Advanced technologies in periodontal tissue regeneration based on stem cells: Current status and future perspectives.

Periodontitis is a progressive inflammation disease, the clinical management of which remains a challenge. The traditional management may control periodontal inflammation, but failed to regenerate functional periodontium. This review summarizes the most advancing regenerative techniques regarding stem cell culture and scaffold fabrication, such as cell sheeting, spheroid culture, electrospinning and 3D printing. The applications of different techniques manifest tremendous potential of regenerating the complete and functional periodontium. Albeit promising, new technologies have met with their own drawbacks such as insufficient vascularization and precision, which necessitate deeper modification. Thus, this review also points out the potential perspectives and methods aiming at their disadvantages, illuminating the directions of future researches to successful clinical scenarios.

Introduction

Chronic periodontitis is a sort of prevalent disease characterized by the nonreversible chronic inflammatory destruction of the periodontium, which eventually leads to the premature tooth loss. The morbidity of chronic periodontitis ranges from 30% to 40% in individuals over 35 years old, which makes periodontitis a global concern in oral diseases. Therefore, the proper treatments remain an urgent public health issue (see Table 1).

Table 1

The comparison of different technologies in advantages and disadvantages.

	Advantages	Disadvantages
Cell sheeting	Minimalize cell loss	Costly
	Keep cell viability	Longer culture period
	Lessen damage to function	Attach weakly to hard tissue
		Insufficient vascularization
Spheroid culture	Similar microenvironment	Spheroid size decrease
	Producing more ECM Enhanced stemness	Lack of oxygen and nutrient at the core
Electrospinning	Similarity with natural ECM	Hinder cell infiltration
	Larger surfaceGuide fiber directions	Difficulties in controlling the degradation rate
3D printing	Custom-made scaffolds	High temperature
	Maintain great accuracy	Limited medical application
	Enable specific functions

The purpose of periodontal treatment is to suppress the inflammation and regenerate functional periodontium, including the cementum, alveolar bone, and the crucial inserted periodontal ligament. Some approaches have been clinically implemented to achieve better periodontal regeneration, such as GBR, bone grafting, and the localized delivery of growth factors. These techniques have shown some improvement in periodontal regeneration in intra-bony defects, but a lack of predictability in cases of advanced ones. Since the isolation of PDLSC(periodontal ligament stem cell) from the extracted third molar, researches based on stem cell in periodontal regeneration have surged exponentially. In light of the low efficacy in traditional tissue engineering strategies, a series of new technologies have been established. In this review, we mainly focus on advanced technologies in periodontal tissue engineering with regard to stem cell culture and scaffold fabrication and shed a light on their drawbacks and the relevant future perspectives to be put into clinical use.

Traditional strategies

Periodontal tissue engineering, by definition, incorporates culture-expanded progenitor cells into prefabricated scaffold, which is then transplanted into the defect sites to realize periodontal regeneration. The traditional tissue engineering method is to inject the cells or build a composite cell-material structure in vitro, then transplanting into the defect site.

Cell injection

Mesenchymal stem cells, a crucial element in tissue engineering, are capable of performing self-renewal, differentiating into various cell lines and modulating immune response by secreting anti-inflammatory factors. The traditional cellular therapy utilizes single-cell suspension from the isolated cells in vitro to directly inject into the damaged site. Compared with cell-material design, cell injection is less invasive for no flaps and suitable for those with systemic diseases and not in the stage of severe periodontitis. The bone marrow mesenchymal stem cell injection in a rat model exerts immunomodulatory effects at the defect sites and contributed to the periodontal repair. But the drawbacks lie in the cell loss, poor engraftment, inadequate localization, spreading into surrounding tissues and loss of cell fate control.

Cell combined with materials

To limit the localization of stem cells and reduce cell loss, cells are transplanted with various materials. The most commonly used stem cells are periodontal ligament stem cells (PDLSCs), and others are also available including stem cells exfoliated deciduous tissue (SHED), dental pulp stem cells(DPSC) and bone marrow mesenchymal stem cells(BMMSCs) for periodontal regeneration. Among the most frequently used scaffold material are natural origin biomaterials like chitosan, collagen, fibrin sealants and synthetic polymers like hydroxyapatite/tricalcium phosphate(HA/TCP). Lemaitre grafted adipose-derived stromal cells with collagen scaffold, and eventually the cementum regeneration took shape as well as the organization of PDL fiber in a mouse model.

Supported by the safeness of the stem cells and the simplicity in operation, it has potential with respect to clinical use. Yet there exists no histological evidence that functional periodontal complex can be formed with this method and it necessitates invasive surgery.

