Regeneration of the pulp-dentin complex in mature teeth using autologous demineralized dentin matrix and dental pulp stem cells: two case reports

Abstract

Background

Regenerating the true pulp–dentin complex in mature teeth with necrotic pulps or apical lesions remains challenging, and consistent clinical evidence of dentin bridge formation is limited. This report describes two adult cases treated with regenerative endodontic therapy (RET) using autologous dental pulp stem cells (DPSCs) and demineralized dentin matrix (DDM).

Case presentation

In Case 1, a 33-year-old woman developed pulp necrosis with apical periodontitis in a maxillary left central incisor following traumatic subluxation. After canal disinfection, autologous DPSCs isolated from her extracted third molar were transplanted into the canal, and DDM particles were placed at the orifice as a capping scaffold. The contralateral traumatized but still vital incisor was monitored as a control. During 52 weeks of follow-up, the treated tooth regained positive pulp sensibility within 1 week, developed a dentin-like bridge exceeding 3 mm at the orifice, and on 52-week MRI showed high-signal pulp-like tissue comparable to adjacent healthy teeth, whereas the untreated control lost vitality and underwent progressive pulp canal obliteration. In Case 2, a 48-year-old man presented with a maxillary left central incisor that had previously undergone root canal treatment and exhibited severe coronal dentin loss beneath an old crown. Orthodontic extrusion was performed to expose sound supragingival dentin, followed by RET with autologous DPSCs and DDM. Sensibility testing was not feasible due to coronal coverage; however, at 48 weeks CBCT demonstrated a well-defined dentin-like bridge extending from beneath the canal orifice into the coronal pulp chamber. In both cases, quantitative CBCT showed progressive increases in mineral density at the regenerated tissue, reaching approximately 75–80% of adjacent native dentin by the final follow-up. No adverse events were observed, and there were no signs of ankylosis, root resorption, or generalized canal obliteration.

Conclusions

These cases suggest that autologous DPSCs combined with DDM can support dentin-like bridge formation and functional recovery in mature incisors, offering a potential biologically based alternative or adjunct to conventional root canal therapy for preserving structurally compromised teeth.

Background

Dentin chips have been used clinically for nearly a century as pulp-capping agents in vital pulp therapy procedures such as direct pulp capping and pulpotomy. Their high biocompatibility and ability to stimulate natural healing are linked to the induction of hard tissue formation at the pulp interface, forming a biological barrier against microbial ingress [1, 2]. Traditionally, autologous dentin was prepared chairside by grinding extracted teeth into fine particles applied directly to exposed pulp tissue [1]. Later, sterilized preparations using lyophilization and gamma irradiation were developed, achieving a clinical success rate of 89% in 101 cases, as reported by Obersztyn in 1968 [2]. Recent advances in vital pulp therapy have revived interest in dentin-derived regenerative biomaterials such as demineralized dentin matrix (DDM) and treated dentin matrix (TDM) [3, 4]. The dentin matrix is now known to contain a rich repertoire of bioactive proteins, including bone morphogenetic proteins (BMPs), dentin sialophosphoprotein, and dentin matrix protein-1, many of which are more readily released upon demineralization or enzymatic treatment [3–5]. These molecules play pivotal roles in promoting the migration and odontoblastic differentiation of mesenchymal stem cells, thereby facilitating reparative dentin formation [3–5]. In this regard, DDM/TDM is not merely a passive scaffold but a biologically active microenvironment with high regenerative potential. This bio-inductive property distinguishes dentin matrix from conventional endodontic materials such as mineral trioxide aggregate (MTA) and calcium silicate cements, which, despite excellent sealing ability and biocompatibility, lack intrinsic capacity to induce cellular differentiation or robust dentin regeneration [6]. Indeed, combining MTA with dentin matrix has been shown to enhance hard tissue barrier formation and preserve pulp vitality compared to MTA alone [7], and an injectable hydrogel form of TDM has demonstrated promotion of dentin bridge formation, preservation of pulp vitality, and reduced inflammation in direct pulp capping [8].

