Journal of Biomechanics 49 (2016) 3549–3554

Contents lists available at ScienceDirect

journal homepage: www.elsevier.com/locate/jbiomech

Journal of Biomechanics

http://d0021-92

n CorrHarvardFax: þ1

E-m1 Bo

www.JBiomech.com

Short communication

A pediatric animal model to evaluate the effects of disuseon musculoskeletal growth and development

Daniel L. Miranda a,n,1, Melissa Putman b,1, Ruby Kandah a, Maria Cubria c,Sebastian Suarez c, Ara Nazarian c, Brian Snyder c,d

a Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, MA, USAb Division of Endocrinology, Boston Children's Hospital and Harvard Medical School, Boston, MA, USAc Center for Advanced Orthopaedic Studies, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, USAd Department of Orthopedic Surgery, Boston Children's Hospital and Harvard Medical School, Boston, MA, USA

a r t i c l e i n f o

Article history:

Accepted 16 August 2016

Prolonged immobilization in hospitalized children can lead to fragility fractures and muscle contracturesand atrophy. The purpose of this study was to develop a lower-extremity disuse rabbit model with

Keywords:NeurectomyBoneDensityGeometryMuscleCross-sectionRabbit

x.doi.org/10.1016/j.jbiomech.2016.08.01890/& 2016 Elsevier Ltd. All rights reserved.

espondence to: Wyss Institute for BiologicUniversity, 3 Blackfan Circle. 2nd Floor, Room617 432 8252.ail address: daniel.miranda@wyss.harvard.eduth are first authors.

a b s t r a c t

musculoskeletal changes similar to those observed in children subjected to prolonged immobilization.Six-week-old rabbits were randomly assigned to control (CTRL, n¼4) or bilateral sciatic and femoralneurectomy (bSFN, n¼4) groups. Trans-axial helical CT scans of each rabbit's hind limbs were acquiredafter eight weeks. The rabbits were then euthanized and the tibiae and calcanea were harvested fromeach rabbit. μCT imaging was performed on the tibiae and calcanea mid-diaphysis. Four-point bending,gas pycnometry, and ashing were then performed on each tibia. All comparisons reflect the differencesbetween the bSFN and CTRL rabbits. Significant decreases in tibiae bone mineral density (Z9.41%,pr0.006), axial rigidity (Z50.47%, pr0.02), and soft tissue mass (55.25%, p¼0.006) were observedfrom the trans-axial helical CT scans. The μCT results indicated significant detriments in tibia and cal-caneus cortical thickness and bone volume fraction (pr0.011). Significant changes in stiffness, yield load,ultimate load, and ultimate displacement (Z30.05%, pr0.025) were observed from mechanical testing.These data indicate that limb disuse at a time of rapid musculoskeletal growth severely impairs muscleand bone development, reflecting the musculoskeletal complications observed in children with chronicmedical conditions causing immobilization. Interventions to reduce these musculoskeletal complicationsin children are urgently needed. This disuse rabbit model will be useful in pre-clinical studies evaluatingnovel interventions for improving pediatric musculoskeletal health.

& 2016 Elsevier Ltd. All rights reserved.

1. Introduction

Medical treatments and disease pathophysiology can result inprolonged immobilization that places hospitalized and chronicallyill infants and children at risk for serious secondary complicationsincluding fragility fractures (Huh and Gordon, 2013), muscle con-tractures and atrophy (Boonyarom and Inui, 2006). This is due toimmobilization-induced bone loss and impairment of musclefunction secondary to reduced levels of activity or limb disuse(Boonyarom and Inui, 2006; Ward et al., 2004). Pediatric patientsat the highest risk for inpatient fracture include premature infants

ally Inspired Engineering at208, Boston, MA 02115, USA.

(D.L. Miranda).

