**Citation:** Örlygsson, G.; Laxdal, E.H.; Kárason, S.; Dagbjartsson, A.; Gunnarsson, E.; Ng, C.-H.; Einarsson, J.M.; Gíslason, J.; Jónsson, H., Jr. Mineralization in a Critical Size Bone-Gap in Sheep Tibia Improved by a Chitosan-Calcium Phosphate-Based Composite as Compared to Predicate Device. *Materials* **2022**, *15*, 838. https:// doi.org/10.3390/ma15030838

Academic Editor: Alessandro Pistone

Received: 29 December 2021 Accepted: 20 January 2022 Published: 22 January 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

the other group materials have properties that make them suitable to fill bone voids caused by trauma or disease. This second group is by far the larger of the two in terms of product excess, while the group intended for bone induction only consists of a very small number of products. As an example of a material intended for bone induction, synthetic grafts containing bone morphogenic protein (BMP), a recombinant growth factor, are effective in many applications [5]. However they are only approved for a limited number of indications due to safety concerns, such as heterotopic ossification [6,7].

Chitosan is a natural biopolymer that has been shown to promote both differentiation of stem cells into bone-forming osteoblasts and growth of bone colonies in in vitro studies [8–11], suggesting osteogenic properties. Results of in vivo studies indicate that chitosan alone is sufficient to stimulate osteogenesis [10,12,13]. Studies where other materials with osteogenic, osteo-inductive and/or osteoconductive properties were added to chitosan-based composites to further stimulate the bone healing process have also been described in the literature [14,15].

Chitosan is biodegradable and biocompatible and consists of a linear polysaccharide chain comprising randomly distributed β-(1→4)-linked D-glucosamine and N-acetyl-Dglucosamine units. It is derived from chitin, an insoluble linear β-(1→4)-linked N-acetyl-D-glucosamine polysaccharide, by deacetylation. An increased degree of deacetylation increases the solubility of the polymer by exposing positively charged amine groups of the glucosamine units [16]. Chitin itself serves as a structural material in the exoskeleton of arthropods and in the cell walls of fungi and is the second most abundant biopolymer in nature after cellulose.

In a previous study in a rat model the quantitative assessment of osteogenic effect of chitin derivatives was established and used to compare the osteogenic effect of deacetylated chitin derivatives, with degree of deacetylation from 50–96% [17]. The study showed that injectable implants containing 50% and 70% deacetylated highly pure chitin derivatives gave the highest bone volume regeneration of the materials tested. Therefore it was decided to compare the efficacy of a developmental product referred to as BoneReg-Inject™, which contains ChitobiomerTM, and an existing, similar and commercially available product, chronOS Inject®, by DePuy Synthes Inc., which served as a predicate device.

BoneReg-Inject™ is a calcium phosphate-based composition comprising tetra-calcium phosphate (TTCP, Ca4(PO4)2O) and α-tricalcium phosphate (α-TCP, α-Ca3(PO4)2) which reacts in situ to form hydroxyapatite (Ca10(PO4)6(OH)2) without excess heat formation. Calcium phosphate based bone cement was chosen as a base for the implants because of its widespread clinical use as well as biocompatible and osteoconductive properties [18]. ChronOS Inject®, however, is based on β-tricalcium phosphate (β-TCP, β-Ca3(PO4)2), monocalcium phosphate monohydrate (MCPM, Ca(H2PO4)2·H2O) and magnesium phosphate (MgHPO4·H2O), forming brushite (CaHPO4·2H2O) instead of hydroxyapatite. Both products contain acetylated biopolymers; chronOS Inject® contains sodium hyaluronate and BoneReg-Inject™ contains partially deacetylated chitin.

The aim of the study was to compare the performance of BoneReg-Inject™ and chronOS Inject® in a sheep tibia model with respect to new bone formation, calcification and effect on surrounding tissues on the basis of findings from X-ray micro-CT combined with histology after an observation period of 12 weeks, and to study the long-term effect of the BoneReg-Inject™ implant in a similar model with an observation period of 13 months.

#### **2. Materials and Methods**

#### *2.1. Implants*

The chitosan polymer used was produced from chitin from the shells of northern Atlantic shrimps (Pandalus borealis). Deacetylation and purification in multiple steps, involving a series of filtration and precipitation steps, was carried out as described earlier [19]. Extensive analytical work was performed to confirm purity and physico-chemical properties of the material [19,20].

Kits of the BoneReg-Inject™ implants were produced and packaged in the laboratories of Genis hf. The implants consisted of 3.0% *w*/*v* partially deacetylated chitin (chitosan polymer, 50% DDA) (Genis hf., Siglufjörður, Iceland), 30.9% *w*/*v* TTCP/α-TCP (Himed, Old Bethpage, NY, USA), 4.2% *w*/*v* sodium glycerol phosphate (Merck, Darmstadt, Germany), 1.9% *w*/*v* calcium hydroxide (Sigma-Aldrich, St. Louis, MO, USA), 7.7% *w*/*v* phosphoric acid (Merck) and water. These ingredients were packed into a solid and a liquid fraction and sterilized by 20 kGy γ-irradiation. Upon application, solid and liquid components were mixed directly by hand for 3 min; the cement was ready to use 7 min after mixing. The cement was designed to set within 30 min at room temperature but the setting can be delayed up to 2 h by cooling at 2–4 ◦C. The implant material is soft and injectable, and develops into a non-loadbearing solid (unpublished information) within 24 h, with increasing hydroxyapatite content in the further course of the setting reaction.

The predicate device, ChronOS Inject® (product number 710.066S, Lot No, 2538153), was purchased from Synthes AB, Korta Gatan 9, 171 54 Solna, Sweden.

#### *2.2. Experimental Design*

It was decided to use a sheep model and to drill holes (8 mm diameter × 30 mm length) into the proximal epiphyseal area of the tibia to inject a standardized volume of the BoneReg-Inject™ implant or chronOS Inject® into the drill holes in order to compare efficacy, resorption, osteoconductive and bone formation (osteogenic) properties in vivo by these two different injectable implants. One hole was drilled into the right leg and one hole was drilled into the left leg of each sheep. Implants were injected into right leg holes. Left leg holes were left empty to serve as control. This model was intended to serve as a representation of a trabecular bone loss associated with bicondylar fracture of the tibial plateau, involving central impaction and causing substantial loss of trabecular bone. The dimensions of the drill holes were chosen to serve this purpose and at the same time to avoid fracture risk [21,22]. The resulting epiphyseal bone void is typically filled with a bone graft or a bone graft substitute. An important aspect of this model is the involvement of cortical and trabecular bone tissues as well as fat and marrow tissues. The comparison was made by applying X-ray micro-CT and histological evaluation of preserved bone tissue after a live phase of 12 weeks (3 months). During a 13 month live period, the long-term effects of BoneReg-Inject™ were tested using the same model. Long term effects of chronOS Inject® were not assessed in this study which represents a limitation.

For the purpose of the study, 45 healthy female sheep, 5–7 years old and of a pristine Nordic breed, were acquired. Thirty sheep were randomly selected for the comparative 3 months study (15 for BoneReg-Inject™ and 15 for chronOS Inject*®*) and 15 sheep were randomly selected for the long-term 13 months study on BoneReg-Inject™. The randomization procedure took place before the animals arrived at the surgical facilities, and without the team having seen the sheep. Each sheep already had an identification number. Computer generated random numbers were used to divide the sheep into the 3 test groups, maintaining similar age distribution in all groups. One sheep in the 3 months chronOS Inject® group was diagnosed with *Mycobacterium paratuberculosis* infection in the lower intestines and was euthanized 3 weeks postoperatively. The average age of all animals was 5.9 years; 5.9 years in the BoneReg-Inject*™* short term group, 5.9 years in the chronOS Inject*®* group and 5.8 years in the long term BoneReg-Inject™ group. Bodyweight of the animals ranged from 53–86 kg, with an average of 65.6 kg. Average body weight of animals in the BoneReg-Inject™ and chronOS Inject*®* group was 64.8 kg and 66.4 kg respectively. The average body weight of animals in the long term BoneReg-InjectTM study was 60.5 kg. Changes in average body weight during the 13 months experiment was +3.24 kg in the BoneReg-Inject™ group, indicating that the animals were in good condition. The changes in body weight in the 3 months comparative study are not available. All 15 sheep in the 3 months BoneReg-Inject™ group, 14 sheep in the 3 months chronOS Inject® group and 15 sheep in the 13 months long term BoneReg-Inject™ group completed the trial alive and without any apparent morbidity.

Before the experiment, sheep were acclimatized for four weeks in the experimental facility (Institute for Experimental Pathology, University of Iceland, Keldur, 112 Reykjavík). During acclimatization and after the operation, animals were kept 3–4 together in a pen. Before the operation, the general health of the animals was confirmed and individual animals assigned to test groups to maintain similar age distribution within the groups. Before the operation the animals were not fed in the afternoon the day before operation and in the morning of the day of operation, but were allowed free access to water. Postoperatively, the sheep were fed hay twice daily (1–2 kg/day) and water ad-libitum.

All experiments were designed and conducted according to the FELASA guidelines. The study protocol was approved by the Icelandic Committee on Animal Experiments with the identification number 0709–0405/2009.

#### *2.3. Surgical Procedure and Sample Preparation*

At arrival to the operation theater, the sheep were weighed and injected i.v. with Chanazin 10% and Atropine sulfate. Further medication administered during the procedure is listed in Table 1. The sheep were prepared for surgery by shaving both hind upper legs around the knee and the left side of the neck, to expose the jugular vein. An i.v. catheter was inserted in the jugular vein and a bolus of ketamin 100 mg/50 kg administered. Following that a 3–5 mg/kg bolus of propofol was given. When the animal was anesthetized it was positioned on the right side on the operating table. The sheep breathed spontaneously during the operation receiving 6 L 100% oxygen via mask at the nostrils. Every 5–10 min during the operation a bolus of 1–2 mg/kg propofol was given i.v., more often if there were signs of movement. Continous monitoring of the anaesthesia depth was performed by an assigned person with no other duties during the operation. Monitoring of pulse and oxygenation was carried out with pulse oximetry at the tail after shaving it. The right hind leg was fixed to the table and the left leg fixed in an upright position.

**Table 1.** Medication and dosages.

The shaved areas on both hind legs were disinfected with Hibiscrub® and subsequently Betadine® solution. The bone surface was accessed through an incision parallel to and over the posterior border of the tibia. The entrance hole was localized centrally between the anterior and posterior borders of the bone at the level of the tuberositas. A hole of 8 mm in diameter was drilled at a right angle through the cortex whereafter the drill was redirected into a 45◦ upwards direction ending beneath the floor of the tibia plateau. After thorough rinsing of bone debris by flushing with 40–50 mL of sterile, saline water combined with suction, the right leg hole was filled with a standardized amount (1.5 mL) of the experimental test formulation (BoneReg-Inject™ or chronOS Inject®). The drill hole in the left leg was left void for comparison. Prior to application the BoneReg-Inject™ product was mixed according to a procedure described in the "Implants" section above

and kept at 2–4 ◦C until application to delay the setting time. ChronOS Inject® comes with a mixing device which was used to mix the two phases, the liquid and the solid, according to instructions from the manufacturer provided with the product. Within the time frame (3 min) given in product instructions the material was injected to fill the bone void. These materials were injected from a 5 mL sterile syringe mounted with a sterile pipette tip, ensuring that the material would fill the entire space of the drill hole from the tibia plateau down, and outwards, to the opening in the cortex. Thereafter the surgical wounds were closed with 4–0 running Vicryl subcutaneous sutures and the skin with 4–0 Ethilon continuous intracutaneous suture.

After surgery and wake-up, the animals were moved back to the sheep pen for recovery and vital signs monitored until they were ambulatory. All animals survived the anaesthesia and operation without complications.

Postoperatively, the animals received medication according to protocol (Table 1). They were monitored daily concerning behaviour, appearance, and appetite as an expression of general health and well-being. No stabilization such as cast was used and the sheep moved around and loaded their limbs as in normal conditions. In general, a drill hole of this size does not cause instability to the bone or the joint as has been confirmed in the literature [23]. Thus, monitoring through X-ray imaging was not considered necessary. The surgical wounds were regularly evaluated with respect to signs of infection, inflammation and dehiscence.

All animals completing the study appeared healthy and behaved normally throughout the study period. One sheep from the chronOS Inject® group was diagnosed 3 weeks postoperatively with *Mycobacterium paratuberculosis* and was immediately euthanized.

At termination after 3 and 13 months of recovery (short and long term study, respectively), sheep were euthanized by 10 mL/50 kg body weight i.v. injection of T 61. Left and right tibia were explanted at the knee joint and carefully cleaned from soft tissues. The tibia was sawed transversally about 4–5 cm below the proximal end maintaining the entire drill hole within the sample. Samples were placed in plastic jars with a tight lid for fixation in a solution of 3.7% formaldehyde in 50 mM phosphate buffer at pH 7.0. During the period of 11–32 days in the fixation media, samples were scanned in a micro CT-scanner. That is, samples had been between 11 and 32 days in the fixation media when CT-scanning was performed. The sample that had experienced the shortest fixation period had been fixed for 11 days, and the sample that had experienced the longest fixation period had been fixed for 32 days at the timepoint of CT-scanning. After the bones had been kept in the fixation media for 21–35 days and scanning was successfully completed, bone samples were sawed transversely through the epiphysis into 3–4 mm slices with a band saw. That is, the sawing of samples was started at day 21 after initial immersion in fixation media and the sawing was finished at day 35 after initial immersion in fixation media. The slices were photographed and returned into the fixation media and kept there at 22–24 ◦C under occasional stirring until a total fixation time of 73–94 days from initial immersion into the media. Thereafter, the slices were decalcified in 15% EDTA solution at neutral pH for 13 weeks, with 3 changes of solution during the decalcification period, as explained in the following. Selected sections were further prepared for histological evaluation.

