Dramatic increase in cortical thickness induced by femoral marrow ablation followed by a 3-month...

ORIGINAL ARTICLE JJBMR
Dramatic Increase in Cortical Thickness Induced byFemoral Marrow Ablation Followed by a 3-MonthTreatment with PTH in Rats
Qing Zhang,1 Jodi Carlson,2 Hua Zhu Ke,3 Jiliang Li,4 Michael Kim,1 Kieran Murphy,5 Nozer Mehta,6

James Gilligan ,6 and Agnes Vignery1

1Yale University School of Medicine, Departments of Orthopaedics and Cell Biology, New Haven, CT, USA2Yale University School of Medicine, Department of Comparative Medicine, New Haven, CT, USA3Department of Cardiovascular and Metabolic Diseases, Pfizer Global Research and Development, Groton, CT, USA4Department of Biology, Indiana University–Purdue University Indianapolis, Indianapolis, IN, USA5Department of Medical Imaging, University of Toronto, Toronto, Ontario, Canada6Unigene Laboratories, Inc., Boonton, NJ, USA

ABSTRACTWe previously reported that following mechanical ablation of the marrow from the midshaft of rat femurs, there is a rapid and

abundant but transient growth of bone, and this growth is enhanced and maintained over a 3-week period by the bone anabolic

hormone parathyroid hormone (PTH). Here, we asked whether further treatment with PTH or bisphosphonates can extend the half-

life of the new bone formed in lieu of marrow. We subjected the left femur of rats to mechanical marrow ablation and treated the

animals 5 days a week with PTH for 3 weeks (or with vehicle as a control) to replace the marrow by bone. Some rats were euthanized

and used as positive controls or treated with vehicle, PTH, or the bisphosphonate alendronate for a further 9 weeks. We subjected

both femurs from each rat to soft X-ray, peripheral quantitative computed tomography (pQCT), micro-computed tomography (mCT),

dynamic histomorphometry analysis, and biomechanical testing. We also determined the concentrations of serum osteocalcin to

confirm the efficacy of PTH. Treatment with PTH for 3 months dramatically enhanced endosteal and periosteal bone formation,

leading to a 30% increase in cortical thickness. In contrast, alendronate protected the bone that had formed in the femoral marrow

cavity after marrow ablation and 3 weeks of treatment with PTH but failed to promote endosteal bone growth or to improve the

biomechanical properties of ablated femurs. We further asked whether calcium-phosphate cements could potentiate the formation

of bone after marrow ablation. Marrow cavities from ablated femurs were filled with one of two calcium-phosphate cements, and rats

were treated with PTH or PBS for 84 days. Both cements helped to protect the new bone formed after ablation. To some extent, they

promoted the formation of bone after ablation, even in the absence of any anabolic hormone. Our data therefore expand the role of

PTH in bone engineering and open new avenues of investigation to the field of regenerative medicine and tissue engineering. Local

bone marrow aspiration in conjunction with an anabolic agent, a bisphosphonate, or a calcium-phosphate cement might provide a

new platform for rapid preferential site-directed bone growth in areas of high bone loss. � 2010 American Society for Bone and

Mineral Research.

KEY WORDS: BONE REGENERATION; PTH; BISPHOSPHONATE; CALCIUM-PHOSPHATE CEMENT; MARROW ABLATION

Introduction

Bone development and repair are mediated by two distinct

yet complementary mechanisms, namely, endochondral and

intramembranous bone formation. While the former requires

the formation of a cartilage enlagen that is synthesized by

chondroblasts, the latter is laid down directly by osteoblasts. The

Received in original form May 1, 2009; revised form September 3, 2009; accepted

Address correspondence to: Agnes Vignery, Departments of Orthopaedics and Cell

06510, USA. E-mail: [email protected]

All supporting information may be found in the online version of this article.

Journal of Bone and Mineral Research, Vol. 25, No. 6, June 2010, pp 1350–1359

DOI: 10.1002/jbmr.25

� 2010 American Society for Bone and Mineral Research

1350

intramembranous process secures the formation of flat bones,

such as those of the skull, the scapula, and the shaft of long

bones. While the formation of bone during adulthood can be

considered intramembranous, the formation bone during

fracture repair involves both endochondral and intramembra-

nous bone-formation processes, which initiate from the perio-

steum and the endosteum, respectively. Following mechanical

January 4, 2010. Published online February 2, 2010.

