ORIGINAL ARTICLE

Pediatric solid organ transplantation and osteoporosis:a descriptive study on bone histomorphometric findings

Inari S. Tamminen & Helena Valta & Hannu Jalanko &

Sari Salminen & Mervi K. Mäyränpää & Hanna Isaksson &

Heikki Kröger & Outi Mäkitie

Received: 23 September 2013 /Revised: 15 January 2014 /Accepted: 22 January 2014# IPNA 2014

AbstractBackground Organ transplantation may lead to secondaryosteoporosis in children. This study characterized bonehistomorphometric findings in pediatric solid organ transplantrecipients who were assessed for suspected secondaryosteoporosis.Methods Iliac crest biopsies were obtained from 19 children(7.6–18.8 years, 11 male) who had undergone kidney (n=6),

liver (n=9), or heart (n=4) transplantation a median 4.6 years(range 0.6–16.3 years) earlier. All patients had received oralglucocorticoids at the time of the biopsy.Results Of the 19 patients, 21 % had sustained peripheralfractures and 58 % vertebral compression fractures. Nine chil-dren (47 %) had a lumbar spine BMD Z-score below −2.0.Histomorphometric analyses showed low trabecular bone vol-ume (< −1.0 SD) in 6 children (32 %) and decreased trabecularthickness in 14 children (74 %). Seven children (37 %) hadhigh bone turnover at biopsy, and low turnover was found in 6children (32 %), 1 of whom had adynamic bone disease.Conclusions There was a great heterogeneity in the histolog-ical findings in different transplant groups, and the resultswere unpredictable using non-invasive methods. The ob-served changes in bone quality (i.e. abnormal turnover rate,thin trabeculae) rather than the actual loss of trabecular bone,might explain the increased fracture risk in pediatric solidorgan transplant recipients.

Keywords Bone histomorphometry . Cancellous bone .

Organ transplantation . Fracture . Glucocorticoid treatment .

Children

Introduction

Solid organ transplantation is an established treatment forchildren with end-stage renal disease, acute or chronic liverfailure, and with terminal heart diseases. While the overalllong-term patient survival has improved, new health concernshave emerged, i.e., secondary osteoporosis and fragility frac-tures [1–6].

Diagnosis of osteoporosis in children is challenging. Thesignificance of low bone mineral density (BMD) and associ-ation between low bone mass and fractures is less well-established in children than in adults. Dual-energy X-ray

I. S. Tamminen and H. Valta contributed equally to this work.

Electronic supplementary material The online version of this article(doi:10.1007/s00467-014-2771-1) contains supplementary material,which is available to authorized users

I. S. Tamminen (*) :H. KrögerBone and Cartilage Research Unit (BCRU), University of EasternFinland, POB 1627, Mediteknia Building, 70211 Kuopio, Finlande-mail: inari.tamminen@uef.fi

I. S. Tamminen :H. IsakssonDepartment of Applied Physics, University of Eastern Finland, POB1627, 70211 Kuopio, Finland

I. S. Tamminen :H. KrögerDepartment of Orthopaedics, Traumatology, and Hand Surgery,Kuopio University Hospital, POB 100, KYS 70029, Kuopio, Finland

H. Valta :H. Jalanko : S. Salminen :M. K. Mäyränpää :O. MäkitieChildren’s Hospital, Helsinki University Central Hospital andUniversity of Helsinki, POB 281, 00029 HUS, Helsinki, Finland

S. SalminenDepartment of Orthopaedics and Traumatology, University Hospitalof Trondheim, Trondheim, Norway

H. IsakssonDivision of Solid Mechanics, Department of Orthopedics, LundUniversity, Box 118, 221 00 Lund, Sweden

O. MäkitieFolkhälsan Research Center, Biomedicum Helsinki, Helsinki,Finland

Pediatr NephrolDOI 10.1007/s00467-014-2771-1

absorptiometry (DXA) is the method most commonly used tomeasure bone mass, but patients with short stature or delayedskeletal maturation may receive falsely low BMD values [7].Biochemical values and bone turnover markers provide littlehelp in evaluating bone mass and susceptibility to fractures[8].

Because of these difficulties in establishing the diagnosisof osteoporosis in children by non-invasive methods, bonebiopsy should be considered [3, 4]. The diagnosis of osteopo-rosis should be definite before medical therapy is considered.Quantitative histomorphometry of bone sections provides in-formation on bone turnover, mineralization, and volume. It isa valuable and well-established clinical tool for studyingmetabolic bone diseases including osteoporosis and renalosteodystrophy, and to evaluate effects of treatment [9, 10].

Only one previous study has evaluated skeletal findings bybone histomorphometry in children after solid organ trans-plantation. The aim of our study was to characterize bonehistomorphometric findings in pediatric kidney, liver, andheart transplant recipients who were evaluated for suspectedpost-transplantation osteoporosis.

