**2.1. Materials and methods**

The research and all procedures involving live animals were processed after approval by the Clinical Hospital, School of Medicine, University of Buenos Aires, Argentina. Animals were maintained in keeping with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

After exposing the masseter muscle, subperiosteal elevation of the muscles detachment was performed exposing the body of the mandible. A standardized CSBD of 5 mm diameter on each side of the mandible was created with a slowly rotating trephine bur, under constant and copious irrigation with saline [33, 34]. Once the bone was excised, each biomaterial (SBM, Lot No: H13010002 or BO, Lot No 100148) was implanted in the corresponding side of the mandible according to the following groups: Control group: CSBD without bone graft; SBM group: CSBD in the right mandible filled with SBM; BO group: CSBD in the right mandible filed with BO. Animals were sacrificed at 4, 8, and 12 weeks postsurgery by a lethal intrave-

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nous administration of sodium pentobarbital (Euthanyle® Brouwer, S.A., Argentina).

observed and recorded.

*2.1.3. Bone mineral density evaluation in rats*

*2.1.4. Histological and histomorphometrical analysis*

Postoperatively, rabbits were given a 0.5ml subcutaneous injection of penicillin and streptomycin antibiotic (Dipenisol Retard, Bayer Argentina) every 48 hours for 7 days. In order to supplement the analgesia, intramuscular injection of 1 mg/kg of Nalbufina (Nalbufine 10 Richmond S.A, Argentina) was administered as premedication and every 12 hours during 3 days postsurgery. The 5-mm diameter CSBD created in the rabbit jaw for implantation with bone grafts is generally considered to be the appropriate critical size to evaluate bone graft materials [35–37]. Bone remodeling in the rabbit is approximately three times faster than in humans. Therefore, a healing period of 4 weeks was considered appropriate to evaluate the early response to bone healing. Likewise, for the evaluation of delayed bone repair, in relation to the amount of neoformed bone and the reabsorption of the biomaterials used, the period of 8 to 12 weeks was considered [38, 39]. In both experiments, the wound was closed with absorbable sutures, after filling each bone defect. Each animal surgical area was disinfected with iodine, and animal behavior was

Total skeleton bone mineral density and bone mineral content (BMD and BMC, respectively) were measured "in vivo" under light anesthesia at the end of the experiment (day 24) using a total body scanner with software designed specifically for small animals (DPX Alpha 8034, Small Animal Softer, Lunar Radiation Corp, Madison, WI) following a previously described technique [40]. Rats were scanned under light anesthesia. The precision of the software in determining total body BMD was assessed by measuring one rat five times after repositioning between scans, both on the same and on different days [40]. The coefficient of variation (CV) was 0.9% for total skeleton BMD. A specific region of interest (ROI) was manually traced at the site of the critical size bone defect for the first animal evaluated. Once established the ROI for the first animal, we used the same ROI to evaluate the BMD at the site of the bone defect in all the animals. The BMD CV of the studied area was 3.5% for the proximal tibia. All the analyses were carried out by the same technician in order to minimize inter-observer variation.

At the end of the experimental period, tibiae (experiment 1) and mandibles (experiment 2) were removed and cleaned of soft tissue. All specimens were prepared for histological evaluation according to a standard protocol for undecalcified sections, as previously described [41], and stained with hematoxylin-eosin. The analysis was "blinded" performed with respect to

## *2.1.1. Experiment 1 in rats*

A total of 12 young male adult Wistar rats (175 ± 10 g) were housed at room temperature (21 ± 1°C), 55 ± 10% humidity, under 12-hour light/dark cycles. They were fed a standard rodent diet (Ganave SA, Argentina) and deionized water "ad libitum." Body weight was recorded 3 times per week.

## *2.1.1.1. Surgical procedure*

Rats were anesthetized by intraperitoneal injection with ketamine hydrochloride [0.1 mg/100 g body weight (BW)] and acepromazine maleate (0.1 mg/100 g BW), (Holliday-Scott S A, Buenos Aires, Argentina). Hind legs were shaved, and the medial aspect of tibiae was exposed. A critical size bone defect (CSBD) (1.6 mm × 2 mm) was made with a fissure bur under copious irrigation with saline [32]. Bone defects were filled as follows: Group 1 (n = 6): The right tibia of each rat was filled with the bovine bone graft Synergy Bone Matrix (SBM), (Synergy Bone Matrix, Odontit Implant Systems, Argentina, Lot No: E11121216), while the left tibia was unfilled and used as control. Group 2 (n = 6): The right tibia of each rat was filled with the bovine bone substitute SBM (Lot No: E11121216) and the left tibia with the bovine bone substitute Bio-Oss (BO), (Bio-Oss, Geistlich, Switzerland, Lot No 100238).

