Tissue Engineering for Bone Regeneration and Osseointegration in the Oral Cavity

1. Introduction

The alveolar processes of the mandible and maxilla line the alveolus and provide structural support and maintenance for teeth as part of the periodontium, consisting of the periodontal ligament (PDL), cementum, connective tissue, and gingiva. Alveolar bone is especially susceptible to inflammation-induced bone resorption due to high rates of progressive periodontitis—a leading chronic oral inflammatory disease estimated to affect 47.2% of adults in the United States, with a prevalence of 70% for adults aged 65 years and older [1]. Advanced periodontal disease alters alveolar bone morphology and destroys surrounding tooth-supporting tissues, thereby necessitating tooth extraction. Since the existence of alveolar bone is mutually connected to the dentition and other periodontal tissues, the alveolar ridge continues to resorb following tooth removal even if a dental implant is placed into a fresh extraction socket. Physiologically, this is caused by continuous bone remodeling in response to mechanical loading changes that occur with alterations in the applied force and strain distribution to the osseous tissue during mastication, as stipulated by Wolff's Law [2]. Ridge or socket preservation and augmentation using bone grafting materials is a clinically viable approach to maintain any remaining bone following tooth extraction and further condition it in preparation for dental implant placement. Sufficient bone volume, height, and width are necessary to ensure implant stability and osseointegration that can sustain optimal bone-implant contact biomechanical loading. Other dental procedures that involve grafting include maxillary sinus floor augmentation, which is employed for patients with bone loss in the posterior maxilla that houses premolar and molar teeth [3]. Bone defects in the oral cavity resulting from trauma, chronic infection, congenital defects, or surgical resection require clinical intervention, most frequently using autologous bone grafting techniques. However, critical limitations of this approach include donor site morbidity and inadequate supply of graft tissue. Tissue engineering approaches using scaffolds alone or in combination with growth factor, cell and/or gene delivery have the potential to address existing challenges in managing bone loss and increase clinical options for controllable regeneration of intraoral osseous tissues.

2. Scaffolds

2.1 Intraoral bone grafts

An autologous bone graft is considered the gold standard due to low risk of immunogenicity or disease transmission that could be associated with an allograft (genetically different donor from the same species) or xenograft (donor from another species). Most importantly, bone transplanted from the patient is native to its host environment and readily associates with the remnant tissue, providing a pre-established population of viable cells and growth factors necessary for osteogenesis. Local sites such as the maxillary tuberosity or mandibular symphysis can be used for harvesting of small autologous grafts [4]. Nevertheless, there are several key reasons for a critical need of alternative grafts capable of substituting the autograft: limited availability of autologous tissue for larger bone defects, donor site morbidity and potential wound-based infections, as well as prolonged operative times [5]. Although lacking in osteogenicity, allografts and xenografts can be prepared to have osteoconductive and osteoinductive properties. Bone allografts are available as fresh/fresh-frozen, freeze-dried, or demineralized and freeze-dried. The mechanical properties of allografts derived from a living donor or cadaveric tissue are changed substantially during extensive tissue processing involving decellularization, sterilization, and preservation for clinical use [6]. Such tissue treatment removes viable cells that are osteogenic and osteoinductive in nature, leaving behind a structurally supportive framework primarily composed of minerals and proteins-termed the extracellular matrix (ECM). The allograft ECM serves as a scaffold for osteoblasts originating from the bone defect into which the graft is placed to facilitate new bone formation. Depending on the method of processing, an allograft can also be osteoinductive if it retains the biological properties necessary to recruit mesenchymal stem cells to the site and stimulate their differentiation into osteoprogenitor cells. One example is demineralized bone matrix (DMB), which has reduced levels of calcium and phosphorus and is primarily type I collagen, but can be considered osteoinductive if it retains factors such as bone morphogenetic proteins (BMPs) and transforming growth factor-β (TGF-β) [7]. As expected, DMB shows an increased rate of resorption relative to a mineralized bone graft during tissue remodeling in vivo. In addition, derivation of DBM involves grinding of bone to obtain particulates as opposed to processing the allograft in its native structural form, making it useful for small to moderate defects [8].

Xenografts offer another alternative for bone replacement in dental regeneration, with most products derived from coral, porcine, or bovine sources. A recent study comparing implant placement into sinus floors augmented with an autologous mandibular bone graft versus a commercially-available bovine xenograft found equivalent implant survival rates over an observational period of 5 years [9]. However, implant survivability depends on many factors, including patient demographics and surgical technique, thereby warranting longer-term evaluations and more comprehensive consideration of factors that may influence the clinical outcome. Extensive meta-analysis of histomorphometric and bone graft healing time results for sinus floor augmentation described in the literature over a period of 16 years concluded that autologous bone grafts result in higher total bone volume levels compared to other bone grafting materials [10]. Another comprehensive systematic review of treatment modalities used to evaluate dental implant survival rates in maxillary sinus grafts employed statistically robust methodology to correct for study effects. It concluded that application of grafting membranes for guided bone regeneration supplementary to a bone graft was more important for implant survival rate over factors such as which bone substitute material was selected for the surgery [11]. These results indicate the difficulty of identifying specific factors that influence final clinical outcomes and underline the fact that there is no unified consensus on whether non-patient derived grafts can perform at the same level as autografts for not only bone regeneration but also implant performance at augmented bone sites. Each case is patient-specific and requires thorough consideration of all contributing factors, including the health of the patient's native bone and its suitability for grafting procedures.

2.2 Natural and synthetic matrices

In addition to standard grafting procedures using bone-derived materials, a number of natural and synthetic materials have become commercially available for use in oral surgery. Scaffolds that are architecturally and/or biologically compatible for bone regeneration are frequently based on one or more of the bone's naturally-occurring proteins or minerals, including organic (predominantly collagen type I) and inorganic (hydroxyapatite, a calcium phosphate mineral) components [12]. Calcium phosphate (CaP) materials are subdivided into ceramics and cements, which vary in their rate of in vivo degradation, structure, and mechanical strength. Common synthetic CaP bone substitutes include hydroxyapatite (HA) ceramics, β-tricalcium phosphate (β-TCP) cements, and biphasic calcium phosphates (BCPs) [13, 14]. Fragility and poor fatigue resistance of these ceramics and cements requires their use at non-load bearing bone replacement sites or as coatings on load-bearing metal implants for increased bone-to-implant contact. Coating a dental implant surface (i.e., titanium, stainless steel, or an cobalt-chrome alloy) with CaP-derivatives has been extensively investigated using various surface coating deposition techniques to improve implant stability and rate of osseointegration [15, 16]. In addition to containing minerals native to osseous tissue, these biomaterials retain an interconnected porous architecture--allowing adequate space through increased surface area for bone ingrowth via cell infiltration, blood vessel formation, nutrient/oxygen transport, and waste elimination. Ongoing studies are being performed to determine the optimum porosity for bone ingrowth and corresponding bone substitute resorption rate, since an ideal regenerative scenario would consist of a biomaterial resorption rate timed with new osseous tissue ingrowth [17]. Likewise, there are ongoing investigations to confirm the utility of using CaP bone substitutes for implant coating. For example, recent studies of hydroxyapatite coatings on titanium cups for orthopaedic-based implants such as femoral stems did not show significant differences between HA-coated and non-coated stems and may not constitute a clinical advantage [18].