Materials and methods

Patients and study design

This cross-sectional study was conducted at the Children’sHospital, Helsinki University Central Hospital, where all pe-diatric solid organ transplantations and post-operative care arecentralized in Finland. The study protocol was approved bythe institution’s Ethics Committee. Transiliac bone biopsieswere taken from 19 children with a history of kidney (n=6),liver (n=9), or heart (n=4) transplantation as a part of clinicalevaluation for suspected osteoporosis. Suspicion of secondaryosteoporosis was based on low areal BMDmeasured by DXAand/or fracture history. Vertebral fractures were diagnosed byplain spinal radiographs and loss of vertebral height ≥20 %was used for diagnosis. All children had received glucocorti-coids (GCs) since transplantation and were receiving low-dose alternate-day oral GCs (methylprednisolone). Total cu-mulative (mg) and weight-adjusted (mg/kg) GC doses as wellas GC exposure (mg/kg/days) during the previous 3 yearswere calculated as methylprednisolone equivalents. None ofthe children had been treated with aluminum-containing phos-phate binders or bisphosphonates. Prior to transplantation twokidney transplant recipients had had secondary hyperparathy-roidism, with PTH levels exceeding 7 and 11 times the uppernormal limit. In the other kidney transplant recipients thepretransplantation PTH values were only mildly elevated (upto 1.3 times the upper normal limit). None of our patients hadreceived growth hormone treatment. All patients were clini-cally assessed by a pediatric nephrologist (H.V.).

Growth and pubertal assessment

Height was reported as standard deviation (SD) units (Z-score) based on Finnish reference data. Body mass index(BMI) was calculated (kg/m2) and transformed into Z-scoresusing age- and gender-specific reference data [11]. Pubertalmaturation was recorded according to Tanner [12].

Bone biopsies

Transiliac bone samples were obtained from a standardizedsite with a 7.5-mm bone biopsy trephine (Rochester BoneBiopsy®;Medical Innovations,Milpitas, CA, USA). Eighteenchildren received double-labeling with tetracycline (15–20 mg/kg/day, maximum 1,000 mg/day) prior to the biopsy.The interval between the two 2-day tetracycline courses was10 days and the medication was discontinued 4 days beforethe biopsy. The biopsy samples were fixed in 70% ethanol forat least 48 h before embedding in polymethylmethacrylate.Samples were sectioned and stained using Masson Goldnertrichrome staining kit.

Bone histomorphometry

Bone histomorphometry (Bioquant OsteoII®; Bioquant ImageAnalysis, Nashville, TN, USA) was performed, as describedpreviously [13]. Additionally, activation frequency (Ac.F),bone formation rate (BFR/BS), and osteoid maturation time(Omt) are reported. The nomenclature, abbreviations, andunits follow the recommendations by the American Societyfor Bone and Mineral Research (ASBMR) [14]. Regions ofinterests (ROIs) were carefully selected to ensure that onlytrabecular bone was measured. All samples were measured bythe same microscopist (I.T.) using a magnification of ×200(tissue area 3.0–7.6 mm2). An extended label search wasperformed for samples with no tetracycline double labels orno tetracycline labeling in the ROI. Altogether four unstainedsections were taken 300 μm apart. To allow cross-sectionalcomparison of the findings, the histomorphometric parameterswere transformed into SD units by comparing them with theage-specific reference values [15] except for the assessment ofthe eroded surface (ES/BS), which was determined using amethod more commonly used in adults, and values over 10 %were considered abnormal [16, 17].

Histological classification

After quantitative analysis of histomorphometric parameters,patients were divided into subgroups by two experiencedreaders (H.K., I.T.) (Supplementary Fig. 1). Based on cancel-lous bone volume for age [15], the samples were divided intolow (BV/TV <1.0 SD) and normal (BV/TV ≥1.0 SD) cancel-lous bone volume groups (Table 2). Children with high and

Pediatr Nephrol

low turnover were identified; those biopsies with low turnoverwere re-evaluated to detect adynamic bone disease (ABD)[10, 18, 19]. Mineralization was evaluated and defined asosteomalacia if clearly suppressed bone turnover, i.e., nodouble labels in the extended label search, was found [10, 20].

Bone densitometry and radiographic evaluation

Bone age was determined from a plain radiograph of the lefthand [21]. Areal BMD for the lumbar spine (L1–L4) and totalhip were measured as part of the patients’ yearly follow-up byDXA (Hologic Discovery A®; Hologic, Bedford, MA, USA)using pediatric software (version 12.4). Values within6 months of the biopsy were used for the study; they weretransformed into BMD Z-scores by using age- and sex-specific reference data [5]; compressed vertebrae were exclud-ed from the measurement area. Since bone age was delayedfor more than a year in 6 children, the aBMD Z-scores wereadjusted for bone age.