#### *2.4. X-ray Micro CT Analysis*

Scanning was performed in an X-ray micro computed tomography (CT)-scanner (nanotom S, General Electric Inspection Technologies, Wunstorf, Germany). The tibia samples were scanned during the period of 11–32 days postmortem. Samples were fixed in a closed plastic cylinder filled with buffered formalin (3.7% formaldehyde in water), which could be mounted on the rotational table in the CT-scanner. Micro CT scanning was performed by transmitting adjusted dosages of X-rays through the sample while the sample was step wise rotated 360◦ in intervals of 0.5◦ , collecting 720 2D images from each sample. Magnification was 2.5, voxel size 20 µm/voxel edge, with exposure time of 3000 ms, frame averaging of 3, and 1 frame skipped. X-ray settings were 110 kV and 120 µA, using tube

mode 0 and no filter. Volume reconstruction was performed using the Datos-x software accompanying the CT-scanner. Data analysis of X-ray micro CT data was performed using Volume Graphics Studio Max 2.0 from Volume Graphics GmbH, Heidelberg, Germany.

Mineral content was assessed with the help of a hydroxyapatite (HA) phantom from QRM GmbH, Möhrendorf, Germany, which was scanned together with each sample. The phantom has 5 cylindrical inserts of 5 mm diameter with different partial densities of calcium hydroxyapatite of approximately 0, 50, 200, 800 and 1000 mg HA/cm3 . The reconstructed CT-data set from each sample was calibrated according to the HA200 insert, which has a partial HA density of 200.3 mg/cm3 ± 0.5%. In the following quantitative analysis the HA200 density was chosen as a lower limit, as this was found to include all traces of implanted fillers as well as mineralized tissue. The grayscale value obtained from the calibration for each sample was used to separate the grayscale range into two corresponding density ranges, one of lower density and the other of higher density than the HA200 standard. This value marked the border between mineralized and unmineralized material in the sample and was termed "iso-surface". The volume of all voxels within the defined volume under analysis (sphere or cylinder), having a grayscale value equal to or greater than the iso-surface value, were added up and defined as the "mineral phase volume" within the total volume under analysis. Hence, in the numerical mineral content calculations, the volume of all voxels with a density equal to HA200 or higher, divided by the total volume being analyzed, was defined as "mineral volume ratio" (volume with a density of ≥200 mg/cm3 divided by the total volume).

Mineralization was compared in specific volumes defined by virtual cylinders. A cylinder of 8 mm in length and with a radius of 3 mm was defined and carefully and concentrically orientated with the longitudinal axis in the direction of the drill hole (Figure 1). While keeping the orientation of the cylinder fixed, the radius was increased stepwise to 4, 5 and 6 mm and the mineral phase volume in each cylinder measured. This analysis was repeated in all samples, both empty holes and holes with implant (Figure 1). By subtracting the narrower cylinder from the broader, such as radius 3 mm from radius 4 mm (R4-R3), R5-R4 and R6-R5, mineral phase volume of a 1 mm outer shell (tube) of each cylinder was obtained. Mineral phase volume of the shell of all cylinders was normalized to a standard volume of 1 mm3 , obtaining mineral content with a density higher than that of the hydroxyapatite standard used.

To estimate mineral density inside the implants a virtual sphere with a radius of 2 mm was defined in the center of all implants/drill holes (Figure 1). Mineral phase volume (volume of voxels with a density ≥ 200 mg/cm3 ) inside the sphere was calculated. Mineral volume ratio was calculated as mineral phase volume/volume of sphere (33.5 mm3 ; r = 2 mm). For comparison to initial density of the two implant types (chronOS Inject® and BoneReg-Inject™), samples of implant composites were allowed to harden ex vivo and mineral phase volume measured in triplicates in 2–3 separate spheres in each sample. Mineral phase volume was measured after 24 h of setting ex vivo at 37 ◦C in saline water and after 18 months of storage in buffered formaldehyde solution at room temperature in order to exclude storage-influenced changes in mineral phase volume.

#### *2.5. Histology*

Histological analysis was used for qualitative identification of areas showing dense structures in the micro CT data and to characterize tissue responses to the implant. A ranking system was applied in order to extract statistical data form the histological images.

The medial condyle was sawed off in the sagittal plane, thus forming a plan to lay the tibia on to continue sectioning. Then the tibia plateau was axially (transverse) sawed off (2–3 mm) and subsequently 6–8 axial slices (3–4 mm) were made as far as beyond the cortical entrance of the drill hole. Thereafter all slices were put back into the fixative, carefully ensuring good access of the formaldehyde solution to the entire surface of each slice.

**Figure 1.** (**a**–**c**) show micro CT constructs in two different planes of samples after 3 months in vivo. (**a**) Sample derived from the left tibia (negative control, empty hole) of an animal receiving BoneReg-Inject™ in right leg drill hole, still essentially void of mineralized tissue. A rim of increased density integrated with the adjacent trabeculum on the edge surrounding the drill hole. The figure shows how virtual cylinders were created, concentric to the drill hole and with different radiuses. (**b**) BoneReg-Inject™ treated sample from the right tibia. A rim of mineralized tissue, well integrated with the adjacent trabecular bone, surrounding the implant, islands of dense material are scattered within the body of the implant. (**c**) chronOS Inject® treated sample from the right tibia. High density granules, characteristic for chronOS Inject® are apparent in the images. Mineralized new tissue is less pronounced in the chronOS Inject® group compared to the BoneReg-Inject™ group. (**d**) Illustration of virtual cylinders created. Length of all cylinders was 8 mm. The largest cylinder has a radius of 6 mm, the smallest has a radius of 3 mm and in the center is a sphere with radius of 2 mm. The cylinder with radius of 4 mm (8 mm in diameter) shows the border of the drill hole. To estimate mineral density inside the implants a virtual sphere with a radius of 2 **Figure 1.** (**a**–**c**) show micro CT constructs in two different planes of samples after 3 months in vivo. (**a**) Sample derived from the left tibia (negative control, empty hole) of an animal receiving BoneReg-Inject™ in right leg drill hole, still essentially void of mineralized tissue. A rim of increased density integrated with the adjacent trabeculum on the edge surrounding the drill hole. The figure shows how virtual cylinders were created, concentric to the drill hole and with different radiuses. (**b**) BoneReg-Inject™ treated sample from the right tibia. A rim of mineralized tissue, well integrated with the adjacent trabecular bone, surrounding the implant, islands of dense material are scattered within the body of the implant. (**c**) chronOS Inject® treated sample from the right tibia. High density granules, characteristic for chronOS Inject® are apparent in the images. Mineralized new tissue is less pronounced in the chronOS Inject® group compared to the BoneReg-Inject™ group. (**d**) Illustration of virtual cylinders created. Length of all cylinders was 8 mm. The largest cylinder has a radius of 6 mm, the smallest has a radius of 3 mm and in the center is a sphere with radius of 2 mm. The cylinder with radius of 4 mm (8 mm in diameter) shows the border of the drill hole.

mm was defined in the center of all implants/drill holes (Figure 1). Mineral phase volume (volume of voxels with a density ≥ 200 mg/cm3) inside the sphere was calculated. Mineral volume ratio was calculated as mineral phase volume/volume of sphere (33.5 mm3; r = 2 mm). For comparison to initial density of the two implant types (chronOS Inject® and BoneReg-Inject™), samples of implant composites were allowed to harden ex vivo and mineral phase volume measured in triplicates in 2–3 separate spheres in each sample. Mineral For further histological preparation, 3 slices were selected from each sample. The most proximal slice was the first slice under the tibial plateau where the implant could be seen macroscopically, containing the greatest amount of trabecular bone surrounding the implant. The most distal slice contained the cortical entrance of the drill hole. The third slice was selected from the slices between the proximal and the distal slices with marrow or fat tissue surrounding the implant. The three slices from each tibia were decalcified in 15%

EDTA (*w*/*v*) at pH 7.2–8.0 (EDTA (ethylenediaminetetraacetic acid disodium salt dihydrate), Sigma-Aldrich, St. Louis, MO, USA). The process of decalcification was monitored by micro CT analysis. Decalcification period was about 13 weeks, EDTA-solution was renewed every 3–4 weeks. After complete decalcification, the specimens were dehydrated and embedded in paraffin according to conventional histological protocol. To that end, tissue specimens were put into cassettes and tissue rinsed in running tap water for 30 min. The next step was the conventional method of tissue processing overnight in an automatic tissue processor (Tissue Tek VIP, Sakura Finetek, Inc., Torrance, CA, USA) ending with paraffin embedding where the tissue is embedded in paraffin wax at 63 ◦C. Paraffin blocks were then moved over to a cooling plate for hardening and after that to a microtome for sectioning (Leica RM2255, Leica Microsystems, Wetzlar, Germany). Disposable blades were used (Accu-Edge Disposable Blades, Sakura) for cutting sections at 3 µm thickness. Sections were mounted on coated microscope slides (Star Frost 76 mm× 26 mm, Knittel Glas GmbH, Braunschweig, Germany), dried in a 60 ◦C oven for 60 min. Ten sections were taken from each paraffin block at 12 µm intervals each and mounted on 10 Star Frost slides marked with specimen number and Roman numerals from I to X. After drying, H&E staining was performed using Shandon instant Hematoxylin and Eosin solution (Sigma-Aldrich).

For histological ranking samples from 13 sheep from the BoneReg-Inject™ group and samples from 12 sheep from the chronOS Inject® group were used, i.e., 25 sheep in total. Three sections per sample were considered. Two observers blindly examined all samples (25 × 3) separately, a total of 25 × 3 × 2 = 150 observations. This gives 6 observations for each sheep. An average was calculated for each sheep. The average was rounded up to the nearest integer of 0, 1, or 2 and the data analyzed for non-parametrical tests. The ranking (0 = none, 1 = moderate presence or 2 = strong presence) was performed with respect to a bone in implant, signs of cartilage tissue and signs of fibrous tissue.

#### *2.6. Statistical Analysis*

For statistical analysis, SigmaPlot 13 (Systat Software, Inc., San Jose, CA, USA) was used. ANOVA and Student's *t*-test (two-tailed) were used for parametrical comparison of groups. For non-parametrical comparison, ANOVA on ranks and Dunn's or Mann-Whitney test was applied. The level of significance was accepted at *p* < 0.05.

#### **3. Results**

#### *3.1. Short Term Study, 3 Months In Vivo*

3.1.1. Comparison of Mineral Phase Volume inside BoneReg-InjectTM and ChronOS Inject® Implants

Results of mineral phase volume calculations of the 2 mm radius sphere in the center of the implant after 24 h ex vivo and 3 months in vivo, presented in Figure 2a, indicate higher initial mineral phase volume in chronOS Inject® (32.9 mm3 ) compared to BoneReg-Inject™ (23.0 mm3 ). For comparison, the volume of a full sphere is 33.5 mm3 . However, during 3 months in vivo, the mineral phase volume of chronOS Inject® was reduced to 29.6 mm3 while the mineral phase volume of BoneReg-Inject™ was reduced to 18.2 mm3 . Figure 2b shows the same data when the initial mineral phase volume has been normalized to 100% for both implants. Hence, during 3 months in vivo, the mineral phase volume of BoneReg-Inject™ was reduced by 21% and chronOS Inject® by 10%. These data suggest that in this model, BoneReg-Inject™ breaks down significantly faster than chronOS Inject® (Student's *t*-test; *p* < 0.001).

#### 3.1.2. Comparison of Mineral Volume Ratio in the Rim of the Implants

Mean mineral volume ratios in concentric virtual cylinders and tubes (1 mm wall thickness) (Figure 1d) represent a gradient of mineral density reaching from the implant and into adjacent tissues (Figure 3). The higher mineral volume ratio of chronOS Inject® in the center of the implants was discussed in the previous section. The same can be observed comparing the mineral volume ratio within cylinders R3. This is reversed for the volume represented by tube R4-R3, forming the rim of the drill hole, where the mineral volume ratio of the BoneReg-Inject™ group was 53% higher than that for chronOS-Inject® group (Student's *t*-test, *p* < 0.001). On the other hand, no statistical difference was observed between the mineral volume ratio in both materials in the outer cylinders R5-R4 and R6-R5. As expected, the lowest mineral volume ratio was observed in the empty holes (Figure 3). The diameter of the drilling hole is 8 mm (radius 4 mm), suggesting that the BoneReg-Inject™ induced mineralization is a result of bone growth into the drill hole, where the new bone is replacing the implant material as the implant is resolved. A comparison between mineral volume ratio in cylinder R3 on one side and tube R4-R3 on the other side for each type of implant reveals that mineral volume ratio increases on going from R3 to R4-R3 in BoneReg-InjectTM, but decreases in chronOS Inject®. This points to increased bone formation in the BoneReg-InjectTM material compared to chronOS Inject®. Histological examination was used to clarify if the mineralized tissue was new bone, as well as possible differences between the groups in terms of tissue response to the two types of implant.

#### 3.1.3. Comparison of Closure of the Cortical Opening of the Drilled Hole

During the 3 months study period, the drill hole through the cortical bone of the tibia was closed to a different degree by bone growth emerging from the edges and striving to fill the hole. Estimated cortical closure of the drilled hole 3 months postoperatively was scored for all samples (0% = no growth, 100% = complete closure of cortical hole). BoneReg-Inject™ group scored highest (94.0%), then chronOS Inject® group (45.0%) and the empty holes had the lowest score (15.0%). However, a statistical difference was revealed between the BoneReg-Inject™ group and the empty hole group (*p* < 0.05), but no difference was found between the ChronOS Inject® and empty hole groups or between the two groups of implant materials (Figure 4). *Materials* **2022**, *15*, x FOR PEER REVIEW 9 of 18 *3.1. Short Term Study, 3 Months in Vivo*  3.1.1 Comparison of mineral phase volume inside BoneReg-InjectTM and chronOS Inject® implants. Results of mineral phase volume calculations of the 2 mm radius sphere in the center of the implant after 24 h ex vivo and 3 months in vivo, presented in Figure 2a, indicate higher initial mineral phase volume in chronOS Inject® (32.9 mm3) compared to BoneReg-

#### 3.1.4. Histological Examination Inject™ (23.0 mm3). For comparison, the volume of a full sphere is 33.5 mm3. However, during 3 months in vivo*,* the mineral phase volume of chronOS Inject® was reduced to

Figure 5 shows a series of images selected from two animals from each group representing a characteristic appearance of histological images from BoneReg-Inject™ and chronOS Inject®. Histological images are aligned with the corresponding micro CT sections in order to compare how dense structures in the micro CT data align with newly formed bone tissue. Figure 5e represents samples from each of the treatment groups showing the best case observed of neo-osteoid formation inside the BoneReg-Inject™ implant and the chronOS Inject® implant. 29.6 mm3 while the mineral phase volume of BoneReg-Inject™ was reduced to 18.2 mm3. Figure 2b shows the same data when the initial mineral phase volume has been normalized to 100% for both implants. Hence, during 3 months in vivo, the mineral phase volume of BoneReg-Inject™ was reduced by 21% and chronOS Inject® by 10%. These data suggest that in this model, BoneReg-Inject™ breaks down significantly faster than chronOS Inject® (Student's *t*-test; *p* < 0.001).

