The ultimate goal of periodontal therapy is homeostatic regeneration of lost attachment of alveolar bone and gingival connective tissue to the exposed root surfaces with a fully functional and healthy periodontal ligament that is covered with a healthy epithelium. This goal needs a complete understanding of the biological mechanisms inherent to healing and inflammatory processes.
Understanding the current status of the biological basis of periodontal regeneration will enable the adoption of emerging strategies in clinical practice for retaining natural teeth.
Developing therapeutic approaches that will take advantage of tissue biology and physiology.
Predicting the outcomes of periodontal regenerative procedures.
One of the oldest debates in periodontal therapy has been the regeneration of the lost tissues due to the disease process. The basis for this ongoing discussion is the complexity of disease mechanisms of initiation, progression, and resolution. Being an open interface between the soft and the hard tissues of a highly active and functional unit involved in mastication and exposed to microbial elements and environmental factors makes the periodontium one of the most complex biological structures in nature. The infection and inflammatory processes affecting the homeostatic stability of the periodontal tissues lead to its destruction and involves the failure of several cellular and molecular mechanisms of defense. Consequently, substantial resources and efforts are focused on understanding periodontal disease pathogenesis. Restoring periodontal structures and regeneration of the lost soft and hard tissues must be based on the exact same pathways that destroy tissue components. It would not be too ambitious to refer to “periodontal regeneration” as more complex than soft tissue-only regeneration (eg, skin) or hard tissue-only regeneration (eg, bone) because periodontal regeneration involves all of these in a highly contaminated and open wound and a highly functional environment in the oral cavity.
The first detailed experimental evidence for periodontal regeneration can be traced to a publication in 1934 in which Stones presented compelling histologic data supporting that root cementum can regenerate in nonhuman primates. In this seminal paper, various critical observations relevant to the modern understanding of periodontal regeneration were made. First, epithelial cells were shown to repopulate the entire root surfaces denuded of cementum, producing a weak and unstable attachment with dentin, easily separated from the root surface. Second, regeneration of cementum was demonstrated to be possible due to new cementoblasts lining the healing tissues, whereas alveolar bone has the capacity to regenerate. Finally, space was identified as critical to regeneration. This work also showed that pathologic disruption of the periodontal attachment could be reversed and restored by the remaining cells of the periodontium. These observations were confirmed in dog studies in the 1950s and collectively demonstrated that periodontal regeneration was feasible, differentiating the reattachment from new attachment with conclusive evidence.
During the next 2 decades, cellular origins of periodontal regeneration were identified. Melcher proposed the critical cells in the periodontium that could be responsible for the outcomes for treatment. Meanwhile, several surgical techniques have been introduced for periodontal regeneration. The advancements for the past 40 years of research since the early 1980s allowed periodontology to be a pioneer in regenerative medicine. Owing the extensive research at the basic science and translational levels, we now appreciate a better understanding of the processes underlying the clinical therapeutic choices ( Fig. 1 ). This article focuses on the biological mechanisms underlying periodontal regeneration, identifying key molecular and cellular processes and noncellular elements of the periodontal regenerative process.
Repair or regeneration: conceptual to biological wound healing
In the absence of any regenerative techniques, periodontal surgery led to a wound healing that was predominantly a repair. , Early studies were based on the histologic analyses of human models and demonstrated 3 histopathological levels of corona apically positioned layers: epithelium, connective tissue, and cementum-bone interface. Given the limited amount of cementum-bone interface, which was defined as “periodontal attachment,” these studies suggested that periodontal surgical techniques without any regenerative strategy are limited in their capacity to restore lost tissues. This observation led to a major paradigm shift in periodontology. Studies in the 1980s demonstrated periodontal regeneration as a feasible outcome in periodontal treatment using innovative techniques. Most importantly, however, regenerative approaches led to a deeper understanding of biology-based treatment strategies to restore periodontal tissues to their levels before the disease.
