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Biomaterials 35 (2014) 3154e3163

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Biomaterials

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Performance of b-tricalcium phosphate granules and putty, bonegrafting materials after bilateral sinus floor augmentation in humans

Michael Stiller a, Esther Kluk b, Marc Bohner c, Marco A. Lopez-Heredia d,Christian Müller-Mai e, Christine Knabe d,*

aDept. of Oral Surgery, Charité University Medical Center Berlin, 14197 Berlin, GermanybDept. of Experimental Dentistry, Charité University Medical Center Berlin, 14197 Berlin, GermanycRMS Foundation, 2544 Bettlach, SwitzerlanddDept. of Experimental Orofacial Medicine, Philipps University, 35039 Marburg, GermanyeDepartment of Traumatology and Orthopaedics, Hospital for Special Surgery, 44534 Lünen, Germany

a r t i c l e i n f o

Article history:Received 2 December 2013Accepted 20 December 2013Available online 16 January 2014

Keywords:Sinus floor augmentationSplit-mouth designb-TCP bone graftGranulesPutty

* Corresponding author. Tel.: þ49 (0)6421 5863600E-mail address: knabec@med.uni-marburg.de (C. K

0142-9612/$ e see front matter � 2014 Elsevier Ltd.http://dx.doi.org/10.1016/j.biomaterials.2013.12.068

a b s t r a c t

Sinus floor augmentation (SFA) using bone grafting materials, and in particular calcium phosphates (CaP),is a well-established pre-implantology procedure. The use of CaP simplifies SFA procedures. b-tricalciumphosphate (b-TCP) is amply used for SFA. This study evaluated the clinical and osteogenic performance ofb-TCP granules (TCP-G) and a b-TCP putty (TCP-P) bone graft material. TCP-P consisted of TCP-G in ahyaluronic acid (HyA) carrier. Bone formation, volume stability and osteogenic marker expression afterbilateral SFA in patients was assessed. Eight patients were selected for a split-mouth design. Biopsiesobtained six months after SFA, were processed for immunohistochemical analysis of collagen type I(Col I), alkaline phosphatase (ALP), osteocalcin (OC) and bone sialoprotein (BSP). Histomorphometricanalysis determined bone, grafting material and marrow space percentages. Cone-beam computed to-mography was used to calculate the graft volume and its stability. Both materials allowed excellent boneregeneration and volume stability. TCP-P displayed better surgical handling properties, greater boneformation, higher expression of Col I, ALP, OC and BSP; as well as significantly lower grafting volumereduction values. HyA had no adverse effect on TCP-P performance. Due to its clinical and osteogenicperformance, TCP-P can be regarded as excellent bone grafting material for SFA.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

The use of synthetic bone graft materials has received increasingattention in implant dentistry over the last decade. Furthermore,augmentation of the maxillary sinus floor with autogenous bonegrafts has become a well-established pre-implantology procedurefor alveolar ridge augmentation of the posterior maxilla. The clin-ical success rates achieved by the use of calcium phosphate (CaP)bone graft materials for sinus floor augmentation (SFA) proceduresdemonstrate, that these resorbable materials are an excellentalternative to the use of autogenous bone grafts, which has beenconsidered as the gold standard [1e3]. In addition, using syntheticresorbable bone graft materials simplifies SFA procedures since itavoids second-site surgery for autograft harvesting and thereby,eliminates the risk of donor-site morbidity [4e6]. Among the CaPbone grafting materials available, b-tricalcium phosphate (b-TCP)

; fax: þ49 (0)6421 5863606.nabe).

All rights reserved.

has achieved wide-spread use for SFA procedures [7e12]. Recently,the combination of b-TCP, and other CaPs, with polymeric scaffoldsor carriers, has kindled scientific research [13,14]. Hyaluronic acid(HyA) is a natural polymer that has shown to be clinically advan-tageous due to its excellent biological properties [15,16]. HyA is ahigh molecular weight polysaccharide which is a major componentof the extracellular matrix of the skin, tendons, muscles, articularcartilage and the synovial fluid of vertebrates. HyA is able tointeract with binding proteins, such as proteoglycans and otherbioactive molecules, is immunologically inert and has a stimulatoryeffect on angiogenesis [17]. Due to these properties the use of HyAin domains, such as regenerative medicine, traumatology andrheumatology, has increased [18e22]. The physicochemical andbiological properties of HyA make it an attractive material to becombined with b-TCP granules. Combining b-TCP granules withHyA creates a putty bone grafting material and may optimize thebone regenerative potential of the b-TCP. Putty materials displayimproved surgical handling properties for bone reconstruction ofdefects with demanding defect morphologies such as in the SFAprocedures. SFA procedures are used in cases of a severely reduced

Table 1Data of patients and design of the study.

Patient Age (years) Genderb Material and side ofplacementc

TCP-G TCP-P

1a 57 F Left Right2 63 F Left Right3 70 F Right Left4 72 F Left Right5 66 M Right Left6a 67 M Right Left7 63 F Left Right8 70 F Left Right

a Smoker.b “F” stands for Female and “M” for Male.c “TCP-G” stands for b-TCP granules and “TCP-P” stands for b-TCP putty.

