INFLUENCE OF VARIOUS COATINGS OF HYDROXYAPATITE BONE CARRIER ON THE SUCCESS OF BONE REGENERATION IN RABBIT CALVARIAL DEFECTS: HISTOMORPHOMETRIC AND HISTOLOGICAL ANALYSIS

The materials used nowadays for bone replacement do not fully meet the requirements for complete regeneration, which is why new ones are being tested. Despite numerous attempts to improve bone tissue regeneration, no fulfilling material has been found yet. This study investigated the influence of poly-lactide-co-glycolide (PLGA) and poly-ethylene-imine (PEI) as coatings for hydroxyapatite (HAP) bone carrier onto bone tissue regenerative potential in rabbit's calvarial defect. Methods. Calvarial defects measuring 6 mm in diameter were made in 19 skeletally mature rabbits. Defects were filled with one of the following materials: PLGA coated HAP (HAP+PLGA), PEI coated HAP (HAP+PEI) and bovine HAP – Bio-Oss (positive control). Unfilled defects represented negative control. Histological analysis was performed in order to determine the inflammatory response of the host tissue. Formation of the new bone was evaluated by means of histomorphometric analysis. All analysis have been conducted in samples obtained 3, 6 and 9 weeks after implantation. Results. Three weeks post-implantation, a trend toward increased healing in HAP+PLGA group compared to other investigated materials was noticed, with no statistically significant difference between study groups ( p >0.05). However, after six and 9 weeks, significant healing was observed in favour of the HAP coated with PLGA compared to other groups ( p <0.05). Within this group, greater bone healing was observed comparing to HAP+PEI and Bio-Oss. Conclusion. PLGA has demonstrated greater coating potential comparing to PEI with respect to osteogenesis improvement in bone reconstructive surgery does.


Introduction
Bone regeneration is an important issue in oral and maxillofacial surgery.
Autogenous bone still presents a gold standard for bone defects repair. On the other hand, several drawbacks regarding the use of autogenous bone grafts have been described, such as donor site morbidity, limited amount of harvested bone and relatively high resorption rate of the construct. Having in mind these drawbacks, synthetic bone substitutes and xenografts have been introduced 1, 2, 3 . An ideal bone substitute should be non-irritable and non-toxic, providing adequate microenvironment for adhesion, proliferation and differentiation of the cells 4 . In addition, requirements for graft material include not easy achievable mechanic stability and high porosity 5 . Likewise, ideal bone substitute is expected to resorb completely, in proper period, synchronized with new bone synthesis 6 .
Geistlich's Bio-Oss is the most investigated bone substitute, characterized by desirable clinical results in comparison to other commercially available products. Despite positive clinical outcomes obtained with Bio-Oss, this material does not provide complete bone regeneration 7,8 . Furthermore, it has been shown that some particles remain within connective tissue for years 9,10 .
As an effort to obtain material with degradation level synchronized by new bone formation, novel bone tissue substitute (scaffold) based on calcium hydroxyapatite (HAP) was synthesized 11,12 . In order to activate the surface, HAP can be layered with various surface-active substances, such as poly-lactide-co-glycolide (PLGA). It had been suggested that PLGA coating did not induce any inflammatory effects 12 weeks after implantation. In addition, accelerated cell adhesion when HAP was coated with PLGA (HAP+PGLA) has been documented 12 . Through activation of the Runx2=CBFA-1 transcriptional activator, HAP+PLGA promotes osteogenic differentiation of preosteoblastic cell lineage. This combination can be used as a tissue engineering scaffold material and delivery carrier of pro-osteogenic bone morphogenetic protein 2 (BMP2) and pro-angiogenic gene of vascular endothelial growth factor as well 13 . The issue whether new bone formation could be obtained in shorter extent of time remained unclear. Despite the fact that coating provides certain advantages, a choice of adequate coating material is still at the centre of researchers' interests.
Another option for bone substitute coating is poly-ethylene-imine (PEI) 14,15 . This material belongs to the next generation of gene-activated scaffolds, which might include multiple genes to promote synergistic cell-mediated protein production and facilitate neovascularisation of the damaged bone 16 . Linear PEI enriched scaffolds have promoted cell growth by mimicking biological function of the native extracellular matrix 17 . Modified PEI also exhibits a number of key advantages, like low immunogenic, low cytotoxic, noncarcinogenic properties and is considered safe for clinical use 18 . In addition, PEI contains a large number of amino nitrogen atoms in the molecular chain, leading to a strong affinity to cells 19, 20 .
The aim of this study was to assess the influence of PLGA and PEI when used as HAP coatings on osteogenesis improving. The ultimate goal was to determine the ratio of the newly formed bone in rabbits' calvarial defects after implantation of HAP+PLGA and HAP coated with PEI (HAP+PEI).