Taking into account that the remodeling phase in the rat takes approximately 21 days, a healing period of 4 weeks was used to assess the late healing response. Animals were sacrificed after 24 days by carbon dioxide inhalation.

#### *2.1.2. Experiment 2 in rabbits*

A total of 15 adult male New Zealand rabbits (weight 3.0 ± 0.5 kg) were housed in individual cages with grid floating floor of stainless steel in a 12 m<sup>2</sup> insulated and equipped room with a continuous extraction system air renewal. They were maintained at room temperature (21 ± 1°C), 55 ± 10% relative humidity, and under 12-hours light/dark cycles. Cleaning and disinfection of excreta trays are made daily. Prior to surgery, animals had an adaptation period of 10 days. Pelleted commercial diet and deionized water were supplied *ad libitum* throughout the experiment with the exception of the first 3 days after surgery, in which they were fed with fresh leafy vegetables. Body weight was assessed 3 times per week during the morning.

#### *2.1.2.1. Surgical procedure*

All surgical procedures were carried out under aseptic conditions and in accordance with ISO 10993-6: 2007. The experimental surgery was performed under general anesthesia by an intramuscular injection of 1 mg/kg of acepromazine maleate and an intramuscular injection of ketamine hydrochloride and xylazine (Holliday-Scott S A, Buenos Aires, Argentina), at a dose of 35 and 5 mg/kg, respectively. The surgical site was shaved and scrubbed with iodine. A parallel skin incision was made along the inferior border of the mandible on both sides. After exposing the masseter muscle, subperiosteal elevation of the muscles detachment was performed exposing the body of the mandible. A standardized CSBD of 5 mm diameter on each side of the mandible was created with a slowly rotating trephine bur, under constant and copious irrigation with saline [33, 34]. Once the bone was excised, each biomaterial (SBM, Lot No: H13010002 or BO, Lot No 100148) was implanted in the corresponding side of the mandible according to the following groups: Control group: CSBD without bone graft; SBM group: CSBD in the right mandible filled with SBM; BO group: CSBD in the right mandible filed with BO. Animals were sacrificed at 4, 8, and 12 weeks postsurgery by a lethal intravenous administration of sodium pentobarbital (Euthanyle® Brouwer, S.A., Argentina).

Postoperatively, rabbits were given a 0.5ml subcutaneous injection of penicillin and streptomycin antibiotic (Dipenisol Retard, Bayer Argentina) every 48 hours for 7 days. In order to supplement the analgesia, intramuscular injection of 1 mg/kg of Nalbufina (Nalbufine 10 Richmond S.A, Argentina) was administered as premedication and every 12 hours during 3 days postsurgery.

The 5-mm diameter CSBD created in the rabbit jaw for implantation with bone grafts is generally considered to be the appropriate critical size to evaluate bone graft materials [35–37]. Bone remodeling in the rabbit is approximately three times faster than in humans. Therefore, a healing period of 4 weeks was considered appropriate to evaluate the early response to bone healing. Likewise, for the evaluation of delayed bone repair, in relation to the amount of neoformed bone and the reabsorption of the biomaterials used, the period of 8 to 12 weeks was considered [38, 39].

In both experiments, the wound was closed with absorbable sutures, after filling each bone defect. Each animal surgical area was disinfected with iodine, and animal behavior was observed and recorded.