Regenerative endodontic therapy (RET) leverages tissue engineering principles to regenerate pulp tissue and dentin, rather than simply replacing pulp with an inert filling. However, true regeneration of the pulp-dentin complex in mature teeth with closed apices remains rare. We report two clinical cases, one with a previously traumatized, non-vital incisor and one with a re-treated, heavily restored incisor, in which autologous DDM from the patient’s own extracted tooth was applied over transplanted autologous DPSCs following cell-based RET. Remarkably, in both cases, extensive dentin bridge formation was observed on the surface of the regenerated pulp tissue within one year. These findings highlight the dentinogenic and regenerative potential of DDM and support its application as a bioactive scaffold in cell-based RET for mature teeth.

Case presentation

Case 1

Patient

A 33-year-old woman presented three weeks after a traumatic subluxation injury involving both maxillary central incisors. The left central incisor (#21) had been splinted immediately after trauma. At the first visit, this tooth showed no discoloration but was nonresponsive to both electric pulp testing (EPT) and cold testing, and it was tender to percussion. Cone-beam computed tomography (CBCT) revealed a periapical radiolucency indicative of asymptomatic apical periodontitis (Fig. 1Aa, Ab). The contralateral right central incisor (#11) exhibited discoloration (Fig. 1Ac) but remained vital, responding to both EPT and cold testing, with a little periapical pathology on initial radiographic examination. It was therefore diagnosed as having sustained subluxation without pulp necrosis. The splint was removed five days after injury, and both incisors were monitored. Approximately 17 weeks after the trauma, CBCT of the left incisor showed enlargement of the periapical radiolucency (Fig. 1Ba, Bb), indicating progression of pulpal necrosis. Although the tooth still showed no external discoloration, root canal intervention was considered necessary. After discussion of treatment options, including conventional root canal therapy versus RET, the patient chose cell-based RET for the left incisor. A comprehensive medical evaluation, including blood tests, urinalysis, and infection screening, confirmed her suitability for the regenerative procedure.

Fig. 1.

Case 1: Root canal treatment and cell-base regenerative endodontic therapy (RET) with demineralized dentin matrix (DDM) in the left maxillary central incisor with traumatic subluxation and apical periodontitis. A Initial and early visits. (Aa, Ab) Three weeks post-trauma: cone-beam computed tomography (CBCT) sagittal (Aa) and coronal (Ab) views showing periapical radiolucency. (Ac) Three weeks post-trauma: no discoloration in the left incisor; the contralateral control incisor (right) with subluxation but no apical periodontitis shows discoloration. B Fourth visit. (Ba, Bb) Seventeen weeks post-trauma: enlargement of the periapical radiolucency in sagittal (Ba) and coronal (Bb) views. C Root canal treatment. (Ca) Necrotic pulp observed at chamber access. (Cb) Removed necrotic pulp tissue. (Cc, Cd) Negative bacterial cultures obtained 3 days and 2 months after initiation of treatment. D Cell transplantation. (Da) Transplantation of DPSCs into the canal beneath the canal orifice. (Db) Placement of DDM particles mixed with atelocollagen from the canal orifice into the coronal cavity

At follow-up, the control right incisor had regained normal color, remained responsive to vitality testing, and exhibited no periapical radiolucency on serial radiographic examinations. Based on the patient’s preference, the right incisor was managed with continued observation.