M. Stiller et al. / Biomaterials 35 (2014) 3154e3163 3155

residual bone height, i.e. less than 3 mm of the residual partiallyedentulous alveolar ridge, where a significant amount of boneneeds to be replaced in order to facilitate a stable and reliableanchorage and osseointegration of dental implants in the graftedsinus floor. Some bony defects in the alveolar ridge, such as lateralbone defects, are three- or five-wall defects which can be easilyaccessed surgically. However, for SFA procedures there is only onebony wall present, i.e. the sinus floor. In this procedure, the bonegrafting material needs to be introduced via a surgically-createdlateral access window to the sinus and has to be carefully placedunderneath the elevated Schneiderian membrane, which has adelicate and vulnerable structure [23,24]. Special care needs to betaken to ensure that the Schneiderianmembrane remains intact, i.e.without any perforations, during the grafting process. If theSchneiderian membrane is damaged during the surgical procedure,the success of the SFA procedure is severely challenged [25,26].Hence, SFA is a more difficult procedure where a greater challengeis encountered.

From a clinical point of view, it is important to investigate theuse of b-TCP granules and putty bone grafting materials for SFA andto compare their performance in patients. In the present work, aclinical study with a randomized split-mouth study design wasused to evaluate the effect of these two bone graft materials, i.e. b-TCP granules and a b-TCP putty, on bone formation, bone matrixmaturation and osteoblast differentiation six months after SFA. Tothis end, the amount of newly formed bone as well as the biode-gradability of the b-TCP grafting materials was determined histo-morphometrically in combination with immunohistochemicalanalysis of osteogenic marker expression after SFA. In addition, thevolume stability of the augmented bone was assessed by usingcone-beam computed tomographies (CTs). Our hypothesis is that ab-TCP putty, i.e. b-TCP granules in a HyA carrier, may yield reducedsurgical complications; enhanced surgical handling properties,bone formation, volume stability and osteogenic expression,compared to b-TCP granules alone, when used for SFA procedures.

2. Materials and methods

2.1. Bone grafting materials

Two commercially available b-TCP-based bone grafting materials were studied.One consisted of pure, synthetic b-TCP granules (TCP-G; CEROS� TCP Granules,Mathys Ltd, Switzerland) with a grain size of 700e1400 mm. The other was a puttymaterial (TCP-P; CEROS� TCP Putty, Mathys Ltd, Switzerland) composed of pure,synthetic b-TCP granules with two types of grain size ranges, i.e. 125e250 mm and500e700 mm, embedded in a sodiumHyA hydrogel matrix with a b-TCP:HyA ratio of10:1. The porosity of the b-TCP granules used for the TCP-G and TCP-P materials wasthe same, with a value of 60 � 3%. The bulk density was 0.65 � 0.02 g/cm3 and0.93 � 0.02 g/cm3 for the b-TCP granules used for the TCP-G and TCP-P, respectively.

2.2. Patient selection

The study was performed in accordance with the ethical protocols stipulatedand approved by the Charité University Medical Center Berlin. Patients were askedto give their consent prior to the study. Patients were not considered for the study iftheir health was compromised. Patients were not selected if their physical status,according to the American Society of Anaesthesiology, (ASA PS) felt into the category3 or 4, or if they were suffering from any drug abuse, including alcohol, or anysignificant systemic disease. All patients were fully informed about the procedures;this included the surgery, bone grafting materials and implants. A total of 8 patients,i.e. 6 women and 2 men, with an age range between 57 and 72 years and a mean ageof 66 years, participated in the present study. Patient data as well as the (split-mouth) design used for the study are listed in Table 1. Patients were bilaterallypartially edentulous in the post-canine region. Hence, a bilateral SFAwas required inall patients in order to facilitate dental implant placement in the posterior maxilla asthe height of the residual alveolar crest was less than 3 mm. After routine oral andphysical examinations, SFA procedures were planned for the selected patients. Allpatients had good oral health without active periodontitis. Radiological changes ofthe maxillary sinuses were excluded. Six selected patients were non-smokers; twopatients had a history of smoking 5 cigarettes daily and were instructed to stopsmoking at least 2 weeks prior to the first surgery and for 40 weeks after the secondsurgery. These two patients were informed of the increased failure risk due to

smoking. In order to facilitate obtaining biopsies in a safe and easy manner, allselected patients had the alveolar crest wider than 6 mm.

2.3. Radiological examination

2.3.1. Cone-beam CTCone-beam computed tomography (CBCT; KaVo-3D-eXam�, KaVo Dental

GmbH, Germany) was used for three-dimensional (3D) assessment of the sinus flooranatomy and bone volume preoperatively, postoperatively and six months after SFAat the selected regions of interest (ROIs). ROIs were acquired with a voxel size of0.25 mm and an exposure time of 26 s. The preoperative CBCT acquisition wasdenominated primary CBCT. This CBCT was obtained in order to evaluate the re-sidual bone volume of the alveolar ridge, to exclude pathological changes of themaxillary sinuses and to plan the treatment needed. The postoperative CBCT, i.e.secondary CBCT, documented the outcome of the SFA procedure and verifiedpossible entrapment of air bubbles within the grafting material, dislocation of thegrafting material and hemorrhages and mucus retention in the maxillary sinus. Sixmonths after the SFA procedure, and directly after biopsy sampling and implantplacement, a third CBCT was obtained to determine the volume of the grafted boneand to document the outcome of the implant placement surgery. This CBCT wasdenominated the tertiary CBCT.

