RESEARCH Open Access

The effect of autogenous tooth bone graftmaterial without organic matter and type Icollagen treatment on bone regenerationMin-Gu Kim1, Jung-Han Lee1, Gyoo-Cheon Kim2, Dae-Seok Hwang1, Chul-Hun Kim3, Bok-Joo Kim3,Jung-Han Kim3 and Uk-Kyu Kim1*

Abstract

Objectives: The aim of this study is to examine the effect of particulate autogenous tooth graft removed withorganic matter and type I collagen addition on bone regeneration and to validate the possibility of useful allograftmaterial for jaw defects.

Material and methods: Autogenous tooth bone maker (Korean Dental Solution® KOREA) made particulateautogenous tooth not including organic matter. We used to the developed tooth grafts for experiment. Celladhesion test with hemacytometer and energy dispersive X-ray spectroscopy (Supra40 VP®, Carl Zeiss, Germany)analysis about the particulate autogenous tooth and type I collagen were performed. Rabbits were divided intothree groups: bone graft with organic matter (OM) removing particulate autogenous tooth group, bone graft withOM removing particulate autogenous tooth and type I collagen group, and a control group. Bone grafting wasperformed in rabbit’s calvaria. The rabbits were sacrificed at different interval at 1, 2, 4, and 6 weeks after bonegrafting for the histopathologic observation and observed the effect of bone regeneration by SEM, H-E & Massonstains, osteocalcin IHC staining.

Result: In vitro cytopathological study showed affinity for cells, cell attachment pattern, and cell proliferation in theorder of control group, OM-removed and collagen-treated group, OM-removed particulate autogenous toothgroup. The results of the degree of mineralization were opposite to those of the previous cell experimental results,and the OM-removed group, OM-removed group and collagen-treated group were relatively higher than thecontrol group. Histopathologic analysis showed that vascularization and neonatal bone formation were higher inparticulate autogenous tooth group with removing OM and with addition of collagen than control group andgroup of OM removed only. Immunohistochemical analysis showed that osteocalcin (OSC) expression was notobserved in the control group, but at 4 weeks groups, OSC expression was observed the OM removed and OM-removed-collagen-treated particulate autogenous tooth, and the degree of expression was somewhat stronger ingroup of the OM removed and collagen additionally treated particulate autogenous tooth.

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* Correspondence: kuksjs@pusan.ac.kr1Department of Oral and Maxillofacial Surgery, School of Dentistry, PusanNational University, Yangsan 50612, Republic of KoreaFull list of author information is available at the end of the article

Maxillofacial Plastic andReconstructive Surgery

Kim et al. Maxillofacial Plastic and Reconstructive Surgery (2021) 43:17 https://doi.org/10.1186/s40902-021-00302-w

Conclusion: Particles that do not contain organic matter, the saint tooth, was responsible for sufficient bone graftmaterial through the role of space maintenance and bone conduction, and further improved bone formation abilitythrough additional collagen treatment. Therefore, research on various extracellular substrates and autologous bonegrafting materials is necessary, and through this, it is possible to lay the foundation for a new type of autologousbone grafting material with excellent academic and technical utility.

Keywords: Tooth bone graft material, Type I collagen, Autogenous graft

IntroductionIn various fields of oral-maxillofacial surgery, bone graftprocedures for the reconstruction of soft tissue defectsare being performed. At the same time, with the growingpopularity of dental implant procedures, cases wherepatients with alveolar bone deficiency are given acombined treatment of implantation and bone defectreconstruction rather than the conventional denturetreatment, are increasing.Bone graft materials are categorized into autogenous,

allogenic, heterologous, and alloplastic bone based onthe origin and immunological properties. Among them,autogenous bone is the most ideal graft material withrespect to stability as it exhibits all three osteogenic,osteoinductive, and osteoconductive potentials, but thelimitations are the collectible amount, the patientdiscomfort and complications after collection [1–3]. Tocomplement these limitations, many studies focused onallogenic or heterologous bone, whose use increasedconsequently, but these two types also have limitationsincluding the possibility of disease infection and immunerejection, lack of osteogenic potential, and high cost.The disease infection and immune rejection, in particu-lar, are the factors that drive the patients to disfavor thetreatment [4–6]. In the case of alloplastic bone, preparedthrough artificial synthesis of bone-like components, thefact that it has only the osteoconductive function andnot the osteogenic or osteoinductive function poses limi-tations to its clinical application despite partial comple-mentation of the drawbacks of other graft materials andthe low cost.Based on the merits and demerits of the conventional

bone graft materials, an ideal graft material should (i)contain a diversity of bone morphogens with osteoin-ductive property; (ii) not cause immune rejection inpatients; and (iii) maintain the stability of the graftedarea as well as promoting rapid revascularization andosteogenesis. The ease of collection and affordabilityshould also be taken into consideration.Recently, autogenous tooth bone graft material