Preparation for autologous DPSCs and DDM

Autologous DPSCs and DDM were prepared from the patient’s extracted maxillary left third molar under standard operating procedures (SOPs) compliant with Act on the Safety of Regenerative Medicine (ASRM). The tooth was removed in a separate minor procedure and transported to a contracted cell-processing facility (Air Water Aeras Bio Inc., Kobe, Japan). At the facility, DPSCs were isolated from the pulp tissue, expanded under hypoxic conditions, and cryopreserved until use. Quality control and safety testing of the cell product were performed according to established SOPs (Table 1) [9]. In parallel, dentin from the same tooth was processed to obtain DDM. After removal of the crown, cementum, and periodontal ligament, dentin fragments were prepared, and sieved to obtain particles measuring 200–500 μm, which were then demineralized in 0.6 N hydrochloric acid. The particles were rinsed, ultrasonically cleaned, dried, and sterilized by gamma irradiation (15–40 kGy). The final DDM product was confirmed to be sterile, with low endotoxin levels (< 1.0 EU/mL) and no detectable contaminants, as verified by inductively coupled plasma emission spectroscopy. The sterile DDM particles were subsequently cryopreserved at -80 °C until use. The frozen vials of DPSCs and DDM were shipped from the contracted cell-processing facility to the clinic in their cryopreservation medium on dry ice at temperatures below − 70 °C.

Table 1.

The quality control tests including viability, doubling time, expression rate of stem cell markers, and safety tests of dental pulp stem cells (DPSCs) at the fourth passage of culture after thawing and demineralized dentin matrix (DDM)

Quality and Safety Tests	Case 1	Case 2
DPSCs
Viability　%	98.8	98.3
Doubling time (hrs)	18.0	16.2
Stem cell markers
CD29%	99.6	99.7
CD105%	99.2	87.0
CD31%	0.0	0.3
Bacteria (Aerobe, Anaerobe, Fungus)	(-)	(-)
Endotoxin (pg/ml)	< 1.0	< 1.0
Mycoplasma	(-)	(-)
DDM
Bacteria (Aerobe, Anaerobe, Fungus)	(-)	(-)
Endotoxin (pg/ml)	< 1.0	< 1.0

Root canal treatment

At approximately six months after trauma, root canal treatment of the necrotic left incisor was initiated. Under local anesthesia and rubber dam isolation, the necrotic pulp tissue was removed upon accessing the pulp chamber (Fig. 1Ca, Cb). The canal was negotiated to the apex, which was fully closed, and patency was confirmed with an electronic apex locator (TriAuto ZX 2, Morita, Japan) and Super Files (MANI, Tochigi, Japan) under microscopic guidance. Biomechanical preparation was performed to size #50/04 using nickel-titanium rotary instruments. Irrigation was carried out with 6% sodium hypochlorite and 17% EDTA, supplemented by adjunctive disinfecting solutions: nanobubble water (Air Water Aeras Bio Inc.), and nanobubbles containing 0.03% levofloxacin (1.5% ophthalmic solution, Nissin Pharmaceutical Co., Yamagata, Japan), and 2.5 µg/mL amphotericin B (Fungizone, Clinigen K.K., Tokyo, Japan). After irrigation, the access cavity was left empty to allow intracanal medicaments to act and to control any residual infection, and was sealed with a double coronal seal consisting of a temporary filling material (Caviton, GC Corp., Tokyo, Japan) placed over a bonded base (Megabond, Kuraray, Tokyo, Japan) and a bulk-fill composite base (Sun Medical Co., Ltd., Moriyama, Japan) to minimize coronal leakage during the interval before cell transplantation. Bacterial samples were collected from the canal at two additional visits, 3 days and 2 months after initiation of treatment, and were cultured in KBM anaerobic medium tubes (KOHJIN BIO, Sakado, Japan) for 5 days. The first KBM test at 3 days confirmed initial disinfection and allowed us to request additional expansion of the DPSCs, as well as DDM processing and sterility and quality-control testing required for the planned transplantation. Because a single negative culture at 3 days might not fully exclude residual bacteria below the detection threshold or accurately reflect the microbial status at the time of transplantation, the 2-month sampling was performed primarily to verify sustained disinfection and to ensure that no intracanal contamination had occurred during the waiting period. Both cultures were negative (Fig. 1Cc, Cd), indicating that the canal remained microbiologically clean at the time of DPSC and DDM transplantation. In this study, “sterilization” of the root canal system was therefore used in an operational sense, meaning reduction of the intracanal bacterial load to below the detection limit of our KBM assay, with no visible turbidity after 5 days of incubation. In preclinical work using canine teeth with refractory periapical lesions, this KBM-based method showed a sensitivity comparable to PCR and conventional blood agar culture (unpublished data), which supports its use as a practical criterion for determining when to proceed to DPSC/DDM transplantation. Once canal sterility was reconfirmed and renewed informed consent obtained, the cell transplantation phase of RET was scheduled.

