Periodontal Bone-Ligament-Cementum Regeneration via Scaffolds and Stem Cells

Abstract

Periodontitis is a prevalent infectious disease worldwide, causing the damage of periodontal support tissues, which can eventually lead to tooth loss. The goal of periodontal treatment is to control the infections and reconstruct the structure and function of periodontal tissues including cementum, periodontal ligament (PDL) fibers, and bone. The regeneration of these three types of tissues, including the re-formation of the oriented PDL fibers to be attached firmly to the new cementum and alveolar bone, remains a major challenge. This article represents the first systematic review on the cutting-edge researches on the regeneration of all three types of periodontal tissues and the simultaneous regeneration of the entire bone-PDL-cementum complex, via stem cells, bio-printing, gene therapy, and layered bio-mimetic technologies. This article primarily includes bone regeneration; PDL regeneration; cementum regeneration; endogenous cell-homing and host-mobilized stem cells; 3D bio-printing and generation of the oriented PDL fibers; gene therapy-based approaches for periodontal regeneration; regenerating the bone-PDL-cementum complex via layered materials and cells. These novel developments in stem cell technology and bioactive and bio-mimetic scaffolds are highly promising to substantially enhance the periodontal regeneration including both hard and soft tissues, with applicability to other therapies in the oral and maxillofacial region.

Keywords: bone-PDL-cementum; growth factors; periodontal regeneration; scaffolds; stem cells; tissue engineering.

Figure 1

Methods of cell delivery via calcium phosphate cements (CPC). Live-dead staining of cell seeding on CPC (a–c); cell encapsulation in alginate-fibrinogen microbeads (Alg-Fb-MB) (d–i); cell encapsulation in alginate–fibrinogen microfibers (Alg-Fb-MaF) (j–o); synthesis of bone minerals by the encapsulated stem cells. Images of (p–r) hBMSCs, (s–u) BM-hiPSC-MSCs, and (v–x) FS-hiPSC-MSCs stained with Xylenol orange (images of hESC-MSCs, hUCMSCs, and hDPSCs were similar to those of hBMSCs). (y) Alizarin red S (ARS) staining of hBMSCs, BM-hiPSC-MSCs and FS-hiPSC-MSCs in calcium phosphate cements-cell encapsulating alginate-fibrin fibers (CPC-CAF) (images of hESC-MSCs, hUCMSCs, hDPSCs were similar to those of hBMSCs). (z) Xylenol orange mineral staining area and ARS mineral concentration produced by cells in CPC-CAF (mean ± s.d.; n = 6). (Adapted from References [39,41,45], with permission.).

Figure 2

CPC-stem cells construct for bone regeneration. (a) Representative hematoxylin-eosin (HE) images of the CPC-MF-hBMSC group at 12 weeks post-surgery. Bone bridging was achieved in the critical-sized defects. The defect was closed with newly woven bone and trabecular bone. (c) and (d) were high magnification images of the dotted-line rectangle in (b); (e) quantification of new bone and residual CPC area fraction. (f–h) Representative HE images at 12 weeks. The cell-seeded groups showed more new bone than CPC control. The greatest amount of new bone was observed in the tri-culture group; (i) high magnification images of new bone from dotted rectangles in the tri-culture group (h). New blood vessels in the macropores of CPC scaffolds; (j) quantification of the new bone area and vessel density (adapted from References [37,41] with permission).

Figure 3

Periodontal regeneration. (a) inflamed soft tissue and bone resorption in periodontitis; (b) periodontal long junctional epithelium (LJE) repair; (c) ideal periodontal regeneration; (d) schematic of the four compartments from which cells could grow into periodontal wound and repopulate the root surface after periodontal treatment: ➀ oral gingival epithelium; ➁ gingival connective tissue; ➂ bone; ➃ PDL; (e) schematic of guided tissue regeneration (GTR). (f) optical micrograph shows LJE ending at the coronal-most end of the regenerated cementum (C) and dentin (D); (g) LJE and partial periodontal regeneration, indicated by the formation of new cementum (NC) and new bone (NB). The arrowhead indicates the apical end of the junctional epithelium, whereas the arrow shows the apical border of the defect. (h) optical micrograph showing periodontal regeneration, with the formation of new PDL fibers (NPLF) attaching to both NB and NC. R: root (adapted from References [65,66], with permission).

