augmentation of screw fixation with injectable calcium sulfate bone cement in ovariectomized rats

Augmentation of Screw Fixation With Injectable Calcium SulfateBone Cement in Ovariectomized Rats

Xiao-Wei Yu,y Xin-Hui Xie,y Zhi-Feng Yu, Ting-Ting Tang

Department of Orthopedic Surgery, Shanghai Ninth People’s Hospital, Shanghai Jiaotong University School of Medicine,Shanghai 200011, China

Received 27 November 2007; revised 1 May 2008; accepted 26 May 2008Published online 20 August 2008 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.b.31184

Abstract: The objective of this study was to determine the effect of augmenting screw

fixation with an injectable calcium sulfate cement (CSC) in the osteoporotic bone of

ovariectomized rats. The influence of the calcium sulfate (CS) on bone remodeling and screw

anchorage in osteoporotic cancellous bone was systematically investigated using histomorpho-

metric and biomechanical analyses. The femoral condyles of 55 Sprague–Dawley ovariecto-

mized rats were implanted with screw augmented with CS, while the contralateral limb

received a nonaugmented screw. At time intervals of 2, 4, 8, 12, and 16 weeks, 11 rats were

euthanized. Six pair-matched samples were used for histological analysis, while five pair-

matched samples were preserved for biomechanical testing. Histomorphometric data showed

that CS augmented screws activated cancellous bone formation, evidenced by a statistically

higher (p < 0.05) percentage of osteoid surface at 2, 4, and 8 weeks and a higher rate of bone

mineral apposition at 12 weeks compared with nonaugmented screws. The amount of the

bone–screw contact at 2, 8, and 12 weeks and of bone ingrowth on the threads at 4 and 8 weeks

was greater in the CS group than in the nonaugmented group (p < 0.05), although these

parameters increased concomitantly with time for both groups. The CS was resorbed

completely at 8 weeks without stimulating fibrous encapsulation on the screw surface. Also, the

cement significantly increased the screw pull-out force and the energy to failure at 2, 4, 8, and

12 weeks after implantation, when compared with the control group (p < 0.05). These results

imply that augmentation of screw fixation with CS may have the potential to decrease the risk

of implant failure in osteoporotic bone. ' 2008 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl

Biomater 89B: 36–44, 2009

Keywords: calcium sulfate; augmentation; fracture fixation; osteoporosis; ovariectomy

INTRODUCTION

As the population ages, the incidence of osteoporosis will

increase dramatically, along with the risk of fracture. These

fractures can range from vertebral fractures to extremity

fractures, and include fractures of the proximal femur, hu-

merus, and distal radius.1 The treatment of osteoporotic ex-

tremity fractures has become a major challenge for the

orthopedic surgeons. Poor implant anchorage in osteopor-

otic bone requires new treatment solutions. Augmentation

with bone cements or substitutes is one strategy that may

address this challenge.2

Historically, some researchers have used PMMA to

maintain the stability of the fracture–implant construct in

osteoporotic bone.3 PMMA has good strength and stiffness,

so the initial fixation is excellent. However, it is nonde-

gradable and has no biological potential to remodel or

integrate into the surrounding bone.4 Several other disad-

vantages include the release of toxic monomers,5 thermal

necrosis during cement setting,6 and infiltration of fibrous

tissue between the PMMA and surrounding bone.5 To over-

come these disadvantages, several injectable ceramic bone

cements or substitutes made from calcium-phosphate, cal-

cium-sulfate, or bioglass have been developed. These

degradable, biocompatible, and osteoconductive ceramic

materials7–9 have been used as an augment for osteoporotic

fractures fixation in the extremities as well as for vertebral

compressive fractures in the spine.10

For many decades, calcium sulfate (CS) has been widely

used as bone graft substitute in various skeletal sites.11–15

It is more rapidly resorbed than other biomaterials, allow-

ing for an earlier ingress of osteoprogenitor cells.16 It also

mimics the mineral phase of bone and resorbs at the rate

similar to that of bone formation.17,18 CS prevents soft tis-

yBoth authors contributed equally to this work.Correspondence to: T.-T. Tang (e-mail: [email protected])Contract grant sponsor: Shanghai Shuguang Educational Funding Committee

(China)

' 2008 Wiley Periodicals, Inc.

