Periodontal regenerative therapies with dental stem cells: from pulp-fiction to clinical application

Introduction

Musculoskeletal and dental regenerative cell therapies typically rely on mesenchymal stem/stromal cells (MSCs), which are fibroblastic cells that are broadly distributed throughout the body often near blood vessels (1,2). MSCs are tested in clinical trials because they have therapeutic functions that support healing after tissue injury or degeneration, based on their biological plasticity and ability to differentiate into multiple mesenchymal lineages including osteogenic cells (1-3). Current models for the use of MSCs in cytotherapies envision that the cells support tissue healing by stimulating endogenous progenitor cells, and by acting as anti-inflammatory paracrine mediators that attenuate tissue degradation and remodeling (1,2). Bone marrow derived MSCs (BMSCs) and dental pulp derived mesenchymal stem cells (DPSCs) represent MSC subtypes derived from mineralizing microenvironments in, respectively, bone and teeth (4). BMSCs and DPSCs have greater osteogenic potential than other types of (e.g., adipose, synovium or umbilical-cord derived MSCs) in cell culture ex vivo and this property is the rationale for their use in de novo bone tissue engineering strategies (3). Their use in cell therapies for bone restoration may not result in long-term integration into mineralized tissues, but rather rapid clearance of cells as tissue repair progresses (1,2). Consequently, most cell therapies with MSCs, including DPSCs, now assume that the cells function as live immunomodulatory ‘drugs’ (1-3).

Preclinical (5) and clinical studies (6) have examined the safety and efficacy of allogeneic dental pulp stem cell injections in tissue regeneration for periodontitis. Periodontitis is a very prevalent dental disorder that is characterized by the formation of periodontal pockets that form initially due to adhesion of bacterial films (plaque buildup) and their subsequent calcification (tartar) to the external surface of the tooth (4). Interactions of bacteria and tartar with the tooth gums provokes inflammation (gingivitis) and the detachment of connective fibers (periodontal ligaments) from the tooth root. This process then sets a vicious cycle into motion in which a deeper pocket promotes deeper microbial penetration, more inflammation and more ligament detachment. The combination of bacterial toxins and chronic inflammation mediates the destruction of the alveolar bone, the loosening of the tooth and ultimately tooth loss. Prophylactic care (brushing and flossing) prevents tartar build up and plaque formation can be reversed mechanically by periodontal cleaning (scaling and root planing). However, advanced periodontitis is a progressive degenerative disease and current clinical treatments do not sufficiently reverse the disease process, leading to tooth extraction and invasive restorative procedures with dental implants (4).

Dental stem cell therapies for periodontal pocket healing and alveolar bone regeneration with DPSCs are viable clinical approaches (7), independent of whether healing occurs directly by tissue integration of the cells or by their anti-inflammatory effects on the surrounding periodontal tissues. Autologous stem cell therapies are in theory sensible, because the use of stem cells from the same patient avoids graft-versus-host rejection. Yet, autologous stem cells require time to be produced at quantities required for therapeutic use, and the healing quality of the original autologous stem cells themselves could be impaired by a range of biological factors including age, genetic mutation or metabolic disorders of the patient (3). Because of these limitations in autologous stem cells, many clinical strategies are focusing on allogeneic stem cells that are sourced from biobanks that offer cryopreserved (“off-the-shelf”) stem cells for regenerative therapies. The added benefit is that the process of expanding and banking of allogeneic stem cells permits production scaling for clinical use and assessment of biological quality control based on a range of release-criteria prior to transplantation (3).

Investigators who study stem cells appreciate that cells represent complex biologics. The efficacy of MSCs is in large part mediated by a heterogeneous proteome of secreted immunomodulatory factors (i.e., anti-inflammatory secretome) (1,2). The paracrine and juxtacrine effects of MSCs reflect the mechanism of action of these cells as living ‘drugs’. Stem cell therapies are evaluated by the US Food and Drug Administration (FDA) based on a solid regulatory framework for investigational new drug (IND) applications (8,9). Cell based products definitely require regulation under IND if the cell product is combined with a drug. The combined application of culture expansion in media and the subsequent cryopreservation of stem cells does not meet the definition of ‘minimally invasive’ necessitating clinical trials for therapeutic uses of allogeneic stem cells (8,9).

Allogeneic dental pulp stem cell treatment for periodontal pockets: ‘borrowed cells for better gums’?

