Engineered scaffolds and cell-based therapy for periodontal regeneration

Bioceramics

HA, structurally similar to bone apatite, is the most commonly used bioceramic for periodontal engineering, but its slow resorbability and subsequent replacement by new bone may delay periodontal regeneration (14). Recently, HA has been proposed not only for bone regeneration, but also for soft tissue augmentation, and its resorption, in particular, has been fine-tuned by modulating size and crystallinity degree (15). In 1- and 2-wall periodontal defects, HA resulted in limited periodontal regeneration at 6 months, with long junctional epithelium, new cementum formation and fibrous tissue response around HA particles (16). HA has been used in association with autografts to regenerate extensive periodontal defects: at 9 weeks, HA particles were encapsulated in fibrous tissue, and newly formed bone was found just around autograft residues (16).

Bicalcium phosphate is a precursor of HA, which, in combination with enamel matrix derivative (EMD) within periodontal defects, resulted in the formation of newly deposited cementum and inserting fibers, although HA was still encapsulated, with negligible evidence of new bone around it (16).

Among bioceramics, however, the most commonly explored compound is β-tricalcium phosphate (β-TCP), which is completely resorbed within 6-9 months from implantation and substituted with new bone, as demonstrated even in wide bone defects (14). During resorption, β-TCP provides calcium, magnesium and phosphate ions at the ideal ion concentration for the activation of alkaline phosphatase, a pivotal enzyme for the ossification process. β-TCP was investigated to treat 1- to 2-wall periodontal defects: after 6-18 months, it produced long junctional epithelium and, in a few cases, periodontal regeneration with weak signs of cementogenesis and osteogenesis (16). A tunnel-structured β-TCP material has recently been developed to enhance, in particular, bone deposition and it was tested in 1-wall intrabony defects in dogs. At 12 weeks, after surgery, newly formed bone, PDL and cementum-like tissue were observed; new bone and vessels were observed within tunnels (17).

To enhance its regenerative properties, β-TCP has been used in combination with growth factors. β-TCP added to platelet-derived growth factor (PDGF) showed a long junctional epithelium healing (16). Similarly, β-TCP particles were coated with growth differentiation factor-5 to treat intrabony defects, enhancing cementum, bone and PDL regeneration, but with no evidence of ankyloses or root resorption, although particles produced a foreign body reaction (16).

In a further implementation, β-TCP was combined with HA to provide the bioactive core structure with fast degradation giving biphasic calcium phosphate (BCP). BCP showed a tunable degradation rate by adjusting the ratio of HA and β-TCP phases. Clinical improvements in treating periodontal bone defects have been reported only in combination with EMD (18).

Another class of bioceramics includes bioglasses or bioactive glasses (BGCs), brittle materials composed of SiO₂, CaO and P₂O_5, not suitable for load bearing applications, but with high osteoconductive properties, very slow resorption and the interesting capacity to bond directly to soft and hard tissues (14). In the treatment of 1-, 2- or 3-wall defects, BGCs promoted healing with long junctional epithelium (16). Similar findings were reported for grade II furcation defects (19). When used in combination with EMD, instead, BGCs showed signs of periodontal regeneration with mineralized tissue around them (16).

Finally, coral-derived bioceramics, in the form of coralline HA and calcium carbonate, have been investigated as osteoconductive scaffolds, which are degradable by means of carbonic anhydrases of osteoblasts. In a dog model of 1-wall intrabony defects, they produced the same periodontal healing as the control site receiving autograft bone and well-organized PDL fibers were connected to neodeposited alveolar bone, with evidence of cementum regeneration (20). In humans, coralline HA was investigated in 1 clinical trial performed to treat 2-3 osseous wall intrabony defects, and although the type of healing was not clarified, bone formation was visible around graft particles (16).

