trabecular bone response to injectable calcium phosphate (ca-p) cement

Trabecular bone response to injectable calcium phosphate(Ca-P) cement

E. M. Ooms, J. G. C. Wolke, J. P. C. M. van der Waerden, J. A. JansenDepartment of Biomaterials, College of Dental Science, University Medical Center Nijmegen, P. O. Box 9101, 6500 HBNijmegen, The Netherlands

Received 4 April 2001; revised 17 July 2001; accepted 26 July 2001

Abstract: The aim of this study was to investigate thephysicochemical, biological, and handling properties of anew developed calcium phosphate (Ca-P) cement when im-planted in trabecular bone. Ca-P cement consisting of a pow-der and a liquid phase was implanted as a paste into femoraltrabecular bone of goats for 3 days and 2, 8, 16, and 24weeks. The cement was tested using three clinically relevantliquid-to-powder ratios. Polymethylmethacrylate bone ce-ment, routinely used in orthopedics, was used as a control.The Ca-P cement was easy to handle and was fast settingwith good cohesion when in contact with body fluids. X-raydiffraction at the different implantation periods showed thatthe cement had set as an apatite and remained stable overtime. Histological evaluation after 2 weeks, performed on 10mm un-decalcified sections, showed abundant bone apposi-tion on the cement surface without any inflammatory reac-tion or fibrous encapsulation. At later time points, the Ca-P

cement implants were totally covered by a thin layer ofbone. Osteoclast-like cells, as present at the interface, hadresorbed parts of the cement mass. At locations where Ca-Pcement was resorbed, new bone was formed without loss ofintegrity between the bone bed and the cement. This dem-onstrated the osteotransductive property of the cement, i.e.,resorption of the material by osteoclast-like cells, directlyfollowed by the formation of new bone. Histological andhistomorphometrical evaluation did not show any signifi-cant differences between the Ca-P cement implanted at thethree different liquid/powder ratios. The results indicatethat the investigated Ca-P cement is biocompatible, osteo-conductive, as well as osteotransductive and is a candidatematerial for use as a bone substitute. © 2002 Wiley Periodi-cals, Inc. J Biomed Mater Res 61: 9–18, 2002

Key words: calcium phosphate cement; injectable; biocom-patibility; osteotransduction; animal study

INTRODUCTION

Calcium phosphate (Ca-P) materials are widelyused as bone substitute in dentistry and orthopedicand reconstructive surgery because of their biocom-patibility and osteoconductivity.1–3 Production ofthese materials involves processing at high tempera-tures, which results in highly crystalline and denseceramics that are able to form a chemical bond withbone.4 Besides this biological benefit of the Ca-P ce-ramics, there are also some shortcomings. They areavailable to the surgeon only in granules or prefabri-cated blocks. The granules can easily migrate or dis-perse into the surrounding tissue and do not provide

mechanical support to the surrounding tissues. How-ever, prefabricated blocks can give mechanical sup-port but are difficult to shape, resulting in an uncom-pleted filling of the bone defect, which can hamperbone healing. A solution for these problems might befound in injectable Ca-P cements5–11 that can beshaped according the defect dimension and harden insitu. They consist of a powder containing one or moresolid compounds of calcium and/or phosphate saltsand a cement liquid that can be water or an aqueoussolution. If the powder and the liquid are mixed in anappropriate ratio, they form a paste that at room orbody temperature sets by entanglement of the crystalsprecipitated within the paste.12–18 One of the most im-portant characteristics of Ca-P cements is that they aresupposed to be osteotransductive, i.e., they are slowlyresorbed and simultaneously transformed into newbone tissue.8,19–21

Since the first Ca-P cement was synthesized byBrown and Chow,22 several formulations have beenpublished.23 Unfortunately, the properties of currentlyavailable Ca-P cements are still insufficient for reli-able and safe clinical application. Problems have

Correspondence to: J. A. Jansen; e-mail: [email protected]

Contract grant sponsor: Dutch Technology FoundationSTW

Contract grant sponsor: Applied science division of NOWContract grant sponsor: technology program of the Min-

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© 2002 Wiley Periodicals, Inc.

been reported with the setting time, mechanicalstrength, application technique, and final biologicalproperties.24,25