The safety and efficacy of allogeneic dental pulp stem cell therapy for gingival and alveolar bone tissue regeneration in the treatment of chronic periodontitis was recently investigated by Dr. Yi Liu and colleagues (6). With support from both local and national funding sources, this group performed a two-center randomized placebo-controlled clinical trial at two hospitals in Beijing from 2020 to 2023. The two concurrent clinical studies evaluated the effectiveness of dental pulp stem cell injections combined with standard periodontal therapy in patients with different degrees of periodontitis. The primary goal of this phase I clinical trial was to establish absence of serious adverse events, which was monitored for 24 hours post-treatment. The secondary outcome was efficacy and the extent to which teeth were detached from the gums (tooth attachment loss) at 6 months after treatment.

Patients were graded by the severity of periodontitis and received either low or high doses of DPSCs (1 vs. 10 million) via either one or two injections. Saline injections were used as controls. Dental pulp stem cells used for injection were validated for cytomorphology (spindle-shaped cells), cell viability (>90%), standard presence of mesenchymal stem cell markers (CD73, CD90, CD105), as well as osteogenic differentiation potential. These properties are similar to the phenotypes of MSCs from many other sources (3). Beyond analysis of gum pocket formation (i.e., tooth attachment loss) as the primary clinical parameter, the investigators analyzed a range of secondary parameters, including the depth of the gum pocket (probing depth), the extent of gum retraction (gingival recession), bleeding (in response to probing), the looseness of the teeth (tooth mobility) and the amount of bone loss (bone defect depth). The safety components of this phase I study showed the anticipated absence of major adverse events. The only clinically notable findings were development of incidental toothaches and minor swelling at the stem cell injection site that each resolved spontaneously. As is generally understood from animal studies, stem cells of mesenchymal origin typically have low immunogenicity and are well tolerated in cell therapy (3,10,11). The absence of major adverse events in a trial with more than 100 patients (6) emphasizes the relative safety of dental pulp stem cell treatments.

Efficacy of cell therapy for gum pocket healing: ‘pulp fiction or dental pulp stem cell reality’?

While the dental stem cell trial was devoid of major adverse events, the efficacy results were below expectations (6). First, most of the parameters that were examined do not show statistically significant changes upon stem cell treatment. Second, statistically significant effects were only observed for very few parameters and for a very limited group of patients with distinct degrees of periodontitis (e.g., some effects were observed in stage III patients but not stage II patients). Third, for parameters exhibiting statistical significance, stem cell treatment results in only very modest quantitative changes in the extent of attachment loss or the size of bone defect depth (merely fractions of a millimeter). In some cases, the magnitude of these changes in these clinical trial parameters is smaller than the observed pathological variation among all patients in the group. For example, stage III patients (with periodontal pockets deeper than 5 mm) that received stem cell treatment had a modest improvement in attachment loss of 1.7 mm (variation: ±1.5 mm), whereas patients receiving the saline placebo had a comparable improvement of 1.0 mm (variation: ±1.3 mm). Even though this result is considered significant (at a modest P value <0.05), at best the quantitative change seems only a marginal improvement considering the size of the pocket (>5 mm). Stem cell injections show a modest improvement in bone defect depth that is statistically significant, but the clinical relevance is uncertain because the bone defects measure 0.2 (±0.5) mm with and 0.04 (±0.3) mm without stem cells (P<0.02). While these results are positive, they are not overwhelmingly encouraging. Because therapeutic efficacy was not the primary endpoint of this phase I trial, the authors acknowledged that their studies have small sample sizes and are underpowered to observe significant clinical outcomes. Yet, regardless of statistical significance, the modest numerical changes observed in this study should temper the expectations that mere injection of stem cells can easily restore gum pocket defects.

Expectations for future clinical trials in dental regenerative medicine

The stem cell study by Dr. Yi Liu and colleagues study represents a herculean effort involving a large team of investigators, coordination with clinical staff at two different hospitals, patient consenting and follow-up, detailed analyses of dental parameters and extensive data management, while still maintaining a normal daily patient flow. This study reported in accordance with the “DOSES” convention by stem cell therapy experts to improve standardization and transparency for cell therapy studies (12). The authors report on the donor type (i.e., allogeneic), origin of tissue (dental pulp), separation from other cell types (i.e., 90% pure), exhibited cell characteristics (fibroblastic cells with appropriate cell surface markers, self-renewal potential, and differentiation ability), and the site of delivery (periodontal pocket). Key take-aways from the phase I clinical trial by the Liu team are the needs, first, to generate larger cohorts to improve statistical power, and second, to consider strategies that reduce variation within groups (intra-group differences). Each of these limitations reduces the statistical power to measure inter-group differences that establish efficacy.