**Figure 2.** (**a**) Mineral phase volume inside the composites ex vivo (before implantation) and after 3 months in vivo*,* measured in spheres (radius 2 mm). (**b**) Bars show mineral phase volume before implantation normalized to 100% for both composites and their relative reduction during 3 months in vivo. Mineral phase volume in BoneReg-Inject™ group (*n* = 15) is reduced by 21% and in chronOS Inject® (*n* = 14) by 10%. (Student's *t*-test; *p* < 0.001). Means and SEM (BoneReg-Inject™ *n* = 15; chronOS Inject® *n* = 14). **Figure 2.** (**a**) Mineral phase volume inside the composites ex vivo (before implantation) and after 3 months in vivo, measured in spheres (radius 2 mm). (**b**) Bars show mineral phase volume before implantation normalized to 100% for both composites and their relative reduction during 3 months in vivo. Mineral phase volume in BoneReg-Inject™ group (*n* = 15) is reduced by 21% and in chronOS Inject® (*n* = 14) by 10%. (Student's *t*-test; *p* < 0.001). Means and SEM (BoneReg-Inject™ *n* = 15; chronOS Inject® *n* = 14).

thickness) (Figure 1d) represent a gradient of mineral density reaching from the implant and into adjacent tissues (Figure 3). The higher mineral volume ratio of chronOS Inject® in the center of the implants was discussed in the previous section. The same can be observed comparing the mineral volume ratio within cylinders R3. This is reversed for the volume represented by tube R4-R3, forming the rim of the drill hole, where the mineral volume ratio of the BoneReg-Inject™ group was 53% higher than that for chronOS-Inject® group (Student's *t*-test, *p* < 0.001). On the other hand, no statistical difference was observed between the mineral volume ratio in both materials in the outer cylinders R5-R4 and R6- R5. As expected, the lowest mineral volume ratio was observed in the empty holes (Figure 3). The diameter of the drilling hole is 8 mm (radius 4 mm), suggesting that the BoneReg-Inject™ induced mineralization is a result of bone growth into the drill hole, where the new bone is replacing the implant material as the implant is resolved. A comparison between mineral volume ratio in cylinder R3 on one side and tube R4-R3 on the other side for each type of implant reveals that mineral volume ratio increases on going from R3 to R4-R3 in BoneReg-InjectTM, but decreases in chronOS Inject®. This points to increased bone

*3.1.2. Comparison of Mineral Volume Ratio in the Rim of the Implants*

**Figure 3.** Bars indicate the mean mineral volume ratio in one virtual cylinder and 3 tubes in both experimental groups and the empty holes (negative control). The empty holes in both groups were summed up into a single control group. Results for a cylinder with a 3 mm radius and 3 different tubes with fixed volume are shown; R4-R3, R5-R4, and R6-R5 (see Figure 1 and insert above for location and alignment). The BoneReg-Inject™ group shows a significantly higher mineral volume ratio in a 1 mm rim of the implant (R4-R3) compared to the chronOS Inject® group (Student's *t*-test *p* < 0.001, indicated by three asterisks in the figure). Values are means and bars are standard error of the mean (SEM). R3 = radius 3 mm, R4 = radius 4 mm, etc. (BoneReg-Inject™ *n* = 15; chronOS Inject® *n* = 14). **Figure 3.** Bars indicate the mean mineral volume ratio in one virtual cylinder and 3 tubes in both experimental groups and the empty holes (negative control). The empty holes in both groups were summed up into a single control group. Results for a cylinder with a 3 mm radius and 3 different tubes with fixed volume are shown; R4-R3, R5-R4, and R6-R5 (see Figure 1 and insert above for location and alignment). The BoneReg-Inject™ group shows a significantly higher mineral volume ratio in a 1 mm rim of the implant (R4-R3) compared to the chronOS Inject® group (Student's *t*-test *p* < 0.001, indicated by three asterisks in the figure). Values are means and bars are standard error of the mean (SEM). R3 = radius 3 mm, R4 = radius 4 mm, etc. (BoneReg-Inject™ *n* = 15; chronOS Inject®*n* = 14). *Materials* **2022**, *15*, x FOR PEER REVIEW 11 of 18

formation in the BoneReg-InjectTM material compared to chronOS Inject®. Histological examination was used to clarify if the mineralized tissue was new bone, as well as possible differences between the groups in terms of tissue response to the two types of implant.

**Figure 4.** Scoring of percentage closure of cortical entrance hole in the two test groups and the empty holes in both groups 3 months postoperatively shown as box plot based on results from ANOVA on ranks, and only showing a significant difference between BoneReg-Inject™ and empty hole (Empty), Dunn's Method, *p* < 0.05. Boxes show a 50% confidence limit and bars show a 90% confidence limit. The median is indicated by the horizontal line within the boxes. (BoneReg-Inject™ *n* = 15; chronOS Inject® *n* = 14; Control *n* = 29). **Figure 4.** Scoring of percentage closure of cortical entrance hole in the two test groups and the empty holes in both groups 3 months postoperatively shown as box plot based on results from ANOVA on ranks, and only showing a significant difference between BoneReg-Inject™ and empty hole (Empty), Dunn's Method, *p* < 0.05. Boxes show a 50% confidence limit and bars show a 90% confidence limit. The median is indicated by the horizontal line within the boxes. (BoneReg-Inject™ *n* = 15; chronOS Inject® *n* = 14; Control *n* = 29).

Figure 5 shows a series of images selected from two animals from each group repre-

chronOS Inject®. Histological images are aligned with the corresponding micro CT sections in order to compare how dense structures in the micro CT data align with newly formed bone tissue. Figure 5e represents samples from each of the treatment groups showing the best case observed of neo-osteoid formation inside the BoneReg-Inject™ implant

*3.1.4. Histological Examination*

and the chronOS Inject® implant.

**Figure 5.** 3 months postoperative. (**a**) An alignment of histological sections with two-dimensional cross-sections of micro CT constructs in a bone marrow/trabecular bone area in the right legs of sheep with BoneReg-Inject™ implant (left column) and chronOS Inject® implant (right column). The left images in each column show the two-dimensional micro CT cross sections in the plane aligned with the histological section in the right image. The scale bars are 2 mm. BoneReg-Inject™ column: (**b**) Histological section, a close up of the area of the yellow frame shown. (**a**). The rim of neo-osteoid covering the implant is clearly visible (yellow arrows). BM: Bone marrow, FL: Fibrous layer, IP: Implant. (**c**) A close up of the right yellow frame in (**b**). New bone, connected to the bone shell covering the implant, has grown into the implant (yellow arrows). Characteristic blue deposits seen on the interface between new bone and the implant indicate areas of calcification. Osteoblast-like cells (OB) visible on the outer layer of the new bone shell. (**d**) A close up of the left yellow frame in (**b**). The rim of neo-osteoid covering the implant is evident (BL) and individual osteocytes are visible (yellow arrows). Osteoblast-like cells (OB) are visible on the outer layer of the bone shell. (**e**) Trabecular-like neo-osteoid formation inside the BoneReg-Inject™ implant. Blue indicates regions of on-going calcification. chronOS Inject® column: (**b**) A close up of the yellow frame area shown in (**a**). The fibrous tissue covering the implant is characteristic of the chronOS Inject® group. The characteristic granules are apparent in the implant. FT: Fibrous tissue, TB: Trabecular bone. (**c**) A close up of the yellow framed area in (**b**) showing the implant surrounded by fibrous tissue and bone marrow. It is characteristic for the chronOS Inject® group to see this layer of fibrous tissue surrounding the implant (\*). Blood vessels with red blood cells are evident both in the bone marrow and fibrous tissue layer (yellow arrows). (**d**) Implant surrounded by fibrous tissue. (**e**) Trabecular-like neo-osteoid (TB) formed inside the layer of fibrous tissue replacing the chronOS Inject® implant (IP), as it resolves. This formation of new bone was generally rare in the chronOS Inject® group and was poorly integrated with the existing surrounding trabecular bone. **Figure 5.** 3 months postoperative. (**a**) An alignment of histological sections with two-dimensional cross-sections of micro CT constructs in a bone marrow/trabecular bone area in the right legs of sheep with BoneReg-Inject™ implant (left column) and chronOS Inject® implant (right column). The left images in each column show the two-dimensional micro CT cross sections in the plane aligned with the histological section in the right image. The scale bars are 2 mm. BoneReg-Inject™ column: (**b**) Histological section, a close up of the area of the yellow frame shown. (**a**). The rim of neo-osteoid covering the implant is clearly visible (yellow arrows). BM: Bone marrow, FL: Fibrous layer, IP: Implant. (**c**) A close up of the right yellow frame in (**b**). New bone, connected to the bone shell covering the implant, has grown into the implant (yellow arrows). Characteristic blue deposits seen on the interface between new bone and the implant indicate areas of calcification. Osteoblast-like cells (OB) visible on the outer layer of the new bone shell. (**d**) A close up of the left yellow frame in (**b**). The rim of neo-osteoid covering the implant is evident (BL) and individual osteocytes are visible (yellow arrows). Osteoblast-like cells (OB) are visible on the outer layer of the bone shell. (**e**) Trabecular-like neo-osteoid formation inside the BoneReg-Inject™ implant. Blue indicates regions of on-going calcification. chronOS Inject® column: (**b**) A close up of the yellow frame area shown in (**a**). The fibrous tissue covering the implant is characteristic of the chronOS Inject® group. The characteristic granules are apparent in the implant. FT: Fibrous tissue, TB: Trabecular bone. (**c**) A close up of the yellow framed area in (**b**) showing the implant surrounded by fibrous tissue and bone marrow. It is characteristic for the chronOS Inject® group to see this layer of fibrous tissue surrounding the implant (\*). Blood vessels with red blood cells are evident both in the bone marrow and fibrous tissue layer (yellow arrows). (**d**) Implant surrounded by fibrous tissue. (**e**) Trabecular-like neo-osteoid (TB) formed inside the layer of fibrous tissue replacing the chronOS Inject® implant (IP), as it resolves. This formation of new bone was generally rare in the chronOS Inject® group and was poorly integrated with the existing surrounding trabecular bone.

A qualitative pathological evaluation, based on three sections from the right leg of each animal. revealed differences between BoneReg-Inject™ and the predicate device.

BoneReg-Inject™ Group: The implanted material was intensely eosinophilic and finely granular. Around the implant was a rim of neo-osteoid, its thickness approximately that of a normal bony trabecula. In some cases, there were signs of the inner border of the new osteoid undergoing calcification. The outer periphery of the rim was lined by a row of osteoblasts, osteoclasts being virtually absent. In some cases, neo-osteoid at the periphery of the rim was seen to connect with adjacent preexistent bone. No foreign body reaction was noted around the implant.

ChronOS Inject® Group: The implanted material was homogenous, weakly eosinophilic and punctuated by large perfectly round defects. Around the implant, most cases demonstrated signs of marked tissue reaction, consisting of granulation tissue, i.e., newly formed vessels, fibroblasts, and lymphocytes, as well as foreign body reaction, including epithelioid histiocytes and multinucleated giant cells of the foreign body type. No cytoplasmic inclusions were noted in giant cells. Neo-osteoid was inconspicuous, seen only in a minority of cases. Where seen, neo-osteoid was haphazardly present, without an apparent connection to preexistent bone trabeculae.

A histological ranking was used for comparison of samples from both groups. Samples were rated from 0–2 in terms of parameters of interest regarding group differences in the histological images. Significant differences between the two groups were revealed concerning: (1) amount of new bone within the implant (*p* < 0.001), (2) signs of cartilage tissue (*p* = 0.003) and (3) signs of fibrous tissue (*p* < 0.001). Table 2 shows the ranking outcome. The histological ranking showed more bone in implant, more signs of cartilage tissue and less fibrous tissue for BoneReg-InjectTM implants compared to the predicate device.

**Table 2.** Mann-Whitney rank sum test results. A total comparison of 25 sheep tibia, 13 of BoneReg-Inject™ and 12 of chronOS Inject® implants. When ranking histological samples (0, 1 or 2) with respect to a bone in implant, signs of cartilage tissue and signs of fibrous tissue, a statistical evaluation showed significant differences between the two groups. 0 = none; 1 = moderate presence; 2 = strong presence.

*3.2. Long Term Effect of BoneReg-InjectTM, 13 Months In Vivo*

3.2.1. X-ray Micro CT Analysis

Comparison of micro CT data of BoneReg-Inject™ implants at 3 and 13 months postoperative revealed continuous mineralization during the 10 months period. The new mineralized tissue appeared well integrated with the adjacent trabecular bone and appeared to be increasingly migrating into the implanted material.

Mineral volumes obtained using virtual cylinders defining specific volumes inside and surrounding the BoneReg-Inject™ implants were used to compare 3 and 13 months postoperatively (Figure 6).

modeling the implanted scaffold into a new mineralized bone tissue.