Bioactive glass (BG), a silicon oxide with substituted calcium first developed in the 1960s by Professor Larry Hench, is a biocompatible glass-ceramic material approved by the US Food and Drug Administration (FDA) for use as a synthetic intraoral bone graft (termed 45S5 Bioglass^®). Upon exposure to aqueous solutions the highly reactive surface converts to a gel layer that mineralizes to form a osteoconductive hydroxy carbonate apatite layer that chemically binds with osseous tissues [19]. Bioglass^® also has a reported elastic modulus of 35 GPa that is similar to that of cortical bone (E_longitudinal = ∼14-20 GPa) [20, 21]. Comparatively, the elastic moduli of HA single crystals and β-TCP are in the range of 54-79 GPa and 120-162 GPa, respectively [22, 23]. This makes BG an attractive option for metal implant coating, reducing the potential for stress shielding and subsequent bone resorption which occurs with decreased bone loading [24]. To date, studies using dental implants coated with BGs have not conclusively shown significant increases in osseointegration relative to other coatings such as HA [25]. However, novel exploratory combinations for dental implant coatings using BG are promising: one example is the incorporation of BGs with HA and the polymer poly(lactide-co-glycolide) (PLGA), which indicates rapid bone-like apatite formation in vitro, with potential antimicrobial activity on oral bacteria [26].

Polymeric materials that have been commercialized or are currently under investigation for intraoral bone regeneration are either naturally- or synthetically-derived. Natural materials that consist of polymeric networks (i.e., collagen, alginate) have been extensively investigated as composite materials with other bone replacement grafts, including β-TCP and HA [27]. Major advantages of a tissue-sourced polymer such as collagen include its biocompatibility, biodegradability, and ability to readily bind growth factors critical for osteoinduction, including BMPs. Currently, collagen is the most commercially-available natural polymer on the market for use in periodontal bone regeneration as a sponge, membrane, or in particulate form combined with other bone grafts [28]. It is expected that future research will continue to focus on the development of a more diverse array of naturally-derived, fully-resorbable polymeric bone grafts combined with non-immunogenic materials such as alginate and chitosan that can be engineered for growth factor, cell, and/or gene delivery. Such delivery devices may also include synthetically-derived resorbable materials whose physical, mechanical, and degradation properties can be more easily controlled via polymer chemical composition and molecular weight. Historically, oral surgery has utilized non-resorbable synthetic membranes as cell-occlusive barriers for guided bone regeneration, among which the most broadly used material has been expanded polytetrafluoroethylene (ePTFE) [29]. Unnecessary patient discomfort and added cost of a follow-up visit to remove the membrane has increased demand for resorbable alternatives. Examples of widely-investigated resorbable polymers for applications in bone regeneration include poly-α-hydroxy esters such as polyglycolic acid (PGA), polylactic acid (PLA), and PLGA. The ease of processing synthetic materials enables their fabrication into a variety of structurally-diverse forms, including thin films, meshes, fibers, and porous foams. An existing disadvantage of these materials is their bulk erosion in vivo due to hydrolysis that can induce foreign body reactions to acidic polymer degradation products, increasing the potential for fibrous tissue encapsulation during wound healing. Although studies of PLGA utility for guided bone regeneration are still limited, there is evidence that its use as a barrier membrane results in alveolar bone regeneration that is on par with that observed using a collagen-based membrane [30]. Other copolymer combinations that yield useful properties for osseous tissue regeneration include PLGA with poly(ethylene glycol) (PEG), a widely used biocompatible hydrophilic drug delivery carrier. PEG-PLGA thermo-sensitive copolymers can be encapsulated with osteoinductive factors and polymerized into a gel-like structure for delivery into an osseous defect, possibly in combination with other mechanically durable bone grafts [31]. PCL is another biodegradable polymer with significant research indicating its suitability for bone regeneration. An inert material, it shows increased osteoblast adhesion, spreading, and proliferation when coated with CaP or HA [32], and multiple studies have indicated PCL's potential to promote alveolar bone formation in periodontal defects [33, 34]. With greater emphasis placed on materials that can be used for exogenous factor delivery to accelerate and improve existing periodontal tissue defect treatments, polymers such as PCL and PEG which are already FDA-approved as drug delivery devices are expected to be more extensively investigated in pre-clinical models of intraoral bone regeneration in the coming years.

2.3 Engineering scaffolds for intraoral bone regeneration

The development of scaffolds that are optimal for regeneration of osseous tissues requires a design strategy which adheres to established knowledge of the mechanical, chemical, structural, and biological properties of natural bone that make it a functional entity. Therefore, key considerations in such scaffold design include: (1) biocompatibility/non-toxic degradation; (2) bioactivity, enabling cell interaction with material surface; (3) maintenance of a 3-D shape after implantation; (4) adequate porosity and pore diameter/distribution/orientation; (5) mechanical properties similar to tissue targeted for regeneration (i.e., Young's modulus); (6) degradation mechanics (i.e., bulk erosion, surface erosion); (7) degradation rate, ideally matching the rate of tissue regeneration; and (8) osteoconductive/inductive and angiogenic factors to influence infiltrating cell populations and promote blood vessel invasion [35, 36].

While a broad range of instructive carrier materials in various forms have been investigated for bone regeneration, the addition of cells and application of growth factor delivery strategies can significantly influence the regenerative outcome by engineering the environment that closely matches that of the target tissue in its native state (see Figure 1). Current strategies include the use of bone-marrow stromal cells and stem cell varieties including mesenchymal (MSCs), adipose-derived (ADSCs) and induced pluripotent (iPSCs). Analyses of cell-specific markers and transcription factors such as Runx2, alkaline phosphatase (ALP), osteocalcin, osteopontin, and osteonectin allow for the determination of osteogenicity during stem cell differentiation into bone-derived cells [37]. Clinically-applicable cell therapy is focused on the use of patient-derived stem cells that are undifferentiated, given that terminally-differentiated cells are difficult to expand ex vivo relative to more highly proliferative stem/progenitor cells. Likewise, the use of stem cells allows for a more completely physiological repair process that involves the differentiation of MSCs or iPSCs not only into bone-derived cells but also cell types involved in neovascularization, such as endothelial cells. However, despite significant progress and tremendous potential in cell therapy, critical challenges remain in transitioning the use of iPSCs into clinically-applicable approaches. Since the creation of iPSCs involves the reprogramming of somatic cells via transcription factors to produce cells with embryonic stem cell-like properties, there is risk of epigenetic and genetic defect accumulations, immunogenic responses, or tumor formations. While the immunogenic response can be mediated through use of a patient's native somatic cells, overexpression of transcription factors or presence of partially-reprogrammed iPSCs are known to cause teratoma formation, requiring significant efforts to address these safety concerns prior to the consideration of iPSCs for patient-based treatments [38].

Figure 1.

A combination of various approaches in scaffold material selection, cell therapy, and growth factor delivery is required to achieve optimal tissue regeneration that mimics the mechanical, chemical, structural, and biological properties of natural bone that make it a physiologically functional entity. Abbreviations: [Scaffolds] Polyglycolic acid (PGA), polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), poly(ethylene glycol) (PEG), hydroxyapatite (HA), β-tricalcium phosphate (β-TCP); [Cell Therapy] Osterix (Osx), alkaline phosphatase (ALP), osteoprotegerin (OPG), bone sialoprotein (BSP); [Growth Factors] bone morphogenetic protein (BMP), transforming growth factor-beta (TGF- β), insulin-like growth factor (IGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF).