Figure 4

Apical tooth germ cell conditioned medium (APTG-CM) enhanced differentiation of periodontal ligament stem cells (PDLSCs) into cementum/periodontal ligament-like tissues. (a–c) PDLSCs in differentiation (a-modified eagle medium (a-MEM) with 10% fetal bovine serum (FBS), 100 μg/mL penicillin and 100 μg/mL streptomycin, 50 μg/mL of ascorbic acid and 2 mM sodium β-glycerophosphate) without APTG-CM had little mineral. (d–f) ultures with APTG-CM had substantial minerals. (g) Gene expression of PDLSCs co-cultured with APTG-CM. Osteocalcin (OCN), bone sialoprotein (BSP) and cementum-derived protein-23 (CP-23) expressions served as markers for cementoblast differentiation. At 21 days co-culture with APTG-CM, there were elevated expressions of OCN and BSP mRNA in the induced PDLSCs. Untreated PDLSCs lacked OCN and BSP expressions. (h) PDLSCs co-cultured with APTG-CM generated cementum-like minerals (C) on bovine bone (CBB) powders and PDL-like collagen fibers (PDL) connected with the new cementum. (i) There were cementoblast-like cells (Cb) at the cementum–PDL interface and cementocyte-like cells (Cc) in the mineral matrix. (j) Collagen bundles (arrows) were attached to cementum. (k, l) Untreated PDLSCs had little cementum or PDL-like structures. (m) No mineralization or PDL-like tissues were seen in CBB alone. Scale bars = 100 μm. APTG-CM: CT: connective tissue (adapted from Reference [90], with permission).

Figure 5

Harnessing endogenous stem cells. (a) Periodontal regeneration could be achieved via harnessing endogenous stem cells from the host tissues, where biomaterials and molecules coax the recruitment of endogenous stem cells to induce growth. (b) Schematic showing the mobilization of stem cells from their niche using cell-mobilizing factors, directing cell migration using cell homing factors and blood flow, then regulating stem cell fate via materials, growth factors and immunomodulatory cytokines. BMMSCs: bone marrow- mesenchymal stem cells; ECM: extracellular matrix; FGF-2: fibroblast growth factor-2; GDF-5: growth/differentiation factor-5; IL-4: interleukin-4; PDGF-BB: platelet-derived growth factor-BB; SCF: stem cell factor; SDF-1α: stromal-derived factor-1α (adapted from Reference [13], with permission).

Figure 6

3D-bioengineered periodontal complex. (a) Biphasic scaffold with cell sheets. Fabrication of biphasic scaffold and cross-section of the biphasic scaffold by scanning electron microscope (SEM) indicating the fusion of electrospun fibers with fused deposition modeling (FDM) component. Microcomputedtomography (micro-CT) of biphasic osteoblast-seeded scaffold 8 weeks after implantation in a subcutaneous athymic rat model; (b) 3D hybrid scaffold. Micro-CT and 3D-reconstructed hybrid scaffold (scale bar = 50 μm). SEM analysis of each layer. Perpendicular orientation of PDL-like tissues to dentin and along the column-like structures (scale bar = 50 μm). (c) Bone-ligament complexes with fiber-guided scaffolds. Micro-CT mandibular pictures and customized defect-fit scaffolds. Microscopic architectures of the fiber-guiding scaffold with micro-grooves and macropores (scale bar = 500 and 250 μm) and organized fibrous tissues with mineral tissue layers (adapted from References [107,108,109], with permission).

Figure 7

Gene-based technologies. Direct and cell-based delivery of a therapeutic gene increased the regenerative capability and promoted the availability of key bio-factors. The gene of interest was either injected directly into the periodontal defect via a retrovirus, or alternatively, was incorporated into embryonic stem cells (ES) or adult stem cells that were then expanded and delivered into the wound (adapted from Reference [113], with permission).

Figure 8

Layered materials and cells to simulate the multiple tissue layers. (a) An engineered membrane (Bio-Gide collagen membrane seeded with cells on both sides). (b) Two mineralized membranes (a cellular small intestinal submucosa in which cells were seeded on one side and cultured in mineralization-induction medium for 8 days). (c) An layer-by-layer (LBL) complex was produced by placing a cell-seeded periodontal membrane between the two cell-seeded/mineralized membranes. (d) The LBL group showed that regular alveolar bones and cementum had reconstructed completely, with mature periodontal ligaments and a normal periodontal gap. (hematoxylin and eosin, 100×). (e) The trauma group showed disordered fibroblasts and external resorption of teeth, with only a few irregular new bones. (hematoxylin and eosin, 100×). (f) The Bio-Gide collagen membrane group showed some new thin and irregular bones; periodontal fibers were disordered, and the periodontal gap was very wide (hematoxylin and eosin, 100×). (g) 3D-printed seamless scaffold with region-specific microstructure and spatial delivery of proteins. The scaffold had three phases: (h) Phase 1: 100 μm microchannels with 2.5 mm in width, (i) Phase 2: 600 μm microchannels with 500 μm in width, (j) Phase 3: 300 μm microchannels with 2.25 mm in width. (k–m) Poly lactic glycolic acid (PLGA) microspheres encapsulating amelogenin, connective tissue growth factor (CTGF), and bone morphogenetic protein 2 (BMP2) were spatially tethered to Phases 1, 2 and 3, respectively. (n) Schematic of a tri-layered nanostructured composite hydrogel scaffold (each layer had different growth factors) for simultaneous regeneration of multiple periodontal tissues. ab—alveolar bones; pl—periodontal ligaments; d—dentin; CEMP1: cementum protein-1; FGF-2: fibroblast growth factor-2; PRP: platelet-rich plasma; nBGC: nanobioactive glass ceramic; rhCEMP1: recombinant human cementum protein-1; rhFGF: recombinant human fibroblast growth factor (adapted from References [11,13,120,121], with permission).