Histological Evidences of Autograft of Dentin/Cementum Granules into Unhealed Socket at 5 Months after Tooth Extraction for Implant Placement

The aim of this clinical case study was to observe biopsy tissues at 5 months after an autograft of a partially demineralized dentin/cementum matrix (pDDM) into a tooth-extracted socket exhibiting healing failure. A 66-year-old female presented with healing failure in the cavity for 2 months after the extraction (#36). Initial X-ray photos showed a clear remainder of lamina dura (#36), a residual root (#37), and a horizontal impaction (#38). The vital tooth (#38) was selected for pDDM. The third molar crushed by electric mill was decalcified in 1.0 L of 2.0% HNO3 for 20 min and rinsed in cold distilled water. The pDDM granules (size: 0.5–2.0 mm) were grafted immediately into the treated socket. X-ray views just after pDDM graft showed radio-opaque granules. At 5 months after pDDM graft, the surface of regenerated bone was harmonized with the mandibular line, and bone-like radio-opacity was found in the graft region. The biopsy tissue (diameter: 3.0 mm) at 5 months after pDDM graft showed that mature bone was interconnected with the remaining pDDM. The novel histological evidence highlighted that newly formed bone was connected directly with both dentin-area and cementum-area matrix of pDDM. We concluded that pDDM contributed to the regeneration of bone in the unhealed socket, and this regeneration prepared the socket for implant placement. Autogenous pDDM could be immediately recycled as an innovative biomaterial for local bone regeneration.


Introduction
Extracted teeth are usually discarded as potentially infectious medical waste. However, the dental community is now considering human primary and secondary teeth as a useful material resource for patients and their families [1]. The first clinical case of human dentin autograft was reported in 2003 [2], while human bone autograft dates back to 1820 in Italy. There was a very long time-lag between the autografts of dentin and bone. Demineralized dentin/cementum matrix (DDM) is a decellularized matrix prepared through demineralization (<pH 1.0), washing by saline or distilled water, and/or freeze-drying [3]. Recently, many autograft cases were reported from the world [4][5][6][7][8][9]. Additionally, familial grafts between a parent and a child were successfully achieved, and the first successful 2 of 8 procedure occurred in South Korea [10]. In this century, the tooth-derived materials have become a realistic alternative to bone grafting [7,11].
Highly calcified tissues such as fresh cortical bone and dentin do not occur earlier in osteoinduction and bone formation than DDM and demineralized bone matrix (DBM) [12][13][14][15]. In 2016, 70% of demineralized human DDM granules (size: 1.0 mm) had a better performance in bone formation than completely DDM (cDDM) and non-demineralized dentin in rat calvarial bone defects [16]. In 2021, cortical bone plate treated supersonically with acidic electrolyzed water (pH 2.3), so-called partial DBM (pDBM), induced bone at 2 weeks, while fresh cortical bone plate did not induce bone even at 8 weeks [17]. In light of the articles [13][14][15][16][17], we believed that the apatite crystals of both calcified dentin and cortical bone should inhibit a release of bone morphogenetic proteins (BMPs) from matrices. Clinically, the immediate DDM autograft system needs speed, compared to the delayed autograft. Our team, therefore, developed the immediate autograft system of partially demineralized dentin/cementum matrix (pDDM) by using a new electric mill [3,18].
The aim of this case study was to observe the biopsy tissues obtained from the dental implant-placed region at 5 months after pDDM autograft into the socket that had exhibited healing failure (#36) for 2 months. The histological evidence highlighted that new bone was connected directly with not only dentin-area matrix, but also cementum-area matrix of pDDM.

