materials Article Biofunctionalization with a TGFβ-1 Inhibitor Peptide in the Osseointegration of Synthetic Bone Grafts: An In Vivo Study in Beagle Dogs Andrea Cirera 1, * , Maria Cristina Manzanares 2 , Pablo Sevilla 3 , Monica Ortiz-Hernandez 4 , Pablo Galindo-Moreno 1 and Javier Gil 5, * 1 Oral Surgery and Implant Dentistry Department, School of Dentistry, University of Granada, Campus Universitario La Cartuja s/n, 18071 Granada, Spain; pgalindo@ugr.es 2 Human Anatomy and Embryology Unit, DPyTEx, School of Medicine, University of Barcelona, 08036 Barcelona, Spain; mcmanzanares@ub.edu 3 Universitary School Salesiana de Sarrià—EUSS Autonomous University of Barcelona, 08017 Barcelona, Spain; Psevilla@euss.es 4 Biomaterials, Biomechanics and Tissue Engineering group (BBT), EEBE, Technical University of Catalonia (UPC), 08019 Barcelona, Spain; Monica.ortiz-hernandez@upc.edu 5 Bioengineering Institute of Technology, School of Dentistry, Universitat Internacional de Catalunya, 08195 Barcelona, Spain * Correspondence: andreacirera@gmail.com (A.C.); xavier.gil@uic.cat (J.G.)  Received: 8 July 2019; Accepted: 24 September 2019; Published: 27 September 2019  Abstract: Objectives: The aim of this research was to determine the osseointegration of two presentations of biphasic calcium phosphate (BCP) biomaterial—one untreated and another submitted to biofunctionalization with a TGF-β1 inhibitor peptide, P144, on dental alveolus. Materials and Methods: A synthetic bone graft was used, namely, (i) Maxresorb® (Botiss Klockner) (n = 12), and (ii) Maxresorb® (Botiss Klockner) biofunctionalized with P144 peptide (n = 12). Both bone grafts were implanted in the two hemimandibles of six beagle dogs in the same surgical time, immediately after tooth extraction. Two dogs were sacrificed 2, 4, and 8 weeks post implant insertion, respectively. The samples were submitted to histomorphometrical and histological analyses. For each sample, we quantified the new bone growth and the new bone formed around the biomaterial’s granules. After optical microscopic histological evaluation, selected samples were studied using backscattered scanning electron microscopy (BS-SEM). Results: The biofunctionalization of the biomaterial’s granules maintains a stable membranous bone formation throughout the experiment timeline, benefitting from the constant presence of vascular structures in the alveolar space, in a more active manner that in the control samples. Better results in the experimental groups were proven both by quantitative and qualitative analysis. Conclusions: Synthetic bone graft biofunctionalization results in slightly better quantitative parameters of the implant’s osseointegration. The qualitative histological and ultramicroscopic analysis shows that biofunctionalization may shorten the healing period of dental biomaterials. Keywords: osseointegration; biofunctionalization; histomorphometrical analysis; scanning electron microscopy; TGF-β1; synthetic bone graft 1. Introduction Grafting materials are currently a predictable treatment for regenerating bone defects in implant dentistry. There are several types of bone grafts, and autogenous bone grafting is the gold standard for its osteoconductive and osteoinductive properties [1–3]. Despite the properties of autogenous bone Materials 2019, 12, 3168; doi:10.3390/ma12193168 www.mdpi.com/journal/materials Materials 2019, 12, 3168 2 of 23 graft, however, not all cases present the desirable amount of bone available [4] and it is necessary to harvest the graft from a donor area, which may require a second surgical site [5]. Alternatively, synthetic biomaterials have emerged since the 1980s [6]. Reynolds et al. [7] performed a systematic study in which the researchers did not find differences in clinical response between granulate bone allograft and calcium phosphate ceramic biomaterial grafts. Due to bone tissue remodeling, the hard tissue in alveolus presents a certain atrophy. The resorption of alveolar ridge dimensions can occur in 12 months [8]. Many biomaterial strategies have been developed to maintain the maximum amount of natural hard tissues, such as narrow dental implants. It is very important to preserve the bone volume after tooth extraction and supply a stable substrate for implant placement [9,10]. An important factor concerning socket preservation is angiogenesis. Angiogenesis is a complicated mechanism, including extracellular matrix (ECM) degradation, adhesion, proliferation, migration, and differentiation cells. [11]. The use of either autologous platelet-rich fibrin (PRF) or beta-tri-calcium phosphate with collagen (β-TCP-Cl) has been found to be excellent in socket preservation [12]. β-TCP has also been compared to β-TCP with added collagen, resulting in no statistically significant differences [13,14]. To further optimize the osteogenic properties of biphasic calcium phosphates (BCPs), the combination of BCPs with molecules or growth factors is being investigated [15]. Minier et al. [16] utilized local bone morphogenetic protein type 2 (BMP-2) release in BCP ceramic particles in a dog animal model. They observed that BMP-2 loaded grafts induced the formation of lamellar bone tissue with an excellent mineralization, well-differentiated and surrounding the BCP biomaterials and bridging granules/bone tissue interfaces eight weeks after surgical operation. Platelet-rich fibrin has also been used in combination with β-TCP, increasing the bone regeneration in the first two weeks after surgery [17]. BCP bone substitutes appear as an effective vehicle for osteoinductive molecules (bioactive peptides, bone growth factors) or even stem cells in a tridimensional structure, thus facilitating bone regeneration [18,19]. An adequate adjustment of both dosage and release profile of the bioactive molecule is required [20]. On the other hand, inflammatory and immune responses have been observed during the osseointegration process [21,22]. Transforming growth factor beta 1 (TGF-β1) has been signaled as the central mediator of the fibrotic response [23]. Moreover, it increased expression in the tissues around failed dental implants, among other roles [23–26]. A TGF-β1 inhibitor peptide, P144, blocks the interaction of TGF-β1 with its receptor, thus altering its interaction with different cell types [27–29]. An increase of osteoblast and osteoprogenitor cell differentiation was observed as the in vitro results of P144 biofunctionalization of a CP-Ti surface [30]. Previous in vitro studies in MC3T3-E1 stem cells showed improvement of the quality of interfacial interactions during the healing process [31]. Then, inhibiting TGF-β1 can either limit the formation of fibrous tissue around a biomaterial or activate the cells responsible for osseointegration [32,33]. The aim of this study was (i) to evaluate the effect on osseointegration of the biofunctionalization with a TGF-β1 inhibitor peptide (P144) of a well-known biomaterial and then (ii) to compare its osseointegration and histomorphometric values with the ones from the same untreated biomaterial. Osseointegration is considered as the union between bone and a biomaterial with mimetic properties. Osteoconduction according to Weber [34] is a three-dimensional (3D) process of ingrowth of sprouting capillaries, perivascular tissue, and osteoprogenitor cells from a bony bed into the 3D structure of a porous implant used as a guiding cue to bridge a defect with bony tissue. 