To overcome these problems, Driessens et al.26 de-veloped an injectable Ca-P cement by mixing powdersof a-tricalcium phosphate (a-TCP), dicalcium phos-phate anhydrous (CaHPO4, monetite), calcium car-bonate (CaCO3), and precipitated hydroxyapatite withan aqueous solution containing sodium phosphate(Na2HPO4). This resulted in a reduction of the settingtime and improvement of the compressive strength.26

Further, they described how these properties changedby varying the liquid-to-powder ratio (L/P) between0.30 and 0.40. As measured with Gilmore needles,12

the initial setting time increased from 3 to 4.5 min andthe final setting time from 5.75 to 13 min when the L/Pratio was increased from 0.30 to 0.40. The compressivestrength was found to decrease with increasing L/Pratio and ranged from 41 MPa for L/P ratio 0.30 to 33MPa for L/P ratio 0.40 (after 5 days of soaking inRinger solution at 37°C).

The aim of the cement study was to examine the invivo response to this injectable Ca-P cement with dif-ferent L/P ratios, after implantation in trabecular bonein goats.

MATERIALS AND METHODS

Cement

The Ca-P cement powder contained 61% a-TCP, 26%CaHPO4, 10% CaCO3, and 3% precipitated hydroxyapatite.The cement liquid was a 4% aqueous solution of Na2HPO4.The cement powder was used as delivered by Merck (Darm-stadt, Germany) and mixed with cement liquid in three dif-ferent L/P ratios. The physicochemical composition of thecements directly after mixing and storage for 72 h in Ringersolution was characterized by X-ray diffraction (XRD) (Fig.1) and energy dispersive spectroscopy (EDS). The XRD pat-tern of the starting powder showed that the cement wasmainly composed of a-TCP.

Further, the cements were used according to the instruc-tions of the manufacturer. For delivery of the cements, acommercially available syringe (Sherwood medical mono-ject 2-mL syringe) was applied. The syringe was filled with1 g of cement powder. Then, cement liquid (4% aqueoussolution of Na2HPO4) was added. Three different L/P ratioswere used: 1. 0.30 mL liquid + 1 g powder (Ca-P 0.3), 2. 0.35mL liquid + 1 g powder (Ca-P 0.35), and 3. 0.40 mL liquid +1 g powder (Ca-P 0.4). The cement was mixed for 15 s usinga Silamatt mixing apparatus (Vivadent, Schaan, Liechten-stein). After mixing, the cement was immediately injected inthe defects in a retrograde manner. Before use in the animalstudy, the cement powder was always sterilized by g radia-tion with 25 kGy. The cement liquid was filter sterilizedthrough a sterile 0.2-mm filter.

Animal model and implantation procedure

Twenty-nine healthy mature (2–4 years of age) femaleSaanen goats, weighing about 50 kg were used in this study.Before surgery, blood samples of the goats were taken toensure that the animals were CAE/CL arthritis free. Theanimals were housed in a stable. National guidelines for thecare and use of laboratory animals were observed.

The cement paste was delivered into the trabecular boneof the femoral condyles. The operation was performed un-der general anesthesia. The anesthesia was induced by anintravenous injection of pentobarbital and maintained byisoflurane 2.4% through a constant volume ventilator, ad-ministered through an endotracheal tube. The goats wereconnected to a heart monitor. To reduce the risk of periop-erative infection, the goats were treated according to thefollowing doses of antibiotics: during the operation, Albi-pent (Mycopharma, de Bilt, The Netherlands) 15%, 3 mL/50kg subcutaneously, 1 and 3 days after the operation AlbipenLA, 7.5 mL/50 kg subcutaneously.

For insertion of the cements, the animal was immobilizedon its back and the hind limbs were shaved, washed, anddisinfected with povidone-iodine.

