**4. Growth factor delivery systems**

hOP-1 and hTGF-β(3) in Matrigel(®) matrix induced substantial periodontal tissue regenera‐

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

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;

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

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,

The first human clinical trial testing the effect of rhPDGF/rhIGF-I in periodontal defects was

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].

; p = 0.14) were greater

p = 0.26) and bone regeneration area (0.74 ± 0.69 versus 0.32 ± 0.47 mm<sup>2</sup>

tion and cementogenesis.

726 Regenerative Medicine and Tissue Engineering

for bone ingrowth may account for these results.

will be needed to verify these findings.

cementum and periodontal ligament formation *in vivo*.

reported by Howell and colleagues [64] with promising results.

The great potential of GFs in bone regeneration has been discussed by numerous authors [6,31,34-35]. BMP-2 and BMP-7 have a marked effect on bone and cartilage growth and the maintenance of homeostasis during bone remodeling [66]. One of their limitations, on the other hand, seems to be the unpredictable nature of the resulting tissue regeneration *in vivo*. It has been suggested that the clinical efficacy of recombinant human forms of BMPs (rh-BMPs) depends on the carrier system used to ensure an effective delivery of adequate protein concentrations to the site being treated [67]. BMPs are soluble proteins and, delivered in a buffer solution, they undergo rapid degradation, leading to an insufficient bioavailability. Other factors, such as protein competition, enzymatic activity, temperature, pH and salt concentration, may also influence the total amount of active protein available immediately after its administration [68].

In 2007 Giannoudis et al. [69] came up with the "Diamond Concept" to describe the conditions needed for osteogeneration, i.e. mechanical stability at the site of the defect, and osteogenic cells combined with osteoinductive growth factors and a suitable carrier or delivery system.

The main purpose of the delivery system is to ensure adequate protein concentrations at the defect site for as long as it takes to enable the regenerative cells to migrate, proliferate and differentiate [33].

A localized, controlled release is also necessary to prevent any unwanted and uncontrol‐ led ectopic bone formation in non-bony body tissues [70]. Supra-physiological concentra‐ tions resulting from imperfect GF release kinetics have been correlated with severe clinical complications, including generalized hematomas in soft tissues and peri-implant bone resorption. Other potential concerns theoretically include carcinogenicity and teratogenic effects [70].

Few authors have investigated the influence of GF release kinetics on bone regeneration. In physiological bone repair, some growth factors (such as BMP-2) are expressed mainly during the early inflammatory phase. Others are up-regulated during the chondrogenic and osteogenic phases, and have a biphasic expression pattern or are constitutively expressed [33]

In vivo studies demonstrated that higher BMP-2 retention times were more osteoconduc‐ tive [71], and that prolonged BMP-2 delivery enhanced the protein's osteogenic efficacy by comparison with a shorter-term delivery of an equivalent dose in a rat model [72]. Release should preferably be sustained over time, either in large single doses or in multiple smallerdose applications. In evaluating the timing of the protein release, it is important to consider the dynamic nature of the healing zone, which depends on the type, location and appear‐ ance of the defect, the patients' age and gender, their hormone and nutritional state, and any diseases, as well as other parameters influencing release rate, including the protein's size and conformational changes, solubility, polymer/scaffold composition/geometry, and molecular weight [33].

Dose and concentration parameters are available for orthopedic clinical applications, where different anatomical sites require different therapeutic doses depending on the degree of vascularization, defect size and the number of resident responding cells. Supraphysiological dosages range from 0.01 mg/ml in small animal models (e.g. rats) to 0.4 mg/ml in rabbits, to more than 1.5 mg/ml in non-human primates [33].

Growth factor release from a delivery system may be diffusion-controlled, chemical or enzymatic reaction-controlled, solvent-controlled, or controlled by a combination of these mechanisms. Diffusion-controlled release is governed by the protein's solubility and diffusion coefficient in the aqueous medium, protein partitioning between the aqueous medium and the material of the delivery system, protein loading and the diffusional distance. Chemical or enzymatic reaction-controlled systems include erodible systems, in which the protein is physically immobilized in the carrier matrix and released as the carrier undergoes degradation and dissolves. In solvent-controlled systems, the protein is embedded in a carrier matrix and a diffusional release occurs as a consequence of the rate-controlled penetration of the solvent (water) in the system [33].

Several GF delivery systems and carriers have been suggested for use in bone regeneration applications in an effort to find the optimal strategy for optimizing their clinical effectiveness and minimizing complications.

Delivery systems and carriers used for bone GFs should meet general requirements (Table 1) such as biocompatibility, predictable biodegradability, and the ability to provoke appropriate inflammatory responses. They must also have the following features: easy and cost-effective to manufacture; stability; easy handling and storage [33].