With advancements in cell therapy, there has been a simultaneous increase in novel scaffold fabrication techniques that emphasize greater control over surface topography, internal microstructure, and pore interconnectivity. Traditionally, porous scaffolds have been widely explored as bone graft substitutes for cell attachment given the importance of allowing adequate room for tissue ingrowth and vascularization (i.e., pore size of 150-500 μm) [39]. While natural materials retain their bioactivity, synthetic non-immunogenic materials have several advantages for development of clinically-translatable scaffolds, including added flexibility in manufacturing, reproducibility, sterilization, and storage times. Electrospinning and solid freeform fabrication (SFF) are two scaffold fabrication techniques which allow increased control over scaffold morphology: Electrospinning is a polymer-processing technique used for creating polymer fibers on the nano and micron scale to influence cell behavior through structural and physical cues that mimic ECM architecture. In addition to allowing control over a variety of parameters that determine fiber dimension, density, and porosity, electrospinning can readily be used for mass production of fiber-based scaffolds [40]. More recently, 3-D printing technology has been adapted for use in bone tissue engineering via solid freeform fabrication (SFF), a rapid prototyping technique. This process consists of developing a computer-aided design (CAD) file that specifies the exact dimensional features of the desired scaffold which is then transferred to a 3-D printer that reproduces the file to yield a printed version of the design with structural integrity. Selective laser sintering (SLS) is an example of a process that creates objects layer-by-layer using polymeric, ceramic, or metal powders that the machine sinters. During sintering, the powder is heated below the melting point, causing its particle boundaries to fuse together at locations dictated by the CAD-based file. When creating a porous material, the sizes and characteristics of the individual pores within the material are limited by the machine's resolution and ability to support a specific printing material [41]. Studies using SLS for bone regeneration have focused on PCL-based printed scaffolds, showing that these are mechanically-appropriate to support bone tissue formation and can be used as BMP-7 and BMP-2 growth factor carriers following biofunctionalization [42, 43]. This technology is especially applicable for clinically-based studies given that patient-specific anatomical bone defects can be obtained using computed tomography (CT) scans and reproduced to yield a scaffold with appropriate structural dimensions. A recent publication by Park et al proposes a potential future application of image-based PCL SFF-based scaffolds for clinical periodontal regeneration (see Figure 2), indicating that further pre-clinical investigation and verification of these technologies will bring them closer to translation into clinical practice [44].

Figure 2.

Solid free-form fabrication (SFF), or rapid prototyping, is emerging as a clinically-viable approach for tissue engineering of anatomically precise scaffolds for periodontal, including intraoral bone, regeneration. This method is based on obtaining a CT scan of the patient-specific defect, generating a CAD-based file of a scaffold with an appropriate fit to the defect, and 3-D printing the final version to obtain a polymer-based scaffold with structural integrity that can be used as a cell therapy and growth factor delivery platform to enhance the regenerative process. Reproduced with permission from Park CH et al [43].

While novel methods of scaffold development and cell therapy are being explored for bone regeneration, there is already an established array of clinically-applicable therapeutic factors and delivery strategies. In order to better understand existing clinical applications and the nature of ongoing pre-clinical studies, the remainder of this review focuses on the areas of growth factor delivery for oral bone regeneration and highlights some of the key studies that have moved previously experimental therapies into today's mainstream clinical applications in periodontal regeneration.

3. Growth Factors and Protein Delivery

Growth factor delivery (GFD) is of critical importance in mediating the scaffold environment and subsequent cellular response. Growth factors are soluble polypeptides that bind to cell membrane receptors and influence cellular function, and the inclusion of osteoinductive/conductive factors (BMP, TGF-β, IGF, FGF-2, PDGF) guides the cell differentiation and tissue formation process in bone regenerative therapies. Likewise, vascularization is vital for the sustainability of newly-formed tissue, and necessitates the inclusion of GFs with angiogenic properties (PDGF, VEGF, FGF-2, and TGF-β). Scaffold-based GFD as opposed to bolus injection of the therapeutic factors into the defect site has several advantages, including the potential for improved control over GF release kinetics and localization. A major challenge in GFD involves the need for release profiles that mimic those present during natural tissue repair or morphogenesis. Sequential or simultaneous spatiotemporal delivery of multiple GFs can be achieved using scaffolds based on the therapeutic time window during which GF delivery is optimal for tissue regeneration, and can involve the combination of factors involved in both tissue formation and angiogenesis. For example, sequential delivery of GF combinations consisting of BMP-2 and TGF-β or VEGF and BMP showed increased bone formation when compared to single GFD [45, 46]. GF rate of release is governed by how it is bound to the scaffold, which can involve (1) mixing the GF with scaffold particles, (2) physical encapsulation within the scaffold, (3) chemical immobilization, and (4) affinity-based binding. Current clinical application of GFs for bone regeneration typically involves the use of biomaterial carriers, yet the factor is usually mixed in and physically adsorbed onto the graft particles and lacks more sophisticated modes of delivery that would enable spatiotemporal control over release kinetics or dual GFD. Other modes of delivery currently investigated in preclinical studies are promising: Physical encapsulation of GFs within the scaffold, for example, can be achieved using polymeric microparticles (1-100μm) with varying surface areas through which the encapsulated GF diffuses at a rate that is dependent on carrier particle size, with a larger size leading to slower diffusion. GFs can also be conjugated on the scaffold surface via covalent immobilization, while heparin-binding GFs such as BMP-2 can be presented using affinity-based binding by conjugating heparin to biomaterials [47, 48]. These modes of GFD have potential for future applications in clinical studies, allowing for a broad range of release profiles and controlled spatiotemporal presentation of multiple GFs using carriers that serve as temporary support structures for osseous tissue engineering strategies.

Growth factors and proteins act locally on the activity of periodontal cell populations to modulate bone formation and enhance the regenerative response (see Table 1). A diverse range of bone matrix-based proteins have been isolated and are delivered alone or in combination with a synthetically- or naturally-derived bone graft. An overview of some of the factors and proteins that are featured most prominently in oral-based surgery or currently being investigated in preclinical (see Table 2) and clinical studies (see Table 3) is presented here.

Table 1.

Effects of growth factors on periodontal cells in vitro.

Growth factor	Cell type	Effect	Studies
PDGF	Cementoblasts	Increased DNA synthesis and osteopontin mRNA expression	[50]
	Dental follicle cells	Stimulated DNA synthesis and expression of CSF-1 and MCP-1	[173]
	Gingival fibroblasts	Increased mitosis No increase in proliferation or chemotaxis	[72, 174]
	Osteoblasts	Increased proliferation and interleukin-6 transcription Inhibited differentiation Blocked osteopontin, osteonectin	[53-55]
	PDL cells	Stimulated proliferation (with or without allograft) Induced matrix synthesis and increased cell migration and mitosis	[51, 52, 64, 72]
BMP2	Cementoblasts	Inhibited differentiation and mineralization	[175]
	Dental follicle cells	Stimulated osteoblast/cementoblast differentiation Increased mineralization and ALP	[176, 177]
	Gingival fibroblasts	Decreased mitosis At high doses, inhibited mineralization and OCN	[72, 178]
	Osteoblasts	Increased proliferation, mineralization, and expression of ALP and OCN	[179]
	PDL cells	Increased mineralization and expression of mineralization markers At doses > 10ng/mL, induced apoptosis/cytotoxicity Stimulated osteoblast differentiation Decreased mitosis	[71-73]
BMP-7	Cementoblasts	Increased mineralization and mineralized tissue markers	[101]
	Dental follicle cells	Increased mineralization and ALP expression	[176]
	Osteoblasts	Increased proliferation, mineralization, and expression of ALP and OCN	[74, 75, 179]
	PDL cells	Reduced proliferation Induced ALP	[180]
FGF-2	Cementoblasts	Stimulated DNA synthesis Decreased mineralization and expression of OCN Modulated expression of OPN	[181]
	Dental follicle cells	Stimulated DNA synthesis and expression of CSF-1 and MCP-1	[173]
	Gingival epithelial cells	Increased proliferation	[106]
	Gingival fibroblasts	Increased proliferation	[182]
	Osteoblasts	Promoted differentiation Induced proliferation Decreased mineralization gene expression	[104, 183]
	PDL cells	Increased proliferation (alone and combined with DFDBA or FDBA), migration, and extracellular matrix production Maintained differentiation potential Stimulated OPN Inhibited ALP expression, mineralization, and OCN	[102, 103]
GDF-5	CT fibroblasts	Increased proliferation	[184]
	Dental follicle cells	Reduced ALP activity	[185]
	Osteoblasts	Increased early differentiation and matrix production Modulated proliferation	[113, 184]
	PDL cells	Increased proliferation and matrix synthesis Decreased ALP activity	[112]
Teriparatide	PDL cells	Modified proliferation & survival and expression of mineralized markers (dependent on maturation state)	[122, 123]