(*t*-test, *p* < 0.001). This might reflect a response to a weakening of the tissue structure im-

Comparing short and long term effect of BoneReg-Inject™ implants (BoneReg 3 versus BoneReg 13, Figure 6), there is a significant increase in mineral content of the R3 cylinders (*t*-test, *p* < 0.001) as well as for the R4-R3 tubes (*t*-test, *p* < 0.05). The mineral content of the adjacent outer trabecular bone tissue, R5-R4, and R6-R5, is not increased over time. This suggests that the implant possesses both osteoconductive and osteogenic activities, leading to the formation of new mineralized bone tissue in the implanted material, re-

**Figure 6.** Mineral volume ratio in a virtual cylinder (R3) and tubes R4-R3, R5-R4 and R6-R5 in BoneReg-Inject™ samples after 3 months (BoneReg 3) and 13 months (BoneReg 13) and in the empty control (Empty 3 and Empty 13). Mean and SEM (*n* = 15 for each treatment). Mineral content within the implanted scaffold at 13 months postoperatively was significantly higher than at 3 months postoperatively (R3 *t*-test, *p* < 0.001 and R4-R3 *t*-test, *p* < 0.05). In the empty drill holes, mineral content within R3 was significantly higher after 13 months (*t*-test, *p* < 0.001). A significant increase in mineral content was also observed in the trabecular bone outside the empty holes (R6-R5, *t*-test, *p* < 0.001), potentially reflecting a response to counteract weakening of the bone due to the drill hole. **Figure 6.** Mineral volume ratio in a virtual cylinder (R3) and tubes R4-R3, R5-R4 and R6-R5 in BoneReg-Inject™ samples after 3 months (BoneReg 3) and 13 months (BoneReg 13) and in the empty control (Empty 3 and Empty 13). Mean and SEM (*n* = 15 for each treatment). Mineral content within the implanted scaffold at 13 months postoperatively was significantly higher than at 3 months postoperatively (R3 *t*-test, *p* < 0.001 and R4-R3 *t*-test, *p* < 0.05). In the empty drill holes, mineral content within R3 was significantly higher after 13 months (*t*-test, *p* < 0.001). A significant increase in mineral content was also observed in the trabecular bone outside the empty holes (R6-R5, *t*-test, *p* < 0.001), potentially reflecting a response to counteract weakening of the bone due to the drill hole.

3.2.2. Histological Evaluation In the long-term 13 month group, the BoneReg-InjectTM implant is eosinophilic and finely granular in appearance. Around the implant is a rim of new bone with a trabecular structure. Trabecular spaces are filled with non-reactive bone marrow. The outer periphery of the new bone is lined by osteoblasts and in some cases connects with adjacent preexistent bone. Bone in-growth is observed in the implanted material, in many cases includ-When comparing the empty holes (Empty 3 and Empty 13), a significant increase is observed in the innermost cylinder (R3) during the 10 month period (*t*-test, *p* < 0.001). No significant difference is observed in the rim of the drill hole (R4-R3 or R5-R4) but there is a significant increase in the trabecular bone density in R6-R5 during the 10 months period (*t*-test, *p* < 0.001). This might reflect a response to a weakening of the tissue structure imposed by the drill hole.

ing its central part. The border between the implant and the new osteoid is often covered with blue deposits indicating calcification. There were no histological signs of inflammation. An example of a series of histological images is shown in Figure 7. Comparing short and long term effect of BoneReg-Inject™ implants (BoneReg 3 versus BoneReg 13, Figure 6), there is a significant increase in mineral content of the R3 cylinders (*t*-test, *p* < 0.001) as well as for the R4-R3 tubes (*t*-test, *p* < 0.05). The mineral content of the adjacent outer trabecular bone tissue, R5-R4, and R6-R5, is not increased over time. This suggests that the implant possesses both osteoconductive and osteogenic activities, leading to the formation of new mineralized bone tissue in the implanted material, remodeling the implanted scaffold into a new mineralized bone tissue.

#### 3.2.2. Histological Evaluation

posed by the drill hole.

In the long-term 13 month group, the BoneReg-InjectTM implant is eosinophilic and finely granular in appearance. Around the implant is a rim of new bone with a trabecular structure. Trabecular spaces are filled with non-reactive bone marrow. The outer periphery of the new bone is lined by osteoblasts and in some cases connects with adjacent preexistent bone. Bone in-growth is observed in the implanted material, in many cases including its central part. The border between the implant and the new osteoid is often covered with blue deposits indicating calcification. There were no histological signs of inflammation. An example of a series of histological images is shown in Figure 7.

**Figure 7.** BoneReg-Inject™. (**a**) Micro CT image (scale bar = 2 mm) and (**b**) a histological slide showing BoneReg-Inject™ implant at 13 months postoperative (scale bar = 2 mm). New bone is well integrated with the surrounding trabecular bone. Yellow frames on the histological slide refer to regions in focus in (**c**) (176) and (**d**) (177). In this animal, the micro CT image shows relatively welldeveloped colonization of mineralized tissue inside the implant. The histological slide indicates trabecular-like neo-osteoid replacing the resolved outer rim of the implant and integrating into the surrounding trabecular bone. (**c**,**d**) show neo-osteoid replacing the resolved surface of the implant and forming islands scattered inside the mass of the implant. Blue indicates calcification. Samples stained with hematoxylin and eosin. **Figure 7.** BoneReg-Inject™. (**a**) Micro CT image (scale bar = 2 mm) and (**b**) a histological slide showing BoneReg-Inject™ implant at 13 months postoperative (scale bar = 2 mm). New bone is well integrated with the surrounding trabecular bone. Yellow frames on the histological slide refer to regions in focus in (**c**) (176) and (**d**) (177). In this animal, the micro CT image shows relatively well-developed colonization of mineralized tissue inside the implant. The histological slide indicates trabecular-like neo-osteoid replacing the resolved outer rim of the implant and integrating into the surrounding trabecular bone. (**c**,**d**) show neo-osteoid replacing the resolved surface of the implant and forming islands scattered inside the mass of the implant. Blue indicates calcification. Samples stained with hematoxylin and eosin.

#### **4. Discussion 4. Discussion**

A significant difference was observed between the two experimental groups, BoneReg-Inject™ and chronOS Inject®, with more bone regeneration in the BoneReg-Inject™ group. A significant difference was observed between the two experimental groups, BoneReg-Inject™ and chronOS Inject®, with more bone regeneration in the BoneReg-Inject™ group.

The BoneReg-Inject™ group was characterized by the formation of a dense mineralized new bone tissue, surrounding the entire implant with scattered islands of new bone The BoneReg-Inject™ group was characterized by the formation of a dense mineralized new bone tissue, surrounding the entire implant with scattered islands of new bone formation throughout the interior of the implant.

formation throughout the interior of the implant. Quantitative analysis of mineral volume ratio revealed that the BoneReg-Inject™ implant underwent more rapid resolution compared to the chronOS Inject® implant. Higher mineral volume ratio was measured on the periphery of the BoneReg-Inject™ implant pointing to a more pronounced formation of new bone. Histological ranking results sup-Quantitative analysis of mineral volume ratio revealed that the BoneReg-Inject™ implant underwent more rapid resolution compared to the chronOS Inject® implant. Higher mineral volume ratio was measured on the periphery of the BoneReg-Inject™ implant pointing to a more pronounced formation of new bone. Histological ranking results supported this finding.

ported this finding. In the long term group implanted with BoneReg-Inject™, a continuing resolution of the implant was apparent and a continuous bone formation within the implant was observed. This indicates that the BoneReg-Inject™ bone void filler comprises a sustained stimulation of bone formation and is replaced by new bone tissue as the implant is re-In the long term group implanted with BoneReg-Inject™, a continuing resolution of the implant was apparent and a continuous bone formation within the implant was observed. This indicates that the BoneReg-Inject™ bone void filler comprises a sustained stimulation of bone formation and is replaced by new bone tissue as the implant is resolved with time.

solved with time. The results obtained here can be viewed in the light of results from experiments that our group performed in rat mandibular bone [17]. It was found that implants containing 50–70% DDA chitosan polymer may stimulate new bone growth. The main increase in bone volume took place in bone tissue surrounding the drill hole in the mandibular bone. The findings suggested that the increase in bone volume was the combined result of biomechanical stresses around the drill hole, and the implant material or components thereof. In the current experiment the same 50% DDA chitosan polymer was used. Here, a significant difference in mineral volume ratio outside the BoneReg-InjectTM implants compared The results obtained here can be viewed in the light of results from experiments that our group performed in rat mandibular bone [17]. It was found that implants containing 50–70% DDA chitosan polymer may stimulate new bone growth. The main increase in bone volume took place in bone tissue surrounding the drill hole in the mandibular bone. The findings suggested that the increase in bone volume was the combined result of biomechanical stresses around the drill hole, and the implant material or components thereof. In the current experiment the same 50% DDA chitosan polymer was used. Here, a significant difference in mineral volume ratio outside the BoneReg-InjectTM implants compared to the chronOS Inject® implants was not observed, but a non-significant trend

towards an increased mineral volume ratio outside the BoneReg-InjectTM implants could be identified. Less biomechanical stress is expected around the drill hole in the current study as compared to the rat study, as the ratio between the hole and the surrounding bone is lower, thus displaying relatively lower levels of mechanical stress. This might lead to a lower factor of biomechanical stimulation of bone growth. However, extensive formation of new bone was observed within the implant in the current study. This might reflect the better containment of the implant, so that biomechanical stimulation through the implant is more effective than in the rat mandibular model. Another explanation may be found in the differences in the biological nature of the mandibular bone compared to the tibial bone (intramembranous ossification vs. endochondral ossification) leading to different conditions for the evolvement of the effect of the chitosan polymer and thus bone formation within the implant in the tibia.

Various chitosan composites and chitosan coatings for bone tissue engineering materials have been tested and reported. A common feature of the research is that the chitosan used is highly deacetylated, as opposed to the chitosan employed in the current research. Chitosan preparation and dissolution strategies vary and different composite components are used, all potentially influencing the biological properties and activity of the chitosan. This often makes the comparison between results from different research groups complicated. Some researchers have observed a neutral effect of the use of chitosan in bone implants [24,25]. Several recent papers indicate a positive influence of chitosan on bone regeneration. A chitosan/dicarboxylic acid scaffold was shown in vivo to be an effective bone regeneration material with good osteoinductive and osteoconductive properties [26]. In a review paper, titanium surfaces functionalized with chitosan were concluded to have greater osseointegration capacity than uncoated controls [27]. Thitiset et al. [28], used chitoologosaccharide for preparation of composites that did show promising results in cell culture, as well as for new bone formation in vivo. The results from Thitiset et al. are remarkable in the light of our ideas on the mechanism of action of chitosan in bone regeneration where chitooligosachharides play a central role [17].

#### **5. Conclusions**

Our overall conclusion is that, in this non-weightbearing sheep tibia model, BoneReg-Inject™ showed osteogenic and osteoconductive properties without identifiable foreign body reaction. In the same model chronOS Inject® provokes foreign body reactions surrounding the implant with significantly less signs of new bone tissue formation. A longterm study indicates that the BoneReg-Inject™ implant stimulates sustained bone formation and is replaced by normal-looking new bone tissue.

For clinical use in bone defects, the current results point to an opportunity for the application of implant materials containing specific, partially deacetylated chitin derivatives, as an active component. The materials stimulate bone formation within the defect over an extended period and, while slowly degrading, facilitate bone ingrowth into the defect.

**Author Contributions:** Conceptualization, J.G. and H.J.J.; Methodology, E.H.L., S.K., A.D., C.-H.N., J.M.E., J.G. and H.J.J.; Validation, A.D. and J.G.; Formal Analysis, J.M.E.; Investigation, G.Ö., E.H.L., S.K., A.D., E.G., C.-H.N., J.M.E., J.G. and H.J.J.; Resources, E.G., C.-H.N., G.Ö., E.H.L., S.K., H.J.J. and J.G.; Data Curation, J.M.E. and G.Ö.; Writing—Original Draft Preparation, G.Ö., J.G. and J.M.E.; Writing—Review & Editing, G.Ö., E.H.L., S.K. and H.J.J.; Visualization, J.M.E. and G.Ö.; Supervision, J.G. and H.J.J.; Project Administration, J.G.; Funding Acquisition, J.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the Technology Development Fund, managed by the Icelandic Centre for Research [RAN<PHONE_NUMBER>].

**Institutional Review Board Statement:** The animal study protocol was approved by the Committee on Animal Experiments of the Icelandic Food and Veterinary Authority (protocol code: 0709-0405; date of approval: 13 June 2009).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author. The data are not publicly available due to the size of the X-ray microCT datasets.

**Acknowledgments:** We thank Jóhannes Björnsson and Kristrún Ólafsdóttir at the Department of Pathology, Landspítali University Hospital, Reykjavík, Iceland, for assistance with microscopical sectioning, staining, and analysis of histological samples.

**Conflicts of Interest:** Jóhannes Gíslason, Jón M. Einarsson and Chuen-How Ng are employees of Genis hf.

#### **References**

## *Article*

## **Evaluation of Osteoconduction of a Synthetic Hydroxyapatite**/β**-Tricalcium Phosphate Block Fixed in Rabbit Mandibles**

**Luis Carlos de Almeida Pires 1 , Rodrigo Capalbo da Silva 2 , Pier Paolo Poli 3,\* , Fernando Ruas Esgalha 1 , Henrique Hadad 2 , Letícia Pitol Palin 2 , Ana Flávia Piquera Santos 2 , Luara Teixiera Colombo 2 , Laís Kawamata de Jesus 2 , Ana Paula Farnezi Bassi 2 , Carlo Maiorana 3 , Roberta Okamoto 4 , Paulo Sérgio Perri de Carvalho 1 and Francisley Ávila Souza 2**

Received: 16 September 2020; Accepted: 29 October 2020; Published: 31 October 2020

**Abstract:** (1) Background: This study aimed to evaluate the incorporation of hydroxyapatite/β-tricalcium phosphate blocks grafted in rabbit mandibles. (2) Methods: Topographic characterization of biomaterial was performed through scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy (SEM-EDX). Ten rabbits randomly received autogenous bone graft harvested from the tibia (Autogenous Group—AG) or synthetic biomaterial manufactured in β-tricalcium phosphate (Biomaterial Group—BG) at their right and left mandibular angles. Euthanasia was performed at 30 and 60 postoperative days; (3) Results: SEM-EDX showed a surface with the formation of crystals clusters. Histological analyses in BG at 30 days showed a slower process of incorporation than AG. At 60 days, BG showed remnants of biomaterial enveloped by bone tissue in the anabolic modeling phase. Histometric analysis showed that mean values of newly formed bone-like tissue in the AG (6.56%/9.70%) were statistically higher compared to BG (3.14%/6.43%) in both periods, respectively. Immunohistochemical analysis demonstrated early bone formation and maturation in the AG with more intense osteopontin and osteocalcin staining. (4) Conclusions: The biomaterial proved to be a possible bone substitute, being incorporated into the receiving bed; however, it showed delayed bone incorporation compared to autogenous bone.