(Huh and Gordon, 2013; Moyer-Mileur et al., 2000) and childrenwith cerebral palsy (Caulton et al., 2004; Mergler et al., 2009),spinal cord injury (Munns and Cowell, 2005; Ward et al., 2004),neuromuscular disorders such as Duchenne Muscular Dystrophy(Ward et al., 2004) or Spinal Muscular Atrophy (Rudnik-Schöneborn et al., 2008), prolonged post-operative immobiliza-tion such as esophageal atresia (Bairdain et al., 2014; Huh andGordon, 2013), and prolonged dependence on parenteral nutrition(Diamanti et al., 2010). Despite the institution of fracture precau-tions, optimization of parenteral nutrition and vitamin D intake,and regular physical therapy, these patients continue to sufferfrom high rates of fracture during hospitalization (Bairdain et al.,2014; Huh and Gordon, 2013; Moyer-Mileur et al., 2000). There-fore, other approaches must be pursued to optimize musculoske-letal health and reduce fracture rates in hospitalized infants.

Muscle atrophy, osteoporosis, and fragility fractures have longbeen a major concern in the elderly (Johnell and Kanis, 2006), and

D.L. Miranda et al. / Journal of Biomechanics 49 (2016) 3549–35543550

several animal models have been developed to portray disuseatrophy, osteopenia, and fragility fractures in adults (Egermann etal., 2005). However, there are no pediatric models available thatincorporate immature and developing large animals (e.g., rabbits).A large animal model is required to specifically test interventionsto address musculoskeletal complications arising from disuse andlack of weight bearing seen in a variety of pediatric inpatients(Huh and Gordon, 2013). Neurectomy is a promising techniquethat has been used in the past to model musculoskeletal disuse(Komori, 2015). However, these studies have typically focused on asingle nerve ligation that leaves substantial hindlimb musculatureintact. Additionally, these studies have been limited to fully-grownsmall animals, such as mice and rats (Brouwers et al., 2009; Fenget al., 2016; Komori, 2015; Sugiyama et al., 2012). Therefore, thepurpose of this study was to develop a pediatric lower-extremitydisuse rabbit model. We hypothesize that bilateral sciatic andfemoral neurectomies in six-week-old rabbits will affect bone andmuscle development in the lower extremity similar to the mus-culoskeletal changes observed in children who are sedated and/ormuscle relaxed with paralytics such as vecuronium during theirhospitalization.

2. Methods

2.1. Animals

The Beth Israel Deaconess Medical Center Institutional Animal Care and UseCommittee approved all experimental procedures used for this study. Female NewZealand white rabbits were chosen, due to their docile and non-aggressive nature,making them easy to handle and observe. Furthermore, the rabbit's skeletal sizeand bone density is similar to human infants, and skeletal growth is completed by28 weeks. This allows research to be conducted during the growth phase in areasonable time period.

2.2. Surgical procedures

Six-week-old rabbits were randomly assigned to either control (CTRL, n¼4) orbilateral sciatic and femoral neurectomy (bSFN, n¼4) groups. The rabbits in theCTRL group did not undergo any intervention. Rabbits undergoing surgical bSFNwere anesthetized with intramuscular doses of Ketamine (35 mg/kg) and Xylazine(2.5 mg/kg) and then maintained on 1–2% isoflurane delivered through a mask. Thefur was shaved and skin wiped with betadine and alcohol.

After administering anesthesia, each bSFN rabbit was placed in the proneposition with the posterior limbs mildly rotated externally with semiflexion of theknees. An oblique incision distal the posterior superior iliac spine was deepened to

A1 A2 A3

B1 B2 B3

Fig. 1. Top row: healthy control rabbit tibia. Bottom row: bilateral sciatic and femoral neright. Note how the bone is smaller, with spurs, and shape deformities. Note the visible

expose the Gluteus Maximus muscle, which was split by blunt dissection, and thesciatic nerve was identified on top of the piriformis muscle exiting the sciatic notch.Before the nerve was sharply bisected, a small amount (approximately 1–2 ml) ofphenol was applied directly onto the nerve to cause chemical demyelination toprevent nerve regeneration and reduce the incidence of autotomy. The notch wasthen over-sewn with a nylon suture, and the operative incision was closed in ahidden subcuticular pattern with absorbable sutures. These procedures were per-formed for the left and right sciatic nerves following established techniques(Bradley et al., 1998; Jiang et al., 2006; Sames and Benes, 1997; Sung, 2004).