#### *2.1.3. Bone mineral density evaluation in rats*

maintained in keeping with the National Institutes of Health Guide for the Care and Use of

76 Bone Grafting - Recent Advances with Special References to Cranio-Maxillofacial Surgery

A total of 12 young male adult Wistar rats (175 ± 10 g) were housed at room temperature (21 ± 1°C), 55 ± 10% humidity, under 12-hour light/dark cycles. They were fed a standard rodent diet (Ganave SA, Argentina) and deionized water "ad libitum." Body weight was

Rats were anesthetized by intraperitoneal injection with ketamine hydrochloride [0.1 mg/100 g body weight (BW)] and acepromazine maleate (0.1 mg/100 g BW), (Holliday-Scott S A, Buenos Aires, Argentina). Hind legs were shaved, and the medial aspect of tibiae was exposed. A critical size bone defect (CSBD) (1.6 mm × 2 mm) was made with a fissure bur under copious irrigation with saline [32]. Bone defects were filled as follows: Group 1 (n = 6): The right tibia of each rat was filled with the bovine bone graft Synergy Bone Matrix (SBM), (Synergy Bone Matrix, Odontit Implant Systems, Argentina, Lot No: E11121216), while the left tibia was unfilled and used as control. Group 2 (n = 6): The right tibia of each rat was filled with the bovine bone substitute SBM (Lot No: E11121216) and the left tibia with the bovine bone

Taking into account that the remodeling phase in the rat takes approximately 21 days, a healing period of 4 weeks was used to assess the late healing response. Animals were sacrificed

A total of 15 adult male New Zealand rabbits (weight 3.0 ± 0.5 kg) were housed in individual

a continuous extraction system air renewal. They were maintained at room temperature (21 ± 1°C), 55 ± 10% relative humidity, and under 12-hours light/dark cycles. Cleaning and disinfection of excreta trays are made daily. Prior to surgery, animals had an adaptation period of 10 days. Pelleted commercial diet and deionized water were supplied *ad libitum* throughout the experiment with the exception of the first 3 days after surgery, in which they were fed with fresh leafy vegetables. Body weight was assessed 3 times per week during the morning.

All surgical procedures were carried out under aseptic conditions and in accordance with ISO 10993-6: 2007. The experimental surgery was performed under general anesthesia by an intramuscular injection of 1 mg/kg of acepromazine maleate and an intramuscular injection of ketamine hydrochloride and xylazine (Holliday-Scott S A, Buenos Aires, Argentina), at a dose of 35 and 5 mg/kg, respectively. The surgical site was shaved and scrubbed with iodine. A parallel skin incision was made along the inferior border of the mandible on both sides.

insulated and equipped room with

substitute Bio-Oss (BO), (Bio-Oss, Geistlich, Switzerland, Lot No 100238).

after 24 days by carbon dioxide inhalation.

cages with grid floating floor of stainless steel in a 12 m<sup>2</sup>

*2.1.2. Experiment 2 in rabbits*

*2.1.2.1. Surgical procedure*

Laboratory Animals.

*2.1.1. Experiment 1 in rats*

recorded 3 times per week.

*2.1.1.1. Surgical procedure*

Total skeleton bone mineral density and bone mineral content (BMD and BMC, respectively) were measured "in vivo" under light anesthesia at the end of the experiment (day 24) using a total body scanner with software designed specifically for small animals (DPX Alpha 8034, Small Animal Softer, Lunar Radiation Corp, Madison, WI) following a previously described technique [40]. Rats were scanned under light anesthesia. The precision of the software in determining total body BMD was assessed by measuring one rat five times after repositioning between scans, both on the same and on different days [40]. The coefficient of variation (CV) was 0.9% for total skeleton BMD. A specific region of interest (ROI) was manually traced at the site of the critical size bone defect for the first animal evaluated. Once established the ROI for the first animal, we used the same ROI to evaluate the BMD at the site of the bone defect in all the animals. The BMD CV of the studied area was 3.5% for the proximal tibia. All the analyses were carried out by the same technician in order to minimize inter-observer variation.