DPSCs and DDM transplantation

Approximately 11 weeks after the initial root canal procedure (around 39 weeks post-injury), autologous DPSCs were transplanted into the disinfected canal system. Immediately before cell transplantation, the cryopreserved cells (1 × 10⁶ cells/tube) were thawed inside a clean bench located in the operating room, washed with sterile physiological saline, and then resuspended in 100 µL of a clinical-grade atelocollagen scaffold (Koken, Tokyo, Japan) containing 750 ng of granulocyte colony-stimulating factor (G-CSF; Neutrogin, Chugai Pharmaceutical Co. Ltd., Tokyo, Japan), and approximately 20 µL of this cell-collagen suspension (approximately 2 × 10⁵ cells; 1 × 10⁴ cells/µL) was carefully injected into the canal up to the level of the canal orifice (Fig. 1Da), delivering the cells as a cell-collagen suspension rather than as a compact cell pellet. Immediately afterward, DDM particles mixed with clinical-grade atelocollagen (1% Koken atelocollagen implant; Koken, Tokyo, Japan) were placed from the canal orifice into the coronal pulp chamber (Fig. 1Db) thereby capping the transplanted cells with the dentin-derived scaffold. The access cavity was subsequently sealed with Biodentine (Septodont, Saint-Maur-des-Fossés, France), followed by application of the bonding agent and the composite resin restoration.

Follow-up examination

The patient was monitored closely after cell transplantation, with clinical and radiographic evaluations at 1, 4, 12, 22, and 52 weeks post-RET. No adverse events or transplantation-related toxicity were observed. By one week, the treated left incisor responded positively to pulp sensibility tests (both EPT and cold), suggesting early reinnervation. Vital responses were maintained at all subsequent visits through 52 weeks, indicating persistence of functional pulp tissue. CBCT demonstrated minimal hard tissue apposition along the apical root canal wall from 12 to 52 weeks (Fig. 2B-D). By 22 weeks, the periapical lesion had completely resolved (Fig. 2Ca, Cb, Cd), and a uniform radiopaque bridge was visible across the canal orifice where DDM had been placed (Fig. 2Ca-Cc). At 52 weeks (approximately 91 weeks post-trauma), CBCT confirmed a dense dentin-like bridge exceeding 3 mm in thickness at the orifice level (Fig. 2Da–Dd). For quantitative assessment, the region of interest (ROI) was set at the DDM placement site, and Hounsfield unit (HU) values were measured at each follow-up. HU values of adjacent native dentin served as a reference, and the HU ratio of the DDM site to native dentin was calculated over time. This ratio progressively increased, reaching approximately 75% by 52 weeks, consistent with ongoing mineralization (Fig. 2E). MRI at 52 weeks further verified pulp regeneration, with high-signal pulp-like tissue in the treated incisor on STIR and T2-weighted images, comparable to that of adjacent healthy teeth (Fig. 2Fa, Fb). Periodontal status also remained stable, with no pathologic mobility, probing depths < 3 mm, and intact lamina dura. Importantly, there was no ankylosis, inflammatory root resorption, or generalized pulp canal obliteration (PCO) occurred; canal width was preserved except for localized dentin deposition at the orifice. By contrast, the untreated contralateral incisor, which remained vital after trauma, gradually developed PCO, as evidenced by progressive mineralization and narrowing of the pulp canal space on serial CBCT examinations and by very low signal intensity on MRI at 52 weeks (Fig. 2F). In summary, the cell-based RET in Case 1 successfully restored pulp vitality and induced localized dentin-like bridge formation in the necrotic incisor, while avoiding the generalized PCO observed in the naturally healed control tooth.