**Keywords:** biocompatibility; biomaterials; bone augmentation; bone conduction; bone grafting; calcium hydroxyapatite; tissue regeneration

#### **1. Introduction**

The results of alveolar bone remodeling after tooth loss, dentoalveolar trauma, and infections may promote an unfavorable site for dental implant rehabilitation with proper prosthetic planning [1]. Bone defects have different patterns and sizes, and their reconstruction can be classified into vertical and horizontal augmentation [2]. Several techniques have been proposed to treat bone defects, including bone substitutes from different origins such as autogenous, heterologous, and alloplastic materials [3,4].

In particular, block grafting is a recommended treatment in cases of horizontal defects, associated with severe atrophy of the alveolar bone [5–8]. In order to recreate the proper anatomy of the alveolar ridge, autogenous bone is considered the gold standard, being the only graft material that presents osteogenic, osteoinductive, and osteoconductive characteristics simultaneously [9]. On the other hand, the harvesting procedure is not free from drawbacks, including the risk of neurovascular injuries at the donor site, the need for an additional surgical site, an extended surgical time, an increasing post-operative patient morbidity, and a prolonged recovery time occasionally associated with hospitalization of the patient for extraoral donor sites [10]. Thus, research has been directed towards the use of biomaterials as autogenous bone substitutes when reconstructive surgery is needed. Currently, a wide range of synthetic or biological biomaterials is commercially available.

Among the different biomaterials, beta-tricalcium phosphate (β-TCP) ceramic has been introduced as a bone substitute [11]. β-TCP acting as a scaffold promotes bone formation through osteoconduction. As a result of its biocompatibility, bioactivity, favorable incorporation, and osteoconductive properties, synthetic β-TCP has been generally accepted and evaluated as a grafting material [12–14]. Its structural characteristic resembling the mineral phase of bone tissue is another factor that promotes great expectations for this material as a bone substitute [15]. As a disadvantage, biomaterials based on β-TCP present high solubility, which makes them unsuitable in reconstructions that need to maintain and create bone volume [16]. In order to reduce its rapid absorption, the association of β-TCP with hydroxyapatite (HA) in different proportions has been studied [17]. It was found that the biphasic composition consisting of HA and β-TCP provides better osteoconductive capabilities when compared to monophasic ceramics, decreasing osteoclastic activity allowing the formation of a framework for osteoconduction [18]. The search for the ideal ratio of HA and β-TCP, although controversial, is considered important because it modifies the solubility of the biomaterial and influences the resorption time. In a biocompatibility and biomechanical study, the association of 60%/40% HA/β-TCP has been tested and suggested as the ideal proportion of these components [19]. The combination of 60% HA/40% β-TCP has been used in a study to lift maxillary sinus in humans, obtaining favorable results for bone neoformation, proving to be an interesting ratio [20]. Different experimental studies in animals and clinical trials have demonstrated the favorable incorporation of such biphasic composition in the filling of critical defects in the calvarium, maxillary sinus, and alveolar socket preservation after dental extraction [14,21,22]. However, most studies employing β-TCP were performed in the reconstruction of two or more walls filled with cements or granular biomaterials, which may promote bone neoformation by taking advantage of the osteoconductive potential of the material. On the contrary, few studies are currently available that evaluate the incorporation of this biomaterial in the form of blocks for horizontal alveolar ridge augmentation [23,24].

In view of the above, the purpose of the present study was to analyze and compare the process of bone incorporation of autogenous bone graft and 60% HA/40% β-TCP blocks fixed by means of bicortical screws to the mandibular angles of rabbits.

#### **2. Materials and Methods**

#### *2.1. Study Design*

The study protocol was approved by the local Animal Experimentation Ethical Committee (CEEA) with the identification number 00999/2011. The sample size calculation was based on histometric and immunohistochemistry values retrieved from the authors' pilot analyses (unpublished data) before the

commencement of the study. New bone formation was determined as the primary outcome. Thus, a significant difference of 5% (standard deviation of 2%) was considered, and for 80% power and setting alpha at 0.05, five grafts per group were necessary in order to compare two different groups.

The sample consisted of 10 male New Zealand white adult rabbits with a weight ranging from 3 to 4 kg. The animals were kept in individual cages in a humidity- and temperature-controlled environment, on a light-dark cycle (12:12 h), and fed with water and rabbit chow (Procoelho-Primor, São Paulo, SP, Brazil) ad libitum. The surgical procedures were made at the local Rabbit Experimentation Center. After randomization using computer-generated lists on the website Research Randomizer (https://www.randomizer.org/), the animals received the bone blocks in their mandibles according to a split-mouth model as described below.

In the autogenous group (AG), an autogenous bone block harvested from the medial portion of the left tibial metaphysis was randomly grafted into the left or right mandibular angle. In the biomaterial group (BG), the synthetic biphasic calcium phosphate biomaterial (Grafts® BCP, Smart Bone Substitute, Aix-En-Provence, France) in the form of blocks was randomly grafted into the contralateral mandibular angle. The biomaterial was composed of a stable component (HA) and a bioactive bioabsorbable component (β-TCP) in a 60:40 ratio, respectively. The rationale was to provide a good balance between support for cell growth and graft absorption. The pores greater than 100 µm distributed over the entire surface of the material provided a degree of porosity of 70%. The relationship between porosity and the HA/TCP ratio represented a novelty that could provide greater cellular activity and maintenance of the trabecular architecture of the biomaterial.

#### *2.2. Topographic Characterization of the Biomaterial*

After covering the biomaterial with a thin layer of carbon, the surface topography of the biomaterial was analyzed using an electron microscope (SEM ZEISS, model EVO LS15, Oberkochen, Germany) to analyze the biomaterial surface morphology and its porosity, coupled with the X-ray dispersive energy spectroscopy system (Oxford model EDX microanalysis detector, Inca X-act, Oxford instruments, Abingdon, UK), for semi-quantitative analysis of the chemical composition of surfaces.

#### *2.3. Experimental Surgery*

General anesthesia was induced by intramuscular injections of ketamine hydrochloride (Vetaset—Fort Dodge Saúde Animal Ltd., Campinas, São Paulo, Brazil) at a dose of 50 mg/kg body weight associated with xylazine hydrochloride (Dopaser—Laboratório Calier do Brasil Ltd., Osasco, São Paulo, Brazil) at a dose of 5 mg/kg body weight. Subsequently, trichotomy and topical antisepsis were performed in both mandibular angle regions and in the left tibia with iodine solution (PVPI 10%, Riodeine, Rioquímica, São José do Rio Preto, Brazil). In addition, local infiltration of 0.3 mL/kg of mepivacaine hydrochloride (Scandicaine 2% with epinephrine 1:100,000, Septodont, Saint Maur des Fossés, France) was used as local anesthesia. A 2-cm dermal incision was made with a #15 scalpel blade (Feather Industries Ltd., Tokyo, Japan) mounted on a #3 scalpel handle in correspondence with each mandibular angle. Soft tissues were dissected respecting tissue planes reaching the periosteum. The periosteal tissue was finally incised in order to expose the bone tissue of the recipient area (Figure 1a). Bone decortication of the lateral plate of the mandibular angle was performed with a #701 rotary drill (Maillefer Instruments, Ballaigues, Switzerland), mounted on a straight surgical handpiece (Kavo do Brasil, Joinvile, Brazil), under constant irrigation of 0.9% sterile saline solution (Darrow, Rio de Janeiro, Brazil). In the AG, following the exposure of the recipient area, autogenous bone was harvested from the tibia. A 2-cm linear incision was made in correspondence with the left tibial metaphysis. A full-thickness skin flap, including skin, muscle, and periosteum, was raised exposing the surface of the bone. A circular osteotomy was performed (Figure 1b) by means of 8-mm internal diameter trephine bur (Neodent®, Curitiba, Paraná, Brazil) mounted on a contra-angle handpiece with a 20:1 reduction (Kavo® do Brasil, Joinvile, Brazil) at a speed of 1500 rpm under constant irrigation with 0.9% sodium chloride (Darrow, Rio de Janeiro, Brazil). The bone was carefully

removed from the trephine and preserved in a sterile saline solution. In the BG, biphasic calcium phosphate ceramic blocks were harvested using the same trephine size, in order to collect grafts of the same diameter in both experimental groups. Autogenous (Figure 1c) or synthetic block grafts (Figure 1d) were subsequently perforated with a 1.2 mm-diameter drill under copious irrigation with sterile saline approximately in their central portion, and randomly fixed to the left or right mandibular angle by means of compressive 1.6 × 8 mm osteosynthesis bicortical screws (SIN, Sistema de Implante Nacional, São Paulo, Brazil) resulting in appositional onlay-like block graft. The recipient bed was perforated with a 1.0 mm-diameter drill cooled with sterile saline in order to favor nourishment and revascularization of the block graft. removed from the trephine and preserved in a sterile saline solution. In the BG, biphasic calcium phosphate ceramic blocks were harvested using the same trephine size, in order to collect grafts of the same diameter in both experimental groups. Autogenous (Figure 1c) or synthetic block grafts (Figure 1d) were subsequently perforated with a 1.2 mm-diameter drill under copious irrigation with sterile saline approximately in their central portion, and randomly fixed to the left or right mandibular angle by means of compressive 1.6 × 8 mm osteosynthesis bicortical screws (SIN, Sistema de Implante Nacional, São Paulo, Brazil) resulting in appositional onlay-like block graft. The recipient bed was perforated with a 1.0 mm-diameter drill cooled with sterile saline in order to favor nourishment and revascularization of the block graft.

(**a**) (**b**)

**Figure 1.** Experimental surgery procedure: (**a**) Surgical exposure of the recipient bed; (**b**) Autogenous bone block grafting procedure; (**c**) Autogenous block grafted in the autogenous group; (**d**) Biomaterial block grafted in the biomaterial group. **Figure 1.** Experimental surgery procedure: (**a**) Surgical exposure of the recipient bed; (**b**) Autogenous bone block grafting procedure; (**c**) Autogenous block grafted in the autogenous group; (**d**) Biomaterial block grafted in the biomaterial group.

Soft tissues were carefully repositioned and sutured in different layers for primary wound closure using an absorbable suture (Polyglactin 910 – Vicryl 4.0, Ethicon, Johnson Prod., São José dos Campos, SP, Brazil), while a non-resorbable monofilament suture (Nylon 4.0, Ethicon, Johnson, São José dos Campos, SP, Brazil) was used for interrupted skin suturing. Additional antisepsis with PVPI was conducted after the suturing. Intramuscular pentabiotic (0.1 mL/kg, Fort Dodge Saúde Animal Ltda, SP, Brazil) was injected immediately after the surgery. A single dose of sodic dipyrone (1 mg/kg/day, Ariston Indústrias Químicas e Farmacêuticas Ltda, São Paulo, SP, Brazil) was also administered. Neither food nor movement restriction was applied to the animals that remained in individual cages during the experimental period. At 30 and 60 postoperative days, 5 animals in each group, respectively, were sacrificed by a lethal dose of pentobarbital (200 mg/kg). The soft tissues were then dissected, and the mandible of each rabbit was extracted. Osteotomies were performed on each mandible to obtain bone samples with at least 3 cm of margins circumferentially around the grafted area. All samples were immersion-fixed in 10% neutral buffered formalin (Reagentes Analíticos® , Dinâmica Odonto-Hospitalar Ltda, Catanduva, SP, Brazil). Soft tissues were carefully repositioned and sutured in different layers for primary wound closure using an absorbable suture (Polyglactin 910—Vicryl 4.0, Ethicon, Johnson Prod., São José dos Campos, SP, Brazil), while a non-resorbable monofilament suture (Nylon 4.0, Ethicon, Johnson, São José dos Campos, SP, Brazil) was used for interrupted skin suturing. Additional antisepsis with PVPI was conducted after the suturing. Intramuscular pentabiotic (0.1 mL/kg, Fort Dodge Saúde Animal Ltd., SP, Brazil) was injected immediately after the surgery. A single dose of sodic dipyrone (1 mg/kg/day, Ariston Indústrias Químicas e Farmacêuticas Ltd., São Paulo, SP, Brazil) was also administered. Neither food nor movement restriction was applied to the animals that remained in individual cages during the experimental period. At 30 and 60 postoperative days, 5 animals in each group, respectively, were sacrificed by a lethal dose of pentobarbital (200 mg/kg). The soft tissues were then dissected, and the mandible of each rabbit was extracted. Osteotomies were performed on each mandible to obtain bone samples with at least 3 cm of margins circumferentially around the grafted area. All samples were immersion-fixed in 10% neutral buffered formalin (Reagentes Analíticos®, Dinâmica Odonto-Hospitalar Ltd., Catanduva, SP, Brazil).

### *2.4. Histological Laboratorial Processing*

After fixing and washing for 24 h in running water, the descaled were decalcified in 20% EDTA (ethylenediaminetetraacetic acid, Merck, Darmstadt, Germany) dissolved in Milli-Q water, replaced weekly for a period of 6 weeks, at room temperature. Following decalcification, the osteosynthesis screws were carefully removed. The samples were then dehydrated in an ascending series of alcohol concentrations (70, 90, 95, and absolute alcohol), changing the solutions every hour in an orbital shaker (KLine CT—150®, Cientec—Equipamentos para Laboratório, Piracicaba, SP, Brazil). The samples were successively cleared in xylol, paraffin-embedded, and prepared using a precision saw to obtain 5 µm thick sections. The sections were mounted onto slides and stained with hematoxylin eosin (HE Merck & Co., Inc., Kenilworth, NJ, USA) for qualitative and histometric analysis, and stained by osteopontin (OP), osteocalcin (OC), and Tartrate-resistant Acid Phosphatase (TRAP) for the immunohistochemical analysis.

