Designing biomaterials for in situ periodontal tissue regeneration

Abstract

The regeneration of periodontal tissue poses a significant challenge to biomaterial scientists, tissue engineers and periodontal clinicians. Recent advances in this field have shifted the focus from the attempt to recreate tissue replacements/constructs ex vivo to the development of biofunctionalized biomaterials that incorporate and release regulatory signals in a precise and near-physiological fashion to achieve in situ regeneration. The molecular and physical information coded within the biomaterials define a local biochemical and mechanical niche with complex and dynamic regulation that establishes key interactions with host endogenous cells and, hence, may help to unlock latent regenerative pathways in the body by instructing cell homing and regulating cell proliferation/differentiation. In the future, these innovative principles and biomaterial devices promise to have a profound impact on periodontal reconstructive therapy and are also likely to reconcile the clinical and commercial pressures on other tissue engineering endeavors. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2012

Introduction

The periodontium is continually undergoing alterations and physiological adjustments due to the effects of aging, occlusal force and other detrimental changes in the oral environment.1 Particularly, the presence of plaque-induced periodontitis results in destruction and pathological changes to the periodontium, i.e., the loss of alveolar bone, periodontal ligament (PDL) and cementum, that unfortunately show very little tendency for self-repair. Hence, once periodontitis becomes established, only effective therapeutic intervention has the potential to induce substantial periodontal tissue regeneration. To date, however, there has been no appropriate method that is available for clinical choice.2 New insights into the reparative capability of the periodontium in conjunction with progress in stem cell biology, molecular science and biomaterials design will enable us to develop novel therapies that use engineered biological compounds and stem cells for therapeutic periodontal regeneration.

Since Seo et al.3 successfully isolated and characterized PDL stem cells from human PDL tissue in 2004, the use of stem cell delivery/transplantation has become the predominant research strategy in periodontal regenerative medicine.4–12 Experimental results obtained using various animal models have shown that under certain conditions, PDL stem cells,4–6 bone marrow mesenchymal stem cells (MSCs),7–10 adipose-derived stem cells11 and other induced pluripotent stem (iPS) cells12 can potentially induce the formation of new periodontal tissue (Table 1). However, stem cell transplantation requires complicated and specific in vitro cell culture conditions and complex operating procedures. Its clinical translation therefore faces enormous technical and financial hurdles, especially in the treatment of non-fatal diseases, such as periodontitis.13 Although stem cell therapy will undoubtedly lead to new clinical technologies in the future and warrants further basic research and translational investigation, it would be more advantageous if well-designed biomedical devices can be applied to achieve in situ tissue regeneration without the use of exogenously manipulated cells. With the progress of stem cell biology research and recombinant protein biotechnology, a promising approach that has recently been proposed is the use of stem cell migration mechanisms in the body to develop new biofunctionalized biomaterials that actively recruit a patient's own stem cells to a desired location for therapeutics (Figure 1). The promotion of in situ periodontal regeneration is expected to ameliorate the translational difficulties in relation to the use of exogenous stem cells and may have broad prospects and potential for routine clinical applications.13–15

Table 1. The Use of Stem Cells as Exogenous Regenerative “Tools” for Periodontal Tissue Regeneration: Selected Examples and Their Brief Outcomes in Animal Models

Stem Cells	Vehicles	Animal Model	Brief Outcomes	Ref.(s)
ASCs, adipose-derived stem cells; BM-MSCs, bone marrow mesenchymal stromal cells; EMD, enamel matrix derivatives; HA, hydroxyapatite; iPS cells, induced pluripotent stem cells; PDL, periodontal ligament; PDLSCs, periodontal ligament stem cells; PRP, platelet-rich plasma; TCP, tricalcium phosphate.
PDLSCs	HA/TCP	Periodontitis models in miniature pigs	PDLSCs were capable of regenerating periodontal tissues and statistical analysis indicated that both the autologous and allogeneic PDLSCs treatments significantly improved periodontal tissue regeneration compared with control groups, with no difference between the autologous and allogeneic PDLSCs groups	4, 5
	Cell sheets withβ-TCP/collagenscaffold	One-wall intrabony defects in dogs	Periodontal regeneration was significantly observed with both newly formed cementum and well-oriented PDL fibers more in the PDL cell group than in the iliac bone marrow mesenchymal stromal cell and alveolar periosteal cell groups	6
BM-MSCs	Atelocollagen(2% type I collagen)	Experimental Class III periodontal defects in beagle dogs	Four weeks after transplantation, the periodontal defects were almost regenerated with periodontal tissue and transplanted BM-MSCs could survive and differentiate into periodontal tissue-forming cells	7, 8
	Collagenscaffold	Periodontal fenestration defects (5 × 5 mm) in beagle dogs	Both freshly isolated and cryopreserved BM-MSCs induced significantly better periodontal regeneration with newly formed cementum, alveolar bone and PDL compared with the application of collagen scaffold alone	9
	Gelatin beads	Periodontal tissue defect in rats (surgically created on the buccal of the mandibular first molar by removing bone, PDL and cementum)	Implantation of BM-MSCs regenerated significantly greater new bone and more appropriately orientated PDL fibers; and donor-derived BM-MSCs were found integrated in newly formed bone, cementum and PDL	10
ASCs	PRP	Periodontal tissue defect in rats	A small amount of alveolar bone regeneration was observed 2 and 4 weeks after ASC/PRP implantation. Moreover, 8 weeks after implantation, a PDL-like structure was observed along with alveolar bone	11
iPS cells	Silk scaffold+ EMD	Critical-size calvarial bone defects in mice	Histological analysis showed much more new alveolar bone and cementum formation (with regenerated PDL between them) in iPS cell group compared with both control groups (cell-free groups)	12

Figure 1. Schematic representation of two stem cell migration mechanisms in the body that may be regulated for the recruitment of endogenous cells (illustrations not to scale, modified from Chen et al.15).

(Top) Stem cells within the extracellular matrix (ECM) (or located in the neighboring cell niches of a injury) can recognize and obey directional cues and reach site of injury via interstitial migration or active amoeboid movement, which can occur independent of blood flow. (Bottom) Stem cells from distance cell niches (i.e., the bone marrow) can be mobilized into the blood (i.e., by injury) and disseminated throughout the body until they recognize and interact with microvascular endothelial cells at a particular target disease site. A coordinated multistep process ensues, including stem cell rolling, adhesion to the endothelium, transendothelial migration, chemotaxis, and invasion of the ECM, where the cells replenish and maintain the cell niche and hence enhance the regenerative potential of the tissue or directly reach the site of injury; they then participate in tissue repair and regeneration.