Materials synthesis
HAP synthesis and PLGA coating were performed as reported previously 11 . In short, powders of calcium and (NH 4 ) 2 HPO 4 (p.a. Merck), were used for the hydrothermal synthesis of HAP. The precursor solutions were prepared as a combination of corresponding mixtures of Ca(OH) 2 and aqueous solution of (NH 4 ) 2 HPO 4 . Afterward, surface-active substance PEVA/PEVV was added for further hydrothermal processing in the autoclave at a temperature of 120 °C for 2 h. The obtained particles were filtrated through a filter with a pore size of 200 nm. HAP granules were obtained using polyurethane foam template and HAP suspension. After immersion of template in the HAP suspension and its drying, the composition was thermally treated to pyrolyse polyurethane template, followed by sintering of porous HAP after thermal treatment at 1200 0 C. Finally, HAP+PLGA coating was obtained by pouring the PLGA solution in chloroform over the HAP granules.
Coating with PEI included presumably slight PEI modification. Briefly, the solution of modified PEI was prepared by dissolving branched PEI (3 g) in 15 mL water by heating and stirring. Carbon dioxide (CO 2 ) was bubbled into this solution at ambient temperature and stirring was continued for 5 h until the reaction was complete. The contents were transferred to an Eppendorf tube, freeze dried to form solid PEI-CO2, and later dissolved in ethanol. HAP+PEI coatings were obtained by immersion of HAP granules in prepared solution. Amino content and subsequently cytotoxicity of PEI were reduced by modifying with CO 2 . animal. The first defect was filled with HAP+PLGA (also known as ALBO-OS), the second defect with HAP+PEI, the third defect with Geistlich Bio-Oss as a positive control, and forth defect was left empty as a negative control. The first six rabbits were sacrificed after 3 weeks, other 6 rabbits after 6 weeks and seven rabbits were sacrificed 9 weeks following the implantation. The biopsy specimens were obtained from each animal with an oscillating saw, including entire cranial vault for histology and histomorphometric analysis.

Study design and surgical procedures
In addition, the dura mater, galea and periosteum remained intact in each of the animal.

Histological analysis
All specimens were optimally decalcified using formic acid. Each specimen was embedded in paraplast, sectioned in 4 μm thick slices by rotary microtome (Leica SM2000R, Leica Microsystems, Wetzlar, Germany). Thereafter, the preparations were dewaxed, processed to Hematoxylin-Eosin (HE) and Goldner's trichrome staining technique and qualitatively analysed under light microscope to determine the level of host tissue inflammatory response.

Histomorphometric analysis
The histological parameters were evaluated at 40 x magnification under a microscope (Leitz Labor Lux S Fuorescence Microscope, Ernst Leitz Wetzlar GMBH, Germany), with exception of inflammatory cell infiltrates which were counted on total magnification of 400x. 2D images were captured at 40x magnification using digital colour camera (Leica DFC295, Germany) and merged to create a single image for each histological section. Thereafter, images were analysed using software (Leica University Suite, version 4.3, Leica Microsystems, Germany) running on the personal computer. Four sections from central defect region and 4 from peripheral defect region were analysed with a spacing of 50 µm between sections. The following parameters were measured: total bone volume in percentages (TB%), mineralized bone in percentages (MB%), nonmineralized bone in percentages (NMB%), connective tissue in percentages (CT%) and blood vessels in percentages (BV%). Within the connective tissue, macrophages, giant cells, plasma cells, lymphocytes and neutrophil granulocytes were counted.

Statistics
Statistical analysis was performed using SPSS for windows -version 18.0 software (SPSS, Inc., Chicago, IL, USA). All data were presented as the mean ± SD. Two-way ANOVA was performed at 95% level of significance, followed by Tukey post hoc comparisons.

Histological and histomorphometric analysis after 3 weeks post bone replacement material implantation
In all specimens 3 weeks after implantation the demarcation line and area of the defect filled with connective tissue containing the graft particles could clearly be noticed (Figs. 1 and 2). In addition, islands of new bone tissue, mainly localized at the particle surfaces or near them were detected. In HAP+PLGA group particles of the graft were almost completely surrounded by new bone tissue, with trabeculae and osteoblasts.
Histomorphometric analysis of specimens after 3 weeks is shown in Table 1.
Although HAP+PLGA group showed higher percentage of total bone area comparing to Bio-Oss, the difference was not statistically significant (p>0.05). Likewise, there was no statistically significant difference in any other parameter 3 weeks after implantation (Figs.1 and 2, Tab. 1).
Inflammatory infiltrate (macrophages, giant cells, lymphocytes, plasmocytes, neutrophils) was mainly localized in close proximity to the particles of the material. The number of the cells was ranging from 0 to10 in the field of view under the light microscope magnification of 400x. (Tab.2).

Histological and histomorphometric analysis after 6 weeks post bone replacement with implanted material
The results of histological analysis 6 weeks after implantation showed that defects were filled by connective tissue with still unabsorbed particles of the graft and new bone tissue. The amount of newly formed bone was the largest in the HAP+PLGA group, followed by HAP+PEI and Bio-Oss, whilst the lowest amount was observed in empty defects. In all tested groups, newly formed bone had lamellar structure with osteocytes, which indicated on bone vitality. In majority of the samples, the absence of inflammatory cells in connective tissue was detected (Figs. 3 and 4).
Histomorphometric analysis of the specimens obtained after 6 weeks demonstrated that the amount of newly formed bone in HAP+PLGA group was 18.9±1.3 %, which was statistically higher comparing to the other groups (p<0.05) (Tab. 3).
Decreasing tendency of inflammatory reaction has been noted. In 4 out of 12 cases no inflammatory cells were noticed. In the rest of the specimens the number of cells was minimal (Tab.4).

