Bone-Defect Healing with Calcium-Sulfate Particles and Cement:An Experimental Study in Rabbit

Giovanna Orsini,1 John Ricci,2 Antonio Scarano,1 Gabriele Pecora,1 Giovanna Petrone,1 Giovanna Iezzi,1

Adriano Piattelli1

1 Department of Oral Histology and Biomaterials, Dental School, University of Chieti, Italy

2 Department of Biomaterials and Biomimetics, New York University College of Dentistry, New York, New York

Received 4 December 2002; revised 10 June 2003; accepted 24 June 2003

Abstract: Calcium sulfate (CaS) has been shown to be a reasonable alternative to autogenousbone graft for treating bone lesions in dentistry. The aim of this work was an histological studyof the bone healing of defects treated with calcium sulfate in the form of cement or beads, inanimal. Eight New Zealand rabbits, weighing about 2.5 Kg were used in this study. In eachrabbit, four 6 mm bone defects were created in the tibial metaphysis. The 2 defects in the righttibia were filled with calcium sulfate as cement, while the 2 defects in the left one were filledwith calcium sulfate as beads. Four rabbits were killed after respectively 2 and 4 weeks, withan intravenous injection of Tanax, and the block sections, containing the bone defects, wereretrieved. A total of 16 defects filled by cement and a total of 16 defects filled by beads wereretrieved. The specimens were processed to obtain thin ground sections with the Precise 1Automated System. In the first phases of healing it was possible to observe an intenseosteoblastic activity, and in some areas osteoid matrix was present. After two weeks thecalcium sulfate (both cement and beads) was still present, and biological fluids and cells werepresent inside the material. Newly formed bone surrounded the calcium sulfate and filledabout 10% of the defect. After four weeks the calcium sulfate was almost completely resorbedand substituted by new bone. Approximately 34% of the defects were filled by newly formedbone. BEI and XRM evaluations showed the structural components of the filled defects. Innone of the specimens were inflammatory cells present. No significant differences were foundusing both calcium sulfate as cement and beads, and they both have shown a high biocom-patibility, appearing to promote newly bone formation in the rabbit model, and they did notinduce any untoward effect on the bone regeneration processes. © 2003 Wiley Periodicals, Inc. JBiomed Mater Res Part B: Appl Biomater 68B: 199–208, 2004

INTRODUCTION

Several graft materials have been proposed in implant den-tistry. Autogenous bone has been considered the gold stan-dard, but its main disadvantages are a limited amount of graftmaterial, the need of an additional surgical site, increaseddonor-site morbidity, and the need to use general anesthesiafor the extraoral bone harvesting.1,2 The allografts most com-monly used are demineralized freeze-dried, freeze-dried boneallografts, and bovine deproteinized bone. Patients concernsabout disease transmission have encouraged the developmentof alloplastic alternatives. Numerous osteoconductive mate-rials have been successfully employed. Of great benefit toclinicians would be a material that is completely resorbable,

safe, and inexpensive. It should be able to maintain space, toact as a reservoir for calcium ions, and to act as a barrier tocreate a protected space for the organizing blood clot and forthe migration of osteoprogenitor cells into the defect. Cal-cium sulfate (CaS) is a highly biocompatible material that isone of the simplest synthetic bone graft materials with thelongest clinical history, spanning more than 100 years.5–12

Calcium sulfate has been successfully used to treat periodon-tal disease, endodontic lesions, alveolar bone loss, and max-illary sinus augmentation.3,4,13–17 Calcium sulfate may act asa binder, facilitating healing and preventing loss of the graft-ing material; moreover, it is tissue compatible, and does notinterfere with the healing process.18 Calcium sulfate has alsobeen used with other materials, such as autogenous bone,2

DFDBA,18 polymers, or HA;19 in these cases there was afavorable effect on the potential for osteogenesis.2,19 Calciumpowder acts as a direct source of calcium supply,19 or themore rapid rate of resorption of CaS compared to othermaterials may allow an earlier ingress of osteoprogenitor

Correspondence to: Adriano Piattelli, Via F. Sciucchi 63, 66100 CHIETI, Italy(e-mail:apiattelli@unich.it)

Contract grant sponsors: Ministry of Education, University and Research(M.I.U.R.) and National Research Council (C.N.R.)

© 2003 Wiley Periodicals, Inc.

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cells.2 Calcium sulfate rapidly resorbs, leaving a calcium-phosphate lattice that promotes osteogenic activity;20 it mim-ics the mineral phase of bone and is resorbed at the rate ofbone formation.21 Ricci et al.22 demonstrated that CaS in-duced new bone formation after 2 weeks in dogs, and after 1month it was almost completely resorbed. The aim of thisstudy was the histological evaluation of the mechanisms ofcalcium-sulfate replacement by newly formed bone, in arabbit model. Two forms of CaS have been used: granulated(or bead) CaS and cement CaS.

