Evaluation of Pre-hydrated Collagenated Cortico-Cancellous Granules ( Mp 3 ® ) in Augmentation of the Maxillary Sinus ( Preliminary Study )

Background: Bone substitutes, such as allografts, xenografts, and alloplasts, have been proposed in several augmentation procedures. Purpose: The aim of the present study was to evaluate radiographically and histologically the pre-hydrated collagenated cortico-cancellous granules in maxillary sinus augmentation. Materials and Methods: This study conducted on eight sinuses with residual bone height less than five mm using lateral approach technique specimens, retrieved after 6 months from augmented sinuses. The specimens processed to be observed under light microscopy. Radiographic measurements presented as means one standard deviation of bone height in millimeter and density of the newly formed bone in Hu. Results: Most of the particles were surrounded by newly formed bone with large osteocyte lacunae. Some slides showed marrow spaces and residual graft material reveals the beginning of new bone formation. Conclusion: The present results show that pre-hydrated collagenated cortico-cancellous granules is a biocompatible, osteo-conductive biomaterial that can be used for maxillary sinus augmentation procedures without interfering with the normal reparative bone processes.

Mp3 ® (Tecnoss, Italy) is a heterologous origin biomaterial made of 600-1000 μm or 1000-2000 μm pre-hydrated collagenated cortico-cancellous granules, properly mixed with OsteoBiol ® Gel 0. Thus, it is possible both skipping the hydration phase and decreasing the risk of accidental exposure of material to pathogens during manipulation and grafting phases; furthermore, the syringe is flexible and ideal to simplify grafting in the receiving site.The granules are endowed with characteristics very similar to human mineral bone, [15] and can be used as an alternative to autologous bone.
Their natural micro-porous consistency facilitates new bone tissue formation in defect sites and accelerates the regeneration process.
Gradually it preserves the original graft shape and volume (osteo-conductive property) [16].Moreover, due to its collagen content, the product facilitates blood clotting and the subsequent invasion of repairing and regenerative cells.
Therefore, the purpose of the present study was to evaluate radiographically and histologicallythe pre-hydrated collagenated cortico-cancellous granules in augmentation of the maxillary sinus.

Introduction
Autogenous bone has been reported to be the golden standard in bone regeneration procedures because it contains viable osteoblasts, organic and inorganic matrices, and biological modifiers [1].However, the use of autogenous bone has several disadvantages, that is, a limited availability, a tendency to partially reabsorb, the need for an additional surgery, and the increased morbidity.Bone substitutes, such as allografts, xenografts and alloplasts, have been proposed in several augmentation procedures [1,2].Maxillary sinus augmentation procedures have been used to obtain a sufficient volume of bone to allow implant placement.Different biomaterials have been used for procedure to place dental implants were eligible for this study.Patients were reported as healthy and there was no requirement for routine medication.This study was drawn up according to Declaration of Helsinki for experimentation on human subjects.Possible complications of surgical therapy treated following the standard protocols of dental management.
Adigital orthopantomographic radiograph with 1:1 magnification and a Cone Beam Computer Tomography (CBCT) scan of the maxilla were taken preoperatively for each patient.Antral spaces were evaluated at 1 mm serial sections.In these patients, CBCT scans showed residual bone in the lateralposterior segments of the edentulous maxilla below the floor of the auxiliary sinus ranged between 3 mm and at least 5 mm of height.Furthermore, average residual bone width had to be at least 6 mm as measured by the CT scans.

Treatment Timetable
Premedication followed the protocol suggested by Misch and Moore [17].That is, dexamethasone 8 mg preoperatively, 6 mg after 24 hours, and 3 mg after 48 hours, as anti-inflammatory drug.Systemic antibiotics, amoxicillin, were also administered 1 hour preoperatively (2 g) and 500 mg (Quarter in die) for 1 week.As an analgesic agent, Etodolac 600 mg initial dose, and 200-400 mg as needed, was also prescribed.
All the patients were treated with the same surgical technique consisting of sinus floor augmentation via lateral approach [18].
Once the sinus membranes were elevated to obtain the necessary volume for bone grafting, all the maxillarysinuses were grafted using 100% cortico-cancellousporcine bone particles (MP3 ® , Tecnoss, Coazze, Italy).
After 6 months, a biopsy was carried out.Cylindrical bone samples were harvested with a 2.5 mm-internal diameter trephine exactly at the planned location of the dental implant in a vertical direction through the crestal incision to take a core biopsy containing both natural and the newly formed bone and sent to histological examination, then the implant placement completed as planned.