2. Results The postoperative healing was uneventful and peri-implant mucosa appeared to be clinically healthy. A total of 24 dental alveoli were processed, 22 of which were finally analyzed due to artifacts and process mishaps. Materials 2019, 12, 3168 3 of 23 Materials 2019, 12, x FOR PEER REVIEW 3 of 23 Bone index contact results were determined and can be seen in Table 1 for each time after Bone index contact results were determined and can be seen in Table 1 for each time after implantation and each biomaterial. implantation and each biomaterial. Table 1. Bone index contact for biphasic calcium phosphate (BCP) and for bone calcium phosphate Table 1. Bone index contact for biphasic calcium phosphate (BCP) and for bone calcium phosphate biofunctionalized with P-144 (BCP-P-144). Standard deviation in parentheses. biofunctionalized with P-144 (BCP-P-144). Standard deviation in parentheses. Biomaterial 2 weeks 4 weeks 8 weeks Biomaterial 2 weeks 4 weeks 8 weeks BCP BCP 20% (8) 20% (8) 33% (8) 33% (8) 65%(9) 65% (9) BCP-P-144 30% (6) 40% (7) 77% (8) BCP-P-144 30% (6) 40% (7) 77% (8) shows the Figure 1 shows the results results of of percentage percentageof ofbone boneinside insidethethealveolar alveolardefects defectsduring during2,2,4,4,and and8 8 weeks weeks aftersurgery. after surgery. The control biomaterial presents a difference in bone growth between weeks 2 and 4 (p = 0.003, = 0.003, marked as as *), *), as as well well as and88(p(p<<0.001, as22and 0.001,marked markedasas*),*), andand the the difference difference between between weeks weeks 4 and 4 and 8 (p8 (p = 0.004, = 0.004, marked marked as *). as *). The P144-functionalized group shows higher values than the biomaterial alone throughout the experiment. Bone percentage presents experiment. presents statistical statistical differences differencesbetween between22and and88 weeks weeks (p(p < < 0.001, marked as &) and between 4 and 88 weeks (p weeks (p = = 0.004, marked as $). However, no difference was observed between 2 and 4 weeks (p = 0.087). = 0.087). The differences for the same time of of implantation implantation between the control and biofunctionalized biofunctionalized biomaterial have been described with letters. The The results results of of the the bone bone percentage percentage are higher for the biofunctionalized biomaterial. biofunctionalized biomaterial.At At22weeks, weeks,thethedifferences differencespresent presentsignificance significance(p(p = 0.0007, = 0.0007, marked marked as as and a) a) and at 8atweeks 8 weeks = 0.003, (p =(p0.003, marked marked as b). as b). However, However, no no difference difference waswas observed observed at 4atweeks. 4 weeks. Figure 1. Figure Newbone 1. New bonevolume volume(NBV) (NBV)ofofnewly newlyformed formedbone bonewithin within the the region region of of interest interest (ROI) (ROI) at 4, at 2, 2, 4, and 8 weeks after bone grafting. BCP, biphasic calcium phosphate; BCP-P-144, and 8 weeks after bone grafting. BCP, biphasic calcium phosphate; BCP-P-144, biphasic calciumbiphasic calcium phosphate treated with phosphate treated with P144. Statistically significant P144. Statistically significant differences differences are are indicated indicated as as *, *, #, #, and and & & for for the the different implantation times and with a or b for the different biomaterials BCP or BCP-P-144. different implantation times and with a or b for the different biomaterials BCP or BCP-P-144. Figure 22 shows Figure shows percentages percentages of of bone bone ingrowth ingrowth around around the the biomaterial biomaterial fragments. fragments. The control biomaterial fragment values show differences between The control biomaterial fragment values show differences between 2 and 2 and 44 weeks (p = weeks (p 0.005, = 0.005, marked as *), between 4 and 8 weeks (corrected p = 0.029, marked as *), and between 2 and marked as *), between 4 and 8 weeks (corrected p = 0.029, marked as *), and between 2 and 8 weeks 8 weeks (p < (p 0.001, marked < 0.001, marked asas *). *). The P144-biofunctionalized group shows the highest values but no significant differences between 2 and 4 weeks in the growth between times. The significant differences are between 2 and 8 weeks (p < 0.001, marked as &) and between 4 and 8 weeks (p = 0.0025, marked as $). Materials 2019, 12, 3168 4 of 23 The P144-biofunctionalized group shows the highest values but no significant differences between 2Materials and 4 2019, weeks 12, in thePEER x FOR growth between times. The significant differences are between 2 and 8 weeks REVIEW 4 of 23 (p < 0.001, marked as &) and between 4 and 8 weeks (p = 0.0025, marked as $). The differences The differences for for the the same same time timeofofimplantation implantation between between the the control control and andbiofunctionalized biofunctionalized biomaterial have biomaterial have been been described described with with letters. letters. The results of of the the bone bone percentage percentage are are higher higher for for the the biofunctionalized biofunctionalized biomaterial. At 2 weeks, the differences present significance (p = 0.0008, marked At 2 weeks, the differences present significance (p = 0.0008, marked as a) a) as andandat at 8 weeks 8 weeks (p (p = 0.0041, = 0.0041, marked marked as as b).b). However, However,nono difference was difference observed was observed at at 4 weeks. 4 weeks. Figure 2. NBV/Total volume (TVol) of newly formed bone within the region of interest (excluding Figure 2. NBV/Total volume (TVol) of newly formed bone within the region of interest (excluding the the area occupied by the biomaterial) at 2, 4, and 8 weeks after bone grafting. BCP, biphasic calcium area occupied by the biomaterial) at 2, 4, and 8 weeks after bone grafting. BCP, biphasic calcium phosphate; BCP-P-144, biphasic calcium phosphate treated with P144. Statistically significant differences phosphate; BCP-P-144, biphasic calcium phosphate treated with P144. Statistically significant are indicated as *, #, and &. differences are indicated as *, #, and &. Histology and BS-SEM Analysis Histology and BS-SEM Analysis The control group biomaterial is surrounded at a distance by sparse, thin, calcified trabeculae, mainlyThe control group constituted biomaterial by chondroid is surrounded bone at a distance as can be observed by sparse, in Figure 3a. Withthin, calcified higher trabeculae, magnification, mainly constituted by chondroid bone as can be observed in Figure 3a. With higher Figure 3c shows its characteristic big, irregular, confluent cell lacunae (orange dots) immersed in magnification, Figurecalcified barely 3c shows its characteristic extracellular matrix big, (deepirregular, confluent green), lined cell lacunae by a band (orangeextracellular of non-calcified dots) immersed in matrix barely calcified (orange in Figureextracellular matrix 3c) constituting thin(deep green), trabeculae lined that by a band establish of non-calcified sparse contacts between extracellular themselvesmatrix and (orange in Figure 3c) constituting thin trabeculae that establish sparse contacts with the biomaterial granules. The thickness of the trabeculae in contact with the biofunctionalizedbetween themselves and with granules biomaterial the biomaterial is greatergranules. than in the The thickness control group of the 3b,d), (Figure trabeculae and itsinhomogeneous contact withdeepthe biofunctionalized green staining and biomaterial granules cell separated polygonal is greater lacunaethan showin that the they control are group (Figure constituted by 3b,d), and its more mature homogeneous deep green staining and separated polygonal cell lacunae show tissue and woven bone. The extracellular non-calcified matrix is visible around the bone cell lacunae that they are constituted from by more the newly formedmature tissuetissue and woven surrounding bone. The (Figure the trabeculae extracellular 3d). non-calcified matrix is visible around the bone cell lacunae from the newly formed tissue surrounding the trabeculae (Figure 3d). Materials 2019, 12, 3168 5 of 23 Materials 2019, 12, x FOR PEER REVIEW 5 of 23 Figure 3. Histologies after two weeks of bone grafting