(AutoBT) that uses the tooth extracted from the patient,has attained much attention. AutoBT is generally pre-pared based on the demineralization method usinghydrogen chloride (HCl) or nitric acid (HNO3), follow-ing a process of washing and grinding of the extracted

tooth. The strongest advantage of AutoBT is that itshistological structure and components resemble those ofautogenous bone [7, 8]. As it is an autogenous tissue,AutoBT ensures excellent biocompatibility without im-mune rejection, which gives emotional stability and sat-isfaction to the patient. In addition, many recent studieshave reported that AutoBT exhibits not only osteocon-ductive but also osteoinductive potential [9–15].Among the dental components, enamel comprises 95%

inorganic and 0.6% organic matter with 4% water,whereas dentin comprises 70-75% inorganic and 20%organic matter with 10% water. The alveolar bone has asimilar chemical composition to enamel and dentin,comprising 65% inorganic and 25% organic matter with10% water in general. Based solely on the chemical com-position, tooth exhibits relatively higher content of inor-ganic matter and lower content of organic matter thanalveolar bone. The inorganic content in tooth and alveo-lar bone is mostly hydroxyapatite, while the organic mat-ter mostly consists of type I collagen as well as a bonegrowth factor known as bone morphogenic proteins(BMPs) [7, 8]. Considering the individual roles of eachdental component, the type and ratio of the componentsin a bone graft material is a crucial determinant of theproperties of the graft material. Notably, regardingosteoconductive, osteoinductive, and osteogenic poten-tials, the influence is highly significant. To illustrate,among the components of a bone graft material, whenthe ratio of organic matter including type I collagen andBMPs falls, the osteoinductive potential is reduced,thereby increasing the time taken for bone formation.Nonetheless, the relative increase in the ratio ofinorganic matter with high calcium content leads toenhanced osteoinductive potential with the effect ofreduced bone resorption following implantation.Several studies have found that the conventional

AutoBT exhibits an outstanding osteogenic potential [9–15].However, considering the characteristics of the componentsof AutoBT, an alteration in the ratio among the componentsor an addition of an organic matter is anticipated to bringabout an enhanced bone repair effect after the bone graft.This study thus applied a new technique to reduce the ratioof organic content in a novel type of AutoBT compared tothe conventional one, then grafted the novel AutoBT to therabbit calvaria to examine the bone repair capability, and

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during the process, monitored the changes in the bone re-pair process after the supplementary addition of type I colla-gen that composes most of the organic content of tooth.

Materials and methodsPreparation of AutoBT without organic matter (OM)The organic substances in the human dental tissue wereremoved and the tissue was subsequently washed,through the use of autogenous tooth bone maker(Korean Dental Solution®, Busan, Korea) and usingsodium chlorate (NaOCl). Next, lipid and proteincomponents in the dental tissue were removed usingethyl alcohol and chloroform, and the tissue wasbleached and sterilized using hydrogen peroxide. After 5times washing, sterilized AutoBT without OM was prepared.

Cell cultureHuman osteosarcoma cell lines, MG63 and HOS, werepurchased from ATCC (Rockville, MD, USA). The twodifferent cell lines were immersed in DMEM/F12 1:1medium and RPMI1640 medium, respectively, then afterthe addition of 100 μg/ml penicillin/streptomycin, 4 mML-glutamine, and 10% fetal bovine serum (FBS), the cellswere cultured in a 5% CO2 incubator at 37 °C.

Cell adhesion activity measurementThe prepared specimens were sterilized, then after thesubculture of 5 × 103 cells, the cells were cultured for 24h. The specimens were washed twice using phosphate-buffered saline (PBS), after which the attached cells wereleft in trypsin-EDTA for 5 min at 37 °C. Using thehemacytometer, the cells were counted, and the celladhesion rate was calculated.