Each bSFN rabbit was then placed in the supine position to perform bilateralfemoral neurectomy. Through an incision just distal to the inguinal ligament, thefemoral nerve was identified and mobilized in the femoral triangle medial to theSartorius muscle. The femoral artery and vein were carefully retracted. Neurectomywas performed by chemical neurolysis using phenol prior to bisecting it withelectrocautery. These procedures were performed for the left and right sciaticnerves following established techniques (Hanashi et al., 2002; O'Connor et al., 1992;Sung, 2004). The combined bSFN procedure resulted in hind limb disuse, whileallowing the rabbit to move independently using its forelimbs and to eat, void, anddefecate spontaneously.

2.3. Study Procedures

Both CTRL and bSFN groups were housed with their lactating mothers for eightweeks after the surgical procedures. The surgical procedures resulted in the bSFNrabbits having limited activity and reduced mobility. The rabbits were moved,cleaned, and bedding was changed regularly to avoid pressure sores and urinescald. Additionally, monitoring the rabbit's hind limbs daily and bandaging whenappropriate mitigated digit autotomy. The rabbits gained weight as expected on astandard rabbit chow diet supplemented with DietGel Criticare (ClearH2O, Port-land, ME, USA) as needed. After eight weeks, the rabbits were anesthetized throughintramuscular doses of Ketamine and Xylazine and euthanized by a fatal dose ofFatal-Plus (0.22 ml/kg).

2.3.1. Clinical CT scansTrans-axial helical CT scans (0.2 mm3 voxel size, 120 kVp tube voltage, 300 mA

tube current) of each rabbit's hind limbs were acquired (Aquilon 64, Toshiba,Tustin, CA, USA) eight weeks post-surgery. Three hydroxyapatite phantoms ofknown mineral density (0, 500, and 1000 mgHA/cm3) were used to convertHounsfield units (HU) to volumetric bone mineral density (vBMD). Segmented CTimages of the left tibia were re-formatted (Mimics v16; Materialise, Ann Arbor, MI,USA) to generate transaxial images perpendicular to the anatomical axis. Axialrigidity was derived for every cross-section using CT-based Rigidity Analysis (CTRA)to calculate the integral of the modulus weighted pixels distributed in space overthe cross-sectional profile of bone (Gordon et al., 2011; Villa-Camacho et al., 2014;Whealan et al., 2000). The vBMD and axial rigidity were then extracted from thefive axial slices surrounding the 4%, 38%, and 66% of the total length measured fromthe distal end (Fig. 1). These sites were chosen because they represent standardclinical peripheral quantitative CT sites used in children and adolescents (Adamset al., 2014). The soft tissue mass, normalized by body weight, was also calculatedfrom the volume and density of the five axial slices surrounding the 38% total tibialength site. This location was chosen because the gastrocnemius muscle diameter

A1 A2 A3

B1 B2 B3

urectomy rabbit tibia. The 6%, 38%, and 66% total length sites are shown from left tothickness differences in the cortical bone.

D.L. Miranda et al. / Journal of Biomechanics 49 (2016) 3549–3554 3551

in healthy control rabbits was the greatest at this site. Each rabbit's hind limbs wereharvested after the clinical CT scan, and the tibiae and calcanea were dissected andstripped of soft tissue. Each bone was wrapped in gauze soaked in 0.9% saline andstored at �20 °C.

2.3.2. μCT scansPrior to imaging, the tibiae were thawed out at room temperature and re-

hydrated in 0.9% saline for 30 min. The proximal and distal epiphyses of each bonewere then cut using two parallel diamond wafering blades on a low-speed saw(Isomet, Buehler Corporation, Lake Bluff, Illinois) under copious irrigation. Micro-computed tomography (μCT) imaging (μCT 40, Scanco Medical AG, Brüttisellen,Switzerland) was performed on tibial and calcaneal mid-diaphysis sections (100slices, 30 μm isotropic voxel size, 250 ms integration time, 55 kV tube voltage,145 mA tube current) to derive cortical thickness (Ct.Th) and bone area fraction(BA/TA). Hydroxyapatite phantoms of known mineral density (0, 100, 200, 400, and800 mgHA/cm3) were used to convert attenuation coefficients to vBMD.