insertion. (a) Control group sample; Figure 3. Histologies after two weeks of bone grafting insertion. (a) Control group sample; (b) P144- (b) P144-biofunctionalized bone graft; (c) control group showing the framed area in (a); biofunctionalized bone graft; (c) control group showing the framed area in (a); (d) P144- (d) P144-biofunctionalized bone graft showing the framed area in (b). biofunctionalized bone graft showing the framed area in (b). The BS-SEM analysis (Figure 4a,c) reveals a similar bone ingrowth in both groups at two weeks The BS-SEM analysis (Figure 4a,c) reveals a similar bone ingrowth in both groups at two weeks after grafting at lower magnification. No apparent contact is visible between the alveolar cortical after grafting at lower magnification. No apparent contact is visible between the alveolar cortical and and the biomaterial fragments. However, when increasing magnification, it can be observed that the biomaterial fragments. However, when increasing magnification, it can be observed that in the in the peptide group (Figure 4d) most fragments show osseous tissue apposition, all situated near peptide group (Figure 4d) most fragments show osseous tissue apposition, all situated near the the preexisting cortical, whereas only some fragments of the control group present bone apposition preexisting cortical, whereas only some fragments of the control group present bone apposition (Figure 4b). (Figure 4b). Materials 2019, 12, 3168 6 of 23 Materials 2019, 12, x FOR PEER REVIEW 6 of 23 Figure 4. Backscattered scanning electron microscopy (BS-SEM) results after two weeks of bone grafting Figure 4. Backscattered scanning electron microscopy (BS-SEM) results after two weeks of bone insertion. (a) Control group sample; (b) framed area in (a), newly formed bone around one granule; grafting insertion. (a) Control group sample; (b) framed area in (a), newly formed bone around one (c) P144-biofunctionalized bone graft; (d) framed area in (c), newly formed bone in contact with the granule; (c) P144-biofunctionalized bone graft; (d) framed area in (c), newly formed bone in contact biofunctionalized granules near the preexisting cortical. with the biofunctionalized granules near the preexisting cortical. Four weeks after implantation, in the control samples, a dense mesh of thicker and denser Four can trabeculae weeks after implantation, be observed surrounding inthe thebiomaterial control samples, a dense fragments mesh of thicker and establishing andwith contacts denser the trabeculae can be observed surrounding the biomaterial fragments and establishing contacts bone cortical (Figure 5a). The preexisting cortical area also shows evidence of an intense remodeling with the bone cortical (Figure 5a). The preexisting cortical area also shows evidence of an process, consisting in numerous, convoluted vascular channels and new lamellar bone appositionintense remodeling process, within consisting (red arrows in in Figure numerous, 5b). convoluted vascular channels Higher magnification and new shows that new lamellar bone apposition bone formation (orange, within (red arrows in Figure 5b). Higher magnification shows that new bone formation (orange, Figure 5d) is still evident lining the vascular channels (area in Figure 5c) that connect the new trabeculae Figure 5d) is still evident with the cortical alveolar wall. lining the vascular channels (area in Figure 5c) that connect the new trabeculae with the cortical alveolar wall. Materials 2019, 12, 3168 7 of 23 Materials 2019, 12, x FOR PEER REVIEW 7 of 23 Figure 5. Results after four weeks of bone grafting insertion in the control group. (a) Stitching summative imagesafter Figure 5. Results of the control four weekssample; of bone (b)grafting framed area in (a),inorange insertion (red arrows) the control group. non-calcified (a) Stitching extracellular matrix (ECM) near the granules; (c) framed area in (a): vascular channel summative images of the control sample; (b) framed area in (a), orange (red arrows) non-calcified crossing the preexisting cortical; (d) framed area in (c), vascular channel. extracellular matrix (ECM) near the granules; (c) framed area in (a): vascular channel crossing the preexisting cortical; (d) framed area in (c), vascular channel. At this stage, the fragments of the biofunctionalized group adopt the same deep green color as the bone tissues of the trabeculae that surround them (Figure 6a,c). These denser and thicker trabeculae At this stage, the fragments of the biofunctionalized group adopt the same deep green color as present numerous large interconnected vascular channels visible in the bone cortical (framed area in the bone tissues of the trabeculae that surround them (Figure 6a,c). These denser and thicker Figure 6b), in the alveolar space (framed area in Figure 6d), and in the periosteal reaction. trabeculae present numerous large interconnected vascular channels visible in the bone cortical (framed area in Figure 6b), in the alveolar space (framed area in Figure 6d), and in the periosteal reaction. Materials 2019,12, Materials2019, 12,3168 x FOR PEER REVIEW 88of of23 23 Figure 6. Results after four weeks of bone grafting insertion in the experimental group. (a) Stitching Figure 6. Results after four weeks of bone grafting insertion in the experimental group. (a) Stitching summative images of the P144-biofunctionalized sample; (b) framed area in (a), granules in contact summative images of the P144-biofunctionalized sample; (b) framed area in (a), granules in contact with the cortical of the alveolar defect; (c,d) framed area in (a), newly formed bone around the granule with the cortical of the alveolar defect; (c,d) framed area in (a), newly formed bone around the granule (see series 1 and 2). (see series 1 and 2). The higher magnifications reveal that the blood vessels are around the biomaterial granules while The higher maintaining magnifications connections with the reveal that the blood vascularization of thevessels are around surrounding areasthe biomaterial (Figure 7). Thegranules vessels while maintaining connections with the vascularization of the surrounding (erythrocytes (e) are visible in Figure 7c) also invade the spaces within the biomaterial areas (Figure 7). and granules The vesselsthe initiate (erythrocytes (e) are visible bone tissue apposition in Figure process evidenced7c) byalso theinvade the spaces linear deposit within theextracellular of non-calcified biomaterial granules and initiate the bone tissue osseous tissue matrix (orange in Figure 7b,c). apposition process evidenced by the linear deposit of non- calcified extracellular osseous tissue matrix (orange in Figure 7b,c). A different view at the same magnifications gives a more detailed image, with osteoblasts A different (red arrows view8b) in Figure at already the same magnifications immersed gives in the still a more detailed non-calcified image, matrix, extracellular with osteoblasts (red within which arrows in Figure 8b) already immersed in the still non-calcified extracellular matrix, the calcification starts to appear as sparse, still unconnected deep green granules. These images reveal within which the size the calcification startswithin of the vessels to appear the as sparse,ofstill cavities theunconnected deep green biomaterial granules; granules. limited These amounts ofimages reveal erythrocytes the size of the vessels within the cavities of the biomaterial granules; limited amounts (e) are visible in the biggest vessels (Figure 8a,b), whereas in the smaller cavities the erythrocytes of erythrocytes (e) are visible in the biggest vessels (Figure 8a,b), whereas in the smaller cavities the erythrocytes appear piled in very small vessels (Figure 9b). In a similar manner, the tissues deposited show the Materials 2019, 