Cell viability measurementTo measure the cell survival rate, the WST-1 methodwas followed. A flat specimen on a 6-well plate was pre-pared, then after the subculture of 5 × 103 cells on theplate, the cells were cultured for 24, 48, and 72 h. Next,10 μl of WST-1 solution was added to each well for thereaction in an incubator for 2 h, and using the ELISAreader (Tecan, Mannedorf, Swiss), the OD was measuredat 450 nm. The analysis was repeated three times foreach cell line.

SEM observationFor the observation of the adhesion pattern of the cellson the specimens, their images were taken using thescanning electron microscopy (SEM; S3500N, HITACHI, Japan). After the subculture of cells on the dentalspecimens and the addition of medium, the cells werecultured in a 5% CO2 incubator at 37 °C for 24 h. Then,the specimens to which cells were attached were fixedusing 2.5% glutaraldehyde (Sigma-Aldrich, USA) for 30

min. The specimen preparing was described previously[16]. The specimens were treated with 1% osmium tet-roxide (Sigma-Aldrich, USA) for 30 min and dehydratedusing ethanol (Sigma-Aldrich, USA) from low concen-tration to high concentration (70%, 80%, 90%, and100%). They were then dried using hexamethyldisilazane(HMDS; Sigma-Aldrich, USA). All specimens wereimmobilized on an aluminum plate and coated usinggold sputter in vacuum state. SEM was operated at 15kV accelerating voltage. All images were photographedat ×2000 and ×5000 magnification, and the resulting im-ages were selected at random for the observation.

Bone mineralization assayFor the determination of the mineralization ability ofosteoblasts on the specimens through quantificationof the newly formed layers and crystals on the speci-mens, the energy dispersive X-ray spectroscopy (EDS,Supra40 VP, Carl Zeiss, Germany) analysis was car-ried out. The line scan using the EDS and the com-ponent analysis for a defined area were carried out at15 kV, ×40 magnification. The dentin specimens weredivided into the top, bottom, left, and right, to whicha same size line was specified, then the contents ofcalcium (Ca) and phosphorous (P) were measuredalong the line. Also, by defining a same size areaaround the line at the center, the values of Ca and Pwere measured within the area. Based on each set ofCa and P values, the Ca/P ratio was obtained and themineralization ability was assessed.

Animal experimentsTo the calvaria of 12 rabbits (weight 3.0-3.5 kg, NewZealand), four same size defects (8 mm) were madefor the control, experimental group A and experi-mental group B, and after applying human AutoBTwithout OM and type I collagen to the defects, threerabbits were sacrificed each at weeks 1, 2, 4, and 6,for monitoring the level of bone formation on thecalvarial tissue. No treatment was applied to the de-fect site of the control; AutoBT without OM wasgrafted to the defect site of the experimental groupA; AutoBT without OM and 100 mg/ml type I colla-gen were simultaneously grafted to the defect site ofthe experimental group B. The animals used in theexperiments were 8-month-old rabbits between 3.0kg and 3.5 kg that were bred in a standard labora-tory where temperature of 25 ± 1 °C and humidityof 53 ± 5% were maintained, and all experimentsand their plans for animal management and dietcontrol were based on the guidelines and regulationsof the Institutional Animal Care and User Commit-tee (IACUC; Pusan National University. PNU-2017-1446) (Figs. 1, 2).

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H-E stainingThe bone tissue of each rabbit was fixed in 10% neutralbuffered formalin solution for 24 h. The tissue was slicedto 5 μm and hematoxylin and eosin (H&E) staining wasperformed. The changes in each tissue were monitoredusing the optical microscope (Pixel link PL-B686 CU,Canada) for the observation of the H-E stained tissuespecimens with respect to the level of bone tissue repairat the defect site.

Masson’s trichrome stainingThe bone tissue of each rabbit was fixed in 10% neutralbuffered formalin solution for 24 h. The tissue was slicedto 5 μm and Masson’s trichrome staining was per-formed. The changes in each tissue were monitoredusing the optical microscope (Pixel link PL-B686 CU,Canada) for the observation of bone formation in bonetissue repair around the AutoBT graft area at the defectsite.

ImmunohistochemistryThe paraffin section was placed in a 68 °C oven for 1 h,and washed three times with xylene for 7 min each for

paraffin removal, then three times with 100% ethanol for5 min each for xylene removal. The section was thenrinsed with 90%, 80%, and 70% ethanol in this order, for5 min each. The rinsed section was washed with PBSthree times for 5 min each to remove alcohol com-pletely. Next, the Mouse and Rabbit Specific HRP/DAB(ABC) Detection IHC Kit (Abcam Inc., Cambridge, UK)was used. Subsequently, the production of osteocalcinwas monitored using the Slide Scanner (Axio Scan.Z1,Germany).