The study by the Liu team used sub-epithelial injections that target the bone surface and used an overfilling method in the periodontal pocket to ensure allogeneic DPSC retention at the treatment site (6). Future studies may consider the combined use of various scaffold types combined with DPSCs or MSCs to maximize cell retention. Alternatively, cell-free studies that leverage the biological healing properties of the secretome of MSCs may represent another promising strategy (3). Efficacy analysis in future studies will require dose-escalation both in the number of stem cells injected and the frequency of injections. The multiplicity of treatments will impact the sample size for each group. Parsing of a multi-institute patient cohort into multiple different treatments will reduce the number of individuals per group and thus retaining statistical power will be challenging. Hence, strategies to reduce the observed biological variation in treatment parameters among patients could be a viable mitigating approach. The Liu team used inclusive selection criteria for patient participation in their stem cells studies. Patients admitted in the study ranged from young adults (18 years) to advanced age (65 years). The periodontal pockets varied from 4 to 8 mm (a 2-fold difference in pocket depth). Patients were deselected for pre-existing conditions (cancer and diabetes) or modifiable risk factors (heavy smoking). Recruiting more patients will allow subdivision for disease severity, age, sex, and multiple secondary factors.

One possibility that is not discussed by Liu and colleagues is whether human leukocyte antigen (HLA) typing of allogeneic donors and patient matching could improve efficacy. Patient matching by HLA typing will ensure that cell surface markers present on allogeneic donor stem cells are compatible with the recipient patient to avoid discrimination of endogenous (“self”) and exogenous (“non-self”) cells by the immune system. This general principle of patient matching allows for the broad implementation of stem cell and tissue transplantations at large. Of course, it has been well-established that stem cells are characterized by low immunogenicity and considered immunosuppressive (10). The immunogenicity of MSCs is low because these cells express low levels of major histocompatibility complex (MHC) Class I proteins (3). Because MSCs do not express MHC Class II proteins, they do not appear foreign to the immune cells of the donor and they lack costimulatory molecules (3). Furthermore, it is understood that mesenchymal stem cells (including DPSCs) often are rapidly cleared (within weeks after delivery) (1,2) and gum pocket regeneration involves short-term local stem cell applications at a periodontal site. Yet, it is conceivable that HLA mismatched DPSCs could still result in the clearance of cells recognized as non-self and reduce the effective dosing of the injected stem cells. Hence, the magnitude of the healing effects could perhaps be improved by avoiding non-self immune clearance of mismatched stem cells after injection.

The team led by Dr. Yi Liu noted that stem cell injections are perhaps more effective for single-root teeth than multiple-root teeth without furcation involvement. Thus, future studies could perhaps focus on particular types of periodontal tooth pockets to improve assessment of treatment effects. Finally, stem cell treatments are based on their therapeutic ability to attenuate inflammation. Therefore, it would be sound practice to compare the effects of stem cell therapy with other anti-inflammatory treatments including pharmacotherapy with glucocorticoids or non-steroidal anti-inflammatory drugs (NSAIDS).

Conclusions

The phase I study by Dr. Yi Liu does not meet our most optimistic expectations, because the clinical outcomes do not yet provide compelling evidence that dental pulp stem cells significantly enhance periodontal regeneration. However, the most positive message of this study is the ‘negative space’ of what is not observed: the absence of serious adverse effects. The relative safety of dental pulp stem cell injections in periodontal pockets permits expansion of future clinical trials that overcome sample limitations and increase the statistical power of the study design. Because risk for adverse effects is rather low, there is ample room for further research to validate findings and optimize treatment protocols for stem cell trials. Larger cohort sizes will potentially permit stratification of diverse patient populations that are by disease severity, age, sex, modifiable risk factors and comorbidities. Study expansion will also permit optimization of (I) dosing, (II) timing and (III) frequency of stem cell injections, treatments customized to (IV) the stage of periodontitis, and (V) long-term longitudinal studies to assess potential benefits during future disease progression or aging. We conclude that the current story of stem cell therapy is neither pulp-fiction nor reality, but rather a promise and call to action to overcome current challenges and render cell therapy both cost-effective and efficacious for dental applications.

Acknowledgments

We thank our colleagues at the University of Connecticut Health Center, including Ivo Kalajzic, Brya Matthews and David Rowe, as well as Ralph Salvagno (Extensor Bio), for support and stimulating discussions.

Footnote

Provenance and Peer Review: This article was commissioned by the editorial office, Frontiers of Oral and Maxillofacial Medicine. The article has undergone external peer review.

Peer Review File: Available at https://fomm.amegroups.com/article/view/10.21037/fomm-2025-1-38/prf

Funding: This study was supported by NIH/NIDCR (No. R43-D034602).

Conflicts of Interest: Both authors have completed the ICMJE uniform disclosure form (available at https://fomm.amegroups.org/article/view/10.21037/fomm-2025-1-38/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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