Fig. 2.

Case 1: Cone-beam computed tomography (CBCT), and MRI follow-up after cell transplantation with demineralized dentin matrix (DDM) in the maxillary left central incisor. A Approximately thirty-seven weeks post-trauma, immediately after transplantation: sagittal (Aa) and coronal (Ab) CBCT views showing persistent periapical radiolucency (arrows). B Fifty weeks post-trauma (12 weeks post-transplantation): reduction of periapical radiolucency (arrows) (Ba, Bb) and dentin-like radiopaque deposition at the DDM site (Ba-Bc). C Fifty-nine weeks post-trauma (22 weeks post-transplantation): resolution of the periapical lesion (Ca, Cb) and uniform dentin-like radiopacity in the DDM region (Ca-Cc). D Eighty-nine weeks post-trauma (52 weeks post-transplantation): increased radiopacity and density in sagittal (Da), coronal (Db), and axial (Dc) CBCT views, indicating progressive mineralization; periapical radiograph (Dd) showing increasing radiopacity in the coronal portion adjacent to the canal orifice. Axial CBCT (Ac-Dc) obtained along the yellow dotted lines in sagittal views (Aa-Da). E Quantitative CBCT analysis: ratio of Hounsfield units (HU) at the DDM site to adjacent native dentin, demonstrating progressive mineralization over 52 weeks. F Fifty-two weeks post-transplantation: MRI (1.5 T) showing high-signal regenerated pulp tissue (arrows) on STIR (Fa) and T2-weighted (Fb) images in sagittal, coronal, and axial views, comparable to healthy pulp in adjacent teeth (arrow heads), consistent with successful pulp regeneration. Notably, the right central incisor (control) exhibited very low signal intensity, consistent with pulp canal obliteration (PCO)

Case 2

Patient

The second case involved a 48-year-old man with no significant systemic history. The maxillary left central incisor had undergone root canal treatment on 15 years earlier, followed by placement of a metal-ceramic crown. Multiple retreatments due to prosthetic misfit resulted in substantial coronal tooth loss. The patient, seeking long-term preservation, specifically requested RET. On presentation, intraoral examination revealed a deep overbite and an ill-fitting porcelain-veneered cast crown on the left central incisor, with localized gingival inflammation around the crown margin (Fig. 3Aa). CBCT confirmed prosthetic misfit (Fig. 3Da, Db) but showed no periapical radiolucency or signs of active infection. Clinically, the tooth was asymptomatic, with no percussion pain or apical tenderness, and periodontal probing depths were within normal limits (2–3 mm). In Case 2, the tooth was therefore diagnosed as a previously endodontically treated maxillary left central incisor with normal apical tissue but severe structural compromise due to extensive coronal dentin loss beneath a misfitting crown. Given the absence of active infection but significant structural compromise, a staged treatment approach was planned: orthodontic extrusion to expose sound root structure for coronal restoration, followed by RET to regenerate the pulp-dentin complex.

Fig. 3.

Case 2: Cell-based regenerative endodontics (RET) with demineralized dentin matrix (DDM) in the left maxillary central incisor with severe coronal dentin loss, deep overbite, and no periapical pathology. A Initial procedure. (Aa) Ill-fitting porcelain-veneered cast crown. (Ab, Ac) Poor adaptation of the metal core after crown removal. (Ad, Ae) Near-complete loss of supragingival dentin after removal of core and caries. Arrows: left maxillary central incisor. B Root canal treatment. (Ba) Provisional composite resin restoration. (Bb) Complete removal of root canal filling material. (Bc) Thorough canal debridement. (Bd) Negative bacterial culture. C Cell transplantation. (Ca) Transplantation of dental pulp stem cells (DPSCs). (Cb) Placement of DDM particles. (Cc) Coverage with Biodentine. (Cd) Final sealing with composite resin. D Serial cone-beam computed tomography (CBCT) images. Coronal (Da, Dc, De, Dg, Di) and sagittal (Db, Dd, Df, Dh, Dj) views. (Da, Db) baseline, (Dc, Dd) immediately after transplantation, (De, Df) 16 weeks, (Dg, Dh) 32 weeks, (Di, Dj) 48 weeks. Radiopaque hard tissue formation extending from beneath the canal orifice into the coronal pulp chamber (arrows) evident from 16 weeks. E Quantitative CBCT analysis. Ratio of Hounsfield units (HU) at the DDM site to adjacent native dentin increased progressively over 48 weeks

