University of Southern Denmark

Multiple Fractures and Impaired Healing in a Patient with and

Hepp, Nicola; Frederiksen, Anja Lisbeth; Dunø, Morten; Jørgensen, Niklas Rye; Langdahl, Bente; Vedtofte, Poul; Hove, Hanne B.; Hindsø, Klaus; Jensen, Jens Erik Beck

Published in: Calcified Tissue International

DOI: 10.1007/s00223-019-00605-1

Publication date: 2019

Document version: Accepted manuscript

Citation for pulished version (APA): Hepp, N., Frederiksen, A. L., Dunø, M., Jørgensen, N. R., Langdahl, B., Vedtofte, P., Hove, H. B., Hindsø, K., & Jensen, J. E. B. (2019). Multiple Fractures and Impaired Bone Fracture Healing in a Patient with Pycnodysostosis and Hypophosphatasia. Calcified Tissue International, 105(6), 681-686. https://doi.org/10.1007/s00223-019-00605-1

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Download date: 29. Sep. 2021 Multiple fractures and impaired bone fracture healing in a patient with pycnodysostosis and hypophosphatasia

Nicola Hepp1, Anja Lisbeth Frederiksen2,3, Morten Dunø4, Niklas Rye Jørgensen5,6, Bente

Langdahl7, Poul Vedtofte8, Hanne B. Hove9, Klaus Hindsø10 and Jens-Erik Beck Jensen1,11

1 Dept. of Endocrinology, Hvidovre University Hospital Copenhagen, Kettegård Alle 30, 2650

Hvidovre, Denmark

2 Dept. of Clinical Genetics, Odense University Hospital, Winsløws Vej 4, 5000 Odense C,

Denmark

3 Department of Clinical Research, Faculty of Health, University of Southern Denmark,

Winsløwparken 19. 3, 5000 Odense C, Denmark

4 Dept. of Clinical Genetics, University Hospital Copenhagen Rigshospitalet, Blegdamsvej 9,

2100 Copenhagen, Denmark

5 Dept. of Clinical Biochemistry, Rigshospitalet, Valdemar Hansens Vej 13, 2600 Glostrup,

Denmark

6 OPEN, Odense Patient data Explorative Network, Odense University Hospital/Institute of

Clinical Research, University of Southern Denmark, J.B.Winsløws Vej 9, 5000 Odense C,

Denmark

7 Dept. of Endocrinology and Internal Medicine, Aarhus University Hospital, Palle Juul

Jensens Boulevard 99, G317, 8200, Aarhus N, Denmark

8 Dept. of Oral and Maxillofacial Surgery, University Hospital Copenhagen Rigshospitalet,

Blegdamsvej 9, 2100 Copenhagen, Denmark

Abbreviations: ALP, ; ALPL, TNSALP encoding gene; BALP, bone specific alkaline phosphatase; BMC, bone mineral content; BMD, bone mass density; CPAP, continuous positive airway pressure; CT, computed tomography; CTSK, ; CTSK, cathepsin K encoding gene; CTX, c-terminal telopeptide of type I collagen; DXA, dual energy X-ray absorptiometry; ECM, bones extracellular matrix; FGF23, fibroblast growth factor 23; HPP, hypophosphatasia; HR- pQCT, high-resolution peripheral quantitative computed tomography; nr, normal range; OB, ; OC, ; PEA, phosphoethanolamine; Pi, inorganic phosphate; PINP, plasma procollagen type 1 N-terminal propeptide; PLP, pyridoxal-5´-phosphate; PPi, inorganic pyrophosphate; PTH, parathyroid hormone; rd, reference data; TBS, trabecular bone score; TNSALP, tissue-non- specific alkaline phosphatase; TRAP-5b, tartrate-resistant acid phosphatase-5b; vBMD, volumetric bone mineral density

1

9 Center for Rare Diseases, Dept. of Pediatrics, University Hospital Copenhagen, Juliane

Maries Vej 6, 2100 Copenhagen, Denmark

10 Paediatric section, Dept. of Orthopedic Surgery, University Hospital Copenhagen

Rigshospitalet, Blegdamsvej 9, 2100 Copenhagen, Denmark

11 Dept. of Clinical Medicine, University of Copenhagen, Blegdamsvej 9, 2100 Copenhagen,

Denmark

Corresponding Author:

Nicola Hepp, MD, PhD-student

Dept. of Endocrinology, Hvidovre University Hospital

Kettegård Alle 30

2650 Hvidovre

Denmark

Phone: +4553625083

[email protected]

Anja Lisbeth Frederiksen (ALF): [email protected]

Morten Dunø (MD): [email protected]

Niklas Rye Jørgensen (NRJ): [email protected]

Bente Langdahl (BL): [email protected]

Poul Vedtofte (PV): [email protected]

Hanne B. Hove (HBH): [email protected]

Klaus Hindsø (KH): [email protected]

Jens-Erik Beck Jensen (JEBJ): [email protected]

2

Abstract

Pycnodysostosis (PYCD) is a rare recessive inherited skeletal disease, characterized by short stature, brittle bones and recurrent fractures, caused by variants in the Cathepsin K encoding gene (CTSK) that leads to impaired mediated .

Hypophosphatasia (HPP) is a dominant or recessive inherited condition representing a heterogeneous phenotype with dental symptoms, recurrent fractures and musculoskeletal problems. The disease results from mutation(s) in the tissue-nonspecific alkaline phosphate

(TNSALP) encoding gene (ALPL) with reduced activity of alkaline phosphatase (ALP) and secondarily defective mineralization of bone and teeth.

Here, we present the first report of a patient with the co-existence of PYCD and HPP. This patient presented typical clinical findings of PYCD, including short stature, maxillary hypoplasia and sleep apnoea. However, the burden of disease was caused by over 30 fractures, whereupon most showed delayed healing and non-union. Biochemical analysis revealed supressed bone resorption and low bone formation capacity. We suggest that the co-existence of impaired bone resorption and mineralization may explain the severe bone phenotype with poor fracture healing.

Keywords

Pycnodysostosis; hypophosphatasia; CTSK; fracture; bone healing

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Introduction

Pycnodysostosis (PYCD) is an inborn recessive skeletal dysplasia with a prevalence of 1-1.7 per million [1, 2]. The disease is caused by Cathepsin K (CTSK) deficiency encoded by

CTSK [3]. The protease CTSK is essential for the bone-resorbing osteoclasts (OC) to degrade bone matrix proteins, and impairment leads to failure of bone resorption and remodelling [4-7]. The clinical presentation is characterized by brittle bones, small stature, craniofacial and oral abnormalities, including micrognathia and dental crowding [8, 9].

The tissue-non-specific alkaline phosphatase (TNSALP) is an important enzyme for normal skeletal and dental mineralization [10]. Here, deficiency caused by mutation(s) in the

TNSALP encoding gene (ALPL) leads to the rare Hypophosphatasia (HPP)

[11]. Clinical manifestations of HPP in adults are highly variable, including stress and fragility fractures, dental symptoms, muscle weakness, chronic pain in the lower extremities and kidney stones [12-14]. Persistently low levels of the TNSALP and an elevation of its substrates pyridoxal-5´-phosphate (PLP) and phosphoethanolamine (PEA) are biochemical hallmarks [15].

We report a 29-year old woman clinically diagnosed with PYCD in early childhood who has encountered over 30 fractures with delayed healing and most resulted in non-union. At the age of 25 years, we observed persistently low levels of alkaline phosphatase (ALP), an uncommon biochemical finding of PYCD [2, 4, 8, 16-18]. Genetic analysis identified the co- existence of a novel presumed pathogenic heterozygous variant in ALPL. Here, we describe clinical, genetic, biochemical findings as well as results of dual energy X-ray absorptiometry

(DXA) and high-resolution peripheral quantitative computed tomography (HR-pQCT).

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Material and Methods

A detailed description of the material and methods is attached in the supplementary material.

