**3. Clinical application of GFs in dentistry**

Given the biological properties of GFs, a major focus of research has concerned the clinical application of the osteoinductive proteins, such as some BMPs, for enhancing new bone formation. Bone loss involving the teeth may be secondary to diseases such as periodonti‐ tis, cystic diseases or tumors, or the consequence of trauma. Alveolar bone augmentation procedures are often needed for the purpose of inserting dental implants for prosthetic rehabilitation.

Missing teeth can be replaced with prostheses supported on dental implants, which can only be inserted in patients with an adequate alveolar ridge height and/or thickness, so bone augmentation procedures enable implant treatments in cases in which it would otherwise not be an option. Bone augmentation procedures can be performed prior to implant placement (in a two-stage procedure), or during the same surgical procedure (one-stage procedure), using numerous materials and techniques.

Various options have been described [29], including: autogenous bone grafts, allografts, xenografts, alloplastic grafts, barrier membranes for guided bone regeneration (GBR), growth factors (and BMPs in particular), platelet-rich plasma (PRP), inlay grafting, onlay grafting, ridge expansion, and distraction osteogenesis.

and proliferation (mitogenesis) at the site of injury. In particular, *in vitro* and *in vivo* studies have demonstrated that PDGF is a potent chemotactic and mitogenic factor for gingival and

Since the first animal study conducted by Lynch and co-workers [19], extensive *in vitro,* preclinical and clinical studies have been performed using PDGF, alone or in combination with other GFs, for incrementing bone vertically and horizontally, and for treating periodontal and peri-implant defects. The positive outcomes of these studies provide strong evidence of the safety and predictably of rhPDGF combined with specific scaffolds in periodontal and peri-

Although a large body of preclinical and clinical data has been obtained for only a few GFs,

The activity and osteoinductive potential of fibroblast growth factor (FGF) have been the object of various studies [22-24]. FGF signaling reportedly interacts with BMP signaling in bone

Few studies have considered the use of parathyroid hormone (PTH) as a factor for modulating bone augmentation and healing [25]. PTH binding activates PTH1R to stimulate several downstream effectors and also drives the internalization of the PTH1R(PTH type I receptor)- TGFβRII (TGF-β type II receptor) complex, which attenuates both TGF-β and PTH signaling on bone development. The transcriptional factor/cAMP response element binding protein (CREB) mediates PTH signaling in osteoblasts, and the PTH-CREB signaling pathway serves

Transforming growth factor-β (TGF-β) [26-27], vascular endothelial growth factor (VEGF) [24], and insulin-like growth factor (IGF) [28] are also the object of studies regarding the biological

Given the biological properties of GFs, a major focus of research has concerned the clinical application of the osteoinductive proteins, such as some BMPs, for enhancing new bone formation. Bone loss involving the teeth may be secondary to diseases such as periodonti‐ tis, cystic diseases or tumors, or the consequence of trauma. Alveolar bone augmentation procedures are often needed for the purpose of inserting dental implants for prosthetic

Missing teeth can be replaced with prostheses supported on dental implants, which can only be inserted in patients with an adequate alveolar ridge height and/or thickness, so bone augmentation procedures enable implant treatments in cases in which it would otherwise not be an option. Bone augmentation procedures can be performed prior to implant placement (in a two-stage procedure), or during the same surgical procedure (one-stage procedure), using

periodontal ligament fibroblasts, cementoblasts and osteoblasts [18].

716 Regenerative Medicine and Tissue Engineering

implant regeneration, suggesting promising clinical applications [18,20,21].

formation, showing a synergic action on osteogenesis [11].

as an effective activator of BMP-2 expression [11].

**3. Clinical application of GFs in dentistry**

properties of these bioactive molecules.

numerous materials and techniques.

rehabilitation.

others have nonetheless been assessed for possible applications in clinical practice.

Tonetti et al. [30] described various techniques that have been developed to correct inadequate vertical and horizontal bone volumes, such as guided bone regeneration (GBR), sinus lift and onlay bone grafting.

Bone augmentation techniques have also been promoted as a means for treating periodontal and peri-implant diseases in an effort to regenerate lost periodontal or peri-implant soft and hard tissues [31-32].

Autogenous bone grafts are still considered the gold standard for bone repair in most cases, though there are some restrictions in their use in clinical practice because of the morbidity of the harvesting procedures and the limited amount of bone available. Many authors have consequently been studying the biocompatibility and effectiveness of other materials as potential substitutes for autogenous bone grafts.

The most recent and promising approach consists in applying osteoinductive growth factors to promote new bone formation (protein therapy) [33], providing a new alternative to autog‐ enous grafts and other bone substitutes.

Combining growth factors with osteoinductive scaffolds may facilitate a faster and more significant enhancement of new bone formation thanks to the delivery of the growth factors at the site of the graft, and because their three-dimensional stability provides protection during the gradual replacement of the graft with newly-formed bone. Numerous materials have been used in combination with GFs, including inorganic bovine bone, porous hydroxyapatite and demineralized human bone matrix.

Numerous pre-clinical and clinical studies have looked into how GF implantation influences bone augmentation and implant osteointegration, focusing particularly on recombinant human BMP-2 (rhBMP-2), rhBMP-7 and recombinant human growth and differentiation factor-5 (rhGDF-5), combined with a variety of biomaterials used as scaffolds and delivery systems.

Although the potential value of GFs in alveolar bone regeneration and augmentation has been highlighted by numerous authors [6,31,34-35], it is still difficult to assess the different biological potential of each growth factor, because few analyses have compared different growth factors under identical *in vivo* conditions [24].

There is still much to learn about osteogenic growth factors: only a handful of growth and differentiation factors have been the object of clinical evaluation [6,18,25] and further studies are needed to identify predictable clinical outcomes.

#### **3.1. Pre-prosthetic surgery for the purpose of dental rehabilitation with implants**

Several surgical techniques and materials - including the use of GFs - have been introduced with a view to increasing bone volume in order to enable the placement of dental implants.