36

sue invasion by acting as a space filler,19 and can be

replaced by newly formed bone that ultimately remodels

comparably to autogenous bone.20 It has also shown signifi-

cant augment pull-out strength when used for augmentation

of pedicle screw fixation in vitro.21

Generally, calcium-phosphate cement is the more com-

mon ceramic for augmenting osteoporotic fractures.

Although Leung et al. reported the temporal changes invivo of calcium phosphate cement in screw augmentation,22

to date, calcium sulfate cement (CSC) has not been eval-

uated in an osteoporotic model. Most studies found that CS

is resorbed in the body within 8 weeks.17,23 Whether this

relative rapid resorption will be followed by enhanced

osteointegration and improved stability of the fixation

implant, remains unexplored. The aim of this study is to

investigate the effect of the CS on bone remodeling and to

evaluate its effect on screw augmentation in osteoporotic

bone in vivo.

MATERIALS AND METHODS

Osteoporotic Rat Model

Fifty-five 6-month-old female Sprague–Dawley rats were

used in this study.24 Under general anesthesia, the rats

underwent bilateral ovariectomies using a dorsal approach

(OVX). Nonabsorbable suture material was used for the lig-

ature, and the bilateral ovaries were completely excised.

Finally, the wound was closed by suturing. All rats were

housed in a cage with a circadian light rhythm of 12:12 h

light/dark, 60% humidity at 218C, and were fed a normal

diet. Four months after ovariectomization, osteoporosis was

confirmed by dual X-ray absorptiometry (Hologic Discov-

ery-A; Hologic Corporation, Waltham, MA). Both the rear-

ing of these rats and the experiments were approved by the

Animal Ethics Committee of Shanghai Jiaotong University

School of Medicine (reference number: 2004-12-5).

Screw Implantation and CS Augmentation

The femoral condyles were selected as the implantation site

of the screws. Under generalized anesthesia, the bilateral

knees of the rat were prepared with a sterile technique.

Straight incisions of 1 cm were made on the lateral sides

of the knees. The fibers of the tensor fascia lata were cut

longitudinally, and the lateral condyles of the distal femurs

were exposed. A 1.2 mm (diameter) 3 5 mm (depth) hole

was drilled unicortically in the cancellous bone, parallel to

the articular surface of the femoral condyles. The hole was

subsequently washed with saline solution and dried with

gauze. Each rat received a screw augmented with CS in

one leg, while the contralateral limb received the screw

without augmentation. The implants were randomly

assigned. The CS used in the study was a commercially

available surgical grade CS hemihydrate with a high com-

pressive strength.25 This injectable paste hardens in about

10 min when mixed with water (MIIG1 X3 CSC; Wright

Medical Technology, Arlington, TN). The CS cement was

prepared according to the manufacturer’s guidelines, and

about 0.02 mL was injected into each augmented hole.26 A

fully threaded cancellous screw (Shanghai Pu Wei Medical

Appliance Co.) with a thread diameter of 1.7 mm, a core

diameter of 0.8 mm, and 6 mm in length was inserted into

the hole before the cement hardened in situ. The soft tis-

sues were closed by suturing in layers.

At each time interval of 2, 4, 8, 12, and 16 weeks after

screw implantation, 11 rats were euthanized by an abdomi-

nal overdose of barbiturates and the femora were dissected.

All rats were injected intraperitoneally with tetracycline

hydrochloride of 30 mg/kg (Sigma, St. Louis, MO) and

15 mg/kg calcein (Sigma) to label the new bone calcification

on day 12 and 2 days before euthanasia. Six pair-matched

samples were put immediately in 70% ethanol for histologi-

cal analysis, while five pair-matched samples were double

bagged and fresh frozen at 2208C for biomechanical testing.