Results

Clinical, genetic, and biochemical findings of the studied family The patient, a Caucasian woman, was born as the first child to non-consanguineous parents at term after an uneventful pregnancy with a birth weight of 2900 g and a length of 50 cm. At the age of 18 months, she was clinically diagnosed with PYCD. From age 4 until 14 years, the patient underwent growth hormone therapy. At the age of 12, sleep apnoea was diagnosed due to a restricted upper airway, midface retrusion and a long soft palate and treated with continuous positive airway pressure (CPAP). Additional co-morbidities include generalised of unknown origin, diagnosed at the age of 17 years and migraine without aura. Further, the patient receives Levothyroxine due to mild non-autoimmune hypothyroidism.

Genetic analyses of the patient revealed compound heterozygous variants in CTSK, c.121-

1G>A; c.926C>T, p.Leu309Pro respectively. The parents were heterozygote carriers and both variants were previously reported in Danish patients with PYCD [19]. Subsequent sequencing of ALPL identified a novel heterozygous variant in exon 12 (c.1487A>G, p.His496Arg) both in the patient and her father. The variant is not reported in the gnomAD database, which describes more than 265.000 alleles in the area, nor in the ALPL mutation database (http://www.sesep.uvsq.fr/ 03_hypo_mutations.php). In silico analysis predicts the variant to have pathogenic potential.

The patient’s mother has never experienced fractures, or dental problems. Opposite, the father presented several dental symptoms, including severe caries and enamel-defects on all 5 teeth, periodic pain in muscles and bones, as well as traumatic fractures of both and a forearm. The clinical phenotype of the father represents a mild adult form of HPP with predominant odonto-manifestations.

The demographic and biochemical data of the family are shown in Table 1. All family members had normal TSH, ionised calcium, phosphate and renal function parameters. Both parents showed signs of mild secondary hyperparathyroidism due to vitamin D deficiency.

Low levels of ALP and elevated PLP were only recognized in the patient and her father. The plasma collagen type 1 C-telopeptide (CTX) was suppressed and the bone specific alkaline phosphatase (BALP) slightly reduced in in the case patient. However, all other markers including plasma procollagen type 1 N-terminal propeptide (PINP), osteocalcin, tartrate- resistant acid phosphatase (TRAP-5b), sclerostin and fibroblast growth factor (FGF-23) were within the normal range.

Fracture history The patient experienced over 30 fractures before the age of 29, of which most showed delayed bone healing or non-union (see Figure 1). The first fracture, a transverse fracture of the left shaft was caused by a low energy trauma at the age of three years. After seven months, the same type of fracture occurred on the right tibia and both fractures recurred several times within the following years due to low energy traumas or stress (see Figure1).

Both fractures resulted in non-union with pseudoarthrosis and were treated conservative with casts and orthoses (Sarmiento) for several years. Because of persistent non-union, osteosynthesis of the tibia was performed bilaterally and both fractures achieved union, while both fibular bones remained with non-unions after osteotomies (see Figure 2).

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At the age of 23, the patient developed two fractures in the left pelvis (ramus inferior), caused by a convulsive epileptic seizure. The fractures were treated conservatively and after 3.5 years, osteosynthesis was performed due persistently pseudoarthrosis of all fractures. Three months post-surgery X-ray follow up and a CT-scan, performed 2.5 years after osteosynthesis confirmed unaltered pseudoarthrosis of all pelvic fractures (see

Supplementary Figure 1). In addition, multiple metatarsal stress fractures of different age and with signs of insufficient bone healing were detected by a CT-scan in 2013.

Investigation of bone morphology DXA showed high bone mineral content (BMC), bones mass density (BMD) and T-scores of the lumbar spine (+4.4), the total hip (+4.2), the forearm (+3.4) and the whole body (+2.9).

Trabecular bone score (TBS) was evaluated with a value of 1.697, which is remarkably higher than the age adjusted normal range.

HR-pQCT demonstrated increased cortical area and cortical thickness, but normal cortical

BMD of the right radius and the tibia compared with reference data [20]. Trabecular BMD of the radius (427.3 mg/cm³ [rd 160±36]) and the tibia (338.0 mg/cm³ [rd 183±34]) were strikingly high. This was further reflected in higher trabecular number and increased trabecular thickness. Finite element analysis revealed impressively increased bone strength

(failure load) especially at the radius. The complete HR-pQCT data are presented in the supplementary material (see Supplementary Table 1).