Biochemistry

Blood and urine samples were collected before noon. Plasmaconcentrations of ionized calcium (P-Ca-ion), phosphate (P-Pi), alkaline phosphatase (P-ALP), and creatinine (P-Crea)were determined using standard methods. Glomerular filtrationrate (GFR) was measured by 51-labeled chromium ethylenedi-aminetetraacetic acid clearance. Kidney function was classifiedas normal (GFR>90 mL/min/1.73 m2; chronic kidney disease(CKD) stage 1), mild kidney dysfunction (GFR 60–89 mL/min/1.73 m2; CKD stage 2), and moderate kidney failure (GFR30–59 mL/min/1.73 m2; CKD stage 3). Urine samples wereanalyzed for calcium and creatinine ratio (U-Ca/U-Crea ratio,reference value ≤0.7 mmol/mmol). Serum concentration of 25-hydroxyvitamin D (S-25-OHD) and plasma parathyroid hor-mone (P-PTH) were determined, as described previously [8].Hypovitaminosis D was defined as S-25-OHD below 50 nmol/L [22]. The reference range for PTH was 8–73 ng/L.

Statistical analysis

The bone histomorphometric data were calculated as SD unitsusing the age-specific reference values [15]. Median valueswere calculated for biochemical markers and bone densitom-etry; Spearman’s correlation coefficients were calculated forvarious parameters. Samples were analyzed in subgroupsbased on transplantation history. i.e.. kidney, liver, or hearttransplant. For bone histomorphometric parameters, the non-parametric Kruskal–Wallis test was used to test differencesbetween more than two subgroups of the samples, and theMann–Whitney U test was used to test differences betweentwo subgroups of the samples. Chi-squared test was used totest differences when appropriate. Bonferroni correction was

used when appropriate. Statistical analyses were performedusing SPSS® Statistics software (version 19.0.0; SPSS, Chi-cago, IL, USA). A p value below 0.05 was considered statis-tically significant.

Results

Clinical characteristics

The cohort included 19 children (median 15.5 years, age range7.6–18.8 years, 11 male) with a median history of solid organtransplantation of 4.6 years (0.6–16.3 years) earlier (Table 1).The median height Z-score was −0.8 and the Z-score wasbelow −2.0 in three children (16 %). BMI ranged from 14.0to 36.1 kg/m2. The median BMI Z-score was −0.1 (−2.2 to +3.3). Three children were overweight (BMI>+1.0 SD) and 3were obese (BMI>+2.0 SD). Four were prepubertal, 4 puber-tal, and 11 were post-pubertal. All patients were on triple-drugimmunosuppressive medication, including cyclosporin A (n=15), tacrolimus (n=4), azathioprine (n=11), or mycophenolatemofetil (n=8), and on low-dose alternate-day methylprednis-olone and vitamin D substitution (n=19). The levels of cal-cineurin inhibitors were comparable, except in 1 patient whohad a high cyclosporin A concentration at the time of biopsy(CsA 211 μg/L) because of a recent liver transplantation. Inthe others the median tacrolimus level was 5.7 μg/L (4.1–6.3 μg/L) and the median CsA level was 78 μg/L (36–126 μg/L). None of the patients had been on immunosuppres-sive treatment prior to transplantation.

Biochemistry

One child had hypovitaminosis D (S-25-OHD 43 nmol/L);others were vitamin D sufficient (Table 1). None of the pa-tients had significantly elevated P-PTH, and mildly elevatedvalues were present in 2 (74 ng/L and 77 ng/L; reference range8–73 ng/L); ionized calcium was normal in all children. Twokidney transplant children had hypophosphatemia; none hadhyperphosphatemia. Two had mildly elevated P-ALP. Creati-nine was elevated (95–134 μmol/L) in 8 children from differ-ent transplant groups. Only 1 child had normal kidney func-tion and others had mild (CKD stage 2, n=9) or moderate(CKD stage 3, n=9) kidney failure. Kidney transplant recip-ients had more severe kidney dysfunction than the liver trans-plant recipients (p=0.025). Two children had mild hypercal-ciuria (U-Ca/U-Crea 1.01 and 1.00 mmol/mmol).

Bone densitometry and fractures

Nine children (47 %), including 7 liver and 2 heart transplantpatients, had low (<−2.0) lumbar spine BMD Z-score. Lowtotal hip BMD Z-score was observed in 5 (26 %), including 1

Pediatr Nephrol

kidney and 4 liver transplant recipients. One fifth (21%) of thechildren, all liver transplant recipients, had sustained periph-eral fractures (in humerus, radius, fibula, and metatarsus; 1 ineach), whereas vertebral compressions were found in morethan half (58 %) of the children in all transplant groups(Table 1). None of the non-vertebral fractures resulted froma high-energy injury (falling >3 m or a motor vehicle acci-dent). The median time between transplantation and the latestperipheral fracture was 5.3 (range 0.2–15.6) years and be-tween fracture and biopsy 0.5 (range 0.3–2.2) years. The exacttime of the vertebral fractures was not known, as they wereusually asymptomatic. All patients were ambulatory.