**Table 1.** General requirements for BMP delivery systems

Carrier materials have been generally divided into four classes (Table 2): natural-origin polymers (collagen, hyaluronic acid, gelatin hydrogel complex, alginates and chitosan); inorganic materials (synthetic bone grafts, hydroxyapatite, calcium phosphates and bioactive glasses); synthetic biodegradable polymers (polylactic acid PLA, polyglycolide PLG, and their polymers PLGA, cholesterol-bearing pullulan nanogel CHPA), and composites (combinations of materials from the above different classes) [33].

To date, only BMP-2 and BMP-7 have been approved by the US Food and Drug Administration for human use in specific orthopedic applications, delivered using absorbable collagen sponges [33].

#### **4.1. Collagen**

complications, including generalized hematomas in soft tissues and peri-implant bone resorption. Other potential concerns theoretically include carcinogenicity and teratogenic

Few authors have investigated the influence of GF release kinetics on bone regeneration. In physiological bone repair, some growth factors (such as BMP-2) are expressed mainly during the early inflammatory phase. Others are up-regulated during the chondrogenic and osteogenic phases, and have a biphasic expression pattern or are constitutively

In vivo studies demonstrated that higher BMP-2 retention times were more osteoconduc‐ tive [71], and that prolonged BMP-2 delivery enhanced the protein's osteogenic efficacy by comparison with a shorter-term delivery of an equivalent dose in a rat model [72]. Release should preferably be sustained over time, either in large single doses or in multiple smallerdose applications. In evaluating the timing of the protein release, it is important to consider the dynamic nature of the healing zone, which depends on the type, location and appear‐ ance of the defect, the patients' age and gender, their hormone and nutritional state, and any diseases, as well as other parameters influencing release rate, including the protein's size and conformational changes, solubility, polymer/scaffold composition/geometry, and

Dose and concentration parameters are available for orthopedic clinical applications, where different anatomical sites require different therapeutic doses depending on the degree of vascularization, defect size and the number of resident responding cells. Supraphysiological dosages range from 0.01 mg/ml in small animal models (e.g. rats) to 0.4 mg/ml in rabbits, to

Growth factor release from a delivery system may be diffusion-controlled, chemical or enzymatic reaction-controlled, solvent-controlled, or controlled by a combination of these mechanisms. Diffusion-controlled release is governed by the protein's solubility and diffusion coefficient in the aqueous medium, protein partitioning between the aqueous medium and the material of the delivery system, protein loading and the diffusional distance. Chemical or enzymatic reaction-controlled systems include erodible systems, in which the protein is physically immobilized in the carrier matrix and released as the carrier undergoes degradation and dissolves. In solvent-controlled systems, the protein is embedded in a carrier matrix and a diffusional release occurs as a consequence of the rate-controlled penetration of the solvent

Several GF delivery systems and carriers have been suggested for use in bone regeneration applications in an effort to find the optimal strategy for optimizing their clinical effectiveness

Delivery systems and carriers used for bone GFs should meet general requirements (Table 1) such as biocompatibility, predictable biodegradability, and the ability to provoke appropriate inflammatory responses. They must also have the following features: easy and cost-effective

effects [70].

728 Regenerative Medicine and Tissue Engineering

expressed [33]

molecular weight [33].

(water) in the system [33].

and minimizing complications.

more than 1.5 mg/ml in non-human primates [33].

to manufacture; stability; easy handling and storage [33].

Collagen is the protein most abundant in the connective tissue of mammals and the main nonmineral component of bone. It has been prepared in powders, membranes, films and implant‐ able absorbable sponges, as well as in aqueous forms. Although it is versatile and easy to manipulate, the manufacture of collagen carriers is highly sensitive to several factors (includ‐ ing mass, soaking time, protein concentration, sterilization, buffer composition, pH and ionic strength) that directly affect rhBMPs binding [73]. Absorbable collagen sponges (ACS) have been evaluated in numerous in vivo models and clinical trials [6, 38,74-76]. In patients requiring staged maxillary sinus floor augmentation, rhBMP-2/ACS safely induced adequate bone formation for the purpose of placing and functionally loading endosseous dental implants [38]. The use of rhBMP-2/ACS without any concomitant bone grafting materials in critical-size mandibular defects prompted an excellent regeneration in a case review of 14 patients [75]. On the other hand, a recent study by Kao et al. demonstrated a more limited bone formation after a lateral-window sinus augmentation procedure involving rhBMP-2/ACS combined with Bio-Oss than when Bio-Oss was used alone [56].