Clinical Case
A 66-year-old female presented with healing failure in the extracted cavity (#36) for 2 months after the extraction in a private dental clinic. The patient was introduced to our dental clinic in 2009. Clinical diagnosis revealed healing failure in the socket. Initial X-ray photos showed a clear remaining of lamina dura (#36), a residual root (#37), and a horizontal impaction (#38) (Figure 1). Her medical history was unremarkable. grafts between a parent and a child were successfully achieved, and the first successful procedure occurred in South Korea [10]. In this century, the tooth-derived materials have become a realistic alternative to bone grafting [7,11].
Highly calcified tissues such as fresh cortical bone and dentin do not occur earlier in osteoinduction and bone formation than DDM and demineralized bone matrix (DBM) [12][13][14][15]. In 2016, 70% of demineralized human DDM granules (size: 1.0 mm) had a better performance in bone formation than completely DDM (cDDM) and non-demineralized dentin in rat calvarial bone defects [16]. In 2021, cortical bone plate treated supersonically with acidic electrolyzed water (pH2.3), so-called partial DBM (pDBM), induced bone at 2 weeks, while fresh cortical bone plate did not induce bone even at 8 weeks [17]. In light of the articles [13][14][15][16][17], we believed that the apatite crystals of both calcified dentin and cortical bone should inhibit a release of bone morphogenetic proteins (BMPs) from matrices. Clinically, the immediate DDM autograft system needs speed, compared to the delayed autograft. Our team, therefore, developed the immediate autograft system of partially demineralized dentin/cementum matrix (pDDM) by using a new electric mill [3,18].
The aim of this case study was to observe the biopsy tissues obtained from the dental implant-placed region at 5 months after pDDM autograft into the socket that had exhibited healing failure (#36) for 2 months. The histological evidence highlighted that new bone was connected directly with not only dentin-area matrix, but also cementum-area matrix of pDDM.

Clinical Case
A 66-year-old female presented with healing failure in the extracted cavity (#36) for 2 months after the extraction in a private dental clinic. The patient was introduced to our dental clinic in 2009. Clinical diagnosis revealed healing failure in the socket. Initial X-ray photos showed a clear remaining of lamina dura (#36), a residual root (#37), and a horizontal impaction (#38) (Figure 1). Her medical history was unremarkable.
After the removing of epithelium and the cleaning of the socket by curettage, p rations into the dense bone in socket were performed by a curbide bur ( Figure 3A) the pDDM granules were grafted immediately into the treated socket ( Figure 3B). The after relaxation incision was sutured with nylon threads (Figure 3C,D). The patient took antibiotics for 2 days. After the removing of epithelium and the cleaning of the socket by curettage, perforations into the dense bone in socket were performed by a curbide bur ( Figure 3A), and the pDDM granules were grafted immediately into the treated socket ( Figure 3B). The flap after relaxation incision was sutured with nylon threads ( Figure 3C,D). The patient only took antibiotics for 2 days.

Surgical Procedure 2 and Tissue Biopsy
At 5 months after surgery 1, tissue biopsy (hole: 3.0 mm) was carried out from the central area of a fixture placement for the tissue observation under local anesthesia ( Figure 4A-C). Next, a titanium-fixture (HAp coating type: diameter, 4.2 mm; length, 10 mm; POI system, KYOCERA Co., Ltd., Kyoto, Japan) was implanted into the regenerated bone ( Figure 4D), and the flap was repositioned. The patient took antibiotics for 3 days. There were no troubles until the finishing of whole treatments.

Surgical Procedure 2 and Tissue Biopsy
At 5 months after surgery 1, tissue biopsy (hole: 3.0 mm) was carried out from the central area of a fixture placement for the tissue observation under local anesthesia ( Figure  4A-C). Next, a titanium-fixture (HAp coating type: diameter, 4.2 mm; length, 10 mm; POI system, KYOCERA Co., Ltd. Kyoto, Japan) was implanted into the regenerated bone (Figure 4D), and the flap was repositioned. The patient took antibiotics for 3 days. There were no troubles until the finishing of whole treatments ( Figure 4E).