12, 3168 9 of 23 appear Materials piled inx very 2019, 12, small FOR PEER vessels (Figure 9b). In a similar manner, the tissues deposited show9 of REVIEW the 23 morphological characteristics of the woven bone (Figure 9a)—big, isolated cell lacunae in a dense, morphological very characteristics calcified extracellular of the matrix, andwoven bone lamellar bone(Figure 9a)—big, apposition provenisolated by thecell lacunae(red osteoblasts in aarrows dense, very in Figurecalcified extracellular 9a) within matrix, and lamellar the still undecalcified bone bone perivascular apposition matrix proven (orange).byVery the different osteoblastsare (red the arrows inand confluent Figure 9a) within the still irregularly-shaped cell undecalcified perivascular lacunae immersed bone matrix in an extracellular (orange). matrix, Very whose different calcification are is theincomplete still confluent and and slightly irregularly-shaped cell lacunae granular, which immersed is the typical aspectinofan theextracellular matrix, chondroid bone shownwhosein calcification Figures 8b andis still 9b. incomplete and slightly granular, which is the typical aspect of the chondroid bone shown in Figures 8b and 9b. Figure Figure 7. Series 1. 7. Series 1. Results Resultsafter afterfour fourweeks weeksofof grafting grafting bone bone insertion, insertion, framed framed areas areas in Figure in Figure 6b. 6b. (a) (a) Higher Higher magnification magnification area area of of Figure6b6bindicated Figure indicatedbybythe thearrow. arrow. (b) (b) Higher Higher magnification area of of Figure 6b indicated by the arrow. (c) Higher magnification area of Figure 6b indicated by the Figure 6b indicated by the arrow. (c) Higher magnification area of Figure 6b indicated by the arrow arrow with withabundant abundantvessels vesselswith witherythrocytes erythrocytes(e). (e). Materials 2019, 12, x3168 FOR PEER REVIEW 10 of 23 Materials 2019, 12, x FOR PEER REVIEW 10 of 23 Figure 8. Series 2. Results after four weeks of grafting bone insertion, framed areas in Figure 6c of a Figure Figure 8. Series 2. 8. Series 2. Results Results P144-biofunctionalized afterfour after sample. fourFramed (a) weeksofarea weeks ofgrafting grafting bone bone in Figure insertion, insertion, 6c; framed framed (b) framed areas areas area ininFigure in Figure Figure 8a: 6c6cofofa newly a P144-biofunctionalized P144-biofunctionalized formed bone growing from sample. sample. (a) Framed area in Figure 6c; (b) framed area in Figure (a) Framed area in Figure 6c; (b) framed area in Figure 8a: newly the granule. 8a: newly formed bone growing from the granule. formed bone growing from the granule. Figure 9. Series 2. Results after four weeks of grafting bone insertion, framed areas in Figure 6d of Figure 9. Series 2. Results after four weeks of grafting bone insertion, framed areas in Figure 6d of a a P144-biofunctionalized sample. (a) Detail of the framed area in Figure 6d, osteoblasts (red arrows) Figure 9. Series 2. Results P144-biofunctionalized after four sample. weeksof (a) Detail ofthe grafting bone framed areainsertion, in Figureframed areas in Figure 6d, osteoblasts 6d of a (red arrows) inside the non-calcified EMC, erythrocytes (e). (b) Detail of the framed area, cell lacunae with type II P144-biofunctionalized inside sample. the non-calcified EMC, (a) Detail of erythrocytes (e).the (b)framed area Detail of theinframed Figurearea, 6d, osteoblasts cell lacunae(red witharrows) type II non-calcified collagen and matrix deposition around the granule, abundant vessels with erythrocytes (e). inside the non-calcified non-calcified EMC, collagen and erythrocytes matrix deposition(e). (b) Detail around of the framed the granule, area,vessels abundant cell lacunae with type II with erythrocytes non-calcified collagen and matrix deposition around the granule, abundant vessels with erythrocytes (e). (e). Materials 2019, 12, 3168 11 of 23 Materials 2019, 12, x FOR PEER REVIEW 11 of 23 In reference reference to tothe theBS-SEM BS-SEMresults, results,most most of of thethe P144-biofunctionalized P144-biofunctionalized granules granulesshow a solid show and a solid continuous and contact continuous withwith contact the the cortical of the cortical alveolar of the alveolardefect, whereas defect, whereasthethecontrol controlgroup grouphas has aa more discontinuous connection, discontinuous connection,both both between between the fragments the fragments and and the the trabeculae trabeculae and the and between between the trabeculae trabeculae and and the preexisting the preexisting cortical cortical (Figure (Figure 10a,c). In the10a,c). In the experimental experimental group (Figure group (Figure 10d), 10d), trabeculae trabeculae are denser are anddenser show and showadvanced a more a more advanced phase of phase of bone remodeling bone remodeling than thethanonethe one observed observed in the in the non-biofunctionalized non-biofunctionalized graft groups graft groups (Figure(Figure 10b).10b). The The bonebone apposition apposition in the in the peptide peptide group group hasa has a lamellarand lamellar andcontinuous continuousapposition appositionuponuponthetheinitial initialchondroid chondroidbone, bone,whereas whereas thethe control group still shows more chondroid bone and and less less lamellar lamellar bone. bone. BS-SEMresults Figure 10. BS-SEM resultsafter afterfour fourweeks weeksofof bone bone grafting grafting insertion. insertion. (a) Control (a) Control group group sample. sample. (b) (b) Framed Framed area area in Figure in Figure 9a,9a, connected connected trabeculae trabeculae withnewly with newlyformed formedlamellar lamellarbone bonearound aroundaa chondroid chondroid bone core. core.(c)(c) P144-biofunctionalized P144-biofunctionalized bone bone graft. graft. (d) Framed (d) Framed area inarea in Figure Figure 9c, interconnected 9c, interconnected mature mature lamellae of newly formed bone in contact with the biofunctionalized lamellae of newly formed bone in contact with the biofunctionalized granules near granules near the the preexisting preexisting cortical. cortical. Eight weeks after the biomaterials’ insertion, there is a general increase of the osseointegration in Eight weeks after the biomaterials’ insertion, there is a general increase of the osseointegration all study groups, coherent with the tendency showed in the results of the histomorphometric data. in all study groups, coherent with the tendency showed in the results of the histomorphometric data. Light microscopy analysis reveals that the bone architecture around all biomaterial fragments Light microscopy analysis reveals that the bone architecture around all biomaterial fragments (Figure 11) is denser because of the apposition of lamellar bone lining the vascular channels. A higher (Figure 11) is denser because of the apposition of lamellar bone lining the vascular channels. A higher number of vascular channels is visible between the biomaterial fragments in the control group number of vascular channels is visible between the biomaterial fragments in the control group (Figure (Figure 11b) than in the biofunctionalized material (Figure 11e). Moreover, the vessels connecting 11b) than in the biofunctionalized material (Figure 11e). Moreover, the vessels connecting the preexisting cortical (pc in Figure 11b) are bigger and more convoluted in the control samples. When Materials 2019, 12, 3168 12 of 23 Materials 2019, 12, x FOR PEER REVIEW 12 of 23 the preexisting cortical (pc in Figure 11b) are bigger and more convoluted in the control samples. When surrounding surrounding biofunctionalized biofunctionalized fragments, fragments, the bone the bone tissues tissues are denser are denser and and moremore continuous continuous withwith the the preexisting preexisting cortical. cortical. TheThe higher higher magnifications magnifications (Figure (Figure 11c,f)reveal 11c,f) revealthat thatthere thereare are still still numerous numerous vascular vascular channels channels around around the the trabeculae trabeculae surrounding surrounding the the biomaterial biomaterial control control fragments, fragments, whereas whereas the the trabeculae around the functionalized fragments are denser, with less and thinner trabeculae around the functionalized fragments are denser, with less and thinner vascular vascular spaces. It is spaces. It noteworthy, however, that the alveolar space in both groups still contains fragments of biomaterial is noteworthy, however, that the alveolar space in both groups still contains fragments of biomaterial that are thatnot arein contact not with with in contact the recently deposited the recently osseous deposited tissues. osseous tissues. 