2.3.2. Analysis and determination of the bone volumeThe Digital Imaging and Communications in Medicine (DICOM) data sets of the

CBCT acquired ROIs of the samples were processed with a 3D image processingsoftware (VoXim�, IVS Technology GmbH, Germany). The DICOM data sets graydistribution values and limits were set equivalent to the Hounsfield scale(level¼ 665, width¼ 3379 and Hounsfield units¼�1024 to 2354). Between 240 and448 slices for each sample were analyzed. The boundaries of the ROI for each samplewere determined manually. The volume was calculated by adding up the volume ofeach slice within the ROI’s boundaries. The volume of the grafted area generated bythe SFA procedure was compared to the volume after six months of graft healing atimplant placement within the same ROI boundaries. The implants were included inthe ROIs of the tertiary CBCT to determine the bone volume present just prior toimplant insertion. A segmentation threshold value for the bone structures wasperformed in order tomark and visualize the bone boundaries of the maxillary sinusprior to surgery. Then, the data sets of the primary and secondary CBCTs weremerged. This allowed visualizing the bony margins of the maxillary sinus prior tosurgery for the grafted area in the secondary CBCT (Fig. 1a). The same steps werefollowed for calculating the volume of the augmented area six months after SFA atimplant placement. The anatomical structures of interest, such as the grafted areaand surrounding bony structures, were color differentiated. To visualize the differ-ences in volume of the grafted area between the SFA procedure and the implantplacement six months later, the images of the secondary and tertiary CBCTs weresuperimposed for each patient (Fig. 1b).

2.4. Sinus floor augmentation

Bilateral SFA procedures were performed, under local anesthesia, by the samesurgeon for all patients. As mentioned previously, the height of the residual alveolarprocess was less than 3 mm in the posterior maxillary region; hence, a staged sur-gical approach was used. The space created between the maxillary alveolar processand the elevated Schneiderian membrane was filled using a combination of TCP-G,or TCP-P, and autogenous bone chips in a ratio of 10:1. Small amounts of autogenousbone chips were harvested from the tuber maxillae. Then, bone chips were milledinto very small particles and added to the bone graft materials. TCP-G and TCP-Pwere implanted in either the left or the right side of the maxillary sinus floor ac-cording to a randomized protocol (Table 1). When augmenting one sinus with TCP-G, TCP-P was used in the contralateral sinus floor and vice versa. Grafting materials

Fig. 1. Two-dimensional radiographic sagittal view images generated by cone-beam computed tomography (CBCT). (a) After sinus floor augmentation (SFA) with b-TCP putty and(b) at implant placement six months after SFA. The yellow region indicates the augmented sinus floor, the blue regions indicate the osseous anatomical structures of the native sinusfloor and the green region indicates the grafted area after six months of healing. Image (a) was generated by merging the data sets of the primary and secondary CBCTs. Image (b)was generated by merging the data sets of the primary and tertiary CBCTs in combination with superimposing the image of the secondary CBCT. (For interpretation of the referencesto color in this figure legend, the reader is referred to the web version of this article.)

M. Stiller et al. / Biomaterials 35 (2014) 3154e31633156

were mixed with venous blood prior to delivery into the open sinus cavity. Patientsreceived 1200 mg of clindamycin (Clindamycin ratiopharm 600 mg, RatiopharmGmbH & Co., Germany) daily for 7 days to prevent infections and an intravenousinjection of 250 mg prednisolone (Solu-Decortin H 250, Merck KGaA, Germany) incombination with daily oral administration of 800e1200 mg of ibuprofen (IBUratiopharm 400 akut, Ratiopharm GmbH & Co., Germany) to reduce pain andswelling, respectively. Additionally, a local submucosal infiltration of 1 ml dexa-methasone (Dexabene 4 mg/ml, Merckle-Recordati KGaA, Germany) was adminis-tered bilaterally in the vestibular region to reduce postoperative pressure formationon the graft resulting from swelling of the Schneiderian membrane.

2.5. Dental implant surgery and bone biopsy retrieval

Six months after the SFA procedure, the patients received the implants. Dentalimplant placement and bone biopsy sampling were performed under local anes-thesia. For each patient, one biopsy was harvested from each site in which a dentalimplant was to be placed. Biopsies were retrieved by using a trephine burr (Strau-mann, Switzerland) with an outer and inner diameter of 3.5 and 2.5 mm, respec-tively, under copious saline irrigation. The retrieved biopsies were 2.5 mm indiameter and, depending on the mechanical stability of the regenerated tissue, up to8 mm in length. Biopsies were used for histomorphometric and immunohisto-chemical evaluation. The biopsies contained the grafted area and the residual nativecrest, which was approximately 1e3 mm in height and was not included in thehistolomorphometric analysis.

2.6. Preparation of biopsy specimens

The biopsies were prepared to perform immunohistochemical analysis onundecalcified hard tissue sections as previously described [27]. Briefly, the tissue

samples were immersed in a fixative solution (HistoCHOICE�; AMRESCO, U.S.A.) atroom temperature. Fixed samples were dehydrated in acetone solutionwith 0.50% v/v of polyethylene glycol 400 (PEG-400; Merck, Germany) and followed by infiltra-tion in a methylmethacrylate (MMA; Merck, Germany), n-butyl-methacrylate (BMA;Merck, Germany) and PEG-400 (Merck, Germany) solution. Specimens wereembedded in a resin composed of pure MMA and BMA supplemented with benzoylperoxide (BPO catalyst; Merck, Germany), PEG-400 (Merck, Germany) and 1.5 mlN,N-dimethyl-p-toluidine (Merck, Germany). Samples were polymerized in poly-ethylene vials at 4 �C. After polymerization the blocks were removed from the vialsand excess resin was trimmed away. Blocks were glued to acrylic slides using a two-component epoxy resin (UHU, Germany). Sections around 50 mm in thickness wereobtained by cutting the blockwith a sawingmicrotome (Leitz 1600; Leitz, Germany).After sawing, sections were ground and polished. Sections were deacrylated byimmersion in toluene, xylene and acetone to perform further analysis.