2.3.3. Mechanical TestingFour-point bending was performed on tibias using an Instron 8511 (Instron,

Norwood, MA, USA) load frame under displacement control with 50 mm supportand 20 mm loading spans (Fig. 2). Specimens were thawed out to room tempera-ture and re-hydrated in 0.9% for 30 minutes prior to testing. The posterior surfaceof the tibiae were placed facing upward on the support span and lightly securedwith elastic bands to avoid rolling during testing. The loading crosshead wasmounted on a pivot in order to ensure symmetrical loading of the irregularlyshaped rabbit tibiae at all four loading points. Each tibia was loaded to failure at a

Fig. 2. Illustration depicting four-poi

constant rate of 0.1 mm/s. Modulus of elasticity, yield load, ultimate load, ultimatedisplacement, ultimate stress, and flexural modulus were calculated using standardcomposite beam equations in MATLAB (MathWorks, Natick, MA, USA).

2.3.4. Helium pycnometry and ashingEight-millimeter long bone segments, adjacent to the fracture site, were excised

after mechanical testing, in order to assess the following true bone tissue parameters:wet and dry bone densities, mineral and matrix densities, and mineral and matrixcontent. Helium pycnometry is a useful technique to calculate the true density of asolid material, with an unknown volume but a known weight. The pycnometerallows helium to enter the chamber where the sample is placed, and calculates theaverage volume of the sample as the space from which the gas is excluded.

Each bone segment was weighed three times using an Analytical Plus Elec-tronic Balance (AP210s; Ohaus Corporation, Florham Park, NJ, USA), scale readingswere recorded, and mean values were calculated. The volume from each bonesegment was determined using a pycnometer (Accupyc 1330; Micromeritics, Nor-cross, GA, USA). In order to guarantee reproducibility and fidelity to the results, thegas pycnometer was working under a controlled temperature of 2671 °C. Wetbone density was then calculated.

Specimens were then placed in a labeled container and dehydrated at 75 °C for 72hours in a Thermolyne Furnace Type 48,000 (Barnstead International, Dubuque, Iowa,USA) and weighed at 12-hour intervals until no changes in weight were recorded. Thefinal mass measurements after dehydration were used to calculate dry bone density.Specimens were ashed at 600 °C for 12 additional hours and were allowed to cool forone hour with the furnace door open. A final mass measurement was performed, andmineral and matrix densities and content were calculated (Carter et al., 1981).

nt bending experimental set-up.

D.L. Miranda et al. / Journal of Biomechanics 49 (2016) 3549–35543552

2.4. Statistical analysis

The Kolmogorov–Smirnov goodness of fit test was used to assess distributionnormality. A one-tailed unpaired Student's t-test was used to test for differencesbetween the CTRL and bSFN groups for each of the outcome measures described. Aone-tailed test was chosen based on the a-priori hypothesis that bSFN would eitherhave no effect or degrade bone quality and soft tissue mass.

3. Results

Using clinical CT, significant decreases in the tibiae vBMD andaxial rigidity were observed eight weeks after the surgical proce-dures. At the 4%, 38%, and 66% cross-sections, the average tibiaecortical bone vBMD was 9.81% (p¼0.004), 10.18% (p¼0.004), 9.41%(p¼0.006) lower for the bSFN group compared to the CTRL group.At the same cross-sections, the corresponding tibiae axial rigiditieswere 67.56% (p¼0.006), 50.92% (p ¼ 0.011), and 50.47% (p¼0.020)less for the bSFN group compared to the CTRL group. The softtissue envelope surrounding the 38% cross-section was 55.28%(p¼0.006) less for the bSFN group compared to the CTRL group.

On μCT testing, cortical thickness and bone area fraction were31.04% (p¼0.005) and 11.92% (p¼0.056) less for the bSFN rabbitscompared to the CTRL rabbits, respectively (Fig. 3). Calcaneal Ct.Thand BA/TA were 53.20% (po0.001) and 1.61% (p¼0.011) less forthe bSFN rabbits compared to the CTRL rabbits, respectively(Fig. 3).