For implantation of the cement paste in the femur, a lon-gitudinal incision was made on the medial surface of the leftand right femur. After exposure of the femoral condyle, theperiosteum was reflected and a 2.0-mm pilot hole wasdrilled. The hole was gradually widened with drills of in-creasing size until a final diameter of 0.5 cm was reached.The bone preparation was performed with a gentle surgicaltechnique, using low rotational drill speeds and continuouscooling with saline. In this way, two holes were made in themedial condyle (superior and inferior, distance between theholes at least 1 cm). Between the two holes, two titaniumbone markers (Stabilokt; Henry Schein, Utrecht, The Neth-erlands) were placed for localization of the implant sites atthe end of the implantation period. After preparation, theholes were irrigated and then packed with sterile cotton

Figure 1. Powder XRD patterns of the investigated Ca-Pcement: (A) starting powder, (B) after mixing at L/P ratio0.35 and 72 h soaking in Ringer solution and after (C) 2 and(D) 8 weeks of implantation in trabecular bone of goats. Adiffractogram of goat bone (E) is added for comparison.Note that the monetite phase (•) has disappeared after 8weeks, leaving an apatitic structure. Diffractograms of ce-ments after 16 and 24 weeks of implantation were identicalto the 8 weeks’ pattern.

10 OOMS ET AL.

gauze to reduce bleeding. Each femur had two implant sites,resulting in four femoral trabecular bone implant sites ineach goat. A total of 116 holes were drilled: 29 holes werefilled with Ca-P 0.3 material, 29 holes with Ca-P 0.35 mate-rial, and 29 with Ca-P 0.4 material. One hole in each animalserved as a control and was filled with polymethylmethac-rylate (PMMA) bone cement (Palacost; Merck). To ensurecomplete randomization, the various materials were placedaccording to a balanced split plot design.

After insertion of the cements in the holes, the soft tissueswere closed in separate layers using resorbable Vicrylt 3-0sutures. Postoperative X-rays were made to ensure properplacement and filling of the implant locations.

Evaluation of the in vivo behavior of the various materialswas planned at implantation periods of 3 days (n = 1), 2(n = 7), 8 (n = 7), 16 (n = 7), and 24 (n = 7) weeks. Further,three goats of the 24-week group received in vivo fluoro-chromes. These markers were applied subcutaneously attimed intervals (Table I).

Processing of cement specimens

At the end of the predetermined period of the experi-ments, the animals were killed by an overdose of Narcovedt(Apharma, Arnhem, The Netherlands). After killing the ani-mals, the femoral condyles were excised immediately andthe excess tissue was removed. Then, using a diamond saw,the retrieved condyles were divided in smaller specimens,suitable for further processing. Finally, each specimen con-tained one cement material with surrounding bone. Alwaysone of the samples of each material and implantation periodwas used for physicochemical characterization of the ce-ments. The other samples were subjected to histologicalevaluation.

Physicochemical analysis

To determine the physicochemical characteristics of thevarious Ca-P cements, XRD and EDS were used. The speci-mens for evaluation with XRD and EDS were dehydrated inethanol. Subsequently, the Ca-P cement was removed fromthe bone tissue using a small bone curette. Thereafter, thisdetached material was ground for XRD analysis with a mor-tar and a pestle. Finally, the chemical composition of thismaterial was determined with in a JEOL 6310 scanning elec-tron microscope equipped with EDS (Noran voyager II, PGTspectrometer, Mylar window).

Histological and histomorphometrical procedures

The specimens for the histological evaluation were fixedin formaldehyde 4%, dehydrated in ethanol, and embeddedin methylmethacrylate. After polymerization, non-decalcified thin (10 mm) sections were prepared using a dia-mond blade sawing microtome technique.27 The sections

were made in a transversal direction perpendicular to thelong axis of the implant. To be able to evaluate the cement-filling efficacy, two of the 2- and 16-week specimens weresectioned in a longitudinal direction parallel to the long axis.All sections were stained with basic fuchsin and methyleneblue and examined with a light microscope (Leica MZ12;Leica BV, Rijswijk, The Netherlands). In addition, digitalimage analysis software (Leica Quin; Leica MicrosystemsImaging Solutions Ltd., Cambridge, UK) was used for his-tomorphometrical measurements.

The histological and histomorphometrical analysis con-sisted of the following:

1. Subjective description.2. Percentage of bone contact at the interface. The amount

of interfacial bone contact was defined as the percent-age of cement perimeter at which there is direct bone-to-cement contact without intervening soft tissue lay-ers. The total perimeter and the total length of siteswith direct bone-to-cement contact were measured inthe sections at 2.5× magnification.

3. Number of remodeling lacunae in the interface. Theamount of remodeling activity was determined bycounting the remodeling lacunae that were in contactwith the cement at 10× magnification.