In recent years, Mao and coworkers have put this principle into practice by using biomaterials complexed with cell homing factors to recruit the body's own stem cells in vivo.16–18 When an incisor scaffold was implanted orthotopically following mandibular incisor extraction in rats, a putative PDL and new bone regenerated at the interface of a rat incisor scaffold with native alveolar bone provided that stromal-derived factor (SDF)-1 and bone morphogenetic protein (BMP)-7 were delivered to scaffold microchannels.16 Similarly, a chemotaxis-based approach has demonstrated the capability of recellularization and revascularization in endodontically treated root canals in vivo, although in an ectopic model.17 Furthermore, recent data from the same group suggest that the entire articular surface of the synovial joint can be regenerated by collagen hydrogels that are spatially infused with transforming growth factor (TGF)-β3 without cell transplantation in a skeletally mature rabbit model in which the entire articular surface of unilateral proximal humeral condyles has been surgically excised.18 Mao and coworkers' endeavor is a renaissance of use of the host as a bioreactor and recruitment of the host's endogenous cells for regeneration. They have bucked the current trend for use of ex vivo cultivated stem cell preparations for tissue engineering. At the same time, other scientists have successfully regulated the cell recruitment that has demonstrated effective and safe for bone and other tissue regeneration (Table 2).19–30 However, cell homing strategies are currently in the early stages of development, which require further optimization; moreover, there are hurdles to overcome before seriously discussing the adaptation of current tested paradigms to large animals and human beings.

Table 2. Selected Examples of Recruiting Endogenous Cells for Tissue Regeneration

Stimulators/Mediators	Biomaterials	Target Cells	Animal Models/Outcomes	Ref.(s)
bFGF, basic fibroblast growth factor; BM-derived cells, bone marrow-derived cells; BMOPCs, bone marrow-derived osteoblast progenitor cells; BMP, bone morphogenetic protein; BMSCs, bone marrow stromal cells; CTPs, circulating osteogenic connective tissue progenitors; EPCs, endothelial progenitor cells; HA, hydroxyapatite; MCP-1, monocyte chemoattractant protein; NGF, nerve growth factor; PCL, poly-ε-caprolactone; PDGF, platelet-derived growth factor; SDF-1, stromal cell-derived factor-1; 3D, three-dimensional; TGF, transforming growth factor; VEGF, vascular endothelial growth factor.
SDF-1 and BMP-7	Anatomically shaped tooth scaffolds/collagen gel solution	Not shown	SDF-1 and BMP-7 not only recruited significantly more endogenous cells but also enabled greater angiogenesis than growth-factor-free controls, leading to a putative periodontal ligament and new bone regenerated at the interface of rat incisor scaffold with native alveolar bone	16
bFGF, VEGF, or PDGF with a basal set of NGF and BMP-7	Collagen gel solution	Not shown	Chemotaxis-based approach has potent cell homing effects for recellularization and revascularization in endodontically treated root canals in vivo (an ectopic model in mouse)	17
TGF-β3	An anatomically correct bioscaffold (PCL-HA)/collagen gel solution	Not shown	The entire articular surface of the synovial joint can regenerate without cell transplantation in rabbits	18
BMP-2	Collagen pellet	BMOPCs	Recruited BMOPCs contribute to ectopic bone formation in mice.	21, 22
Endogenously produced factors in response to injury (transverse fibular fracture)		CTPs	CTPs are physiologically mobilized into the fracture healing site in the parabiotic mice model	23
SDF-1α	Porous 3D scaffolds made of mineralized collagen	BMSCs	Efficient invasion of BMSCs into internal compartments of the scaffolds both in vitro and in vivo (a mouse model)	24
Endogenously produced EPC-mobilizing cytokines, such as VEGF, SDF-1 and MCP-1		EPCs	Mobilization of EPC population during distraction osteogenesis in human limb-lengthening patients.	25
SDF-1α	A bioactive knitted silk-collagen sponge scaffold	Fibroblast-like cells	Recruitment of fibroblast-like cells for in situ tendon regeneration in a rat Achilles tendon injury model	26
Endogenously produced cytokines (not shown)		BM-derived cells	Inflammation-induced influx of BM-derived cells into the infarction area in rat myocardial infarction models	27
BMP-2	Gelatin hydrogels	BMOPCs	Recruited BMOPCs contribute to the ectopic de novo generation of bone tissue	29
SDF-1 and BMP-2	Gelatin hydrogels	BM-derived cells	Synergistic effects of SDF-1 and BMP-2 on the recruitment of osteogenic cells, enhancing both ectopic and in situ bone regeneration in rats	30

To date, various bioengineering research strategies have sought to induce cell recruitment and direct cell fates toward tissue formation via the incorporation of signaling molecules and the functionalization of scaffolding materials.15 These strategies have become increasingly popular in the field of stem cell research and regenerative medicine. Given that different bioactive factors play different key roles in the different stages of tissue reconstruction, such as cell recruitment, cell proliferation/differentiation and tissue integration/vascularization, one critical issue that must be addressed is the design and development of biomaterials that can provide the controlled spatiotemporal release of various key signaling molecules during different stages of regeneration. An understanding of which factors to deliver and the timing of delivery is emerging from advances in the elucidation of the critical pathways that are involved in the development of integrated periodontal tissues. With modern drug delivery technology, biomaterials can be designed to release a variety of critical exogenous signaling molecules to mobilize/activate the body's stem cell niches and regulate them toward functional regeneration. This is a new research direction for biomaterials design in tissue engineering and regenerative medicine and has important clinical significance and the potential to be applied broadly in the near future.13–15

This article addresses the design of novel medical devices for periodontal regeneration and highlights the development of the state of the art of biofunctionalized scaffolds that incorporate signaling molecules required to capture host own cells and catalyze endogenous repair. Furthermore, it describes the challenges and future directions in this rapidly advancing field. Although our focus in this manuscript is on the regeneration of periodontal tissues, the concept should be more generally applicable.

Clinical Need for Periodontal Regeneration

Periodontitis is a worldwide chronic inflammatory disease that afflict up to 50% of adults worldwide and more than 15% display severe disease concomitant with early the destruction/damage of the periodontium, i.e., the tissues that surround and support the teeth, and if left untreated, can lead to the loosening and subsequent loss of teeth.1 It is also closely related to cardiovascular disease, diabetes and other systemic diseases.31Around the world, the occurrence of periodontal disease results in significant health and quality-of-life complications.32, 33 There may not be any other chronic infectious disease that can affect as many people globally as periodontal disease does. Therefore, it has become an important public health issue.34, 35 The ultimate aim of periodontal therapy in perspective is the restoration of lost tissue to its original form and function, i.e., the reformation of an epithelial seal, deposition of new acellular extrinsic fiber cementum and insertion of functionally oriented connective tissue fibers into the root surface and restoration of alveolar bone height. Current treatment, however, is generally unable to achieve this goal.34 The complete regeneration of the periodontal attachment is a highly desirable outcome of periodontal therapy that is directed at undoing the harms that this disease causes to periodontal structures. Although conventional periodontal treatment (e.g., debridement/scaling and flap surgery) can control further development of periodontal disease and arrest further loss of periodontal support tissue to a certain extent, it cannot properly regenerate lost periodontal tissue. The wide development and clinical application of bone grafting and guided tissue/bone regeneration techniques have greatly improved the efficacy of clinical periodontal therapy, but the goal of appropriately restoring the lost structure and function of periodontal tissue has not yet been achieved in many, if not all, cases.36, 37