Histological and histomorphometric analysis 9 weeks post bone replacement with implanted material
The results of histological and histomorphometric analysis 9 weeks after implantation showed the highest amount of newly formed bone in the HAP+PLGA group and the lowest in the Bio-Oss group and empty defect. The difference between HAP+PLGA group and Bio-Oss group was found to be statistically significant (p˂0.001) (Figs. 5 and 6; Tab.5).
Nine weeks after the implantation of the materials, in 10 out of 12 specimens no histological signs of inflammatory reaction were discovered (Tab.6).

Discussion
The present study evaluated regenerative potential of various coatings for HAP bone substitutes. In HAP+PLGA group, the greatest amount of new formed bone was noticed, accompanied with the lowest number of inflammatory cells in all the investigated time cut offs.
Obtained results are the extension of the previous investigation that evaluated HAP+PLGA influence on the calvarial defect healing in period of 12 weeks after implantation 12 . The objectives of the current study were to determine whether bone healing could be achieved in three to nine weeks post-implantation, as well as to assess the influence of PEI coating on regeneration capacity of previously designed HAP scaffold. It has been demonstrated that the type of coating exhibits considerable impact on regeneration potential of the bone substitute. For instance, it should be observed that the newly formed bone ratio 6 weeks following the implantation was 3 times higher in the PLGA than in PEI coated group (4.3±0.7 % vs. 1.4±0.3 %, respectively). Similarly, the rate of mineralized bone at 9 weeks cut-off was almost 2 times higher in HAP+PLGA comparing to HAP+PEI group (28±4 % vs. 16.9±0.8 %, respectively). The results of the current study are promising: when compared to the "gold standard", Bio-Oss, ~50% more mineralized bone is observed in the HAP+PLGA group after six weeks and ~70% more mineralized bone at week 9. The HAP used in this study reaches adequate balance between material resorption and new bone formation. It is previously demonstrated that HAP coated with PLGA promotes adequate bone healing 12 weeks after implantation 16,21,22 . This study went a step further by introducing PEI as a novel coating substance. PEI is a typical poly-cationic polymer that contain a high density of protonated secondary amines. Despite the fact that cytotoxic effects of free PEI on many cells were documented, the protonated form has been most widely used as a gene delivery agent due to its high charge density 23 . In our investigations, CO 2 -modified PEI coatings were used in order to decrease the toxic properties of PEI 19, 24 .
Although the results obtained for HAP coated with PEI are inferior when confronting to HAP+PLGA, HAP+PEI showed superior healing capacity in comparison to Bio-Oss 6 and 9 weeks following implantation. In addition, all above mentioned results stand in line with the outcomes achieved by Tang et al. 29 , who used bio-inspired trimodal macro/micro/nano-porous scaffold loaded with recombinant human BMP-2 (TMS/rgBMP-2). They assumed that osteogenic promotion of TMS/rhBMP-2 mainly occurred in the first 8 weeks after implantation. Later on, tissue maturity was mostly depended on self-remodeling of the newly formed bone tissue. In the current study, the greatest amount of newly formed bone was found in the HAP+PLGA group after 6 weeks of regeneration, with the same trend prolonged to week 9 in both studies. Extensive angiogenesis and osteogenesis noticed in our specimens after 3 weeks of regeneration are in agreement with primary bone formation stage in Tang's et al study 29 . In the present study, lamellar bone was formed after 9 weeks of regeneration, which is close to 8 weeks found in Tang's et al investigation. Exogenous rhBMP-2 was important, but probably not the crucial factor for bone regeneration process in the mentioned study. Results of our experiments for periods of 6 and 9 weeks after implantation indicate that precise biological mechanism of bone forming after implantation remains unresolved. It is well known that bone regeneration imply biological events including bone induction and conduction, as well as several cell types and signalling pathways. Bone grafting includes osteoinduction (BMPs and other growth factors), osteogenesis (osteoprogenitor cells) and osteoconduction (scaffold) 30 . The used scaffold has a key role in supporting cell growth and tissue formation, as well as in providing appropriate microenvironment and structural integrity; additionally, it can support during cellular colonization and tissue regeneration. Suitable scaffold should also support direct cell growth and tissue formation by many growth factors, cytokines and signal molecules.
Biological mechanisms, which occur after scaffold implantation, e.g. scaffold biodegradation, still have to be elucidated.

Conclusion
The efforts of the current research were focused on modifying surface topography of novel HAP by means of PLGA and PEI coating in order to accelerate new bone tissue forming. Results of the study suggest that PLGA presents a superior coating option capable to considerably improve the bone regenerative potential of the synthetic HAP.