ALP: alkaline phosphatase, BMP: bone morphogenetic protein, CSF-1: colony stimulating factor-1, FGF-2: fibroblast growth factor-2, GDF-5: growth/differentiation factor-5, MCP-1: macrophage chemotactic protein-1, OCN: osteocalcin, OPN: osteopontin, PDGF: platelet-derived growth factor, PDL: periodontal ligament

Table 2.

Preclinical animal models of growth factor delivery for periodontal and implant applications.

Growth factor	Model	Animal	Results	Studies
PDGF-BB	Furcation defect	Canine	Stimulated PDL formation (early stage) Promoted periodontal regeneration (late stage)	[63]
	GBR at implants	Canine	With IGF-1, significantly increased histologic bone-implant contact and peri-implant bone fill With IGF-1, increased early (3 week) bone formation at immediate implants With ePTFE membrane and IGF-1, increased bone gain and histologic parameters versus membrane alone or membrane + DFDBA	[56, 64, 65]
	Periodontal defect	Canine	After flap surgery, increased new bone, cementum and PDL With IGF-1, promotes periodontal regeneration	[57-59, 62]
		Non-human primate	Significantly increased new attachment	[60-62, 76]
	Ridge augmentation	Canine	With block graft, increased histologic bone formation With bone mineral and collagen membrane, supports lateral bone formation With biphasic calcium phosphate and collagen membrane, supports GBR With xenograft scaffold, promoted bone regeneration similar in qualityto native bone With xenograft, improved radiographic results when used without collagen membrane	[67, 68] [66]
rhBMP-2	Extraction socket	Rat	Increased speed and quantity of bone formation Induced proliferation and differentiation of mesenchymal cells	[83]
	Furcation defect	Feline	Early ankylosis may resolve with polymer carrier	[186]
	GBR at implants	Canine	Resorbable and ePTFE membranes delay early (1 month) bone formation but may result in increased or similar 3-month bone formation vs. no membrane Increased bone augmentation for implants placed in extraction sockets and for implant-site circumferential and fenestration defects BMP2-coated implants provide increased bone formation, histologic bone apposition, and osseointegration Improved late (3-month) bone formation for ACS versus PLGA carrier	[82, 95, 132, 147, 187]
	Periodontal defect	Non-human primate	Increased bone and cementum regeneration	[76]
		Canine	Increased quantity and speed of bone formation Bone quantity formed correlated with residual bone height Limited cementum regeneration Induced ankylosis and root resorption No benefit for calcium phosphate cement carrier	[77-79, 81, 82]
	Ridge augmentation	Rat	Significant horizontal and vertical bone augmentation ePTFE membranes improve bone contour Increased bone formation with ACS+ bone graft material Potential carriers: absorbable collagen sponge, hyaluronic acid polymer, collagen-calcium hydroxyapatite-TCP complex, PLGA/gelatin sponge	[89, 91-93]
		Canine	Increased histologic and radiographic bone formation, +/- bone graft Increased incidence of seromas and wound failure Possible decreased bone quality when combined with bone graft With xenograft block, supported bone formation Late-stage implant stability comparable to native bone	[80, 90, 91, 121]
		Non-human primate	Increased ridge width and bone quality in TCP/HA/ACS and CaP cement carriers	[188]
	Sinus augmentation	Canine	Enhanced histologic bone formation	[84]
		Rabbit	Increased bone volume for collagenated BCP/BCP carriers	[85]
		Goat	Increased radiographic bone formation	[88]
		Non-human primate	Increased vertical bone gain	[86]
		Mini-pig	BMP-2 coatings did not improve peri-implant bone gain	[87]
BMP-4	Ridge augmentation	Rat	Improved bone quality and comparable quantity versus BMP-2	[89]
BMP-7	Furcation defect	Canine	Significantly increased histologic bone, cementum, and new attachment in class III furcations	[94]
	GBR at implants	Canine	Implant-coating applications	[95]
		Non-human primate	Stimulated cementum formation	[98]
	Ridge augmentation	Rat	Increased bone formation in xenograft block versus control	[189]
	Sinus augmentation	Non-human primate	Comparable radiographic and histologic bone formation and residual lateral wall defect reduction versus bone graft	[97, 190]
		Mini-pig	With xenograft, increases speed and quality of osseointegration at simultaneous implants	[99, 100]
	Socket augmentation	Rabbit	Histologic increased speed of healing by 4-6 weeks Significantly increased ALP activity and calcium	[96]
FGF-2	Furcation defect	Canine	Significantly increased regeneration of cementum, PDL, and bone vs. controls	[109, 110]
GDF-5	GBR at implants	Canine	Improves peri-implant GBR (β-TCP) GDF-5 coating may increase bone formation (dose-dependent)	[191, 192]
	Implant coating	Rabbit	Improved implant stability as determined by pull-out test	[118]
	Periodontal defect	Canine	Significantly increased perio. regeneration in PLGA (dose-dependent), β-TCP, ACS carriers, with bone formation for β-TCP stable up to 24 wks Beta-TCP/PLGA carrier may cause ankylosis	[115-117, 193]
		Non-human primate	Supported periodontal regeneration with β-TCP carrier	[114]
	Ridge augmentation	Canine	With xenograft block, supported bone regeneration	[121]
	Sinus augmentation	Mini-pigs	Enhanced bone formation with β-TCP	[119, 120]
Teriparatide	Extraction socket	Osteopenic rats	Increased bone mineral density and anabolic effects	[124]
	GBR at implants	Canine	Significantly improved bone formation	[126, 127]
	Periodontal defect	Ovarectimiz ed rats	Preventative effects on periodontal bone loss	[125]

ACS: absorbable collagen sponge, ALP: alkaline phosphatase, BCP: biphasic calcium phosphate, BMP: bone morphogenetic protein, bTCP: beta tricalcium phosphate, ePTFE: expanded polytetrafluoroethylene, FGF: fibroblast growth factor, GBR: guided bone regeneration, GDF: growth/differentiation factor, HA: hydroxyapatite, IGF: insulin-like growth factor, PDGF: platelet-derived growth factor, PDL: periodontal ligament, PLA: polylactic acid, PGA: polyglycolic acid

Table 3.

Clinical application of growth factor and protein delivery in periodontics, osseointegration, and pre-prosthetic surgical procedures.