Bone biopsy findings

Structural bone parameters

Normal trabecular bone volume (BV/TV) was observed in 12children (63 %; Fig. 1a). Six children (32 %) had a BV/TV Z-score below −1.0, and one child (5 %) above +1.0 (Table 2).Only 2 children, both liver transplant recipients, had a BV/TVZ-score below −2.0. There was no difference in BV/TV Z-

score between the different transplant groups. Thin trabeculaeand increased trabecular number were observed in 14 children(74 %; Fig. 1b, c) so that increased trabecular separation wasfound only in 1 child. Pubertal stage was not associated withhistomorphometric Z-score values.

Bone turnover and mineralization

Abnormal turnover findings were common in pediatric trans-plant patients: 7 children (37 %) had high turnover and 6(32%) had low turnover (Table 2). Increased osteoid thickness(>+2.0 SD) was found in 4 children (21 %) 1 of whom hadreceived kidney and 3 liver transplant (Fig. 1e). These chil-dren showed double labels. No mineralizing defect or osteo-malacia was found. Among the subjects with low bone turn-over, 2 with appropriate tetracycline administration had nolabels in the region of interest (ROI). In the extended labelsearch patient #8 had one subcortical double label and thehistological classification remained as low bone turnover.Patient #19 had no labeling, even in the extended label search,and the histological diagnosis was ABD. In addition, in 1child labels were lacking because no tetracycline had been

Table 1 Characteristics of the pediatric solid organ transplant recipients

All (n=19) Kidney transplantation(n=6)

Liver transplantation(n=9)

Heart transplantation(n=4)

p value

Age (years) 15.5 (7.6–18.8) 17.6 (13.6 to 18.8) 15.5 (14.1 to 18.2) 11.1 (7.6 to 17.6) NS

Males/females (n) 11/8 4/2 5/4 2/2 NS

Time between transplantation and biopsy (years) 4.6 (0.6 to 16.3) 12.3 (4.3 to 15.6) 1.7 (0.6 to 16.3) 3.5 (1.4 to 6.2) NS

Height Z-score −0.8 (−2.4 to 0.9) −1.9 (−2.4 to −1.0) −0.3 (−1.7 to 0.9) −0.7 (−2.1 to 0.5) 0.019

BMI Z-score −0.1 (−2.2 to 3.3) −0.9 (−2.2 to 1.7) −0.1 (−1.8 to 3.3) −0.7 (−2.0 to 2.4) NS

Lumbar BMD Z-score −2.0 (−3.9 to 0.4) −1.3 (−2.0 to 0.4) −2.5 (−3.9 to −0.4) −1.8 (−2.7 to −1.4) 0.042

Total hip BMD Z-score −1.5 (−2.9 to 1.5) −1.3 (−2.9 to 1.5) −1.8 (−2.6 to −0.2) −1.4 (−1.7 to −0.7) NS

Daily GC dose at time of biopsy (mg) 2.0 (1.0 to 5.0) 2.0 (1.0 to 3.0) 2.0 (1.0 to 5.0) 2.0 (1.0 to 3.0) NS

Cumulative weight-adjusted GC dose (mg/kg) 61 (14 to 207) 47 (33 to 95) 89 (28 to 207) 101 (14 to 157) NS

Total GC exposure (mg/kg/days) 0.07 (0.01 to 0.30) 0.04 (0.03 to 0.09) 0.12 (0.03 to 0.20) 0.11 (0.01 to 0.30) NS

Children with non-vertebral fracture(s) (n) 4 0 4 0 NS

Children with vertebral compression(s) (n) 11 3 7 1 NS

S-25-hydroxyvitamin D (nmol/L) 62 (43 to 167) 81 (54 to 115) 62 (50 to 167) 57 (43 to 88) NS

P-parathyroid hormone (ng/L) 42 (17 to 77) 69 (30 to 77) 31 (17 to 63) 42 (23 to 66) NS

P-ionized calcium (mmol/L) 1.28 (1.20 to 1.34) 1.33 (1.24 to 1.34) 1.25 (1.20 to 1.29) 1.28 (1.21 to 1.30) 0.045

P-phosphate (mmol/L) 1.29 (0.66 to 1.65) 1.28 (0.66 to 1.38) 1.35 (1.00 to 1.43) 1.26 (1.03 to 1.65) NS

P-alkaline phosphatase (U/L) 125 (74 to 321) 109 (81 to 173) 125 (75 to 321) 197 (74 to 310) NS

P-creatinine (μmol/L) 77 (34 to 134) 117 (76 to 134) 65 (47 to 119) 47 (34 to 104) 0.018

GFR (mL/1.73 m2) 66 (31 to 96) 38 (31 to 55) 77 (41 to 96) 73 (44 to 85) 0.011

U-Ca/U-Crea ratio (mmol/mmol) 0.21 (0.07 to 1.01) 0.17 (0.07 to 1.01) 0.30 (0.11 to 1.00) 0.22 (0.11 to 0.46) NS

