Osseous Healing in Surgically Prepared Bone Defects Using Different Grafting Materials: An Experimental Study in Pigs.

Regeneration of large jaw bone defects still remains a clinical challenge. To avoid incomplete bone repair, bone grafts have been advocated to support the healing process. This study comparatively evaluated new bone formation among a synthetic graft substitute, a human bone derivative, and a bovine xenograft. Materials were placed in 3 out of the 4 bone cavities, while 1 deficit was left empty, serving as a control, in mono-cortical defects, surgically prepared in the porcine calvaria bone. Animals were randomized in 2 groups and euthanized at 8 and 12 weeks. Harvested tissue specimens were qualitatively evaluated by histology. New bone formation was quantitatively measured by histomorphometry. Maximum new bone formation was noticed in defects grafted with beta-tricalcium phosphate b-TCP compared to the other bone substitutes, at 8 and 12 weeks post-surgery. Bovine and human allograft induced less new bone formation compared to empty bone cavity. Histologic analysis revealed that b-TCP was absorbed and substituted significantly, while bovine and human allograft was maintained almost intact in close proximity with new bone. Based on our findings, higher new bone formation was detected in defects filled with b-TCP when compared to bovine and human graft substitutes.


Introduction
Regeneration of jaw bone deficits caused by accidents (traffic, occupational, sports, or shooting accidents), surgery (removal of benign lesions, malignant neoplasms, tooth extraction), congenital abnormalities (clefts or visceral skull bones hypoplasia), periodontal inflammation, and finally jaw atrophy (advanced age, systemic disease) still remains a clinical challenge [1].
To avoid the unfavorable outcome of incomplete bone repair, bone grafts have been advocated to support the healing process.
In general, the ideal grafting material should ultimately be fully replaced by high quality host bone, exhibiting synchronized degradation rate in relation to new bone development for complete regeneration [2]. cavity left to follow natural bone healing were all compared, both qualitatively and quantitatively, in surgically prepared osseous cavities using histologic and histomorphometric analysis.

Animals
This protocol meets the guidelines and rules for research on animals as stated by the Institutional Animal Care and Use Committee of the Veterinary Department, Greek Ministry of Rural Development and Veterinary, Attica Prefecture, Greece (1265/ 11-05-2015).
Sixteen adult male pigs (Landrace/Large White) were included in the study, acclimatized under the same environmental conditions for a period of 7 days before initiation of the experiment and fed with a balanced diet in individual cages to "N. S. Christeas", Laboratory of Experimental Surgery and Surgical Research, Medical School, University of Athens, Greece.

Surgical Procedure and Experimental Design
For induction of anesthesia, ketamine hydrochloride (Imalgene, Merial, Lyon, France) at a dosage of 25 mg/kg body weight combined with intramuscular injection of 2 mg/kg body weight xylazine (Rompun, Bayer Hellas AG, Athens, Greece) was applied. General anesthesia by orotracheal intubation was maintained with iv propofol (Diprivan, AstraZeneca, London, UK).
At the surgical field of interest, disinfection of the skin of the skull was achieved using povidone iodine (Betadine, Lavipharm, Athens, Greece) solution. Topical infiltration of local anesthesia with 4% articaine hydrochloride combined with 1:100,000 epinephrine (Ubistesin Forte, 3M ESPE, Athens, Greece) was also performed to induce hemostasis. Throughout the procedure, the animals were provided 100% oxygen and monitored for vital signs.
A semicircular incision was made in the skin over the top of the cranial vault, with subsequent elevation of a cutaneous full-thickness flap, exposing the calvaria bone ( Figure 1A). During the next step, 4 mono-cortical defects (10 mm depth/10 mm diameter) of cylindrical shape were performed-as previously described by Schlegel et al.-using a trephine bur (Meisinger, Neuss, Germany) with rotation speed of 250 rpm and flushing of 0.9% physiologic saline, with a 10 mm distance between them to avoid possible biologic interactions ( Figure 1B) [15]. The cylindrical bone segments were gradually mobilized and detached using a thin chisel ( Figure 1C). All sites were thoroughly debrided from soft tissues and rinsed with sterile saline. Bone grafts of synthetic (Calc-i-oss classic, Sunstar Guidor, Switzerland) bovine (Bio-Oss, Geistlich Pharma AG, Wolhusen, Switzerland) and human origin (Demokritos, National Centre for Scientific Research tissue bank, Athens, Greece) were placed in 3 out of the 4 defects, while 1 defect was left to heal without graft implantation, serving as a control (Table 1, Figure 1D). Wound closure was successfully accomplished using 3/0 silk sutures (Ethicon, Johnson & Johnson, Somerville, NJ, USA), followed by povidone iodine (Betadine, Lavipharm, Athens, Greece) solution, applied on the skin.  Animals were monitored for 2 h in the recovery room, further given appropriate intramuscular antibiotic prophylaxis with cefuroxime 750 mg (Zinacef, Glaxosmithkline, Athens, Greece) as well as an analgesic suppository of paracetamol (Depon, Bristol-Myers Squibb, Athens, Greece).