Tissue engineering, often used synonymously with regenerative medicine, encompasses a broad range of strategies to replace or regenerate severely damaged or irreversibly affected tissues or organs. In recent years, stem cell biology and tissue engineering research have made rapid advances. The use of stem cell therapy, together with tissue engineering principles, to promote periodontal regeneration has attracted increasing attention and concern and has become the focus of research in this arena.1, 34 This requires the exploitation of these cells either by transplanting them from an exogenous source3–11 or by activating endogenous stem cells pharmacologically.13, 15 However, several technical and clinical challenges must be overcome before ex vivo cultured stem cells can be used to treat patients. This is because there are many ‘bottleneck’ problems surrounding the clinical translation of stem cell delivery-based therapy that have not yet been resolved.38 Potentially useful cell populations already exist in the body, and attracting these cells to the diseased site (e.g., the damaged periodontium) by designing instructive biomaterials has the potential to provide new therapeutic options for in situ regeneration (Figure 2). The cost and complexity should be reduced in terms of approaches that use ex vivo bioreactors and cell delivery.39 Therefore, the ability to recruit these host cells and to stimulate and enhance the body's own ability to regenerate lost periodontal tissue has important clinical significance and the potential to accelerate clinical translation.14

Figure 2. Schematic representation of the use of cell-instructive biomaterials in capturing endogenous cells for tissue regeneration (in vivo tissue engineering strategy).

This schematic assumes that there is already an existing degree of periodontal dysfunction that cannot be restored by endogenous mechanisms alone. The aim of cell-instructive biomaterials is to use biological stimuli and biomimetic scaffolds to harness the body's innate ability to heal through strategies that avoid in vitro cell manipulation and ex vivo cell delivery. Such biomaterials must be designed to bear complex information influences (encapsulation of cues) coded within their physical and chemical structures to mimic, as closely as possible, a natural extracellular milieu to recruit/capture enough endogenous cells from extracellular matrices (ECM) and/or blood and thereafter influence their behavior in vivo for regeneration of lost/damaged periodontal tissues.

Endogenous Regeneration of Periodontal Tissues

It is in the light of improvements in our understanding of wound repair, concerted research efforts based on the concepts of tissue engineering and regenerative medicine have begun to harness advances in materials science and recombinant DNA technology to yield next generation of biomaterials-based medical devices for replacing tissue function and new treatment approaches designed to stimulate or augment endogenous repair mechanisms. This development is also true for the regeneration of tooth-supporting tissues that have been lost to periodontal disease.14 Keeping this in mind, an alternative, perhaps simpler, regenerative strategy to periodontal regeneration therefore is to promote endogenous repair/regeneration through design of instructive biomaterials and delivery of growth factors (GFs) and/or other signaling molecules that stimulate/recruit host endogenous cells, including stem/progenitor cells, to invade an injury site and direct robust extracellular matrix (ECM) synthesis and in situ tissue regeneration.

Roles of growth factors

Broadly speaking, even without any therapeutic intervention, living tissues can have a staggering capacity for regeneration, normally termed self-repair or endogenous regeneration.15 Endogenous periodontal regeneration requires an orchestrated process involving coordinated migration, proliferation and differentiation of cells from host tissue resources (i.e. the healthy portion of the periodontium or the remaining alveolar bone) specifically contributing to restoring the tooth-supporting apparatus. The innate abilities of organization and self-repair unfortunately do not offer a universal regenerative solution.14 Over the past several decades, the discovery of GFs has led to much hope and speculation about the use of these potent peptides to augment the regenerative potential of tissue in the treatment of wounds that are difficult to heal. In terms of periodontal therapy, studies have shown that the local application of biologically active factors, such as platelet-derived growth factor (PDGF), TGF-β, acidic and basic fibroblast growth factors (a- and bFGF), insulin-like growth factors (IGF-I and -II), cementum-derived growth factor (CGF) and BMPs, can stimulate self-repair and the regeneration of body tissues and can result in the endogenous regeneration of the periodontium.2, 14, 15, 40, 41

GFs are found either as matrix bound proteins attached to the ECM or as soluble molecules secreted by cells or cleaved from the matrix by certain enzymes or proteases. They are large polypeptides that modulate cellular activities including proliferation, differentiation, migration, adhesion and gene expression and regulate the mechanisms and pathways that govern wound healing and tissue regeneration.42 It is now well recognized that GFs play crucial roles in the complex cascade of biological events of wound healing, by stimulating chemotaxis and cellular proliferation, by transferring information among cells of the same and different type and their microenvironment, by controlling ECM formation and angiogenesis, by regulating the process of contraction and by re-establishing tissue integrity.43–48 GFs initiate their action by binding to specific receptors on the surface of target cells, and their effects are concentration-dependent, often in a complex non-monotonic way.43 The availability of GFs from the conditioned medium of cultured human cells, their expansion through recombinant technologies and improved understanding of their functions and clinical applications has increased the need for pharmaceutical forms.44–46 Unfortunately, although many recombinant GFs are now available for research purposes and some have also been tested in humans, the clinical experience so far has been disappointing.47, 48

Because exogenous GFs are expensive, have a short half-life and are quickly diluted and metabolized when used locally or are inactivated through in vivo enzymatic degradation, their conventional routes of administration are unlikely to be effective.42 Concerns and complications associated with burst release of high doses of GFs, rapid degradation and thus low local availability following bolus injection have motivated extensive recent study into sustained delivery strategies that may provide efficacy at lower doses.45, 46 From a careful examination of the literature, it is soon realized that only with an appropriate carrier/delivery system can locally applied GFs play a sustained and efficient role in promoting tissue regeneration.39, 40, 42–44 Particularly, it seems that any treatment aimed at mimicking the natural tissue regeneration processes that occur in precise space and time during the development of the periodontium should not be limited to the provision of a single GF but should deliver multiple agents in an optimized ratio and a specific spatiotemporal pattern.39, 40, 48 Hence, dual/multiple GF delivery is the focus of GF application in periodontal healing via endogenous regeneration.44–48 Currently, however, well-established strategies and techniques are lacking with respect to multiple delivery and clinical application. In this regard, the research and development of controlled-release systems of GFs have been and still are important research focuses in periodontal tissue engineering and regenerative medicine.39, 40, 42

Design of material vehicles

For over two decades, a wide range of materials such as natural polymers (e.g., alginate,49 collagen,50 and chitosan51), synthetic polymers (e.g., polylactides, polyglycolides, polyurethanes, and polycaprolactones),52–54 ceramics (e.g., silicate-based glasses, calcium sulfate hemihydrate/dihydrate and calcium phosphates)55 and more often their composites56–60 have been intensively studied and widely used as delivery vehicles for GFs (Table 3), However, the release of GFs from conventional vehicles is typically governed by molecular diffusion and material degradation, which do not allow for precise and near-physiological delivery of therapeutics to sites of injury. Hence, there is a need to devise more advanced delivery systems to deliver GFs in a controlled, sustained and localized fashion.42

Table 3. Selected Examples of Material Vechicle for Periodontal and Bone Regeneration