Natural polymers

Naturally occurring degradable polysaccharides are commonly used in surgical reconstructions and in periodontal tissue engineering (11). They are derived from algae, as sodium alginate and agarose (21), from animals, as chitosan (22) or from polypeptides, as collagen and hyaluronic acid (16, 23). Recently, a biomimetic electrospun matrix in silk fibroin nanofiber was successfully proposed for oral mucosa repair, as evaluated in a rat model, revealing a promising alternative to acellular dermal matrix (24).

Collagen represents one of the most widely applied natural polymers, and in humans, it is the core protein of extracellular matrix found in several connective tissues. Collagen is thus the most abundant polypeptide in mammals and, as a biomaterial in periodontics, has largely been applied in the form of a biodegradable barrier during GTR. Collagen barriers allow periodontal regeneration (with defect resolution, new cementum and new inserting fibers), without leaving material residues 5 months after surgery; instead, no coronal bone overgrowth was reported (16). Collagen scaffold, as hydrogel, also facilitates periodontal wound healing (18), but to date, evidence of periodontal regeneration is limited (16).

Chitosan, a polysaccharide formed from D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit), is a natural biomaterial able to induce antiviral activity and innate immunity in plants (25). It is a linear polysaccharide, made by treating the chitin shells of crustaceans with alkaline agents. Chitosan has recently been introduced in periodontal engineering for its biocompatibility, antibacterial and mucoadhesive properties, in the form of gel or freeze-dried scaffolds to enhance alveolar bone regeneration (26, 27). A further application is in the form of a biodegradable membrane for GTR) and for guided bone regeneration (22).

Chitosan scaffold, as hydrogel, was reported to lead to periodontal healing within intrabony defects (26). Yeo et al, in particular, found that chitosan porous membrane stimulated new deposition of bone and cementum within 1-wall intrabony defects, in beagle dogs (28).

One major concern is related to chitosan sterilization, since autoclaving showed a reduction in molecular weight, viscosity and gelification. Thus, to overcome this issue, a thermosensitive chitosan hydrogel, proposed as an injectable scaffold, was produced via autoclave treatment and β-glycerophosphate, resulting in suitable physicochemical and biocompatible properties, with evidence of periodontal regeneration in class III furcation defects in dogs (29).

Chitosan and collagen have been used in combination to form sponge-like scaffolds, used to treat 1-wall intrabony defects in dogs (30). Histology found that scaffolds enhanced the formation of bone and cementum (30).

Less frequently, alginate (a natural polysaccharide) and gelatin (a natural polypeptide) have been investigated, mainly as GTR or guided bone regeneration barriers rather than as scaffolds for periodontal regeneration. In vivo evidence of their efficacy in this field remains limited (31, 32).

Some interesting results can be found using gelatin combined with platelet-rich plasma (PRP) and blood clots, revealing successful results in bone regeneration and probing depth (PD) reduction (33, 34). Gelatin was used to obtain a “periodontal inspired” scaffold, synthesized via the freeze-casting technique (35). Despite having low resistance to compression, gelatin possesses important biological properties; among others, gelatin contains tripeptide Arg-Gly-Asp (RGD) motifs which mediate cell attachment (35). Composite PCL/gelatin nanofibrous scaffolds were electrospun to obtain a directionally oriented nanofiber membrane able to drive PDL stem cells (PDLSCs) stem cells under mechanical-stress conditions (36). In rat premaxilla periodontal defect models, these scaffolds were successfully integrated into the defect, enhancing bone formation (36).

Scaffold multicompartmentalization and micropatterning

Compartmentalization allows controlling of the spatiotemporal biological processes, which result in effective regeneration of the periodontal apparatus, preventing tooth ankylosis, and enhancing deposition of bone and of PDL fibers directed in a functional manner. Indeed, RPT aims at a synchronized reestablishment of both soft and hard tissues. Thus a successful approach putatively includes spatial organization achieved by multicompartmental (or multilayer or multiphasic) 3D scaffolds. Here, the architecture and the chemical composition of each compartment match the organization and the cellular, mechanical and biochemical composition of those tissues to be regenerated (27, 39). Mechanobiology and the contact guidance concepts support the rationale underlying this approach, since cells can be guided by mechanical stress during their migration and differentiation. Nonetheless, a macroporous architecture with channels promoting transport phenomena during the delivery of nutrients and in the removal of waste products to and from the interior of cell-populated 3D scaffolds, may promote cell growth (40).