Sacrifice of Animals
The animals were randomly divided into 2 groups (8 animals per group) and sacrificed with an intravenous injection of sodium thiopental (Pentothal, Abbott Hellas, Athens, Greece) on the 8th and 12th post-operative week, respectively, to evaluate contribution of bone grafts to bone regeneration.
This was followed by the use of a mechanical saw to remove the skull bones containing the bone defects that were further cut into 4 mm-thick frontal sections. The 2 most representative sections from the central part of the deficits were selected for each bone defect and were embedded in tissue cassettes while the samples were further processed.

Histologic Analysis
The block specimens were fixed in 10% formalin for 2 days and subsequently decalcified in bone decalcification solution (Diapath S.p.a., Martinengo, Italy) for 21 days with a liquid change every 7 days. Later on, tissue specimens were embedded in paraffin and 3 μm-thick sections were prepared and stained with hematoxylin and eosin, properly examined under a light microscope (Nikon Eclipse 80, Nikon, Tokyo, Japan) at a minimum ×40 magnification. The entire section was evaluated for macroscopic parameters only qualitatively. Animals were monitored for 2 h in the recovery room, further given appropriate intramuscular antibiotic prophylaxis with cefuroxime 750 mg (Zinacef, Glaxosmithkline, Athens, Greece) as well as an analgesic suppository of paracetamol (Depon, Bristol-Myers Squibb, Athens, Greece).

Sacrifice of Animals
The animals were randomly divided into 2 groups (8 animals per group) and sacrificed with an intravenous injection of sodium thiopental (Pentothal, Abbott Hellas, Athens, Greece) on the 8th and 12th post-operative week, respectively, to evaluate contribution of bone grafts to bone regeneration.
This was followed by the use of a mechanical saw to remove the skull bones containing the bone defects that were further cut into 4 mm-thick frontal sections. The 2 most representative sections from the central part of the deficits were selected for each bone defect and were embedded in tissue cassettes while the samples were further processed.

Histologic Analysis
The block specimens were fixed in 10% formalin for 2 days and subsequently decalcified in bone decalcification solution (Diapath S.p.a., Martinengo, Italy) for 21 days with a liquid change every 7 days. Later on, tissue specimens were embedded in paraffin and 3 µm-thick sections were prepared and stained with hematoxylin and eosin, properly examined under a light microscope (Nikon Eclipse 80, Nikon, Tokyo, Japan) at a minimum ×40 magnification. The entire section was evaluated for macroscopic parameters only qualitatively.

Histomorphometric Analysis
Examinations were performed under a light microscope (Nikon Eclipse 80, Nikon, Tokyo, Japan) at a magnification ×40. From every pair of selected bone section, the most appropriate was selected and this area was acquired with a digital camera microscope unit (Nikon DS-2MW, Nikon, Tokyo, Japan). The sections were merged and processed using image analysis software (Image-Pro Plus v. 5.1, Media Cybernetics, Rockville, MD, USA) and the appropriate mask was created for the area of newly formed bone.
Histomorphometric evaluations consisted of measurements regarding the percentage of new-formed bone to the total volume of the site of interest (BV/TV) (bone volume/tissue volume).

Statistical Analysis
Pairwise comparisons were performed using the Bonferroni test. In case of non-normal data distribution, the Mann-Whitney test was used. Data were expressed as mean ± standard deviation. All tests were two-sided, and statistical significance was set at p < 0.05.