11. Results Figure 11. Resultsafter aftereight eightweeks weeksofof bone bonegrafting insertion. grafting (a) (a) insertion. Control group Control sample. group (b) Framed sample. area (b) Framed in Figure area 10a; pc, 10a; in Figure preexisting cortical. (c)cortical. pc, preexisting Higher magnification of Figure 10b. of (c) Higher magnification (d) Figure P144-biofuncionalitzed 10b. (d) P144- sample. (e) Framed biofuncionalitzed area in(e) sample. Figure 10d.area Framed (f) Higher magnification in Figure 10d. (f) Higherof Figure 10e. magnification of Figure 10e. BS-SEM analysis shows BS-SEM analysis shows aa more more mature mature stage stage ofof bone bone formation formation in in both both experimental experimental groups. groups. Figure Figure 12a,c shows a dense and compact bone formation connecting the granules; in the treated group, 12a,c shows a dense and compact bone formation connecting the granules; in the treated group, this this linking linking seems seems more more consistent consistent because because the the trabecular trabecular structure structure connects connects both both corticals corticals (framed (framed areas areas in in Figure Figure 12a,c) 12a,c) of of the the alveolar alveolar defect defect and and thethe grey grey color color of of the the lamellar lamellar bone bone apposition apposition is is predominant. However, in the control group, the trabecular web is less continuous, predominant. However, in the control group, the trabecular web is less continuous, and osteonal and osteonal remodeling remodeling is is visible visible mainly mainly inin the the preexisting preexisting cortical cortical (asterisks (asterisks in in Figure Figure 12b). 12b). The The peptide peptide group group shows osteonal apposition mainly within the biomaterial granules (Figure shows osteonal apposition mainly within the biomaterial granules (Figure 12d). 12d). Materials 2019, 12, 3168 13 of 23 Materials 2019, 12, x FOR PEER REVIEW 13 of 23 Figure 12. BS-SEM Figure12. results after BS-SEM results after eight eightweeks weeksofofbone bonegrafting graftinginsertion. insertion. (a)(a) Control Control group group sample. sample. (b) (b) Framed area in Figure 11a, * osteonal bone tissue. (c) P144-biofunctionalized bone graft. (d) Framed area in Figure 11a, * osteonal bone tissue. (c) P144-biofunctionalized bone graft. (d) Framed Framed area areain inFigure Figure11c, 11c,**osteonal osteonalbone bonetissue. tissue. 3. Discussion 3. Discussion The purpose of this investigation was to compare the effects on bone ingrowth and osseointegration The purpose of this investigation was to compare the effects on bone ingrowth and of a biofunctionalized synthetic bone graft with respect to a well-known synthetic bone graft. The bone osseointegration of a biofunctionalized synthetic bone graft with respect to a well-known synthetic grafts were inserted in the fresh extraction sites of the mandibular premolar area of beagle dogs the bone graft. The bone grafts were inserted in the fresh extraction sites of the mandibular premolar area same day of the atraumatic teeth extraction. The aim of bone grafting is to limit ridge resorption and of beagle dogs the same day of the atraumatic teeth extraction. The aim of bone grafting is to limit bone loss, as well as to preserve as much bone mass as possible for implant placement. ridge resorption and bone loss, as well as to preserve as much bone mass as possible for implant In vivo studies proved that calcium phosphates (CaPs) are biocompatible and offer an effective placement. possibility to allograft biomaterial without the common problems such as an immune reaction and In vivo studies proved that calcium phosphates (CaPs) are biocompatible and offer an effective disease transmission [35,36]. The use of alloplastic materials can offer a useful procedure to avoid these possibility to allograft biomaterial without the common problems such as an immune reaction and risks [5]. The osteoconductive properties of BCP bioceramics have been enhanced with osteoinductive disease transmission [35,36]. The use of alloplastic materials can offer a useful procedure to avoid features via the modulation of its physicochemical properties [35]. The implanted calcium phosphate these risks [5]. The osteoconductive properties of BCP bioceramics have been enhanced with surface adsorbs the proteins that attract the osteoblasts, inducing the bone cells to differentiate and osteoinductive features via the modulation of its physicochemical properties [35]. The implanted promoting the mineralization process [37]. calcium phosphate surface adsorbs the proteins that attract the osteoblasts, inducing the bone cells to Bone remodeling takes place around the BCP grains starting with the resorption of its calcium differentiate and promoting the mineralization process [37]. phosphate components. Thus, the integration of the granules of the biomaterial with the bone Bone remodeling takes place around the BCP grains starting with the resorption of its calcium structure is continuously in progress. This interface is highly dynamic, involving the bone biology and phosphate components. Thus, the integration of the granules of the biomaterial with the bone structure is continuously in progress. This interface is highly dynamic, involving the bone biology and physiopathology, the underlying biomechanical factors, and the niche factor (the surrounding Materials 2019, 12, 3168 14 of 23 physiopathology, the underlying biomechanical factors, and the niche factor (the surrounding vessels, biochemical factors, nutritional status, etc.). Bone remodeling is aimed at a “restitutio ad integrum”, resulting in an organized and mineralized bone ingrowth at the expense of the artificial bone [38]. As a final result, bioactive bone graft materials form a direct, living bond with the host bone. The healing periods used for our study, that is, 2, 4, and 8 weeks, were selected to demonstrate early and later stages of bone remodeling and to determine the progression of the bone while interacting with the different bone grafts. An adverse correlation between the presence of TGF-β1 and bone formation was described in in vivo experiments [39,40]. Moreover, a fibrotic layer around the surface was found after the insertion of dental implants with added TGF-β1 [41]. In vitro experiments, however, proved that TGF-β1 suppressed the osteoblastic differentiation of bone marrow cells, whereas its inhibition released the stromal cells from their differentiation arrest, facilitating the formation of terminally differentiated osteoblasts [42]. Furthermore, TGF-β1 has been related to cellular processes for wound healing as well as for the regulation of inflammatory processes [21]. That fact led our group to explore the biofunctionalization of titanium dental implants with TGF-β1 inhibitor peptides, which resulted in accelerating the osteoprogenitor cell