Gingiva regeneration has rarely been investigated through a multilayer approach. Lotfi et al developed a bilayer scaffold able to achieve gingival augmentation in dogs lacking keratinized gingiva (41). It consisted of a first dense layer made of a mixture of chitosan and polyvinyl alcohol, able to increase the scaffold strength and to suture the scaffold to its adjacent tissue, and of a second layer with a sponge-like structure made of chitosan (41).

Most researchers have attempted especially to restore the physiological bone-PDL architecture by promoting ligament integration with surrounding tissues. Microchanneled scaffolds for PDL regeneration, in particular, have been investigated, following the concept of contact guidance, where the cells adherent to the substrate follow its morphology (Fig. 2).

In the literature there are proposals to use several different types of micropatterned PCL structures. Park and colleagues developed a 3D printed PCL scaffold with a microchanneled structure that guided PDL fiber orientation (42-43-44). In this work, the fiber-guiding PCL compartment for PDL was computer-designed using 3D printing, although the same group lately proposed using a freeze-casting method to control pore architecture, mimicking the topographies of alveolar bone and PDL fibers (45). The same fiber-guiding PCL scaffold was further enriched with a compartment for delivery of recombinant human PDGF, and then used for filling the human periodontal osseous defect, without acute signs of chronic inflammation or dehiscence (46). This remained covered until the 12-month follow-up visit, showing a 3-mm gain of CAL and partial root coverage; after 13 months, however, the scaffold became exposed, and ultimately it had to be removed altogether (46).

Vaquette et al proposed a bilayer construct supporting cell sheets: It incorporated bone and PDL compartments made of PCL containing β-TCP (47). They demonstrated, in a rat periodontal ectopic model, the successful, simultaneous and spatially controlled in situ localization of those cells required for regenerating bone, PDL and cementum (47).

Recently, a bilayer scaffold composed of PCL electrospun membrane (to mimic PDL) and a chitosan-based construct (to mimic bone tissue) was developed, reporting in vitro differentiation of fibroblast and osteoblasts, respectively (48). A similar biphasic scaffold, used in combination with cell sheets, was developed by adding calcium phosphate to the bone compartment and by substituting the electrospun membrane for PDL with a thin melt electrospun scaffold with larger pores to enhance cellular interaction and neo-vascularization (49). In vivo, the revised construct produced functional PDL orientation (49).

Along the same lines, Lee et al investigated multicompartment PCL/HA scaffolds fabricated using 3D printing: it possessed a 100-μm microchannel compartment to regenerate the interface between cementum and dentin, a 600-μm microchannel compartment was designed for PDL, while a 300-μm microchannel compartment was intended for bone regeneration (50). The scaffold was used to support the adhesion of stem cells in combination with the delivery of 3 proteins (connective tissue growth factor, amelogenin and bone morphogenetic protein-2 [BMP-2]). In vivo results showed formation of oriented PDL fibers, which were inserted into bone, and dentin/cementum-like tissues (50).

The bone–PDL ligament interface has represented a further challenging aspect of periodontal regeneration. PLGA-based scaffolds were proposed for its regeneration. A semirigid PLGA/CaP bilayered construct had externally a membrane acting as barrier and helping to avoid collapse, while inside, a micropatterned compartment retained the blood clot. This was successfully tested in dog class II furcation defects (51). More recently, a bilayered system for supporting the adhesion of PDLSCs was made in the form of a platelet lysate/PLGA-based construct (52). In a rat 3-wall intrabony defect, the scaffold favored periodontal regeneration, independently from the seeding of PDLSCs, with significant connective tissue attachment formation and bone deposition (52).