Surgical Procedure 2 and Tissue Biopsy
At 5 months after surgery 1, tissue biopsy (hole: 3.0 mm) was carr central area of a fixture placement for the tissue observation under local a 4A-C). Next, a titanium-fixture (HAp coating type: diameter, 4.2 mm; le system, KYOCERA Co., Ltd. Kyoto, Japan) was implanted into the regen ure 4D), and the flap was repositioned. The patient took antibiotics for 3 no troubles until the finishing of whole treatments ( Figure 4E).

Radiographic Evaluation
Initial X-ray photos including CT were taken in 2009 before pDDM graft. Next, X-ray photos were taken just after the pDDM graft ( Figure 3D) (Surgical Procedure 1), and before and after the fixture placement (Surgical Procedure 2).

Tissue Preparation
Biopsy tissue was fixed with 10% neutral buffered formalin, decalcified for 7 days with 10% formic acid, and embedded in paraffin. Sections (with a thickness of 4 µm) were stained with hematoxylin and eosin (HE).

Gross View and Radiographic Evaluation
Before the pDDM graft (Surgery 1), the lamina dura (#36) remained clear. (Figure 1A,C). Just after the pDDM graft, pDDM were seen like radio-opaque granules in the treated socket ( Figure 3D). At 5 months (Surgery 2), a smooth surface line was seen on the graft site after the opening of mucoperiosteal flap ( Figure 4A). The grafted pDDM was harmonized with the mandible, and a bone-like radio-opacity was found in the graft region ( Figure 4B). A titanium-fixture was placed properly after the biopsy ( Figure 4C,D). The final crown was set ( Figure 5A,B) and functionality was well maintained. During the whole follow-up period, a complication, such as marginal bone loss, did not occur ( Figure 5C).

Radiographic Evaluation
Initial X-ray photos including CT were taken in 2009 before pDDM gra photos were taken just after the pDDM graft ( Figure 3D) (Surgical Proced fore and after the fixture placement (Surgical Procedure 2).

Tissue Preparation
Biopsy tissue was fixed with 10% neutral buffered formalin, decalcif with 10% formic acid, and embedded in paraffin. Sections (with a thickness stained with hematoxylin and eosin (HE).

Gross View and Radiographic Evaluation
Before the pDDM graft (Surgery 1), the lamina dura (#36) remained 1A,C). Just after the pDDM graft, pDDM were seen like radio-opaque g treated socket ( Figure 3D). At 5 months (Surgery 2), a smooth surface line w graft site after the opening of mucoperiosteal flap ( Figure 4A). The grafte harmonized with the mandible, and a bone-like radio-opacity was found gion ( Figure 4B). A titanium-fixture was placed properly after the biopsy The final crown was set ( Figure 5A,B) and functionality was well maintain whole follow-up period, a complication, such as marginal bone loss, did no 5C).

Histological Findings of Biopsy Tissue at 5 Months after pDDM Autograft
The biopsy tissue showed that mature bone was interconnected with pDDM granules (Figure 6A,B). pDDM included a small patch of cementum ated bone was connected directly with dentin-and cementum-area matrix dues ( Figure 6C,D). The dentin-derived areas revealed dentinal tube spac matrix, while the cementum-derived area showed Sharpey's fibers structu The boundary line between the dentin-and cementum-area matrices wa ( Figure 6C,D).

Histological Findings of Biopsy Tissue at 5 Months after pDDM Autograft
The biopsy tissue showed that mature bone was interconnected with the remained pDDM granules (Figure 6A,B). pDDM included a small patch of cementum. The regenerated bone was connected directly with dentin-and cementum-area matrix of pDDM residues ( Figure 6C,D). The dentin-derived areas revealed dentinal tube spaces and acellur matrix, while the cementum-derived area showed Sharpey's fibers structure ( Figure 6D).
The boundary line between the dentin-and cementum-area matrices was seen clearly ( Figure 6C,D).