Every form of wound healing includes regeneration. The distinction between “repair” and “regeneration” is conceptual rather than biological. Repair has been used to refer to an incomplete or lack of regeneration where cellular elements populate the wound area depending on their proliferation rates and activities after an injury inflicted by periodontal surgery. Therefore, cells with higher multiplication, growth, and proliferation capacity would be the first to arrive at the injury site. Not surprisingly, similar to the other wound models elsewhere in mammals, epithelial cells originating from the gingiva have the highest migration and proliferation capacity. Thus, wound healing by repair starts with epithelial cells covering the wound area. This stage should be seen as a defensive action to protect the site from being infected by excluding the injury site from the environment. On the other hand, it also prevents an actual attachment of the hard tissues and complete regeneration of periodontal ligament on the exposed root surfaces. Therefore, periodontal epithelization has been perceived as a protective but limiting step in regeneration, leading to disruption of homeostatic wound healing during the repair.
Based on these fundamental observations, “guiding” periodontal regeneration has been introduced as a clinical technique. Guided regeneration refers to intentionally directing the specific cells to where they should be located to restore tissues. To this end, it differs from the random/natural repair process after surgical wounding, where regeneration is limited and unpredictable. Many guided strategies included the prevention of epithelization on the root surfaces. Physical prevention of epithelialization along the denuded root surface was thought to allow dental and periodontal cell sources to proliferate and populate, leading to a new attachment. The first generation of guided periodontal regeneration involved the use of nonresorbable barriers, which required removal. The need for a second surgery was overcome by introducing resorbable barriers, which provided varying degrees of resorption times and sources to the clinicians. The operator’s case selection and technical skills were critical in the success of guided periodontal regenerative approaches, , which became the gold standard in periodontal therapy. In the absence of an epithelial seal around the tooth, it has also become apparent that the spatiotemporal reconstruction of periodontal ligament and hard tissues with an optimal attachment of alveolar bone to cementum through fibers was not always predictable.
Precision and timing are critical for periodontal regeneration. Periodontal wound healing is not a linear process. All cell types are present and functional at the same time. Regeneration is the outcome of an orchestrated proliferation, differentiation, synthetic activity, and apoptosis of individual cell lineages.
Cellular regulation of periodontal regeneration
Early research suggested the involvement of epithelial cells, gingival fibroblasts, periodontal ligament fibroblasts, cementoblasts, osteoblasts, osteoclasts, and osteocytes in the healing after periodontal treatment. In addition to these primary cells with structural roles, endothelial cells, immune cells, and neuronal cells are critical in regulating periodontal regeneration. Wound healing was initially thought to follow similar stages of development of dentoalveolar and dentogingival structures. However, we now know that there are profound differences in the origin of cells involved in the healing of damaged tissues of the periodontium. , For example, the oral epithelium replaces the odontogenic epithelium in forming the junctional epithelium at the dentogingival junction. Gingival and periodontal ligament fibroblasts become the primary stromal cells during wound healing, replacing the dental follicle. Progenitor stem cells in the periodontal ligament and mesenchymal stem cells from the endosteal spaces differentiate into cementoblasts, fibroblasts, and osteoblasts, replacing the dental follicle and dental papilla. These observations suggested 2 critical points: (1) wound healing is different in adult periodontal and dental tissues than the neonate and (2) cells regulating wound healing in the adult need to be identified to predict periodontal regeneration.
On the other hand, identifying prenatal and postnatal processes during the tooth eruption and formation of the periodontium led to the first completely biological regenerative strategy in periodontal therapy. Enamel matrix proteins were introduced as mediators of periodontal regeneration owing to their multifaceted functions. While increasing epithelial cell attachment for optimal wound closure, these embryonic proteins stimulate fibroblastic proliferation and activity of cells in the gingiva and periodontal ligament. Most importantly, they regulate the endothelial function by chemotactic stimulation of endothelial cells, which is essential for homeostatic wound healing. The effect of enamel matrix proteins is not equal on all cell types; proliferation of periodontal fibroblasts is favored over gingival fibroblasts and epithelial cells, which allows periodontal attachment to take place before the denuded root surfaces could be populated by the gingival connective tissue cells and epithelium. Simultaneously, osteogenic cell proliferation is stimulated, preparing an osseous wall for the new attachment to connect. Mechanistically, enamel matrix proteins were shown to stimulate various transcriptional factors, growth factors, and mediators involved in osteoblastic, cementoblastic, and odontoblastic cell function. These studies demonstrated the role of biological mediators as proregenerative strategies in the absence of physical barriers. Thus, enamel matrix proteins and other growth factors that have been introduced for periodontal regeneration represent the third generation of guided regenerative approaches.