Results are given as median values and range for clinical characteristics, densitometry, glucocorticoid treatment, and fractures. The differences betweenvarious transplantation groupswere tested and a p value below 0.05was considered to be statistically significant (Kruskal–Wallis test except Chi-squaredtest for fractures)

NS non-significant, n number of patients, BMI body mass index, BMD bone mineral density, GC glucocorticoid, P plasma, S serum, GFR glomerularfiltration rate, U-Ca/U-Crea urinary calcium-to-creatinine ratio

Pediatr Nephrol

given prior to the biopsy. The patient with ABD was a 17-year-old heart-transplanted male with vertebral fractures andmoderately decreased kidney function (CKD stage 3, trans-plantation 2.4 years earlier). Despite kidney insufficiency, hehad normal S-25-OHD and P-PTH. His P-ALP (74 U/L) wasnormal, but the lowest in the study and his cumulative GCdose (10,133 mg) was the highest in the study.

After excluding the children without labeling (n=3), themineralizing surface (MS/BS Z-score median −1.1, range−2.7–2.3), mineral apposition rate (MAR Z-score median−2.1, range −4.3–1.4; Fig. 1g), bone formation rate (BFR/BS Z-score median −1.4, range −3.0–1.1), and activationfrequency (Ac.F Z-score median −0.9, range −2.5–2.0;Fig. 1h) all tended to be low to normal; there were no signif-icant differences between the different transplant groups. Sixchildren (32 %) had a normal turnover rate in the biopsy.

Correlations with clinical findings, biochemical parameters,densitometry, and skeletal characteristics

Children with low turnover or ABD (n=6) had higher P-PTHthan children with normal turnover (n=6; p=0.016), but onlyone of the P-PTH values was slightly above the referencerange and all others were within the reference range. Theyalso had lower P-ALP than subjects with normal or highturnover (n=13; p=0.039), but no other differences in bio-chemistry were found (Table 3). Children with low BV/TV (<−1.0 SD, n=6) where compared with those with normal BV/TV (≥−1.0 SD, n=13). There were no significant differencesin age, time from transplantation, GC treatment, DXA, frac-tures, or biochemistry between the groups. Lumbar BMD Z-score correlated positively with trabecular number (r=0.49,p=0.034) and negatively with Tb.Sp (r=−0.50, p=0.029);

Fig. 1 Bone histomorphometryresults for each transplant group(kidney, liver, heart). Age-specificreference values (mean valuesolid line, 2× standard deviation(SD) dashed lines) were obtainedfrom Glorieux et al. [15], exceptfor ES/BS, where ≤10 % wasconsidered normal and for BV/TV where ±1 SD was considerednormal. Age is given in years

Pediatr Nephrol

Table2

Bonehistom

orphom

etry

findings

inpediatricsolid

organtransplant

recipients

Patient

#Sex

Transplanto

rgan

Age

(years)