MATERIALS AND METHODS

Eight New Zealand rabbits weighing about 2.5 kg were usedin this study, upon approval of the Ethical Committee forHuman and Animal studies at the School of Medicine, Uni-versity of Chieti, Italy. The animals were anesthetized with adose of ketamine (Ketalar, Parke-Davis S.p.A., Milan, Italy)and xylazine (Rompum, Bayer AG, Leverkusen, Germany).The ketamine dose was 44 mg/kg and the xylazine dose was6–8 mg/kg. A local injection of 1.8 ml of Lidocaine withoutvasoconstrictor was performed (Lidocaine, Astra, Sodertalje,Sweden). A full-thickness incision was performed to exposethe upper anterior portion of the tibia. A slow-speed dentaldrill equipped with a carbide round burr was used to create

two 8-mm-wide defects in each tibial metaphysis, undermanual saline irrigation. Defects in the right tibia were filledwith calcium sulfate (CaS) cement (Group 1). This cementwas prepared by mixing together CaS powder and Fast liquid,consisting of 4% potassium sulfate solution (Surgiplaster,Classimplant, Rome, Italy). Defects in the second group (lefttibiae) were packed with calcium-sulfate (CaS) beads (Group2; Surgiplaster, Classimplant, Rome, Italy). The surgicalwounds were sutured with stainless-steel monofilament wire3.0 (Ethicon, J & J, Somerville, NJ). After the surgicalprocedure a single dose of antibiotic was performed (0.25 gr,Cefazolin IM). No postoperative complications or deathsoccurred. Four animals were killed after 2 weeks, and fourafter 4 weeks. A total of 32 defects were retrieved. Thesetreated defects were compared with unfilled defects used as acontrol in previous studies.23–25 All the specimens were fixedwith 10% buffered formalin for 1 day and then washed insodium phosphate, pH 7.2. They were then dehydrated ingraded alcohols and embedded in LR White resin (LondonResin, Berkshire, UK). From the blocks, semithin sectionswith a thickness of about 40 �m were produced with the useof the Precise 1 Automated System (Assing, Rome, Italy),26

and then stained for light microscopy with toluidine blue andacid fuchsin. Histological evaluation at the different timeintervals and histomorphometry were performed. Histomor-phometry was carried out with the use of a light microscope

Figure 1. (a) Micrograph showing a 15-day CaS-cement (Group 1) –filled defect (4 �). (b) Highermagnification (40 �). There is newly formed bone in contact with the calcium-phosphate–rich layer. B:bone, CaS: calcium sulfate, CaP: calcium phosphate.

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(Laborlux S, Leitz, Wetzlar, Germany) connected to a high-resolution video camera (3CCD, JVC KY-F55B) and inter-faced to a monitor and PC (Intel Pentium III 1200 MMX).This optical system was associated with a digitizing pad(Matrix Vision GmbH) and a histometry software packagewith image-capturing capabilities (Image-Pro Plus 4.5, MediaCybernetics Inc., Immagini & Computer Snc Milano, Italy).All data were reported with means � standard deviations(SD). Single-factor analysis of variance (ANOVA) was usedto assess the statistical significance of results. Statisticallysignificant differences were set at p � .05. After histologicanalysis, selected blocks were polished, coated with a verythin layer of gold, and examined for mineral content. Thesystem used for backscattered electron imaging (BEI) andx-ray microprobe (XRM) analysis was a Hitachi S2500 scan-ning-electron microscope (Tokyo, Japan) equipped with aGW Electronics backscattered electron imaging (NorcrossU.S.) and a Princeton Gamma Tech IMIX digital imaging andx-ray microprobe system (Princeton, NJ).

RESULTS

Histological results indicated in vivo formation of a calcium-phosphate (CaP) -rich layer on the periphery of both CaSformulations. There appears to be a combination of solution-mediated dissolution/cell-mediated degradation of the cal-cium sulfate,20 with subsequent surface conversion or precip-itation. The end result was the formation of this consistentlatticework of the CaP-rich layer, which was stable in theshort term, and acted as an osteoconductive trellis for newbone formation.

After 15 days, there was an intimate bone appositionaround CaS, showing different patterns in the two groups. Inthe CaS-cement–filled defects (Group 1), the newly formedbone showed a ring pattern. From the CaS material, whichappeared dark gray in color, pale grayish colored deposits ofcalcium phosphate formed. These regions were in strict con-tact with acid fuchsin-positive bands that represented theinitial new bone formation (Figure 1). No inflammatory re-

Figure 2. Backscattered electron image (BEI) of the 14-days CaS-cement (Group 1) –filled defect.