Although they do away with the need to harvest autologous bone (with the associated pain), the use of animal-derived collagens is limited by their xenogenic nature: anti-type I collagen antibodies reportedly developed in almost 20% of patients treated with rhBMP-2/ACS [6]. In addition, collagen sponges are usually sterilized with ethylene oxide prior to soaking the


**Table 2.** Major classes of carrier materials

sponge in the BMP solution, and this can affect the GF release kinetics or the protein's bioactivity [73].

#### **4.2. Alginate and chitosan**

Alginate is a non-immunogenic polysaccharide used in a wide range of tissue engineering applications for its gel-forming properties. Alginate hydrogels allowing for a controlled, prolonged release of BMPs have only been studied in the preclinical phase, with promising results in vitro [72,77]

Chitosan is a cationic glucopolymer well known for its biological, chelating and adsorbing properties, and has been used as a BMP-2 carrier in a rat critical-size mandibular defect model, with positive results on histological and histomorphometric analysis [78].

#### **4.3. Hyaluronic acid**

Hyaluronic acid is a naturally-occurring biopolymer that plays a significant part in wound healing. It has been associated with an improved bone formation in mandibular defects by comparison with collagen sponges, when both were used to carry rhBMP-2 [79].

#### **4.4. Hydroxyapatite**

sponge in the BMP solution, and this can affect the GF release kinetics or the protein's

**Class Types Advantages Disadvantages**

Biocompatible,

with BMPs

biodegradable, soluble in physiological fluids, natural affinity with BMPs

Osteoconductive, affinity

Easy to process and sterilize, flexible to tailor and reproducible, excellent chemical and mechanical properties

Depending on the combination of the different materials' characteristics

Immunogenicity (xenogenic), pathogen transmission, sensitivity to sterilization process

formulations are exothermic

Brittle, difficult mold, some

Inflammatory response, localized pH drop and limited biological function

Complex to manufacture

Natural polymers Collagen (gels, nano fibers,

730 Regenerative Medicine and Tissue Engineering

Inorganic materials Synthetic bone grafts

scaffolds and films) Fibrin glue

Alginate and chitosan

CPC (calcium phosphate

hydroxyapatite, hyaluronic

cement) Bioactive glasses,

sulfate

copolymers CHPA

Synthetic polymers PLLA and PGLA and their

Composites Collagen-HA and titanium PLLA

acid, tricalcium phosphates, metal, ceramics and calcium

Alginate is a non-immunogenic polysaccharide used in a wide range of tissue engineering applications for its gel-forming properties. Alginate hydrogels allowing for a controlled, prolonged release of BMPs have only been studied in the preclinical phase, with promising

Chitosan is a cationic glucopolymer well known for its biological, chelating and adsorbing properties, and has been used as a BMP-2 carrier in a rat critical-size mandibular defect model,

Hyaluronic acid is a naturally-occurring biopolymer that plays a significant part in wound healing. It has been associated with an improved bone formation in mandibular defects by

with positive results on histological and histomorphometric analysis [78].

comparison with collagen sponges, when both were used to carry rhBMP-2 [79].

bioactivity [73].

**4.2. Alginate and chitosan**

**Table 2.** Major classes of carrier materials

results in vitro [72,77]

**4.3. Hyaluronic acid**

Hydroxyapatite (HAP) is well known for its osteoconductivity and has been widely used as a bone substitute material in clinical practice since the 1970s because of its ability to bond directly with bone [80]. Synthetic HAP comes in ceramic or non-ceramic, cementable forms, and has been evaluated as a scaffold and a controlled-release carrier, demonstrating lack of resorption and limited bone induction [6]. It has been combined with tri-calcium phosphates, collagen and other materials to form rigid, resorbable, porous carriers, in which case delivery and bone formation were generally found better than when HAP was used alone [81,82].

#### **4.5. Synthetic biodegradable polymers**

Unlike natural polymers and collagen, synthetic polymers pose no problem of immunogenicity or risk of disease transmission.

The most commonly-used polymers are polylactic acid (PLLA) and polyglycolic acid (PLGA). Bioresorbable PLLA/PLGA copolymers have been found superior to collagen when used to deliver rh BMP-2 to mandibular defects in the rat [83].

#### **4.6. Bone grafts and derived composite materials**

Bone grafts act as scaffolds for the ingrowth of vessels and bone-forming cells. During this osteoconductive bone regeneration process, the scaffold allows for bone to grow on its surface and inside the pores in the material. Given the biological limitations of other osteoconductive materials and the donor site morbidity after bone harvesting, the combination of osteocon‐ ductive scaffolds with osteoinductive proteins, such as BMPs, has been a major focus of research. [13,84]

Bone substitutes for use in dental and maxillofacial surgery are classified in three groups according to their origin. Allogenic bone grafts are derived from human donors, xenogenic bone grafts from other species (mostly bovine, but also equine, porcine and coralline), and the last group comprises the synthetically-produced materials. Synthetic bone grafts aim to imitate the natural bone's structure. The most widely used are the calcium phosphates, including hydroxyapatite, tri-calcium phosphates (TCP) and composites of the two. By means of a thermal treatment (sintering) and subsequent cooling they can be transferred into ceramics with a very solid but porous structure and a rough surface closely resembling human bone.

