A new biphasic osteoinductive calcium composite material with a negative Zeta potential for bone augmentation

Due to generalized horizontal bone loss and chronic periodontitis in the upper and lower jaw (Fig. 3), standardized maxillary sinus floor augmentation was performed prior to the placement of dental implants in a 61 year old male patient. The molars of the upper jaw and the second premolar in the first quadrant were missing. Due to a bone thickness of the sinus floor of less than 4 mm, a two-stage surgery was indicated. Both the right and left sinus were augmented according to the method of Boyne and James [1]. In brief, a full thickness mucoperiosteal flap was detached; a rectangular window was reamed into the lateral sinus wall and infractured. The internal mucosa that covers the maxillary sinus was carefully elevated and displaced inwards (towards medial) together with the bone window. The space beyond the prepared membrane and bone window was filled with the BCC bone graft (Fig. 4). The material was compacted and the mucoperiosteal flap was readapted and sutured. The postoperative period was uneventful. Six months after the augmentation of both sinuses, two 11 × 4 mm implants (Osseotite Certain; 3i Implant Innovations; West Palm Beach, FL, USA) were placed on each side in the augmented regions corresponding to the first right upper molar, second right upper premolar and first and second left upper molars. Prior to the insertion of the implants, dental computed tomography (CT; Twin Elsent, Maconi Philips, Best, Netherlands) was made in order to evaluate the dimension and density of the augmented sinus floors.

Patch clamp physiological techniques were used to characterize the voltage-activated calcium currents expressed in the plasma membrane of osteoblastic cells which are influenced by the surface loading of the bone substitute. Preparations enriched with osteoblasts were isolated by collagenase digestions of newly formed bone and cultured under different conditions which affected cell proliferation. The Zeta potential was measured by using the Zetasizer 3000 device (Malvern, Herrenberg, Germany). Ethanol, isopropanol and methanol were used as liquid phases in reagent grade. Measurements were performed in saturated calcium phosphate solution, in 0.05 mol/l sodium phosphate solutions and in deionized water. The potential was determined six times; the mean values and standard deviations were calculated. The initial setting time of the cements was measured according to the Gilmore needle test in a humidity chamber at 37°C and a humidity of >90% [19].

During implant placement, a bone biopsy was taken from the augmented area in the first quadrant using a trephine bur. In order to focus on the augmented region, only the cranial bone portion of the bone cavity for the 4 × 11 mm implant was harvested resulting in a 4 × 6 mm sample which was available for histologic evaluation. The specimen was immediately rinsed in saline, fixed in 4% formalin in phosphate buffer for 24 hours at room temperature and demineralized by using EDTA solution. The samples were cut (1–2 μm thickness) and stained in hematoxylin-eosin and Ladewig stain. Microscopic evaluation was carried out on light microscopy in the magnifications 25×, 100× and 400×.

The BCC material demonstrated a negative Zeta potential in organic media (-5.2 ± 1.3 mV in ethanol and -3.4 ± 1.4 mV in isopropanol), as well as in aqueous antibiotic solution [-20.4 ± 2.1 mV in gentamicine and -24.6 ± 1.8 mV in amoxicillin]. For comparison, it demonstrated -19.6 ± 1.1 mV in water. The particle surfaces only showed a slight electrostatic charge in the solvents. The mean particle size was 1.9 to 2.1 (mm) depending upon the organic suspension medium. According to the value of the Zeta potential, the minor particle size correlated with an increasing electrostatic charge of the particle surface.

During the procedure of implant site preparation in the first quadrant, a tissue specimen of 4 × 6 mm was taken and stored for further analysis in 4% formalin in phosphate buffer. The sections of the biopsy showed a portion of the bone tissue, characterized by marginally compact trabecular bone with regular lamellar and woven structure as well as a fatty non-fibrosing bone marrow (Fig. 5). No BCC particles could be identified. However, sporadic osteoclasts indicated a preceding resorption activity (Fig. 5c) and little cementum lines showed the formation of new mineralized bone (Fig. 5). A thin strand of osteoid, stained in blue color in Ladewig stain (Fig. 5), represented the margins of the bony trabecules and was surrounded by osteoblasts. Singular lymphocytes were also visible in the bone marrow. There were no signs of acute inflammatory or foreign body reactions.

The bone quality of the augmented sinus floor was evaluated in a dental CT of the upper jaw prior to implant placement (Fig. 6). Radiological signs of marginal osseointegration of the graft could be shown. Bone density was assessed by using the Hounsfield classification. The measurements corresponded to a bone quality of Q3 to Q2. There were no radiological signs of inflammation visible. In the region of the roots of the upper jaw, a regular bone structure was found. Also, sinus maxillaris was free of inflammation and was normally ventilated.

A postoperative panoramic radiograph (orthopantomogram) was made five months after the insertion of the implants prior to crown restoration on the implants (Fig. 7). The radiograph shows gain of sufficient bone volume in the sinus floor area of both sides surrounding the osseointegrated implants. The augmented sinus floors were characterized by homogenous bone with a regular trabecular structure and similar density compared to the residual bone. No signs of inflammation were evident.

The surface potential of bone substitutes in direct contact to bone is of great interest in the recent time. However, until now the exact system of the implant to bone bond can not completely be explained. Surface functional groups such as carboxyl and hydroxyl ions determine the surface charge, the degree to which such surface groups alter the resulting bone substitute to osteoblast or precursor cells or bone is not fully understood [20]. To our knowledge this is the first report of biphasic calcium composite material for sinus floor augmentation. The clinical, radiographic, histological and patch clamp results of our study suggest that this biomaterial might be a more effective bone substitute for maxillary sinus augmentation than other alloplastic bone substitutes. The histological analysis six months after sinus augmentation showed osteoblasts and evidence of new bone formation in the augmented regions. Moreover, enhanced presence of osteoclasts revealed the tendency of ongoing accelerated resorption processes. Interestingly, there were no BCC particles visible anymore, suggesting a fast and complete remodeling of the material.