Discussion
The biopsy tissue at 5 months after pDDM graft showed that the pDDM were received by the host and were harmonized with bone. Very interestingly, the histological evidence highlighted that newly formed bone was connected directly with dentin-and cementum-area matrices of the pDDM. This is novel evidence related to the direct bonding of new bone and demineralized cementum in clinical cases. We found that pDDM facilitated its adaption of the grafted site and was slowly absorbed as new bone began to form. As pDDM granules contain non-demineralized core (calcified matrix), the absorption of pDDM should take much more time than that of cDDM.
Dentin and bone are mineralized tissues and almost similar in chemical components. As bone and dentin consist of fluid (10%), collagen (20%), and apatite (70%) in weight volume [2,11], our attention for biomaterials focus on natural components such as collagen and apatite materials [19]. Both freeze-dried cDDM and cDBM are predominately composed of type I collagen (95%) and the remaining of non-collagenous proteins that contain a small amount of growth factors [20]. Both mature and immature types of bone morphogenetic protein-2 (BMP-2) were detected in human dentin and dental pulps by western-blotting [21]. Even after the demineralization of dentin and bone, active types of BMPs bind to acid-insoluble and collagen-rich matrices [11,22]. Interestingly, the strong acid treatment for bone-and dentin-derived materials increased their osteoinductivity and decreased their antigenicity [23]. Reduction and alkylation of BMP molecules resulted in the total loss of biological activity [24]. Additionally, after the processing system with 2.0%HNO3-demineralization as shown in Figure 2, bacteria free of pDDM granules were confirmed in the blood-agar medium [25]. From clinical points of view, sterilization by strong acid and safety are very important evidences for the processing procedures of hard

Discussion
The biopsy tissue at 5 months after pDDM graft showed that the pDDM were received by the host and were harmonized with bone. Very interestingly, the histological evidence highlighted that newly formed bone was connected directly with dentin-and cementumarea matrices of the pDDM. This is novel evidence related to the direct bonding of new bone and demineralized cementum in clinical cases. We found that pDDM facilitated its adaption of the grafted site and was slowly absorbed as new bone began to form. As pDDM granules contain non-demineralized core (calcified matrix), the absorption of pDDM should take much more time than that of cDDM.
Dentin and bone are mineralized tissues and almost similar in chemical components. As bone and dentin consist of fluid (10%), collagen (20%), and apatite (70%) in weight volume [2,11], our attention for biomaterials focus on natural components such as collagen and apatite materials [19]. Both freeze-dried cDDM and cDBM are predominately composed of type I collagen (95%) and the remaining of non-collagenous proteins that contain a small amount of growth factors [20]. Both mature and immature types of bone morphogenetic protein-2 (BMP-2) were detected in human dentin and dental pulps by western-blotting [21]. Even after the demineralization of dentin and bone, active types of BMPs bind to acidinsoluble and collagen-rich matrices [11,22]. Interestingly, the strong acid treatment for bone-and dentin-derived materials increased their osteoinductivity and decreased their antigenicity [23]. Reduction and alkylation of BMP molecules resulted in the total loss of biological activity [24]. Additionally, after the processing system with 2.0% HNO 3 -demineralization as shown in Figure 2, bacteria free of pDDM granules were confirmed in the blood-agar medium [25]. From clinical points of view, sterilization by strong acid and safety are very important evidences for the processing procedures of hard tissuederived graft materials [23][24][25]. After the immediate autograft of pDDM, thw patient was successfully restored to health with a dental implant. In the near future, ultrasound imaging will support histological findings and/or radiographic appearances as a noninvasive diagnostic tool [26].

Conclusions
The biopsy tissue at 5 months after immediate autograft of pDDM demonstrated novel histological evidences that new bone was connected directly with dentin-and cementumarea matrix of pDDM. A vital tooth-derived pDDM granules contributed to regenerate bone in an unhealed socket in a 66-year-old woman. After the pDDM autograft, the patient was successfully restored with a dental implant. Autogenous pDDM could be immediately recycled in our system as an innovative biomaterial for local bone engineering.

Institutional Review Board Statement:
The study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committee of Health Sciences University of Hokkaido (approval code No.29) for studies involving humans.