Statistical analysisStatistical analysis data were expressed ± SD from atleast three independent experiments. Statistical analysesused GraphPad Prism version 5.0 for Windows (Graph-Pad Software, San Diego, CA). A one-way ANOVA wasused for Dunnett’s multiple-comparison test in the stat-istical analysis.

ResultsCell adhesion rateFirst, a cell adhesion test was conducted to examine theaffinity for cells of the specimens of human AutoBTwithout OM prepared using the autogenous tooth bonemaker. HOS cell adhesion rate was 100%, 69%, and81.3%, respectively, in the control with cell adhesion totooth surface, in the OM-removed group with cell adhe-sion to AutoBT without OM, and in the OM-removedgroup to which type I collagen was added. MG63 celladhesion rate was 100%, 61.4%, and 74.3%, respectively,in the control, in the OM-removed group, and in theOM-removed-collagen-treated group (Fig. 3).

Cell adhesion morphologySEM was used for the observation of cell adhesionmorphology. MG63 and HOS cells were each dividedinto three groups: the control, the OM-removed group,and the OM-removed, collagen-treated group, then cul-tured on tooth specimens. HOS cells showed good sur-face adhesion in the control specimens, where the cellswere seen to maintain a large number of spindle-likeprotrusions for adhering to the surface, whereas theOM-removed specimens showed weak surface adhesionand only a small number of adhering cells. The OM-removed group to which type I collagen was addedshowed the cells with spindle-like protrusions but manycells appeared steric in volume due to weak adhesion.MG63 cells, despite different morphology of adheringcells, displayed a similar adhesion pattern to HOS cells(Fig. 4).

Cell proliferation rateThe time-dependent cell proliferation rate of HOS cellswas 100%, 150%, and 220% in the control; 54%, 124%,

Fig. 1 A photo showing the rabbit calvaria after dissection

Fig. 2 A photo showing four same size defects (8 mm) at thedissected rabbit calvaria after the graft of AutoBT and type I collagen

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and 150% in the OM-removed group; 61%, 135%, and170% in the OM-removed-collagen-treated group. Asignificantly higher rate was exhibited by the OM-removed-collagen-treated group in comparison to theOM-removed group (Fig. 5a).The time-dependent cell proliferation rate of MG63

cells was 100%, 161%, and 232% in the control; 52%, 93%,and 133% in the OM-removed group; 90%, 221%, and232% in the OM-removed-collagen-treated group. Similarto the HOS cells, the rate was higher in the OM-removedgroup to which type I collagen was added (Fig. 5b).

Mineralization assayThe ratio between calcium (Ca) and phosphorous (P)contents in HOS and MG63 cells was estimated, to

determine the degree of mineralization in the threegroups: control, OM-removed group, and OM-removed-collagen-treated group. HOS cells showed 100% in thecontrol; 176% in the OM-removed group; 180% in theOM-removed-collagen-treated group, while MG63 cellsshowed 100% in the control; 139% in the OM-removedgroup; 141% in the OM-removed-collagen-treated group.The degree of mineralization in both cell lines was shownto be higher in the OM-removed and the OM-removed-collagen-treated groups than in the control (Fig. 6).

Histological findingsH-E stainingIn the control, the neonatal bone formation was negli-gible in weeks 1 and 2, while in week 4, a large number

Fig. 3 A graph showing the cell adhesion activity of HOS cells (A) and MG63 cells (B). Each value represents the mean ± S.D. *p < comparedwith control

Fig. 4 AutoBT-grafted cell adhesion morphology of HOS cells (A) and MG63 cells (B)