Preparation for autologous DPSCs and DDM

Because the patient resided in a distant region, his mandibular left third molar was extracted at an affiliated clinic and transported to the certified cell-processing facility (Air Water Aeras Bio Inc.) for preparation of DPSCs and DDM, following the same protocols as in Case 1. After approximately 8 weeks, sufficient numbers of DPSCs were expanded for transplantation, and quality and safety testing confirmed that both the DPSC and DDM products met all release criteria (Table 1).

Root canal treatment

At the first treatment visit, the existing crown and metal core were removed. Almost no supragingival dentin remained, as caries and repeated preparations had resulted in near-total loss of the clinical crown (Fig. 3Ab-Ae). To enable restoration, orthodontic extrusion was initiated immediately. A bracket and elastic chain were applied, extruding the root by several millimeters over approximately 8 weeks. After sufficient extrusion and soft tissue recontouring, a provisional composite resin restoration (Clearfil Majesty ES Flow and Gracefil LoFlo; Kuraray Noritake Dental Inc.) was placed as an interim coronal barrier (Fig. 3Ba).

At the fourth and fifth visits, endodontic retreatment of the left incisor was undertaken. The root canal was re-accessed through the provisional restoration, and the previous gutta-percha filling was completely removed (Fig. 3Bb). The canal was then instrumented to size #60 to ensure thorough debridement of residual obturation material and necrotic debris. Irrigation was performed using 2.5% sodium hypochlorite, 17% EDTA, and levofloxacin-containing nanobubble water (Fig. 3Bc). The canal was medicated and sealed between visits with a double layer of temporary materials (Caviton and resin filling). By the sixth visit, the tooth was asymptomatic, and bacterial culture of canal samples in KBM anaerobic medium was negative (Fig. 3Bd), confirming successful disinfection.

DPSCs and DDM transplantation

After confirming canal sterility, autologous DPSC transplantation was carried out similar to Case 1. The DPSCs were delivered into the canal space, and autologous DDM particles were placed at the canal orifice (Fig. 3Ca-Cd). The access cavity was sealed with Biodentine and composite, as described previously. Because the entire coronal portion was covered by composite resin, EPT could not be reliably performed in this case post-treatment (there was no exposed tooth structure for electrode contact). Therefore, pulp vitality could not be directly assessed by EPT or cold testing during follow-up.

Follow-up examination

The treated incisor was evaluated immediately after RET and at 16, 32, and 48 weeks. Clinically, the tooth remained asymptomatic and functional throughout the follow-up period. At each recall, there were no signs of periapical disease, and the gingiva around the extruded tooth remained healthy under the definitive crown, which had been placed after initial healing. Because sensibility testing was not feasible, CBCT imaging was used to monitor tissue regeneration. At 16 weeks, CBCT revealed an early radiopaque area beneath the canal orifice, consistent with initial dentin bridge formation (Fig. 3De, Df). By 32 weeks, this mineralized bridge-like tissue had increased in thickness and density (Fig. 3Dg, Dh). At 48 weeks, CBCT demonstrated a well-defined hard tissue bridge extending from the intracanal region below the canal orifice into the coronal pulp chamber (Fig. 3Di, Dj). This finding was consistent with the deposition of new mineralized matrix and indicated progressive maturation of the regenerated tissue. Quantitative CBCT analysis of Hounsfield unit (HU) values at the DDM placement site showed a steady rise in mineral density from baseline to 32 weeks, plateauing by 48 weeks (Fig. 3E). At that time, the density of the bridge region reached approximately 80% of adjacent native dentin, suggesting substantial maturation. No complications were observed: the tooth showed no pathological root resorption, no new periapical radiolucency, and no ankylosis. In summary, RET in Case 2 achieved regeneration of a mineralized dentin bridge beneath the canal orifice, contributing to structural reinforcement of the tooth without any adverse outcomes noted during the first year of follow-up.