Recent studies have reported successful bone regeneration after grafting on periodontal defects, using sinus floor elevation techniques, and in post-extraction socket defects using TCP carriers [58,65, 85].

Clinical studies reporting results of GFs delivery systems in oral surgery are revised in Table 3.

Some authors have also investigated the application of GFs to dental implant surfaces to stimulate local bone formation and osteointegration. In preclinical studies, functionalized titanium implant surfaces coated with rhBMP-2 have been shown to be able to stimulate bone formation around implants [35, 86]



**Table 3.** Clinical studies on GF delivery systems applicable in oral surgery

#### **4.7. Gene delivery methods**

**References Study**

Jung et al. 2003

Boyne et al. 2005

Herford and Boyne 2008 [75]

Van den Bergh et al. 2000 [76]

Kao et al., 2012 [56]

Alonso et al. 2010 [89]

Stavropoulos et al. 2011 [63]

Nevins et al. 2011 [49]

Stavropoulos et al. 2011 [58]

Jayakumar et al. 2011 [65]

[40]

[38]

**design**

732 Regenerative Medicine and Tissue Engineering

Case review

Clinical trial

Cohort study

**Total number of patients**

RCT 11 rhBMP-2 Xenogenic

**Protein Carrier Application Main findings**

Maxillary implant placement

elevation

elevation

closure in cleft lip and palate patients

periodontal defects

RCT 31 rhGDF-5 TCP Sinus floor elevation Comparable amount and similar quality of bone

periodontal defects

14 rhBMP-2 ACS Mandibular defect Bone formation could be identified

therapy

implants

rhBMP-2 has the potential to predictably improve and accelerate guided bone regeneration

rhBMP-2/ACS safely induced adequate bone for the placement and functional loading of dental

radiographically after 5 to 6 months

Sinus floor elevation Less bone formed in patients treated with the

reduced morbidity

statistically significant

Socket preservation No statistically significant differences were observed

formation as in controls

rhBMP-2/ACS/xenogenic bone device

Satisfactory bone healing at 6 months and

Greater alveolar regeneration, differences not

Increased bone formation and soft tissue healing

Potential for initiating bone formation in the human maxillary sinus within 6 months after a sinus floor elevation, but its behavior is currently not sufficiently predictable in this application

bone (Bio-Oss)

3 rhBMP-2 Type I collagen Maxillary sinus floor

xenogenic bone

rhBMP-2 ACS and

RCT 16 rhBMP-2 Collagen Alveolar defect

RCT 20 rhGDF-5 β-TCP Regeneration of

rhPDGF Mineral

RCT 54 rhPDGF TCP Regeneration of

collagen scaffold

RCT 48 rhBMP-2 ACS Maxillary sinus floor

The potential applications of gene therapy have recently expanded to include the local treatment of bone defects. Gene transfer methods may circumvent many of the weaknesses of protein delivery to soft tissue wounds. The application of growth factors or soluble forms of cytokine receptors by means of gene transfer offers a greater sustainability than the use of a single protein application. Gene therapy may make growth factors more readily bio-available.

Gene transfer is accomplished by using viral and non-viral vectors. Examples of viral vectors are retroviruses, adenoviruses (Ads), and adeno-associated viruses (AAV), and non-viral vectors include plasmids and DNA polymer complexes.

Some authors have studied gene delivery via adenoviral or liposomal vectors carrying information for encoding recombinant human GFs combined with a collagen matrix in animal models [87,88].

### **5. Conclusion**

The role of growth factors for alveolar bone regeneration in dentistry is a recent field of research, with a relative paucity of clinical studies. Findings seem to demonstrate a positive effect of GFs on intraoral hard and soft tissues healing, and the bone regeneration associated with implant therapy represents one of the main scenarios of interest. For the time being, however, the application of GFs in this field is limited by the dubious results, complications and side effects encountered so far. In particular, one of the main problems seems to be the relationship between the GF delivery and the timing of the healing process. Among the delivery systems tested to date, only collagen matrices have correlated with successful clinical results, albeit with some limitations. Other potential delivery systems have been studied only in a few animal models, and the currently available data are not enough for any final conclu‐ sions to be drawn. The development of dedicated and more "sophisticated" GF delivery systems is probably the most interesting area of research for the future.