Growth Factors	Biomaterial Carriers	Animal Model	Conclusion(s)	Ref.(s)
ACS, absorbable collagen sponge (type I bovine collagen); Bio-Oss, bovine deorganified crystalline bone matrix; BMP-2, bone morphogenetic protein-2; DBM, demineralized bone matrix; Drilac, polylactic acid granules; GDF-5, growth/differentiation factor-5; IGF-I, insulin-like growth factor I; NGF, nerve growth factor; PDGF-BB, platelet-derived growth factor BB; PLGA, poly(L-lactide-co-glycolide).
BMP-2	Peptide-modified alginate hydrogel	Critically-sized segmental bone defects in rats	The hybrid alginate/nanofiber mesh system is a promising growth factor delivery strategy for the repair of challenging bone injuries	49
BMP-2 and β-NGF	Biodegradable collagen hydrogel	Class III furcation defects in beagle dogs	a topical application of rhBMP2 and rh beta-NGF may improve the quality and quantity of regenerated bone in artificially created Class III furcation defects of beagle dogs	50
PDGF-BB	Chitosan sponge	Calvarial defects in rats	PDGF-BB-loaded chitosan sponge may be beneficial to enhance periodontal bone regeneration	51
BMP-2	canine DBM, Bio-Oss, ACS, PLGA or Drilac	Routine critical size supra-alveolar periodontal defects in dogs	DBM and Bio-Oss performed well as carriers for rhBMP-2-driven periodontal regeneration	52
PDGF-BB and/or IGF-I	A methylcellulose gel vehicle	Ligature-induced periodontitis in non-human primates	The combined delivery of PDGF-BB and IGF-I resulted in significant increases in periodontal tissue regeneration	53
BMP-2	A sponge-type carrier material made of gelatin and polylactic acid polyglycolic acid copolymer	Horizontal circumferential defects created by experimental periodontitis	Suitable application of rhBMP-2 can produce considerable periodontal tissue regeneration, even in cases of horizontal circumferential defects	54
BMP-2	ACS or calcium phosphates putty	3-wall intrabony periodontal defects in the baboon	Although both carriers loaded with BMP-2 appear to enhance periodontal regeneration, the refinement in carrier systems may provide the key to enhancement of the regenerative potential	55
GDF-5	Composite matrix constituted by bioresorbable PLGA and additives forming in situ	Periodontal defects in dogs	Characteristics of the PLGA composite makes it an attractive matrix to support native wound healing and rhGDF-5-enhanced periodontal regeneration	56
GDF-5	Injectable PLGA composite carrier	Bilateral 4 × 5 mm (width × depth) one-wall, critical-size, intra-bony periodontal defects	The PLGA composite appears to be an effective ease-of-use candidate for delivery of growth factors to the periodontium	57
BMP-2	Dextran and collagen composite hydrogel	Class III furcation defects in beagle dogs	Glycidyl methacrylate derivatized dextran/gelatin microsphere/scaffold system was promising to delivery growth factors for catalyzing periodontal regeneration	59, 61
BMP-2 and dexamethasone	PLGA and alginate	In vitro rat BMSCs culture model	The present coaxial system was very effective in manufacturing PLGA core-alginate shell microcapsules and in encapsulating multiple biomolecules essential for stem cell differentiation	60

Hydrogels, due to their unique biocompatibility, flexible methods of synthesis, range of constituents and desirable physical characteristics, have been one of the most attractive materials of choice for GF delivery applications in regenerative medicine, and myriad hydrogel-based formulations fabricated from natural and synthetic polymers have been proposed for controlled delivery systems.41, 42 However, the lack of space provision by hydrogels may be one of the major issues that needs to be addressed in design of material vehicles for tissue engineering applications.58, 61 Therefore, a hydrogel-based carrier need to be modified to provide support for new tissue formation and maturation during the more advanced stages of healing. In this regard, Wikesjö et al. (2003)62 proposed a space-providing macroporous expanded polytetrafluoroethylene (ePTFE) device to control volume and geometry of rhBMP-2/absorbable collagen sponge (ACS)-induced alveolar bone augmentation.

In terms of cell penetration and tissue ingrowth, the porosity and pore size of most hydrogels may not meet the requirements for tissue engineering scaffolds, which is another critical design consideration for hydrogels in regenerative medicine.61 To develop a hydrogel vehicle with proper porosity and pore size for periodontal regeneration, we have recently designed a macroporous glycidyl methacrylate derivatized dextran (Dex-GMA)/gelatin (Gtn) hydrogel system that may incorporate BMP-loaded microspheres into its porous structures (Figure 3).59 The envelopment of two types of microspheres, each preloaded with different GFs, into the inner pores of Dex-GMA/Gtn hydrogels enables the integration of drug delivery carriers and tissue engineering scaffolds into a single device and, to some extent, allows for the controlled release of dual GFs.63 While significant work has been dedicated to the development of material vehicles that deliver GFs directly, a relatively simpler approach involves the conversion of cells into protein-producing factories, normally termed gene delivery.64–66 However, we must be aware of that material vehicles are also required for in vivo gene delivery. For example, a chitosan/collagen scaffold was used to delivery adenoviruses expressing either BMP-7, PDGF-B, or a combination of both BMP-7 and PDGF-B in surgically induced defects on both sides of the dog mandible.65 This study demonstrates the promising potential of biomaterial expression of combinations of growth factors such as BMP-7 and PDGF-B for bone regeneration in tissue engineering applications. In another impressive investigation, a collagen matrix containing an adenoviral vector encoding PDGF-B demonstrated regenerative and safety capabilities for bone tissue engineering and osseointegration in alveolar bone defects that are comparable with recombinant human PDGF-BB protein delivery in vivo.66 The use of gene delivery for periodontal regeneration is a relatively new strategy because non-viral vectors are continually optimized for clinical therapeutic purposes, and significant reservations about viral vectors have increasingly dampened their appeal.67 Novel gene delivery systems that act as localized gene depots, providing prolonged maintenance of the therapeutic level of encoded proteins, thus have great potential for future clinical periodontal regeneration.68 Furthermore, we should be aware that novel delivery systems developed for bone or other tissue regeneration can also be applied to periodontal research directly or with slight modification.48, 69–72

Figure 3. Representative scanning electron microscopy images of two types of glycidyl methacrylate derivatized dextran (Dex-GMA)/gelatin(Gtn) scaffolds developed in our laboratory.

(A) Dex-GMA/Gtn scaffolds with an interconnected pore structure containing microencapsulated growth factors; (B) a particulate-based scaffold obtained from the chemical combination of batches of Dex-GMA/gelatin microspheres loaded with growth factors (unpublished data).