The systematic literature review conducted by Jung and coworkers [25] assessed the clinical, histological and radiographic outcomes after BMP-2, BMP-7, GDF-5, PDGF, and PTH had been used for localized alveolar ridge augmentation. Altogether, 74 studies met the authors' inclusion criteria, including 6 on the outcome of BMP-2 for localized alveolar ridge augmen‐ tation in humans; the remainder were pre-clinical studies involving BMP-2, BMP-7, GDF-5, PDGF, and PTH. For all the GFs other than BMP-2, no human studies met the inclusion criteria. Concerning the animal studies, most of those on BMP-2 (43 out of 45) showed a positive effect of this growth factor. Six of 8 studies reported a positive effect of BMP-7. The one animal study on GDF-5 spoke of a statistically significant increase in bone volume. Five of 10 studies involving the use of PDGF also reported a statistically significant increase in bone volume. Four animal studies identified a significantly greater bone regeneration in cases treated with PTH than in controls. In the six human studies, BMP-2 influenced local bone augmentation, with a dose-dependent increase in bone volume. The dose of BMP-2 delivered seemed to have an impact on treatment outcome, local bone regeneration being greater for higher BMP-2 doses [36-38], with a smaller decrease in bone height at extraction socket sites [39]. Four of these six human studies were designed as randomized-controlled clinical trials (RCT) [37-40], the other two as prospective cohort studies [36,41]. The locally-applied dose of BMP-2 ranged from 0.5 to 1.75 mg/ml, or 0.12 to 3.4 mg/patient, respectively. An absorbable collagen sponge (ACS) was used in five studies, while Jung et al. [40] used a demineralized bovine bone matrix (DBBM) as a carrier. The treatments included sinus floor augmentation [38,41], extraction socket preservation [36-37,39], augmentation of localized ridge defects [36], and lateral ridge augmentation combined with simultaneous implant placement [40].

The 16-week open-label study conducted by Boyne and coworkers [41] assessed the safety and efficacy of implanting BMP-2 delivered on an absorbable collagen sponge (rhBMP-2/ACS) for two-stage maxillary floor sinus augmentation. The dose of rhBMP-2 ranged from 1.77 to 3.40 mg per patient. Significant bone growth was documented by computed tomographic (CT) scans in all evaluable patients (11/12), with an overall mean response of 8.51 mm in height (±4.13 mm). Histology on core bone biopsies obtained when the dental implant was inserted confirmed the good quality of the bone induced by rhBMP-2/ACS.

In a more recent RCT, Boyne and colleagues [38] found no statistically significant differences in terms of the increase in ridge height, as measured using CT scans, between their treatment and control (bone graft) groups, and even a narrower ridge width in the former after using BMP-2/ACS in two-stage maxillary floor sinus augmentations.

Bianchi et al. [37] investigated the efficacy of different concentrations of rhBMP-2 in regener‐ ating bone in alveolar defects in the anterior maxilla, reporting a positive outcome in terms of bone volume augmentation.

Another RCT [39] compared the efficacy of rhBMP-2 in two different concentrations, delivered on ACS, with placebo ACS alone in 80 patients requiring local alveolar ridge augmentation for buccal wall defects (> or =50% buccal bone loss around the extraction socket) immediately after tooth extraction of the maxillary bicuspids. They found no statistically significant effects of BMP-2 on the treatment outcome when a lower dose was used, but a statistically significant positive effect of a higher dose (1.50 mg/ml rhBMP-2/ACS). In addition, bone density and histology revealed no differences between newly-induced and native bone.

The systematic literature review conducted by Jung and coworkers [25] assessed the clinical, histological and radiographic outcomes after BMP-2, BMP-7, GDF-5, PDGF, and PTH had been used for localized alveolar ridge augmentation. Altogether, 74 studies met the authors' inclusion criteria, including 6 on the outcome of BMP-2 for localized alveolar ridge augmen‐ tation in humans; the remainder were pre-clinical studies involving BMP-2, BMP-7, GDF-5, PDGF, and PTH. For all the GFs other than BMP-2, no human studies met the inclusion criteria. Concerning the animal studies, most of those on BMP-2 (43 out of 45) showed a positive effect of this growth factor. Six of 8 studies reported a positive effect of BMP-7. The one animal study on GDF-5 spoke of a statistically significant increase in bone volume. Five of 10 studies involving the use of PDGF also reported a statistically significant increase in bone volume. Four animal studies identified a significantly greater bone regeneration in cases treated with PTH than in controls. In the six human studies, BMP-2 influenced local bone augmentation, with a dose-dependent increase in bone volume. The dose of BMP-2 delivered seemed to have an impact on treatment outcome, local bone regeneration being greater for higher BMP-2 doses [36-38], with a smaller decrease in bone height at extraction socket sites [39]. Four of these six human studies were designed as randomized-controlled clinical trials (RCT) [37-40], the other two as prospective cohort studies [36,41]. The locally-applied dose of BMP-2 ranged from 0.5 to 1.75 mg/ml, or 0.12 to 3.4 mg/patient, respectively. An absorbable collagen sponge (ACS) was used in five studies, while Jung et al. [40] used a demineralized bovine bone matrix (DBBM) as a carrier. The treatments included sinus floor augmentation [38,41], extraction socket preservation [36-37,39], augmentation of localized ridge defects [36], and lateral ridge

augmentation combined with simultaneous implant placement [40].

confirmed the good quality of the bone induced by rhBMP-2/ACS.

BMP-2/ACS in two-stage maxillary floor sinus augmentations.

bone volume augmentation.