Several growth factors have been tested for periodontal regeneration. Recombinant human platelet-derived growth factor, insulinlike growth factor, bone morphogenetic proteins, fibroblast growth factor, growth/differentiation factor are among those that have shown promising clinical results with strong preclinical and in vitro supporting data. , Platelet preparations offer packaged growth factors and other components of wound healing (eg, lipoxins) involved in the regenerative process. These preparations can be used in combination with other regenerative procedures and continue to evolve.
Biologics offer more directed stimulation of cells and molecules to be recruited to the site of the surgical injury. However, the issue of spatiotemporal guiding of periodontal regeneration has still not been addressed. Current regenerative strategies involve the controlled release of growth factors and other biologically active molecules using various scaffolds and other tissue-engineered constructs that offer a prolonged delivery while maintaining the wound space. , Another regenerative approach uses nanoparticles that are loaded with biologics. In addition, gene therapy presents many opportunities for regenerative medicine, including periodontal regeneration. These “smart” regenerative techniques allow the recruitment of different cell types and molecules needed for tissue regeneration in the periodontium and, when used in combination, will address the programmed regeneration at the site of injury.
At the cellular level, stem cells in the periodontium have received substantial attention as critical regulators of types of cells involved in the regenerative process. As periodontal ligament stem cells are multipotent progenitor cells, they can proliferate and differentiate into required cell types of the periodontium. This observation was ground-breaking because it demonstrated that periodontal tissues did have the capacity to regenerate if the stem cells could be mobilized and directed toward a predictable periodontal regeneration. Indeed, in response to various growth factors, adhesion molecules, and extracellular matrix components, progenitor multipotent periodontal ligament stem cells can differentiate into precementoblasts, prefibroblasts, and preosteoblasts and subsequently commit to the mature cells of these lineages (ie, cementoblasts, fibroblasts, osteoblasts).
Under the optimal conditions and in the absence of any inflammation or infection, periodontal regeneration can be seen as an orchestration of these cell types along the exposed root surfaces. Inflammation, however, is an impactful determinant of the outcomes of healing along with the immunologic factors. Systemic causes of inflammation, such as diabetes and cardiovascular diseases, result in an impaired regenerative process.
Molecular basis of wound healing and periodontal regeneration
In healthy tissues, gingival connective tissue presents similar characteristics to the skin. Collagen and noncollagenous matrix (eg, proteoglycans, fibronectin, elastin) are produced by gingival fibroblasts at varying degrees depending on the topographic and functional features. Oral mucosal wound healing follows 4 phases: (1) hemostasis, (2) inflammation, (3) new tissue formation and cell proliferation, and (4) maturation and matrix remodeling. The hemostasis phase involves coagulation, enrichment of platelet and other hematological elements in coagulum, and fibrin meshwork with coagulum to generate “provisional extracellular matrix.” This phase is followed by the inflammatory recruitment of immune cells and other cellular components to regulate tissue turnover. Neutrophils and monocytes arrive in the wound area; macrophages are differentiated from monocytic precursors into proinflammatory M1 phenotype or proresolutive M2 phenotype. The transition of the inflammatory phase to the proliferative phase is the most critical step and requires an infection-free environment. During this transition, granulation tissue derived from the periodontal ligament induces epithelial cells to form a keratinized masticatory gingiva. Granulation tissue is a healing vasculature and fibroblast-rich extracellular matrix regulating endothelial cell recruitment for new capillary vessels. At this step, fibroblasts are recruited from wound edges and pericytes. Epithelial-mesenchymal transition involves the formation of epithelium from keratinocytes, whereas the soft connective tissue determines the characteristics of the overlaying oral epithelium. In return, oral epithelium determines connective tissue healing. Therefore, epithelial-mesenchymal communication is a central event in wound healing and regeneration. Although there is an overlap between epithelial and connective tissue regeneration, there is also a gap in timing. Typically, epithelial healing takes 7 to 14 days following nonsurgical and surgical periodontal therapy. Functional stability between the denuded root and soft tissue is achieved approximately 14 days after surgery. Therefore, the timing of these events is crucial for optimal healing.