Overall
Surgical intervention was uneventful and during post-operative course till sacrifice, 15 animals exhibited no complication except 1 that died before completion of the 12 weeks healing period. Also, at the time points of euthanasia, no signs of local inflammation-infection around the skin of the skull were detected.
After debridement of the soft tissues, it was interesting that the areas of the bone defects appeared macroscopically similar, fulfilled with bone, exhibiting only few residual deficits. However, after frontal gross sectioning of the calvaria bone, it was noticed that at areas implanted with the grafting products, residual particles could be identified to a greater or lesser extent. As to empty cavities, it was observed that a part of the defect was fulfilled with a relatively soft tissue ( Figure 2).

Ηistology
Light microscopy allowed qualitative analysis as to bone healing and regeneration process within the defects. Comments are listed below: A. 8 weeks

Histology
Light microscopy allowed qualitative analysis as to bone healing and regeneration process within the defects. Comments are listed below: A. 8 weeks At 8 weeks post-surgery, histologic analysis exhibited presence of new bone tissue in all cavities filled with bone substitutes, woven and partially lamellar, while no signs of inflammatory response, necrosis, or foreign body reaction were observed. The graft particles were surrounded by or were in contact with lamellar bone, demonstrating good osteoconductivity and biocompatibility.
Spaces between the graft particles and the newly formed bone were characterized by copious mesenchymal cells and vessels while osteoblasts and multi-nucleated osteoclasts could be identified on the periphery of the grafts surface. Presence of residual graft was observed in the surgical defects filled with alloplastic material as well as the bovine and human graft. In particular, granules of bovine graft appeared stable, almost intact, without resorption lacunae in the context of the absorption process. In the empty deficits, small amounts of new bone tissue were formed at the margins of the defect with a tendency of extension towards the central part where the greater proportion was filled with a layer of fibrous connective tissue ( Figure 3A,C,E,G).

B. 12 weeks
At 12 weeks, more pronounced bone formation was noted, with lamellar bone being more prominent, surrounding all graft types, whereas, fibrous connective tissue was found to be less compared to mesenchymal tissue observed at 8 weeks.
It was obvious that b-TCP granules were deconstructed to a sufficient degree, with signs of both dissolution of the b-TCP particles and direct cellular resorption. These granules were predominantly B. 12 weeks At 12 weeks, more pronounced bone formation was noted, with lamellar bone being more prominent, surrounding all graft types, whereas, fibrous connective tissue was found to be less compared to mesenchymal tissue observed at 8 weeks.
It was obvious that b-TCP granules were deconstructed to a sufficient degree, with signs of both dissolution of the b-TCP particles and direct cellular resorption. These granules were predominantly embedded in the newly formed bone which was more interconnected than at 8 weeks, with signs of bone remodeling. In addition, the bovine and human allograft exhibited osseous integration with mature bone, to a greater extent, compared to 8 weeks, though particles seemed relatively intact, almost without evidence of absorption and bone substitution. In non-grafted cavities, bone maturation had increased significantly, mainly in the periphery of the deficits ( Figure 3B,D,F,H).

Histomorphometry
Results of histomorphometric analysis are shown in Table 2. Based on these findings, new bone formation was noticed, to a greater extent, in defects grafted with b-TCP compared to other substitutes in 8 and 12 weeks. An interesting finding was that while b-TCP exhibited less new bone compared to empty cavity at 8 weeks, this material surpassed unfilled defect bone formation at 12 weeks. Less bone formation volume was noticed in bovine and human allograft compared to empty cavity, in 8 and 12 weeks.