Mechanical testing (Fig. 4) indicated significant reductions instiffness (52.25% lower, po0.001), yield load (41.82% lower,

CTRLbSFN

0

0.5

0.6

0.7

0.8

BA

/TA

*

CTRLbSFN

0

0.950

0.975

1.000

BA

/TA

*

TIB

CALC

Fig. 3. Calcaneal and tibial bone area fraction and cortical thickness. T

po0.001), ultimate load (39.01% lower, p¼0.001), and an increasein ultimate displacement (30.05% higher, p¼0.025).

Matrix density (p ¼ 0.042) was the only true bone tissueoutcome where the bSFN group had a significant decrease com-pared to the CTRL group (Fig. 5).

4. Discussion

The goal of this study was to develop a disuse rabbit model byperforming bilateral sciatic and femoral neurectomies in six-week-old rabbits with the hypothesis that it would result in markeddecreases in the bone and muscle development of the lowerextremity. Overall, the data indicate that limb disuse at a time ofrapid growth of the musculoskeletal system severely impairs bothmuscle and bone development.

Structural and material property indices measured in-vivo fromhelical CT images and ex-vivo by μCT and mechanical testing showthat bone andmuscle properties decrease, as reflected by a decrementin the cross-sectional bone geometry and reduction in the bone tissuematerial properties related to diminution in vBMD. The lack of dif-ference in the true bone tissue outcomes between the groups, com-bined with a decline in vBMD, implicates under mineralization of thebone tissue, an observation further supported mechanically by theincrease in ultimate displacement. Importantly, the predicted changesin bone structure and function calculated from the analysis of non-invasive in-vivo helical CT images were confirmed by mechanicaltesting and compositional analysis of post-mortem specimens.

Interventions to reduce the musculoskeletal complications oflimb disuse and/or immobilization in infants and children are

CTRLbSFN

0

0.75

1.00

1.25

1.50

1.75

Cor

tical

Thi

ckne

ss (m

m)

*

CTRLbSFN

0

0.25

0.50

0.75

1.00

1.25

Cor

tical

Thi

ckne

ss (m

m)

*

IAE

ANEA

he (*) denotes a significant difference between groups (po0.05).

CTRLbSFN

0

100

200

300

400

500

Stiff

ness

(N/m

m)

*

CTRLbSFN

0

100

200

300

400

Yiel

d Lo

ad (N

)

*

CTRLbSFN

0

100

200

300

400

500

Ulti

mat

e Lo

ad (N

)

*

CTRLbSFN

0

0.75

1.00

1.25

1.50

1.75

Ulti

mat

e D

ispl

acem

ent (

mm

)

*

Fig. 4. Tibial bone mechanical property results. The (*) denotes a significant difference between groups (po0.05).

CTRLbSFN

0

69.5

70.0

70.5

71.0

71.5

72.0

Min

eral

Con

tent

(%)

CTRLbSFN

0

28.4

29.0

29.6

30.2

Mat

rix C

onte

nt (%

)

CTRLbSFN

0

1.14

1.16

1.18

1.20

1.22

1.24

1.26

1.28

Min

eral

Den

sity

(g/c

m3 )

CTRLbSFN

0

0.48

0.49

0.50

0.51

0.52

Mat

rix D

ensi

ty (g

/cm

3 )

*

CTRLbSFN

0

1.60

1.65

1.70

1.75

1.80

Bon

e D

ensi

ty (g

/cm

3 )

Fig. 5. True tibial bone tissue results from pycnometry. The (*) denotes a significant difference between groups (po0.05).

D.L. Miranda et al. / Journal of Biomechanics 49 (2016) 3549–3554 3553

urgently needed. For example, episodes of musculoskeletal disusefrom sedation appear to be the most significant risk factor forfractures in pediatric patients who underwent treatment for Long-Gap Esophageal Atresia (Bairdain et al., 2015). Fracture rates of up to40% were observed in these patients, and clinically they show evi-dence of increased bone resorption and decreased bone formationattributed to their prolonged immobilization. In general, diseasepathophysiology, medical treatments, nutritional status, andimmobilization are all considered risk factors for fragility fracturesin hospitalized children (Huh and Gordon, 2013). It is critical todevelop novel treatments to prevent morbidity associated withfractures and to optimize immediate and future bone health inthese at-risk patients. This pediatric paralytic rabbit model will beuseful for understanding the role of normal muscle function andfluid flow on modulating musculoskeletal growth. Additionally, thismodel will permit pre-clinical tests of innovative treatments thatameliorate bone resorption and muscle atrophy in afflicted children.Moreover, the bone structural indices calculated from in-vivo helicalCT images were sensitive enough to effectively and non-invasivelyquantify changes in bone function, allowing for in-vivo evaluationsof the rabbits as well as human clinical populations.