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of blood vessels and compact connective tissue were ob-served at the defect site with the start of bone fragmentformation upon intramembranous ossification. In week6, most of the space at the defect site was filled withbone tissue, and compared to the original bone tissue inthe vicinity; the neonatal tissue was thin due to insuffi-cient bone matrix. A neat alignment of osteoblasts wasseen around the neonatal bone tissue, and an abundanceof adipose tissue was prominently observed (Fig. 7).In the OM-removed experimental group A, compact

connective tissue was observed surrounding the graftmaterial at the defect site in week 1, and a large numberof blood vessels were seen to have started forming. Inweek 2, a large number of osteoclasts were observedaround the graft material, with the steady formation ofneonatal bone tissue in the area. In week 4, far more

concentrated compact connective tissue was observed,compared to week 2, with osteoclasts and osteoblastsaround the graft material and neonatal bone formation.In week 6, a lot more blood vessels have formed, and theneonatal bone tissue around the graft material could beseen to have thickened (Fig. 8).In the OM-removed-collagen-treated experimental

group B, neonatal bone formation and vascularizationare predominantly seen under the periosteum at the de-fect site in week 1. The most substantial difference inweek 2 is the high level of vascularization around thegraft material in comparison to the other groups, withthe neonatal bone tissue under the periosteum havingmatured further. In week 4, the density of the compactconnective tissue around the graft material filling the de-fect site, could be seen to have increased, while a large

Fig. 5 A graph showing the time-dependent cell proliferation rate of HOS cells (A) and MG63 cells (B). Each value represents the mean ± S.D. *p< 0.05, **p < 0.01 compared with control

Fig. 6 A graph showing the mineralization ratio of HOS cell secretion (A) and MG63 cell secretion (B). Each value represents the mean ± S.D. *p< 0.05, **p < 0.01 compared with control

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number of osteoclasts were observed around the graftmaterial and at the same time, the neonatal bone forma-tion at the end of the graft material. In week 6, manydents were seen to have formed around the graft mater-ial, with a high level of neonatal bone formation in thevicinity. A pattern where myriads of osteoblasts were ad-hering to the area surrounding the graft material couldbe seen, and the neonatal bone formation was even moreelaborate than experimental group A (Fig. 9).

Masson’s trichrome stainingIn the control, the neonatal bone formation was negli-gible in weeks 1 and 2, while in week 4, the defect sitewas filled mostly with red, immature bone tissue, and in

week 6, the site was filled mostly with blue, mature bonetissue (Fig. 10).In the OM-removed experimental group A, the graft

material was seen to be reliably filling the defect sitewith compact connective tissue immersing the graft ma-terial. A similar pattern of neonatal bone formation wasseen in weeks 2, 4, and 6. The blue expression of colla-gen among the compact connective tissue could be ob-served, but the growth into mature, mineralized bonetissue had not yet occurred (Fig. 11).In the OM-removed-collagen-treated experimental

group B, the difference in comparison to other groupswas not significant in week 1, whereas in weeks 2 and 4,neonatal bone formation began to be observed in the

Fig. 7 Microphotograph at 1 week (A), 2 weeks (B), 4 weeks (C), and 6 weeks (D) after bone graft in control rabbit. H-E. ×50

Fig. 8 Microphotograph at 1 week (A), 2 weeks (B), 4 weeks (C), and 6 weeks (D) after bone graft with organic matter removed particulateautogenous tooth in experimental rabbit. H-E. ×50

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area surrounding the graft material, and in week 4, colla-gen expression among the compact connective tissueimmersing the graft material could be seen. In week 6,more intensive neonatal bone formation was detected,with stronger expression of collagen among the compactconnective tissue (Fig. 12).

Osteocalcin immunohistochemical stainingIn the control, the expression of osteocalcin (OSC) wasnot observed in weeks 1 and 2 as neonatal bone forma-tion was negligible, and OSC expression was still not ob-servable in weeks 4 and 6. In addition, blood vessels andred blood cells in the form of brown OSC expressionwere observed (Fig. 13).In the OM-removed experimental group A, OSC ex-

pression was absent in weeks 1 and 2, then it appeared

in week 4, but disappeared in week 6. OSC expression inneonatal bone tissue was weak overall, and it was ob-served in parts of blood and the implanted bone graftmaterial (Fig. 14).In the OM-removed-collagen-treated experimental

group B, OSC expression was absent in weeks 1 and 2,then it appeared in week 4, while in week 6, OSC ex-pression in neonatal bone tissue was observed althoughthe level of expression was weak (Fig. 15).