Specimen processing and analyses
Core biopsy specimens fixed at 10% naturally buffered formalin, decalcified in formic acid (10%) that renewed frequently until we ensured adequate decalcification reached by the end of the 48 hours.Then specimens embedded longitudinally into paraffin blocks and oriented in a standardized way for labeling and differentiating the newly formed bone end of the native bone.
Specimens were cut into longitudinal sections using a manual rotary microtome, and stained with Mayer's hematoxylin and eosin for histological analysis under light microscope with magnification of 20X.Representative slide samples were recorded and digitized.The slides evaluated blindly away from data and orientation for the presence of cortical and/or trabecular bone, thickness of osseous trabeculae, and the presence of lamellar and woven bone, the amount of osteoblasts and osteoclasts found the presence of fibrosis and vasculature of marrow spaces and evidence of mononuclear cell or mixed inflammatory cell infiltration.
This accomplished by using a subjective histological scoring system, which typically involves the assignment of a categorical grade according to the percentage of the tissue that is estimated visually by bone histology experienced observer to represent the quality of the newly formed bone under higher magnification (200X).It is a semi quantitative and subjected to inter-and/or intra-observer variability.

Patient positioning
Standardization during imaging achieved through adjusting the patient positioning lightin the cone beam machine as follows: The seat height adjusted to position the Region of Interest (ROI) vertically within the Field of View (FOV), and the upper light beam indicated the top of the FOV and the lower light beam indicated the bottom of the FOV.
The sagittal light (vertical front light) positioned in the center of the FOV from sagittal direction so that it is in the center of the ROI.
The lateral light (vertical side light) positioned in the center of the FOV in the lateral direction so that it is in the center of the ROI.
The patient instructed not to move during the duration of exposure, which performed at eight mA, 85 KV and at a field of view.The image reconstruction performed using special software , and the obtained data subjected to statistical analysis.
On the second day and at 6 months postoperatively; Cone Beam Computed Tomography (CBCT) was performed on the operated side to ensure the proper graft position inside the sinus cavity, evaluate the density of the newly formed bone inside the sinus cavity, and bone height evaluation at the sub-sinus area.Natural bone height measured on immediate postoperative CBCT.Intraoral radiograph (IOR) and profile were the tools in this cone beam C.T software that we used to diagnose and record the bone height & density.

Radiographical results
On second day postoperative CBCT, all radiographs revealed proper position and increase in both bone height due to presence of the graft material (9.1 mm average) &density of the grafted material (494.6 Hu average) (Figures 1&2).
On six months postoperative CBCT, there was increase in the mean bone height (9.4 mm average) and bone density with the presence of bone deposition (666.3Hu average).This appeared as increased radio-opacity above the subsinus bone (Figures 3&4), except one case showed that a decrease in bone height & density six months postoperatively (Figures 5-7).
Bone height: Second day post-operative, there was a non-statistically significant increase in mean bone height in comparison and preoperative phase.
From second day post-operative period to 6 months, there was a statistically significant increase in mean bone height.
Through the whole study period (Pre-operative to 6 months), there was a statistically significant increase in mean bone height (Table 1).

Bone density:
Through the whole study period (second day postoperative to 6 months), there was a statistically significant increase in mean bone density and the newly formed bone later on.

Histological results
Under light microscope, all the histological samples of core biopsy showed areas of lacunae containing viable osteocytes.Other areas showed irregular woven bone trabecules formation surrounded by a loosely arranged and disorganized connective tissue stroma, with areas of hyalinization, and condensation of collagen fibers around areas of dystrophic calcification with granular pattern between bone tissues (Figure 8).
One of the slides showed infiltration of the connective tissue stroma with some chronic inflammatory cells as neutrophils around areas of hemorrhage (Figure 9), another showed some reversal lines denoting bone resorption followed by bone formation surrounded by disorganized connective tissue stroma (Figure 10).At this stage (six months), some remnants of the graft material was seen, while the collagen membrane was completely resorbed.

Discussion
Maxillary sinus augmentation surgical techniques as well as osteoconductive potential of various bone substitutes have greatly evolved over the last few years, allowing predictable placement of dental implants in the regenerated maxillary premolar and molar areas.
Although the use of autogenous bone remains "the golden standard," other various bone substitutes have greatly evolved over the last few years in order to perform a predictable regeneration of maxillary posterior area with low morbidity for the patients [19].
The current study has taken under examination pre-hydrated corticocancellous porcine bone to evaluate the osteoconductive properties and the re-absorption rate after maxillary sinus