Biology, Yale University School of Medicine, 330 Cedar Street, New Haven, CT

ablation of the marrow from long bones, bone transiently forms

inside the marrow cavity independent of cartilage, hence via the

intramembranous mechanism. This bone-formation phase is

complete 7 days after ablation, when osteoclasts differentiate in

synchrony and resorb the new bone formed to recreate the

marrow cavity into which bone marrow cells rehome.(1,2) We had

previously questioned whether the bone anabolic hormone

parathyroid hormone (PTH), which is a calcium-regulating

hormone first reported as bone anabolic nearly 30 years ago

by Reeve and colleagues,(3,4) could extend the bone-formation

phase induced by marrow ablation to promote the formation of

additional bone. We reported that the amount of bone formed in

the marrow cavity of ablated rat femurs treated with PTH far

exceeded that achievable with either marrow ablation or an

anabolic hormone alone in a 3-week time period.(5) Sincemarrow

ablation alone results in the transient formation of bone, which is

resorbed by osteoclasts within 2 weeks, it is the combination of

the two treatments that was novel, and we called that biologic

phenomenom the bone bioreactor. Therefore, PTH not only

extended the half-life of the new bone but also increased its

abundance over a 3-week period. This indicated that PTH

transiently promotes the formation of bone independent of

osteoclasts. Here, we ask whether PTH can maintain the new

bone formed beyond 3 weeks, which was the endpoint of our

previous study. We also ask whether molecules that inhibit

osteoclasts, such as bisphosphonates, can protect the new

bone formed 3 weeks after mechanical ablation of the marrow

and treatment with PTH. Furthermore, we ask whether the

implantation of bone-formation-promoting calcium-phosphate

cements(6) inside the ablated marrow cavity can provide a

scaffold for the maintenance of the newly formed bone and

whether PTH treatment following such cement implantation

synergistically modifies that new bone.

We report that 3 months of treatment with PTH after marrow

ablation results in an increase in cortical bone-formation rate and

thickness. We also report that the bisphosphonate alendronate

protects the new bone formed in themarrow cavity after marrow

ablation and treatment with PTH for 3 weeks, but it is less potent

than PTH at increasing cortical thickness and improving the

biomechanical properties of ablated femurs. Further, we report

that calcium-phosphate cements integrate in the new bone

formed after marrow ablation and extend its half-life and that

PTH treatment following cement implantation enhances its

abundance.

Materials and Methods

Animals

Fisher 344 male rats weighing an average of 200 g (8 to 10 weeks

old) were obtained from Charles River (Kingston, NY, USA). All

rats were allowed to rest for 2 weeks after arrival at the Yale

Animal Care Facility and housed under controlled temperature

(248C) and light (12/12-hour light/dark) with food and water

available ad libitum. The care and treatment of the experimental

animals complied with NIH guidelines and were approved by the

Institutional Animal Care and Use Committee at Yale University.

Bone marrow ablation was performed as described pre-

viously.(5) In brief, rats were anesthetized with a combination of

SITE-SPECIFIC BONE FORMATION IN RATS

ketamine (50mg/kg) and xylazine (10mg/kg). Hair over the left

knee joint was shaved, and the shaved area was cleaned with

Betadine scrub and then washed with ethanol. A 1.0-cm-long

longitudinal skin incision was made across the medial aspect of

the knee joint. The distal femur was exposed by lateral luxation of

the patella, which was accomplished by release of the medial

ligamentous structures. A 1.0-mm-hole was drilled through the

femoral intracondylar notch above the tendon by a smooth

0.035-inch K-wire drill bit into the marrow cavity. The drilling

motion was performed five times. The drill was stopped and

pushed further into the marrow cavity to ensure that it went

through the growth plate. Drilling then was repeated using a

threaded 0.045-inch K-wire drill bit. The content of the bone

marrow cavity was backflushed by injection of 5mL of normal

saline solution into the femur using a syringe attached to a 21G

needle. A pipe cleaner (Sharn, Inc., Tampa, FL, USA) was used to

remove cells and debris from the bone marrow cavity. Calcium-

phosphate cements (Pepgen-p15 from CeraPedics, Lakewood,

CO, USA, and Cementek from Teknimed, Vic en Bigorre, France)

were placed in the marrow cavity of some rats. The medial

ligamentous structures were sutured with a 4-0 Dexon thread.