Limitations of this study include the small number of rabbitsstudied; however, the observed striking differences in bone andmuscle outcomes suggest that the study was sufficiently powered.Additionally, no sham surgeries were performed in the CTRLgroup. Young rabbits are notoriously fragile and pain sensitive. Wesought to avoid subjecting additional rabbits to the risks of,complications from, and pain associated with the surgical proce-dures. Finally, due to the paucity of trabecular bone present inrabbit tibiae and calcanei, specific changes in trabecular bonecould not be assessed. However, we can infer from the observedcortical bone loss that significant trabecular loss also likelyoccurred (Parfitt, 1983) based on studies evaluating disuse-associated bone loss in other animal models (Jiang et al., 2007;LeBlanc et al., 1985; Liu et al., 2008; Yarrow et al., 2014).

In conclusion, bSFN in young rabbits leads to reductions inbone density, structure, and strength, and a decrease in musclemass that reflect the musculoskeletal complications observed ininfants and children with chronic medical conditions causingimmobilization. This disuse rabbit model will be useful in pre-

clinical studies evaluating interventions for improving muscu-loskeletal health in immobilized pediatric patients.

Conflict of interest statement

The authors have no financial or personal relationships thatcould bias this work.

Acknowledgments

This work was supported by Center for MusculoskeletalResearch Pilot Grant (1P30AR066261-01), Thrasher Research FundEarly Career Award, Boston Children's Hospital InnovestmentAward, the NIH LRP (AN - L30 AR0566-6) and the Wyss Institute.

Appendix A. Supporting information

Supplementary data associated with this article can be found inthe online version at http://dx.doi.org/10.1016/j.jbiomech.2016.08.018.

References

Adams, J.E., Engelke, K., Zemel, B.S., Ward, K.A., International Society of ClinicalDensitometry, 2014. Quantitative computer tomography in children and ado-lescents: the 2013 ISCD pediatric official positions. J Clin. Densitom. http://dx.doi.org/10.1016/j.jocd.2014.01.006

Bairdain, S., Dodson, B., Zurakowski, D., Rhein, L., Snyder, B.D., Putman, M., Jennings, R.W., 2015. High incidence of fracture events in patients with Long-Gap EsophagealAtresia (LGEA): a retrospective review prompting implementation of standardizedprotocol. Bone Rep. 3, 1–4. http://dx.doi.org/10.1016/j.bonr.2015.06.002.

Bairdain, S., Kelly, D.P., Tan, C., Dodson, B., Zurakowski, D., Zurakowksi, D., Jennings, R.W.,Trenor, C.C., 2014. High incidence of catheter-associated venous thromboembolicevents in patients with long gap esophageal atresia treated with the Foker process.J. Pediatr. Surg. 49, 370–373. http://dx.doi.org/10.1016/j.jpedsurg.2013.09.003.

Boonyarom, O., Inui, K., 2006. Atrophy and hypertrophy of skeletal muscles:structural and functional aspects. Acta Physiol. 188, 77–89. http://dx.doi.org/10.1111/j.1748-1716.2006.01613.x.

Bradley, J.L., Abernethy, D.A., King, R.H., Muddle, J.R., Thomas, P.K., 1998. Neural archi-tecture in transected rabbit sciatic nerve after prolonged nonreinnervation. J. Anat.192 (Pt 4), 529–538. http://dx.doi.org/10.1046/j.1469-7580.1998.19240529.x.

Brouwers, J.E.M., Lambers, F.M., van Rietbergen, B., Ito, K., Huiskes, R., 2009. Com-parison of bone loss induced by ovariectomy and neurectomy in rats analyzed

D.L. Miranda et al. / Journal of Biomechanics 49 (2016) 3549–35543554

by in vivo micro-CT. J. Orthop. Res. 27, 1521–1527. http://dx.doi.org/10.1002/jor.20913.