#### *2.5. Qualitative Histological Analysis*

Five slides of each sample were obtained for hematoxylin and eosin staining in the most central region of the piece. The qualitative evaluation was performed by a researcher, blinded with respect to the experimentation through a binocular optical microscope JENAMED 2 (Carl-Zeiss, Oberkochen, Germany), considering the internal part of the bone graft, the interface between the bone graft and the recipient bed, and the newly formed tissue.

#### *2.6. Histometric Analysis*

The capture of the region of interest involving the bone substitute/autogenous bone block, the interface between the graft and the recipient bed, and the recipient bed, was performed with a 40× magnification, considering the most central region identified by the contour of the fixation screw and the limits of the graft. The capture generated an image of the bone substitute, the recipient bed, and their interface. After obtaining the images, the quantitative analysis was performed using dedicated software (Image J version 1.53, NIH Image, Bethesda, MD, USA). The image and respective percentage value generated by the capture using the "rectangle" tool was considered as the total area to be evaluated. After that, through the integrated tool "polygon", the bone substitute, connective tissue, and newly formed bone tissue areas were delimited and the respective percentage areas proportional to the total area were calculated and submitted to statistical tests.

#### *2.7. Immunohistochemical Analysis*

The immunohistochemical analysis was performed in the Department of Basic Sciences, Araçatuba Dental School—UNESP (FAPESP, 2015/14688-0). The histological slices obtained after 30 and 60 days from the surgical procedure were used.

The immunohistochemical procedure started by deparaffinization and rehydration of the slices, and then endogenous peroxidase activity was inhibited with hydrogen peroxide. Endogenous biotin was blocked with skimmed milk. The primary antibodies (Santa Cruz Biotechnology, Inc., 10410 Finnell Street, Dallas, TX 75220 USA) were used against Osteopontin (OP—SC10593), Osteocalcin (OC—SC18319), and Tartrate-resistant Acid Phosphatase (TRAP—SC30832). OP and OC proteins are expressed during the mineralization process, where OP marks osteoblasts at the beginning of the mineralization process and OC is expressed in the late stages of the mineralization process. TRAP, on the other hand, is expressed by osteoclast activity, allowing the evaluation of the resorption process. The rabbit anti-goat IgG (H+L) secondary antibody, Biotin (Pierce Biotechnology, Waltham, MA, USA) was used and the reaction signal was amplified by streptavidin (Dako North America, Inc., 6392 Via Real Carpinteria, CA 93013, USA). The Diaminobenzidine (Dako North America, Inc., 6392 Via Real Carpinteria, CA 93013, USA) ends the reaction and then the slices were counterstained with Meyer Hematoxylin.

For each antibody used, the expression of proteins was evaluated semi-quantitatively by assigning different "scores" in accordance with the immunostained cells in the wound-healing process. The analysis was performed with an optical microscope (LeicaR DMLB, Heerbrugg, Switzerland) by means of scores (ordinal qualitative analysis); when the scores were light labeling (++), moderate labeling (+++), and intense labeling (++++) it was considered positive for diaminobenzidine, taking care to hold negative controls to evaluate the specificity of the antibodies. These scores and the methodology were established according to previous studies [25–27], where light labeling represented about 25% of the immunolabeling area in the blades, moderate labeling represented about 50%, and intense labeling represented about 75%. *Materials* **2020**, *13*, x FOR PEER REVIEW 6 of 17 process. The analysis was performed with an optical microscope (LeicaR DMLB, Heerbrugg, Switzerland) by means of scores (ordinal qualitative analysis); when the scores were light labeling (++), moderate labeling (+++), and intense labeling (++++) it was considered positive for diaminobenzidine, taking care to hold negative controls to evaluate the specificity of the antibodies. These scores and the methodology were established according to previous studies [25–27], where light labeling represented about 25% of the immunolabeling area in the blades, moderate labeling represented about 50%, and intense labeling represented about 75%.

#### *2.8. Statistical Analysis 2.8. Statistical Analysis*

An independent statistician performed the statistical analysis using IBM SPSS Statistics 24.0 (IBM Corp., Armonk, NY, USA). The results were initially submitted to the Shapiro–Wilk test to assess data distribution. After testing for normality, Student's *t*-test was performed for parametric data, while the Mann–Whitney U test was applied in cases of non-parametric data. A significance level of 0.05 was adopted for all tests. An independent statistician performed the statistical analysis using IBM SPSS Statistics 24.0 (IBM Corp, Armonk, NY, USA). The results were initially submitted to the Shapiro–Wilk test to assess data distribution. After testing for normality, Student's t-test was performed for parametric data, while the Mann–Whitney U test was applied in cases of non-parametric data. A significance level of 0.05 was adopted for all tests.

#### **3. Results 3. Results**

#### *3.1. Topographic Characterization of Biomaterial 3.1. Topographic Characterization of Biomaterial*

The SEM-EDX analysis evidenced a surface with the formation of crystals clusters and high macroporosity > 100 µm between it. The pores showed a crater shape and approximately 100 µm to 200 µm in diameter in multidirectional ways (Figure 2a). It was possible to observe regions with different heights on the same block surface (Figure 2b). The biomaterial base showed irregularities of the surface accompanied by its porosity, as well as the crystal clusters in different shapes and sizes (Figure 2c–e). The X-ray Dispersive Energy Spectroscopy analysis showed the amount of phosphorus (467), oxygen (372), and calcium (365) ions present, respectively, followed by a low percentage of magnesium (Figure 2f). The SEM-EDX analysis evidenced a surface with the formation of crystals clusters and high macroporosity > 100 μm between it. The pores showed a crater shape and approximately 100 µm to 200 µm in diameter in multidirectional ways (Figure 2a). It was possible to observe regions with different heights on the same block surface (Figure 2b). The biomaterial base showed irregularities of the surface accompanied by its porosity, as well as the crystal clusters in different shapes and sizes (Figure 2c–e). The X-ray Dispersive Energy Spectroscopy analysis showed the amount of phosphorus (467), oxygen (372), and calcium (365) ions present, respectively, followed by a low percentage of magnesium (Figure 2f).

**Figure 2.** *Cont.*

**Figure 2.** SEM-EDX analysis: (**a**–**e**) SEM views of the biomaterial surface at ×100, ×5,000, ×25,000, ×50,000, and ×100,000 magnifications, respectively. (**f**) EDX spectrometry values obtained before the surgery. The elements found were phosphorus, oxygen, calcium, and magnesium. **Figure 2.** SEM-EDX analysis: (**a**–**e**) SEM views of the biomaterial surface at ×100, ×5000, ×25,000, ×50,000, and ×100,000 magnifications, respectively. (**f**) EDX spectrometry values obtained before the surgery. The elements found were phosphorus, oxygen, calcium, and magnesium.

#### *3.2. Qualitative Histological Analysis 3.2. Qualitative Histological Analysis*

#### Autogenous group • Autogenous group

In the AG, after 30 healing days (Figure 3a), it was possible to observe the autogenous bone graft (A), and bone trabeculae under the receiving bed (B). The space between the graft and the recipient bed was occupied by bone tissue with a relevant number of trabeculae in the maturation stage with high cellular activity and good vascularization (C). Focusing on the graft (Figure 3b), autogenous bone (A) and newly formed bone (B) could be identified. In the inner part of the autogenous bone graft, small areas of resorption (black arrows) might indicate a phase of remodeling. In the AG, after 30 healing days (Figure 3a), it was possible to observe the autogenous bone graft (A), and bone trabeculae under the receiving bed (B). The space between the graft and the recipient bed was occupied by bone tissue with a relevant number of trabeculae in the maturation stage with high cellular activity and good vascularization (C). Focusing on the graft (Figure 3b), autogenous bone (A) and newly formed bone (B) could be identified. In the inner part of the autogenous bone graft, small areas of resorption (black arrows) might indicate a phase of remodeling.

**Figure 3.** Histological analysis of the autogenous group (AG) at 30 days. (**a**) Autogenous bone graft (A) positioned over the recipient bed (B), trabeculae in maturation stage and good vascularization (C); space previously occupied by the osteosynthesis screw is recognizable in (D). Hematoxylin and eosin stain at a magnification 40×; (**b**) Autogenous bone (A) newly formed bone (B), and small areas of resorption (black arrows). Hematoxylin and eosin stain at a magnification 125×. **Figure 3.** Histological analysis of the autogenous group (AG) at 30 days. (**a**) Autogenous bone graft (A) positioned over the recipient bed (B), trabeculae in maturation stage and good vascularization (C); space previously occupied by the osteosynthesis screw is recognizable in (D). Hematoxylin and eosin stain at a magnification 40×; (**b**) Autogenous bone (A) newly formed bone (B), and small areas of resorption (black arrows). Hematoxylin and eosin stain at a magnification 125×. **Figure 3.** Histological analysis of the autogenous group (AG) at 30 days. (**a**) Autogenous bone graft (A) positioned over the recipient bed (B), trabeculae in maturation stage and good vascularization (C); space previously occupied by the osteosynthesis screw is recognizable in (D). Hematoxylin and eosin stain at a magnification 40×; (**b**) Autogenous bone (A) newly formed bone (B), and small areas of resorption (black arrows). Hematoxylin and eosin stain at a magnification 125×.

(**a**) (**b**)

In the AG, after 60 post-operative days (Figure 4a), it was possible to observe the autogenous bone graft (A) in contact with the newly formed bone (B). Internally, the presence of connective tissue areas might suggest a remodeling phase of the bone. The identification of anucleated cells could suggest the presence of osteoblasts (red arrows). At the microscopic examination (Figure 4b), it was possible to distinguish the autogenous bone graft (A) and newly formed bone (B) separated by a cement line (black arrow). In the inner part of the autogenous bone graft, connective tissue areas were noticed, confirming the remodeling phase of the bone. Osteocytes were clearly visible (red arrows). In the AG, after 60 post-operative days (Figure 4a), it was possible to observe the autogenous bone graft (A) in contact with the newly formed bone (B). Internally, the presence of connective tissue areas might suggest a remodeling phase of the bone. The identification of anucleated cells could suggest the presence of osteoblasts (red arrows). At the microscopic examination (Figure 4b), it was possible to distinguish the autogenous bone graft (A) and newly formed bone (B) separated by a cement line (black arrow). In the inner part of the autogenous bone graft, connective tissue areas were noticed, confirming the remodeling phase of the bone. Osteocytes were clearly visible (red arrows). In the AG, after 60 post-operative days (Figure 4a), it was possible to observe the autogenous bone graft (A) in contact with the newly formed bone (B). Internally, the presence of connective tissue areas might suggest a remodeling phase of the bone. The identification of anucleated cells could suggest the presence of osteoblasts (red arrows). At the microscopic examination (Figure 4b), it was possible to distinguish the autogenous bone graft (A) and newly formed bone (B) separated by a cement line (black arrow). In the inner part of the autogenous bone graft, connective tissue areas were noticed, confirming the remodeling phase of the bone. Osteocytes were clearly visible (red arrows).

(**a**) (**b**) **Figure 4.** Histological analysis of the AG at 60 days. (**a**) Autogenous bone graft (A) in contact with the newly formed bone (B), and anucleated cells suggesting the presence of osteoblasts (red arrows). Hematoxylin and eosin stain at a magnification 40×; (**b**) Autogenous bone graft (A) and newly formed bone (B) separated by a cement line (black arrow) and osteocytes (red arrows). Hematoxylin and eosin **Figure 4.** Histological analysis of the AG at 60 days. (**a**) Autogenous bone graft (A) in contact with the newly formed bone (B), and anucleated cells suggesting the presence of osteoblasts (red arrows). Hematoxylin and eosin stain at a magnification 40×; (**b**) Autogenous bone graft (A) and newly formed bone (B) separated by a cement line (black arrow) and osteocytes (red arrows). Hematoxylin and eosin stain at a magnification 125×. **Figure 4.** Histological analysis of the AG at 60 days. (**a**) Autogenous bone graft (A) in contact with the newly formed bone (B), and anucleated cells suggesting the presence of osteoblasts (red arrows). Hematoxylin and eosin stain at a magnification 40×; (**b**) Autogenous bone graft (A) and newly formed bone (B) separated by a cement line (black arrow) and osteocytes (red arrows). Hematoxylin and eosin stain at a magnification 125×.

#### stain at a magnification 125×. Biomaterial group • Biomaterial group

 Biomaterial group In the BG, after 30 healing days (Figure 5a), it was possible to observe the biomaterial (A) in close contact with the newly formed bone (B), and the residual recipient site (C). In the surrounding areas of the neoformed bone, it was possible to identify a high number of lining cells compatible with osteoblasts (blue arrows) suggesting a phase of matrix synthesis. At higher magnification (Figure 5b), the contiguous contact between the biomaterial (A) and the neoformed bone (B) was appreciated. In the BG, after 30 healing days (Figure 5a), it was possible to observe the biomaterial (A) in close contact with the newly formed bone (B), and the residual recipient site (C). In the surrounding areas of the neoformed bone, it was possible to identify a high number of lining cells compatible with osteoblasts (blue arrows) suggesting a phase of matrix synthesis. At higher magnification (Figure 5b), the contiguous contact between the biomaterial (A) and the neoformed bone (B) was appreciated. Lining osteoblast cells were identified in the periphery of the newly formed bone (blue arrows). In the BG, after 30 healing days (Figure 5a), it was possible to observe the biomaterial (A) in close contact with the newly formed bone (B), and the residual recipient site (C). In the surrounding areas of the neoformed bone, it was possible to identify a high number of lining cells compatible with osteoblasts (blue arrows) suggesting a phase of matrix synthesis. At higher magnification (Figure 5b), the contiguous contact between the biomaterial (A) and the neoformed bone (B) was appreciated. Lining osteoblast cells were identified in the periphery of the newly formed bone (blue arrows).

Lining osteoblast cells were identified in the periphery of the newly formed bone (blue arrows).