Structural

Form

ation

Resorption

Interpretatio

n

BV/TV

Tb.Th

Tb.N

Tb.Sp

OV/BV

OS/BS

O.Th

Ob.S/BS

MS/OS

BFR

/BS

MAR

Ac.F

ES/BS(%

)Oc.S/BS

Bonevolume

Turnover

1Male

Kidney

13.6

−0.7

−3.0

4.7

−2.2

0.0

−1.1

−0.4

−1.4

−3.0

−2.0

−4.3

−1.9

3.8

−0.7

Normal

Low

TO

2Female

Kidney

14.7

0.6

−3.7

13.3

−3.9

3.6

0.6

1.1

−1.5

−1.5

−1.4

−2.1

−0.7

4.1

2.3

Normal

HighTO

3Female

Kidney

17.1

0.9

−1.8

4.1

−2.2

−0.8

−2.2

3.8

−2.0

−3.1

−2.3

−3.4

−2.2

2.8

4.4

Normal

Low

TO

4Male

Kidney

18.1

0.3

−2.6

5.1

−2.4

0.5

0.3

−1.1

−1.4

−3.9

−2.2

−2.1

−2.1

7.7

2.0

Normal

Normal

5Male

Kidney

18.2

−1.1

−2.3

1.7

−0.9

−0.1

−0.8

−0.7

−1.6

−1.1

−1.3

−2.4

−1.3

2.0

− 1.0

Low

Normal

6Male

Kidney

18.8

1.0

−0.7

2.0

−1.5

2.2

1.5

0.7

3.3

−1.2

−0.3

−2.5

0.0

4.8

−0.2

Normal

HighTO

7Male

Liver

14.1

−1.1

−2.2

1.1

−0.2

1.8

−0.5

2.9

2.9

−0.5

−1.2

−1.6

−0.7

4.6

2.0

Low

HighTO

8Male

Liver

14.2

−3.7

−5.0

−1.9

3.5

3.3

−0.3

−1.6

−1.9

N/A

N/A

N/A

N/A

5.7

−1.5

Low

Low

TOa

9Female

Liver

14.8

−2.2

−3.1

−0.1

1.0

2.4

−0.8

4.6

−1.0

−0.5

−1.5

−1.5

−1.0

6.0

−0.5

Low

HighTO

10Female

Liver

15.4

−0.1

−1.4

2.2

−1.2

−1.0

−1.5

0.4

−1.7

N/A

N/A

N/A

N/A

0.8

−1.5

Normal

Low

TOb

11Male

Liver

15.5

0.2

−2.1

5.1

−2.3

0.5

−1.1

1.8

0.4

2.1

0.4

−0.1

0.6

5.4

−1.5

Normal

Normal

12Female

Liver

15.8

−0.5

−2.7

4.6

−1.9

1.9

−0.7

5.2

−0.8

−0.4

−1.3

−1.5

−0.8

3.5

1.1

Normal

HighTO

13Female

Liver

16.9

0.1

−1.7

3.5

−1.8

−1.8

−2.6

−1.2

−1.8

2.2

−3.0

−4.2

−2.5

0.8

−0.9

Normal

Low

TO

14Male

Liver

17.3

0.4

−1.6

2.7

−1.7

0.8

−0.3

1.7

1.4

2.7

1.1

−1.4

2.0

17.9

14.2

Normal

HighTO

15Male

Liver

18.2

−0.3

−2.6

4.1

−2.0

0.1

−0.7

−0.6

−1.6

−3.2

−2.2

−4.2

−2.0

6.2

3.3

Normal

Normal

16Male

Heart

7.6

−1.2

−3.9

6.3

−2.6

−0.6

−1.5

0.2

−1.8

1.2

−1.4

1.4

−0.6

12.2

0.5

Low

Normal

17Female

Heart

10.4

0.9

−2.1

5.2

−2.7

0.5

−0.9

0.7

−1.2

−2.8

−1.7

−0.8

−1.8

5.3

1.5

Normal

Normal

18Female

Heart

11.7

1.9

−2.1

7.4

−3.3

2.7

1.2

0.7

2.0

0.2

0.2

−2.5

1.0

8.1

3.8

Normal

HighTO

19Male

Heart

17.6

−1.1

−3.2

4.2

−1.9

−0.9

−1.9

−0.8

−2.0

N/A

N/A

N/A

N/A

4.8

−0.2

Low

Low

TO–ABDc

Mean

15.3

−0.3

−2.5

4.0

−1.6

0.8

−0.7

0.9

−0.6

−0.8

−1.2

−2.1

−0.9

5.6

1.4

SD2.9

1.3

1.0

3.2

1.6

1.6

1.1

2.0

1.7

2.1

1.1

1.5

1.3

4.0

3.6

Median

15.5

−0.1

−2.3

4.1

−1.9

0.5

−0.8

0.7

−1.4

−0.8

−1.4

−2.1

−0.9

4.8

0.5

Reference

values

forchild

renwereobtained

from

Glorieuxetal.[15],except

forerosionsurface(ES/BS,n

ormal≤1

0%).Tableshow

sstandard

deviationZ-scoresforeach

parameter.T

rabecularbone

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Pediatr Nephrol

other structural bone histomorphometry parameters showedno correlation with BMD.

Children with a higher present GC dose (≥3 mg/day) hadsignificantly lower Oc.S/BS (p=0.018) and osteoid matura-tion time (Omt, p=0.028) than children with the lower GCdose; no differences were seen in the osteoblast number of thebone resorption surface between the groups. A negative cor-relation was found between GC dose at the time of biopsy andOc.S/BS Z-score (r=−0.55, p=0.016). Oc.S/BS Z-score cor-related positively with the time from transplantation to thebiopsy (r=0.66, p=0.002) and lumbar BMD Z-score (r=0.47,p=0.043). Children with normal or mild kidney dysfunctionwere younger and had more recently undergone transplanta-tion, had a lower lumbar BMD Z-score, S-25-OHD, and P-Calevel, but similar serum phosphate level, compared with chil-dren with moderate kidney dysfunction (p<0.05). Bonehistomorphometry (Z-scores) did not differ between thegroups.

Discussion

In this study bone histomorphometry was assessed in 19children and adolescents who had undergone kidney, liver,or heart transplantation. All had received GC treatment andhad suspected secondary osteoporosis. More than half (58 %)had had vertebral compression fractures and 21 % had ahistory of peripheral fractures, suggesting impaired strengthof both trabecular and cortical bone. Analysis of bonehistomorphometry showed that abnormal bone biopsy find-ings in various parameters reflecting bone quantity, quality, ordynamics are common after pediatric solid organtransplantation.