Histologic and Histomorphometric Analysis

The distal femurs were dehydrated in graded series of etha-

nol and finally in xylene, then infiltrated and embedded in

methylmethacrylate.22 These specimens were longitudinally

sectioned into 220-lm-thick slices parallel to the axis of

the screw, using a diamond saw (Leica SP1600; Leica

Instruments, Nussloch, Germany). The sections were hand-

ground to 50 lm and stained with Stevenel’s blue and Van

Gieson’s picro-fuchsin for histomorphometric analysis of

the bone–screw contact and bone ingrowth, and again were

ground to 20–30 lm for fluorescence microscopy. After

assessing the fluorescence microscopic images, the sections

were stained again for histomorphometric analysis of the

osteoid surface.

Four histomorphometrical parameters, as described by

Tadjoedin, Fini, and Nkenke et al., were blindly measured

as illustrated in Figure 1 by a single observer.27–29 Briefly,

the osteoid surface in the surrounding area of the screw

was determined in Eq. (1) as

Figure 1. Schematic diagram of histomorphometrical parameters(bone–screw contact perimeter, thread area, and the surrounding

area of screw).

37AUGMENTATION WITH CALCIUM SULFATE BONE CEMENT

Journal of Biomedical Materials Research Part B: Applied Biomaterials

Osteoid perimeter

Trabecular perimeter3 100 ð%Þ ð1Þ

The bone–screw contact was defined in Eq. (2) as

Bone contact with the screw surface perimeter

Screw surface perimeter3 100 ð%Þ

ð2ÞThis bone–screw contact measurement started from the

most coronal thread down to the most apical thread. The

bone ingrowth was defined in Eq. (3) as

Area of bone between the screw threads

Area between the screw threads3 100 ð%Þ ð3Þ

Finally, the bone mineral apposition rate (MAR) (lm/

day) in the surrounding area of the screw was expressed in

Eq. (4) as

Mean distance between adjacent fluorochromemarkers ðlabelsÞNumber of days ð10 daysÞ

ð4Þ

All image analysis used BioQuant OSTEO II software

(BioQuant Image Analysis Corporation, Nashville, TN).

Biomechanical Testing

The femurs were progressively thawed and rehydrated in

phosphate-buffered saline for 48 h before biomechanical

testing (Instron model 4411; Instron, Canton, MA). Each

femur was mounted in acrylic cement, and clamped as

shown in Figure 2. The screw head was grasped by a cus-

tom-made jig which connected into the actuator of the test

frame. The screw was then pulled to failure along the long

axis of the screw at a constant speed of 0.1 mm/s. During

testing, the force-displacement curve was displayed on the

monitor by using a testing software program (Series IX

software; Instron). The pull-out force (N) was determined

as the maximum point on the curve where failure occurred.

The energy to failure (J) was calculated based on the area

under the curve. Five specimens from each group were

evaluated at each time point.

Statistical Analysis

The results of both the histological analysis and the biome-

chanical test were expressed as means 6 SD. Paired t testwas used to compare histomorphometrical parameters and

biomechanical results between the CS and control groups.

All the data analyses were carried out using SAS (version

6.12; SAS Institute, Cary, NC). The level of significance

was set at p\ 0.05 (two-tailed).

RESULTS

Overall Observation

No animals encountered complications after surgery, and

all wounds healed without signs of infection. Radiographs

obtained postoperatively confirmed good placement of the

screws. There was no observed inflammation or adverse

soft tissue reaction to the CS implantation. Additionally, no

screw-loosening was observed at the surgical site in all

femurs.

Histologic Results

At 2 weeks, residual bone fragments were observed

between the bone bed and screw interface, because of oper-

ative pilot hole drilling. New woven bone was observed on

the surface in both groups. In the CS group, most of the

space surrounding each screw was filled with graft material

and appeared homogeneous [Figure 3(A)]. There was much

more void space in the control group [Figure 3(F)], and

some fibrous tissue infiltration was detected. Connective

tissue began to separate the screw from bone interface in

this group. At this time point, few fluorescent markers were

present in either group [Figure 4(A,F)]. As highlighted in

Figure 5, a higher frequency of active osteoblasts with cu-

boidal morphology was observed in the augmented speci-

mens compared with the control specimens as evidenced

by the area of osteoid matrix. These cells were more con-

centrated in areas in close proximity to the CS surface.