The skin incision was closed with surgical metallic clips. The rats

were injected ip with a 5-ml bolus of saline and were given

Carprofen (5mg/kg/day) for the first 24 hours after surgery. A

recombinant analogue of human PTH [PTH(1-34) NH2; 40mg/kg/

day] was provided by Unigene Laboratories, Inc. (Boonton, NJ,

USA). PBS and PTH were injected sc in the dorsal neck region of

the animals. Injections were initiated on the day of surgery

(day 1) and were performed for 5 consecutive days per week

(Monday through Friday, between 5:00 and 8:00 p.m). Alen-

dronate (20mg/kg; Sigma, St. Louis, MO, USA) was delivered sc

twice a week. At the time of euthanization, rats were anest-

hetized, and blood was collected by cardiac puncture. Rats

received four sc injections of calcein (10mg/g of body weight;

Merck, Darmstadt, Germany) on days 9, 8, 2, and 1 before

euthanization.

Bone radiography

Both excised femurs from each rat were subjected to X-ray on a

cranial-caudal view using an MX-20 system (Faxitron X-ray

Corporation, Wheeling, IL, USA) at 30 kV for 3 seconds. X-ray films

were scanned using an Epson Perfection 4870.

Bone densitometry

Bone density was determined as we described previously(7) by

peripheral quantitative computed tomography (pQCT) with a

Stratec scanner (Model XCT Research, Norland Medical Systems,

Fort Atkinson, WI, USA). Routine calibration was performed daily

with a defined standard that contained hydroxyapatite crystals

embedded in Lucite, provided by Norland Medical Systems. We

scanned 1-mm-thick slices located midway between epiphyses

at the center of the femoral shaft. The voxel size was set at

0.1mm. Scans were analyzed with a software program supplied

by the manufacturer (XCT 520, Version 5.1). Bone density and

geometric parameters were estimated by loop analysis. The low-

and high-density threshold settings were 1300 and 2000,

respectively. Separation of soft tissue from the outer edge of

Journal of Bone and Mineral Research 1351

Table 1. Study Design: Number of Rats per Group

0 day 21 days 84 days

Control 6 (baseline) 6 10

Bmx 6 10

Bmxþ 21 days PTH,

then 63 days PBS

10

Bmxþ 84 days PTH 6 10

Bmxþ 21 days PTH,

then 63 days alendronate

10

Bmxþ Pepgen p-15 3

Bmxþ Pepgen

p-15þ 84 days PTH

3

BmxþCementek 4

BmxþCementekþ 84 days PTH 3

bone was achieved using contour mode 1. Cortical (high-bone-

density) and trabecular (low-bone-density) bone were separated

to obtain trabecular data using peel mode 3.

Computed tomography on a microscale (mCT)

Both femurs from each rat were scanned with a mCT scanner

(MicroCT40, Scanco, Bassersdorf, Switzerland) with a 2048� 2048

matrix and isotropic resolution of 9mm3 (12-mm voxel size). 3D

trabecular measurements in the medullary cavity were made

directly.

Biomechanical testing of the femoral midshaft:Three-point bending test

Right and left femurs were subjected to three-point bending to

record the ultimate force, the stiffness, and the energy to failure

of femurs as we previously published.(5) The anterior-to-posterior

diameter at the midpoint of the femoral shaft was recorded

using an electronic caliper. Femurs were placed on the lower

supports of a three-point bending fixture with the anterior side

facing downward in an Instron Mechanical Testing Instrument

(Instron 4465 retrofitted to 5500, Norwood, MA, USA). The span

between the two lower supports was set at 14mm. The upper

loading device was aligned to the center of the femoral shaft. The

load was applied at a constant displacement rate of 6mm/min

until the femur broke. The locations of maximal load, stiffness,

and energy absorbed were selected manually from the load.

Histology

Femurs were dehydrated in a graded ethanol series and

embedded, without decalcification, in methyl methacrylate, as

we described previously.(8) Transversal 7-mm-thick sections of

the femoral shafts were obtained using an Autocut microtome

equipped with a tungsten-carbide blade (Jung, Reichert,

Germany). Sections were kept unstained or stained with

toluidine blue or decalcified and observed under polarized

light. Unstained sections were viewed with epifluorescence

illumination for dynamic histomorphometry analysis, as we

described previously.(7) Given the difficulty in generating well-

preserved thin undecalcified transverse sections of the femoral

shafts, we were unable to complete the static part of the

histomorphometric analysis.