201BONE-DEFECT HEALING WITH CALCIUM-SULFATE PARTICLES

action was present. The mean percentage � SD of newlyformed bone was 10.3 � 1.7%. Remnants of CaS representeda percentage of 15.2 � 2.1%.

The BEI and XRM analysis evaluated mineral structuresof the specimens, mapping for elements such as calcium,phosphorus, and sulfur. After 15 days, CaS cement specimens(Group 1) demonstrated that the opening in the cortex hasbeen sealed with a dense layer of calcium phosphate thatprobably corresponded to the original location of the calcium-sulfate cement (Figures 2 and 3).

On the other hand, in the CaS-beads specimens (Group 2)there was an initial new-bone formation all around and in thecore of the beads (Figure 4). In particular, at the periphery ofeach granule there was a diffuse band of calcium phosphatethat appeared grayish in color in close contact with a regionpositive for the acid fuchsin, representing the newly formed

bone. In the center of each bead, a grayish granular polycrys-talline material was evident; there were small patches of acidfuchsin positivity, showing that osteoid was also forming inthe center of the defect [see Figure 4(a)]. The percentage ofthe newly formed bone reached 10.5 � 1.8%. The CaSrepresented a percentage of 16.1 � 2.4%.

During the same 15-day period, BEI and XRM analysis, inthe specimens filled by granules (Group 2), showed that someof the most superficial granules had been converted to cal-cium phosphate and showed no remaining sulfate, whereasthe deeper material still consisted of calcium sulfate (Figures5 and 6).

After 30 days, in both forms, calcium sulfate was almostcompletely resorbed and large regions of new bone wereobserved. The newly formed osseous tissue showed lamellarstructures and appeared more mature than in the specimens

Figure 3. X-ray microprobe (XRM) image of the same field of the 14-day CaS-cement–filled defect,which shows the mapping of elements. Phosphorus appears in yellow, sulfur in blue. There issignificant calcium sulfate left. CaS: calcium sulfate.

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Figure 4. (a) Micrograph showing a 15-day CaS-granule (Group 2) –filled defect (4 �). (b) Highermagnification of a CaS granule (arrows), which is promoting new bone formation (40 �). B: bone.

Figure 5. Backscattered electron image (BEI) of the 15-day CaS-granules (Group 2) –filled defect.

observed at 2 weeks. No inflammatory cells were observed.The newly formed bone filled the 34.1 � 2% of the defect inGroup 1 and generally presented a concentric ring pattern(Figure 7). In Group 2, the newly formed bone filled 33.5 �1.7 %. Calcium-sulfate remnants represented a percentage of3.3 � 0.4% in Group 1, and 3.6 � 1% in Group 2.

After a 4-week period, BEI and XRM results showed thatin both formulations there was very little calcium-sulfate left,mainly in the deepest center of the defect. All the rest of themineral was demonstrated to be calcium phosphate, and therewas bone attaching to it in multiple spots (Figures 8 and 9).This analysis showed that there were some small differencesbetween the cements and granules that may be related to thetiming of dissolution of the calcium sulfate and the patterns ofcalcium-phosphate formation.

The above-mentioned results showed that at these timeintervals, defects of about 8 mm demonstrate more new boneformation than unfilled defects.23–25 At both the examined

time intervals (15 and 30 days), statistical evaluation ofhistomorphometric results of newly formed bone and CaSremnant percentages did not show significant differencesbetween the two groups (at p � .05).

Furthermore, both groups showed that the remainder of thedefects presented the amorphous Ca-P–rich layer, which wasuneven, more dense at 15 days than at 30 days. There werelarge marrow spaces colonized by biologic fluids and cells.At 30 days osteogenic cells were more distinguished than at15 days (Figures 10 and 11).

DISCUSSION

Calcium sulfate has been used in the treatment of dehiscencesand fenestrations, in immediate postextraction implants, insinus-augmentation procedures, in the filling of cysts or de-fects left after removal of impacted wisdom teeth, in end-

Figure 6. X-ray microprobe (XRM) image of the same field of the 15-days CaS-granule–filled defect,which shows the mapping of elements. Note the presence of calcium sulfate in blue. CaS: calciumsulfate.

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Figure 7. (a) Micrograph showing a 30-day CaS-cement (Group 1) –filled defect, (4 �). (b) Detailedview of the characteristic disposition in concentric rings of the newly formed bone (10 �).

Figure 8. Backscattered electron image (BEI) of the 30-day CaS-cement–filled defect.