Growth factor/protein	Indication	Evidence level	Efficacy and safety	Referenc es
PDGF-BB	Periodontal defects	★★★	Gain in PD, CAL and BOP Increasing of radiographic bone level	[49, 134-137]
	Furcation involvements	★	Gain in PD, CAL and BOP Increasing of radiographic bone level
	Alveolar bone augmentation/preservation	★	Accelerate the healing process
BMP-2	Alveolar bone augmentation/preservation	★★	Accelerate the healing process Histological findings high proportion of newly formed bone
BMP-7	Sinus augmentation	★★	In combination with allogeneic/autologous bone graft: histologic findings similar to autologous bone and adequate vertical bone gain (BMP-2) In combination with bovine-derived xenogeneic graft: histologic findings show less newly-bone formation compared to xenograft alone but adequate vertical bone gain (BMP-2, BMP-7)	[140-143, 145-148]
GDF-5	Periodontal defect	★	Gain in CAL	[149, 150]
	Sinus augmentation	★	Histologic results similar to autologous bone Adequate vertical bone gain
FGF-2	Periodontal defects	★	Early radiographic bone fill	[151]
Teriparatide	Peri-implant bone	★	Gain in PD and CAL Increasing radiographic bone level	[153, 154]
			Greater bone-to-implant contact in the periosteal and medullary compartment Minimal effect on cortical compartment Slight higher bone-volume-per-tissue-volume

Evidence level= (★) Slight clinical evidence; (★★) Moderate clinical evidence; (★★★) Robust clinical evidence; Clinical parameters= (PD) pocket depth; (CAL) clinical attachment level; (BOP) bleeding on probing

3.1 Pre-clinical Studies using Growth Factor and Protein Delivery

3.1.1 Platelet-derived growth factor (PDGF)

PDGF's primary role is the promotion of soft-tissue healing. It was introduced to improve healing of diabetic ulcers, and was later approved for periodontal regeneration [49, 50]. In vitro studies show that PDGF stimulates cell populations key for periodontal regeneration, increasing cementoblast DNA synthesis and regulating osteopontin expression [51]. PDGF stimulates PDL cell chemotaxis and mitosis, and has synergistic proliferative effects when combined with allografts [52, 53]. PDL cells may modulate bone formation by increasing osteoblast proliferation and blocking osteoblast differentiation and expression of the mineralized tissue markers osteopontin and osteonectin [54-56]. In canine and primate models, application of PDGF, often combined with insulin-like growth factor (IGF), to periodontal defects resulted in increased bone, cementum, and PDL formation [57-63]. In a canine class III furcation defect model, guided tissue regeneration (GTR) with PDGF-BB and ePTFE membranes stimulated PDL formation in early stages followed by total periodontal regeneration [64]. PDGF/IGF-1 for guided bone regeneration (GBR) at implants placed into extraction sockets showed increased early (3-week) histologic and clinical bone formation [57, 65, 66]. When combined with xenograft, PDGF at immediate implants in canines resulted in enhanced radiographic bone gain when used without a collagen membrane [67]. PDGF also enhanced lateral GBR in dogs when used with a collagen membrane and xenograft or alloplastic graft material. In another canine model testing for vertical ridge augmentation, PDGF in a xenogeneic block graft showed increased histologic bone gain when used without a collagen membrane [68, 69].

3.1.2 Bone morphogenetic proteins

BMPs are members of the transforming growth factor-beta (TGF-β) superfamily and have strong osteoinductive properties, especially BMP-2, -4, -6, -7, and -9 [70, 71]. BMP-2 and BMP-7 stimulate PDL cell differentiation into osteoblasts and increase expression of mineralized tissue markers when combined with PDL cells or osteoblasts in vitro, although doses greater than 10 ng/mL may be toxic to cells [72-76]. BMPs have also been shown to downregulate proliferation and mineralization of cementoblasts and gingival fibroblasts. They are used primarily to enhance bone formation for implant site development. When applied to periodontal intrabony defects, BMPs showed enhanced speed and quantity of bone formation but limited cementogenesis, complicated by ankylosis and root resorption [77-83]. Commercially, rhBMP-2 is approved for extraction socket and sinus augmentation. In rat extraction sockets, BMP-2 was shown to increase the speed and quantity of bone formation via its osteoinductive effects [84]. In sinus augmentation models in various species, BMP-2 consistently improved histologic and radiographic bone gain [85-89] and there are indications that it may provide significant vertical and horizontal ridge augmentation. Delivery of BMP-2 via grafting materials may modulate its effects on bone formation, contour, and quality [90-94]. Possible complications of BMP-2 delivery shown in canine ridge augmentation models include increased incidence of seromas and wound failure [91]. When compared to BMP-2, BMP-4 has been shown to have comparable effects on bone gain and improved bone quality in a rat ridge augmentation model [90]. BMP-7, also known as osteogenic protein-1 (OP-1), has similar applications as BMP-2 [95-101]. BMPs may be suitable for periodontal regeneration as demonstrated in canine class III furcations and its modulatory effects on cementoblast mineralization in vitro [95, 102].

3.1.3 Fibroblast growth factor-2

FGF-2 was initially found to stimulate proliferation of bovine fibroblasts. It has effects in soft tissues by inducing proliferation of gingival epithelial cells, gingival and connective tissue fibroblasts, and PDL cells [103-107]. Interestingly, FGF-2 inhibits mineralization and ALP expression by PDL cells but allows them to maintain their differentiation potential and express bone regulatory compound osteopontin [104, 108]. These features, coupled with its strong angiogenic potential, may allow FGF-2 to promote an environment favoring periodontal regeneration [104, 109]. FGF-2 applied topically has been studied in primate and canine class II furcation defect models, where it significantly increased regeneration of PDL, cementum, and bone without adverse effects [106, 110]. GTR with FGF-2 and a collagen membrane resulted in more defect fill in dog class III furcations versus control groups [111].

3.1.4 Growth/differentiation factor-5

Like BMPs, GDF-5 is another member of the TFG-β superfamily and shares a similar structure [112]. It stimulates PDL cell proliferation, early osteoblast differentiation, and extracellular matrix synthesis by both cell types [113, 114]. It has been shown to significantly increase periodontal regeneration in canines and primates with a β-TCP carrier [115, 116]. Various other carriers tested in dog periodontal regeneration showed a primarily positive effect [117, 118]. In implants, GBR with GDF-5/β-TCP carrier showed increased peri-implant bone, while GDF-5 coated implants had increased stability determined by pull-out test in rabbits [119]. GDF-5 has also been used in mini-pig sinus augmentation, where it enhanced bone formation with a β-TCP carrier [120, 121]. In lateral ridge augmentation with a coated Bio-Oss^® block, GDF-5 resulted in increased mineralized tissue formation [122].

3.1.5 Teriparatide

The osteoporosis medication teriparatide consists of parathyroid hormone's first 34 amino acids. In vitro and depending on cell state, teriparatide influences PDL cell survival and causes osteoblast-like behavior with increased osteoprotegerin expression [123, 124]. It has been tested in rats with induced osteoporosis, where it caused increased bone mineralization and formation in extraction sockets and prevented periodontal bone loss [125, 126]. In canine GBR, teriparatide improved bone formation around implants [127, 128].

3.2 Clinical Applications of Growth Factor and Protein Delivery

Periodontal regenerative therapies focus on bone regeneration to provide implant site development. Guided bone/tissue regeneration is the most well-documented technique for targeted bone regeneration and is designed to exclude undesired cells using barrier membranes [129]. Under certain circumstances (i.e., defect shape and size) both procedures have shown high predictability; nonetheless, due to the non-osteogenic characteristics of available biomaterials (xeno-/allo-geneic), some shortcomings have arisen for challenging situations (i.e., vertical bone augmentation). The application of growth factor and protein delivery might overcome these limitations by inducing the proliferation of MSCs to achieve bone formation [130, 131]. Indeed, this approach has shown very promising results from countless pre-clinical studies [132, 133] (see Table 3), which has aroused enthusiasm among clinicians.