Carter, D.R., Caler, W.E., Spengler, D.M., Frankel, V.H., 1981. Uniaxial fatigue ofhuman cortical bone. The influence of tissue physical characteristics. J Biomech.14, 461–470.

Caulton, J.M., Ward, K.A., Alsop, C.W., Dunn, G., Adams, J.E., Mughal, M.Z., 2004. Arandomised controlled trial of standing programme on bone mineral density innon-ambulant children with cerebral palsy. Arch. Dis. Child. 89, 131–135. http://dx.doi.org/10.1136/adc.2002.009316.

Diamanti, A., Bizzarri, C., Bizzarri, C., Basso, M.S., Gambarara, M., Cappa, M., Daniele,A., Noto, C., Castro, M., 2010. How does long-term parenteral nutrition impactthe bone mineral status of children with intestinal failure? J. Bone Miner.Metab. 28, 351–358. http://dx.doi.org/10.1007/s00774-009-0140-0.

Egermann, M., Goldhahn, J., Schneider, E., 2005. Animal models for fracture treat-ment in osteoporosis. Osteoporos. Int. 16, S129–S138. http://dx.doi.org/10.1007/s00198-005-1859-7.

Feng, B.O., Wu, W., Wang, H., Wang, J., Huang, D., Cheng, L., 2016. Interactionbetween muscle and bone, and improving the effects of electrical muscle sti-mulation on amyotrophy and bone loss in a denervation rat model via sciaticneurectomy. Biomed. Rep. 4, 589–594. http://dx.doi.org/10.3892/br.2016.637.

Gordon, C.M., Gordon, L.B., Snyder, B.D., Nazarian, A., Quinn, N., Huh, S.Y., GiobbieHurder, A., Neuberg, D., Cleveland, R., Kleinman, M., Miller, D.T., Kieran, M.W.,2011. Hutchinson–Gilford progeria is a skeletal dysplasia. J. Bone Miner. Res. 26,1670–1679. http://dx.doi.org/10.1002/jbmr.392.

Hanashi, D., Koshino, T., Uesugi, M., Saito, T., 2002. Effect of femoral nerve resectionon progression of cartilage degeneration induced by anterior cruciate ligamenttransection in rabbits. J. Orthop. Sci. 7, 672–676. http://dx.doi.org/10.1007/s007760200119.

Huh, S.Y., Gordon, C.M., 2013. Fractures in hospitalized children. Metab. Clin. Exp.62, 315–325. http://dx.doi.org/10.1016/j.metabol.2012.07.018.

Jiang, S.-D., Jiang, L.-S., Dai, L.-Y., 2007. Changes in bone mass, bone structure, bonebiomechanical properties, and bone metabolism after spinal cord injury: a 6-month longitudinal study in growing rats. Calcif. Tissue Int. 80, 167–175. http://dx.doi.org/10.1007/s00223-006-0085-4.

Jiang, S.-D., Jiang, L.-S., Dai, L.-Y., 2006. Spinal cord injury causes more damage tobone mass, bone structure, biomechanical properties and bone metabolismthan sciatic neurectomy in young rats. Osteoporos. Int. 17, 1552–1561. http://dx.doi.org/10.1007/s00198-006-0165-3.

Johnell, O., Kanis, J.A., 2006. An estimate of the worldwide prevalence and disabilityassociated with osteoporotic fractures. Osteoporos. Int. 17, 1726–1733. http://dx.doi.org/10.1007/s00198-006-0172-4.

Komori, T., 2015. Animal models for osteoporosis. Eur. J. Pharmacol. 759, 287–294.http://dx.doi.org/10.1016/j.ejphar.2015.03.028.

LeBlanc, A., Marsh, C., Evans, H., Johnson, P., Schneider, V., Jhingran, S., 1985. Boneand muscle atrophy with suspension of the rat. J. Appl. Physiol. 58, 1669–1675.

Liu, D., Zhao, C.-Q., Li, H., Jiang, S.-D., Jiang, L.-S., Dai, L.-Y., 2008. Effects of spinalcord injury and hindlimb immobilization on sublesional and supralesional

bones in young growing rats. Bone 43, 119–125. http://dx.doi.org/10.1016/j.bone.2008.03.015.