205BONE-DEFECT HEALING WITH CALCIUM-SULFATE PARTICLES

Figure 9. X-ray microprobe (XRM) image of the same field of the 30-day CaS-cement–filled defect,which shows the mapping of elements. There are small remnants of calcium sulfate in the center ofthe defect. CaS: calcium sulfate.

Figure 10. After 15 days the marrow spaces of the defect (Group 2) are colonized by cells of thehematopoietic system (arrows). B: bone; CaP: calcium phosphate (100 �).

odontic procedures, in periodontal defects, and as a barriermembrane in regenerative procedures. Yoshikawa et al.,4 inan experimental study in dogs with the use of e-PTFE mem-branes and CaS after apicectomy, found that bone regenera-tion with the use of CaS was similar to that obtained usinge-PTFE and superior to controls. Pecora et al.,3 in a studyusing CaS in the surgical treatment of a through and throughperiradicular lesion, found that the addition of calcium sulfateto conventional surgical treatment of this type of endodonticlesions may contribute to an improvement of the clinicaloutcome. Calcium-sulfate facilitates complete closure ofwounds when primary closure was not obtainable, and thiscan probably be explained by the fact that calcium sulfate, invitro, facilitated fibroblast migration to a greater degree thandid either e-PTFE or polylactic acid.27

One of the major concerns regarding calcium sulfate isrelated to its fast resorption.1 Calcium sulfate seems to becompletely resorbed in 4–10 weeks, depending on the vas-cularity of the grafted site.18 Yoshikawa et al.4 found that CaShad completely disappeared at 16 weeks. Kelly et al.28 foundthat, at 6 months postoperatively, radiographic results for allpatients showed that 99% of the calcium sulfate had beenresorbed. Pecora et al.17 found that complete resorption ofCaS had occurred 9 months after the augmentation procedure.

It has been recently reported25 that, at early time intervals,untreated bone defects do not show detectable new bone forma-tion if compared with calcium-sulfate treated ones, in rabbit. Thepresent study corroborates previous findings showing that bonehealing is highly improved by calcium sulfate and that bothcement and beads are effective.25 The differences between thetwo formulations are mainly seen in the distribution of the newlyformed bone but not in its quantity, and bone formation alwayshappens through an intermediate, which is the calcium-phos-phate–rich layer.20,22 The BEI system produces an image basedon atomic number and density, so it shows bone structure andmineral structure very well, and does not show soft tissues at all.The XRM is used to scan the same field as the BEI image andproduce corresponding maps for the presence of elements. TheCaS granule formulation shows the same activity as the cement,but the granules individually convert to calcium phosphate atdifferent times, possibly depending on their location and expo-sure. It can be speculated that one may use cement or beadformulations depending on the clinical situation. Guarnieri andBovi formulated the hypothesis that a careful stratification anddry compaction of calcium sulfate may be effective in reducingthe resorption rate and the extent of mass contraction duringhealing.1 Thus, the compact CaS granules seem to resorbslightly slower than cement and they could be a space maintainer

Figure 11. High-power micrograph at 30 days: An osteoblastic rim (arrows) is close to the bonesurface. B: bone (100 �).

207BONE-DEFECT HEALING WITH CALCIUM-SULFATE PARTICLES

for a longer period, allowing a more complete osteogenesis.Further analyses at different time intervals are needed to evalu-ate the possible mechanism at later stages.

Moreover, it can be suggested that granule employmentcan have advantageous results in bone defect sites, whichshow significant bleeding. Setting of CaS-cement can beconsiderably retarded when organic agents, such as bloodproteins, are present.22 Using a slightly dry cement or bettersolid preset beads works well in these spaces because somemoisture is usually absorbed from the environment.

Finally, because CaS is easily absorbed, it makes a gooddelivery vehicle for osteoinductive materials such as thegrowth factors.22,29 In particular, CaS granules might repre-sent a good carrier for these bioactive molecules because, likebiological apatites, they can be easily impregnated with os-teoconductive proteins, then released in the environment.This prospective needs to be supported by future studies thatwill evaluate the efficacy of this delivery system.

In conclusion, calcium sulfate has been shown to be aresorbable material that promotes bone formation in bonedefect. The present results showed that both CaS cement andCaS beads acted as bioactive materials when placed in a bonyenvironment. This study can be a relevant contribution for thefuture clinical use of cement and granule formulation. Bothforms have shown to be safe, well-tolerated, biodegradable,osteoconductive bone graft substitutes that may represent areasonable alternative to autogenous bone graft for fillingbone defects. The compact CaS granules formulation mightshow some clinical advantages, but further research is neededto corroborate this hypothesis.

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