There are no established cut-offs (e.g., SD values) for thevarious histomorphometric parameters to define osteoporosisin children. In our study, low cancellous bone volume wasdefined as a BV/TV Z-score below −1.0 compared with age-matched reference values [15]. Similar cut-off limits have

Table 3 Pediatric transplant patients grouped based on the histological turnover rate

Adynamic bone disease(n=1)

Low turnover(n=5)

Normal turnover(n=6)

High turnover(n=7)

p value

Age (years) 17.6 15.4 (13.6–17.1) 16.8 (7.6–18.2) 14.8 (11.7–18.8) NS

Interval between transplantation and biopsy (years) 2.4 2.5 (0.6–15.4) 6.7 (1.3–15.6) 4.3 (1.2–16.3) NS

BMI Z-score 2.4 1.2 (0.2–3.3) −0.7 (−1.9–2.3) −1.5 (−2.2– −0.1) 0.007

Lumbar BMD Z-score −1.5 −2.3 (−3.3–0.4) −2.3 (−2.7– −1.0) −1.9 (−3.9– −0.4) NS

Total hip BMD Z-score −1.7 −1.7 (−2.9– −0.9) −1.5 (−2.1– −0.7) −1.5 (−2.4–1.5) NS

GC dose at time of biopsy (mg) 3.0 2.0 (1.0–5.0) 2.0 (1.0–4.0) 2.0 (1.0–4.0) NS

Total GC exposure (mg/kg/days) 0.14 0.12 (0.04–0.19) 0.06 (0.01–0.20) 0.06 (0.03–0.30) NS

Children with nonvertebral fracture(s) (n) 0 1 0 3 NS

Children with vertebral compression(s) (n) 1 4 4 2 NS

P-parathyroid hormone (ng/L) 66 48 (31–74) 30 (23–42) 58 (17–77) 0.047

P-ionized calcium (mmol/L) 1.30 1.29 (1.25–1.33) 1.27 (1.20–1.33) 1.25 (1.21–1.34) NS

S-25-hydroxyvitamin D (nmol/L) 88 65 (54–115) 63 (43–97) 59 (50–167) NS

P-alkaline phosphatase (U/L) 74 94 (75–173) 140 (81–235) 170 (86–321) NS

BV/TV (%) 22.8 24.9 (6.3–32.1) 26.7 (17.5–29.1) 28.7 (14.3–32.5) NS

OV/BV (%) 1.0 1.3 (0.5–5.3) 2.0 (1.5–2.7) 4.0 (2.1–5.6) 0.013

OS/BS (%) 6.1 13.8 (4.5–23.4) 13.9 (10.3–18.4) 22.1 (14.8–31.6) 0.011

O.Th (μm) 5.9 6.0 (4.7–11.5) 6.1 (5.5–8.1) 8.9 (7.4–11.5) 0.042

ES/BS (%) 4.8 2.8 (0.8–5.7) 5.8 (2.0–12.2) 4.8 (3.5–17.9) NS

Ob.S/BS (%) 0.0 0.5 (0.0–1.0) 1.1 (0.3–9.4) 9.0 (1.6–19.9) 0.002

Oc.S/BS (%) 1.0 0.4 (0.0–2.8) 1.6 (0.0–2.4) 2.4 (0.8–6.7) NS

MAR (μm/day) N/Aa 0.43 (0.0–0.48) 0.68 (0.37–1.05) 0.64 (0.52–0.68) 0.007

Ac.F (1/year) N/Aa 0.03 (0.0–0.15) 0.22 (0.05–1.01) 0.63 (0.55–1.19) 0.004

Median values and range are presented for clinical characteristics, densitometry, glucocorticoid treatment, fractures, biochemistry, and bonehistomorphometry. Statistical differences between various turnover stages were tested and a p value below 0.05 was considered to be statisticallysignificant (Kruskal–Wallis test except for the Chi-squared test for fractures). For the analysis, the patient with adynamic bone disease was combinedwith patients with low turnover findings in the biopsy

BMD bone mineral density, GC glucocorticoid, P plasma, S serum, N/A not availablea No tetracycline labels in the extended label search

Pediatr Nephrol

been used before [8, 13]. By histomorphometry, low BV/TVwas observed in only one third of the children (32 %) whereaslow lumbar BMD Z-score by DXA (<−2.0) was found in halfof the patients (47 %). The lumbar spine has significantlymore trabecular bone than the iliac crest where the bonebiopsy was obtained, which may explain the differences.Despite normal cancellous bone volume, the trabecular archi-tecture was often altered and was characterized by thin tra-beculae and an increased trabecular number. Since most of thepatients had normal height and skeletal maturation, thesechanges are likely to reflect true alterations of bone quality.Sanchez and coworkers observed no differences in trabecularthickness or number in children after renal transplantation[23]. In our previous study apparently healthy, fracture-prone children had low-normal trabecular thickness and a highto normal trabecular number, but the changes were less sig-nificant than in the present study [8]. The observed changes inthis study are likely to have an adverse effect on bone strengthand fracture resistance.