DiscussionIn the reconstruction of hard tissue defects, the mostideal bone graft material is the autogenous bone. Theautogenous bone exhibits osteogenic, osteoinductive,and osteoconductive potentials simultaneously, while itcauses no immune rejection and has the benefit of rapid

Fig. 9 Microphotograph at 1 week (A), 2 weeks (B), 4 weeks (C), and 6 weeks (D) after bone graft with organic matter removed particulateautogenous tooth and type 1 collagen in experimental rabbit. H-E. ×50

Fig. 10 Microphotograph at 1 week (A), 2 weeks (B), 4 weeks (C), and 6 weeks (D) after bone graft in control rabbit. Masson. ×50

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healing. However, the downside is the limitation of col-lectible amount and the need for a donor site [17]. Toresolve the limitations of allogenic, heterologous, andalloplastic bones, there is an ongoing research and devel-opment on various methods, and furthermore, it is ne-cessary to develop an ideal bone graft material like theautogenous bone [4–6].Recently, a bone graft material based on extracted au-

togenous tooth has been developed and applied to clin-ical practice, as the material overcoming the demerits ofallogenic, heterologous, and alloplastic bones. The au-togenous tooth bone graft material (AutoBT) has been

developed based on the close correspondence betweendental components and bone components. The elaboratecollagen structure of tooth provides an ideal environ-ment for the differentiation and proliferation of cells es-sential in new bone formation, such as bonemesenchymal stem cells and osteoblasts. As an autogen-ous tissue, AutoBT ensures excellent biocompatibilitywithout immune rejection, and together with the factthat there is no concern for disease infection or need fora donor site [2, 3], the emotional rejection patients usedto display before allogenic or heterologous bone graftcan be eliminated. AutoBT has also been reported to

Fig. 11 Microphotograph at 1 week (A), 2 weeks (B), 4 weeks (C), and 6 weeks (D) after bone graft with organic matter removed particulateautogenous tooth in experimental rabbit. Masson. ×50

Fig. 12 Microphotograph at 1 week (A), 2 weeks (B), 4 weeks (C), and 6 weeks (D) after bone graft with organic matter removed particulateautogenous tooth and type 1 collagen in experimental rabbit. Masson. ×50

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exhibit not only osteoconductive but also osteoinductivepotential [9–15], and it can be produced in diverse sizesand forms to allow easy application.The early research on the use of tooth as a graft

material mainly focused on inorganic matter, especiallyon hydroxyapatite. The high-temperature treatment oftooth led to the collection of hydroxyapatite and cre-ation of organic matter-removed particulate dentin thatwas reported to be suitable as a graft material for in-creasing the alveolar bone. The burnout of toothremoves the organic matter to leave the inorganic hy-droxyapatite and β-TCP as the main components, andthis is referred to as particulate dentin. The graft ofparticulate dentin mediates rapid resorption and substi-tution by a bone tissue at an early stage, followed by a

pattern of reduced resorption. This is due to the com-position of particulate dentin; the early rapid resorptionis due to β-TCP and the later slow resorption pattern ismainly due to hydroxyapatite. Such burnout method re-sembles the method of processing heterologous bone,and a high-temperature treatment of tooth forms highcrystalline carbonic apatite to cause low resorption inthe body, which may have an adverse effect on bone for-mation [10, 11].Hydroxyapatite is the main component of the inor-

ganic matter that composes 95% of enamel and 70-75%of dentin, and it plays a crucial role in maintaining thevolume of bone graft material and in osteoconduction.The organic matter in dentin is approximately 90% col-lagen, mostly type I collagen, which plays a key role in

Fig. 13 Microphotograph at 1 week (A), 2 weeks (B), 4 weeks (C), and 6 weeks (D) after bone graft in control rabbit. Osteocalcin. ×50

Fig. 14 Microphotograph at 1 week (A), 2 weeks (B), 4 weeks (C), and 6 weeks (D) after bone graft with organic matter removed particulateautogenous tooth in experimental rabbit. Osteocalcin. ×50

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calcification. The rest of the organic matter comprisesnon-collagenous proteins, carbohydrate, lipid, citrate,and lactate [9]. It is known that proteins have variousbone growth factors including BMPs that play significantroles in the osteoinductive potential of AutoBT [14].Recent studies have focused on the method of

demineralization that removes the inorganic matteramong the dental components of AutoBT while leavingthe organic matter. Rather than the heat treatmentmethod to produce particulate dentin, a reagent is usedto demineralize the tooth to remove the inorganic mat-ter, with the product being used in a form of powder orblock. The reduced proportion of inorganic content inthe graft material induces neonatal bone formation aswell as the process of resorption and substitution of thegrafted material by a bone tissue, which may improvethe beneficial effect of the graft material on bone regen-eration. However, the rapid resorption of demineralizedgraft material may lead to inadequate ability to maintainthe space and a drawback of excessive resorption of thegraft material [18].This study thus set out to examine the osteogenic po-