2.7. Histomorphometry and immunohistochemistry

Histomorphometric analyses were performed in a rectangular area of approxi-mately 6 mm2 in size (H-ROI) and defined in each section at 3 mm from the nativealveolar crest and extending in the apical direction. The areas (mm2) of newlyformed bone, the graft material and the marrow spaces were measured for each H-ROI and their percentage was calculated in terms of the total measurable H-ROI area.Histomorphometric analyses were performed on sections by using a light micro-scope (Vanox-T AH2; Olympus, Germany) in combination with a digital camera(Colorview IIIu; Olympus, Deutschland) and an analysis software (Analysis�;Olympus, Germany).

Immunohistochemical staining was performed as described by Knabe et al. [2].Primary mouse monoclonal antibodies were used for alkaline phosphatase (ALP;

Table 2Results of the cone-beam computed tomography (CBCT) assessment of the volumeat the grafted region after the sinus floor augmentation procedure (A) and 6 monthsafter sinus floor augmentation (B).

Patienta A, Right sideb

(ml)B, Right sidec

(ml)A, Left sideb

(ml)B, Left sidec

(ml)

1 2.14 1.97 3.41 3.122 1.22 0.82 2.27 1.983 1.77 1.73 2.09 1.944 2.78 1.33 3.43 2.475 5.90 4.18 5.65 3.596 3.68 3.17 4.77 3.437 3.66 2.42 3.25 3.02

a One female patient dropped out of the study after successful sinus flooraugmentation. Reasons of the drop out were unrelated to the study; patient 1 andpatient 6 were smokers.

b Secondary CBCT.c Tertiary CBCT.

M. Stiller et al. / Biomaterials 35 (2014) 3154e3163 3157

SigmaeAldrich, Germany) and osteocalcin (OC; Abcam, UK) while with rabbitpolyclonal antibodies were used for type I collagen (Col I; LF-39, NIH, USA) and bonesialoprotein (BSP; LF-84, NIH, USA). Some sections were stained with a prefabricatedkit (SigmaeAldrich, Germany) for Tartrate Resistant Acid Phosphatase (TRAP) ac-tivity, to identify cells with osteoclastic activity [28]. Mayer’s hematoxylin was usedas a counterstain. Sections obtained from experimental fracture healing sites in maleWistar rats were used as positive controls, since osteoclasts are known to be presentthere. Non-immunized mouse (PP54; Millipore, USA) and rabbit (PP64; Millipore,USA) IgG were applied as negative controls. This ruled out the non-specific reactionsof mouse and rabbit IgG to human tissues, as well as non-specific binding of thesecondary antibodies and/or peroxidase labeled polymer to human tissues. Incu-bation with a peroxidase labeled dextran polymer conjugated to goat anti-mouseand anti-rabbit immunoglobulins (DAKO DakoCytomation Envision þ Dual linksystem peroxidase; DAKO, Denmark) were performed and a liquid 3-amino-9-ethylcarbazole system (DAKO, Denmark) for color development. Mayer’s hematox-ylin was also used here as a counterstain. Semi-quantitative analyses of theimmunohistochemically stained sections were performed as described previously[2,29,30]. In brief, stained sections were analyzed with a light microscope (Vanox-TAH2; Olympus, Germany) by two investigators blinded to the staining. For theimmunohistochemical assessment, the H-ROI was divided in a central and an apicalarea of equal size, i.e. 3mm2 each. Tissues were examined for antibody decoration ofcellular and matrix components. The cellular components examined included fi-broblasts, osteoblasts, osteocytes and osteoclasts. The matrix components includedtrabecular bone, osteoid seams, bone marrow spaces and fibrous matrices. All thesehistological components were identified on morphological grounds. A scoring sys-tem to quantify the amount of staining observed by light microscopy was used. Ascore of “þþþ”(¼5), “þþ”(¼4), and “þ”(¼2) corresponded to strong, moderate andmild staining, respectively, in the general overview. Whereas for the localized areas,a score of “þþþ”(¼4), “þþ”(¼3), and “þ”(¼1) corresponded to strong, moderateand mild staining, respectively. A score of (0) corresponded to “no staining”. Thescores for the amount of staining of a given cellular or matrix component for a

Fig. 2. (a) Reduction of the grafting volume observed six months after the sinus floor augpercentage of volume reduction of the grafting volume six months after SFA for the graftingthe drop out were unrelated to the study. TCP-G ¼ b-TCP granules. TCP-P ¼ b-TCP putty.

respectivemarkerwere averaged for the apical and central areas. For a given graftinggroup, average expression scores from 5 to 3.5 were considered as strong, from 3.4 to2.3 as moderate, from 2.2 to 1 as mild and from 0.9 to 0.1 as minimal. For each givenmarker, the average of the scores of all examined cell and matrix components in thecentral and apical areas of a given site were used to determine an overall “immunescore”.

2.8. Statistical analysis

Collected data were analyzed by a paired student t-test to determine the sta-tistical significance (SPSS Statistics 21; IBM, USA). Values of p < 0.05 were consid-ered to be significant.

3. Results

3.1. Clinical performance

After SFA, no postoperative complications occurred in any of thepatients. One female patient dropped out of the study after suc-cessful SFA. The reasons of the drop outwere unrelated to the study.Normal wound healing was observed after SFA and after implantplacement surgeries. Six months after augmentation, all patientshad sufficient bone levels for placement of the implants withadequate primary stability. A total of 40 dental implants wereinserted into the augmented maxillary sinus floors of 7 patients. Nodifferences were noted intraoperatively, in terms of drilling resis-tance, when preparing implant beds in sites filled with TCP-G orTCP-P. CBCTs did not reveal any pathological changes in theaugmented sinuses or the surrounding tissues. No incidence ofsinusitis or perforations of the Schneiderian membrane wereobserved for any of the patients. No implant failures were noted upto 2e3 years after implant placement. As mentioned before, bi-opsies varied in length between patients depending on the me-chanical stability of the regenerated tissue.