718 Regenerative Medicine and Tissue Engineering

The 16-week open-label study conducted by Boyne and coworkers [41] assessed the safety and efficacy of implanting BMP-2 delivered on an absorbable collagen sponge (rhBMP-2/ACS) for two-stage maxillary floor sinus augmentation. The dose of rhBMP-2 ranged from 1.77 to 3.40 mg per patient. Significant bone growth was documented by computed tomographic (CT) scans in all evaluable patients (11/12), with an overall mean response of 8.51 mm in height (±4.13 mm). Histology on core bone biopsies obtained when the dental implant was inserted

In a more recent RCT, Boyne and colleagues [38] found no statistically significant differences in terms of the increase in ridge height, as measured using CT scans, between their treatment and control (bone graft) groups, and even a narrower ridge width in the former after using

Bianchi et al. [37] investigated the efficacy of different concentrations of rhBMP-2 in regener‐ ating bone in alveolar defects in the anterior maxilla, reporting a positive outcome in terms of

Another RCT [39] compared the efficacy of rhBMP-2 in two different concentrations, delivered on ACS, with placebo ACS alone in 80 patients requiring local alveolar ridge augmentation for buccal wall defects (> or =50% buccal bone loss around the extraction socket) immediately after tooth extraction of the maxillary bicuspids. They found no statistically significant effects of BMP-2 on the treatment outcome when a lower dose was used, but a statistically significant

Finally, Jung et al. [40] tested whether adding rhBMP-2 to a xenogenic bone substitute mineral could improve guided bone regeneration in the case of bone defects requiring lateral bone augmentation procedures and simultaneous implant placement. Following implant insertion (baseline), the peri-implant bone defect height was measured from the implant shoulder to the first implant-bone contact. The authors reported a positive, but statistically insignificant effect of BMP-2 on the amount of newly-formed bone (37±11.2%) compared with the control group (30± 8.9%). On the other hand, they found more mature lamellar bone (76±14.4% versus 56±18.3%) and a greater area of bone-to-graft contact (57±16.2% versus 30±22.6%) at the BMP-2 treated sites.

Various methods have been described for increasing bone volume before or at the time of positioning implants [25], one of the best-documented of these methods being GBR for intraoral bone augmentation. To overcome some of the drawbacks of this technique, e.g. a long treatment time, the difficulty of predicting any vertical bone augmentation, the risk of infection after membrane exposure, research has concentrated on the use of bioactive molecules that induce local bone formation. Using the GBR technique, the width and height of the alveolar ridge is increased in areas of insufficient bone volume by applying barrier membranes, alone or in combination with bone grafts or substitutes.

Misch [42] published a human case series of atrophic posterior mandible augmentation prior to implant insertion, using recombinant human BMP-2 2/absorbable collagen sponge (rhBMP-2/ACS) and titanium mesh. All the 10 implants involved in the study, inserted after a 6-month healing period, became integrated and were restored with single crowns.

Many *in vivo* studies used critical-size supra-alveolar peri-implant defect models and other bone augmentation methods simultaneously with implant insertion. In an animal study, Sigurdsson et al. [43] found that defect sites implanted with rhBMP-2⁄ACS showed signs of a statistically significant and clinically relevant vertical alveolar bone augmentation by com‐ parison with controls (ACS). Although the titanium implant was osseointegrated after a 16 week healing interval, the BIC (bone-to-implant contact) was lower than in resident bone, as was to be expected; the newly-induced bone was often in a thin layer on the implant surface, probably due to the unpredictability of ACS in providing adequate space for new bone formation.

Wikesjö and colleagues [44] subsequently used a critical-size supra-alveolar peri-implant defect model to study the efficacy of an ePTFE GBR device in supporting rhBMP-2⁄-induced bone formation in dogs. The space-providing macro-porous membrane was characterized by the ability to prevent the compression of the rhBMP-2/ACS construct, while allowing for vascularization via the gingival connective tissue. The authors compared GBR alone with rhBMP-2(0.4 mg)⁄ACS and rhBMP-2(0.4 mg)⁄ACS combined with GBR. Histometric analysis on block biopsies after an 8-week healing interval revealed the best results in the third sample, i.e. the GBR-rhBMP-2⁄ACS combination, which revealed bone formation filling the domeshaped GBR device, with a vertical bone gain at the turned implants averaging 4.7 ± 0.2 mm, and an induced bone area of 9.6 ± 0.7 mm2 , generating a highly-significant correlation between the induced bone area and the space provided by the GBR device. This study highlighted the crucial importance of providing space in order to obtain clinically significant benefits from a BMP construct.

Jung et al. [45] ran a randomized-controlled clinical trial with a split-mouth design, in which implants were placed in sites exhibiting lateral bone defects and patients were randomly selected for treatment with demineralized bovine bone mineral and bioresorbable collagen membrane, with (test) or without (control) the addition of rhBMP-2. After an average healing period of 6 months, a reentry operation was performed for abutment connection and prosthetic reconstruction. At the 3-year follow-up, all 34 implants in all 11 patients were clinically stable and radiologically osseointegrated. At the 5-year follow-up, 32 implants were stable and functioning, while 2 were not re-examined because the patient had moved away. The survival rate of the implants examined at 3 and 5 years was therefore 100% for both the test and the control sites. The periapical radiographs of the test and control sites also showed no periimplant radiolucency at the 3- and 5-year follow-up examination, demonstrating healthy periimplant tissues with minimal marginal bone loss, and only minor prosthetic complications were recorded. In short, both the test and the control sites revealed excellent clinical and radiological outcomes after 3 and 5 years, with no statistically significant differences in any of the parameters examined (though the authors emphasized the need for a larger group of patients in future studies).

In a micro-CT study in dogs, Al-Hazmi and co-workers [20] assessed the efficacy of using PDGF-BB and xenografts, with or without collagen membranes, for GBR around immediate implants with buccal dehiscence defects. They concluded that using PDGF and xenografts resulted in greater BBT (buccal bone thickness), BBV (buccal bone volume), VBH (vertical bone height) and BIC (bone-to-implant contact) when used alone rather than in combination with a collagen membrane. Their results are consistent with the report from Simion et al. [46], who said that barrier membranes may interfere with the chemotactic effect of GFs on periosteal pluripotential mesenchymal cells.

Further studies are nonetheless warranted to investigate the influence of barrier membranes on the periosteal pluripotential mesenchymal cells [20].