Within osseous regeneration, PDGF-BB has been tested for infrabony defects and alveolar bone regeneration. Recently, a multicenter randomized double-masked clinical trial aimed to evaluate the long-term stability of periodontal defects in patients with localized severe periodontitis filled with rhPDGF-BB in a β-TCP scaffold vs. scaffold alone. Results from this study showed that the use of PDGF-BB (0.3 mg/mL) after a 36-month follow-up achieved 87% of bone gain compared to the control group (53.8%). It was also found that, albeit not reaching statistical difference, the steady increase of clinical attachment level and linear bone gain suggest the long-term stability when using this growth factor [134]. These findings were in agreement with results obtained by Jayakumar et al. in a shorter follow-up clinical trial [135]. These demonstrated that after 6 months, a significant increase in bone fill (65.6%) was achieved when using 0.3 mg/mL of PDGF-BB vs. β- TCP alone (47.5%) [50]. Nevins et al. also reported that the use of PDGF-BB in combination with demineralized freeze-dried allogeneic bone graft is capable of attaining complete regeneration of the attachment apparatus for infrabony and Class II furcation defects [136]. Therefore, PDGF-BB seems to promote periodontal regeneration in a safe and effective manner. However, the available data is still limited to draw clear conclusions about its potential. Likewise, PDGF-BB has also been recently used for increasing the predictability of alveolar bone regeneration. Nevins et al. evaluated the effect of PDGF-BB compared with enamel matrix derivative (EMD) and two other grafting materials upon newly formed bone in socket regeneration [137]. Due to the small sample size and weak defect standardization it was not possible to draw a clear line between groups; nevertheless, ridge morphology for implant placement was more convenient for the PDGF-BB group. More recently, the same group [138] studied in a case-series the effect of PDGF-BB in combination with equine/bovine grafting materials on bone regeneration of large extraction site defects. Histologic results revealed new bone formation in association with remaining graft particles and no evidence of inflammatory cell infiltration. Accordingly, horizontal/vertical alveolar bone augmentation might become a more predictable technique with the use of PDGF-BB.

In 2007, rhBMP-2 was approved by FDA as an alternative to autologous bone grafting in alveolar ridge augmentation and sinus elevation procedures [139] due to its osteoinductive potential [140]. Results obtained for sinus augmentation indicate that when compared to autogenous bone, no differences can be observed by means of vertical bone gain and density [141]. Nonetheless, if rhBMP-2 is grafted with autologous bone it seems to increase cell activity, osteoid lines and vascular supply [141, 142]. Contrasting these findings, Kao et al. showed that when rhBMP-2 was blended with bovine-derived xenogeneic graft, less bone formation was found. Authors claimed that this may be due to the enhancement of osteoclast differentiation by the adjustment of RANKL, a protein involved in bone remodeling and regeneration [143]. Additionally, the safety of the growth factor was demonstrated by lack of an immune response in any of the studies [141-143]. Therefore, agreement was found between human clinical trials and preclinical studies [144]. However, more clinical studies need to be conducted focusing on the effect of the carrier and dose-dependent responses [145]. Other findings indicate that rhBMP-2 preserves alveolar ridge heights while also increasing their horizontal dimensions [146-148]. Fiorellini et al. noted the dose-dependence of the protein, showing that a 1.5 mg/mL dose was more optimal than 0.75 mg/mL. Nevertheless, it is worthy to note that although overall safety of rhBMP-2 was demonstrated, moderate signs of local inflammation were present, which may be a trigger for an impaired healing process.

BMP-7 has been predominantly studied for sinus augmentation. Using a small sample size case study, Corinaldesi et al. compared the use of rhBMP-7 with deproteinized bone (0.5g) vs. deproteinized bone alone (2g). Although there were no observed differences by means of bone gain, newly formed bone was statistically greater for the control group (19.9% vs. 6.6%) 4 months after grafting [149].

Another recently studied growth factor is GDF-5, which has been used for periodontal regeneration, alveolar bone and sinus augmentation procedures. A pilot randomized clinical trial was conducted to study the effect of GDF-5 embedded in β-TCP for infrabony periodontal defects of chronic periodontitis patients. Six months after therapy the clinical attachment gain for the test group was almost double compared to the level gained in the control group (3.4mm vs. 1.7mm), although this was not statistically significant [150]. To date, only one study has appraised the use of rhGDF-5 for sinus augmentation: Koch et al. found that at 4 months GDF-5 behaved similarly to an autologous graft in terms of bone formation (28% vs. 32%) [151]. Notably, larger bone augmentation occurred in the composite group of GDF-5 combined with β-TCP regardless of the time point assessed.

After being evaluated in pre-clinical model and showing its effectiveness in regenerating periodontal defects, FGF-2 has also been tested for human use due to its robust angiogenic and mitogeneic potential [110]. Kitamura et al. showed in a Phase 2B multicenter randomized clinical trial that at 36 weeks after periodontal surgical therapy, bone fill was 35% greater when used with 0.3% FGF-2 compared to the carrier alone [152]. An insignificant increase occurred from 36 up to 72 weeks, which may be explained by different patterns of healing (regeneration vs. long-junctional epithelium).

Currently, teriparatide is being investigated for craniofacial regeneration [153]. One clinical trial assessing its effect on periodontal regeneration in moderate to severe chronic periodontitis patients showed that the adjuvant of teriparatide administration combined with vitamin D daily with periodontal surgery had a favorable influence on attachment levels and bone repair. In addition, it is worthy to mention that improvements were correlated with baseline levels of 1.25 dihydroxyvitamin D3 (better results with baseline levels >20ng/dL) [154]. The use of teriparatide might increase the reliability in cases of poor bone density. Kuchler et al. conducted a randomized controlled feasibility study to appraise the effect of 20 μg of teriparatide daily during 28 days on mandibular dental implants, finding that after a healing period of 9 weeks, new bone-volume-per-tissue-volume for the teriparatide group was 17.6% and 15.4% for the control group. In addition, it was shown that bone-to-implant contact was shown to be higher in the periosteal and medullary compartment for the test group, but not for the cortical compartment (5% vs. 4.4%, respectively) [155]. Based on these findings, the safety and efficacy of several growth factors have been documented, but more clinical trials are necessary to validate these preliminary studies.

4. Cell and Gene Delivery

To enhance the therapeutic potential of growth factor delivery, gene therapy has emerged as a method of establishing sustained growth factor expression through the transduction of cell populations via viral and non-viral delivery mechanisms that alter gene expression. Currently investigated methods of vector delivery for intraoral bone regeneration and existing pre-clinical studies of their success are addressed below:

4.1 Methods of Gene Delivery

4.1.1 Therapeutic viral gene delivery

Targeted gene delivery can be achieved using viral vectors engineered to infect cells with genetic material that will compensate for defective gene(s) or produce a beneficial protein product, without causing disease. This highly effective cellular uptake and favorable intracellular trafficking abilities make recombinant viral vectors suitable for both in vivo and ex vivo application. Gene expression following transfection is generally efficacious and long-lasting, ranging from several weeks to months depending on the vector type and target tissue [156]. Furthermore, the genetic material can either be integrated into the host chromosome or transported into the nuclei of infected cells without chromosomal integration.

Recombinant viral vectors include adenovirus, adeno-associated virus (AAV), and retrovirus, among others. Adenoviruses are large, double-stranded DNA viruses that infect non-dividing cells without integrating into the host chromosome, resulting in relatively short-term gene expression. AAVs are ssDNA viruses with a similar biological profile as adenoviruses, but with the primary added benefit of integration competence. Although AAVs have a broad tropism, they require adenovirus co-infection for replication cycle completion. In contrast, lentiviruses – part of the retrovirus family – will infect both dividing and non-diving cells to provide stable gene expression by integrating into the host genome.