Mobilization of endogenous cells

Tissue regeneration requires the presence of a sufficient number of stem cells at the injury site. With the stimulation of local GFs and the support of bioscaffolds, stem cells differentiate into different “tissue-forming cells”, which is critical to the formation of new tissue.73–75 Previous studies have mainly focused on the transplantation of high concentrations of ex vivo cultured pluripotent stem cells. In addition to the low rate of engraftment and other unsolved problems, stem cell transplantation requires in vitro cell manipulation and ex vivo cell delivery, according to strict operating procedures. Furthermore, its clinical application faces enormous technical and financial challenges. Given the current options for periodontal disease treatment and its non-fatal nature, the benefits of conventional stem cell therapy may not outweigh these drawbacks.13, 16, 17 Therefore, the ability to mobilize the body's own stem cells and avoid cell culture and transplantation to stimulate self-repair in patients is very promising and may accelerate clinical translation.14, 15 Recently, researchers have shown that with the help of GF induction, bone marrow-derived precursor cells in peripheral blood circulation can be recruited for ectopic bone formation.20–22, 24, 25 Kim et al.16, 17 have also confirmed that by recruiting the body's own stem cells it may be possible to achieve a certain degree of odontogenic tissue regeneration. Further exploration of delivery strategies and release criteria for exogenous induction factors in the design of novel functional biomaterials as delivery systems may ultimately lead to endogenous regeneration of targeted tissues.14, 15 In addition, the identification of different key factors at different stages of the wound healing cascade (i.e., cell recruitment, cell proliferation/differentiation and tissue remolding and integration/vascularization) is also very important in the development of safer and more effective approaches of using bioactive factors in the future, which will have far-reaching effects on the repair and regeneration treatment of a variety of tissue defects.13–15, 39

Crucial Barriers to Progress

Not surprisingly, given the pressing clinical need, the market for biomaterials-based treatments in periodontitis is growing at a rapid rate. Similarly, an emerging philosophy in periodontal regenerative medicine is that rather than attempting to recreate tissue replacements of living periodontium ex vivo, we should aim to develop synthetic materials that establish key interactions with cells in ways that unlock the body's innate ability to heal.13, 14 As described above, endogenous regeneration involves the coordination of a variety of biological signaling molecules. It is the result of in vivo stem cell recruitment, proliferation and differentiation until vascularized tissue forms.75 This process requires a well-designed functional scaffold that can support cell penetration and tissue ingrowth and a drug delivery system (DDS) that can provide the sequential release of multiple factors to the defect region.76–79 Although promising, the current solutions do not feature medical devices that meet general the requirements of clinical application.

Barriers in release technology

There is increasing evidence that maximizing the potency of tissue regenerative therapies will require design and development of release technology that provides controlled spatiotemporal delivery of multiple key signaling molecules. Richardson et al.80first reported the use of microspheres and scaffolds to coordinate the release of two GFs in 2001. Since then, many scientists have used different strategies to achieve various GF release profiles to meet the needs of different tissue regeneration applications. To date, the simultaneous delivery,63, 81 sequential delivery82, 83 and spatiotemporal delivery70, 84, 85 of different GFs have all been achieved in laboratory studies. Delivery strategies include, but are not limited to, the following86: direct incorporation (Figure 4), layer-by-layer technology, multiphase loading and particulate-based delivery systems (Figure 3). The introduction of various controlled-release microsphere systems that are preloaded with various bioactive factors into well-designed scaffolds makes the release of several GFs a possibility (Figure 5).86–89 Alongside these positive developments, progress in biomaterial design and drug delivery is converging to produce a new generation of materials for endogenous regeneration.90 To date, however, there is no true GF controlled-release system that has successfully been applied in clinical practice. This is, at least in part, because many difficulties and challenges are associated with the delivery of multiple factors, such as the selection and combination of different factors, the establishment of their dose and release criteria and the identification of dose-effect relationships.86 These technical difficulties have greatly restricted the application of delivery systems and release technology for the treatment of disease.86, 89

Figure 4. A selected representative scanning electron microscopy image of a freeze-dried particulate vehicle incorporated with soluble growth factors (sample from a large-sized batch of microparticles, as shown in Chen et al.,59 Fig. 2D).

Figure 5. Schematic of the sequential release of multiple growth factors from an assumed particulate-based delivery systems.

Each growth factor is pre-encapsulated into a polymer microsphere system that has unique release kinetics. Sequential release may be achieved by the incorporation of multiple microsphere systems into hydrogel scaffolds.

Barriers in biomaterial design

Biomaterials created today for tissue regeneration are routinely information-rich, and recently, their degree of sophistication has increased significantly because scaffolding biomaterials must be designed to bear complex information influences that are coded within their physical and chemical structures to mimic, as closely as possible, a natural extracellular milieu to influence cell behavior and control cell fate in vivo.90, 91 However, for commercialization and clinical use, therapeutic biomaterials must not only be scientifically sound and practically efficacious but must also be technically simple and economical; these requirements introduce a potential dichotomy between the need for sophistication and the ease of production.91 The success of a therapeutic material device in the market will finally depend on a combination of its safety, effectiveness, accessibility and cost-effectiveness.91

It is worth mentioning that current major research in the field of tissue engineering and regeneration focuses on the use of DDS directly or in combination with other biomaterials as cell vehicles for cell transplantation.87, 88 The translational difficulties faced by cell transplantation, as described previously in this manuscript, also affect the clinical use of DDSs as medical devices. Based on a careful examination of the literature, it can be understood that cell-recruiting designs based on endogenous regeneration have not received enough attention in existing biomaterials (DDS or scaffold) research and development.13–15 The development of cell-recruitment devices to carry exogenous signaling molecules that can stimulate the body's own repair and regeneration mechanisms after implantation have great potential for clinical application, which may accelerate the application of novel biomaterials to modern clinical practice and hence warrants further explorations.89

Biofunctionalization of Biomaterials

Biomaterials play an essential role in the design of medical devices and delivery systems for periodontal therapy. However, in nearly every case, these materials were adopted from other areas of medical science without substantial redesign for periodontal regenerative use. Further synergism of biologists, materials scientists and periodontists promises to deliver sophisticated biomaterials that display and release multiple selected key signals to meet the physiological requirements of the periodontal tissue repair processes. Those biofunctionalized biomaterials may have a profound impact on periodontal regenerative medicine and will advance clinical translation of tissue engineering-based therapeutics.

Cell recruitment design

The stem cell homing concept in endogenous tissue regeneration research broadly includes two basic forms of cell recruitment in the body15: (i) stem cells reach the target tissue or organ in a manner similar to hematopoietic stem cells (HSCs) homing to bone marrow after bone marrow transplantation; and (ii) under the influence of stem cell homing inducers, stem/progenitor cells from the stem cell niche around the tissue damage zone react to a particular microenvironment and, through amoeba-style movement or interstitial migration, finally settle at the target site (Figure 1). The latter method relies mainly on cell surface receptors to receive molecules along a vascular extracellular signal concentration gradient, resulting in directional, non-blood-flow-dependent movement.15, 91 Although the molecular mechanisms of stem cell homing are not yet completely clear, the phenomenon of stem cell homing in tissue repair and regeneration has received great attention from a number of scientists. It has become the core of in situ regeneration technology14, 15, 89 and has been successfully demonstrated in certain animal models.16–30