Microscopy

Microscopy was performed using an IMT-2 Olympus microscope

equipped with ultraviolet (UV) light and an OM-4 camera.

Biochemical parameters

Blood was collected by cardiac puncture at the time of

euthanization, and the concentration of serum osteocalcin was

determined by radioimmunoassay, as described previously.(7)

Statistical analysis

Data represent the mean� 1 SD. Treatment groups were

compared using the analysis of variance. Pair-wise comparison

p values between the treatment groups were adjusted using the

Tukey multiple-comparison procedure. Statistical significance

1352 Journal of Bone and Mineral Research

was declared if the two-sided p value was less than .05. All

computations were performed using SPSS.

Results

We previously demonstrated a synergistic effect of bone

marrow ablation and PTH treatment in the rapid formation of

new bone in the femoral shaft of rats. However, it remained to

be established if the newly formed bone could be maintained

over a long period of time. We have therefore investigated the

long-term effect of PTH treatment after marrow ablation. We

subjected the left femur of young adult rats to marrow ablation

and treated the rats 5 days a week with PTH for a period of 84

days (Table 1). Control rats with left ablated femurs received

PBS for the same length of time. To ask whether a bispho-

sphonate could protect the new bone formed in the marrow

cavity from rats treated with PTH for a period of 21 days, we

treated some rats 2 days a week with alendronate for an

additional period of 63 days. We included control rats that were

subjected to femoral marrow ablation and treated with PBS or

PTH for a period of 21 days or PBS for a period of 84 days. We

also included baseline rats that were euthanized on the day of

surgery and control rats that were euthanized on day 21 or day

84 after surgery. All rats gained weight during the course of the

experiment, independent of surgery or hormone or drug

treatment (data not shown), hence confirming that marrow

ablation does not adversely affect animal health. As anticipated,

PTH increased the serum concentration of osteocalcin, which

was determined at the time of sacrifice (Table 2). PTH also

augmented the total density of ablated femoral shafts from rats

treated for 21 days that were used as positive controls

(Supplemental Fig. 1).

High-resolution radiographic imaging of the ablated femurs

confirmed an increase in radiopacity of the medullary cavity

21 days after surgery when compared with contralateral femurs

and femurs from control rats (Fig. 1A). This intramedullary

radiopacity had dissipated 84 days after surgery and was

indistinguishable from that of control and contralateral femurs

(Fig. 1A), thereby confirming the transient nature of the new

ZHANG ET AL.

Table 2. Serum osteocalcin

Group

Osteocalcin

(mg/mL) SD Level of significance

Baseline day 0 129.7 29.5

Control day 21 97.5 12.7 p< .03 vs baseline

Bmx PBS 21 days 106.7 15.5

PTH 21 days 129.3 29.6 p< .04 vs control 21 days

Control day 84 61.8 8.8 p< .001 vs baseline

Bmx Day 84 54.7 6.3

PTH 21 days,

then 63 days PBS

70.4 8.2 p< .002 vs bmx 84 days

PTH 84 days 89.4 14.3 p< .0001 vs control 84 days

p< .0001 bmx 84 days

p< .005 bmxþ PTH 21 days

then 63 days PBS

PTH 21 days,

then 63 days

alendronate

53.1 16.0 p< .0001 vs bmx PTH 84 days

p< .02 bmx PTH 21 days

then 63 days PBS

Pepgen-p15 82.3 10.4 p< .01 vs control 84 days

p< .001 bmx 84 days

Cementek 77.2 9.7 p< .01 vs control 84 days

p< .001 bmx 84 days

Pepgen-p15þ PTH 110.0 23.4 p< .001 vs control 84 days

p< 0.0002 bmx 84 days

p< 0.002 bmxþ PTH 21 days

then 63 days PBS

Cementekþ PTH 86.0 18.4 p< 0.01 vs control 84 days

p< 0.002 bmx 84 days

Note: In addition to PTH, cements placed in the medullary shaft after marrow ablation increase serum osteocalcin.

bone formed in response to marrow ablation. By contrast,

radiopacity was substantially increased in ablated femurs from

rats treated with PTH for a period of 84 days when compared

with control and contralateral femurs, revealing the efficacy of

long-term PTH. Femoral radiopacity from rats treated for a 21-

day period with PTH followed by PBS for a 63-day period was

indistinguishable from that of control rats. In contrast, femoral

radioopacity from rats treated with PTH followed by alendronate

was substantially more pronounced, suggesting that the newly

formed bone was maintained by treatment with the bispho-

sphonate (Fig. 1A).