differentiation in vitro [30]. P144 (NH2-TSLDASIWAMMQN-OH) is a TGF-β inhibitor peptide developed by CIMA (University of Navarra) that excels in the arrest of the TGF-β1, inhibiting its interaction with fibroblast cells. Due to this inhibition, the osteoblast cells migrate to the calcium phosphate and consequently favor the osseointegration. P144 is a peptide derived from the TGF-β type III receptor. This peptide is in the cytoskeleton of many cell types. P144 interacts with TGF-β1 in the area where the cytokine connects with the cell receptor and blocks the cell signaling pathway [43–45]. Several techniques have been proposed for socket preservation after teeth extraction, although none has yet been proven as the gold standard. Moreover, a higher loss of bone can occur because of endodontic pathology, periapical granuloma infection, periodontitis, or traumatic extraction [10]. Bouwman et al. proved in 2010 the osteoconductive properties of BCP material histomorphometrically and histologically in a sinus floor experiment [4]. Bone maturation was evident by the presence of lamellar bone [10]. Our results prove that the biofunctionalization with P144 of a BCP biomaterial has an additive effect, since it stimulates both osteoconduction and osteoinduction by avoiding the loss of bone mass due to teeth extraction. There is a notable histomorphometric similarity of beagle and human bone [9,46,47]. Despite the considerable variability in the methods to measure the contact between the osseous tissues and the profile of the implants [48–50], BIC (Bone index contact) represents a quantitative measurement that has been used by many authors as a measure for osseointegration. The observations with BS-SEM offer a simpler, less resource-consuming, and highly discriminative method for BIC determination [51], constituting a non-subjective, systematic measurement of the bone contact of all the active implant surface. No standardized, objective, and systematic measurement has been proposed to date to assess the bone tissues in living contact with the biomaterial fragment’s surface. Alt et al. [52] and Ding et al. [53] propose that the histomorphometric analysis be made with histological images, but different parameters and regions of interest (ROIs) are proposed. Our analysis was made also on the whole perimeter of the alveolar defect as represented in a stitched complete image of the mandible. The region of interest (ROI) was determined by using the profile of the alveolar cortical as a limit. For each sample, image segmentation and binarization with the ImageJ program was carried out. Then, the biomaterial, the osteogenesis, and the non-calcified tissues were quantified, as represented in Figure 13. Materials 2019, 12, 3168 15 of 23 Materials 2019, 12, x FOR PEER REVIEW 15 of 23 Figure 13. Image processing of biomaterials for quantification using ImageJ and determination of ROI. Figure 13. Image processing of biomaterials for quantification using ImageJ and determination of ROI. (a) Original image; (b) contrasted image; (c) bone tissue segmentation; (d) segmentation of the grafted (a) Original image; (b) contrasted image; (c) bone tissue segmentation; (d) segmentation of the grafted biomaterial; (e) image binarization of (d); (f) image binarization of (c). biomaterial; (e) image binarization of (d); (f) image binarization of (c). The ROI selected corresponded to the contour of the alveolar defect, excluding the bone tissues The ROI selected corresponded to the contour of the alveolar defect, excluding the bone tissues constituting the alveolar wall cortical profile. Then, the biomaterial granule’s area was measured constituting the alveolar wall cortical profile. Then, the biomaterial granule’s area was measured and and the new osseous tissues around the granules were quantified. The values that reached statistical the new osseous tissues around the granules were quantified. The values that reached statistical significance were the new bone volume (NBV), defined as the percentage (%) of newly deposited significance were the new bone volume (NBV), defined as the percentage (%) of newly deposited bone tissue within the ROI, and NBV/Total volume (TVol), that is, the ratio of mineralized new bone bone tissue within the ROI, and NBV/Total volume (TVol), that is, the ratio of mineralized new bone to the ROI after subtracting the area corresponding to the biomaterial granules, estimated from the to the ROI after subtracting the area corresponding to the biomaterial granules, estimated from the analyzed sections. The results of this quantitative analysis were obtained by stitching histological analyzed sections. The results of this quantitative analysis were obtained by stitching histological images, following a method similar to the one described by Bakhshalian et al. [54]. These authors images, following a method similar to the one described by Bakhshalian et al. [54]. These authors excluded from their analysis the cortical bone, and subtracted the profile of the biomaterial’s granules excluded from their analysis the cortical bone, and subtracted the profile of the biomaterial’s granules for calculating the total volume of the ROI. Moreover, these calculations were made without taking into for calculating the total volume of the ROI. Moreover, these calculations were made without taking account valuable information that could eventually be applied to the analysis of the osseointegration into account valuable information that could eventually be applied to the analysis of the of granulated biomaterials, such as the trabecular number (Tb.N), trabecular separation (Tb.Sp), osseointegration of granulated biomaterials, such as the trabecular number (Tb.N), trabecular trabecular thickness (Tb.Th) and especially the trabecular bone pattern factor (Tb.Pf) as reported by separation (Tb.Sp), trabecular thickness (Tb.Th) and especially the trabecular bone pattern factor Blouin et al. [55] by using the “Standardized Nomenclature for Bone Histomorphometry” that was (Tb.Pf) as reported by Blouin et al. [55] by using the “Standardized Nomenclature for Bone established by Parfitt in 1987 and Parfitt et al. [56,57] to evaluate bone remodeling [58]. Histomorphometry” that was established by Parfitt in 1987 and Parfitt et al. [56,57] to evaluate bone Our histomorphometric results showed a better behavior of our P144-biofunctionalized grafts with remodeling [58]. respect to the untreated samples. However, our histological and BS-SEM observations demonstrated Our histomorphometric results showed a better behavior of our P144-biofunctionalized grafts the importance of the contact between the preexisting alveolar cortical bone and the new bone tissues with respect to the untreated samples. However, our histological and BS-SEM observations demonstrated the importance of the contact between the preexisting alveolar cortical bone and the Materials 2019, 12, 3168 16 of 23 deposited around the biomaterial granules. The connections between the newly formed bone trabeculae are relevant to assess the quality of the bone filling the alveolar space and thus the bone anchorage for the future implant placement. Our results at the early stages of healing (two weeks) showed higher values for the experimental group. While the control group showed a lineal increase of bone ingrowth, our experimental group showed decreased values at four weeks; this decrease could be explained by our qualitative results (BS-SEM and histology), where at higher magnification the peptide biofunctionalized grafts showed more vascular spaces within the alveolar defect. This neovascularization permits granules to become invaded by vessels and facilitates protein adsorption. Indeed, this can be observed by the color change of the fragments that adopted the same green