The use of stem cell homing to achieve in situ tissue regeneration has unique advantages compared to current research on stem cell transplantation: (i) homing overcomes the limitations of stem cell transplantation and is beneficial to clinical transformation;13–15 (ii) stem cell homing induction factors and the GFs that promote stem cell proliferation and differentiation or other factors that play key roles in organizational integration can also be combined with tissue-engineering-based porous scaffold materials86 to achieve an orderly, coordinated release and can be developed into commercial products for clinical use. The molecular and physical information coded within extracellular matrices is guiding the development of advanced biomimetic scaffolds for robust stem cell homing and tissue regeneration. Several powerful extracellular influences that have been biologically identified have already found their way into cell-instructive scaffolds, while many others remain largely unexploited and are in need of further investigation.90Further synergy between cell biological and biomaterial technologies promises to enable the development of new generations of therapeutic biomaterials that can recruit endogenous stem/progenitor cells and subsequently guide their differentiation to form new tissues.92 Thus, bioscaffold materials with cell recruitment properties have broad application and translational potential. This is not only the focus and key of tissue engineering research but also highlights essential scientific issues that must be addressed in endogenous regenerative medicine.91

Selection and combination of different factors

The factors required for the regeneration of different tissues can vary. Guidance on which factors to deliver and the timing of delivery is emerging from advances in understanding of critical pathways involved in the development of integrated periodontal tissues. The selection and combination of factors are the first important issues to be considered for the in vitro design of biomaterials for in situ periodontal tissue regeneration (Table 4). In terms of instructing stem cell homing, SDF-1 probably requires primary consideration. SDF-1 is a cytokine that belongs to the CXC class chemokine protein, with the system named CXCL12 (CXC chemokine ligand-12). It is the only known natural chemokine in the body that is activated by binding to CXCR4 receptors.93, 94 Recent studies have shown that the SDF-1/CXCR4 complex plays a key role in stem cell homing. Under normal circumstances, SDF-1 is primarily released from bone marrow stromal cells to ensure that the CD34+ HSCs are chemotactically adhered to the bone marrow. Results have shown that intravenous injection of SDF-1α can promote the mobilization of HSCs and that the neutralizing antibody anti-CXCR4 and SDF-1 can inhibit chemotaxis.94–97 HSCs, endothelial progenitor cells, MSCs and the like are capable of expressing the specific receptor CXCR4. The increase in local SDF-1 in damaged tissues is a key factor that regulates stem cell homing and recruitment to injured tissues. Therefore, SDF-1 can serve as an important induction factor for stem cell homing; its physiological function and effect have also been confirmed by many experiments.16, 24, 29, 30, 93–97

Table 4. Potential Cell Homing Factors for Periodontal Tissue and Bone Regeneration

Homing Factors	Target Cells (Determined thus Far)	Ref. (s)
bFGF, basic fibroblast growth factor; BMP, bone morphogenetic protein; PDGF, platelet-derived growth factor; SDF-1, stromal cell-derived factor-1; VEGF, vascular endothelial growth factor.
SDF-1	A wide range of cell types including stem/progenitor cells derived from bone marrow and circulating system	16, 24, 30
bFGF	Cells derived from periodontal ligament and dental pulp	17,100,101
BMP-2	Bone precursor cells including bone marrow-derived cells	21, 29, 30
VEGF	Endothelial cells	106
PDGF-BB	Smooth-muscle cells and pericycles	80, 84

bFGF is a heparin-binding mitogenic protein that can induce mesenchymal cell proliferation and differentiation. It is an important factor in promoting bone and periodontal tissue regeneration,98 which has been confirmed in clinical trials at multiple centers.99 Recent studies have also shown that bFGF stimulates the directed migration of PDL cells via PI3K/AKT signaling and CD44/hyaluronan interaction.100 More recently, bFGF was found to induce recellularization and re-vascularization in endodontically treated human teeth implanted into the dorsum of rats, suggesting that endogenous dental pulp cells, including stem/progenitor cells, may be recruited and subsequently differentiated by the chemotaxis of selective cytokines in the regeneration of dental pulp.101

BMP belongs to the TGF-β superfamily. As an osteogenic GF, BMP-2 is known to strongly stimulate bone formation, mainly by inducing recruitment to bone precursor cells21, 22, 29, 102 and by promoting the angiogenesis and differentiation of MSCs. Results have demonstrated that BMP-2 can achieve complete bone regeneration in bone defect regions and has a strong ability for ectopic bone induction.29, 30, 102 Thus, in the process of new bone formation, BMP-2 is both the inductor of cell recruitment and the stimulating factor that promotes cell differentiation. BMP-7, also known as osteogenic protein (OP)-1, is highly efficient at promoting osteoinductive activity. It can promote the regenerative repair of defects and the creation of hard tissue such as bone, cartilage, dentin, etc. At the same time, it can induce undifferentiated MSCs into bone cells, cartilage cells and cementum.103, 104 In the process of periodontal tissue regeneration, BMP-2 and BMP-7 can play a synergistic role; the former significantly promotes the formation of new bone, while the latter can induce the regeneration of cementum.105

In the process of angiogenesis, different GFs play important roles at different stages. Although vascular endothelial growth factor (VEGF) is the initiating factor for early angiogenesis, VEGF alone cannot form a mature vascular system.91 In contrast, PDGF-BB can recruit smooth muscle cells to promote vascular maturity and stability.106 Results have demonstrated that the controlled release of VEGF and PDGF-BB can effectively promote neovascularization (Figure 6).80, 84

Figure 6. Schematic of the sequential release of vascular endothelial growth factor (VEGF) and platelet-derived growth factor BB (PDGF-BB) for vascularization (modified from Place et al.91).

New blood vessels initially form by endothelial cell organization into tubes with the help of VEGF. However, VEGF alone results in the formation of unstable, leaky vessels. The subsequent release of PDGF-BB helps to recruit smooth muscle cells and pericytes, which favor vessel maturation.

Analysis of data published to date indicates that SDF-1 is the most widely used homing inducer. The combined application of SDF-1 and BMP-2 has become the primary strategy used to induce MSC homing to promote bone regeneration;29 in the bone healing process, bFGF may stimulate the proliferation of early osteoblasts, and BMP-2 can enhance bone mineralization.107, 108 Following periodontal wounding, rhBMP-2 may stimulate cell recruitment by increasing the proliferation and migration of cells from the adjacent unwounded PDL into the wounded area, thus promoting periodontal regeneration by stimulating the formation of new cementum.109 Studies have also shown that the orderly release of BMP-2 and BMP-7 may be coordinated in development and enhances new bone formation,83, 105 and angiogenesis is the synergistic result of VEGF and PDGF-BB. In the process of angiogenesis, bFGF and PDGF-BB can also play a synergistic role in promoting blood vessel maturation110 or in inducing vascular endothelial cells to differentiate into vascular forming cells.111 Given the various roles of key factors in periodontal tissue regeneration at different stages, the combination and screening of different factors are important areas of future research. By addressing the problem of cell recruitment design and the orderly release of multiple factors, we can apply periodontal tissue regeneration scaffold designs to the development of a new generation of biomaterials (Figure 7).

Figure 7. Sequential release of multiple factors from hypothetical material devices designed for periodontal regeneration (medical hypotheses in need of further investigation).