Trilayered scaffolds intended to support the simultaneous regeneration of cementum, alveolar bone and PDL have been developed, although in most cases only animal evidence has been provided. A porous hydrogel was made with 3 compartments and enriched with growth factors as follows: (i) chitin-PLGA/nanobioactive glass ceramic (nBGC), loaded with cementum protein 1 (CEMP1), for the cementum regeneration; (ii) chitin-PLGA, loaded with fibroblast growth factor 2, for the PDL regeneration and (iii) chitin-PLGA/nBGC, loaded with PRP-derived growth factors, for the alveolar bone regeneration (53). After 1 and 3 months from implantation in periodontal defects in rabbits, the trilayered scaffold demonstrated complete periodontal healing and bone neodeposition, as evaluated with micro-CT and histology (53).

Furthermore, Varoni et al developed a micropatterned chitosan-based trilayer scaffold (27). It exploited different molecular weights of chitosan (low vs. medium) to obtain a bilayer structure for gingiva and alveolar bone healing. This was then assembled with a further layer intended for PDL, obtained by electrochemical deposition of the same polymer and showing an highly oriented micropatterning (27). The latter, having 450-µm-large pores, had been tested previously to provide evidence of enhanced neovascularization within the scaffold (10).

Scaffold 3D manufacturing

In recent decades, 3D printing has received a great deal of attention for the manufacture of customized scaffolds with fine-tuned architecture mimicking periodontal multitissue complexes and accurately adapting to the shape of patient defects. Three-dimensional printing overcomes the high heterogeneity of scaffold porosity and architecture related to conventional methods of synthesis, such as particle leaching, freeze-drying and solution casting (54), and enables personalized medicine intended to produce a scaffold that fits the individual periodontal defect.

The revolutionary introduction of computer-aided design (CAD) and computer-assisted manufacturing (CAM) have allowed the development of solid freeform fabrication techniques, referred to as rapid prototyping (RP). RP makes it possible to obtain, in a highly reproducible manner, complex scaffolds composed of both natural and synthetic biomaterials, with precise external and internal 3D architecture. Solid freeform fabrication includes additive manufacturing technologies, producing scaffolds layer by layer via 3D printing. Among others, these include laser-assisted printing (such as stereolithography and selective laser sintering), inkjet printing and extrusion printing (fused deposition modeling) (55). When the process involves not only scaffolds (usually hydrogels), but also the contextual deposition of living cells, the technique is called bioprinting, and it allows cell encapsulation and cell-based therapies with a high degree of control of the position of cells within the scaffold. Furthermore, it mimics the tissue interface and the surrounding microenvironment.

In a clinical scenario, CAD models for 3D printed scaffolds will be based on images taken from radiographs of a patient-specific bone defect, particularly using CBCT in periodontal regeneration. Recent studies have demonstrated that CBCT can be applied in periodontology to determine the size and shape of the periodontal defect and then fabricate a scaffold, which is tailored to the specific patient. In periodontics, CBCT has been proposed for both diagnosis and treatment planning, as well as evaluation of treatment outcomes. Several in vitro and in vivo studies have shown that CBCT is a much more sensitive diagnostic tool than intraoral radiography for the detection of periodontal defects (56-57-58-59). A minimum voxel size of 0.150 mm³ seems to be required for the detection of periodontal defects (57). A recent systematic review (60) looked at CBCT utility in periodontology (from diagnostics to patient management) and found it provides the most accurate assessment of vertical bony defects; however, the recommendation is to use it on a case-by-case basis. Authors of that review discourage routine use because of the high irradiation risk and not always favorable cost-benefit balance. According to the review, furcation-involved second maxillary molars stand to benefit the most from CBCT to evaluate the degree of furcation involvement, as intraoral radiographs often fall short in this area (60).

CBCT has even been applied to assess bone level changes following RPT. It showed the potential to replace surgical reentry as the technique of choice for assessing therapy outcomes (61). Regarding geometric accuracy, a systematic review (62) has reported mean measurement errors ranging from 0.19 ± 0.11 mm to 1.27 ± 1.43 mm, with no consistency among studies regarding whether the deviation is due to overestimation or underestimation of the defect. A difference in measurement error depending on the position of the defect has also been reported, with vestibular sites having a greater measurement error (62). The authors of that review were unable to perform a meta-analysis due to incomplete information, and they warned that given the high heterogeneity between studies, the results cannot currently be used as the basis for any recommendation of the use of CBCT for assessment of periodontal defect geometry when high precision is needed (62).