4. Cement area. The total area of cement present in thetransversal sections was determined and compared be-tween the different implantation periods to determinethe rate of cement resorption.

All quantitative measurements were performed for threedifferent sections per specimen. The presented data arebased on the average of the three measurements.

The histological specimens, retrieved from the animalsthat received fluorochromes, were prepared as describedabove. In addition to thin sections (10 mm), thicker sections(20 mm) were made and reflectant fluorochrome microscopywas used for their descriptive evaluation

Statistical analysis

All measurements were statistically evaluated using atwo-way analysis of variance and a multiple comparisonprocedure (Tukey).

RESULTS

Macroscopical evaluation

During the experimental period, all goats remainedin good health. At sacrifice, no macroscopical signs ofinflammation or adverse tissue reaction were apparentaround the implantation sites. All implant sites could

11INJECTABLE CALCIUM PHOSPHATE BONE CEMENT

be easily located. Unfortunately, one 24-week samplefilled with Ca-P 0.35 material was lost at retrieval dur-ing the division of the condyles in smaller specimens.

Physicochemical analysis

XRD revealed that no differences in diffraction pat-terns existed between the cement mixed at the threedifferent L/P ratios. Apparently, at 3 days and 2weeks after implantation, the a-TCP phase had disap-peared and an apatite-like structure with superposi-tion of the peaks of CaHPO4 was formed. At laterimplantation times, except for the disappearance ofthe CaHPO4 peaks, no further changes were observed(Fig. 1).

EDS measurements showed that the calcium-to-phosphate ratio in the cements for all Ca-P materialsand implantation periods varied between 1.53 and1.65.

Histology

The Ca-P cement was clearly visible in the createdbone defects. However, the PMMA had dissolved dur-ing the histological preparation and embedding pro-cess. Subsequently, the bone cavity became filled withthe methylmethacrylate embedding material. Never-theless, this dissolution and refilling process did notinterfere with the final light microscopical assessment.This evaluation revealed a very uniform bone reactionfor all implantation times and Ca-P materials. For ex-ample, no clear differences in healing response to thethree Ca-P cement compositions could be observed.Differences in morphological appearance were onlyfound between the various implantation times.

Implantation time: 2 weeks

All drilled holes were found to be completely filledwith either Ca-P or PMMA cement. Only occasionally,macropores were seen in the center of the materials.No penetration of any of the cements into intertra-

becular voids was seen. The Ca-P cements were neverassociated with an inflammatory response. The sur-face of the Ca-P cements was already almost com-pletely covered with a thin layer of woven bone [Fig.2(A)]. This newly formed bone was characterized bythe presence of a layer of osteoid lined with osteo-blasts [Fig. 2(B)]. A tight contact existed between thenew bone and cement surface. No intervening fibroustissue layer was present. At some places, mineraliza-tion of the newly deposited bone had started. Then,osteocytes at a distance of less than 10 mm to the ce-ment surface could be seen. Their canaliculi seemed toreach and contact with the cement [Fig. 2(C)]. The sur-face of the Ca-P cements had a granular appearance.The sections clearly showed that these cements con-sisted of small particles disseminated in an amor-phous mass. The outer layer of the cement looked lessdense than the central part [Fig. 2(C)]. Nevertheless,no loosening of particles was observed. The PMMAmaterial was completely surrounded by a fibrousmembrane (Fig. 3).

No direct contact between trabecular bone andPMMA cement was seen. Occasionally, macrophagesand giant cells were seen in between the cement sur-face and fibrous membrane.

Finally, around all implanted materials, the trabec-ular bone showed increased remodeling activity up to2.5 mm distance to the interface.


At 8 weeks of implantation, bone coverage of theCa-P cements had proceeded (Fig. 4). All Ca-P cementsurfaces, including pores and deep crevices, werecompletely covered with a very uniform bone layer.The remodeling process of this new bone had alreadystarted. The bone was in very close contact with thecement surface, without any sign of intervening fi-brous tissue layer. Also, no inflammatory cells wereseen. The surface zone of the Ca-P cements still had aless dense appearance compared with the central part.Occasionally, pores were seen in the cement. When thepores had an opening to the outer bone environment,the cement surface inside the pore became completelycovered with a thin layer of bone (Fig. 5).