hue as the osseous tissues around them. Two weeks after implantation, the calcified tissues surrounding the biomaterials revealed by the light and electronic microscopy analysis could help to explain the numerical results of the analysis presented in Figures 1 and 2. Thus, the osteoconductive properties of the biomaterial allowed the granules to approach the preexisting cortical; the granules were in contact with new bone starting to grow around them. BS-SEM examination permits the observation of calcified tissues according to different levels of whiteness, which depends on their calcium concentration [50,59–63]. At four weeks, the biofunctionalized granules showed a dense bone structure in contact with the preexisting corticals; the granules were being integrated through the entire alveolar defect and the newly formed bone seemed more structured and compact than in previous weeks. These results are consistent with the numerical analysis presented in Figures 1 and 2, taking into account that our initial quantitative analysis did not consider the preexisting cortical bone. At eight weeks, the values of bone ingrowth were similar for both groups. These results confirm the promoting properties of the biofunctionalization technique, at least partially designed to attract mesenchymal stem cells in order to produce their phenotype to osteogenic cell lines. The backscattered scanning electron microscopy analysis of the alveolar defect sections depicted the bone ingrowth at the expense of the granules; trabeculae were thicker and lamellar bone apposition was denser in the biofunctionalized grafts. The tissues of the first trabeculae that surround the granules as well as the ones formed as a response to the periosteal injury are mainly constituted by chondroid bone [64]. It has a faster calcification and contains types I and II collagen fibers [65,66]. Woven, lamellar, and osteonal bone are the calcified tissues that replace chondroid bone in our histological and BS-SEM findings, in a similar manner to the one seen in other endomembranous ossification-based processes, such as mandibular symphysis closure [62], skull suture growth and closure [67–69], tooth eruption [70,71], fracture repair [59–67], and, more recently, the osseointegration of non-metallic biomaterials [14,35]. Our histological analysis demonstrated the presence of lining cells in the surface perimeter of the granules at the early stages of osseointegration. The lining cells remained active in the samples at the end of the experimentation; the active deposition of non-calcified bone matrix in the border of the vascular channels and granules was observed in greater measure in the biofunctionalized bone grafts. The differences in the coloring of the lamellar bone showed the maintenance of the lamellar apposition throughout the experimental period. The positive effects of biofunctionalization on the osseointegration of synthetic bone graft could be not only immediate but also in the medium or long term, thanks to trabecular apposition and lamellar remodeling, as described by Martino et al. [72] and Biguetti et al. [73]. Our TGF-β1 inhibitory peptides have proven to be able to adhere to Cp-Ti surfaces and keep their bioactivity [67] by evoking an in vitro cell response with a reduction of fibroblast differentiation and an increase of osteoblastic markers [30]. The heterogeneity of the morphology of the alveolar defects, surgical procedure, trauma, and duration have to be taken into account because they present an important influence on the resorption and biological response. The limited literature available with respect to TGF-β1 inhibitor peptides regarding bone organization obliges us to take these results with caution. Further research on Materials 2019, 12, 3168 17 of 23 Materials 2019, 12, x FOR PEER REVIEW 17 of 23 biofunctionalization biofunctionalization with with bioactive bioactive peptides peptides suchsuch as this as this TGF-β1 TGF-β1 inhibitor inhibitor peptide peptide andand its its effects effects on on osseointegration is necessary osseointegration is necessary [36]. [36]. In In relation relation toto P144’sstability, P144’s stability,Sevilla Sevilla et et al. [74] [74] determined determinedthat thatonce oncethe thecalcium calcium phosphates phosphates have thethe have P144, the P144, theactivity activityisisimmediate immediateand and the peptide peptide isisnot notdenaturalized denaturalizedunderunder normal normal conditions. conditions. TheThe mechanical mechanical stability stability was was determined determined by by sonication. sonication. TheThe samples samples were were introduced introduced in ainsolution a solution of of NaOHNaOH 0.10.1 MM with with streptavidin-FITC streptavidin-FITC conjugate conjugate andand sonicated sonicated until until thethe surface surface released released all all thethe fluorescence. fluorescence. Fluorescence Fluorescence intensity intensity of the of the samples samples waswas measured measured using using an an Olympus Olympus E600 E600 upright upright microscope, microscope, equipped equipped withwith a digital a digital camera camera Olympus Olympus DPDP 25 25 (Olympus, (Olympus, Tokyo, Tokyo, Japan). Japan). TheThe samples samples were were sonicatedfor sonicated for 30 30 hh in inwater waterand thethe and fluorescence fluorescenceintensity was measured intensity after different was measured sonication after different periods.periods. sonication The results Theshowed a good mechanical results showed stability stability a good mechanical of the biofunctionalized calcium phosphate, of the biofunctionalized calcium revealingrevealing phosphate, that the peptides that thepresented high bond high peptides presented forcesbond due to the covalent forces character due to the covalentof the bond to character ofthe thecalcium bond tophosphate the calcium after 30 h of sonication phosphate after 30 h of[70]. sonication [70]. 4. Materials 4. Materials andand Methods Methods 4.1. Description of Bone Grafts 4.1. Description of Bone Grafts A synthetic bone graft was used for this study and biofunctionalization was carried out A synthetic bone graft was used for this study and biofunctionalization was carried out for ® Botiss Klockner (n = 12) and (ii) Maxresorb® Botiss for comparison, namely, (i) Maxresorb comparison, namely, (i) Maxresorb® Botiss Klockner (n = 12) and (ii) Maxresorb® Botiss Klockner Klockner biofunctionalized with P144 peptide (n = 12). A schematic comparison can be seen in biofunctionalized with ® P144 peptide (n = 12). A schematic comparison can be seen in Figure 14. Figure 14. Maxresorb is a 100% synthetic bone graft substitute with controlled resorption properties. Maxresorb® is a 100% synthetic bone graft substitute with controlled resorption properties. This This biomaterial shows a homogenous and biphasic composition of 60% hydroxyapatite (HA) and 40% biomaterial shows a homogenous and biphasic composition of 60% hydroxyapatite (HA) and 40% beta-tricalcium phosphate (β-TCP) [60–63]. beta-tricalcium phosphate (β-TCP) [60–63]. Figure 14. Comparison between calcium phosphate Maxresorb® and the same calcium phosphate Figure 14. Comparison biofunctionalized between with P144 in calcium phosphate relation to Maxresorb® and the same calcium phosphate osseointegration. biofunctionalized with P144 in relation to osseointegration. Dilution of P144 peptide in sterile PBS (1 mmol/mL concentration) was done. Afterward, a 0.5 mL Dilution portion wasofadded P144 to peptide 2 mg ofinMaxresorb ® and sterile PBS (1 mmol/ml concentration) mixed just was done. before introducing Afterward, the graft a 0.5 into the alveolar mLdefects. portionThe wasconcentration added to 2 mg of Maxresorb of the ® andthe peptide inside mixed just before biomaterial was introducing 10 mg/cc. the graft into the alveolar defects. The concentration of the peptide inside the biomaterial was 10 mg/cc. 4.2. Animal Model and Animal Selection Criteria 4.2. Animal Model For this and Animal research, Selection Criteria six one-year-old male beagle dogs (body weight 12–15 kg) were used. The project wasForapproved by the this research, sixUniversity one-year-oldEthical maleCommittee beagle dogs(CEEHA 3256 DMAH (body weight 8890) 12–15 kg) and were all animal used. The procedures were performed under the European Community Guidelines (Directive project was approved by the University Ethical Committee (CEEHA 3256 DMAH 8890) and all animal 2010/63/EU). Prior to surgery, procedures animals were were performed acclimatized under to theCommunity the European local environment for two Guidelines weeks. 