Both the essential growth factors that determine the fate of multiple periodontal tissues and the criteria used to establish the doses demonstrate the molecular complexity of these devices. A few of the challenges facing the field involve improving the pharmacokinetics and biodistribution of the growth factors released and developing tailored products for various pathological situations (SDF-1: stromal-derived factor-1; bFGF: basic fibroblast growth factors; BMP-2/7: bone morphogenetic protein-2/7; VEGF: vascular endothelial growth factor; PDGF-BB: platelet-derived growth factor BB).

Sequential release of multiple factors

Studies have shown that the administration of SDF-1 can significantly extend the release time of drugs, thereby increasing the effect of cell recruitment and ensuring that they safely and effectively perform their biological functions.112–115 After the local or systemic administration of SDF-1α, a problem arises involving its rapid diffusion and degradation by enzymes such as matrix metalloproteinase -2/9 or CD26/dipeptidyl peptidase IV (DPPIV). Hydrolyzed products such as nerve toxin SDF-1 (5-68) may be related to the pathogenesis of dementia.94 Meanwhile, cell-recruiting scaffold material must be designed with the ability to promote stem cell proliferation/differentiation and stimulate the formation of new blood vessels; only then can functional tissue regeneration occur.114, 115 On the other hand, based on an analysis of the wound healing cascade, homing induction factors should function before proliferated and differentiated GFs, while vascular formation should occur during new tissue regeneration. Different factors should execute their functions during specific periods to then effectively increase the biological efficacy of biologically active drugs and reduce possible side effects. Unfortunately, no study has taken all of these factors into consideration thus far; there is also no research involving the remote activation of stem cells in the body to enhance local cell recruitment and thereby promote tissue regeneration (Figure 8). It is, of course, a substantial way to go before such a dream is to be realized, but there is room for hope. In recent years, the systemic administration of SDF-1 has been shown to mobilize and activate the body's stem cell niche,95–97 pointing to an important adjunctive strategy in the design of a future endogenous regeneration strategy that is worth deeper exploration.

Figure 8. Schematic of the systemic delivery of directional cues (e.g., stromal cell-derived factor-1) that instruct stem cells from distant cell niches such as bone marrow to find their way home (illustrations not to scale, modified from Chen et al.15).

Drug delivery system (DDS) composed of various natural and synthetic biocompatible polymers can provide controlled drug delivery by differing mechanisms. One technically simple DDS involved the encapsulation of cues into DDS (e.g., microparticulates), and the subsequent release of cues is programmed according to the design of the delivery vehicles (normally via cue diffusion and polymer degradation). After systemic administration of DDS, the sustained release of cues guides the mobilized stem cells' journey through the blood and to the microvasculature at a target site (normally over distance), where the cells replenish and maintain the cell niche; hence they enhance the regenerative potential of the tissue or directly reach the site of injury and thereafter participate in tissue repair and regeneration.

Consideration of foreign body reaction

Implanted biomedical devices, such as those based on polymeric materials, provoke the body to initiate an inflammatory reaction to the surface of the implanted material. This is known as the foreign body reaction/response (FBR) and is potentially affected by the physical and chemical properties of implanted biomaterials; moreover, it has proven to be a hindrance to the functionality of biomaterials.116, 117 Hence, FBR must be carefully considered in the design of biomaterials for in situ periodontal regeneration. The events in the FBR include protein adsorption, monocyte/macrophage adhesion, macrophage fusion to form foreign body giant cells (FBGCs), consequences of the FBR on biomaterials and cross-talk between macrophages/FBGCs and inflammatory/wound healing cells. The FBR begins as wound healing, which includes the accumulation of exudate at the site of injury, infiltration of inflammatory cells to debride the area and the production of particulates or other foreign bodies that inhibits full healing. The success of implanted medical devices is therefore dependent on the ability to direct this reaction via the material-dependent control of key cellular components, macrophages and FBGCs.118 Macrophages interact with the surfaces of implanted materials and secrete intercellular signals, including cytokines and GFs, which direct the actions of immune cells in the surrounding tissue. Understanding these complex inter-relationships is the key to designing a biomaterial that stimulates regeneration and induces tolerance in tissue engineering applications. It is now clear that the type and quantity of cytokines and GFs produced by macrophages at an implant surface could be indicators of the outcome of the FBR.119 Thus, it can be understood that macrophage adhesion to biomaterial surface is one of the early steps of the inflammatory response to an implanted material, which indicates that a surface that modulates macrophage adhesion may serve to direct the FBR.120 Other variables that affect the host response include manufacturing processes, the rate of scaffold degradation and the presence of cross-species antigens.121 Although inflammation is usually viewed as detrimental, it has unexpected and potentially beneficial effects on fibrosis and transplant rejection. For example, the magnitude of inflammation due to a biomaterial does not determine the extent of fibrosis. Similarly, biomaterials do not always show adjuvancy.122 Although there remains much more to be done, the innate and adaptive immune responses of the host must be controlled to enable the positive function of the implanted biomaterials. The host response is also central to the remodeling that enables biomaterials to be integrated by the host. An in-depth understanding of how the immune system interacts with biomaterials and how biomaterials influence these interactions may prove pivotal to the safety, biocompatibility and function of the device or system under consideration for any medical use.123 There is a long road ahead, but the benefits warrant the effort.

Importance of instructive design

In addition to information transfer between different cell types, cells derive a vast wealth of information from their in vivo milieu, including the biomaterials that surround and separate them within tissues.91 The native ECM surrounded, and produced, by cells is instructive, providing a dynamic and spatially heterogeneous constellation of microstructural, compositional and mechanical cues that can influence cell behavior by binding and clustering cell surface integrins, which act as receptors for triggering signal transduction pathways.42 Implantable biomaterials must take on this instructive role to some degree in order to mimic the natural molecular, mechanical and architectural components of the ECM, and hence, to maintain cell viability and control cell fate toward safe and effective tissue regeneration.91

The ECM provides an important model for biomaterials design. Most ECM components such as collagen, elastin, laminin, fibronectin, glycoproteins and proteoglycans have small bioactive domains for cell binding, proteolytic degradation and/or GF-binding. Usually, from these domains, short peptide sequences can be identified as motifs that are responsible for these biofunctions.124 To this end, bioactive biomaterials have been achieved typically by incorporating short peptide sequences into suitable materials to emulate key molecular features of the ECM either by surface modification or by bulk incorporation, and clues for their design come from naturally bioactive scaffolds such as collagen and fibrin.125 Unlike the entire protein structure, which is subject to denaturation and degradation, short peptide sequences have the advantage of being relatively stable for modification, tunable for cell binding and easy to be synthesized in a large scale. The modification of biomaterials with adhesive peptides can provide biological ligands for cell-scaffold interactions that promote cell adhesion, proliferation, differentiation, osteogenesis and osseointegration, wherein the integrin-binding arginine-glycine-aspartic acid (RGD) sequence found in many ECM proteins, including fibronectin, laminin, collagen type IV, tenascin and thrombospondin is by far the most effective and most often employed peptide sequence for stimulated cell adhesion on synthetic surfaces.126 This is based upon its widespread distribution and use throughout the organism, its ability to address more than one cell adhesion receptor and its biological impact on cell anchoring, behavior and survival.126 Substantial evidence suggests that RGD modification is sufficient to transform artificial construct from a relatively inert biomaterial into a substance that supports the formation of convincing tissue-like structures in vitro and in vivo.127–130 The effects of RGD, however, can depend on the specific application and might diminish during its inevitable interaction with blood and tissue proteins upon implantation. In terms of periodontal and bone regeneration, other adhesive factors such as heparin-sulfate-mediated factors, consensus-heparin-binding motifs, and arginineglycine-aspartate-serine may be more specific for osteoblast adhesion than RGD.131, 132

