the high density region. Two lengths from each scan were measured: length 1, the distance from the anterior edge of the vertebral body to the anterior edge of the high density region; and length 2, the distance from the anterior edge of the vertebral body to the posterior edge of the high density region. The extensor muscle moment arm was estimated as the distance from the center of the vertebral body to the middle lamina. Thus, we calculated the moment arm (d) as the average of length 1 and length 2 minus half of the middle vertebral body depth; that is, d = (length 1 + length 2 − midvertebral body depth)/2. The CV for these measurements ranged from 1.6% to 1.9% (Fig. 3).
The data were expressed in absolute terms and as SD scores. The T score is the observed value minus the young mean value divided by the SD in young normal subjects and expresses the group mean as the number of SDs above or below the young normal mean (of zero). The Z score is the observed value minus age-predicted mean divided by the SD in the age-matched controls and expresses the group mean as the number of SDs above or below the age-matched normal mean (of zero). The age-related changes in vertebral body vBMD, dimensions, and stress (load per unit area) were determined by linear regression analysis. One-sample t-tests were used to determine whether the T or Z scores differed from zero. Two-sample t-tests were used to determine the significance of any trait differences in patients with fractures relative to controls. Results are presented as mean ± SEM and were regarded as significant at the 5% level (two tailed). In the Results section, only mean values are presented for the sake of brevity; SEMs and p values are shown in the Table 1.
Table Table 1.. Age, Height, Weight, Third Lumbar Vertebral Body Width, Depth, Height, CSA, Volume, BMC, aBMD, vBMD, Muscle Moment Arm, and Load Per Unit Area (Stress) in Healthy Men and Women and Patients With Spine Fractures
Young men had greater vertebral body width, depth, and height than young women; CSA was 24.8% greater in young men than in women (Table 1). During aging, periosteal bone formation increased vertebral body width and depth, increasing CSA three times more in men than in women (9.8% vs. 3.3%; slopes, 0.0274 cm2/year vs. 0.0106 cm2/year; p < 0.001) so that in old age, men had 32.6% greater vertebral body CSA than women (Fig. 4).
Peak vertebral body BMC was higher in men than in women (8.98 g vs. 6.74 g) (because men had a bigger vertebral body volume; Table 1 and Fig. 4). Using the calculation described in the Materials and Methods section, from young adulthood to old age, periosteal bone formation in men resulted in an absolute gain of 3.1 g bone mineral and endosteal resorption resulted in 3.7 g absolute bone loss, producing 0.6 g net bone loss. Periosteal bone formation in women resulted in an absolute gain of 1.2 g bone mineral and endosteal resorption resulted in 3.1 g absolute bone loss, producing 1.9 g net bone loss. Thus, net bone loss was less in men than in women (0.6 g vs. 1.9 g) because absolute periosteal bone formation was greater in men than in women (3.1 g vs. 1.2 g), not because absolute endosteal bone resorbed was less. On the contrary, absolute amount of bone resorbed was greater in men than in women (3.7 g vs. 3.1 g) and only slightly less expressed as a percent of the (higher) peak BMC in men than in women (41% vs. 46%).
The results were similar when expressed as vBMD estimated from DXA. The observed peak vBMD was no different in young men and women (0.253 g/cm3 vs. 0.250 g/cm3), but was 0.222 g/cm3 and 0.176 g/cm3 in elderly men and women, respectively. Thus, the observed age-related fall in vBMD from young adulthood to old age was 12.1% in men and 29.8% in women (slopes, −0.0009 g/cm3 per year vs. −0.0020 g/cm3 per year; p < 0.001; Fig. 4). However, the adjusted vBMD derived in elderly men and women, had no periosteal bone formation taken place, was 0.150 g/cm3 versus 0.135 g/cm3, respectively, lower than the respective observed values. Therefore, without compensatory periosteal bone formation, vBMD of the whole vertebral body (the amount of bone within the confines of the periosteal envelope) declined more greatly (by 40.7% in men and by 46.0% in women). The results expressed as BMC or vBMD were similar when calculations were confined to the midvertebral body (data not shown). Thus, periosteal bone formation both increased bone size and offset the fall in vBMD in both genders but more greatly in men.
Men and women with spine fractures
Men and women with spine fractures had reduced vertebral body CSA and height relative to controls. vBMD was similar in men and women with spine fractures (0.149 g/cm3 vs. 0.140 g/cm3; Table 1). These values were lower than observed in age- and gender-matched controls but no different from the adjusted vBMD in age-matched controls derived assuming no periosteal bone formation occurred (Fig. 5).