tential of AutoBT after the removal of organic matter inthe dental tissue using sodium chlorate (NaOCl), ethylalcohol, and chloroform, as well as that after the supple-mentary addition of type I collagen constituting over90% of the organic matter of tooth. The in vitrocytological experiments showed that the affinity for cellswas the highest in the control, followed by the OM-removed-collagen-treated group and the OM-removedgroup, while cell adhesion morphology also showed adecrease in adhesion in the above order. A similar resultwas obtained for the cell proliferation rate. This is a con-sequence of cell induction effect from the addition of

type I collagen, and combining AutoBT with theaddition of type I collagen was shown to have led to en-hanced osteoinductive potential. The estimated degreeof mineralization showed a contrasting result, wherecomparatively higher values were obtained for the OM-removed group and the OM-removed-collagen-treatedgroup than the control group. This is determined to bedue to the increased bone density from the removal oforganic matter, which is likely to help AutoBT toenhance the osteoconductive potential and maintain thebone volume.In addition, this study monitored the changes in rabbit

calvarial tissue after having created four same size de-fects (8 mm) and applied AutoBT without OM and typeI collagen. In the control, no treatment was given to thedefect site, whereas in the experimental group A, an in-dependent treatment of a graft using AutoBT withoutOM was given to the defect site, and in the experimentalgroup B, a combined treatment of a graft using AutoBTwithout OM and a simultaneous addition of 100 mg/mltype I collagen was given to the defect site. At the end ofweeks 1, 2, 4, and 6, the rabbits were sacrificed and thelevels of bone formation were comparatively observed.Histopathologic and immunohistochemical analyseswere also conducted.The result of histopathologic analysis showed that, in

the experimental group grafted with AutoBT withoutOM, the formation of connective tissue and blood vesselwas observed around the graft material at an earlierperiod than the control, while neonatal bone formationwas also detected at an early stage. In the experimentalgroup grafted with AutoBT without OM and addition-ally administered with type I collagen, more intensevascularization was observed at an earlier period,

Fig. 15 Microphotograph at 1 week (A), 2 weeks (B), 4 weeks (C), and 6 weeks (D) after bone graft with organic matter removed particulateautogenous tooth and type 1 collagen in experimental rabbit. Osteocalcin. ×50

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compared to the other experimental group and the con-trol, with neonatal bone formation detected at an earlierperiod. In week 4, a characteristic collagen expression inthe compact connective tissue immersing the graftmaterial began to be detected, and the expression inten-sified toward week 6. Also, in week 6, a high level ofneonatal bone formation could be seen, compared to theother experimental group and the control, with a largenumber of osteoblasts observed in the area around thegraft material. These findings indicated that, comparedto the control, the experimental group grafted withAutoBT exhibits neonatal bone formation at an earlierperiod, and the additional collagen further promotes theformation. In week 6, the experimental group thatreceived the treatment combining AutoBT and type Icollagen led to the detection of a larger number of oste-oblasts, which may implicate the possibility of furtherbone formation at a later stage. To conclude, thecombined treatment of AutoBT without OM and typeI collagen facilitates the neonatal bone formationwhile increasing the level of bone formation further,whereby the advantages of AutoBT as a graft materialare maximized.This study went on to conduct immunohistochemical

analysis to monitor the expressed level of osteocalcin(OSC), a protein among BMPs, in the tissue of the con-trol and each experimental group, in order to examinethe effects of AutoBT without OM and type I collagentreatment on bone formation. OSC is a non-collagenousprotein engaged in the accumulation of inorganic matterproduced in bone or tooth [19]. Previous studies havefound OSC expression in osteoblasts of the periodontalligament close to the alveolar bone upon tooth move-ment in rats, which was characterized by the expressionof OSC in the bone formation area but a lack of OSC inthe resorption area [20]. In humans, OSC is founduniquely in osteoblasts and dentinoblasts, and consider-ing this characteristic, OSC expression can serve as amarker for determining osteogenic potential in theexpressed area [21–23]. In this study, OSC expressionwas not observed in the control, but the expression wasdetected in both experimental groups A and B in week4. The expressed level was, however, far higher in theexperimental group B with additional type I collagentreatment. This was indicative of the strong osteogenicpotential by week 4 upon the combined treatment ofAutoBT without OM and type I collagen, and the fall inOSC expression in week 6 showed that neonatal boneformation was accompanied with a gradual decrease inosteogenic potential.The findings of this study suggested that AutoBT