2010/63/EU). (Directive Prior to surgery, animals were acclimatized to the local environment for two weeks. Materials 2019, 12, 3168 18 of 23 4.3. Surgical Procedures All experimental surgeries were performed at the Hospital Universitari de la Facultad de Veterinaria of the Universitat Autònoma de Barcelona (Barcelona, Spain). Regarding the surgical process, beagle dogs were preanesthetized using medetomidine and methadone. The anesthesia used was propofol and diazepam injected intravenously. The dogs were maintained with inhaled isoflorane in an oxygen carrier. During the surgical operation, all dogs received an intravenous saline isotonic solution and an intravenous cefazolin injection. Local dental anesthesia was achieved with articaine 4% 1:100,000 epinephrine using the infiltrative technique at the site of extractions and surgery in the mandibles. Root canal treatment of second and third premolars (P2, P3) was carried out two months prior to surgery. Extractions of both premolars of each hemimandible were carried out following an atraumatic technique and bone grafts were inserted in the alveolar defects at the same surgery. Flap margins were adapted using a tension-free suture with bioresorbable polyglactin 910 Vycril©4/0. After surgery, all dogs received cefovecin subcutaneously as a prophylactic long-acting antibiotic as well as methadone and meloxicam as pain relief. A non-steroidal anti-inflammatory drug was given subcutaneously to the animals for a week to prevent inflammation and pain during the postoperative time. The healing was uneventful and peri-implant mucosa appeared to be clinically healthy [75]. Two dogs were euthanized at 2, 4, and 8 weeks after implantation, by an intravenous injection of an overdose of pentobarbital sodium. Before the euthanasia, the dogs were sedated by medetomidine in order to see to the animals’ welfare. 4.4. Histology and Sample Preparation All samples were processed following the same protocol [32]. To preserve tissues, mandibles were immediately immersed in a 10% formalin solution (pH = 7) for three weeks. Afterward, the jaws were dehydrated by immersion in aqueous solutions with increasing ethanol concentrations (30%, 50%, 70% 96%, and twice 100%). Once totally dehydrated, samples were embedded in MMA resin Technovit 7200 VLC (Kulzer-Heraeus, Hanaus, Germany) by immersion with increasing resin concentrations (30%, 50%, 75%, and 100%), using benzoic peroxide as initiator. Then, resin polymerization was carried out with a photopolymerization unit EXAKT 520 (Exakt), for 12 h with white light and 12 h with UV light. After the polymerization process, samples were stored in a heater (37 ◦ C) to evaporate resin vapors and cut in coronal sections using an irrigated diamond saw EXAKT 300 (Exakt, Oklahoma City, OK, USA). All samples were polished by a grinding machine EXAKT 400 CS (Exakt). Then, the block samples were sectioned into two blocks. One block was sectioned and the slices polished using SiC abrasive papers with different index mesh (P800, P1200, and P4000, Buelher, Lake Bluff, IL, USA) until a thickness of 50 µm was obtained, and then stained with Masson-Goldner trichrome (Merk, Barcelona, Spain). Slices from all samples were processed for optical microscopy; the alternate samples from the blocks selected for further analysis were reprocessed to be observed by backscattered scanning electron microscopy (BS-SEM). The half-blocks were polished with the same SiC papers and coated with carbon sputtering to be observed under scanning electron microscopy (SEM, Jeol J-6510® , Tokyo, Japan). 4.5. Image Acquisition and Quantification Optical microscopy was performed using a Nikon E600 optical microscope (Nikon, Tokyo, Japan) at different magnifications (×5, ×10, ×20, ×50, ×100). A stitching procedure was made to obtain an image of the whole sample for quantification. Samples were observed at higher magnifications for quality and cell identification. Quantification was carried out with Photoshop (2017, Adobe Systems, San José, CA, USA) and ImageJ (150.i, National Institutes of Health (NIH), Bethesda, MD, USA) to determine the quantity of Materials 2019, 12, 3168 19 of 23 osteogenesis at the zone of the alveolar defect. The region of interest (ROI) was determined by using the profile of the alveolar cortical as a limit. For each sample, by image segmentation and binarization, the biomaterial, the new bone, and the non-calcified tissues were quantified. NBV (%) was defined as the area occupied by new bone within the ROI; TVol (%) was the result of the subtraction of the area corresponding to the biomaterial from the ROI. NBV/TVol (%) was the ratio of mineralized new bone to the total alveolar volume estimated from the analyzed section. 4.6. Statistical Analysis The statistical study was carried out using the linear mixed model, in which the repeated factor was the time since surgery (2, 4, and 8 weeks) and the between factor was the biomaterial, the hemimandible being the covariate of no interest. We used a scaled identity covariance matrix. Each dependent variable was analyzed separately. For the percentage variables (proportions), we used the logit transformation. The Bonferroni correction was applied in order to control the effect of multiple comparisons, when it was necessary. Means and standard error of the mean were provided after adjusting for the covariate of no interest. All the analysis was carried out using SPSS V20.0 (IBM, New York, NY, USA). 5. Conclusions The parameters, values, and calculations proposed to assess bone biomaterial granules’ osseointegration must be reviewed. In terms of blood vessels (vascular channels) and bone tissues present in the early stages of healing (2 and 4 weeks) and remaining active in direct contact with the grafts until the end of the experiment, the biofunctionalized samples presented better results than the control ones. The biomaterial biofunctionalization maintained a stable endomembranous bone formation, more advanced than in the control samples, throughout the experiment timeline. The peptide biofunctionalization may shorten the healing period of osseointegrated biomaterials. The results of this study may serve as a useful reference for further studies oriented toward the analysis of the mechanisms behind biofunctionalization. Author Contributions: Conceptualization: J.G., P.G.-M., Methodology, M.C.M., P.S., M.O.-H., P.G.-M., J.G. Formal analysis J.G, P.S., P.G.-M. Investigation A.C., P.S., M.C.M., M.O.-H. Data curation. A.C., P.S., M.O.-H., M.C.M., Writing and supervision: M.C.M., J.G., A.C. Funding: The present study was supported entirely by the Research Cathedra Klockner-University of Granada. Acknowledgments: The authors kindly acknowledge the collaboration of many individuals and institutions in the present manuscript. The authors are grateful to the Spanish Government and the European Union FEDER for the concession of the project RTI2018-098075-B-C22. 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