Most of the clinical studies on rhPDGF have focused on periodontal and peri-implant regen‐ eration, and only a few human studies have investigated ridge preservation for implant placement in extraction socket defects [47], or three-dimensional ridge augmentation [48].

In a pilot study, Nevins et al. [47] tested whether mineralized collagen bone substitute (MCBS) combined with recombinant human platelet-derived growth factor-BB (0.3 mg/mL) could generate enough viable bone in buccal wall extraction defects to enable implant placement.

In a more recent clinical study, Nevins and colleagues [49] focused on human buccal plate extraction socket regeneration with recombinant human platelet-derived growth factor BB or enamel matrix derivative. Buccal plate resorption is a critical issue when it comes to implant placement. They compared four groups: A (mineral collagen bone substitute [MCBS] scaffold alone), B (MCBS with recombinant human platelet-derived growth factor BB [rhPDGF-BB; 0.3 mg/mL]), C (MCBS with enamel matrix derivative [EMD]), and D (a combination of EMD with bone ceramic). Grafting was done at the time of extraction, advancing the buccal flap for primary closure. Histology on trephine core biopsies of the implant site performed 5 months later, at the time of implant placement, identified new bone healing around the biomaterial scaffolds with no statistically significant differences between the four treatment groups. There was a histomorphometric trend towards a greater quantity of new bone in the rhPDGF-BBtreated group, with the most favorable ridge morphology for the purposes of an optimal implant placement at reentry surgery.

Simion et al. [48] reported on two human cases of patients who underwent three-dimensional ridge augmentation using a xenograft combined with rhPDGF-BB. In the first patient, a deproteinized bovine block infused with rh-PDGF was attached to the alveolar crest with two screws to obtain a horizontal ridge augmentation. The second patient underwent a vertical ridge augmentation procedure involving deproteinized bovine bone particles embedded in a collagen matrix soaked in rhPDGF-BB. Three titanium dental implants were placed in each patient 5 months later with excellent clinical and histological outcomes, mean that rhPDGF-BB in combination with a deproteinized bovine graft has promise in applications for regener‐ ating large three-dimensional alveolar defects in humans.

#### **3.2. Dental implant surface coatings with GFs**

and an induced bone area of 9.6 ± 0.7 mm2

720 Regenerative Medicine and Tissue Engineering

BMP construct.

patients in future studies).

pluripotential mesenchymal cells.

on the periosteal pluripotential mesenchymal cells [20].

, generating a highly-significant correlation between

the induced bone area and the space provided by the GBR device. This study highlighted the crucial importance of providing space in order to obtain clinically significant benefits from a

Jung et al. [45] ran a randomized-controlled clinical trial with a split-mouth design, in which implants were placed in sites exhibiting lateral bone defects and patients were randomly selected for treatment with demineralized bovine bone mineral and bioresorbable collagen membrane, with (test) or without (control) the addition of rhBMP-2. After an average healing period of 6 months, a reentry operation was performed for abutment connection and prosthetic reconstruction. At the 3-year follow-up, all 34 implants in all 11 patients were clinically stable and radiologically osseointegrated. At the 5-year follow-up, 32 implants were stable and functioning, while 2 were not re-examined because the patient had moved away. The survival rate of the implants examined at 3 and 5 years was therefore 100% for both the test and the control sites. The periapical radiographs of the test and control sites also showed no periimplant radiolucency at the 3- and 5-year follow-up examination, demonstrating healthy periimplant tissues with minimal marginal bone loss, and only minor prosthetic complications were recorded. In short, both the test and the control sites revealed excellent clinical and radiological outcomes after 3 and 5 years, with no statistically significant differences in any of the parameters examined (though the authors emphasized the need for a larger group of

In a micro-CT study in dogs, Al-Hazmi and co-workers [20] assessed the efficacy of using PDGF-BB and xenografts, with or without collagen membranes, for GBR around immediate implants with buccal dehiscence defects. They concluded that using PDGF and xenografts resulted in greater BBT (buccal bone thickness), BBV (buccal bone volume), VBH (vertical bone height) and BIC (bone-to-implant contact) when used alone rather than in combination with a collagen membrane. Their results are consistent with the report from Simion et al. [46], who said that barrier membranes may interfere with the chemotactic effect of GFs on periosteal

Further studies are nonetheless warranted to investigate the influence of barrier membranes

Most of the clinical studies on rhPDGF have focused on periodontal and peri-implant regen‐ eration, and only a few human studies have investigated ridge preservation for implant placement in extraction socket defects [47], or three-dimensional ridge augmentation [48]. In a pilot study, Nevins et al. [47] tested whether mineralized collagen bone substitute (MCBS) combined with recombinant human platelet-derived growth factor-BB (0.3 mg/mL) could generate enough viable bone in buccal wall extraction defects to enable implant placement. In a more recent clinical study, Nevins and colleagues [49] focused on human buccal plate extraction socket regeneration with recombinant human platelet-derived growth factor BB or enamel matrix derivative. Buccal plate resorption is a critical issue when it comes to implant placement. They compared four groups: A (mineral collagen bone substitute [MCBS] scaffold alone), B (MCBS with recombinant human platelet-derived growth factor BB [rhPDGF-BB; 0.3 Another interesting approach to enhancing alveolar ridge augmentation with a view to dental implant placement involves using implants coated with GFs.

Wikesjo and colleagues [35] reviewed the literature on implants coated with a bone-inductive factor capable of stimulating local bone formation and osseointegration. They concluded that rhBMP-2 can be delivered successfully for the purposes of inducing local bone formation and osseointegration by using screw-type endosseous oral implants with titanium oxide surfaces with open pores as a carrier. They also found that purpose-designed implant surfaces coated with rhBMP-2 resulted in the formation of Type II bone and significant osseointegration without any need for biomaterials or devices for GBR.