Discussion

This report describes two clinical cases: one involving pulp necrosis following traumatic subluxation (Case 1), and another with a previously root canal-treated incisor presenting with substantial coronal tooth structure loss (Case 2). Both cases highlight the potential of cell-based RET using autologous DPSCs combined with DDM in mature permanent teeth.

Pulp necrosis and pulp canal obliteration (PCO) are distinct but well-recognized sequelae of dental trauma, with pulp necrosis occurring in roughly one-third and PCO in about one-quarter of permanent teeth [10, 11]. Although most teeth with PCO remain asymptomatic and vital for many years, severe canal calcification makes any future root canal treatment technically challenging and increases the risk of procedural complications [12], and a small but clinically relevant proportion (7%–27%) may ultimately develop late pulp necrosis and apical periodontitis [11]. Conventional non-surgical root canal treatment, when undertaken, effectively controls infection but eliminates the native pulp, predisposing the tooth to long-term structural weakening and fracture [13, 14]. In Case 1, the contralateral incisor exemplified this trauma-related PCO pathway on the “non-regenerated” side, with progressive canal narrowing on serial CBCT examinations and low pulp signal on MRI during follow-up. By contrast, cell-free RET in both immature and mature teeth, although capable of relieving symptoms and promoting periapical healing, often results in fibrous or periodontal ligament-like tissue with cementum-/bone-like apposition and generalized canal calcification, rather than regeneration of an organized, innervated pulp-dentin complex [15–18].

In the present Case 1, although a small periapical radiolucency was detected three weeks after trauma in the left incisor, there was no crown discoloration and no other signs of pulp necrosis apart from a negative pulp test. Because such a radiolucency may reflect post-traumatic bone remodeling or periodontal ligament changes rather than infected necrosis, we initially chose close radiographic and clinical follow-up instead of immediate root canal therapy, in line with current evidence-based recommendations. At approximately 17 weeks after trauma, CBCT showed enlargement of the radiolucency. Anticipating that endodontic therapy might become necessary, we extracted the patient’s left maxillary third molar two weeks later to obtain pulp tissue for DPSC isolation, recognizing that DPSC culture is not always successful. After confirming successful DPSC expansion and quality-control results, and because the apical lesion showed clear progression, we initiated endodontic treatment at six months post-trauma, at which point the diagnosis of pulp necrosis with asymptomatic apical periodontitis was definitive. Taken together, the risks of structural weakening after conventional root canal treatment, the technical challenges and potential late complications associated with PCO, and the tendency toward generalized canal mineralization after cell-free RET provided the rationale for attempting a cell-based RET strategy in Case 1.

We employed a cell-based strategy in which transplanted DPSCs provided odontoblastic progenitors [19], while DDM served as a bioactive scaffold capable of releasing dentin-embedded growth factors such as TGF-β1 and BMPs, thereby supporting cell differentiation and dentin bridge formation [20, 21]. In Case 1, this approach successfully restored pulp sensibility and resolved apical periodontitis without inducing generalized PCO. Consistent with a previous report of RET in an autogenously transplanted tooth [22], no ankylosis or root resorption was observed. Quantitative CBCT demonstrated a progressive increase in the HU ratio at the DDM site relative to adjacent native dentin, consistent with ongoing mineralization over time. In contrast, the untreated control incisor developed marked PCO, suggesting that cell-based RET may help preserve canal space while still inducing a localized hard-tissue barrier at the orifice.