The periodontal ligament is dissimilar to other tissues in the mammalian body. It is primarily a connective tissue and therefore contains collagen types I and III as the major proteins with Sharpey fibers attaching to the cementum. Another unique tissue is the periodontium is the root cementum. Although structurally similar to other hard tissues, alveolar bone and cementum are uniquely attached through predominantly collagen fibers. All these structures are vascularized through the vessels in the gingiva and periodontal ligament.
Degradation of the extracellular matrices can occur through various pathways, including activation of matrix metalloproteinases, the release of reactive oxygen species cytokine release, and prostaglandin synthesis cell activation. Upon injury, 3 types of wound healing may occur, all of which are regulated by inflammation. The ideal healing is by full regeneration after a complete resolution of inflammation. However, when the inflammation cannot be resolved and becomes chronic because of the inability to eliminate the injurious agent or insufficient coping by the immunoinflammatory process, 2 scenarios may occur: (1) tissue destruction and (2) fibrosis. Both tissue responses also represent healing but not an ideal regeneration. Thus, in almost all types of healing, there will be areas of destruction and fibrotic changes. At the same time, minimal regenerative sites emphasize the role of inflammation as a master regulator of healing. Clinically, however, the 3 distinct models are recognized as pathologic conditions (chronic inflammation associated with periodontal tissue destruction or fibrotic expansion of periodontal tissues) or regenerative healing.
In addition to the inflammation as a process, several critical molecular events and mediators are actively involved after the injury; these include growth factors, cytokines, chemokines, lymphokines, and various tissue factors released from inflammatory cells and damaged tissue. These molecules trigger a cascade of signaling reactions as a prelude to their action and lead to the production of further molecular mediators. Although these markers of inflammation were thought to be detrimental, they were also involved in wound healing. Therefore, inflammatory resolution and reprogramming are key for regenerative wound healing. Likewise, extracellular matrix proteins can be used as regenerative biologics when combined with scaffolds.
Periodontal regenerative medicine is a predictable procedure when the underlying biological process is understood. Many surgical techniques and materials have been used to deliver this clinical outcome. Bone grafts, growth factor preparations, platelet derivatives and preparations, scaffolds, nanoparticle-based delivery systems, stem cells, and gene therapies are currently being tested for the spatiotemporal predictability of periodontal regeneration. Sufficient and convincing evidence is accumulating to support several applications such as growth factors. Meanwhile, the inflammatory resolution is being tested as a tool to allow the cells of the periodontium to repopulate on the exposed root surfaces. Reprogramming the stemness of periodontal ligament stem cells under inflammatory conditions may offer a wider capability for regenerative periodontal therapy in clinical practice. Cells may become irrelevant when we can obtain and deliver the small extracellular vesicles of exosomes of stem cells for periodontal regeneration. The field has moved to a more biological-based understanding of periodontal regeneration to address the mechanism of healing. Recruitment of specific cell types to the site of injury is feasible. A customized strategy with vascularization of the regenerated tissue and innervation of the newly formed structures fully integrated with the native tissues and mechanoresponsive will focus on future studies to accomplish the ultimate goal of maintainable regeneration.
Clinics care points
Although dental implants offer excellent solutions for restoring edentulism, the goal of periodontal therapy is to restore and retain natural teeth as a functional and healthy unit. Periodontal regeneration is feasible and predictable, offering a wide variety of treatment options to clinicians.