At 4 weeks, bone was observed in closer proximity to

the screw surface than in 2 week specimens. While the

bone in both groups exhibited spongy architecture, the

bone present in the augmented group was more mature

[Figure 3(B,G)]. The diffuse presence of fluorescent

markers for new bone calcification near the screw surface

showed an evidence of continued remodeling [Figure

4(B,G)]. In the experimental group, CS presence had

Figure 2. Biomechanical testing configurations showing that the

screw was pulling out to failure along the long axis of the screw at

a constant speed of 0.1 mm/s. [Color figure can be viewed in theonline issue, which is available at www.interscience.wiley.com.]

38 YU ET AL.


decreased from previous time points and abundant new

immature woven bone was found in the spaces between the

CS [Figure 6(A)]. There was an evidence of direct bone

apposition without fibrous encapsulation on screw surfaces

in the experimental group; however, the control group

screw surfaces were marked by fibrous tissue apposition

[Figure 6(B)].

At 8 weeks, increased areas of woven bone with greater

surface apposition to screw surfaces were observed in both

groups compared with the previous time points [Figure

Figure 3. Representative photomicrographs of six sections in each group showing the cancellous

bone around the screw, starting with the most apical thread to the third thread, augmented with

(A–E) and without (F–J) CSC at 2, 4, 8, 12, and 16 weeks after implantation from the same animal.

White arrows, CSC; black arrows, fibrous tissue (Stevenel’s blue and Van Gieson’s picro-fuchsinstaining; 350). [Color figure can be viewed in the online issue, which is available at www.interscience.

wiley.com.]

Figure 4. Fluorochrome-marked cancellous bone demonstrating bone deposited adjacent to the

screw augmented with (A–E) and without (F–J) CSC at 2, 4, 8, 12, and 16 weeks after implantation,tetracycline (yellow) and calcein (green) is shown, as arrows indicated (350). [Color figure can be

viewed in the online issue, which is available at www.interscience.wiley.com.]



3(C,H)]. In the experimental group, CS was no longer

observed in the sections. Higher concentrations of fluores-

cent markers were present indicating intense remodeling

[Figure 4(C,H)].

At 12 weeks, thicker, more mineralized, mature bone

was observed in the surrounding area of the screw in both

groups when compared with previous time points [Figure

3(D,I)]. In the CS group, a greater presence of properly

aligned and mature bone along the screw surface was

observed compared with the control group [Figure 3(D)].

In some screw threads, the new bone completely filled the

thread area. The osteointegration of the control group was

improved over previous time points, with a reduction in fi-

brous tissue and increased contact between the screw and

bone surfaces [Figure 3(I)].

Finally, at 16 weeks more mature bone was apparent in

both groups, with no visible difference in appearance

[Figure 3(E,J)].

Histomorphometric Results

Histomorphometric data indicated that the osteoid surface

surrounding the screws in the CS group was significantly

higher than that in control group at 2, 4, and 8 weeks

Figure 5. At 2 weeks, numerous active osteoblasts with cuboidal morphology were observed, as

evidenced by the area of osteoid in close proximity to the CS group (A). Meanwhile, much more

void space was observed in the control group, with fibrous tissue infiltration detected (B). CSC, cal-cium sulfate cement; NB, new bone; FT, fibrous tissue (Stevenel’s blue and Van Gieson’s picro-

fuchsin staining; 3100). [Color figure can be viewed in the online issue, which is available at

www.interscience.wiley.com.]

Figure 6. At 4 weeks, new bone, predominantly in the form of early woven bone, was being depos-

ited in contact with the screw in the CS group (A), while fibrous tissue infiltration was observed

around the screw in the control group (B). CSC, calcium sulfate cement; NB, new bone; FT, fibroustissue; S, screw (Stevenel’s blue and Van Gieson’s picro-fuchsin staining; 3100). [Color figure can

be viewed in the online issue, which is available at www.interscience.wiley.com.]