Case 2 presented a different challenge, with extensive coronal tooth loss following multiple prosthetic procedures. Orthodontic extrusion was performed to expose sound tooth structure before RET. At one-year follow-up, CBCT showed a distinct hard tissue bridge from the intracanal area below the orifice toward the coronal pulp chamber, consistent with new mineralized matrix deposition. This “dentin roof” may provide biomechanical reinforcement, in line with evidence that preservation of coronal dentin enhances fracture resistance in endodontically treated teeth [23]. Rather than merely occupying space like inert filling materials, the biological approach is designed to re-establish living tissue with inherent capacities for defense and self-repair.

This report has limitations. Long-term functional outcomes and structural durability remain to be determined, and sensibility testing was possible only in Case 1 because coronal coverage precluded testing in Case 2. Ongoing clinical and radiographic monitoring of pulp vitality and structural integrity is therefore warranted. In addition, the two incisors presented here represent part of a larger, prospectively monitored cohort treated with the same ASRM-compliant DPSC/DDM-based RET protocol, for which mid- and long-term analyses (e.g. 3- and 5-year outcomes) focusing on tooth survival, maintenance of sensibility, radiographic healing, and structural failures are planned. Preclinical canine studies using the same protocol with DPSC and DDM support the biological plausibility of our clinical findings, showing dentin bridge formation by CBCT as well as histologic evidence of odontoblast-like layers and osteo-/tubular dentin (Iohara & Nakashima, unpublished). Larger clinical studies with standardized protocols and extended follow-up are needed to further validate the efficacy of this approach.

Conclusions

These two cases suggest that autologous DPSCs combined with DDM scaffolds can support dentin-like bridge formation and functional recovery in mature incisors. This approach has the potential to restore pulp vitality, contribute to periapical healing, and promote localized dentin bridge formation, thereby supporting biological function and structural reinforcement. Our findings provide preliminary clinical evidence that cell-based RET may represent a viable alternative to conventional root canal therapy in selected cases. Confirmation through larger clinical series with extended follow-up, including teeth with extensive apical lesions, will be essential to establish standardized protocols, evaluate potential complications, and verify long-term success. If validated, this biologically based strategy could advance the treatment paradigm toward true pulp-dentin regeneration and improve long-term preservation of structurally compromised teeth in daily clinical practice.

Authors’ contributions

MN conceived the treatment concept and clinical procedures, including developing and providing the DDM preparation protocols, and drafted and critically revised the manuscript. NY (Case 1) and HT (Case 2) performed the clinical treatments and patient follow-up for their respective cases, and contributed to scientific oversight and manuscript revision. KI contributed to the analysis of mineral density in the dentin bridge and assisted in the design of the dentin regeneration protocol. All authors read and approved the final manuscript.

Funding

This case report did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data availability

All data generated or analyzed during this study are included in this published article. Additional details (e.g., deidentified clinical datasets or imaging) are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

All procedures performed in studies involving human participants were conducted in accordance with the ethical standards of the institutional and national research committees and with the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. Our provisional plan of pulp regenerative cell therapy as a clinical treatment was approved by a Category II Certified Committee for Regenerative Medicine and was officially accepted by the Japanese Ministry of Health, Labour and Welfare (approval numbers #PB3220153, and #PB4210005, approval data: 11 January 2023, and 7 July 2021) under the Act on the Safety of Regenerative Medicine. Clinical trial number: not applicable. Written informed consent was obtained from each patient after discussing the risks and benefits compared to alternative treatments (including conventional root canal therapy).

Consent for publication

Written informed consent was obtained from both patients for publication of their case details and any accompanying images.

Competing interests

MN, NY and KI declare no competing interests. HT is a director of Air Water Aeras Bio Inc. These affiliations did not influence the conduct or outcomes of this work.