Mergler, S., Evenhuis, H.M., Boot, A.M., De Man, S.A., Bindels-De Heus, K.G.C.B., Huijbers,W.A.R., Penning, C., 2009. Epidemiology of low bone mineral density and fracturesin childrenwith severe cerebral palsy: a systematic review. Dev. Med. Child. Neurol.51, 773–778. http://dx.doi.org/10.1111/j.1469-8749.2009.03384.x.

Moyer-Mileur, L.J., Brunstetter, V., McNaught, T.P., Gill, G., Chan, G.M., 2000. Dailyphysical activity program increases bone mineralization and growth in pretermvery low birth weight infants. Pediatrics 106, 1088–1092.

Munns, C.F., Cowell, C.T., 2005. Prevention and treatment of osteoporosis inchronically ill children. J. Musculoskelet. Neuronal. Interact. 5, 262–272.

O'Connor, B.L., Visco, D.M., Brandt, K.D., Myers, S.L., Kalasinski, L.A., 1992. Neuro-genic acceleration of osteoarthrosis. The effects of previous neurectomy of thearticular nerves on the development of osteoarthrosis after transection of theanterior cruciate ligament in dogs. J Bone Jt. Surg. Am. 74, 367–376.

Parfitt A.M., 1983. The physiologic and clinical significance of bone histomorpho-metric data, In: Recker R.R., (Ed), Bone Histomorphometry: Techniques andInterpretation, CRC, Boca Raton, FL, 143–223.

Rudnik-Schöneborn, S., Heller, R., Berg, C., Betzler, C., Grimm, T., Eggermann, T.,Eggermann, K., Wirth, R., Wirth, B., Zerres, K., 2008. Congenital heart disease isa feature of severe infantile spinal muscular atrophy. J. Med. Genet. 45,635–638. http://dx.doi.org/10.1136/jmg.2008.057950.

Sames, M., Benes, V., 1997. Surgical approach to the rabbit sciatic nerve. Tech. note.Acta Chir. Plast. 39, 65–67.

Sugiyama, T., Meakin, L.B., Browne, W.J., Galea, G.L., Price, J.S., Lanyon, L.E., 2012.Bones' adaptive response to mechanical loading is essentially linear betweenthe low strains associated with disuse and the high strains associated with thelamellar/woven bone transition. J. Bone Mineral. Res. 27, 1784–1793. http://dx.doi.org/10.1002/jbmr.1599.

Sung, D.H., 2004. Locating the target nerve and injectate spread in rabbit sciaticnerve block. Reg. Anesth. Pain. Med 29, 194–200.

Villa-Camacho, J.C., Iyoha-Bello, O., Behrouzi, S., Snyder, B.D., Nazarian, A., 2014.Computed tomography-based rigidity analysis: a review of the approach inpreclinical and clinical studies. Bonekey Rep. 3, 587. http://dx.doi.org/10.1038/bonekey.2014.82.

Ward, K., Alsop, C., Caulton, J., Rubin, C.T., Adams, J., Mughal, Z., 2004. Low mag-nitude mechanical loading is osteogenic in children with disabling conditions.J. Bone Miner. Res. 19, 360–369. http://dx.doi.org/10.1359/JBMR.040129.

Whealan, K.M., Kwak, S.D., Tedrow, J.R., Inoue, K., Snyder, B.D., 2000. Noninvasiveimaging predicts failure load of the spine with simulated osteolytic defects*†.J. Bone Jt. Surg. Am. 82. http://dx.doi.org/10.1007/BF00401809.

Yarrow, J.F., Ye, F., Balaez, A., Mantione, J.M., Otzel, D.M., Chen, C., Beggs, L.A.,Baligand, C., Keener, J.E., Lim, W., Vohra, R.S., Batra, A., Borst, S.E., Bose, P.K.,Thompson, F.J., Vandenborne, K., 2014. Bone loss in a new rodent modelcombining spinal cord injury and cast immobilization. J. Musculoskelet. Neu-ronal. Interact. 14, 255–266.