Odontogenic and maxillo-facial manifestations. The patient was referred to the Dept. of Oral and Maxillofacial Surgery at the age of 12 years for treatment of dentofacial anomaly and malocclusion. Surgery performed in 2010 included

7 a high midface osteotomy with advancement and downward movement of the upper incisor.

In addition, mandibular asymmetry was corrected by a left sided mandibular advancement.

Bone healing of the midface was without complications. In contrast, bone healing of the osteotomy in the mandible was delayed. Six years after the initial osteotomy, a necrotic piece of bone exfoliated spontaneously and a pathologic fracture in the previous osteotomy site occurred later in the same year.

Discussion

To the best of our knowledge, this is the first report of a patient with the co-incidence of

PYCD and adult HPP. Furthermore, we found that the fracture healing complications of the patient appear to be more severe compared with other published cases of PYCD [17, 21,

22]. Recurrent fractures and delayed fracture healing have been reported in patients with

PYCD as well as HPP [21-23]. However, detailed data about the impact of CTSK and

TNSALP deficiency on fracture healing is not available. During facture healing CTSK is assumed to play a key role in osteoclast (OC) mediated bone resorption of cartilaginous callus and remodelling of regenerated bone tissue [17]. In contrast, (OB) activity contributes to the formation of mechanically competent callus by mineralization of the cartilagous callus [24, 25]. The TNSALP is an essential component in this process and is expressed on the cell membrane of OBs. TNSALP hydrolyses inorganic pyrophosphate

(PPi), a potent inhibitor of bone mineralization, and provides phosphate (Pi) for the formation of calcium and phosphate compounds and , which contributes to mineralization of the bones extracellular matrix (ECM) [10, 25]. The primary mineralization, processed during fracture healing is followed by remodelling where the hard callus is 8 resorbed by OCs and lamellar bone is produced by OBs [24]. Elevated ALP with a peak approximately four weeks after fracture occurrence has been measured in the serum of humans [26] and human callus tissue [27]. In addition, Komnenou, A. et al could show that dogs with bone union within two months had higher levels of ALP in comparison with dogs that showed delayed fracture healing or non-union, leading to the assumption that an increased activity of OBs and ALP is important for sufficient bone mineralization and fracture healing [28].

Bone marker analysis confirmed a decreased bone turnover with low bone resorption and bone formation capacity. The supressed bone resorption (CTX) can be explained by CTSK impairment [29]. Furthermore, a previous study reported both reduced CTX and PINP levels in a cohort of heterozygous ALPL variant carriers [30]. However, mechanisms linking ALP impairment and reduced bone turnover remain unclear. Deficiency of ALP leads to accumulation of PPi, which is a biological analogue of bisphosphonate, the most common drug in treatment that inhibits OC mediated bone resorption [14, 31]. We speculate if increased levels of PPi, due to ALP impairment cause a similar effect, resulting in reduced bone resorption and possibly also to impaired bone formation [30].

HRpQCT revealed that the increase in BMD was due to increased cortical thickness and increased number of trabeculae and thickness of the trabeculae. Both are explained by the insufficient bone resorption due to the lack of CTSK. Finite element analysis revealed impressively increased bone strength. This finding in a patient with multiple fractures underscores that BMD derived measures of bone strength is not reliable when bone quality is impaired. 9

Conclusion

In conclusion, a patient with the co-incidence of pathogenic variants in ALPL and CTSK, presented with high bone mass, reduced bone turnover and multiple fractures with severely delayed fracture healing and non-union. The detailed role of ALP and CTSK impairment in bone regeneration and fracture healing requires further studies.

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Ethics statement

Genetic analysis, blood sample analysis, DXA and X-ray evaluation were performed during clinical diagnostic investigations.

Human Rights

The research was conducted in accordance with the ethical standards.

Informed Consent

Informed consent was obtained from the subjects to publish their data.

Declaration of interest

NH, PV, HBH, KH, ALF, MD, NRJ have nothing to disclose.