Discussion
During this project, histomorphometrιc evaluation revealed that bone defects filled with b-TCP were characterized by a more pronounced bone formation compared to other grafts in both 8 and 12 weeks post-surgery. These results seem to be in agreement with many other research protocols that described optimum bone regeneration in alloplastic bone substitutes containing b-TCP. A recent study measuring newly formed bone volume in intact extraction sockets fulfilled with a resorbable alloplastic biomaterial, consisting of b-TCP and polylactide-co-glycolide, concluded that osseous regeneration was more pronounced compared to the empty unassisted sites where natural healing occurred. This finding, however, was not statistically significant, possibly, according to authors, due to the minimum number of animals used and presence of factors such as maintenance of buccal bone plate after extraction and flapless surgery [16]. Also, another experiment using micro computed tomography-CT described that the highest vital bone content was noticed in defects grafted with b-TCP and calcium sulfate (b-TCP/CS), followed by sockets with no graft material and a bovine xenograft in rabbit calvaria defects, 8 weeks after implantation [17]. The objective of another recent article was to evaluate an in situ hardening biphasic hydroxyapatite/b-TCP bone graft substitute for ridge preservation after tooth extraction without primary wound closure or a barrier membrane. Histomorphometric analysis of core biopsy samples showed that using this substitute results in an effective preservation of the ridge contour and sufficient new bone formation [18]. A systematic review analyzed the outcomes of randomized controlled trials of socket grafting procedures performed with flapless extraction of teeth after a minimum healing period of 12 week. The mean histologic outcomes at or beyond the 12-week re-entry period revealed the highest vital bone content for sockets grafted with alloplasts, followed by sockets with no graft material, xenografts, and allografts [19]. According to another publication, b-TCP/CS implanted in cranial defects in rabbits resulted in 26.28% and 38.47% median values for new bone volume at 3 and 6 weeks, characterized by the authors as a biocompatible and osteoconductive material [20]. Yang et al., using micro-CT analysis to study the performance of a b-TCP/CS bone substitute compared to available polymethylmethacrylate cement and empty cavity in a sheep vertebral bone defect model, reported a higher new bone development of b-TCP/CS at 8, 16, and 36 weeks of observation [21]. In vivo histologic evaluations estimating the osteogenic potential of b-TCP/CS and human demineralized bone matrix putty in the mandible of rabbits confirmed that both grafts possess osteogenic activity and can support new bone formation, although at a slower rate than the spontaneous healing response [22]. Podaropoulos et al., evaluating with histomorphometric analysis b-TCP/CS in comparison with pure b-TCP in surgically prepared bone defects on the iliac crest of Beagle dogs, reported that b-TCP/CS produced 49.38% new bone compared to 40.31% using b-TCP alone and 17.77% on empty cavity, 4 months after implantation [23]. Our histologic findings confirm earlier animal studies documenting adequate resorption of b-TCP, while decrease of granules seems to be a combination of dissolution and direct cell-mediated resorption [24]. One possible hypothesis for relatively fast absorption of this bone substitute is its chemical composition, with a Ca/P ratio of 1.5, as well as its porous ceramic structure [3].
The fact that 2 observation periods were used in this study enabled the opportunity to identify changes in bone formation potential at the tissue level.
Indeed, an interesting finding of our study was that while at 8 weeks, b-TCP produced less new bone compared with empty cavity, at 12 weeks this grafting material was found to surpass empty cavity as to bone formation. Based on the results of our study, Hong et al. reported that the production of new bone in post-extraction sockets in dogs where b-TCP was inserted was more pronounced in the advanced maturation phase of bone healing at 8 weeks, while this material exhibited low bone formation at 2 and 4 weeks [24]. A relative analogous result is also described in a study where b-TCP exhibited delay compared to autograft in the early stages, but at 8 weeks presented more pronounced bone production [25]. These results, although initially raising questions about the efficacy of grafting materials, can be answered by considering the potential changes that these materials may have in the healing process. As already reported in previous studies, cellular growth processes with the addition of grafts may complicate, inhibit, or delay bone healing [26,27]. At this point, it is worth noting that at the molecular level, inflammatory cytokines such as IL-1b and TNFa may impair bone regeneration by inhibiting osteoblast differentiation and maturation as well as inducing osteoclast activity [28,29]. Trying to interpret results in our analysis, it seems that while inflammatory absorptive process for b-TCP is more acute at 8 weeks, it begins to cease during the maturation phase of bone healing at 12 weeks, allowing for more intense new bone formation.