The field of CBCT imaging is changing quickly, and while this imaging modality is a good diagnostic tool for the detection of periodontal defects, higher radiation doses and measurement errors can still pose a challenge for its widespread use. More up-to-date research is needed, as studies regarding accuracy performed using earlier generation devices may not apply to the current technology.

Custom-made scaffolds for periodontal regeneration, however, are possibly not far from being produced. As described in the previous paragraphs, 3D RP was recently applied to synthesize multicompartmentalized PCL-based scaffolds having a PDL fiber-guiding side and custom made on periodontal defects (47, 49, 50, 63), although this was unsuccessful in clinical application (46, 50). Authors suggest that 3D printing’s low resolution and the very slow degradation rate of PCL, as well as low cell affinity and low osteoconductivity, may explain the adverse outcomes. Current RP techniques, indeed, are not always able to control precisely the overall geometrical design and porosity – depending on the machine’s resolution and material repertoire. The combination of RP with other fabrication methods, such as electrospinning, may allow more and more efficient constructions. In addition, despite the addition of bioceramics in the construct modulating synthetic polymers’ biodegradation, the amount of HA, in these reports, might not have been sufficient to accelerate the PCL degradation profile (35). Furthermore, the biological variations between species (rodents and humans) may have further hampered the straightforward transfer of findings from preclinical to clinical trials (35).

Cell sheet technology

Poly(N-isopropylacrylamide) (PIPAAm) is a thermoresponsive synthetic polymer, which allows cell adhesion or detachment from the surface, according to temperature of the environment (78). At 37°C, a PIPAAm surface is slightly hydrophobic, enabling cell adhesion and proliferation; under 32°C, the PIPAAm surface becomes hydrophilic, where cells, forming a sheet, can easily detach. This process allows harvesting of the cell sheets, avoiding proteolytic enzymes (such as trypsin), thus preserving the structure of cell membrane proteins which mediate adhesion, mainly laminin 5 and E-cadherin. Cell sheet technology, with PDLSCs and human umbilical vein endothelial cells (HUVECs), was successfully tested for periodontal regeneration in an ectopic periodontal model in nude mice (79). A coculture of PDLSCs and jaw BMSCs was further investigated (80).

Cell sheet technology has also been verified as a part of a biphasic β-TCP/PCL construct for the regeneration of both periodontal and bone tissues in rats. The PCL construct should provide, during the healing, mechanical support to cells (47). Fused deposition modeling was applied to produce β-TCP/PCL compartment for bone, which was further integrated with a PCL membrane, synthesized by electrospinning, intended for hosting PDL cell sheets. The authors demonstrated the successful regeneration of cementum, alveolar bone and PDL (47). Since no functional PDL fiber orientation was detectable, the same group synthesized a further implementation of the scaffold, adding a functional micropatterning to the PDL compartment with concentric rings (49). In this case, the scaffold resulted in enhanced bone formation with evidence of oriented PDL fibers and neovascularization (49).

Similarly, Dan et al described bone- and PDL-derived cell sheets, which when placed on a electrospun PCL scaffold enriched with calcium phosphates (CaP-PCL), favored in particular periodontal attachment and alveolar bone formation (81).

Cell transfer technology

Cell transfer technology is a new method of cell manipulation, which enables the in situ delivery of cells using scaffolds, composed of an amniotic membrane in overlapping bilayers. This showed a high capacity to provide growth factors and elements for osteogenic differentiation (82). A plethora of adherent “cell population blends” have been used with this intention, including PDLSCs and osteoblasts, and Akazawa et al emphasized that their structure was not altered by material deformations, but significantly improved bone deposition (83).