Vertebral body CSA was larger in young men than in young women, but men were taller and weighed more. Because of this scaling, the load per unit area (stress) on the vertebral body was no different in young men and women (261.9 N/cm2 vs. 265.9 N/cm2). Because age-related periosteal bone formation increased the CSA 3-fold more in men than in women, the load imposed on the vertebral body per unit CSA decreased more greatly in men than in women (13% vs. 5%, respectively; Fig. 4). The load per unit area in patients with spine fractures was reduced relative to controls (not increased) because body weight and height were significantly lower in fracture cases than in controls (Table 1).
During aging, men and women form bone on the periosteal (outer) surface of bone and lose bone on the endosteal (inner) surface. Periosteal bone formation offsets endosteal bone loss in both genders but more so in men than in women, so that the net amount of bone lost across age is less in men than in women. In addition, periosteal bone formation increases the CSA of the vertebral body in both genders but more so in men than in women, so that the load per unit area imposed on the vertebral body decreases in both genders but more so in men than in women. Thus, the vertebral body adapts better in men than in women during aging by greater periosteal bone formation, resulting in lower stresses on the vertebral body and less net bone loss.
Net bone loss is less in men than in women because men form more absolute periosteal bone
It is commonly believed that men lose less bone than women during aging.(16, 33, 34) This notion is based on measurements of net bone loss using bone densitometry. Net bone loss during aging is indeed less in men than women. However, this is not because absolute bone loss is less in men than in women. On the contrary, estimated absolute L3 vertebral bone loss caused by resorptive removal of endosteal bone is greater in men than in women (3.7 g vs. 3.1 g) and is only slightly less when expressed as a percentage decline from peak in young adulthood (41% vs. 46%). Net bone loss is less in men than in women because men form more bone on the periosteum than women (3.1 g vs. 1.2 g).
There is a great deal of published literature supporting these data.13-15, 23, 35-37) Ruff and Hayes report greater gender differences in periosteal bone formation than endosteal bone resorption at the tibia.(14) Per decade, tibial CSA (reflecting absolute periosteal bone formation) increased more in men than in women (2.5% vs. 1.1%, respectively). Endosteal area (reflecting absolute bone resorbed) increased similarly in men and women (7% vs. 8%, respectively). Garn et al. reported that men had greater periosteal bone gain and less endosteal resorptive bone loss than women at the metacarpal.(15) Mosekilde et al. reported that vertebral body CSA increased by 25-30% across age in men, not in women.(23) By contrast, and as reviewed in the introduction, the amount of trabecular bone lost at the spine and iliac crest across age is similar in men and women.17-24) Thus, there are many studies independently showing that the CSA of the axial and appendicular skeleton increased more greatly in men than in women during aging because of greater periosteal bone formation in men, while the absolute amount of endosteal bone lost is similar in men and women. Consequently, the sexual dimorphism in net age-related bone loss is caused by the greater periosteal bone formation, not less endosteal bone loss, in men than in women.
This study and the aforementioned literature do not diminish the importance of bone loss. On the contrary, this study emphasizes that bone loss is important; both men and women lost almost half of their peak bone mass in absolute terms from the endosteal surface of bone. This bone loss produces architectural disruption such as cortical thinning and porosity, trabecular thinning, and perforation that is likely to explain why fractures occur in both genders. However, fractures occur less often in men than in women because the greater concomitant periosteal bone formation does two things: it offsets bone loss more greatly in men than in women, maintaining vBMD of the whole vertebral body more greatly in men than in women, and it increases the CSA of the vertebral body more greatly in men than in women, reducing the imposed load per unit area more greatly in men than in women.
The possible role of reduced periosteal bone formation in the pathogenesis of spine fractures
Men and women with spine fractures have both smaller vertebral body size and reduced vBMD (or less bone within the periosteal envelope of the smaller bone).(25, 32, 38-41) Generally, it is believed that the reduced vBMD is caused by excessive bone loss. Our observation that vBMD in the patients with spine fractures was no different to the adjusted vBMD in controls derived assuming no periosteal apposition had occurred during aging, has led us to speculate that reduced periosteal bone formation may be responsible for both reduced vertebral size and in part for the reduced vBMD in men and women with spine fractures.