In an *in vivo* animal model, Susin et al. [50] used the critical-size supra-alveolar peri-implant defect model to assess the potential of a purpose-designed porous titanium oxide implant surface coated with rhBMP-7 for inducing alveolar bone formation and enhancing osseointe‐ gration. The animals received implants coated with rhBMP-7 at 1.5 or 3.0 mg/ml randomized to the contralateral jaw quadrants. The authors found clinically relevant bone formation and osseointegration with no statistically significant differences in terms of bone formation between the sites treated with rhBMP-7 at 1.5 or 3.0 mg/ml. Histology showed an increase in the height and area of the bone, and the newly-formed bone exhibited the same characteristics as the contiguous resident bone. Their observations support the significant clinical value of rhBMP-7 in inducing bone regeneration, but the authors made the point that higher concen‐ trations were associated with some local side effects.

Other authors [e.g. 51-52] have investigated *in vivo* the potential of an rhGDF-5 coating on an oral implant with a porous titanium oxide surface for stimulating local bone formation, including osseointegration and vertical augmentation of the alveolar ridge.

Polimeni and co-workers [51] examined a bilateral critical-size, 5 mm, supra-alveolar periimplant defect model in dogs. Six animals received implants coated with 30 or 60 μg rhGDF-5, and another six animals received implants coated with 120 μg rhGDF-5 or left uncoated (controls). The implants coated with rhGDF-5 displayed only limited peri-implant bone remodeling in the resident bone, as measured using fluorescent bone markers, with the 120 μg dose coinciding with a more advanced remodeling than the 60 and 30 μg doses. These results suggest a dose-dependent osteoinductive and/or osteoconductive effect of rhGDF-5 coated oral implants. Leknes et al. [52] performed an *in vivo* study in dogs that consisted in placing different kinds of implant in the alveolar ridge of the posterior mandible following the surgical extraction of the premolars and reduction of the alveolar ridge. Six animals were treated with implants coated with rhGDF-5 in doses of 30 or 60 μg/implant in contralateral jaw quadrants, while six received implants coated with rhGDF-5 at 120 μg/implant or uncoated implants (for control purposes), using a split-mouth design. The radiographs showed a dosedependent formation of mineralized tissue significantly greater than around the uncoated implants, the greatest increase corresponding to the implants coated with 60 μg and 120 μg of rhGDF-5, and amounting to approximately 2.2 mm in both cases at 8 weeks. The authors also reported no adverse events, such as peri-implant bone remodeling, implant displacement, or seroma formation.

The above-mentioned studies indicate that these GFs have great potential for stimulating clinically relevant local bone formation, though it should be emphasized that further studies are essential to address their most appropriate dosage, carriers, and applications, as well as the long-term prognosis of GF-coated titanium implants.

#### **3.3. Maxillary sinus lift procedure**

Sinus floor elevation with immediate or delayed dental implant placement is a well-known technique for dental rehabilitation in cases of severe atrophy of the posterior maxilla due to the extension and pneumatization of the maxillary sinus. Many materials, such as autografts, xenografts, and synthetic bone substitutes, have been shown to achieve acceptable clinical results when used in maxillary sinus floor augmentations [53]. The use of GFs with various carriers and dosages has recently been investigated in combination with sinus augmentation procedures too.

Ho and colleagues [54] assessed the efficacy of various bioimplants used in maxillary sinus lift procedures with the lateral window approach in a rabbit model. They compared particulated autogenous bone, demineralized bone matrix (DBM), DBM combined with purified BMP-7 (BMP-7/DBM bioimplants), and bioimplants consisting of a poloxamer gel with BMP-7 in two different doses. In their animal model, BMP-containing bioimplants had produced more new bone and a greater new bone surface area at 2 weeks than autografts, but the advantage of these bioimplants subsequently seemed to be lost, since the differences between the bioim‐ plants and the autografts had disappeared by 8 weeks. The authors concluded that BMP- containing bioimplants prompt a more rapid bone formation, possibly offering a greater implant stability earlier in the healing period, and therefore enabling clinicians to place osseointegrated implants in augmented maxillae sooner after grafting.

Other authors [e.g. 51-52] have investigated *in vivo* the potential of an rhGDF-5 coating on an oral implant with a porous titanium oxide surface for stimulating local bone formation,

Polimeni and co-workers [51] examined a bilateral critical-size, 5 mm, supra-alveolar periimplant defect model in dogs. Six animals received implants coated with 30 or 60 μg rhGDF-5, and another six animals received implants coated with 120 μg rhGDF-5 or left uncoated (controls). The implants coated with rhGDF-5 displayed only limited peri-implant bone remodeling in the resident bone, as measured using fluorescent bone markers, with the 120 μg dose coinciding with a more advanced remodeling than the 60 and 30 μg doses. These results suggest a dose-dependent osteoinductive and/or osteoconductive effect of rhGDF-5 coated oral implants. Leknes et al. [52] performed an *in vivo* study in dogs that consisted in placing different kinds of implant in the alveolar ridge of the posterior mandible following the surgical extraction of the premolars and reduction of the alveolar ridge. Six animals were treated with implants coated with rhGDF-5 in doses of 30 or 60 μg/implant in contralateral jaw quadrants, while six received implants coated with rhGDF-5 at 120 μg/implant or uncoated implants (for control purposes), using a split-mouth design. The radiographs showed a dosedependent formation of mineralized tissue significantly greater than around the uncoated implants, the greatest increase corresponding to the implants coated with 60 μg and 120 μg of rhGDF-5, and amounting to approximately 2.2 mm in both cases at 8 weeks. The authors also reported no adverse events, such as peri-implant bone remodeling, implant displacement, or

The above-mentioned studies indicate that these GFs have great potential for stimulating clinically relevant local bone formation, though it should be emphasized that further studies are essential to address their most appropriate dosage, carriers, and applications, as well as

Sinus floor elevation with immediate or delayed dental implant placement is a well-known technique for dental rehabilitation in cases of severe atrophy of the posterior maxilla due to the extension and pneumatization of the maxillary sinus. Many materials, such as autografts, xenografts, and synthetic bone substitutes, have been shown to achieve acceptable clinical results when used in maxillary sinus floor augmentations [53]. The use of GFs with various carriers and dosages has recently been investigated in combination with sinus augmentation

Ho and colleagues [54] assessed the efficacy of various bioimplants used in maxillary sinus lift procedures with the lateral window approach in a rabbit model. They compared particulated autogenous bone, demineralized bone matrix (DBM), DBM combined with purified BMP-7 (BMP-7/DBM bioimplants), and bioimplants consisting of a poloxamer gel with BMP-7 in two different doses. In their animal model, BMP-containing bioimplants had produced more new bone and a greater new bone surface area at 2 weeks than autografts, but the advantage of these bioimplants subsequently seemed to be lost, since the differences between the bioim‐ plants and the autografts had disappeared by 8 weeks. The authors concluded that BMP-

the long-term prognosis of GF-coated titanium implants.