Only one third of the children (32 %) had normal boneremodeling. However, 2 of the children with a normal turn-over had low trabecular bone volume. Consequently, only 4children (21 %) had normal biopsy findings. High turnoverwas present in 37 %; low turnover was present in 32 %,including 1 with ABD. The prevalence of ABD in childrenis highly dependent on the reference values used, and thedefinitions of ABD vary greatly in both adult and pediatricpatients [24–26]. ABD is often characterized by an absence ofremodeling activity [18] and dramatically decreased boneformation with relatively increased or decreased bone resorp-tion [19]. The amount of bone cells is decreased [18, 19].Some previous studies have included samples with a normalrange of bone cells and osteoid [25, 27], whereas we carefullyevaluated the low turnover cases histologically to confirm thesimultaneous presence of both absent remodeling and a dra-matic decrease in bone formation. None of the children hadovert osteomalacia. The abnormal turnover rate in combina-tion with altered bone architecture rather than actual loss oftrabecular bone volume might explain the increased fracturerisk in children after solid organ transplant; true ABD seems tobe rare.

The prevalence of osteoporosis in children after kid-ney transplantation, despite the continuous GC treatment,has been lower than expected [5]. Our findings supportthis since only one third of the children (32 %) hadhistologically confirmed low cancellous bone volume.GCs are a risk factor for ABD [28, 29]. However, only1 patient in this study had ABD. Children with vertebralcompressions had a higher present and cumulative GCdose than those without vertebral compressions. Al-though extensive GC exposure did not result in reducedtotal bone volume or ABD, GCs seem to play a role inthe development of post-transplantation bone fragility.

As one half of our children had normal or mild, and theothers moderate (stage 3 CKD) kidney insufficiency, the roleof CKD-MBD in skeletal pathology is likely to be smaller inthis group compared with those with more severe kidneyfailure. Further, most of our patients were vitamin D sufficientand had good control of PTH levels. Recent studies suggestthat alterations in skeletal mineralization and mineral homeo-stasis may be present already at very early stages of CKD [30,31]. We did not find any differences in histomorphometrybetween those with mild and those with moderate kidneyinsufficiency.

Our previous studies have shown that liver transplant re-cipients are at a higher risk of post-transplantation peripheraland spinal fractures and low BMD than other transplant re-cipients [3–5]. In line with this, liver transplant recipientstended to have lower BV/TV, higher Oc.S/BS, lower Tb.N,and higher Tb.Sp than kidney or heart transplant recipients.The structural changes rather than a mineralization defectmight explain the greater propensity to fractures in livertransplant patients compared with other groups.

Our study has some limitations. The number of sampleswas relatively low. However, to our knowledge there havebeen no previous histomorphometric studies on children afterliver or heart transplantation. Ideally, the histomorphometricfindings should be compared with age- and gender-matchedhealthy controls whose biopsies are prepared and analyzed ina similar manner to the patients’ biopsies. We compared ourresults with the literature controls, using the only normativedata that have been published [15] and also used in previouspediatric studies [8, 32, 33]. Our study cohort was fairlyheterogeneous and the interval from transplantation to thebiopsy varied; in only one child the biopsy was taken duringthe first post-transplantation year. The small number of sub-jects may have prevented us from observing potential effectsof pubertal timing on histomorphometric parameters.We havepreviously shown that pubertal bone mass gain is disturbedafter liver transplantation and that older age at transplantationis associated with a higher risk of vertebral fractures [4], but itremains to be evaluated in future studies whether these effectscan be seen at a tissue level.

We found histological evidence for low bone volume inone third of the pediatric solid organ transplant recipients(32 %). In addition, several other abnormal findings relatedto bonemicroarchitecture and turnover were observed, despiterelatively good kidney function and controlled P-PTH level;the histomorphometric findings correlated poorly with BMD.The observed changes in bone quality (i.e., abnormal turnoverrate and thin trabeculae) rather than the actual loss of trabec-ular bone, may explain the increased bone fragility in pediatricsolid organ transplant recipients. There was a great heteroge-neity in the histological findings in different transplant groups,and the results were unpredictable by non-invasive methods.The degree of kidney insufficiency did not explain this

Pediatr Nephrol

variability, but it is possible that some of the skeletal changeswere caused by mild to moderate CKD. Thus, bonehistomorphometry may provide important information in clin-ical practice to characterize skeletal status after solid organtransplantation. However, further studies in larger cohorts andwith prospective follow-up are needed to confirm our findingsand to define the long-term consequences and clinical impli-cations of these histomorphometric abnormalities.

Acknowledgements The authors would like to acknowledge Ms. AijaParkkinen and Ms. Ritva Sormunen for their assistance with samplepreparation, and financial support from Kuopio University Hospital, theAcademy of Finland, National Doctoral Programme of MusculoskeletalDisorders and Biomaterials, the Finnish Cultural Foundation, the Päivikkiand Sakari Sohlberg Foundation, the Foundation for Pediatric Research, theSigrid Juselius Foundation, Emil Aaltonen Foundation, the FolkhälsanResearch Foundation, the Helsinki University Hospital research funds,and the strategic funding of the University of Eastern Finland.

Conflict of interest All authors state that they have no conflicts ofinterest.

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