JEBJ: Board member: Eli Lilly, Amgen, MSD. Funding: Eli Lilly and Amgen. Consulting fees:

MSD, Giliad and Amgen

BL: Research grants: Novo Nordisk and Amgen. Advisory boards and lecturing: UCB,

Amgen, Eli Lilly and TEVA.

Author contributions

NH, PV, HBH, KH, JEBJ: Examination and treatment of the patient. ALF: Supervision and genetic counselling. MD: Implementation of genetic analyses. NRJ: Implementation of biochemical analyses. BL: Provided data from HR-pQCT evaluation. All authors contributed to the interpretation of data, development and critical revision of the manuscript, and approved the final version for submission.

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Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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Table

Table 1 Demographic and biochemical data of the case patient and the parents

Case patient Father Mother

Age (years) 29 53 52

Height (cm) 152.6 187.0 163.7

Weight (kg) 58.9 108 69.7

BMI 24.9 30.9 26.0

25-OH-Vitamin D (> 50 nmol/L) 82 19 ↓ 25 ↓

PTH (1.1-7.1 pmol/L) 7.1 8.2 ↑ 7.5 ↑

ALP (35-105 U/L) 18 ↓ 23 ↓ 87

PLP (15-73 nmol/L) 162 ↑ 125 ↑ 65

PEA (0-3.5 µmol/L) 4 ↑ 3 3

CTX (*ng/L) < 33.0 ↓ 139.8 781.3

PINP (**µg/L) 24.8 29 108.1

BALP (***µg/L) 5.8 ↓ 7.5 28.0

FGF23 (23.2 – 95.4 pg/mL) 39.6 75 94

* CTX: M (40-125 years): 90.0-1086 ng/L; W (25-30 years): 91.0-1318 ng/L; W (50-125 years): 125-1477 ng/L ** PINP M (40-125 years): 19.0-82.0 µg/L; W (25-30 years): 16.0-142.0 µg/L W (50-125 years): 22.0-114 µg/L *** BALP M (40-125 years): 7.5-25.1 µg/L; W (25-30 years): 5.9-30.0 µg/L; W (50-125 years): 8.3-29.4 µg/L M=Men W=Women

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Figures

Figure 1 Fractures of the case patient documented during lifetime. All fractures, including re- fractures are visualized with the year of occurrence and the fracture localization

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Figure 2 Re-fractures of the right tibia and fibula resulted in non-union. a;b X-ray controls two and three years after fracture debut (2008) showed pseudoarthrosis of stress-fractures (tibia and fibula). c Post-operative follow-up one day after osteosynthesis of the tibia and osteotomy of the fibula. d Three months postoperative control showed intact and well-positioned osteosynthesis material and complete healing of the tibia, while the fibula did not achieve union

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Supplementary material

Material and Methods

Genetic analysis The CTSK gene (NM_000396.3) was sequenced through an NGS protocol, after the patient had sought genetic counselling in 2017. The coding and intron flanking sequences of ALPL

(NM_000478.5) were subsequently screened by Sanger sequencing (primers and PCR conditions are available upon reasonable request). In silico prediction of the novel ALPL variant was carried out using Mutation Taster (http://www.mutationtaster.org) and PolyPhen2

(http://genetics.bwh. harvard.edu/pph2). Control population frequency analysis was performed by reviewing gnomAD (http://gnomad.broadinstitute.org). In addition, the ALPL mutation database (http://www.sesep.uvsq.fr/ 03_hypo_mutations.php) was applied to review if the detected variant has been described previously.

Biochemical evaluation Blood sampling was performed in fasting state, between 8.00 and 9.00 in the morning.

Calcium and vitamin B6 supplements were paused for two weeks. Blood samples were centrifuged at 4˚C and frozen at - 80 ˚C. Bone markers such as plasma collagen type 1 C- telopeptide (CTX), plasma procollagen type 1 N-terminal propeptide (PINP) and osteocalcin were analysed using chemiluminescence immunoassays. To measure bone specific alkaline phosphatase (BALP) and tartrate-resistant acid phosphatase (TRAP-5b), a spectrophotometric assay was performed. Bone markers described above were carried out using an automated analyser, IDS iSYS (Immunodiagnostic Systems Ltd., Boldon, England), according to the manufacturer’s instructions. Sclerostin was analysed using TECOmedical