3.2. Radiological results

The results of the assessment of the volume by using CBCT toanalyze the grafted region after the SFA procedure and six monthsafter this procedure are depicted in Table 2. Volumes for TCP-G andTCP-P after the SFA, and six months after the SFA, were notsignificantly different. A decrease in volume of the grafted area sixmonths after the SFA procedure was present for both types ofgrafting materials and for all patients. The reduction in volumewasvisible in the periphery of the grafted areawith a shrinkage towardsthe center. Sites filled with TCP-G showed a greater reduction in

mentation (SFA) for each of the seven patients and (b) mean values (�SEM) for thematerials. One female patient dropped out of the study after successful SFA. Reasons of

Fig. 3. Histomicrographs of resin embedded biopsies stained immunohistochemically for osteocalcin after deacrylation. The biopsies were sampled six months after sinus flooraugmentation with (a) b-TCP granules (TCP-G) and (b) b-TCP putty (TCP-P); (a) the residual TCP-G particles displayed a scalloped morphology, while with the residual TCP-Pparticles of the putty material the smaller grain size compared to the TCP-G material was evident (B ¼ bone, FM ¼ fibrous matrix). Undecalcified sawed sections counterstained withhematoxylin. Bar ¼ 2000 mm.

Fig. 4. Immunodetection of bone sialoprotein in deacrylated sawed section of biopsysampled six months after sinus floor augmentation with b-TCP putty (TCP-P). Intensestaining of osteoblasts (white arrows) which have migrated into the degrading TCP-particles is visible in combination with moderate staining of the osteoid (OS) liningthe newly formed trabecular bone (B) in contact with the TCP-P particles, whichexhibit excellent bone-particle contact, i.e. bone-bonding behavior. Furthermore, boneformation within the degrading particles is visible (arrowheads). Undecalcified sawedsection counterstained with hematoxylin. Bar ¼ 200 mm.

M. Stiller et al. / Biomaterials 35 (2014) 3154e31633158

volume of the grafted area compared to sites filled with TCP-P.Fig. 2a shows mean values of the percentage of volume reductionof the grafted area for each patient. Only for patient 1, the reductionof volume between using TCP-G or TCP-P was comparable. Fig. 2bshows the mean values (�SEM) for the volume reduction whenusing TCP-G and TCP-P. Mean values for TCP-G and TCP-P were(mean � SEM) 28.4 � 6.1% and 14.5 � 3.9%; these differences werestatistically significant. Secondary CBCTs taken directly after SFArevealed air entrapment in 4 out of 7 sites where TCP-G was used,but only in 1 out of 7 sites were TCP-P was used. However, sixmonths after SFA and at implant placement, no air entrapment wasobserved in any patient in the tertiary CBCTs. The Schneiderianmembrane did not show any change in 9 out of 14 grafted sinusfloors six months after augmentation. A slight swelling of theSchneiderian membrane was present in the remaining 5 sites, outof 14. Two of these remaining sites were filled with TCP-G and threewith TCP-P.

3.3. Histology, histomorphometry and immunohistochemicalanalysis

Six months after the SFA, the b-TCP granules of the graft ma-terials, i.e. TCP-G or TCP-P, presented an achromatic scalloped shapedepending on the phase of resorption (Fig. 3a and b). The size ofthese residual particles seemed larger for TCP-G (Fig. 3a) comparedto TCP-P (Fig. 3b); this reflected the granule grain size used for theTCP-G and TCP-P. Newly formed bone was visible at the surface ofthese residual particles (Fig. 4). Complete resorption of the graftingmaterials, i.e. TCP-G or TCP-P, did not occur within six months afterSFA. Residual granules were partially embedded in newly formedbone, which was predominantly lamellar bone. No signs of in-flammatory tissue response were observed in any SFA site.

A mesenchyme rich in osteogenic cells, i.e. osteoblasts, wasperceived preceding bone formation. This mesenchyme’s presenceand the positive expression of Col I, BSP and OC, in cell and matrixcomponents, indicated a progressing matrix mineralization (Fig. 3).Good bone bonding, as well as bone formation within thedegrading particles, was observed for both groups of materials.However, the degradation seemed more advanced for TCP-P. Bone

Table 3Histomorphometric results of the assessment of biopsies six months after sinus flooraugmentation. Results indicate the type of graft material (GM), i.e. b-TCP granules(TCP-G) or b-TCP putty (TCP-P), and the percentage of bone (Bone), b-TCP particles(Particle) left for both materials and the marrow spaces (MS) in the ROIs.

Patienta Right side maxillary sinus Left side maxillary sinus

GM Bone(%)

Particle(%)

MS(%)

GM Bone(%)

Particle(%)

MS(%)

1 TCP-P 25.89 26.40 47.72 TCP-G 18.34 26.33 55.332 TCP-P 37.43 39.48 23.09 TCP-G 35.02 27.81 37.173 TCP-G 20.76 29.22 50.03 TCP-P 42.42 18.38 39.204 TCP-P 33.22 26.40 40.38 TCP-G 10.02 45.42 44.565 TCP-G 11.14 32.17 56.69 TCP-P 29.19 23.33 47.476 TCP-G 12.77 33.41 53.82 TCP-P 20.70 39.30 39.997 TCP-P 21.66 32.92 45.42 TCP-G 14.02 35.93 50.05

a One female patient dropped out of the study after successful sinus flooraugmentation. Reasons of the drop out were unrelated to the study; patient 1 andpatient 6 were smokers.