40 YU ET AL.


(Figure 7). In addition, the bone-screw contact of the CS

group was significantly superior to that of the control group

at 2, 8, and 12 weeks (Figure 8). Bone ingrowth of the CS

group was also significantly higher than that of the control

group but only on 4 and 8 weeks (Figure 9). Although all

CS group’s MAR values of the newly formed bone in the

surrounding area of the screw experimental groups was

greater than the control group except at week 2 (which

could not be determined because of lack of fluorescent

markers), a significant difference was only exhibited at the

12 weeks time point (Figure 10).

Biomechanical Testing Results

There were three major failure modes observed in the pull-

out testing of the CS group. At 2 weeks, failure occurred at

the cement–cancellous bone interface. At 4 weeks, fracture

of the CS cement was observed. At 8 weeks, the cancellous

bone itself failed, with residual bone strongly attached to

the screw surface. In contrast, there were only two major

modes of failure in the control group. At 2 weeks, fixation

failure happened at screw–cancellous bone interface with

little bone–screw interference. But after 8 weeks, failure

mode of control group was similar to that of the CS group.

The screw pull-out force of the CS group was signifi-

cantly higher than the control, at all time points except the

16 weeks time point (Figure 11). Most notably, at week 2,

the pull-out force of the control group was only half of the

CS group value. Similarly, the energy to failure in the CS

group was significantly higher than the control, at all time

points except at 16 weeks (Figure 12).

DISCUSSION

This investigation was designed to evaluate the effects of

screw augmentation with injectable CSC in osteoporotic

fracture fixation. In this study, we augmented screw fixa-

tion in vivo using OVX aged rat model. The model is still

reliable for postmenopausal osteoporosis after 8 months

postovariectomization, as confirmed by earlier studies.30

The increase in screw pull-out force and the energy to fail-

Figure 7. Comparison of the osteoid surface of the CS and control

groups. Differences were significant at 2, 4, and 8 weeks (p\ 0.05).

Figure 8. Comparison of the bone–screw contact of the CS andcontrol groups. Differences were significant at 2, 8, and 12 weeks (p

\ 0.05).

Figure 9. Comparison of the bone ingrowth of the CS and control

groups. Differences were significant at 4 and 8 weeks (p\ 0.05).

Figure 10. Comparison of the bone mineral apposition of the CSand control groups. Differences were significant at 12 weeks (p \0.05).



ure after cement augmentation showed the cement to be

highly biocompatible and osteoconductive.

In the control groups, higher quantities of fibrous tissue

were observed. It is postulated that the fibrous tissue layer

resulted from excessive micromotion at the implant-bone

interface. Such tissue hampers bony ingrowth at the screw–

bone interface, making this interface the weak point during

biomechanical pull-out testing. Micromotion may stimulate

precursor cells differentiation into fibroblasts rather than

osteoblasts, hindering the revascularization process.22 As

reported by Fini et al., impairments in bone–screw contact,

bony ingrowth, and extent of bone mineralization are res-

ponsible for poor implant stability in osteoporotic bone.28

We also observed increased osteoid levels at early time

points in the control group. This may be due to the effect

of localized trauma caused by the drilling of the pilot hole,

implantation of the screw, and/or the influence of implant

loading on the tissue.31

Our results showed that the CS provided significant rein-

forcement of screw fixation in early time points, because

little bony ingrowth and bone–implant contact were pres-

ent. As indicated through the biomechanical pull out

testing, there were different failure modes between two

groups. During the earliest postoperative phases, fixation of

the CS implant depended solely on mechanical interface of

the CS and surrounding cancellous bone. The CS-bone

interface was the mechanically weak point because the CS-

metal bond is quite strong. In contrast, the interface

between the screw threads and cancellous bone was the

mechanically weak point in the control group. It is theor-

ized that the thorough infiltration of CSC into the surr-

ounding cancellous bone creates a composite structure

improving interfacial shear strength through increased sur-

face area contact. Moreover, it may serve as conduit for

bony ingrowth by physically halting soft connective tissue

proliferation.32 At 4 weeks, new bone formation occurred

at the expense of the CS resorption making the degraded

CS surface the mechanically weak point. Even so,

increased levels of new bone ingrowth and bone–screw

contact within the CS group offered significantly greater

pullout force and energy absorption values compared with

the control group. Additionally, the abundant immature wo-

ven bone formed around the CS at 4 weeks had begun

to demonstrate a more mature trabecular alignment at

8 weeks; most likely due to the load bearing of the animal.