To visualize the 3D architecture of the new bone formed in

ablated femurs, we subjected femoral shafts to mCT analysis. As

expected, new bone was found in the ablated marrow cavity

from rats treated with PTH for 3 weeks (Fig. 1B). While the new

bone was no longer present 84 days after surgery in rats that had

received PBS, it was partially retained in femurs from rats that

had received PTH. Unexpectedly, the cortical thickness of these

femurs appeared significantly augmented. In contrast, femurs

from rats that had been treated with PTH followed by

alendronate demonstrated abundant intramedulary bone

(Fig. 2A).

To quantify bone density, we subjected femurs to pQCT

analysis. Long-term treatment with PTH alone was more potent


than PTH followed by alendronate at increasing cortical

thickness (Fig. 3A). This increase in cortical thickness resulted

from a profound reduction in endosteal circumference (Fig. 3B)

combined with a moderate increase in periosteal circumference

(Fig. 3C), which suggests that marrow ablation promotes

periosteal growth, whereas PTH favors endosteal growth after

marrow ablation.

To ask whether bone formation was still active, we observed

nondecalcified 7-mm-thick transverse sections from the femoral

shafts under UV light. Such an analysis confirmed that the new

bone formed in the marrow cavity from rats treated with PTH for

a 21-day period is labeled by fluorescent calcein, reflecting active

bone formation (Fig. 1C). Of note, small segments of the

intramedullary bone exhibited fluorescent labeling 84 days after

marrow ablation and treatment with PTH (Fig. 1C). In addition,

both endosteal and periosteal surfaces from these femurs

exhibited fluorescent labeling, which reflects active bone

formation (Fig. 1C). However, calcein labels had merged by

then, suggesting an overall reduction in the rate of bone

formation. In contrast, femurs from rats that had been treated

with PTH followed by alendronate demonstrated abundant

intramedullary bone (Fig. 2A). This bone, however, was only

discretely labeled with calcein when compared with the 84-day

PTH-treated group (Fig. 1C). To further assess the thickness of the


Fig. 1. Femoral marrow ablation followed by treatment with PTH for 3 months (84 days) increases cortical bone thickness: results of radiographic, pQCT,

mCT, and histologic analysis. (A) High-resolution radiograms of the left operated and right unoperated femurs from control, sham-operated, and bone

marrow–ablated (bmx) rats that were treated 5 days a week with PBS or PTH for a duration of 21 or 84 days. Two groups of rats were treated with PTH for

21 days and then with PBS or alendronate for 63 days. Note that the representative marrow-ablated femur from rats that were treated with PTH for 84 days

is more intensely radiopaque than any other femur and is alsomore intensely radiopaque than its contralateral femur, which is itself more radiopaque than

the control femur. X-ray images are representatives of the overall data. (B) mCT analysis of femoral shafts from control and marrow-ablated femurs from

rats treated with PBS or PTH for 21 or 84 days. Note the dramatic increase in cortical thickness of the ablated femur from rats treated with PTH for 84 days.

(C) Histologic analysis following calcein labeling. The intramedullary bone formed in response to marrow ablation followed by 21 days of treatment with

PTH is undergoing calcification, as demonstrated by the regular fluorescent signal from calcein that has been incorporated into mineralizing bone. Note

that after 84 days of treatment with PTH, most of the calcein label highlights the endosteal and periosteal bone surfaces.

new cortical bone formed, we observed cross sections from

ablated shafts under polarized light. Under these conditions,

long-term PTH appeared to have a more dramatic effect on

cortical thickness than short-term PTH followed by alendronate

(Fig. 2B). These observations were confirmed by histologic

analysis of toluidine blue–stained sections, which confirmed the

presence of osteoblasts lining the endosteal surfaces of femurs

from rats treated for a period of 21 days with PTH. Osteoblasts

appeared less well developed in femurs from rats that had been

treated with PTH for a period of 84 days and were absent in

femurs from rats treated with PTH followed by alendronate

(Fig. 2C, D). No intramedullary bone was present and cortical

thickness remained unchanged in ablated femurs from rats that

were treated with PTH followed by PBS (Fig. 2A–C).