M. Stiller et al. / Biomaterials 35 (2014) 3154e3163 3159

formation was more abundant in the crestal and central areas thanin the apical area. Osteoblasts present in the newly formed boneand in contact with both b-TCP particles expressed Col I, ALP, BSPand OC (Fig. 4).

Table 3 and Fig. 5 present the results of the histomorphometricanalysis six months after SFA procedures. Using TCP-G for the SFAresulted in bone, particle and marrow spaces values (mean � SEM)of 17.4 � 3.3%, 32.9 � 2.4% and 49.7 � 2.6%, respectively. Sites for

Fig. 5. Histomorphometric results obtained from the biopsies, i.e. six months after the sinusP). (a) Histogram showing the (mean values) results for the bone, material particle and maoccupied by (b) newly formed bone, (c) particles of material still present from the TCP-P a

which TCP-P was used gave values (mean � SEM) of 30.1 � 3.1%,29.5 � 3.0% and 40.5 � 3.2% for bone, particle and marrow spaces,respectively. The bone amount and the marrow space values weresignificantly higher and lower, respectively, for TCP-P six monthsafter SFA. The values for the residual b-TCP particles six monthsafter SFA procedures, were not statistically different between thetwo materials, i.e. TCP-G and TCP-P.

Results of the immunohistochemical assessment of the osteo-genic marker expression of the biopsies for the different graftmaterials in the central and the apical region are presented inTables 4 and 5, respectively. Osteogenic marker expression for Col I,ALP, OC and BSP were scored for the cellular components, i.e. os-teoblasts, osteocytes and fibroblasts, and for the matrix compo-nents, i.e. fibrous matrix, bone matrix and osteoids.

A more enhanced staining for TCP-P, compared to TCP-G, wasobserved for ALP, OC and BSP in the central region (Table 4) and forCol I, ALP, OC and BSP in the apical region (Table 5). In general,higher overall immune score values were observed for the centralregion than for the apical (Fig. 6). These overall immune scorevalues were also higher for the TCP-P group compared to the TCP-G.Immune score values (mean � SEM) for TCP-G in the central regionwere 10.4 � 1.3, 11.0 � 1.5, 6.7 � 1.6 and 6.3 � 0.9 for Col I, ALP, OCand BSP, respectively. TCP-P overall immune score values(mean � SEM) for Col I, ALP, OC and BSP in the central region were10.7 � 1.9, 11.4 � 1.8, 9.1 � 1.8 and 7.3 � 1.4, respectively. Overallimmune scores values (mean � SEM) for TCP-G in the apical region

floor augmentation procedures with b-TCP granules (TCP-G) and with b-TCP putty (TCP-rrow space percentages. Detailed results (mean � SEM) of the percentage of the areand TCP-G grafting materials and (d) marrow spaces.

Table 4Results of the immunohistochemical assessment, six months after sinus floor augmentation, of the osteogenic marker expression in the central region of the biopsies for thedifferent type of graft material (GM), i.e. b-TCP granules (TCP-G) or b-TCP putty (TCP-P). Marker scores are indicated for the cellular components, i.e. osteoblasts (OB), osteocytes(OCy) and fibroblasts (FB), and for the matrix components, i.e. fibrous matrix (FM), bone matrix (BM) and osteoid (OS). Col I ¼ collagen I; ALP ¼ alkaline phosphatase,OC ¼ osteocalcin and BSP ¼ bone sialoprotein.

Marker GM Cellular components Matrix components

OB OCy FB FM BM OS

Col I TCP-G 2.1 mild 0.6 minimal 1.0 mild 3.0 moderate 1.4 mild 2.3 moderateTCP-P 1.9 mild 0,7 1.4 mild2,0 1.9 mild 3.3 moderate 1.3 mild 1.0 mild

ALP TCP-G 2.1mild 0.9 minimal 2.7 moderate 3.9 strong 0.4 minimal 1.0 mildTCP-P 2.3 moderate 1.3 mild 2.4 moderate 3.1 moderate 0.7 minimal 1.6 mild

OC TCP-G 1.3 mild 0.1 minimal 1.3 mild 2.7 moderate 0.3 minimal 1.0 mildTCP-P 2.0 mild 0.9 minimal 1.7 mild 3.0 moderate 0.4 minimal 1.1 mild

BSP TCP-G 0.9 minimal 0.1 minimal 1.0 mild 2.7 moderate 0.3 minimal 1.3 mildTCP-P 2.3 moderate 1.1 mild 0.6 minimal 1.6 mild 0.6 minimal 1.1 mild

M. Stiller et al. / Biomaterials 35 (2014) 3154e31633160

were 4.8� 1.6, 5.0� 1.7, 3.9� 2.1 and 2.1�0.9 for Col I, ALP, OC andBSP, respectively. TCP-P overall immune score values (mean� SEM)in the apical region for Col I, ALP, OC and BSP were 7.0 � 1.2,7.1 � 1.5; 6.4 � 2.2 and 4.3 � 1.7, respectively. As previouslymentioned, overall immune score values were higher for the TCP-Pgroup compared to the TCP-G, but not statistically different. Allsections stained for non-immunized mouse and rabbit IgGremained negative.