Human Sclerostin HS ELISA (TECOmedical group, Sissach, Switzerland). Serum 19 concentrations of fibroblast growth factor (FGF23) were determined using a three-step sandwich chemiluminescence immunoassay (Liaison FGF23 assay) on the Liaison XL automated analyser (DiaSorin, Saluggia, Italy). All bone markers were measured via batch analysis. PLP was determined by high pressure liquid (HPLC) analysis (Chromosystems

Instruments and Chemical GmbH, Germany). Plasma levels of PEA were analysed by

UPLC-UV technique. ALP was determined by photometric measurement on the Cobas 8000

(Roche Diagnostics, USA). 25-OH-d vitamin (D3 + D2) was analysed via chemiluminescence immunoassay (Cobas 8000, Roche Diagnostics, USA).

Bone parameters and body composition DXA (Hologic, Inc., Bedford, MA, USA) was performed of the whole body, left forearm, right hip (because of osteosynthesis in the left hip) and the lumbar spine (L1-L4) to evaluate BMD.

Body composition and fat mass (%) were assessed by whole-body DXA. Lumbar spine T- score was calculated using reference values and STD from the HOLOGIC study [32]. T- scores of the hip and the whole body were determined using values from the NHANES phase III study [33]. In addition, trabecular bone score (TBS) was derived from the texture of the DXA-image of the lumbar spine (TBS version 3.0.2.0, Hologic Horizon A).

To investigate bone geometry, microarchitecture and volumetric bone mineral density

(vBMD), HR-pQCT (XtremeCT, Sanco Medical, Switzerland) was performed. Non-dominant distal radius and tibia were investigated by this technique. The following parameters were obtained: total, trabecular and cortical BMD, trabecular thickness, cortical thickness, trabecular number, stiffness and finite element failure load. The HR-pQCT protocol according

20 to the image acquisition as well as the analysis of microarchitecture and the bone strength by finite element analysis have been described previously [34, 35].

21

Supplementary Figure 1 Pelvic fractures – a follow up over 4 years. At fracture debut (day 0), two fractures in the ramus inferior were identified (yellow arrows). After 27 and 31 months, a control X-ray revealed an additional fracture in the ramus superior and non-union of the ramus inferior fractures.

Due to non-union, osteosynthesis was performed in 2016 (Post-op day 1). The x-ray control three months after operation (Post-op 3 months) showed insufficient healing with pseudoarthrosis of all fractures

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Supplementary Table 1 Geometry, volumetric bone mineral density, microarchitecture and estimated bone strength, evaluated by HRqCT in the case patient

Case Patient Women 20-35 years * (mean ± SD)

Radius

Total area (mm²) 262.9 254 ± 44

Cortical area (mm²) 107.1 57 ± 11

Trabecular area (mm²) 164.1 194 ± 46

Total BMD (mg/cm³) 603.7 342 ± 72

Cortical BMD (mg/cm³) 896.8 898 ± 49

Trabecular BMD (mg/cm³) 427.3 160 ± 36

Cortical thickness (mm) 1.55 0.94 ± 0,20

Trabecular thickness (mm) 0.127 0.069 ± 0.013

Trabecular number (1/mm) 2.81 1.92 ± 0.26

Stiffness (kN/mm) 157 79 ± 17

FEA failure load (N) 71532 3993 ± 731

Tibia

Total area (mm²) 729.7 672 ± 111

Cortical area (mm²) 171.8 125 ± 22

Trabecular area (mm²) 565.8 545 ± 115

Total BMD (mg/cm³) 479.2 323 ± 54

Cortical BMD (mg/cm³) 966.6 902 ± 33

Trabecular BMD (mg/cm³) 338.0 183 ± 34

Cortical thickness (mm) 1.64 1.26 ± 0.24

Trabecular thickness (mm) 0.099 0.007 ± 0,013

Trabecular number 2.86 1.99 ± 0.31

Stiffness (kN/mm) 339 218 ± 35

FEA failure load (N) 16046 10923 ± 1.721

* HR-pQCT reference data of adult Danish women between 20-35 years 23

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