without OM played a sufficient role as a bone graft ma-terial in the spatial retention and osteoconduction, andthat the osteogenic potential was enhanced by additional

type I collagen treatment. Nevertheless, the difference inthe ability as a bone graft material was not significant,which was presumed to be due to the low level of otherbone morphogens such as BMPs. Thus, it is necessarythat further studies focus on whether the supplementaryaddition of bone morphogens and AutoBT without OMcould carry out the role of a mediator.In this study, a graft material based on human au-

togenous tooth rather than rabbit tooth was used. Forthe animal experiments, human autogenous tooth bonegraft material was used unlike the previous studieswhere animal tooth was used, because it was predictedthat the graft of pre-treated human AutoBT withoutOM as in conventional cases of allogenic or heterol-ogous bone graft, would not cause immune rejection inrabbit calvaria, in addition to the issue of nutrient supplydue to extraction [24]. Based on the findings of thisstudy, AutoBT without OM and the addition of type Icollagen is likely to play a sufficient role as a bone graftmaterial in clinical practice. Nevertheless, studies shouldcontinue to focus on the immune rejection upon an allo-genic graft of AutoBT without OM.

ConclusionIn present study, we were to determine the osteogenicpotential of AutoBT from which organic matter of den-tal tissue was removed using sodium chlorate (NaOCl),ethyl alcohol, and chloroform through the use of the au-togenous tooth bone maker.

1. The affinity for cells was highest in the controlgroup, followed by OM-removed-collagen-treatedgroup, and OM-removed group in vitro. This is aresult of the cell-inducing effect of type I collagentreatment, and it has been shown that AutoBTcombined with the addition of type I collagen canenhance the bone induction potential. Theestimated degree of mineralization showedcontrasting results with the OM-removed group,the OM-removed-collagen-treated group showingrelatively higher values than the control group.

2. In the histopathological analysis, compared to thecontrol group, neonatal formation was observed inthe experimental group in which AutoBT wastransplanted without OM and the experimentalgroup in which AutoBT was grafted without OMand type I collagen was additionally administered,compared to the control group and otherexperimental groups, and also osteocalcin (OSC)was found to be slightly higher in the groupreceiving AutoBT and type I collagen.

Taken together, AutoBT without OM can be seen agraft material that exerts a positive effect on the grafted

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area by enhancing the osteogenic potential and the add-itional administration of type I collagen further enhancesthe osteogenic potential. Notably, a positive effect onneonatal bone formation was shown 4 weeks after thebone graft, suggesting a contribution to the possiblereduced bone resorption that may arise in the long runafter the graft, thereby ultimately leading to increasedbone volume.

AbbreviationsAutoBT: Autogenous tooth bone graft material; OM: Organic matter;SEM: Scanning electron microscopy; EDS: Energy dispersive X-ray spectros-copy; OSC: Osteocalcin

AcknowledgementsNot applicable

Authors’ contributionsMGK participated in the design of this study, contributed to conception, anddata collection, interpretation, analysis, drafting and writing manuscript. JHLand JHK participated in data collection, analysis and drafting and writingmanuscript. GCK participated in histopathological measurement andcontributed to vitro study’s design, vitro data analysis, and revision ofmanuscript. DSH, CHK, and BJK participated in reviewing and editing of thismanuscript. UKK participated to original thesis’s conception, design of thisstudy and critically revised the manuscript. All authors gave final approvaland agree to be accountable for all aspects of the work. The authors readand approved the final manuscript.

FundingNot applicable

Availability of data and materialsAll data generated or analyzed during this study are included in thispublished article.

Declarations

Ethics approval and consent to participateThis study protocol was approved by IRB of Pusan National University (IRBApproval No. PNU-2017-1446).

Consent for publicationAll authors were consented for publication of this article.

Competing interestsThe authors declare that they have no competing interests.

Author details1Department of Oral and Maxillofacial Surgery, School of Dentistry, PusanNational University, Yangsan 50612, Republic of Korea. 2Department of OralAnatomy, School of Dentistry, Pusan National University, Yangsan 50612,Republic of Korea. 3Department of Oral and Maxillofacial Surgery, College ofMedicine, Dong-A University, Pusan 49201, Republic of Korea.

Received: 22 March 2021 Accepted: 6 June 2021

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