4. Discussion

The hypothesis of this study was that TCP-P, compared to TCP-G,may facilitate an enhanced performance for SFA procedures, i.e. lesssurgical complications, enhanced surgical handling properties aswell as greater bone volume, bone formation, stability and osteo-genic expression. Since TCP-G and TCP-P had identical chemicalcomposition, the two main differences were the b-TCP size rangeand the presence of the HyA carrier in TCP-P. HyA created a bonegrafting dough, or putty, which facilitated its handling and dis-played a more advantageous surgical handling as filling the sinusfloor. Also, compacting this bone grafting material into thedesigned defect was performed in an easier manner. CBCT facili-tated the three-dimensional visualization of the maxillary sinuseswith a limited radiation exposure. CBCTallowed obtaining values tofollow the changes in volume of the grafted area occurring duringthe six month healing period.

The process and the resin selected for the immunostainingmaintained the antigenicity of the tissue and provided adequatematerial properties for cutting sections of 50 mm in thickness.Immunohistochemical analysis showed that with both graft ma-terials bone matrix synthesis and matrix mineralization, and thusbone formation, were still actively progressing six months afterSFA. A tendency towards a higher activity was present in the central

Table 5Results of the immunohistochemical assessment, six months after sinus floor augmentatdifferent type of graft material (GM), i.e. b-TCP granules (TCP-G) or b-TCP putty (TCP-P)osteocytes (OCy) and fibroblasts (FB), and for the matrix components (MC), i.e. fibrousphosphatase, OC ¼ osteocalcin and BSP ¼ bone sialoprotein.

Marker GM Cellular components

OB OCy FB

Col I TCP-G 0.1 minimal 0.1 minimal 1.7TCP-P 0.9 minimal 0.7 minimal 2.0

ALP TCP-G 0.7 minimal 0.1 mild 1.0TCP-P 1.6 mild 0.1 minimal 1.4

OC TCP-G 0.6 minimal 0.0 none 0.6TCP-P 0.9 minimal 0.3 minimal 1.6

BSP TCP-G 0.1 minimal 0.0 none 0.4TCP-P 1.4 mild 0.6 minimal 0.6

area as well as a greater bone formation and stronger osteogenicexpression of markers for the TCP-P group. The amount of bonewassignificantly higher for TCP-P. TCP-P had a more cohesive andmoldable structure, while the b-TCP granules displayed a moreopen structure which favored granule migration during theirplacement. Applying several layers of the TCP-P graft material, andcompacting them, was easy and possible. Due to its better coher-ence, the surgeon had a better control of the TCP-P material whenintroducing it into remote areas (obstructed from view) and at thesame time preventing a wash out effect of the material due tobleeding and blood flow during surgery, whose intensity cannotalways be exactly anticipated [31]. In addition, migration of thematerial through the Schneiderian membrane, especially in pa-tients with thin Schneiderian membranes, can be avoided whenusing a putty instead of granules. Consequently, using a putty mayalso facilitate the grafting of sites where granules can only be usedin combination with membranes and pins for securing the graftmaterial into place. Hence, in terms of surgical handling, TCP-Pperformed better than TCP-G without diminishing the osteo-conductive and osteogenic properties of the b-TCP particles. TRAPpositive osteoclasts were neither observed in TCP-G nor TCP-Pgroups. Positive controls, i.e. fracture healing sites, presentednumerous multinucleate TRAP positive cells. Since b-TCP cannot bedissolved passively in physiological conditions, these results sug-gest that other biological mechanisms, different from an osteo-clastic dissolution, are involved in the resorption process of TCP-Gand TCP-P [32,33].

The use of autogenous bone is still considered the gold standardfor SFA due to its excellent osteogenic potential [9,12,34,35]. Theuse of b-TCP bone grafting materials has been well documented innumerous clinical cases in implant dentistry, as well as in oral andmaxillofacial surgery, and excellent success rates have beendemonstrated [2,9]. b-TCP is currently regarded as a bone grafting

ion, of the osteogenic marker expression in the apical region of the biopsies for the. Marker scores are indicated for the cellular components (CC), i.e. osteoblasts (OB),matrix (FM), bone matrix (BM) and osteoid (OS). Col I ¼ collagen I; ALP ¼ alkaline

Matrix components

FM BM OS

mild 2.1 mild 0.4 minimal 0.3 minimalmild 1.4 mild 1.0 mild 1.0 mildmild 2.7 moderate 0.0 none 0.4 minimalmild 2.9 moderate 0.1 minimal 1.0 mildminimal 2.3 moderate 0.0 none 0.4 minimalmild 2.3 moderate 0.1 minimal 1.3 mildnone 1.3 mild 0.0 none 0.3 minimalminimal 1.3 mild 0.0 none 0.4 minimal

Fig. 6. Histogram showing the mean values (�SEM) of the overall immune score for the osteogenic markers expressed in (a) the central and (b) the apical area of biopsies. Valueswere assessed six months after the sinus floor augmentation procedures with b-TCP granules (TCP-G) and with b-TCP putty (TCP-P).