There are only limited data available on the dynamics of resorption of bone replacement materials after maxillary sinus floor augmentation. For bovine hydroxyapatite, a slow resorption is known to occur after maxillary sinus floor augmentation. Although in animal models [21, 22] and in humans [6] an increase of new bone formation in an osteoconductive manner has been shown [23], in most studies available bovine hydroxyapatite particles were not or not fully absorbed after a period of six months [24], twelve months [6, 21, 25] or even later [25]. Similar findings were made with the use of conventional tricalcium phosphates. However, data suggest a faster resorption rate with these materials [23, 26, 27]. In a case control study, Eggli et al. showed a TCP resorption of 85% compared to 5.4% for hydroxyapatite six months after implantation in cancellous bone of rabbits [28]. In a comparative histomorphometric analysis, Artzi et al. reported a better resorption rate of TCP than bovine hydroxyapatite [29]. However, a complete resorption of calcium phosphate particles with simultaneous osteoconductive bone formation six months after sinus augmentation as shown in the present study with the BCC has not been reported yet. The reason for this observation might be based on special surface properties of this biomaterial. Both components of the BCC, the porous calcium phosphate and implant grade calcium sulfate, biodegrade within weeks to months. Biodegradation of calcium sulfate leads to a developing macroporosity. To replace the initial microporosity in between calcium phosphate particles, cells and nutrient fluids draw in [30]. Calcium phosphate particles are then able to interact with osteogenic cells. In this interaction, the ZPC™ principle plays an important role [13].

Based on this unique concept, the surface of the material will be charged negatively in an aqueous environment. A number of studies have shown that a material that has an electronegative surface charge (negative Zeta potential) is more accessible for the attachment and proliferation of osteoblasts than surfaces with no or even positive electric charge [31‐34]. The Zeta potential as one of the surface properties is strongly influenced by the reactivity of the calcium phosphate particles. It plays an important role due to the manufacturing and application process of the fine grain powder mixtures of BCC. Suspension media leading to a high surface charge of the Zeta potential and particle sizes of this material ground in several organic media particles lead to electrostatic stabilization of the particle surface, minimized agglomerization resulting in the splitting of the particles instead of agglomerates. As a reaction, small rapidly diffusing positively charged proteins initially attach to the graft material. These are then replaced by larger, positively charged proteins with a strong affinity to the surface that show chemotactic and adhesive properties. Through this mechanism of adsorption of positively charged proteins at the surface, the material is accessible for a rapid attachment of osteoblasts [35] as the negatively charged species (mesenchymal cells and osteoblasts) are drawn into intimate contact [36]. Once the osteoblasts have attached, they undergo the process of cellular adhesion which is slower than the adsorption of charged proteins. During cellular adhesion, the osteoblasts accomplish a true biological attachment, enable cell proliferation and benefit bone formation (Fig. 2).

For the BCC material presented in this study, the formation of a negative Zeta potential had recently been demonstrated in vivo and in vitro: Cooper and Hunt evaluated the expression of selected osteogenic markers (alkaline phosphatase, osteocalcin, osteopontin, core binding factor alpha-1 (CBFA1) and collagen type 1) in vitro by reverse transcription-polymerase chain reaction (RT-PCR) in a culture of osteoblasts in contact to different calcium phosphate materials with positive and negative Zeta potential values [37, 38]. They demonstrated a strong correlation of a negative Zeta potential with the expression of several osteogenic markers. Other authors recently reported on the significance of relative Zeta potentials of bone and different biomaterials and their influence on protein adsorption [34]. They demonstrated that the adsorption of specific extracellular matrix proteins onto biomaterial surfaces provided sites for an integrin-mediated osteoblast attachment. In the case of BCC with its negatively charged surface, all osteogenic markers were expressed while the conventional pure phase TCP with a positively charged surface only induced the expression of osteopontin and alkaline phosphatase. Moreover, it is well known that rapid resorption of the calcium sulfate matrix alone promotes osteogenic activity due to formation of a calcium phosphate lattice [39, 40] promoting osteogenic activity. It mimics the mineral phase of bone and is resorbed at the rate of bone formation [41].

The density of the newly formed bone corresponded to a quality of Q2 to Q3 according to the Hounsfield classification, as verified by determination of the specific Hounsfield units by CT scans. This quality of bone can be compared with the local origin trabecular bone and is even denser than the original bone matrix of the posterior maxillary region. This confirms our histologic findings and the stable osseointegration of the implants, which are surrounded by sufficient bone as seen in the postoperative panoramic radiograph.

The authors are aware of the limitations of the present study, whose data base on the analysis of samples from one patient. Therefore, we regard this report as to indicate a trend. We also cannot draw general conclusions or definitive statements about the possible shrinkage of the grafted volume due to degradation of BCC, which has been observed with other bone replacement materials [42, 43]. From a practical point of view, however, we felt that careful but sufficient compaction of the material during sinus augmentation might contribute to minimize a possible shrinkage due to resorption processes. Nevertheless, this study indicates previously unknown properties of a new composition of two materials with a special biodegradation dynamic and surface potential as this could be demonstrated by the patch clamp technique. Nevertheless, the presented experiences are encouraging since the final aim of the maxillary sinus floor augmentation procedure is osseointegration of implants into vital bone.