Evaluation of Pre-hydrated Collagenated Cortico-Cancellous Granules (Mp3®) in Augmentation of the Maxillary Sinus (Preliminary Study)
Copyright: © 2014 Noami et al.In this study the sinus floor elevation and antral augmentation by the grafting material done first, then the core biopsy and implant placement performed six months later due to the presence of less than five mm of bone between the crest of the ridge and the floor of the maxillary sinus, which cannot give primary stability to the implant [20].
At second day postoperative CBCT, it was found a significant increase in the relative density and bone height due to the radio opacity of the grafting material.At six months postoperatively, the radiographic result showed significant increase in the bone density and bone height due to more deposition & calcification of the newly formed bone.These radiographic results give an idea about the rapid resorption process of the selected graft and allow newly formed bone to be completely mature before complete resorption of the graft.
All the patients scheduled in for this study showed a residual ridge height within 5 and 3 mm; this was considered be an important clinical because the residual ridge height might be a variable to evaluate the final outcome of the procedures [21].Therefore, in this study all patients with similar residual ridge height were treated with an identical surgical protocol.Six months after augmentation, the bone core specimens harvested vertically from the alveolar ridge where native bone is present.This is of paramount importance, since the histological results

Evaluation of Pre-hydrated Collagenated Cortico-Cancellous Granules (Mp3®) in Augmentation of the Maxillary Sinus (Preliminary Study)
Copyright: © 2014 Noami et al.        shown, a greater amount of newly formed bone close to the residual crest (natural bone) compared with the amount in the whole biopsy sample.This is because the healing and maturation of the graft is derived from the cellular and vascular supply of the residual maxillary bone [22].
The histological findings supported the idea that porcine bone had good biocompatibility and excellent osteoconductive properties.Histological results indicated that both osteogenesis and angiogenesis followed ordinary periods; moreover, osteoblasts were observed apposing osteoid matrix directly on graft particles.In addition, the presence of multinucleated cells in resorption lacunae along the surface of porcine bone particles and the presence of bone metabolizing units within granules indicated that a remodeling/resorption processes were taking place.This agree with the result of bone density obtained from the six months postoperative CBCTs, which indicated the increase in the bone density.This is due to the presence of new bone formation [23,24].The clinical success observed with porcine bone depends on the surface topography of the graft material [25].The macro and micro-porosity have a decisive role in osteoconduction, because both sufficient pore size and an interconnecting pore structure are required for osteoblast to grow into graft biomaterial [26].Some authors observed that pores of 100-300 mm would be necessary for vascularization and osteoblast migration [27].

Conclusion
In conclusion, findings from the present study supported the hypothesis that collagenated porcine bone has excellent osteoconductive properties and can be partially reabsorbed.
Moreover, the collagenated porcine bone allows an increase in the percentage of new bone and, at the same time, a reduction in the percentage of residual grafting material.This is very important because it is still under discussion whether the different biomaterials, such as bovine bone, will be reabsorbed with time.However, more studies in which collagenated corticocancellous porcine bone is involved are needed before routine clinical use can be recommended.

Data analyses :
Paired t-test is used to compare between mean bone height and density measurements through different periods Data presented as mean and Standard Deviation (SD) values.The significance level was set at p ≤ 0.05.Statistical analysis performed with IBM ® SPSS ® Statistics Version 20 for Windows.

Figure 1 :
Figure 1: (Left) A photoradiograph of immediate postoperative CBCT showing increase in bone height due to presence of graft material.

Figure 2 :
Figure 2: (Right) A photoradiograph of second-day postoperative CBCT showing the bone density.

Figure 3 :
Figure 3: A photoradiograph of six months postoperative CBCT showing increase in bone height six months postoperatively.

Figure 4 :
Figure 4: A photoradiograph of six months postoperative CBCT showing increase in bone density due to presence of the newly formed bone.

Figure 5 :
Figure 5: A photoradiograph of six months CBCT of one of the cases showing decrease in bone density of the grafted material.

Figure 6 :
Figure 6: (Left) Bar chart representing comparison between bone heights measurements at different periods.

Figure 7 :
Figure 7: (Right) Bar chart representing comparison between bone density measurements at the two periods.

Figure 8 :
Figure 8: A photomicrograph of living woven bone tissue showing lacunae containing viable osteocytes (red arrow).Another area shows beginning of woven bone formation (area of hyalinized tissue around osteocytes) (yellow arrow).Another area shows condensation of collagen fibers around an area of dystrophic calcification (blue arrow) (H&E 200X).

Figure 9 :
Figure 9: Higher magnification of the previous photomicrograph showing inflammatory cell infiltration around an area of hemorrhage (yellow arrow) (H&E 200X).

Figure 10 :
Figure 10: Photomicrograph of dense woven bone tissue showing some reversal lines denoting bone resorption followed by bone formation (white arrows) surrounded by disorganized connective tissue stroma (H&E 200X).

Table 1 :
Bone Height.The mean, Standard Deviation (SD) values and results of paired t-test for comparison between bone height measurements at the three periods.

Table 2 :
Bone Density.The mean, Standard Deviation (SD) values and results of paired t-test for comparison between bone density measurements at the two periods.