M. Stiller et al. / Biomaterials 35 (2014) 3154e3163 3161

material well suited for various clinical applications, such as SFAprocedures. As the vascularization and the surface area of the sinusfloor, from which osteoprogenitor cells can migrate and interactwith the grafting material, are limited compared to other bonedefects, there is a greater clinical need for this particular applicationto add a given amount of autogenous bone to the synthetic bonegrafting material. In the present study 10 vol % of autogenous bonechips were added to the grafting materials to ensure a sufficientsupply of osteogenic cells, such as osteoblasts, and osteoprogenitorcells. This addition sustains new bone formation and graft consol-idation via the osteoprogenitor cells from the sinus floor interactingwith the material into the grafted area [34e36]. Even if notcompletely omitted, the use of autogenous bone was limited to aminimum; this made it possible to study the effect of TCP-G andTCP-P on bone formation without a major effect from the addedautogenous bone chips. Both grafting materials were able toregenerate bone and to form a sufficient bone volume for facili-tating reliable and stable dental implant placement and anchorage.The evaluation of the application characteristics of the graftingmaterials was only possible to be done indirectly, i.e. not clinically,by assessing the volume stability of the grafted area by analyzingthe radiological data. There are a number of in vitro methodsavailable for volume assessment that do not take adequately intoaccount the postoperative variability of tissues such the Schnei-derian membrane, air entrapment in the grafting material andothers [37]. The method presented in the current study for pro-cessing the CBCT data sets and determining the graft volume,facilitated visualizing and assessing the available bone volume andits morphology preoperatively in a precise manner. Moreover, thecomputational procedure developed in this study enabled, in areproducible manner, determining the volume of the grafted areain the anatomical situation present clinically. TCP-G and TCP-P haddifferent volume graft reductions within a same patient. This is ofgreat clinical importance as the insertion of implants of a specificlength requires a minimal volume for a successful anchorage andstability. Volume graft reductions proved to be statistically signifi-cant. Due to safety and ethical considerations regarding the amountof radiation exposure for the patient, it was only possible to acquireone CBCT at a single time point, i.e. preoperatively, postoperativelyand six months after SFA. Hence, it was not possible to assess theshrinkage of the graft volume occurring at early stages within thefirst postoperative days and to differentiate that occurring due to

the bone regeneration and to the graft resorption. The b-TCP withsmaller grain size present in the TCP-P may have had a positiveeffect on damping the graft shrinkage as these particles are packedin a more dense way when introduced into the sinus floor [38]. Inaddition, the bond between the hyaluronic acid and the b-TCPparticles, or the viscoelasticity of this interaction, might havecontributed to reduce the shrinkage due to the HyA resorption,which can vary from days to weeks [39e41]. Nevertheless, the in-fluence and weight of these factors on the graft shrinkage needs tobe further studied.

Histologically and histomorphometrically, both materialsshowed excellent new bone formation and bone bonding behavior.They were capable of providing an environment for the synthesisand mineralization of the bone matrix which lead to an active andprogressive bone formation six months after SFA. The behavior ofTCP-P in terms of bone formation and osteogenic marker expres-sion, i.e. greater activity with respect to bone matrix formation andmaturation, is in agreement with the findings by Chazono et al.[42]. They observed a slightly greater bone formation for TCP incombination with HyA, versus TCP blocks, after implantation inNew Zealand white rabbits. Chanozo et al., contrary to our findings,noted the presence of osteoclasts. This may be related to the shorterimplantation periods used in their study. Osteoclastic activity ispresent at early staged and later decreases in favor of differentbiological mechanism mediating the degradation process. In addi-tion, long bones are formed by endochondral ossification and cra-nial bones by desmal ossification. Aslan et al. [43] demonstrated thestimulatory effect on osteogenesis of HyA per sewhen implanted inthe rat tibia without any TCP. In vitro studies have also demon-strated the stimulatory effect of HyA on osteoblasts derived fromneonatal rat calvaria; this stimulatory effect was dose as well asmolecular weight dependent [44]. HyA enhances the effect ofgrowth factors as well as the osmotic properties which lead to anenvironment in the grafted area which favors osteoblast prolifer-ation and differentiation [39,45]. The present study demonstratedthat TCP-G as well as TCP-P supported bone formation and osteo-blast differentiation. The greater osteogenic potential displayed byTCP-P may be related to the physicochemical properties of HyA.Previous studies which used 3D-visualization for biopsies sixmonths after SFA with TCP granules, showed that bone formationprogressed from the sinus floor in an apical direction; while theSchneiderian membrane exhibited only a minor osteogenic

M. Stiller et al. / Biomaterials 35 (2014) 3154e31633162

potential [2,46]. This explains the greater bone formation, osteo-genic marker expression and particle degradation noted in thecentral area compared to the apical area of the biopsies. Theenhanced staining for osteogenic markers observed in the apicalregion -which exhibits fewer osteogenic cells compared to thecentral region- for TCP-P compared to TCP-G supports the hy-pothesis of the stimulatory effect of HyA on osteoblast differenti-ation. TCP-P displayed better surgical handling properties than TCP-G, without any adverse effect on bone formation, bone tissuematuration or graft volume stability, confirming positively ourhypothesis. Considerable differences in the amount of bone formedand the osteogenic marker expression were detected between in-dividual patients. These differences emphasize the need forexamining the effect of age and gender factors such as hormonestatus, body mass index, etc., on bone regeneration after SFA.

5. Conclusion

Both TCP grafting materials actively supported bone formationand matrix mineralization six months after SFA. This resulted infavorable bone regeneration and degradation of the graft materialwhich created a sufficient volume of osseous tissue to facilitatestable dental implant placement. The TCP-P material, compared toTCP-G, displayed better surgical handling properties. The HyAmatrix had no adverse effect on bone formation, bone tissuematuration or graft volume stability. Histomorphometric andradiologic results of TPC-P expressed statistically significant dif-ferences, compared to TCP-G, in terms of volume reduction, volumestability and bone formation. Immunohistochemical osteogenicmarker expression displayed also a higher tendency for TCP-P.Hence, TCP-P can be regarded as excellent grafting material forSFA in a clinical setting. The influence of the b-TCP particle size,within the HyA, as well as the patient’s factors, i.e. age and genderfactors such as hormone status, body mass index, etc., on thegrafting material performance, need to be clarified in furtherstudies.

Acknowledgments

The authors are grateful for the support and materials providedby the RMS foundation. The authors thank Ms. A. Kopp and Mr. R.Struck for their excellent technical assistance.

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