New tissue engineering technologies

As material, bioengineering, and regenerative medicine improves by leaps and bounds, a lot of new technologies in tissue engineering are emerging nowadays, giving the field of periodontal regeneration a boost. Herein, we reviewed the new technologies in cell culture and scaffold manufacturing.

New technologies in cell culture

The stem cell culture has always been a hurdle in regenerative medicine because one of the critical terms is to keep such features as stemness and differentiation capacities. Obstacles are met in integrally maintaining characteristics in vivo by mimicking microenvironment. Thus, various techniques have surfaced such as cell sheeting and spheroid culture.

Cell sheeting

As an alternative to cell suspension, cell sheeting is an advanced technology in cell culture, which equals scaffold-free cell delivery. When cells are cultured, they develop cellular junction and extracellular matrix (ECM). In this context, confluent cultured stem cells can be harvested from the culture dish without any enzyme in monolayer or multilayers together with intact ECM and cellular junction as a whole(Fig. 1A). Cell sheets can attach to the adjacent one, piling in layers to fabricate thick tissues and 3D structures. Compared to the above-mentioned techniques, the complete preservation of extracellular matrix and cell-to-cell junction without enzymes such as trypsin minimalize the cell loss as well as the cell viability and lessen damage to function.

Figure 1

The principle of cell sheeting and electrospinning. (A) Cells attach to the surface at 37 °C, and detach from it at temperature lower than 32 °C. (B) Under electrical field, polymer deposits into electrospinning fibers. (C)The scheme of pellet culture and liquid overlay in spheroid culture. (D) Extrusion-based techniques in 3D printing. Adapted from References,,, with permission.

A type of temperature-responsive polymer named N-isopropylacrylamide was developed to form cell sheets. Attached onto the dish surface, it is dehydrated and hydrophobic at 37 °C, which cells favor and expand on. But at temperature lower than 32 °C, it is hydrated and hydrophilic, yielding cell sheets with complete ECM. In addition, other methods are also available based on magnetic force and polyelectrolytes.

Hu et al. transplanted human dental pulp stem cells(hDPSC) using cell injection or cell sheeting respectively, both of which significantly facilitated regenerated bone whereas hDPSC sheets group showed more bone regeneration capacity than hDPSC injection. In many researches, stem cell sheets are combined with bone substitutes to stabilize stem cells within defects like tricalcium phosphate, hydroxyapatite, and hydrogel. Iwata et al. delivered multilayered PDL cell sheets to the nude dentin supported with woven polyglycolic acid(PGA) in the middle and osteo-inductive material porous β-tricalcium phosphate in the bone defect. This structure successfully facilitate both cementum and bone regeneration linked to well-oriented periodontal fibers. Chen et al. used autologous PDLSCs sheets combined with Bio-oss in a randomized clinical trial but the results showed no statistical difference, which may owe to the restricted validity of the cells from the biomaterials. Hence further researches should be implemented to better build structure between cell sheets and materials.

Regarding the drawbacks of cell sheeting, it is costly and require longer culture period as it will be fragile when not confluent enough. Besides, cell sheets attach weakly on such hard tissues as the root surface. As mentioned, combining cell sheets with materials is at play but we should also assure no limitation of cell viability simultaneously. And cells may necrotize if the sheets are too thick and lacking in vascularization. Of late some researchers have focused on pre-vascularization by coculturing endothelial cells in multilayer sheets. Better approaches should be developed to promote the combination of cell sheets and various materials to allow supreme attachment and pre-vascularization.

Spheroid culture technique

Apart from the aforementioned cell sheeting technology, multiple cell culture techniques have been developed. Different from 2D culture of monolayer, 3D culture reconstitute in vivo environment, including cell–cell and cell-ECM interactive networks. Spheroid culture, an emerging and promising method, works by cell aggregates and self-assembly. Compared to cell suspension or cell sheeting, spheroid cells are capable of producing ECM and other growth factors at a higher level.

The principles of spheroids depend on the culture microenvironment wherein cell–cell adhesion outweigh cell-material adhesion. In periodontal tissue engineering, various spheroid forming methods have been established such as hanging drop, pellet culture, membrane-based aggregation and microwell chips approach(Fig. 1B). Pellet culture utilizes centrifugal force, and hanging drop utilizes surface tension and gravitational forces. Microwell chips can better control spheroid size by changing the size of microwell scale. Spheroids of hPDLMSCs (human periodontal ligament mesenchymal stem cells) were formed using microwell chips, showing greater stemness and enhanced osteogenic potential, which may be beneficial for hard tissue repair. Iwasaki et al. utilized pellet culture in polypropylene tubes to form PDLSC spheroids, which upregulated the anti-inflammation and angiogenesis expression, promoting periodontal regeneration theoretically. Yet it showed a failure of periodontal regeneration probably owing to the dead cells contained in the central necrotic zone.