To determine the rate of cortical bone formation at the end

time point, we subjected femoral shafts to dynamic histomor-

phometric analysis. Such an analysis revealed a potent effect of

PTH on both periosteal and endosteal bone-formation rate

(Fig. 3D, E). It also revealed a potent inhibitory effect of

alendronate on the endosteal bone-formation rate.

To assess the functional consequences of such dramatic

changes in bone architecture, we subjected femurs to biomecha-

nical testing using three-point bending. Such an analysis revealed


that in contrast to long-term treatment with PTH after marrow

ablation,which augmentedultimate force and intrinsic stiffness to

failure (Fig. 4A, B), short-term treatment with PTH followed by PBS

or alendronate decreased the energy to failure (Fig. 4C); hence

ablated bones required less energy to break. Together these data

revealed that the bone anabolic potentiating effect of marrow

ablation extends over a 3-month period. These data also

suggested that alendronate protects the new bone formed in

response tomarrowablationandPTH treatment, but thequality of

the bone differs from that of rats treated with PTH alone.

Calcium-phosphate compounds are becoming of increasingly

greater importance in the field of biomaterials and, in particular,

as bone substitutes. In this way, donor tissue-induced morbidity

can be avoided.(6) Today, calcium-phosphate cements are placed

routinely in alveolar bone following tooth extraction prior to

tooth implant(9) and are being developed for augmentation of

fractures in the extremities as well as for vertebral compressive

fractures in the spine.(10) We therefore asked whether a calcium-

phosphate cement placed in the ablated marrow cavity could

affect the new bone formed and possibly extend its half-life.

Cementek is a self-setting bone cement composed of a solid

phase and a liquid phase. After mixing these two phases, it sets in

situ to form hydroxyapatite as the only end product. The cement

ZHANG ET AL.

Fig. 2. Femoral marrow ablation followed by treatment with PTH for 3 weeks and then by PBS or alendronate for 2 months fails to increase cortical bone

thickness. (A) mCT and histologic analysis of ablated femoral shaft from baseline rats and rats treated with PTH for 21 days and then with PBS or

alendronate for 63 days. Note that the residual bone present in the marrow cavity of alendronate-treated rats is weakly labeled by the fluorochrome

calcein. (B) Polarized light imaging of femoral shafts highlights the new layer of cortical bone (white bar) formed in response to marrow ablation and

84 days of treatment with PTH. Note that treatment with PTH for 21 days followed by PBS does not lead to an increase in cortical thickness. In contrast,

treatment with PTH for 21 days followed by alendronate for 63 days leads to an increase in cortical thickness. (C, D) Histologic analysis of femurs stresses

the cellular difference between the cells that line bone in PTH- versus PTH-alendronate-treated rats, which lack osteoblasts (bar¼ 100 mm).

Pepgen P-15 includes a 15-amino-acid portion of human

collagen type I, which is used to improve tooth implantation.(11)

Immediately following marrow ablation, we injected some

femurs with commercially available calcium-phosphate cements

(Pepgen p15 or Cementek) and treated the rats with PTH or PBS

five times a week for a period of 84 days. Radiopacity appeared

increased by both cements and further enhanced by PTH

(Fig. 5A). mCT analysis revealed the presence of a bone-like

structure in the shafts of the ablated femurs that was augmented

dramatically in femurs from rats treated with PTH. All femurs

exhibited fluorescent calcein labels, suggesting that bone was

being calcified (Fig. 5C). Indeed, histologic analysis of the shafts

confirmed the presence of intramedullary bone, the abundance

of which appeared further augmented in the PTH-treated group,

where remnants of cement could be detected integrated into

calcified bone (Fig. 5D). Polarized light analysis revealed the


abundance of orderly orientated collagen fibers (Fig. 5E). When

we compared the total bone density between all femurs from

rats euthanized on day 84, femoral shafts from rats that had

received Pepgen p15 alone demonstrated the greatest total

density, followed by femurs that had received Cementek from

rats treated with PTH (Fig. 6). These data indicated that cements

had not only integrated in the calcified bone from which they

were poorly distinguishable but also had promoted the anabolic

effect of PTH.