Carter et al. discussed that the amount of regenerated bone

should create a biomechanical balance where the stiffness

of the composite (residual bone graft 1 newly formed

bone construct) equals the physiological properties of the

adjacent bone tissue.33 Fini et al. also found that bony

ingrowth of the bone–screw interface was enhanced by the

presence of an osteoconductive biomaterial compared with

a bone–screw interface alone.28 Both the screw pull-out

force and energy to failure increased little in CS group at

4 weeks, which may suggest that immature woven bone is

the biomechanically weak link in the screw–cement–bone

system when cement begins to resorb. From then on, bio-

mechanical stability rose again concomitantly with time

because of the qualitative gain of new bone surrounding

the screw.

In this study, CS was found to activate new bone forma-

tion. At 2 weeks, large numbers of actively proliferating

osteoblasts and osteoid secretion were found at the surface

of cancellous bone, which allowed direct osteoblastic appo-

sition and led to faster and more abundant bone formation.

This activation suggests that CS may have a simulative

effect on the individual activity of osteoblasts, and was

effective in bone regeneration as evidenced by the results

of other authors.23,34,35 The MAR results revealed that CS

may increase cancellous bone turnover, especially at

12 weeks, which indicated that the increased concentration

of calcium ions may stimulate bone mineralization.23 Some

authors suggest that the bony response may be related to

the local acidity and subsequent demineralization of adja-

cent bone and release of matrix bound BMPs resulting in a

stimulatory effect.34

Figure 11. Comparison of the screw pull-out force of the CS and

control groups. Differences were significant at 2, 4, 8, and 12 weeks

(p\ 0.05).

Figure 12. Comparison of the energy to failure of the CS and con-trol groups. Differences were significant at 2, 4, 8, and 12 weeks (p

\ 0.05).

42 YU ET AL.


In earlier studies, other authors found that it took more

than 12 weeks for CS to disappear completely.36,37 In the

present study, CS was resorbed completely at 8 weeks,

which was similar to other studies, and most likely due to

the small volume of implanted cement surrounding screw.23

At 2 weeks, the cement appeared homogeneous and dense,

and then disintegrated into granules surrounded by new wo-

ven bone at 4 weeks, and finally disappeared at 8 weeks.

This suggests that CS was resorbed mainly by chemical

dissolution at first; then by osteoclast-mediated degradation,

as suggested by Sidqui et al.38

We also found that interconnectivity between adjacent

bone trabeculae was better in the CS group than in the con-

trol group, which implies that this biomaterial is conducive

to reestablishing the three-dimensional pattern that was lost

with osteoporosis. Further experiments are needed to quan-

tify this phenomenon and determine whether the newly

restored interconnectivity has some mechanical signifi-

cance. Also, a dynamic fatigue test should be conducted

because CS has relatively low shear strength, brittleness,

and susceptible to fatigue failure.

CONCLUSION

In summary, the CS used in the study was highly biocom-

patible and had a favorable effect on the osteogenic poten-

tial in osteoporotic bone. Though the pull-out force, the

energy to failure, bone ingrowth, and bone–screw contact

values were not significantly different after 16 weeks for

the two groups; a screw augmented with CS could stimu-

late new bone formation and withstand more load than a

nonaugmented screw in the first 12 weeks. Thus this con-

struct could provide a temporary support during a critical

period of fracture healing which may be applicable in the

treatment of osteoporotic fracture in humans.

The authors are deeply indebted to Shuang-Yan Zhang for thetechnical assistance in processing undecalcified sections, De-ZhiWang (Orthopaedic Research Lab/Center of Metabolic Bone Dis-ease Lab, University of Alabama at Birmingham) for the valuablesuggestions about histomorphometric analysis, and Ren-Guo Xie(Department of Hand Surgery, Affiliated Hospital of Nantong Uni-versity) for the biomechanical measurements.

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augmentation of screw fixation with injectable calcium sulfate bone cement in ovariectomized rats

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