Over 50 years ago, Fuller Albright suggested that osteoporosis was a disease of reduced bone formation,(42) a notion that has been ignored in favor of the view that increased bone resorption is largely responsible for bone loss. We suggest that reduced periosteal (cortical) bone formation may form the structural basis of reduced bone formation. One small study of cadaveric specimens reports reduced vertebral body cortical thickness in patients with spine fractures.(43) However, many investigators report that cortical thickness is reduced by about 20-40% in patients with spine fractures based on iliac crest histomorphometry.44-46) Both Kimmel et al. and Foldes et al. report that the reduced cortical thickness was likely to be caused by reduced periosteal apposition, not increased endocortical resorption, because the bone biopsy specimen core thickness (the distance between the periosteal surfaces of the outer and inner iliac cortices) was reduced while the medullary width was normal relative to controls.(44, 45)
Most biochemical or histomorphometric measures of bone resorption and bone formation show a wide scatter, with normal, reduced, or increased values for bone remodeling, bone resorption, and bone formation.22, 47-49) These varying biochemical and histomorphometric observations can be understood if the pathogenesis of bone fragility is regarded as being heterogeneous.
We suggest that patients with one or more spine fractures are unlikely to represent a single homogeneous group of individuals with one unifying cause of bone fragility. A more parsimonious view fitting the observed biochemical and structural diversity is that the underlying bone fragility leading to spine fractures is heterogenous. Both increased endosteal bone resorption and reduced periosteal bone formation are important. Excessive endosteal bone resorption is responsible for reduced bone mass in some patients; reduced periosteal bone formation during growth or aging is responsible for reduced bone mass and bone size in others.
The observations made here explain why men fracture less frequently than women. The greater increase in CSA in men than in woman lowered the load imposed per unit area more greatly in men, while the lesser fall in vBMD in men than in women maintained vertebral strength more greatly in men than in women. Thus, fewer men than women fracture because a lower proportion of men are at risk by virtue of the compensatory effects of periosteal bone formation in offsetting the fall in vertebral strength produced by bone loss, by both increasing bone size and offsetting the fall in vBMD, the two important determinants of vertebral strength.
In summary, this study emphasizes (i) the neglected role of periosteal bone formation in determining bone size and offsetting the fall in vBMD because of endosteal bone resorption in men and women, (ii) that absolute endosteal bone loss is similar in men and women, (iii) that net bone loss is less in men because periosteal bone formation is greater than in women, and (iv) the role of gender differences in periosteal bone formation in determining gender differences in bone fragility. This study generates the need to test the hypothesis that reduced vertebral body size and reduced vBMD in men and women with spine fractures may be in part because of reduced periosteal bone formation during growth or aging.
This study has several limitations. First, the work is cross-sectional. However, secular increases in bone size are well documented; earlier born persons (forming the elderly in the study sample) were smaller in their youth than the more recently born persons (forming the young normal subjects in the study sample) so that finding older persons having increased bone size is likely to produce a conservative error. Second, an increase in bone size may be the result of osteophytosis. However, measurements were made for the whole vertebra and in the middle of the vertebra, a region relatively free of osteophytosis. The results were consistent for both analyses, suggesting that osteophytes were not a confounding factor. Third, as bone density falls the edge detection error may produce a smaller bone area. However, bone size increased as bone density declined with age so that any error is likely to be conservative. Fourth, although the estimated vertebral body volume correlates with vertebral body volume determined by submersion, it is an estimate only. Thus, the BMCadj that would be predicted to have occurred without periosteal apposition in old age is dependent on the validity of the assumptions used to calculate the vertebral body volume, the BMCadj, and the average value of 1.2 g/cm3. Finally, whether the difference in body composition between men and women may influence the estimates of bone size and bone mass is uncertain.(50)
Within the constraints imposed by the methodological limitations mentioned previously, we suggest that the pathogenesis of bone fragility in old age is likely to be heterogenous. Reduced bone size may be the result of reduced periosteal expansion during growth, aging, or both, and reduced vBMD may be the result of reduced peak mineral accrual, age-related bone loss, or reduced periosteal apposition (which will reduce the age-related increase in CSA of bone and fail to offset the age-related fall in vBMD produced by endosteal bone loss). We conclude that advances in our understanding of the structural basis of bone fragility require the study of the periosteal and endosteal surfaces of the skeleton. The absolute changes that occur on periosteal surface during growth and aging determine the size of the skeleton in old age. The changes that occur on the endosteal surface relative to the periosteal surface determine its mineral mass and architecture. Together, these macro- and microarchitecture changes throughout life establish the strength of the skeleton in old age in health and in disease in men as well as in women.
The authors thank Sister Jan Edmonds for her assistance with subject recruitment during this study and senior technologists Patricia D'souza and Vanessa De Luca for their technical assistance.