Discussion

A primary finding of this study was an unexpected and

significant increase in the cortical thickness of rat femoral shafts

as a result of mechanical marrow ablation followed by 3-month


Fig. 3. Marrow ablation and treatment with PTH alone or PTH followed by alendronate increases cortical thickness and bone formation rate: pQCT and

dynamic histomorphometric analysis. (A) Marrow ablation increases cortical thickness, which is further augmented by treatment with PTH and, to a lesser

degree, by PTH followed by alendronate. (B) Marrow ablation potentiates endosteal bone formation induced by treatment with PTH for a period of 84 days

and, to a lesser degree, by PTH treatment followed by alendronate. (C) Marrow ablation alone leads to an increase in periosteal circumference, which

appears partially prevented by short-term treatment with PTH. (D, E) Marrow ablation tends to augment the periosteal bone-formation rate, whereas PTH

augments both the periosteal and the endosteal bone-formation rate (n¼ 3 to 4).

treatment with PTH. We had reported previously that PTH

potentiates the formation of intramembranous bone in the

medullary cavity in response to marrow ablation over a 3-week

period.(5) To investigate the long-term effect of our interven-

tional approach, we extended the treatment with PTH to 3

months. While we had expected the new bone formed in the

ablated cavity from rats treated with PTH for 3 weeks to be in part

resorbed, we had not anticipated that the resorption of this new

bone would be coupled with a potent bone-formation response


on the endosteal surface of the shafts. Previous studies have

indicated that intermittent or pulsatile PTH treatment is known

to markedly increase trabecular bone volume owing to a

dominant stimulation of trabecular bone formation and to cause

a small loss of cortical bone.(12,13) Here, we demonstrate that

both short- and long-term treatment with intermittent PTH after

marrow ablation induces the sequential formation of new bone,

first intramedullary and then endosteal. Indeed, PTH continues to

increase the bone-formation rate after 84 days of treatment. Of

ZHANG ET AL.

Fig. 4. Differential biomechanical consequences of PTH and alendronate treatment on marrow-ablated femurs. Right and left femurs were subjected to

three-point bending to record (A) the ultimate force, (B) stiffness, and (C) energy to failure of femurs (n¼ 4 to 5).

note, both short- and long-term treatment with PTH leads to

improved biomechanical properties of the femoral shafts

independent of the location of the new bone formed. Since

cortical bone represents about 80% of the entire skeletal mass,

and because cortical volume and thickness are major predictors

of bone strength and fracture risk,(14) long-term treatment with

PTH after marrow ablation potentially could lead to fracture

prevention.

Although the bone mineralization rate mediated by endosteal

cells appeared decreased based on calcein labeling at 84 days

compared with that of the 3-week PTH cohort, marrow cells

appear morphologically healthy. This is in contrast to endosteal

cells from alendronate-treated animals, which appeared rela-

tively unhealthy. Endosteal bone formation in alendronate-

treated femurs was nearly abolished. In addition, the shafts from

alendronate-treated rats were not endowed with improved

biomechanical properties. Indeed, these shafts demonstrated

decreased energy to failure in the three-point bending test,

which suggests an undesirable effect provoked by alendronate.

In addition, alendronate was less potent than PTH at increasing

the cortical thickness of the shafts, indicating that alendronate

inhibits the resorption of the new intramedullary bone formed,

yet, unlike PTH, alendronate also inhibits the formation of

endosteal bone. Therefore, the question as to whether

protecting the new intramedullary bone formed in response

to marrow ablation and short-term PTH with a bisphosphonate is

appropriate remains open.


While it is well established that calcium-phosphate cements

are osteoconductive and are used widely to induce alveolar bone

growth prior to tooth implantation, the combination of marrow

ablation and cement appears to extend the half-life of

intramedullary bone independent of PTH. Yet PTH appears

not only to potentiate the formation of intramedullary bone but

also to promote the formation of endosteal bone, which

eventually merges with the remodeled intramedullary bone,

leading to a dramatic increase in cortical bone density associated

with a drastic reduction in the size of the marrow cavity. The

question as to whether calcium-phosphate cement–bone

complexes improve the biomechanical properties of the femurs

remains to be investigated.