Considering the PMMA cement, a fibrous tissuelayer still surrounded this material (Fig. 6). However,we noticed that the thickness of this layer had de-creased and the inflammatory response had almostcompletely receded. The decrease in thickness wasdue to the maturation of the fibrous layer. The amountof fibroblast cell layers in the fibrous layer remainedthe same as after 2 weeks of implantation time.

The increased remodeling activity in the surround-ing bone was now restricted to about 1 mm to theinterface.

TABLE ITime Intervals at Which Three of the Six Goats in the24-Week Implantation Period Received Subcutaneous

Fluorochrome Markers Indicating Remodeling Activityafter Cement Implantation

Weeks BeforeSacrifice Fluorochrome Color

Dose(mg/kg)

23 Tetracycline Yellow 2516 Calcein Green 258 Alizarin-complexone Red 255 Tetracycline Yellow 251 Calcein Green 25

12 OOMS ET AL.


After 16 weeks of implantation, the bone layer thatcovered the Ca-P cements had almost completely re-modeled [Fig. 7(A)]. Numerous remodeling lacunae

could be seen close to the cements. Frequently, thecutting cone of these remodeling lacunae was direc-tional and penetrated into the cement surface [Figs.7(B,C)]. In these situations, osteoclast-like cells werepresent at the cement outside, whereas bone apposi-tion occurred at the other side of the lacuna [Fig. 7(B)].The content of these osteoclast-like cells had a granu-lar-like appearance [Fig. 7(C)].

The PMMA cement was still surrounded by a fi-brous tissue layer without any sign of direct bone–cement contact. No further changes in this fibrouslayer were observed.

The increased remodeling activity in the surround-ing bone close to the implants had disappeared after16 weeks of implantation.


Remodeling of bone, formed at the Ca-P cements,had proceeded. The bone was mature and could

Figure 4. Remodeling of surrounding bone and Ca-P ce-ment surface at 8 weeks. A surface zone (SZ) in the Ca-Pcement has a less dense appearance. L/P ratio = 0.35. Bar =500 mm.

Figure 2. Photomicrographs showing the tissue responseof Ca-P cement after 2 weeks of implantation. (A) The ce-ment surface is covered with a thin layer of newly formedbone (arrowheads). A trabecular bone fragment (BF) is takenup in the bone remodeling process. L/P ratio = 0.35. Bar =500 mm. (B) Detail of bone formation at the interface. Ob,osteoblasts; Oc, osteocyte; *, osteoid layer. L/P ratio = 0.30.Bar = 125 mm. (C) Osteocytes (Oc) in newly formed bone atclose distance to the Ca-P cement surface. L/P ratio = 0.35.Bar = 250 mm.

Figure 3. PMMA control after 2 weeks of implantation.The PMMA has dissolved because of the embedding pro-cess. A fibrous membrane (arrowheads) is present betweenthe PMMA and the bone tissue. Bar = 500 mm.


not be discerned from original trabecular bone (Fig. 8);the major part of the Ca-P cement was still present inthe produced bone defects. Only occasionally, largerparts of the cement had disappeared and became sub-sequently completely filled with bone (Fig. 9). Remod-eling activity in the interface, as characterized by thepresence of remodeling lacunae and osteoclast-likecells, was still seen. The PMMA implants were stillsurrounded by a fibrous membrane that did not differfrom that observed after 16 weeks of implantation.Further, the bone surrounding the PMMA had thesame appearance as that of the Ca-P specimens.

Finally, the fluorochrome-labeled sections of the 24-week specimens confirmed that, indeed, active remod-eling around and in the Ca-P cements had occurred[Fig. 10(A)].

Of the five sequential fluorochrome labels that thegoats received throughout the implantation period,unfortunately only the last three were visible as col-

ored bands. This prevented determination of wherebone formation had started, i.e., at the Ca-P cement orbone surface. Nevertheless, in the remodeling lacunae,at the interface between bone and cement, bone appo-

Figure 6. The fibrous membrane (arrowheads) between thePMMA implant and bone after 8 weeks of implantation. Bar= 250 mm.

Figure 5. Because of air entrapment, a pore of approxi-mately 2.5 mm in diameter was formed in this 8-week Ca-Pimplant. Note that the inner side of the pore is fully coveredby a thin layer of bone (arrowheads). L/P ratio = 0.35. Bar =1500 mm.