To conclude, spheroid culture has various advantages associated with the similarity in vivo microenvironment, such as the enhanced stemness and osteogenic potential as well as the immunomodulatory effects, making it promising conditioned medium for periodontal regeneration. However, the spheroid size shows a time-dependent decrease probably due to the smaller cell size in spheroids, the reduced proliferating cells and the increased death cells. Lack of oxygen and nutrients at the core of spheroids accounts for the limitations of spheroid culture. Hence, further researches should be established to illustrate the optimization of cell size and number.

New technologies in creating multiphasic scaffold

The regeneration of the periodontal complex is in need of coordinated process among each individual component. Inspired from the anatomical structure of the periodontium, multiphasic scaffolds are comprised of hierarchical structures to facilitate regeneration by managing a series of complicated spatiotemporal interactions occurring in periodontal remodeling procedure.

Electrospinning

Electrospinning mimics natural ECM to provide suitable environment for cell development, standing out as a universal micro to nanofiber manufacturing technique. A typical machine consists of a syringe that carries polymer solution or the polymer melt, a pump to pulse the polymer, a source of electrical field and a collector plate. Under the electrical difference between the syringe and the collector, polymer deposits in the form of micro to nanofibers(Fig. 1C). Solution electrospinning fibers are inorganized nanofibers with smaller pores, while melt electrospinning fibers are organized microfibers with larger pores.

Due to the similarity with natural ECM, electrospinning scaffolds favors cell adhesion, proliferation, migration and differentiation. Electrospinning, a resourceful and cost-effective way to fabricate nanofiber, has the advantages of larger surfaces that can improve protein interaction and offer binding sites. For tissue engineering, the electrospinning fibers, whether in random or in alignment, are capable of guiding the growth direction of fibers or cells topographically, benefiting the specifically oriented tissues such as the hierarchical periodontal fibers.

Promising as electrospinning is, there exist a few limitations standing in the way in the further progress in periodontal regeneration. For solution electrospinning fibers, the smaller fiber diameter is, the bigger superficial area to volume ratio will be, which supplies more protein and molecule binding sites but may exert detrimental effects on the cell infiltration due to the reduced pore sizes. Hence, it is of great significance to control the different pore sizes in accordance with various structures. The drawbacks also lies in the difficulties in controlling the degradation rate synchronizing with the periodontal tissue remodeling. If the degradation begins ahead of tissue maturation, the scaffold may collapse and 3D network vanish, hindering the growth and remodeling of the fiber.

3D printing

3D printing, also acknowledged as rapid prototyping or additive manufacturing, emerges as a pioneering technique in tissue engineering.

In the context, scaffolds are produced in a layer-by-layer manner compared with subtractive manufacturing. Bioprinting takes advantage of cell-laden bioinks to yield functional tissue and organ, wherein cells are embedded in matrix. Nowadays the bioprinting technologies involve stereolithography, inkjet, laser-induced forward transfer(LIFT) and extrusion. Among these techniques, extrusion bioprinting is a common way for fabricating scaffolds in periodontal regeneration. In most cases, controlled extrusion is driven by the piston, screw or pneumatic system in the form of filament or struct from the printer head to the collector(Fig. 1D). As a common example of this technique, fused deposition modeling utilizes thermoplastic polymer melted at the nozzle at elevated temperature, empowering the deposition of semi-liquid struct above the collector. The frequently-used materials for this technique include polycarbonate (PC), acrylonitrile butadiene styrene (ABS), polycaprolactone (PCL), and polylactic acid(PLA) owing to the relatively low melting points.

3D bioprinting, relying on data from computed tomography in digital imaging system, makes it easy to fabricate custom-made scaffolds, maintain great accuracy in details and enables specific functions. Regarding FDM, it has the advantages of low-cost, high speed printing due to the simultaneous printing from multi-nozzle. Yet the high temperature eliminates FDM as an alternative for biomedical applications.

Application of multiphasic scaffolds

Multiphasic scaffolds are fabricated in many manners to show optimized spatiotemporally control over the periodontal regeneration process.

Biphasic scaffolds with cell sheets

Some studies combined the cell sheeting with biphasic scaffolds. Vaquette et al. fabricated a biphasic scaffold, composed of a 3D printed PCL scaffold for bone area via fused deposition modeling with osteoblast culture and a solution electrospinning membrane for the PDL compartment integrated with multiple PDL cell sheets. Applied on the dentin block, scaffolds gained better attachment of cell sheets to the dentin surface, owing to the thin mineralized tissue. But the periodontal fibers remained mainly parallel to the dentin surface. One reason may be that the solution electrospinning membrane had small pores partially hindering the further insertion of new periodontal ligament fibers into the bone. Another reason may be lack of fiber guiding structure.