**3.3. Maxillary sinus lift procedure**

722 Regenerative Medicine and Tissue Engineering

including osseointegration and vertical augmentation of the alveolar ridge.

seroma formation.

procedures too.

In a clinical study, Boyne and colleagues [38] compared different concentrations of rhBMP-2 (0.75 and 1.5 mg/mL), delivered on an absorbable collagen sponge (ACS) carrier, with bone grafts to identify a safe and effective concentration of rhBMP-2 for use in maxillary sinus floor augmentation procedures. Judging from density measurements on CT scans obtained before and 4 months after treatment, and 6 months after functional loading of the dental implants, and from core biopsies obtained at the time of placing the dental implant, they established that the 1.5 mg/mL dose of rhBMP-2/ACS was more appropriate in a pivotal, randomized, multicenter study to compare rhBMP-2/ACS with conventional bone graft for staged maxillary sinus floor augmentation to support dental implants for long-term functional loading.

These data prompted a randomized, parallel evaluation of rhBMP-2/ACS and autogenous bone grafts for two-stage maxillary sinus floor procedures [55]: 160 individuals with less than 6 mm of native bone height in the posterior maxilla were randomized for treatment with 1.5 mg/mL rhBMP-2/ACS or an autograft. Height and density measurements were obtained on CT scans, and core biopsies obtained at the time of dental implant placement underwent histological examination. A significant amount of new bone had formed by 6 months postop‐ eratively in both treatment groups, but there was a significant difference in the density of the newly-induced bone at the 6-month follow-up, which was denser in the bone graft group than in the group treated with rhBMP-2/ACS. Six months after dental restoration (functional loading), however, the bone induced in the rhBMP-2/ACS group was significantly denser than in the bone graft group. No major differences emerged between the two groups in terms of the histological parameters. 17% of the patients in the autograft group experienced long-term parasthesia, pain, or gait disturbance relating to the bone graft harvest. Adverse reactions frequently recorded in the rhBMP-2/ACS group related to excessive facial swelling, and this edema was attributed to the chemotactic cellular recruitment to the site of rhBMP-2 implan‐ tation and neovascularization of the grafted area; although it was severe, this edema did not adversely affect the outcome. This study confirmed the efficacy and safety of rhBMP-2/ACS by comparison with bone grafting for sinus floor augmentation, given the morbidity, cost, and increased surgical time associated with the harvesting of autogenous bone.

Kao and coworkers [56] measured the bone formation after a lateral window sinus augmen‐ tation with recombinant human BMP-2/ absorbable collagen sponge (rhBMP-2/ACS) in combination with Bio-Oss by comparison with the results achieved with a Bio-Oss graft alone. Histology demonstrated that less new bone formed in patients treated with rhBMP-2/ACS + Bio-Oss than in those treated with Bio-Oss alone, pointing to a negative effect on bone formation of combining rhBMP-2 with Bio-Oss for maxillary sinus augmentation.

Gruber and coworkers [57] studied a GF closely related to the BMP family - the recombi‐ nant human growth and differentiation factor-5 (rhGDF-5) - in an *in vivo* study involving the use of different materials in sinus floor augmentation procedures in Goettingen miniature pigs. They demonstrated that associating rhGDF-5 with β-tricalcium phosphate (β-TCP) enhanced bone formation by comparison with the results obtained using the β-TCP carrier material alone.

In a further study using a split-mouth study design, the same authors [13] compared rhGDF-5 coated β-TCP with particulated autogenous bone grafts combined with the scaffold material (β-TCP). In each minipig, the sinus floors were augmented (simultaneously inserting the dental implants) with β-TCP mixed with autogenous cortical bone chips on one side, and using β-TCP coated with two different concentrations of rhGDF-5 on the contralateral side. Histology and histomorphometric analyses demonstrated that rhGDF-5-coated β-TCP not only enhanced new bone formation, but also - by comparison with a combination of β-TCP and autogenous bone chips - induced a significant increase in VD (volume density) and BIC (bone-to-implant contact) in the augmentation material.

Stavropoulos et al. [58] ran a prospective, multicenter, randomized clinical trial to examine the histological outcome of maxillary sinus lifting with rhGDF-5/β-TCP or β-TCP and autogenous bone (β-TCP/AB) composite. Thirty-one patients requiring unilateral maxillary sinus floor augmentation with a residual alveolar bone height <5 mm were treated using a lateral window approach. Cylindrical biopsies were harvested with a trephine bur during implant site preparation 3 or 4 months after sinus floor augmentation (three groups (a) rhGDF-5/b-TCP and a 3-month healing period, (b) rhGDF-5/b-TCP and a 4- month healing period, and (c) b-TCP/AB and a 4-month healing period). Histological and histometric analyses showed that sinus augmentation with rhGDF-5/β-TCP resulted in new bone in comparable amounts and of similar quality to the bone obtained with a β-TCP/AB composite graft, suggesting that rhGDF-5/β-TCP could eliminate the need for AB grafting in sinus lift procedures.

Though these favorable regenerative findings are encouraging, further studies are needed to ascertain the influence of GFs on the amount and quality of new bone formation, and on the implant survival rate after sinus lift procedures.