PTH stimulates osteoblasts and augments bone mass, and the

abundance of cortical bone formed in response to marrow

ablation and 3 months of treatment with PTH is far more than

that formed in response to hormone alone without marrow

ablation. Indeed, PTH alone does not induce the formation of

new cortical bone in the nonablated marrow. Hence we have

shown that it is the synergistic effect of mechanical marrow

ablation and PTH, with or without a calcium-phosphate cement,

that triggers the increase in cortical thickness. Therefore, our data

support a potent and sustained anabolic effect of PTH when

given 5 days a week for 3 consecutive months after marrow

ablation. Such a potent anabolic effect of PTH after marrow

ablation might be used to stimulate implant anchorage in low-

density trabecular bone.(11)


Fig. 5. Anabolic effect of calcium-phosphate cements after femoral marrow ablation: results of radiographic, mCT, and histologic analysis. (A) High-

resolution radiograms of the left operated and right femurs. Left femurs were ablated and injected with one of two different cements, Pepgen p15 or

Cementek. Rats were kept untreated or were treated with PTH for 3 months. Note the radiopacity of the right femurs from rats treated with PTH. Note also

the radiopacity of the femurs injected with cement and the potentiating effect of PTH. (B) mCT analysis of the shafts from the left femurs shown in panel A.

(C) Marrow cavities from ablated femurs filled with cement show active calcification, as demonstrated by calcein label. (D) Transverse sections of the

femurs demonstrate abundant bone in the marrow cavities from ablated femurs and indicate the presence of lamellar bone; the deposition of new bone

appears further potentiated by treatment with PTH. (E) Polarized light imaging of femoral shafts highlights the strong light refraction hence the regularity

of the new bone formed in response to marrow ablation and implantation of calcium-phosphate cement, with and, to some extent, without 84 days of

treatment with PTH.

We reported previously that the intrinsic nature of the new

bone formed after ablation of the marrow in rats is mature and

regular and hence referred to as lamellar bone. In support of our

former observation, the bone that forms during the last week of a

3-month treatment with PTH after marrow ablation is also

regular, based on the regularity of calcein labels and the bone-

formation rate. Here again, and unlike trabecular bone, which

occupies the epiphyseal marrow cavity, this new bone is not

modeled on a cartilageneous anlagen; rather, it undergoes

intramembranous development. Hence the fate of this new

intramedullary bone on extended treatment with PTH is

surprising and opens avenues to improve cortical density to

prevent fractures.

While the molecular switch that triggers osteoclast differ-

entiation, which initiates 7 to 10 days after marrow ablation,

remains poorly understood, the predictable timing of differ-

entiation might offer a unique model system to investigate the


programming of the endogenous gene circuitry that initiates

osteoclastogenesis. Our observation expands the role of PTH in

bone and might open new avenues of investigations to the field

of regenerative medicine and tissue engineering. These findings

also might be potentially useful for investigations on the

molecular mechanisms that mediate intramembranous bone

formation and remodeling. In addition, local bone marrow

removal at selected sites in conjunction with pharmacologic

intervention with an anabolic agent may provide a technique for

rapid preferential site-directed bone growth in areas of high

bone loss.

Disclosures

QZ contributed to the design of the experimental plan,

performed the surgeries, injected the hormone, subjected the

ZHANG ET AL.

Fig. 6. PTH potentiates the increase in femoral bone density impacted by cements: pQCT comparative analysis between all groups.

femurs to pQCT and mCT, and completed the dynamic

histomorphometry. JC contributed to the design of the

experimental plan and performed the surgeries. MK contributed

to the design of the experimental plan and helped with the

surgeries.

KM helped with the study design. JL completed the

biomechanical analysis and the interpretation of results in the

context of the study design. NM and JG contributed to the study

design, interpretation of the data, and writing of the manuscript.

HK worked with QZ on the pQCT and mCT analyses. AV was the

project director/manager and wrote the manuscript in colla-

boration with the coauthors. AV has a conflicting financial

interest because her laboratory receives funds from Unigene

Laboratories.

Supplemental Fig. 1. Total bone density of femoral shafts from

positive control animals euthanized on day 21: pQCT analysis.

Acknowledgments

The authors are grateful to the Comparative Medicine Surgery

Team at Yale School of Medicine and to the Yale Core Center for

Musculoskeletal Diseases—in particular, to Dr Caren Gunberg for

her assistance in determining the concentration of osteocalcin.

We are grateful to Dr James Benedict from CeraPedics for

providing us with Pepgen-p-15. This work was supported by

funds from Unigene Laboratories, Inc. Fairfield, NJ.

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