Each of the recombinant viral vectors has its advantages and disadvantages (see Table 4). Risks for potential immunogenicity and insertional mutagenesis continue to raise safety concerns with some recombinant viral vectors, which will be circumvented through an improved understanding of our immune response and the viruses themselves.

Table 4.

Viral and non-viral vectors utilized in tissue engineering [159].

Vector	Type	Advantages	Disadvantages	Pre-Clinical Studies
Adenovirus	viral	High transduction rate; transfection of wide range of cell types	High immunogenic potential; transient gene expression [159, 194, 195]	Chang et al., 2009 [160]; Jin et al., 2003 [162]
Lentivirus	viral	Non-immunogenic; sustained gene expression	Risk for insertional mutagenesis; transduction limited to dividing cells [159, 194, 195]	Xiang et al., 2014; [165], Logan et al., 2002 [166]
Adeno-associated virus	viral	Little/no immunogenicity; transduction in both dividing and non-dividing cells; sustained gene expression	Accommodates only very small size transgenes; production of high titers is difficult [159, 194, 195]	Cirelli et al., 2009 [163]; Warrington et al., 2006 [164]
Plasmid	non-viral	Non-immunogenic; transfection of wide range of cell types; localized gene expression	Very low transfection efficiency; transient gene expression [159, 195]	Liu et al., 2004 [196]; Huang et al., 2005 [167]
Nucleic acid/polymer complexes	non-viral	Targeted down regulation of gene expression	Low transfection efficiency; very transient effects on gene expression; potential for immunogenicity [157, 197]	Iwanaga et al., 2007 [198]; Kotopoulis et al., 2013 [199]

4.1.2 Therapeutic non-viral gene delivery

Non-viral methods rely on the combination of nucleic acids with synthetic or natural vectors, as well as physical forces to deliver genetic information to a target cell population [157]. In general, non-viral gene delivery can be utilized to introduce new genetic material or down-regulate the expression of abnormal genes at the mRNA level. Compounds such as cationic lipids and polymers form condensed complexes with negatively charged nucleic acids to protect and facilitate their cellular uptake and intracellular transport. Additionally, physical forces, including but not limited to electroporation, form transient defects in the plasma and nuclear membranes to further facilitate the transport of genes into the nucleus. Major advantages of non-viral gene delivery include delivery of significantly larger fragments of genetic information and lack of a risk for immunogenicity and infection [158-160]. Further research is required to improve the variability of gene expression and specificity of the cell types being transfected using non-viral methods for gene delivery.

4.2 Safety and Regulatory Considerations of Cell and Gene Therapy

The translation of cell- and gene-based therapies to the clinic is intrinsically linked with reducing or eliminating significant risks associated with the treatment to the patient and improving the clinical outcome. Regulatory policies established by the FDA stipulate that the safety and efficacy of a given treatment must be verified prior to its approval for clinical use. In addition to the safety issues associated with processing cells ex vivo, other considerations involve the selected cell source, handling and expansion protocols, including best practices for maintaining cells during transportation, scaffold seeding, and transplantation [161]. While the current safety profile of iPSCs still presents significant barriers to clinical translation, cell-based therapy using MSCs has successfully been applied for treatment of degenerative diseases and investigated as a potential clinically-viable approach for dental-based tissue engineering strategies. For example, human dental pulp-derived mesenchymal stem cells (DPSC) can be processed using current good tissue practices (cGTP) within a 5 day period post-isolation from an extracted tooth, banked via cryopreservation, and recovered prior to treatment [162]. In the presence of a scaffold that serves as a carrier for cell delivery, additional considerations for safe application to the clinic include adherence to good manufacturing practices (cGMP) for scaffold fabrication and sterilization [163]. Additional steps involving scaffold cell-seeding, maintenance, and transfer to the clinic require stringent quality control measures and comprehensive protocols to ensure safety and efficacy. To minimize the associated treatment costs and improve practicality of cell-based therapy in the clinic with or without scaffold carriers, these processes must be streamlined and simplified to avoid unnecessary complications and reduce regulatory burdens.

Clinically-applicable procedures for the regeneration of oral and craniofacial bone using gene therapy are highly dependent on the selection of vectors with appropriate safety profiles. Primary concerns with viral vectors include their potential to illicit an immune response, exhibit insertional mutagenesis, and activate oncogenes, as has been observed with lentiviruses and retroviruses. Safer alternatives are adeno-associated vectors (AAVs) and adenoviruses: AAVs are non-pathogenic, while adenoviruses do not integrate into the cell genome and thereby are not replicated during cell division [164]. While these vectors are currently being tested in FDA-approved human clinical trials for rare disorders, the development of more efficient non-viral gene therapy methods involving DNA, mRNA, or siRNA delivery can be more promising from a regulatory approval perspective for regeneration of common tissue defects [165].

4.3 Pre-clinical Studies for Regeneration of Oral Tissues using Gene Therapy

Adenoviruses have been used experimentally for tissue engineering of tooth-supported bony defects. One example is the use of an adenovirus encoding the PDGF-B gene (Ad-PDGF-B) on a collagen matrix to treat periodontal lesions in vivo. Clinical, hematological and blood chemical tests were done without any significant histopathological changes when Ad-PDGF-B was used [166, 167]. Jin et al. demonstrated Ad-BMP7 induced rapid chondrogenesis, osteogenesis and cementogenesis in bridging periodontal alveolar bone defects without significant inflammatory responses [168]. This is an example of the potential that gene therapy-based vectors such as adenoviral BMP have for successfully engineering bone in a preclinical model (see Figure 3) when combined with a transduced cell carrier such as gelatin that provides three-dimensional support for tissue growth during the regenerative process.

Figure 3.

Successful regeneration of alveolar bone and surrounding periodontal tissues using gene therapy vectors such as adenoviral BMP-7 (Ad-BMP-7) has been achieved in animal models. Bone regeneration and bridging was observed with the use of ex vivo BMP-7 gene transfer using a gelatin-based cell carrier in a rat wound model consisting of a large mandibular alveolar bone defect. Comparatively, transduction of syngeneic dermal fibroblasts using green fluorescent protein (Ad-GFP) or noggin (Ad-noggin) did not result in ectopic bone formation (left panel). Mature cartilage and newly-formed bone was observed at Day 21 using Ad-BMP-7 gene transfer (right panel). Reproduced with permission from Jin Q-M et al [168].

AAVs have been used to prevent periodontal disease progression in rats. Cirelli et al., demonstrated the use of pseudotyped adeno-associated virus vector based on serotype 1 (AAV2/1) to deliver TNF receptor-immunoglobulin Fc (TNFR:Fc) to rats subjected to Porphyromonas gingivalis (Pg) lipopolysaccharide (LPS)[169]. Animals treated with AAV2/1-TNFR:Fc showed sustained levels of serum TNFR protein and sustained levels of Pg-LPS-mediated bone loss. AAVs have also been successfully used for the treatment of neurological, cardiovascular and autoimmune diseases [170].

Lentiviral transfection was used to investigate follicular dendritic cell secreted protein (FDC-SP) on the inhibition of osteogenic differentiation of human PDL cells. Xiang et al. used FDC-SP transfection on hPDL cell proliferation, osteogenic and fibrogenic phenotypes [171]. Cell proliferation and cell cycle tests indicated that transfection with FDC-SP did not affect hPDLC proliferation. Moreover, expression levels of type 1 collagen were upregulated while osteocalcin, osteopontin and bone sialoprotein were downregulated in the transfected cells. Lentiviral vectors are considered excellent genetic vector systems by being both efficient and stable in gene delivery and therapy [172].