As to bovine and human allograft used in our project, new bone formation was found to be less compared to b-TCP, possibly due to the slow resorption rate of the formers. These results reconfirm previous findings that long-term presence of residual non-resorbable or slowly resorbable particles of the graft might deregulate bone healing and the remodeling mechanism depending on the biodegradation pattern, having negative results on the overall quality, quantity, and architecture of the reconstructed bone [30]. These opinions raise dilemmas as to whether bovine graft is actually resorbable, an issue requiring further research. In line with this possibility, on our histological sections in the bovine graft, osteoclast-like multinucleated cells were identified in close approximation to graft granules, however, with absence of resorption lacunae. Multinucleated giant cells were also observed in relation to b-TCP, but, in contrast to the bovine graft, they were not arranged along the b-TCP remnants, rather being actively phagocytizing b-TCP particles, as described in previous studies [31]. Based on these findings, Artzi et al. found total resorption of b-TCP particles compared with inorganic bovine bone 24 months post-surgery in a canine model [32]. Mordenfeld et al., performing histologic and histomorphometric analysis of bone harvesting 11 years after sinus floor augmentation with deproteinized bovine and autogenous bone, stated that the xenograft particles were not resorbed but were well-integrated in lamellar bone, with no significant changes in particle size [33]. According to a recent study, evaluating bovine graft absorption after sinus elevation in rabbits, 8 weeks post-surgery, reported that this material is resorbed very slowly, leading to low new bone formation since biodegradation is an important factor for bone regeneration [34]. A relatively conflicting result in studies with bovine bone grafts is the fact that although new bone formation is not the maximum in this type compared to other grafts, total fractions of mineralized tissue (graft and bone) is sometimes found to be higher in relation to other grafts. One explanation might be that complete incorporation of the bovine graft particles within new bone, is leading to a dense, hard tissue network overall. This intermixed, dense, hard tissue element was also identified in our histologic sections, where osseous development was in direct contact with bovine graft particles, exhibiting lack of inflammation or connective tissue development. With respect to the human allograft as already reported, decreased bone regeneration and absorption rate of the fragments was observed in our analysis compared to b-TCP. A potential mechanism for these phenomena is the increased immune response that could inhibit bone regeneration [35]. According to another possible explanation, freeze-dried mineralized human allografts, like in our project, exhibit a slow rate of biodegradation and remain trapped within the surrounding newly formed bone, while after mineralization, resorption capability is suspended [36,37].
Based on our histologic observations, it is possible to argue that graft replacement and bone regeneration follow a different pattern in each of the grafting material examined at tissue level, a comment described by previous studies as well [26,38].
Though larger new bone production observed in our study within the empty cavity, compared to all bone grafts at 8 weeks and bovine and human graft substitute at 12 weeks, seems initially puzzling, it can be explained by the theory that the relatively reduced absorptive process of these materials delays the production of new bone. Drawbacks including the weakness of the empty cavity to support the structural integrity of bone deficit from soft tissue penetrance as well as incomplete fulfillment of the deficit but only in the periphery since growth factors are relatively less in concentration in such defects reinforce the use of grafting materials even if they exhibit some delay regarding bone regeneration.
The present study compared the osteogenic potential of bone grafts of synthetic, bovine, and human origin as well as natural bone healing of an empty cavity, in a previously established pig model by qualitatively and quantitatively analyzing new bone formation using histologic and histomorphometric analysis.
The results of this project are listed below: (1) b-TCP graft exhibited more pronounced new bone formation compared to bovine and human graft substitutes at 8 and 12 weeks; (2) While b-TCP induced less bone compared to empty cavity at 8 weeks, its bone production overcame empty defect during bone healing maturation at 12 weeks; (3) Bovine and human materials developed less bone in comparison to the control at 8 and 12 weeks; (4) According to histologic analysis, b-TCP was found to be characterized by the most significant rate of resorption between 8 and 12 weeks, compared to human allograft and bovine xenograft, where particles were spotted to be among these time moments almost intact, without signs of significant absorption ability. It seems that bone regeneration among these grafting materials follows different patterns.
Author Contributions: All of the named authors were involved in the work leading to the publication of this paper and have read the paper before this submission. Conceptualization, S.T. and E.E.; methodology, S.T., T.K. and E.E.; writing -original draft preparation, S.T., L.P. and D.K.; funding acquisition, S.T.; project administration, T.K., D.P. and G.A.; investigation, S.T. and G.A.; software, G.A.; data curation, G.A. and C.K.; writing-review and editing, S.T., L.P., G.A., D.K. and C.K. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.