Figure 7. Cement resorption and replacement by bone at16 weeks. (A) Bone ingrowth after resorption of the Ca-Pcement. A thin shell of cement (arrowheads) indicates theborder of the cement at the time of implantation. L/P ratio= 0.30. Bar = 500 mm. (B) Remodeling lacuna (RL) with thecutting cone headed toward the cement. Osteoclast-like cells(black arrowheads) resorb bone and Ca-P cement (white ar-rowheads). L/P ratio = 0.35. Bar = 250 mm. (C) Two osteo-clast-like cells resorbing bone (OCL1) and Ca-P cement(OCL2). OCL2 shows intracellular-located particles, suggest-ing degradation of the cement by phagocytosis as well as byextracellular resorption. L/P ratio = 0.30. Bar = 25 mm.

14 OOMS ET AL.

sition was mainly seen in the cement–bone direction[Fig. 10(B)].

This was different for the PMMA implants, wherebone apposition was shown to take place in the bone–cement direction.

Histomorphometrical evaluation

Percentage of bone contact

The results of the bone contact measurements forthe Ca-P cement at the three different L/P ratios anddifferent implantation periods are depicted in Figure11(A).

Bone-to-implant contact for the PMMA material isnot given. All PMMA samples were completely sur-rounded by a fibrous membrane without any bone–PMMA contact. Statistical analysis revealed that no

significant differences existed in percentage of bonecontact (p > 0.05) between the three Ca-P cements andtheir respective implantation times.

Number of interfacial remodeling lacunae

The results regarding the number of remodeling la-cunae in contact with the Ca-P cements are presentedin Figure 11(B). The 2-week specimens were excludedfrom analysis. Bone formation had just started and too

Figure 9. Low magnification photomicrograph of a trans-versal section of the Ca-P cement implant after 24 weeks ofimplantation. Parts of the cement mass have been resorbedfollowed by bone ingrowth. L/P ratio = 0.30. Bar = 1500 mm.

Figure 8. Continued resorption of the Ca-P cement andsubsequent bone ingrowth after 24 weeks of implantation intrabecular bone. L/P ratio = 0.40. Bar = 250 mm.

Figure 10. Fluorescence micrographs of the Ca-P cementafter 24 weeks of implantation. (A) Active bone remodelingaround and in the Ca-P cement implant. L/P ratio = 0.40. Bar= 250 mm. (B) The different fluorochrome labels indicatebone apposition in the Ca-P cement-to-bone direction. (G)Calcein green, (Y) tetracycline yellow, (R) alizarin-complexone red. L/P ratio = 0.30. Bar = 250 mm.


much bony debris of the surgical procedure was seento allow a reliable counting. The PMMA samples werenot evaluated, because the PMMA cement was com-pletely covered with a fibrous membrane.

Although a tendency appeared to exist for increasedremodeling activity at 24 weeks, no significant differ-ences (p > 0.05) between the three Ca-P cements andtheir respective implantation periods were found.

Cement area

The measurements suggested a decrease in Ca-P ce-ment area at 16 and 24 weeks of implantation [Fig.11(C)]. Nevertheless, this was not confirmed by statis-tical analysis (p > 0.05). Also, no significant difference(p > 0.05) existed between the three Ca-P cementswithin an implantation period.

DISCUSSION

The aim of the present study was to investigate thetrabecular bone response to Ca-P cement prepared ac-cording to three different L/P ratios. In these studies,PMMA cement was used as a reference material.

In our experiment, the Ca-P cement was appliedinto the created bone defects by using a syringe. In-jection of the cement paste offers the opportunity tofill the defect in a retrograde way, which can preventsignificant air entrapment. Besides, pressure can begenerated during the injection. This provides a goodcement fill of the bone bed and a tight initial contactwith the defect walls. Our histological results con-firmed these advantages of the injection technique. In-clusion of large macropores was only very occasion-ally observed and the cement was always in close con-tact with the surrounding bone.

A new mechanical mixing technique was selected toprepare the Ca-P cement paste directly in the syringe.The method was derived from the mixing of dentalrestorative cements. Shaking for 15 s in the machinewas already sufficient to achieve a homogenous inject-able paste. Consequently, the method used providesan easy way to standardize the mixing procedure forCa-P cements. Spill of cement, compositional varia-tions, and poorly controllable setting times, as mightoccur because of hand mixing, can now be avoided.