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**Figure 5.** Histological analysis of the biomaterial group (BG) at 30 days. (**a**) Biomaterial (A) in close contact with the newly formed bone (B), and the residual recipient site (C); osteoblast-like lining cells are indicated with blue arrows. Hematoxylin and eosin stain at a magnification 40×; (**b**) Biomaterial (A), newly formed bone (B), and lining osteoblast cells in the periphery of the newly formed bone (blue arrows). Hematoxylin and eosin stain at a magnification 125×. **Figure 5.** Histological analysis of the biomaterial group (BG) at 30 days. (**a**) Biomaterial (A) in close contact with the newly formed bone (B), and the residual recipient site (C); osteoblast-like lining cells are indicated with blue arrows. Hematoxylin and eosin stain at a magnification 40×; (**b**) Biomaterial (A), newly formed bone (B), and lining osteoblast cells in the periphery of the newly formed bone (blue arrows). Hematoxylin and eosin stain at a magnification 125×. **Figure 5.** Histological analysis of the biomaterial group (BG) at 30 days. (**a**) Biomaterial (A) in close contact with the newly formed bone (B), and the residual recipient site (C); osteoblast-like lining cells are indicated with blue arrows. Hematoxylin and eosin stain at a magnification 40×; (**b**) Biomaterial (A), newly formed bone (B), and lining osteoblast cells in the periphery of the newly formed bone (blue arrows). Hematoxylin and eosin stain at a magnification 125×.

In the BG, after 60 post-operative days (Figure 6a), the biomaterial (A) was enwrapped by neoformed bone (B). In the inner part of the biomaterial, it was possible to observe islands of newly formed bone, highlighting the osteoconductivity capability of the bone substitute. At the same time, areas of resorption associated with volume loss were noticed, indicating high solubility of the biomaterial. At higher magnification (Figure 6b), it was possible to observe the bone substitute (A) on the recipient bed (B), and neoformed bone tissue (C). In the BG, after 60 post-operative days (Figure 6a), the biomaterial (A) was enwrapped by neoformed bone (B). In the inner part of the biomaterial, it was possible to observe islands of newly formed bone, highlighting the osteoconductivity capability of the bone substitute. At the same time, areas of resorption associated with volume loss were noticed, indicating high solubility of the biomaterial. At higher magnification (Figure 6b), it was possible to observe the bone substitute (A) on the recipient bed (B), and neoformed bone tissue (C). In the BG, after 60 post-operative days (Figure 6a), the biomaterial (A) was enwrapped by neoformed bone (B). In the inner part of the biomaterial, it was possible to observe islands of newly formed bone, highlighting the osteoconductivity capability of the bone substitute. At the same time, areas of resorption associated with volume loss were noticed, indicating high solubility of the biomaterial. At higher magnification (Figure 6b), it was possible to observe the bone substitute (A) on the recipient bed (B), and neoformed bone tissue (C).

(**a**) (**b**)

(**a**) (**b**) **Figure 6.** Histological analysis of the BG at 60 days. (**a**) Biomaterial (A) enwrapped by newly formed bone (B). Hematoxylin and eosin stain at a magnification 40×; (**b**) Biomaterial (A) on the recipient bed **Figure 6.** Histological analysis of the BG at 60 days. (**a**) Biomaterial (A) enwrapped by newly formed bone (B). Hematoxylin and eosin stain at a magnification 40×; (**b**) Biomaterial (A) on the recipient bed (B), and newly formed bone (C). Hematoxylin and eosin stain at a magnification 125×. bone (B). Hematoxylin and eosin stain at a magnification 40×; (**b**) Biomaterial (A) on the recipient bed (B), and newly formed bone (C). Hematoxylin and eosin stain at a magnification 125×.

#### (B), and newly formed bone (C). Hematoxylin and eosin stain at a magnification 125×. *3.3. Histometric Analysis*

*3.3. Histometric Analysis* The mean values of the neoformed bone tissue in the AG were 6.56% and 9.70% at 30 and 60 postoperative days, respectively, while the mean values of connective tissue were 47.83% and 45.34% in the same periods evaluated. Additionally, the mean values of autogenous graft remnants were 45.61% and 44.96%, respectively. For the BG, the mean values of newly formed bone tissue were 3.14% and 6.43% at 30 and 60 postoperative days, respectively, while the mean values of connective tissue were 41.58% and 45.34% in the same periods analyzed. The mean remaining values of the synthetic biomaterial based on β-tricalcium phosphate were 55.28% and 40.26%, respectively. The registered data were submitted to statistical analysis, namely t-test for parametric data (neoformed bone tissue, and bone substitute in both periods of analysis, and connective tissue at 60 days period), *3.3. Histometric Analysis* The mean values of the neoformed bone tissue in the AG were 6.56% and 9.70% at 30 and 60 postoperative days, respectively, while the mean values of connective tissue were 47.83% and 45.34% in the same periods evaluated. Additionally, the mean values of autogenous graft remnants were 45.61% and 44.96%, respectively. For the BG, the mean values of newly formed bone tissue were 3.14% and 6.43% at 30 and 60 postoperative days, respectively, while the mean values of connective tissue were 41.58% and 45.34% in the same periods analyzed. The mean remaining values of the synthetic biomaterial based on β-tricalcium phosphate were 55.28% and 40.26%, respectively. The registered data were submitted to statistical analysis, namely t-test for parametric data (neoformed bone tissue, and bone substitute in both periods of analysis, and connective tissue at 60 days period), and Mann–Whitney test for non-parametric data (connective tissue at 30 days period). The mean **Figure 6.** Histological analysis of the BG at 60 days. (**a**) Biomaterial (A) enwrapped by newly formed The mean values of the neoformed bone tissue in the AG were 6.56% and 9.70% at 30 and 60 postoperative days, respectively, while the mean values of connective tissue were 47.83% and 45.34% in the same periods evaluated. Additionally, the mean values of autogenous graft remnants were 45.61% and 44.96%, respectively. For the BG, the mean values of newly formed bone tissue were 3.14% and 6.43% at 30 and 60 postoperative days, respectively, while the mean values of connective tissue were 41.58% and 45.34% in the same periods analyzed. The mean remaining values of the synthetic biomaterial based on β-tricalcium phosphate were 55.28% and 40.26%, respectively. The registered data were submitted to statistical analysis, namely *t*-test for parametric data (neoformed bone tissue, and bone substitute in both periods of analysis, and connective tissue at 60 days period), and Mann–Whitney test for non-parametric data (connective tissue at 30 days period). The mean values of neoformed bone tissue in the AG were statistically higher when compared to the mean values

and Mann–Whitney test for non-parametric data (connective tissue at 30 days period). The mean

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of neoformed bone tissue in the BG at both experimental periods. Conversely, there was no statistically significant difference between groups in the amount of connective tissue and bone substitute in both experimental periods. The data of newly formed bone tissue, connective tissue, and remnants of autogenous graft and biomaterial for both groups are reported in Table 1.

**Period Group Bone Substitute Connective Tissue Newly Formed Bone** 30 days Biomaterial Autogenous 55.28% 4.61% 41.58% 47.83% 3.14% \* 6.56% \* 60 days Biomaterial Autogenous 40.26% 44.96% 53.31% 45.34% 6.43% \* 9.70% \*

**Table 1.** Analysis of the comparison between the biomaterial and autogenous bone at each experimental period.

\* Statistically significant difference between BG and AG.

## *3.4. Immunohistochemical Analysis*

At 30 postoperative days, in both groups, it was possible to observe bone tissue in the ongoing maturation phase, with neoformed bone trabeculae, and the presence of osteoblastic lineage cells. Already at 60 days, in both groups, organized, mature bone tissue, with the presence of osteocytes inside the gaps surrounded by a mineralized bone matrix, was noticed.

In the AG, at 30 days, it was possible to observe an intense osteopontin staining, showing a very intense (++++) initial bone mineralization activity, with positivity for the cells (osteoblasts) around the trabeculated bone, as well as the staining itself in the mineralized extracellular matrix (Figure 7a). In contrast, in the same period, in the BG, a light marking (++) was observed (Figure 7d), with a scarce mineralized extracellular matrix. *Materials* **2020**, *13*, x FOR PEER REVIEW 11 of 17

**Figure 7.** Osteopontin (OP), Osteocalcin (OC), and Tartrate-resistant Acid Phosphatase (TRAP) immunostaining at 30 days for the experimental groups. Immunolabeling is indicated by orange arrows. (**a**) OP immunostaining at 30 days in the AG; (**b**) OC immunostaining at 30 days in the AG; (**c**) TRAP immunostaining at 30 days in the AG; (**d**) OP immunostaining at 30 days in the BG; (**e**) OC immunostaining at 30 days in the BG; (**f**) TRAP immunostaining at 30 days in the BG. **Figure 7.** Osteopontin (OP), Osteocalcin (OC), and Tartrate-resistant Acid Phosphatase (TRAP) immunostaining at 30 days for the experimental groups. Immunolabeling is indicated by orange arrows. (**a**) OP immunostaining at 30 days in the AG; (**b**) OC immunostaining at 30 days in the AG; (**c**) TRAP immunostaining at 30 days in the AG; (**d**) OP immunostaining at 30 days in the BG; (**e**) OC immunostaining at 30 days in the BG; (**f**) TRAP immunostaining at 30 days in the BG.

At 60 days, in the AG, a moderate (+++) osteopontin staining was observed (Figure 8a), with the presence of positively stained cells adjacent to the bone tissue. It is worth noting the organization of the bone tissue visible in the region of interest. At the same time, osteocalcin was markedly intense (++++), with areas of precipitation on mature bone tissue (Figure 8b). Positively marked osteocytes were noted, characterizing the maturity of the bone tissue in this period. TRAP protein was lightly Regarding osteocalcin, a protein related to the late stage of mineralization of the extracellular matrix, a moderate (+++) staining was observed in the AG (Figure 7b), with cells of the osteoblastic lineage and the extracellular matrix presenting positive immunostaining for this protein. When the osteopontin and osteocalcin marking was correlated for this experimental group, it was observed that the bone mineralization activity was in the initial stages, compatible with the evaluation period

For the BG, light (++) staining was observed for osteopontin, osteocalcin, and TRAP, showing few cells stained for each of these proteins (Figure 8d–f). Regarding osteopontin and osteocalcin,

(++) marked, with few osteoclasts in resorption activity (Figure 8c).

stained in the region of interest.

listed in Table 2 and Table 3, respectively.

*Materials* **2020**, *13*, x; doi: FOR PEER REVIEW www.mdpi.com/journal/materials

The scores obtained from the immunohistochemical analysis in the periods of 30 and 60 days are

**Figure 8.** Osteopontin (OP), Osteocalcin (OC), and Tartrate-resistant Acid Phosphatase (TRAP) immunostaining at 60 days for the experimental groups. Immunolabeling is indicated by orange arrows. (**a**) OP immunostaining at 60 days in the AG; (**b**) OC immunostaining at 60 days in the AG; (**c**) TRAP immunostaining at 60 days in the AG; (**d**) OP immunostaining at 60 days in the BG; (**e**) OC

immunostaining at 60 days in the BG; (**f**) TRAP immunostaining at 60 days in the BG.

(30 days). In the BG, it was possible to notice positive osteocalcin light (++) staining in the pre-existing bone tissue, with osteoblast lineage cells and a positively stained extracellular matrix (Figure 7e). At 60 days, in the AG, a moderate (+++) osteopontin staining was observed (Figure 8a), with the presence of positively stained cells adjacent to the bone tissue. It is worth noting the organization of

**Figure 7.** Osteopontin (OP), Osteocalcin (OC), and Tartrate-resistant Acid Phosphatase (TRAP) immunostaining at 30 days for the experimental groups. Immunolabeling is indicated by orange arrows. (**a**) OP immunostaining at 30 days in the AG; (**b**) OC immunostaining at 30 days in the AG;

*Materials* **2020**, *13*, x FOR PEER REVIEW 11 of 17

The TRAP expression, showing osteoclasts in bone resorption activity, was light (++) in the AG (Figure 7c), with low osteoclast activity, and was moderate (+++) for the BG (Figure 7f), indicating resorption activity. the bone tissue visible in the region of interest. At the same time, osteocalcin was markedly intense (++++), with areas of precipitation on mature bone tissue (Figure 8b). Positively marked osteocytes were noted, characterizing the maturity of the bone tissue in this period. TRAP protein was lightly

At 60 days, in the AG, a moderate (+++) osteopontin staining was observed (Figure 8a), with the presence of positively stained cells adjacent to the bone tissue. It is worth noting the organization of the bone tissue visible in the region of interest. At the same time, osteocalcin was markedly intense (++++), with areas of precipitation on mature bone tissue (Figure 8b). Positively marked osteocytes were noted, characterizing the maturity of the bone tissue in this period. TRAP protein was lightly (++) marked, with few osteoclasts in resorption activity (Figure 8c). (++) marked, with few osteoclasts in resorption activity (Figure 8c). For the BG, light (++) staining was observed for osteopontin, osteocalcin, and TRAP, showing few cells stained for each of these proteins (Figure 8d–f). Regarding osteopontin and osteocalcin, which are extracellular matrix proteins presenting staining on the mineralized matrix, the low mineralization activity for both proteins in this later period stands out. There were also few TRAP stained in the region of interest.

**Figure 8.** Osteopontin (OP), Osteocalcin (OC), and Tartrate-resistant Acid Phosphatase (TRAP) immunostaining at 60 days for the experimental groups. Immunolabeling is indicated by orange arrows. (**a**) OP immunostaining at 60 days in the AG; (**b**) OC immunostaining at 60 days in the AG; (**c**) TRAP immunostaining at 60 days in the AG; (**d**) OP immunostaining at 60 days in the BG; (**e**) OC immunostaining at 60 days in the BG; (**f**) TRAP immunostaining at 60 days in the BG. **Figure 8.** Osteopontin (OP), Osteocalcin (OC), and Tartrate-resistant Acid Phosphatase (TRAP) immunostaining at 60 days for the experimental groups. Immunolabeling is indicated by orange arrows. (**a**) OP immunostaining at 60 days in the AG; (**b**) OC immunostaining at 60 days in the AG; (**c**) TRAP immunostaining at 60 days in the AG; (**d**) OP immunostaining at 60 days in the BG; (**e**) OC immunostaining at 60 days in the BG; (**f**) TRAP immunostaining at 60 days in the BG.