#### **3.4. Periodontal regeneration**

Periodontitis is a widely prevalent inflammatory disease of the tissues supporting the teeth, characterized by a progressive loss of bone and attachment.

The ultimate goal of periodontal therapy is the regeneration of periodontal tissues, which consists in stimulating new cementum formation, new alveolar bone apposition, and a functionally-oriented periodontal ligament reconstruction. Various techniques have been suggested for promoting periodontal tissue regeneration, using different bone graft materials that have gained clinical acceptance in the treatment of periodontal defects.

To overcome the weaknesses of conventional regenerative procedures, the predictability of which may be limited to selected case types, using GFs with biocompatible scaffolds to promote tissue regeneration may represent a new and promising periodontological approach.

After preliminary *in vitro* experiments, extensive *in vivo* preclinical studies have been per‐ formed to assess the potential and safety of using various GFs, alone or in combination, to treat periodontal defects.

A recent animal study by Oortgiesen et al. [23] investigated the regenerative potential of an injectable macroporous calcium phosphate cement (CaP) combined with BMP-2 or fibroblast growth factor-2 (FGF-2) in intrabony defects. After 12 weeks, only the CaP revealed limited effects on both periodontal ligament (PDL) and bone healing, while a good response in terms of bone healing was also seen with CaP/BMP-2 and CaP/FGF-2. The best PDL healing scores coincided with the combined CaP/FGF-2 treatment, suggesting that associating a topical application of FGF-2 with an injectable CaP might be a promising treatment for the purposes of periodontal regeneration.

(β-TCP) enhanced bone formation by comparison with the results obtained using the β-

In a further study using a split-mouth study design, the same authors [13] compared rhGDF-5 coated β-TCP with particulated autogenous bone grafts combined with the scaffold material (β-TCP). In each minipig, the sinus floors were augmented (simultaneously inserting the dental implants) with β-TCP mixed with autogenous cortical bone chips on one side, and using β-TCP coated with two different concentrations of rhGDF-5 on the contralateral side. Histology and histomorphometric analyses demonstrated that rhGDF-5-coated β-TCP not only enhanced new bone formation, but also - by comparison with a combination of β-TCP and autogenous bone chips - induced a significant increase in VD (volume density) and BIC (bone-to-implant

Stavropoulos et al. [58] ran a prospective, multicenter, randomized clinical trial to examine the histological outcome of maxillary sinus lifting with rhGDF-5/β-TCP or β-TCP and autogenous bone (β-TCP/AB) composite. Thirty-one patients requiring unilateral maxillary sinus floor augmentation with a residual alveolar bone height <5 mm were treated using a lateral window approach. Cylindrical biopsies were harvested with a trephine bur during implant site preparation 3 or 4 months after sinus floor augmentation (three groups (a) rhGDF-5/b-TCP and a 3-month healing period, (b) rhGDF-5/b-TCP and a 4- month healing period, and (c) b-TCP/AB and a 4-month healing period). Histological and histometric analyses showed that sinus augmentation with rhGDF-5/β-TCP resulted in new bone in comparable amounts and of similar quality to the bone obtained with a β-TCP/AB composite graft, suggesting that

rhGDF-5/β-TCP could eliminate the need for AB grafting in sinus lift procedures.

Though these favorable regenerative findings are encouraging, further studies are needed to ascertain the influence of GFs on the amount and quality of new bone formation, and on the

Periodontitis is a widely prevalent inflammatory disease of the tissues supporting the teeth,

The ultimate goal of periodontal therapy is the regeneration of periodontal tissues, which consists in stimulating new cementum formation, new alveolar bone apposition, and a functionally-oriented periodontal ligament reconstruction. Various techniques have been suggested for promoting periodontal tissue regeneration, using different bone graft materials

To overcome the weaknesses of conventional regenerative procedures, the predictability of which may be limited to selected case types, using GFs with biocompatible scaffolds to promote tissue regeneration may represent a new and promising periodontological approach.

After preliminary *in vitro* experiments, extensive *in vivo* preclinical studies have been per‐ formed to assess the potential and safety of using various GFs, alone or in combination, to treat

that have gained clinical acceptance in the treatment of periodontal defects.

TCP carrier material alone.

724 Regenerative Medicine and Tissue Engineering

contact) in the augmentation material.

implant survival rate after sinus lift procedures.

characterized by a progressive loss of bone and attachment.

**3.4. Periodontal regeneration**

periodontal defects.

Ishii and colleagues [22] investigated the effect of the combined use of basic FGF-2 and beta tricalcium phosphate (β-TCP) on root coverage in a dog model, finding that FGF-2/β-TCP enhanced the formation of new bone and cementum without any significant root resorption.

Kitamura et al. [59] undertook a multi-center, randomized, double-blind, placebo-control‐ led, dose-finding study on the potential of local applications of FGF-2 in periodontal regeneration. Modified Widman periodontal surgery was performed, during which 200 μL of the investigational formulation containing 0% (vehicle alone), 0.2%, 0.3%, or 0.4% FGF-2 was administered to 2- or 3-walled vertical bone defects in 253 adult patients with periodontitis. The primary outcome was the percentage of bone fill visible on radio‐ graphs 36 weeks after administering the treatment. All the doses of FGF-2 were significant‐ ly superior to the vehicle alone (p < 0.01) in terms of the percentage of bone fill, and this percentage peaked in the 0.3% FGF-2 group. No significant differences were observed between the four groups in terms of the regained clinical attachment (CAL), with all patients scoring around 2 mm (this was judged to be due to the different healing pat‐ terns between the FGF-2 groups and the 'vehicle alone' group). Conventional periodontal surgery (which corresponds to the 'vehicle alone' group) usually gives rise to long junctional epithelial attachments, but manual probing cannot precisely distinguish fibrous from epithelial attachments, so the difference in healing pattern cannot be reflected in the CAL regained by the different treatment groups. This limitation could have been over‐ come by histology, but this was not done for ethical reasons. No clinical safety issues emerged in this study. These results support the efficacy and safety of topical FGF-2 applications for periodontal regeneration in humans.