In previous studies with Ca-P cements, loss of co-hesion of the cement after contact with body fluids hasbeen observed.24,28,29 Although we stopped seriousbleeding out of our bone defects by packing withgauzes, the currently used Ca-P cements did set per-fectly in all cases. Apparently this is due to the re-duced (2–3 times as short) setting time of this cement

Figure 11. Results of histomorphometrical measurementsperformed on light microscopical sections of the cements atdifferent implantation periods (in weeks). L/P ratio at whicha Ca-P cement is prepared. (A) Bone contact measurement.(B) Number of remodeling lacunae in the interface. (C) Totalcement area.

16 OOMS ET AL.

compared with other materials.5,26,30 Nevertheless, wehave to emphasize that a very short setting time canalso result in application problems. For example, in-jectable Ca-P cement can be used in transcutaneousfracture repair.31–34 The combination of a short settingtime and complicated surgical treatment will hamperthe proper delivery of the Ca-P cement.

The histological and histomorphometrical examina-tions confirmed the excellent bone biocompatibility ofthe used Ca-P cement. The material did not evoke anyinflammatory response. The Ca-P cement appeared tofavor strongly new bone formation. Already in all2-week specimens, the cement surface was almostcompletely covered with newly deposited bone. Thisin contrast to the PMMA-cement, which was at alltime periods surrounded by a fibrous membrane with-out any evidence of PMMA–bone contact. These find-ings corroborate with several other studies dealingwith Ca-P cement.8,20,21,24,35 However, occasionally anunfavorable bone healing of Ca-P cement is described,as characterized by fibrous encapsulation and thepresence of inflammatory cells.36–39 The occurrence ofsuch a reaction is supposed to be due to the use ofacidic cements or the supplement of additives to im-prove the handling properties of the cement.

Light microscopy also proved the osteoconductiveproperties of the used Ca-P cements. Evidently, thebone was guided over the cement surface. Conse-quently, deep crevices and pores, even at some dis-tance of the original defect walls, became completelycovered with bone.

At the end of the study, most of the cement materialwas still present in the bone defects. Yet, we observedthe presence of remodeling lacunae in the interfacebetween cement surface and contacting bone. In thelacunae, osteoclast-like cells in contact with the ce-ment were observed. In various studies already, cell-mediated resorption is described as a mechanism forthe degradation of Ca-P cement.8,14,19–21,24,40 The rateat which the material finally degrades varies betweenweeks19 to years.8

Complete degradation of Ca-P cement is dependenton physicochemical material properties, like crystal-linity, density, porosity, as well as animal model andimplantation site.8,14,41,42 The maintenance of our ma-terial during the 24-week implantation period also ex-cludes the occurrence of passive resorption of the Ca-Pcement due to dissolution. This suggestion is con-firmed by another in vivo study, in which similar com-posed cement was implanted subcutaneously in ratsfor 8 weeks, while excluding cellular contact by pack-ing the cement in stainless gauzes.43 The resultsshowed that almost no dissolution had occurred.

As mentioned above, our cement appears to be re-sorbed by osteoclast-like cell activity. The kind of cellsinvolved in the degradation of Ca-P cements differsbetween the variously used cements. Generally, for

fast-degrading cements, macrophages and giant cellsare the cell types involved in the resorption process.This is in contrast to very slow degrading cements(months to years). Then, osteoclast-like cells are heldresponsible for the degradation.8,20 Evidently, this isalso true for the currently investigated Ca-P cement.At last, the cement will completely disappear and bereplaced by bone. However, this will take consider-able time.

Finally, we noted that the three different L/P ratiosdid not influence the bone response to the cement.This observation benefits the final clinical use of Ca-Pcement, because cement characteristics as setting timeand viscosity can be adjusted without any effect on thebiological behavior of the cement.

CONCLUSION

In summary, we conclude that the investigated Ca-Pcement is a bone biocompatible and osteoconductivematerial that is easy to handle and can be regarded asa promising material for use as injectable bone substi-tute.

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18 OOMS ET AL.

trabecular bone response to injectable calcium phosphate (ca-p) cement

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