#### *2.1.4. Histological and histomorphometrical analysis*

At the end of the experimental period, tibiae (experiment 1) and mandibles (experiment 2) were removed and cleaned of soft tissue. All specimens were prepared for histological evaluation according to a standard protocol for undecalcified sections, as previously described [41], and stained with hematoxylin-eosin. The analysis was "blinded" performed with respect to the rendered treatment. Images of the histological sections were captured by a digital camera (Olympus DP 10; Olympus Optical, Tokyo, Japan) connected to a light microscope (Olympus CX 31; Olympus Optical). Digital images were saved for static histomorphometrical analysis (experiment 2) using Image-Pro Plus 4.5 software. The following criteria were used to standardize the analysis: the total area (TA) to be analyzed was delineated on the composite image. The TA (mm<sup>2</sup> ) corresponded to the area of the mandible where the surgical defect was previously created, and it was considered 100% of the area to be analyzed. The newly formed bone area (NFBA) and the remaining graft particles area (RGPA) were then delineated. Both, NFBA and RGPA, were located entirely within the confines of the TA. The NFBA and RPGA were also calculated in mm<sup>2</sup> and the percentage calculated according to the following formula: 100-NFBA/TA. Values obtained from each animal were used to calculate the means and SD of each control and experimental group. We evaluated bone volume (% BV/TV): the percentage of cancellous bone within the total measured area and the percentage of remaining particles (% RP/TV) of either SBM or BO.

#### *2.1.5. Biomechanical tests in rabbits*

Biomechanical measurements were performed using a three-point bending test (Instron 4411, Universal Testing Materials). The equipment consists of a load frame in which is placed the material to be test (test tube) and a control console that provides calibration controls, programming, and test operation. The installed load cell allows the measurement of compressive forces exerted on the crosshead specimen. IX Series software was used to pick up data analysis of testing bone material. Control, SBM, and BO specimens were cleaned of soft tissues and cut in squares of 20 × 20 mm in the area where the bone defect was done. Bones were weighed and measured. Then they were placed one by one on the rollers, and the bone fracture was performed to evaluate elastic modulus (Mpa) and shear modulus (Mpa). Compression test was performed to evaluate compressive strength (KgF/mm<sup>2</sup> ).

*3.1.2. Histological analysis*

bone matrix groups (SBM) (\*p < 0.05 vs. control).

**3.2. Experiment 2 in rabbits**

*3.2.1. Histological analysis*

Cross-sections of tibiae showed remaining particles of each bovine bone graft in the area of the CSBD. Multiple particles of either SBM or BO, of different shapes and sizes, surrounded by laminar bone tissue were observed in the bone medullary space (**Figure 2A**, **B** and **C**). This finding indicates that both bone substitutes were osteoconductive. Proper bone healing was observed in tibiae from both groups. No signs of inflammation were observed; this result suggests biocompatibility (**Figure 2A**, **B** and **C**). After 4 weeks, blood vessels with small angiogenesis and revascularization foci formed in the CSBD implanted with either SBM or BO. The samples also showed mature Haversian systems forming a thin interface within the NBF represented by bone growth and surfaces covered by osteoblasts and fibroblast-like cells surrounding the bone grafts, which implied active osteogenesis. The present study suggests that the bovine bone graft SBM presented similar properties of biocompatibility without inflammatory signs to that of BO. Moreover, SBM also exhibited similar osteoconductive

**Figure 1.** Total skeleton bone mineral content (A) and bone mineral density (B) in control, Bio-Oss (BO) and synergy

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properties to BO, allowing a normal bone formation surrounding the particles.

As expected, control rabbits did not exhibited NBF at any of the studied times. Instead histological samples exhibited normal development of fatty bone marrow with occasional remnants of hematopoiesis foci in the site of the CSBD (**Figure 3A**, **D** and **G**). CZBD filled with each of the bovine bone grafts did not evidenced "foreign body" reaction at any of the studied times. Bone healing over time was accompanied by a progressive inflammatory response consistent with the expected histological stages of repairing. In addition, SBM and BO groups presented NBF characterized by trabecular bone growth and the presence of osteoblasts and fibroblastlike cells (**Figure 3**). Both samples exhibited blood vessel formation with small angiogenesis and revascularization foci and Haversian mature systems the implanted grafts, forming a

#### *2.1.6. Statistical analysis*

Results were expressed as mean ± standard deviation (SE). Data were analyzed using parametric tests according to data distribution and "a posteriori" tests. Data were analyzed using one-way analysis of variance (ANOVA). The Bonferroni multiple comparisons test was performed when significant differences were found. Student's t-test for independent samples was used to compare both bone grafts at each end point. Statistical analyses were performed using SPSS version 19 (SPSS Inc., Chicago, IL, USA). p < 0.05 was considered significant.