When implanted in furcation defects exposed surgically or by inflammatory processes in *Papio ursinus*, recombinant human osteogenic protein-1 (hOP-1) or BMP-7 tends to induce cemen‐ togenesis with the insertion of *de novo* generated Sharpey's fibers. Long-term studies on *P. ursinus* after hOP-1 implantation show a highly-organized periodontal ligament space with periodontal ligament fibers cursing from the newly-formed and mineralized cementum to the regenerated alveolar bone, with a multitude of supporting capillaries throughout the perio‐ dontal ligament space [60].

In an experimental study by Teare et al. [27], binary applications of hOP-1 and hTGF-β(3) were implanted in Class II furcation defects of the mandibular molars of Chacma baboons (*P. ursinus*) to induce periodontal tissue regeneration. Sixty days after implantation, the animals were killed and histological and histomorphometric studies led the authors to conclude that hOP-1 and hTGF-β(3) in Matrigel(®) matrix induced substantial periodontal tissue regenera‐ tion and cementogenesis.

In their review, Ripamonti et al. [61] emphasized the induction of bone formation by the osteogenic proteins of the TGF-beta superfamily in the nonhuman primate, *P. ursinus*.

In a recent study in beagle dogs, Kim and co-workers [62] compared a candidate β-tricalcium phosphate (β-TCP) carrier technology with the absorbable collagen sponge (ACS) benchmark for supporting rhGDF-5-stimulated periodontal wound healing/regeneration in intrabony periodontal defects. Both solutions stimulated the formation of functionally-oriented perio‐ dontal ligament, cellular mixed-fiber cementum, and woven/lamellar bone, but bone regen‐ eration (height and area) was significantly greater for the rhGDF-5/β-TCP construct. The structural integrity of the β-TCP carrier preventing compression while providing a framework for bone ingrowth may account for these results.

A phase IIa randomized controlled clinical and histological pilot study was conducted to assess rhGDF-5/β-TCP for periodontal regeneration [63]. Twenty chronic periodontitis patients participated in the study, each with at least one tooth scheduled for extraction with a probing depth (PD) ≥6 mm and an associated intrabony defect ≥4 mm following basic periodontal therapy. Participants (one defect/patient) were randomized to receive open flap debridement (OFD) + rhGDF-5/β-TCP (n = 10) or OFD alone (control; n = 10). Both protocols resulted in statistically significant clinical improvements. Descriptive statistics showed a greater reduc‐ tion in PD after OFD with rhGDF-5/β-TCP than after OFD alone (3.7 ± 1.2 versus 3.1 ± 1.8 mm; p = 0.26), as well as less gingival recession (0.5 ± 0.8 versus 1.4 ± 1.0 mm; p < 0.05) and a greater CAL gain (3.2 ± 1.7 versus 1.7 ± 2.2 mm; p = 0.14) at the deepest aspect of the defect. Block biopsies of the defect sites were collected 6 months after surgery and prepared for histology. Five biopsies (1 rhGDF-5/β-TCP; 4 OFD) were deemed unsuitable for histological or histo‐ metric evaluation. Bone regeneration height (2.19 ± 1.59 versus 0.81 ± 1.02 mm; p = 0.08) and PDL (2.16 ± 1.43 versus 1.23 ± 1.07 mm; p = 0.26), cementum (2.16 ± 1.43 versus 1.23 ± 1.07 mm; p = 0.26) and bone regeneration area (0.74 ± 0.69 versus 0.32 ± 0.47 mm<sup>2</sup> ; p = 0.14) were greater at sites treated with rhGDF-5/β-TCP compared to controls. These differences failed to reach statistical significance, however, and the authors said that further studies on larger samples will be needed to verify these findings.

The potential of PDGFs for promoting new bone formation and/or periodontal wound healing/ regeneration has been examined in a variety of pre-clinical animal models. *In vivo* experimental studies have been performed using PDGF-BB alone or in combination with other GFs, such as insulin-like growth factor (IGF), and shown that these growth factors promoted new bone, cementum and periodontal ligament formation *in vivo*.

The first human clinical trial testing the effect of rhPDGF/rhIGF-I in periodontal defects was reported by Howell and colleagues [64] with promising results.

Early human clinical studies used rhPDGF-BB combined with bone allografts. An alternative is to use a synthetic system, such as β-tricalcium phosphate (β-TCP). Since rhPDGF applica‐ tions have proved clinically effective in the treatment of intrabony defects, this growth factor has also been considered for the treatment of soft tissue recession defects [18].

Jayakumar and coworkers [65] ran a double-blind, prospective, parallel, active-controlled, randomized, multi-center clinical trial on the efficacy and safety of rhPDGF-BB with β-TCP in human intraosseous periodontal defects. Fifty-four patients with periodontal osseous defects were randomly grouped for treatment with rhPDGF-BB/β-TCP or β-TCP alone. A total number of 50 defects in 25 patients in the rhPDGF-BB/β-TCP group and 25 in the β-TCP group were ultimately available for statistical analysis. The radiographic parameters considered were linear bone growth (LBG) 6 months after surgery and percent bone fill (% BF), both of which were found significantly higher in the rhPDGF-BB/β-TCP group than in the β-TCP group. There also emerged a significantly higher area under the curve for clinical attachment level gain from 0 to 6 months, and a greater reduction in PD at the third and sixth month than after β-TCP treatment alone. The implantation of rhPDGF-BB/β-TCP for the treatment of intraoss‐ eous periodontal defects was safe and well tolerated, and resulted in clinically and statistically significant improvements in bone formation parameters and soft tissue outcomes.

Preliminary investigations thus indicate that GFs have great potential for improving perio‐ dontal regeneration, but randomized clinical trials must be conducted to gain a better under‐ standing of the role of GFs in periodontal treatments, focusing particularly on establishing the safety and efficacy of their application.