Hence, following on this study, Costa et al. applied a calcium phosphate layer in the bone area to increase the osteo-conductivity and a melt electrospinning membrane with larger pore sizes to allow the attachment of functionally oriented periodontal ligament.

Biphasic scaffolds

Park et al. developed compartmentalized biphasic scaffolds with fiber-guiding properties via computer-aided manufacturing and 3D wax printing. They fabricated polycaprolactone (PCL) bone region and polyglycolic acid (PGA) PDL region respectively with human dentin slices before subcutaneous implantation. After the cell-seeding process into the ectopic mice model, this biphasic scaffold successfully demonstrated bone and periodontal ligament regeneration along with the formation of parallel and obliquely oriented fiber.

However, the periodontal model was geometrically highly standardized. To enhance adaptability, Park et al. designed scaffolds according to micro-computed tomographic imaging of the defects. This time, biphasic scaffolds were formed with perpendicularly oriented microchannels by casting a PCL solution in the mold. Finally, the microchannels enhanced the controlled orientation of fiber. Based on the image-based and fiber-guidance system, they advocated the manufacture of customized scaffolds by 3D printing.

Triphasic scaffolds

As a further evolution of biphasic scaffolds, a triphasic scaffold surfaced via fused deposition modeling. Different sizes of microchannels are fabricated for each region. Phase A, the cementum–dentin interface, had 100-μm channels; phase B, the PDL compartment, had 600-μm channels; phase C, the bone region, had 300-μm channels. By combining spatially released bioactive cues, oblique fibers were yielded and inserted into the newly formed bone and cementum. Albeit promising, one of the limitations may be the stiffness of PCL scaffolds, making it difficult to adapt into the complex anatomical structures. And it's also unclear how the cementum component can attach onto the root surface regarding clinical applications.

Clinical trials

Clinically, Rasperini et al. reported the PCL scaffold via selective laser sintering (SLS) to treat periodontal defect. A customized scaffold was fabricated by a computed tomography scan of the periodontal defect. In the internal region, there are ports for delivering growth factors and pegs perpendicular to the root surfaces, while the external region would resorb over time. However, the constructs became exposed 13 months post-implantation, leading to a failure from a clinical perspective. The exposure may result from the slow-degrading and stiff bulky polymer that created misfit in the soft tissue. In another study, Vaquette et al. designed PCL melt electrospinning scaffolds for the bone region with PCL solution electrospun membrane for PDL region. The melt electrospinning scaffold made it possible for a construct of macropore size with aligned microfibers and lower stiffness than the previous study, which ultimately regenerated newly formed obliquely periodontal ligament fibers and successful insertion. Thus, it is presumed that the changes in porosity and stiffness contribute to the integrity of soft tissue and reduces the risk of exposure.

Prospects and conclusions

In the aforementioned researches, the fiber guiding features lacked consistency and regularity. Of late, studies related to micropattern and microgroove of scaffolds have mounted up. To enhance control over fiber orientation, Pilipchuck et al. designed PCL periodontal ligament scaffolds where organized grooved pillars in various sizes can be seen in a 3D patterned PCL thin film. Park et al. utilized high resolution printers to produce micro-grooved molds via digital slicing process with parallel, oblique and perpendicular orientations. The assessment in vitro presented high efficacy in aligning human PDL cells.

In brief, there still exist insufficient accuracy in terms of the multiphasic scaffolds. Even though micro-to nano-structures can be manufactured, the whole size of scaffolds reaches up to millimeter scale, marking a striking gap between the scaffold and the natural periodontium. Additionally, further endeavors should be made in designing biomimetic angulations and precise microstructures in the scaffolds to mimic the natural periodontal ligament anatomical structure.

Conclusion

Undoubtedly, tissue engineering based on stem cells have made evident progress in both cell culture and scaffold fabrication. But it's still not sufficient to put into clinical scenarios. Of necessity is the optimization of cell culture methods and related standard protocol as a whole. Multiphasic scaffolds should be improved in accuracy and adaptation regarding the angulation and three-dimension formation of micro-structures. The limitation also lies in the scarce source of stem cells as well as ethic and safe issues when using allogenic stem cells. Currently, the traditional clinical managements are still the options of most cases. It's concluded that stem-cell based tissue engineering warrants further researches and investigations in an effort for clinical use.

Declaration of competing interest

The authors have no conflicts of interest related to this article.