With regard to the application of non-viral vectors for growth factor delivery, plasmids have been used successfully to enhance cell survival and engraftment with IGF-1 in smooth muscle cells. Huang et al. used plasmid DNA encoded BMP-4 in critical-sized cranial defects in rats and demonstrated bone regeneration significantly increased both on the edges and center of the defects as compared to the control group of the scaffold only [173].

4.4 Clinical Applications of Cell Delivery

Cell and gene therapy have the potential to greatly improve current methods of bone regeneration through increased bioactivity of scaffolds and localized growth factor delivery. This concept is being comprehensively studied to provide a more predictable armamentarium of available treatment options for local alveolar bone loss. Reviewed here are documented clinical studies using cell-based therapy to the craniofacial complex. Cell-based therapy pre-clinical studies are in the process of being transitioned into clinically-applicable approaches that are safe and effective in regenerating periodontal tissues.

4.4.1 Alveolar bone augmentation/preservation

Existing studies have investigated the safety of cell-based therapy for alveolar bone augmentation: Filho-Cerruti et al. studied the association of platelet-rich plasma (PRP) and mononuclear cells from bone marrow aspirate and bone scaffold for bone augmentation in the maxillae [174]. An overall graft success rate of 94.7% was reported with enough ridge dimension obtained for proper dental implant placement. Documented graft failures were due to infection of the maxillary sinus and lack of integration into the host cortical bone. Bone formation with the presence of osteoblasts scattered throughout the trabeculae were noted histologically, with minimal marginal bone loss noticed during the 4-year study follow-up. Subsequently, Pelegrine et al. evaluated in a case series study the clinical and histomorphometric behavior of upper anterior extraction sockets treated with an autologous bone marrow graft versus no graft material in the control group [175]. Clinical results showed that the use of bone marrow stem cells (BMSCs) minimized alveolar bone loss after tooth extraction compared to control. Nevertheless, 6 months after grafting, similar outcomes were found by means of mineralized bone. More recently, Kaigler et al. conducted a randomized controlled feasibility trial to compare the use of tissue repair cells (BMSCs) with conventional GBR (membrane only with gelatin carrier) for alveolar bone preservation. At the time of implant placement, a second need of grafting was more frequent (6-fold greater implant exposure) for sockets treated with GBR. Additionally, it is also important to note that the regenerated bone in the test group exhibited greater density and higher vascularization with significant acceleration of osteogenesis at 6 weeks.

4.4.2 Sinus augmentation

Vertical bone augmentation in the sinus antrum is oftentimes an imperative to achieve implant stability due to progressive alveolar bone resorption. The use of stem cell therapy as a complement to conventional graft and scaffold materials could improve bone formation and accelerate regeneration. In a histomorphometric study, Gonshor et al. compared bone formation following sinus augmentation procedures using either an allograft cellular bone matrix containing native mesenchymal stem cells or a conventional allograft. Results of the test group revealed a mean of 32.5% and 4.9% for vital bone and remaining graft material, respectively, over a follow up healing period of 3.7 months. Contrastingly, for the control group, only 18.3% of vital bone content was found while an increase of up to 25.8% was noticed of remaining graft [176]. These results were consistent with the results obtained by Rickert et al., who aimed to compare bovine-derived mineral bone seeded with mononuclear stem cells with bovine-derived mineral bone mixed with autogenous bone. Significantly greater bone formation was observed in the test (17.7%) when compared with the control group (12.0%) at 14 weeks [177].

Overall, stem cells seeded in allograft or xenograft scaffold particles are capable of inducing sufficient new bone volume formation to achieve primary implant stability. Within these limitations, the high percentage of vital bone content after a relatively short healing period might encourage clinicians to consider implant therapy at an earlier stage post-grafting [178]. However, the clinical significance in small defects is very finite and therefore, the use of this approach should be further studied in more challenging scenarios with standardized randomized clinical trials.

5. Existing Limitations

Major advances have been made in the reconstruction of intraoral bone defects as a result of improvements in scaffolding matrices and application of bioactive factors that enhance the regenerative response. However, there are existing limitations in the development of optimized scaffolding matrices that meet all the necessary criteria for the regeneration of structurally and physiologically functional osseous tissues. As described in this review, current technologies such as 3-D printing are being adapted for use in the design and development of natural and synthetic matrices that are architecturally similar to bone that allow for controllable features such as chemical composition, porosity, and rate of degradation. Likewise, the selection of materials in scaffold development for intraoral bone regeneration is limited by the attempt to match the properties and rate of regrowth of the developing tissues to the degradation properties of the supporting biomaterial. It remains a challenge to balance the preference for FDA-approved materials that have been more thoroughly investigated for use in humans with materials that may be more mechanically-appropriate for bone regeneration but have been biologically/chemically modified or release degradation products which may hinder the regenerative process in situ. This further relates to the established regulatory requirements for translating novel treatments to the clinic, with more complex strategies involving combinations of materials with cell, growth factor, or gene delivery yielding a biologic-device combination product that is more difficult to evaluate as opposed to a stand-alone biologic or device product. While current clinical applications of growth factors in combination with commercialized material carriers are showing significant improvements in clinical treatment, further optimization of delivery systems in necessary to ensure that time-dependent biofactor dose and release kinetics are appropriate for the regenerative repair process. Gene therapy is promising in this area of research and is in need of further evaluation to determine the most appropriate vectors for growth factor gene delivery that is safe and clinically acceptable for further implementation in patient-based studies.

6. Future Directions

There are many exciting opportunities that lie ahead for the reconstruction of craniofacial deficiencies including periodontal, alveolar ridge and large mandibular/maxillary discontinuity defects. The innovations that are ongoing in materials science and in biology have offered many potential avenues in the laboratory and clinic to extend the field of tissue engineering of oral structures such as alveolar bone and soft tissues of the teeth and dental implants. In particular, recent advances in biomaterial design, drug delivery and biologic agents offer less invasive technologies to accelerate and more predictably promote tissue repair and regeneration. The ability of rapid prototyping, three-dimensional printing, electrospinning, and enhanced drug delivery strategies to personalize patient therapies is allowing a more customized approach for oral tissue engineering to benefit both clinicians and patients. Development of scaffolds that act as delivery vehicles for drugs that can be controllably released to counter bone degenerative processes is an example of a future focus for intraoral tissue engineering strategies, as recently evidenced in a study by Ji et al [179] that focused on the incorporation of hydrophilic naringin into elecrospun amphiphilic copolymer nanofibers for bone resorption treatment. Further improvements to existing materials with a history of use in dental and craniofacial applications, such as calcium phosphate-based cements, are a promising venue for addressing their existing limitations through the improvement of mechanical integrity and incorporation of growth factor delivery vehicles. Lee et al [180] present such an approach through the reinforcement of macroporous calcium phosphate cement (CPC) with absorbable fibers and simultaneous delivery of rhBMP2 in the CPC matrix with VEGF or TGF-β1 in alginate hydrogel microbeads within the matrix. Such combinational approaches to address the need for controlled delivery of growth factors at critical stages of the regenerative process are important in advancing our ability to predictably regrow lost or damaged tissue. Further improvement and streamlining of existing processes and technologies for addressing these tissue engineering strategies will enable their continued entrance into the clinical arena to provide more predictable regenerative medicine therapeutics for enhanced patient care.

Highlights.

• Bone regeneration using scaffolds with cell/growth factor delivery is reviewed.

• Pre-clinical/clinical results using bone regenerative therapies are discussed.

• Novel methods of scaffold fabrication and growth factor delivery highlighted.

• Intraoral bone regeneration requires strategic scaffold design parameters.