*Materials* **2020**, *13*, x; doi: FOR PEER REVIEW www.mdpi.com/journal/materials The scores obtained from the immunohistochemical analysis in the periods of 30 and 60 days are listed in Table 2 and Table 3, respectively. For the BG, light (++) staining was observed for osteopontin, osteocalcin, and TRAP, showing few cells stained for each of these proteins (Figure 8d–f). Regarding osteopontin and osteocalcin, which are extracellular matrix proteins presenting staining on the mineralized matrix, the low mineralization activity for both proteins in this later period stands out. There were also few TRAP stained in the region of interest.

The scores obtained from the immunohistochemical analysis in the periods of 30 and 60 days are listed in Tables 2 and 3, respectively.

**Table 2.** Osteopontin (OP), Osteocalcin (OC), and Tartrate-resistant Acid Phosphatase (TRAP) immunostaining at 30 days for the experimental groups.

**Table 3.** Osteopontin (OP), Osteocalcin (OC), and Tartrate-resistant Acid Phosphatase (TRAP) immunostaining at 60 days for the experimental groups.

#### **4. Discussion**

The ability of bioceramics to interact with osteoblastic cells is essentially related to their structural properties. Synthetic β-calcium-phosphate-based biomaterials present a porosity of approximately 70%. This type of conformation provides a considerable surface area supplied with several protein-binding sites available for a stable adhesion with a high number of osteoblasts [28]. Another factor that must be considered is the pore diameter. A previous study showed that wide pores with an accessible volume (>200 µm) exhibited an incremented proliferation and differentiation of osteoblasts within the scaffold, as a result of the revascularization process supplying oxygen and nourishment [29]. The same concept can be applied to calcium-phosphate-based biomaterials, in which the presence of pores allows the migration and proliferation of osteoblasts and mesenchymal cells along with the revascularization process needed to promote bone formation. In addition, a porous surface enhances the interconnection between the biomaterial and the surrounding bone tissue, creating high mechanical stability in the recipient bone–biomaterial interface [30]. The SEM showed a macroporosity > 100 µm of the synthetic biomaterial employed in the present study, which constitutes approximately 45% of its composition, thus facilitating the biological exchanges needed for the osteogenic process. Conversely, such porosity led to technical difficulties during the stabilization of the graft to the recipient bed during the experimental surgery. Higher strength was needed to tighten the screws while fixing the graft, with the risk of developing cracks and fractures of the blocks. Consequently, special attention was paid while fixing the biomaterial to the recipient bone in order to prevent block fractures during the intra- and post-operative periods. Indeed, the drilling technique in cases of such small-sized blocks is extremely challenging, especially for the biomaterial group, as the bone substitute is very friable. In addition, from an anatomical standpoint, the mandibular angle is particularly fragile, and requires care to obtain an effective fixation. Thus, the perforations were made in a direction that guaranteed the preservation of the block and the recipient bed in order to avoid complications such as the fracture of the block or the loss of stability in the fixation. This, however, often resulted in a non-centered position of the osteosynthesis screw, which may be considered a technical limitation during the histomorphometric analysis, as a central position of the screw would have been more helpful by serving as a reference guide during the cutting process. Energy-dispersive X-ray spectroscopy showed the presence of a low percentage of magnesium on the biomaterial surface. This component can promote the osteoimmunomodulatory properties, by improving osteoinduction by BMP signaling [31], being considered essential for bone tissue [32,33].

Autogenous bone graft is characterized by a relevant number of advantages with respect to most of the bone substitutes, particularly in terms of biocompatibility and regenerating potential. Autogenous bone is considered the gold standard due to its osteogenic properties, in addition to osteoinductive and osteoconductive capabilities, promoting the apposition of new bone tissue from the pre-existing bone as a model for new bone formation. In the present study, such properties have been validated when analyzing the microscopic images of the autogenous block graft at 60 post-operative days. Close contact between the recipient bed and the grafted autogenous block was observed, together with the presence of cement lines, newly formed bone associated with a high number of trabeculae, and vital osteocytes populating the inner portions of the graft. These biological events were promoted by the bone matrix, which induced the differentiation of osteoprogenitor cells of the recipient region into osteoblasts in order to stimulate bone formation. Those advantages and characteristics are supported by the expected results of the histometric analysis, where the AG presented a statistically significant difference in bone neoformation when compared to the BG. However, the autogenous graft needs a donor site, which contributed to the development of biomaterials, such as the HA-β-TCP used in the present experimental study, and although the histometric findings showed a greater amount of newly formed bone (bone incorporation) for the AG, the biomaterial was incorporated into the recipient bed and allowed bone formation by osteoconduction, which was expected. A previous study compared autogenous bone grafts with allogeneic grafts in block form through histological, histometric, and immunohistochemistry analyzes, showing the superiority of the autogenous bone in new bone formation and maturation of bone tissue during the incorporation process [34]. This highlights the need to search for an ideal biomaterial as an alternative to autogenous grafts.

Histological findings were confirmed by the immunostaining of OP and OC proteins. The marking of OP was intense at 30 days in the AG and light in the BG, showing earlier osteoblastic activity in the former. A delayed incorporation is confirmed within 60 days, when AG showed intense osteocalcin expression indicating bone maturation, whereas BG showed light expression of this protein, which represents the formation of an immature tissue in this period. In a previous study assessing critical-size defects in rat calvaria filled with autogenous bone or beta-tricalcium phosphate biomaterial, the authors evaluated the immunostaining of osteocalcin at 30 and 60 postoperative days [13]. It was observed that the AG showed higher protein expression at 60 days, indicating a greater maturation of the bone tissue formed compared to the biomaterial, which is in agreement with the results obtained herein.

Considering the donor site, the rabbit tibia was used to collect the autogenous graft. The reason is related to low post-operative morbidity, and ease of the surgical technique with respect to other donor sites. Harvesting bone from the rabbit calvaria leaves the dura-mater exposed due to an inability to separate the external and the internal cortical plates. The tibia is mainly characterized by cortical bone, with the presence of an intra-medullary canal. Hence, due to its composition, the tibia could be safely used to retrieve bone tissue in the form of blocks, with an adequate thickness for bone reconstructions [35]. On the other hand, in order to reduce the morbidity associated with the harvesting procedure, the research has been directed toward the use of bone substitutes. Among the wide range of biomaterials, synthetic bioceramics are gaining space in clinical scenarios as an alternative to autogenous bone grafts in maxillo-facial reconstructions. This study evaluated the incorporation of a biomaterial based on beta-tricalcium phosphate with hydroxyapatite compared to autogenous bone grafted in rabbit mandibles, in order to simulate critical clinical situations requiring horizontal augmentation in mandibular alveolar ridges, in which the use of biomaterial blocks may be indicated. However, for vertical defects, as an alternative to autogenous bone grafts, these biomaterials may not be the best clinical choice, or even contraindicated.

The biocompatibility and bifunctionality of calcium phosphate bioceramics in the substitution of bone tissue have been evaluated in vivo [36]. It was concluded that HA and β-TCP were progressively resorbed and substituted with newly formed bone, acting as a scaffold that could be favorably colonized by osteogenic cells. β-TCP has been compared to biphasic calcium phosphate ceramic containing HA/β-TCP in a 75:25 ratio in the process of trabecular bone repair in an animal model [30]. The histomorphometric analysis demonstrated that 36% of β-TCP, used alone, underwent resorption in 26 weeks, whereas the HA/β-TCP group healed undisturbed. Bone neoformation occurred in both groups; however, a higher amount of peripheral bone volume was observed in the biphasic ceramic group. Therefore, it was concluded that both bone substitutes were biocompatible and were characterized by a satisfying osteoconductive potential; nevertheless, the biodegradation of β-TCP was faster, losing the volume that should be maintained. A previous study evaluated β-TCP as a bone substitute in preserving the alveolar ridge for subsequent implant insertion in humans [37]. The ridge dimensions were measured before the grafting procedure and after a 6-month healing period at the second stage surgery for tissue sample harvesting and implant placement. Resorption of approximately 9% occurred during the healing time. After 6 months, the histological evaluation showed a complete resorption of the biomaterial, replaced by vital bone of good quality and quantity, which allowed a proper implant insertion. This might underline the osteoconductive properties of the biomaterial

associated with a high level of solubility. Accordingly, the same pattern has been observed during the histological analysis of the present study. However, it must be noticed that the biomaterial has been used in a more challenging clinical situation in the present study, namely, as an appositional block graft to increase the bone thickness, and not as a filling material in self-containing defects during socket preservation procedures. In addition, a recent study evaluated HA/β-TCP in the form of blocks [38]. The study assessed and compared qualitatively autogenous bone grafts versus a synthetic biomaterial based on HA/β-TCP in a 60:40 proportion. The biomaterial underwent gradual resorption followed by the apposition of bone tissue. Nonetheless, even at 60 post-operative days, it was possible to detect a certain amount of bone substitute in contact with newly formed bone and the recipient bed.

### **5. Conclusions**

In conclusion, the biomaterial based on β-tricalcium phosphate combined with hydroxyapatite evaluated in the present experimental study served as a scaffold, promoting bone formation in the grafted area and highlighting its osteoconductive property. However, there was a delay in the incorporation process when compared to the autogenous block graft.

**Author Contributions:** Conceptualization, F.A.S., A.P.F.B., P.S.P.d.C., R.O., C.M.; methodology, L.C.d.A.P., R.C.d.S., L.K.d.J., P.P.P., F.R.E., L.T.C., R.O.; validation, H.H., L.P.P., F.R.E., A.F.P.S.; formal analysis, L.C.d.A.P., R.C.d.S., L.K.d.J., F.Á.S., A.P.F.B., R.O.; investigation, P.S.P.d.C., C.M., H.H., F.R.E., F.Á.S.; resources, P.P.P., L.P.P., A.F.P.S., L.T.C.; data curation, H.H., P.P.P., L.C.d.A.P., R.C.d.S., L.K.d.J., L.P.P., A.F.P.S., L.T.C., R.O.; writing—original draft preparation, L.C.d.A.P., R.C.d.S., L.K.d.J., H.H., L.P.P., A.F.P.S., F.R.E., P.S.P.d.C.; writing—review and editing, P.P.P., L.T.C., C.M., F.Á.S., A.P.F.B., R.O.; visualization, L.C.d.A.P., R.C.d.S., L.K.d.J., H.H., A.F.P.S., L.P.P.; supervision, P.P.P., L.T.C., C.M., R.O.; project administration, F.Á.S., A.P.F.B., and P.S.P.d.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

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## *Article* **Sol–Gel Synthesis and Characterization of a Quaternary Bioglass for Bone Regeneration and Tissue Engineering**

**Ricardo Bento 1 , Anuraag Gaddam 1,2 and José M. F. Ferreira 1,\***

**\*** Correspondence<EMAIL_ADDRESS>Tel.: +351-234-370-242

**Abstract:** Sol–gel synthesis using inorganic and/or organic precursors that undergo hydrolysis and condensation at room temperature is a very attractive and less energetic method for preparing bioactive glass (BG) compositions, as an alternative to the melt-quenching process. When properly conducted, sol–gel synthesis might result in amorphous structures, with all of the components intimately mixed at the atomic scale. Moreover, developing new and better performing materials for bone tissue engineering is a growing concern, as the aging of the world's population leads to lower bone density and osteoporosis. This work describes the sol–gel synthesis of a novel quaternary silicate-based BG with the composition 60 SiO2–34 CaO–4 MgO–2 P2O5 (mol%), which was prepared using acidified distilled water as a single solvent. By controlling the kinetics of the hydrolysis and condensation steps, an amorphous glass structure could be obtained. The XRD results of samples calcined within the temperature range of 600–900 ◦C demonstrated that the amorphous nature was maintained until 800 ◦C, followed by partial crystallization at 900 ◦C. The specific surface area—an important factor in osteoconduction—was also evaluated over different temperatures, ranging from 160.6 ± 0.8 m2/g at 600 ◦C to 2.2 ± 0.1 m2/g at 900 ◦C, accompanied by consistent changes in average pore size and pore size distribution. The immersion of the BG particles in simulated body fluid (SBF) led to the formation of an extensive apatite layer on its surface. These overall results indicate that the proposed material is very promising for biomedical applications in bone regeneration and tissue engineering.

**Keywords:** bioactive glasses; alkali-free; sol–gel; bone regeneration; tissue engineering

## **1. Introduction**

Noticeable increases in life expectancy have been achieved over the past two centuries, as well documented in several literature reports [1,2]. The overall scientific progress made in the medical field, especially over the most recent decades, has greatly contributed to an increase in the population's quality of life and life expectancy. Such progresses together brought an unavoidable increase in the average age of the population, with social, economic, and medical consequences [3,4]. Older people are less capable of physical exertion, and the reduction of the mechanical stimuli of the bones, along with the degradation of the tissues due to a lifetime of use, increases the risk of fractures [5,6]. The consequent increased incidence of bone-related diseases (e.g., osteoporosis, trauma fractures, removal of tumours, etc.) challenges researchers to provide new therapeutic solutions to prevent the onset of osteopathy, and to develop bone grafts to treat these ailments. Autografts, although still considered the gold standard due to their optimal osteogenic, osteoinductive, and osteoconductive properties, have multiple drawbacks, including the donor site morbidity and their limited availability [7]. On the other hand, allografts are often associated with risk of infection and a high non-union rate with host tissue [8,9]. Therefore,

**Citation:** Bento, R.; Gaddam, A.; Ferreira, J.M.F. Sol–Gel Synthesis and Characterization of a Quaternary Bioglass for Bone Regeneration and Tissue Engineering. *Materials* **2021**, *14*, 4515. https://doi.org/10.3390/ ma14164515

Academic Editor: Gigliola Lusvardi

Received: 30 June 2021 Accepted: 4 August 2021 Published: 11 August 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

tissue engineering—and bone tissue engineering in particular—presents itself as a most promising alternative solution to the current bone grafting approaches [10].