Besides, the degradation rate of biomaterials represents one of most important considerations and once implanted, it is highly desirable if the degradation rate matches with new tissue regeneration at site of injury.124 Keeping this in mind, synthetic biomaterials have been crosslinked by enzyme-degradable peptide sequences to mimic the invasive characteristics of a native provisional ECM, where a combination of integrin-binding sites and substrates for matrix metalloproteinases (MMP) was required to render the networks degradable and invasive by cells via cell-secreted MMPs, allowing the cells to migrate through the scaffold in a process reminiscent of tissue remodeling.133 Cell-mediated degradation is more likely to generate a biomaterial temporal profile in tune with the generation of new tissue. These biomaterials may be useful in tissue engineering and cell biology as alternatives for naturally occurring ECM-derived materials such as fibrin or collagen.133 Bioactive peptides for molecular engineering of instructive biomaterials can be obtained from biological or chemically synthesized sources, increasing their specific cellular responses for tissue growth and development. Compared to using an entire protein in regenerative therapy, these peptides demonstrate potential advantages such as overcoming possible immunogenicity, being less susceptible to degradation and producing fewer tumor-related side effects.124 The promise of biosynthetic approaches to biomaterials design, however, must be weighed against the fact that very little is known about the in vivo performance of systems prepared in this way.

Although the chemical composition has been the focus of biomaterials design for the past few decades, there is growing appreciation of the importance of other properties (e.g. topological, mechanical and electrical cues) that are important in instructing a biological response effectively for a given tissue; each tissue has its own characteristic mechanical properties that vary over the microscale and influence cellular behavior.134 The cellular response in vitro and in vivo can drastically alter depending on the mechanical properties of biomaterials. On a basic level, the scaffold must usually provide some level of physical support from the moment of implantation, to assist tissue function while new matrix is being deposited.91 Ideally, the scaffold should have mechanical properties consistent with the anatomical site into which it is to be implanted and, from a practical perspective, it must be strong enough to allow surgical handling during implantation. Key roles in molecular pathways are played by adhesion complexes and the actin-myosin cytoskeleton, whose contractile forces are transmitted through transcellular structures.135 Therefore, the mechanical properties of the substrate to which the cells are attached are critical to the regulation of cellular mechanotransduction and subsequent cellular behavior.136 Advances in biomaterials will include extensive development of more sophisticated medical devices with respect to biofunctionality and the expanded application of developed biomaterials into new biomedical fields such as Periodontology. In future, understanding the way in which complex dynamic behaviors are accomplished in nature and rethinking fundamentally the way in which inspiration is drawn from biology may lead to the design of novel biomaterials that mimic nature not through presenting active motifs replicated exactly from biological molecules but rather through reproducing the functional behavior of these biomaterials to obtain properties that are currently unavailable.134, 137

Future perspectives

The development of functional biomaterials/scaffolds for cell delivery is a critical research focus of tissue engineering and regenerative medicine.45, 84, 136, 138–141 However, to date, the majority of cell-based therapies have not yet been applied to patients. Most studies remain at the laboratory stage of research, and their translation to clinical practice faces severe difficulties and challenges.37 Mao and coworkers have recently developed a new path using biofunctionalized biomaterials to instruct host stem cell homing to the site of injury,16–18 which provides a classic example of the recruitment of a patient's own cells for endogenous tissue regeneration.

This strategy promises major improvements and the potential to be adapted to the regeneration of pulp and periodontal tissue.13–16, 142 Not only is there the controversial prospect of the clinical application of current stem cell therapy and tissue engineering technology in the treatment of non-fatal diseases, but there is also a concern over whether high-risk technology that requires significant investment should be applied to treat a disease that can be treated with current alternative methods (such as tooth extraction and dental implants).14 The emergence and development of cell recruitment regeneration technology will surely bring new vitality to regenerative dentistry. Although the mechanisms of current biological signaling molecules and functional biological materials that promote stem cell homing require further study, the results of previous studies indicate that this line of research has certainly become an important strategy for endogenous regenerative medicine.15

The research of Mao and coworkers only combined stem cell homing induction factors with biomaterial scaffolds directly, and the results were only obtained for small animals.15–18 Improvements in the induction of directional migration, recruitment, settlement, differentiation and molding of the patient's own functional cells are still required. Indeed, the formation of new functional tissues in large animals and even in human patients has yet to be demonstrated.15 In further studies in endogenous periodontal regeneration, the optimal combination of factors and dose criteria and the design of cellular scaffolds must be investigated; additionally, these studies should take into account the physical and chemical properties and structural features of the scaffold to achieve the sequential controlled release of multiple factors (Figure 9).86 All of these issues must be concisely integrated into research efforts in the future, with a focus on circumventing each issue one after another.143, 144 Ultimately, clinicians will have commercialized products to aid patients with periodontal disease.

Figure 9. Implantable biomaterials developed in our laboratory for the management of periodontal defects (unpublished data).

(A) An image of a biofunctional scaffold fabricated from glycidyl methacrylate derivatized dextran (Dex-GMA) and gelatin (Gtn), wherein Dex-GMA/Gtn microparticles loaded with growth factors are incorporated into its macroporous pore structures; (B) cutaway view of the highly porous interconnected structures and the microencapsulated growth factors entrapped within the inner pores of the hybrid scaffold.

Conclusions

In summary, the design of biomaterials for cell recruitment, the selection and combination of multiple factors with biomaterials and their dosages and controlled release criteria are important foundations of the new field of functionalized scaffold research in periodontal regenerative medicine. The use of drug delivery methods to enable the coordinated and orderly controlled release of stem cell homing inducers, stem cell proliferation and differentiation factors and new blood vessel formation stimulators from next generation of medical devices are expected to allow robust mobilization and homing of resident stem cells to periodontal defect areas, where the coordinated interactions with various signaling molecules, other cells and their artificial microenvironments may define a local biochemical and mechanical niche with complex and dynamic regulation that leads to safe and effective regeneration of multiple tooth-supporting tissues. Research on the design of new biofunctionalized scaffolds will provide an effective basis for the study of endogenous regeneration of various tissues and in vivo tissue engineering. Furthermore, it will provide new treatments for the reconstruction of complex tissues and the structure and function of physiological organs, which has important clinical significance and wide application potential.

Acknowledgements

Fundings from the National Natural Science Foundation of China (81071253/H1818; 31170912/C1002) for some of the work mentioned in the review is gratefully appreciated.